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Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. Acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation. DGAT is an integral membrane protein that has never been purified to homogeneity, nor has its gene been cloned. We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate. Expression of a mouse cDNA for this expressed sequence tag in insect cells resulted in high levels of DGAT activity in cell membranes. No other acyltransferase activity was detected when a variety of substrates, including cholesterol, were used as acyl acceptors. The gene was expressed in all tissues examined: during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in DGAT activity. The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism and its regulation

Dof is a novel family of plant proteins that share a unique and highly conserved DNA binding domain with one C2-C2 zinc finger motif. Although multiple Dof proteins associated with diverse gene promoters have recently been identified in a variety of plants, their physiological functions and regulation remain elusive. In maize, Dof1 (MNB1a) is constitutively expressed in leaves, stems, and roots, whereas the closely related Dof2 is expressed mainly in stems and roots. Here, by using a maize leaf protoplast transient assay, we show that Dof1 is a transcriptional activator, whereas Dof2 can act as a transcriptional repressor. Thus, differential expression of Dof1 and Dof2 may permit leaf-specific gene expression. Interestingly, in vivo analyses showed that although DNA binding activity of Dof1 is regulated by light-dependent development, its transactivation activity and nuclear localization are not. Moreover, in vivo transcription and in vitro electrophoretic mobility shift assays revealed that Dof1 can interact specifically with the maize C4 phosphoenolpyruvate carboxylase gene promoter and enhance its promoter activity, which displays a light-regulated expression pattern matching Dof1 activity. We propose that the evolutionarily conserved Dof proteins can function as transcriptional activators or repressors of tissue-specific and light-regulated gene expression in plants

A putative yeast sn-2 acyltransferase gene (SLC1-1), reportedly a variant acyltransferase that suppresses a genetic defect in sphingolipid long-chain base biosynthesis, has been expressed in a yeast SLC deletion strain. The SLC1-1 gene product was shown in vitro to encode an sn-2 acyltransferase capable of acylating sn-1 oleoyl-lysophosphatidic acid, using a range of acyl-CoA thioesters, including 18:1-, 22:1-, and 24:0-CoAs. The SLC1-1 gene was introduced into Arabidopsis and a high erucic acid-containing Brassica napus cv Hero under the control of a constitutive (tandem cauliflower mosaic virus 35S) promoter. The resulting transgenic plants showed substantial increases of 8 to 48% in seed oil content (expressed on the basis of seed dry weight) and increases in both overall proportions and amounts of very-long-chain fatty acids in seed triacylglycerols (TAGs). Furthermore, the proportion of very-long-chain fatty acids found at the sn-2 position of TAGs was increased, and homogenates prepared from developing seeds of transformed plants exhibited elevated lysophosphatidic acid acyltransferase (EC 2.3.1.51) activity. Thus, the yeast sn-2 acyltransferase has been shown to encode a protein that can exhibit lysophosphatidic acid acyltransferase activity and that can be used to change total fatty acid content and composition as well as to alter the stereospecific acyl distribution of fatty acids in seed TAGs

The activities of critical enzymes in fatty acid and triacylglycerol biosynthesis are tightly controlled by different nutritional, hormonal, and developmental conditions. Feeding previously fasted animals high-carbohydrate, low-fat diets causes a dramatic induction of enzymes--such as fatty acid synthase (FAS) and mitochondrial glycerol-3-phosphate acyltransferase (GPAT)--involved in fatty acid and triacylglycerol synthesis. During fasting and refeeding, transcription of these two enzymes is coordinately regulated by nutrients and hormones, such as glucose, insulin, glucagon, glucocorticoids, and thyroid hormone. Insulin stimulates transcription of the FAS and mitochondrial GPAT genes, and glucagon antagonizes the insulin effect through the cis-acting elements within the promoters and their bound trans-acting factors. This review discusses advances made in the understanding of the transcriptional regulation of FAS and mitochondrial GPAT genes, with emphasis on elucidation of the mechanisms by which multiple nutrients and hormones achieve their effects

The polyketides are a diverse group of natural products with great significance as human and veterinary pharmaceuticals. A significant barrier to the production of novel genetically engineered polyketides has been the lack of available heterologous expression systems for functional polyketide synthases (PKSs). Herein, we report the expression of an intact functional PKS in Escherichia coli and Saccharomyces cerevisiae. The fungal gene encoding 6-methylsalicylic acid synthase from Penicillium patulum was expressed in E. coli and S. cerevisiae and the polyketide 6-methylsalicylic acid (6-MSA) was produced. In both bacterial and yeast hosts, polyketide production required coexpression of 6-methylsalicylic acid synthase and a heterologous phosphopantetheinyl transferase that was required to convert the expressed apo-PKS to its holo form. Production of 6-MSA in E. coli was both temperature- and glycerol-dependent and levels of production were lower than those of P. patulum, the native host. In yeast, however, 6-MSA levels greater than 2-fold higher than the native host were observed. The heterologous expression systems described will facilitate the manipulation of PKS genes and consequent production of novel engineered polyketides and polyketide libraries

Regulation of gene expression by nutrients is an important mechanism in the adaptation of mammals to their nutritional environment. This is especially true for enzymes involved in the storage of energy, such as the lipogenic and glycolytic enzymes in liver and adipose tissue. Transcription of the genes for lipogenic and glycolytic enzymes is stimulated by glucose in adipose tissue, liver, and pancreatic beta-cells. Several lines of evidence suggest that glucose must be metabolized to glucose-6-phosphate to stimulate gene transcription. In adipose tissue, insulin increases the expression of lipogenic enzymes indirectly by stimulating glucose uptake. In the liver, insulin also acts indirectly by stimulating the expression of glucokinase and, hence, by increasing glucose metabolism. Glucose response elements have been characterized for the L-pyruvate kinase and S 14 genes. They have in common the presence of a sequence 5'-CACGTG-3', which binds a transcription factor called USF (upstream stimulatory factor). Another glucose response element, which uses a transcription factor named Sp1, has been characterized in the gene for the acetyl-coenzyme A carboxylase. The mechanisms linking glucose-6-phosphate to the glucose-responsive transcription complex are largely unknown

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes the irreversible conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate and is considered a key regulatory step controlling isoprenoid metabolism in mammals and fungi. The rate-limiting nature of this enzyme for isoprenoid biosynthesis in plants remains controversial. To investigate whether HMGR activity could be limiting in plants, we introduced a constitutively expressing hamster HMGR gene into tobacco (Nicotiana tabacum L.) plants to obtain unregulated HMGR activity. The impact of the resulting enzyme activity on the biosynthesis and accumulation of particular isoprenoids was evaluated. Expression of the hamster HMGR gene led to a 3- to 6-fold increase in the total HMGR enzyme activity. Total sterol accumulation was consequently increased 3- to 10-fold, whereas end-product sterols such as sitosterol, campesterol, and stigmasterol were increased only 2-fold. The level of cycloartenol, a sterol biosynthetic intermediate, was increased more than 100-fold. Although the synthesis of total sterols appears to be limited normally by HMGR activity, these results indicate that the activity of one or more later enzyme(s) in the pathway must also be involved in determining the relative accumulation of end-product sterols. The levels of other isoprenoids such as carotenoids, phytol chain of chlorophyll, and sesquiterpene phytoalexins were relatively unaltered in the transgenic plants. It appears from these results that compartmentation, channeling, or other rate-determining enzymes operate to control the accumulation of these other isoprenoid end products.

Alcaligenes eutrophus genes encoding the enzymes, beta-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB), and polyhydroxyalkanoate synthase (phaC) catalyze the production of aliphatic polyester poly-D-(-)-3-hydroxybutyrate (PHB) from acetyl-CoA. PHB is a thermoplastic polymer that may modify fiber properties when synthesized in cotton. Endogenous beta-ketothiolase activity is present in cotton fibers. Hence cotton was transformed with engineered phaB and phaC genes by particle bombardment, and transgenic plants were selected based on marker gene, beta-glucuronidase (GUS), expression. Fibers of 10 transgenic plants expressed phaB gene, while eight plants expressed both phaB and phaC genes. Electron microscopy examination of fibers expressing both genes indicated the presence of electron-lucent granules in the cytoplasm. High pressure liquid chromatography, gas chromatography, and mass spectrometry evidence suggested that the new polymer produced in transgenic fibers is PHB. Sixty-six percent of the PHB in fibers is in the molecular mass range of 0.6 X 10(6) to 1.8 X 10(6) Da. The presence of PHB granules in transgenic fibers resulted in measurable changes of thermal properties. The fibers exhibited better insulating characteristics. The rate of heat uptake and cooling was slower in transgenic fibers, resulting in higher heat capacity. These data show that metabolic pathway engineering in cotton may enhance fiber properties by incorporating new traits from other genetic sources. This is an important step toward producing new generation fibers for the textile industry

A rapid, efficient, and reproducible system to generate large numbers of independently transformed, self-fertile, transgenic barley (Hordeum vulgare L.) plants is described. Immature zygotic embryos, young callus, and microspore-derived embryos were bombarded with a plasmid containing bar and uidA either alone or in combination with another plasmid containing a barley yellow dwarf virus coat protein (BYDVcp) gene. A total of 91 independent bialaphos-resistant callus lines expressed functional phosphinothricin acetyltransferase, the product of bar. Integration of bar was confirmed by DNA hybridization in the 67 lines analyzed. Cotransformation frequencies of 84 and 85% were determined for the two linked genes (bar and uidA) and for two unlinked genes (bar and the BYDVcp gene), respectively. More than 500 green, fertile, transgenic plants were regenerated from 36 transformed callus lines on bialaphos-containing medium; albino plants only were regenerated from 41 lines. T(0) plants in 25 lines (three plants per line) were analyzed by DNA hybridization, and all contained bar. Most contained the same integration patterns for the introduced genes (bar, uidA, and the BYDVcp gene) as their parental callus lines. Transmission of the genes to T(1) progeny was confirmed in the five families analyzed by DNA hybridization. A germination test of immature T(1) embryos on bialaphos-containing medium was useful for selecting individuals that were actively expressing bar, although this was not a good indicator of the presence or absence of bar. Expression of bar in some progeny plants was indicated by resistance to the herbicide Basta. The T(1) plants were in soil approximately 7 months after bombardment of the immature embryo