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.

Triglycerides (or triacylglycerols) represent the major form of stored energy in eukaryotes. Triglyceride synthesis has been assumed to occur primarily through acyl CoA:diacylglycerol transferase (Dgat), a microsomal enzyme that catalyses the final and only committed step in the glycerol phosphate pathway. Therefore, Dgat has been considered necessary for adipose tissue formation and essential for survival. Here we show that Dgat-deficient (Dgat-/-) mice are viable and can still synthesize triglycerides. Moreover, these mice are lean and resistant to diet-induced obesity. The obesity resistance involves increased energy expenditure and increased activity. Dgat deficiency also alters triglyceride metabolism in other tissues, including the mammary gland, where lactation is defective in Dgat-/- females. Our findings indicate that multiple mechanisms exist for triglyceride synthesis and suggest that the selective inhibition of Dgat-mediated triglyceride synthesis may be useful for treating obesity.

Detecting and determining the relative abundance of diverse individual sequences in complex DNA samples is a recurring experimental challenge in analyzing genomes. We describe a general experimental approach to this problem, using microscopic arrays of DNA fragments on glass substrates for differential hybridization analysis of fluorescently labeled DNA samples. To test the system, 864 physically mapped X clones of yeast genomic DNA, together representing >75% of the yeast genome, were arranged into 1.8-cm x 1.8-cm arrays, each containing a total of 1744 elements. The microarrays were characterized by simultaneous hybridization of two different sets of isolated yeast chromosomes labeled with two different fluorophores. A laser fluorescent scanner was used to detect the hybridization signals from the two fluorophores. The results demonstrate the utility of DNA microarrays in the analysis of complex DNA samples. This system should find numerous applications in genome-wide genetic mapping, physical mapping, and gene expression studies. As a general approach to this problem, we have developed a system for making microarrays of DNA samples on glass substrates, probing them by hybridization with complex fluorescentlabeled probes, and using a laser-scanning microscope to detect the fluorescent signals representing hybridization. Fluorescent labeling allows for simultaneous hybridization and separate detection of the hybridization signal from two or more probes. This in turn allows very accurate and reliable measurement of the relative abundance of specific sequences in two complex samples. with a mixed probe consisting of lissaminelabeled DNA from the 6 largest yeast chromosomes together with fluorescein-labeled DNA from the 10 smallest yeast chromosomes. A red color indicates that yeast sequences present in the lissamine-labeled hybridization probe hybridized to an array element. A yellow-green color indicates that yeast sequences present in the fluorescein-labeled hybridization probe hybridized to an array element. An orange color indicates cross-hybridization of both chromosome pools to an array element (e.g., dispersed repetitive elements, such as Tyl elements). RESULTS Array Hybridization PatternEach clone was spotted twice, resulting in duplicate hybridization patterns in adjacent quadrants of the array. Control DNA spots, which were randomly amplified in the same manner as the X clone array elements, are located in the bottom corner of each quadrant. "A" points to a pair of spots containing total yeast genomic DNA. These spots appear orange because both chromosome pools hybridized to yeast genomic DNA. The negative controls are as follows: "B" points to a pair of spots of wild-type X DNA, "C" points to a pair of human genomic DNA spots, and "D" points to a pair of ~X174 DNA spots. The lack of a hybridization signal at these three negative control spots indicates that the hybridization was specific for yeast sequences.