New data and collaborations at the Saccharomyces Genome Database: updated reference genome,alleles, and the Alliance of Genome Resources

Saccharomyces cerevisiae is used to provide fundamental understanding of eukaryotic genetics, gene product function, and cellular biological processes. Saccharomyces Genome Database (SGD) has been supporting the yeast research community since 1993, serving as its de facto hub. Over the years, SGD has maintained the genetic nomenclature, chromosome maps, and functional annotation, and developed various tools and methods for analysis and curation of a variety of emerging data types. More recently, SGD and six other model organism focused knowledgebases have come together to create the Alliance of Genome Resources to develop sustainable genome information resources that promote and support the use of various model organisms to understand the genetic and genomic bases of human biology and disease. Here we describe recent activities at SGD, including the latest reference genome annotation update, the development of a curation system for mutant alleles, and new pages addressing homology across model organisms as well as the use of yeast to study human disease.     

Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Purification and properties.

Procedures were developed for the optimal solubilization of D-lactate dehydrogenase, D-mandelate dehydrogenase, L-lactate dehydrogenase and L-mandelate dehydrogenase from wall + membrane fractions of Acinetobacter calcoaceticus. D-Lactate dehydrogenase and D-mandelate dehydrogenase were co-eluted on gel filtration, as were L-lactate dehydrogenase and L-mandelate dehydrogenase. All four enzymes could be separated by ion-exchange chromatography. D-Lactate dehydrogenase and D-mandelate dehydrogenase were purified by cholate extraction, (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and chromatofocusing. The properties of D-lactate dehydrogenase and D-mandelate dehydrogenase were similar in several respects: they had relative molecular masses of 62 800 and 59 700 respectively, pI values of 5.8 and 5.5, considerable sensitivity to p-chloromercuribenzoate, little or no inhibition by chelating agents, and similar responses to pH. Both enzymes appeared to contain non-covalently bound FAD as cofactor.     

Demography of the western harvest mouse,Reithrodontomys megalotis,in eastern Kansas

Summary Reithrodontomys megalotis was live-trapped on three open field grids in eastern Kansas from August 1979 to August 1982. One grid was a control on which normal demography was monitored, and two were experimental grids where periodic removal of residents allowed the investigation of the demographic and fitness consequences of emigration. Popullations on the control grid showed an annual cycle in numbers, reaching peaks in density during the winter of each year, falling to low densities during the summer. Low summer densities were attributed, at least partially, to low trappability of R. megalotis during periods of high resource abundance. Reproduction was initiated in the spring of each year at approximately the same time as the emergence of new vegetative growth, and ceased in late fall of each year. The trappable population was composed almost entirely of adults, and the sex ratio was skewed significantly toward females. A statistically significant negative association between the number of M. ochrogaster residents and the reproductive activity of female R. megalotis residents was found. A canonical correlation analysis revealed a common seasonal component to the demography of the two species, and possible interspecific affects. Emigrating R. megalotis were a nonrandom sample of the population, with emigrants more likely to be subadult and juvenile males when compared to residents. No association was detected between the numbers of M. ochrogaster colonizing the removal grid and the numbers of R. megalotis colonizing the same removal grid, or between the number of M. ochrogaster residents on the control grid and the numbers of R. megalotis colonizing the removal grids. However, the number of R. megalotis residents on the control grid is positively correlated with the number of R. megalotis on the removal grids.     

Substrate independent ATPase activity may complicate high throughput screening

Inorganic phosphate release, [Pi], is often measured in an enzymatic reaction in a high throughput setting. Based on the published mechanism, we designed a protocol for our screening for inhibitors of SAICAR synthetase (PurC), and we found a gradual increase in [Pi] in positive control samples over the course of the day. Further investigation indicated that hydrolysis of ATP catalyzed by PurC, rather than substrate-related phosphate release, was responsible for a partial contribution to the signals in the control samples. Thus substrate-independent ATPase activity may complicate high throughput screening.     

Improved calculations of compactness and a reevaluation of continuous compact units

A new method for calculating compactness (Z) that uses look-up table-based algorithms for area and volume computations is introduced. These algorithms can be used in any iterative area orvolume calculation, are more than 1000 times faster than the originalalgorithms, and have equal or better precision. With the faster algorithms it is now possible to calculate the compactness of all continuous units in a protein, and to precisely locate the optimal compact units without the screening functions and limited resolution used previously. These methods have been incorporated into a fully automatic domain finding algorithm, and this method has been applied to the 21 proteins originally analyzed as well as 12 additional proteins. This method is robust, and yields similar units even when applied to coordinates of protein crystals grown under different experimental conditions. © 1993 Wiley-Liss, Inc.     

Exocellular phosphatidylethanolamine production and multiple-metal tolerance in Pseudomonas fluorescens

Abstract Pseudomonas fluorescens appeared to circumvent the challenge imposed by millimolar amounts of metals (5 mM Al³⁺, 5 mM Fe³⁺, 2 mM Ca²⁺, 1 mM Ga³⁺ and 3 mM Zn²⁺) by the formation of phosphatidylethanolamine. This lipid moiety constituted an important organic component of an insoluble gelatinous residue in which most of the test metals were immobilized at stationary phase of growth. Ultracentrifugation and dialysis experiments showed that the metals were associated with phosphatidylethanolamine from early stages of growth. Transmission electron microscopy revealed metal rich bodies in the cytoplasm prior to their secretion in the spent fluid. These results demonstrate a role of phosphatidylethanolamine in multiple-metal homeostasis.     

The use of stable isotope-labeled glycerol and oleic acid to differentiate the hepatic functions of DGAT1 and -2

Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triglyceride (TG) synthesis. There are two isoforms, DGAT1 and DGAT2, with distinct protein sequences and potentially different physiological functions. To date, the ability to determine clear functional differences between DGAT1 and DGAT2, especially with respect to hepatic TG synthesis, has been elusive. To dissect the roles of these two key enzymes, we pretreated HepG2 hepatoma cells with (13)C(3)-D(5)-glycerol or (13)C(18)-oleic acid, and profiled the major isotope-labeled TG species by liquid chromatography tandem mass spectrometry. Selective DGAT1 and DGAT2 inhibitors demonstrated that (13)C(3)-D(5)-glycerol-incorporated TG synthesis was mediated by DGAT2, not DGAT1. Conversely, (13)C(18)-oleoyl-incorporated TG synthesis was predominantly mediated by DGAT1. To trace hepatic TG synthesis and VLDL triglyceride (VLDL-TG) secretion in vivo, we administered D(5)-glycerol to mice and measured plasma levels of D(5)-glycerol-incorporated TG. Treatment with an antisense oligonucleotide (ASO) to DGAT2 led to a significant reduction in D(5)-glycerol incorporation into VLDL-TG. In contrast, the DGAT2 ASO had no effect on the incorporation of exogenously administered (13)C(18)-oleic acid into VLDL-TG. Thus, our results indicate that DGAT1 and DGAT2 mediate distinct hepatic functions: DGAT2 is primarily responsible for incorporating endogenously synthesized FAs into TG, whereas DGAT1 plays a greater role in esterifying exogenous FAs to glycerol.     

Synthesis and structure-activity relationship studies of 1,3-disubstituted 2-propanols as BACE-1 inhibitors

A library of 1,3-disubstituted 2-propanols was synthesized and evaluated as low molecular weight probes for β-secretase inhibition. By screening a library of 121 1,3-disubstituted 2-propanol derivatives, we identified few compounds inhibiting the enzyme at low micromolar concentrations. The initial hits were optimized to yield a potent BACE-1 inhibitor exhibiting an IC(50) constant in the nanomolar range. Exploration of the pharmacological properties revealed that these small molecular inhibitors possessed a high selectivity over cathepsin D and desirable physicochemical properties beneficial to cross the blood-brain barrier.