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Metabolomics in Yeast.

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Metabolomics in Yeast.

Cold Spring Harb Protoc. 2017 Sep 01;2017(9):pdb.top083576

Authors: Caudy AA, Mülleder M, Ralser M

Abstract
Budding yeast has from the beginning been a major eukaryotic model for the study of metabolic network structure and function. This is attributable to both its genetic and biochemical capacities and its role as a workhorse in food production and biotechnology. New inventions in analytical technologies allow accurate, simultaneous detection and quantification of metabolites, and a series of recent findings have placed the metabolic network at center stage in the physiology of the cell. For example, metabolism might have facilitated the origin of life, and in modern organisms it not only provides nutrients to the cell but also serves as a buffer to changes in the cellular environment, a regulator of cellular processes, and a requirement for cell growth. These findings have triggered a rapid and massive renaissance in this important field. Here, we provide an introduction to analysis of metabolomics in yeast.

PMID: 28864573 [PubMed - indexed for MEDLINE]



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Spectrophotometric Analysis of Ethanol and Glucose Concentrations in Yeast Culture Media.

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Spectrophotometric Analysis of Ethanol and Glucose Concentrations in Yeast Culture Media.

Cold Spring Harb Protoc. 2017 Sep 01;2017(9):pdb.prot089102

Authors: Caudy AA

Abstract
Fermentative growth on glucose is one of the most widely studied conditions of yeast growth in the laboratory. The production of ethanol from sugars is relevant to the wine, beer, and bread industries and to production of biofuels. Assaying the levels of glucose and ethanol in yeast growth medium allows the experimenter to determine the consumption of the carbon source glucose and the production of ethanol. This protocol describes enzyme-coupled assays for determination of glucose and ethanol concentrations in a sample of cell-free culture medium. Enzymes convert glucose or ethanol into other compounds through chemical reactions that reduce NAD(P)+ to NAD(P)H, and the production of NAD(P)H is measured using a spectrophotometer. The methods presented are highly sensitive, with a detection limit of ∼0.4 mg/L of glucose and 50 mg/L of ethanol, and also have the advantage of high specificity. For example, glucose and fructose have identical chemical formulas and thus cannot be distinguished by a mass spectrometer, but the enzyme assay presented here is specific for glucose. The glucose assay can be coupled to other assays to determine the quantity of additional carbohydrates such as fructose, trehalose, and glycogen.

PMID: 28864566 [PubMed - indexed for MEDLINE]



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Metabolite Extraction from Saccharomyces cerevisiae for Liquid Chromatography-Mass Spectrometry.

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Metabolite Extraction from Saccharomyces cerevisiae for Liquid Chromatography-Mass Spectrometry.

Cold Spring Harb Protoc. 2017 Sep 01;2017(9):pdb.prot089086

Authors: Rosebrock AP, Caudy AA

Abstract
Prior to mass spectrometric analysis, cellular small molecules must be extracted and separated from interfering components such as salts and culture medium. To ensure minimal perturbation of metabolism, yeast cells grown in liquid culture are rapidly harvested by filtration as described here. Simultaneous quenching of metabolism and extraction is afforded by immediate immersion in low-temperature organic solvent. Samples prepared using this method are suitable for a range of downstream liquid chromatography-mass spectrometry analyses and are stable in solvent for >1 yr at -80°C.

PMID: 28864564 [PubMed - indexed for MEDLINE]



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First critical repressive H3K27me3 marks in embryonic stem cells identified using designed protein inhibitor.

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First critical repressive H3K27me3 marks in embryonic stem cells identified using designed protein inhibitor.

Proc Natl Acad Sci U S A. 2017 09 19;114(38):10125-10130

Authors: Moody JD, Levy S, Mathieu J, Xing Y, Kim W, Dong C, Tempel W, Robitaille AM, Dang LT, Ferreccio A, Detraux D, Sidhu S, Zhu L, Carter L, Xu C, Valensisi C, Wang Y, Hawkins RD, Min J, Moon RT, Orkin SH, Baker D, Ruohola-Baker H

Abstract
The polycomb repressive complex 2 (PRC2) histone methyltransferase plays a central role in epigenetic regulation in development and in cancer, and hence to interrogate its role in a specific developmental transition, methods are needed for disrupting function of the complex with high temporal and spatial precision. The catalytic and substrate recognition functions of PRC2 are coupled by binding of the N-terminal helix of the Ezh2 methylase to an extended groove on the EED trimethyl lysine binding subunit. Disrupting PRC2 function can in principle be achieved by blocking this single interaction, but there are few approaches for blocking specific protein-protein interactions in living cells and organisms. Here, we describe the computational design of proteins that bind to the EZH2 interaction site on EED with subnanomolar affinity in vitro and form tight and specific complexes with EED in living cells. Induction of the EED binding proteins abolishes H3K27 methylation in human embryonic stem cells (hESCs) and at all but the earliest stage blocks self-renewal, pinpointing the first critical repressive H3K27me3 marks in development.

PMID: 28864533 [PubMed - indexed for MEDLINE]



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An atlas of alternative splicing profiles and functional associations reveals new regulatory programs and genes that simultaneously express multiple major isoforms.

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An atlas of alternative splicing profiles and functional associations reveals new regulatory programs and genes that simultaneously express multiple major isoforms.

Genome Res. 2017 Oct;27(10):1759-1768

Authors: Tapial J, Ha KCH, Sterne-Weiler T, Gohr A, Braunschweig U, Hermoso-Pulido A, Quesnel-Vallières M, Permanyer J, Sodaei R, Marquez Y, Cozzuto L, Wang X, Gómez-Velázquez M, Rayon T, Manzanares M, Ponomarenko J, Blencowe BJ, Irimia M

Abstract
Alternative splicing (AS) generates remarkable regulatory and proteomic complexity in metazoans. However, the functions of most AS events are not known, and programs of regulated splicing remain to be identified. To address these challenges, we describe the Vertebrate Alternative Splicing and Transcription Database (VastDB), the largest resource of genome-wide, quantitative profiles of AS events assembled to date. VastDB provides readily accessible quantitative information on the inclusion levels and functional associations of AS events detected in RNA-seq data from diverse vertebrate cell and tissue types, as well as developmental stages. The VastDB profiles reveal extensive new intergenic and intragenic regulatory relationships among different classes of AS and previously unknown and conserved landscapes of tissue-regulated exons. Contrary to recent reports concluding that nearly all human genes express a single major isoform, VastDB provides evidence that at least 48% of multiexonic protein-coding genes express multiple splice variants that are highly regulated in a cell/tissue-specific manner, and that >18% of genes simultaneously express multiple major isoforms across diverse cell and tissue types. Isoforms encoded by the latter set of genes are generally coexpressed in the same cells and are often engaged by translating ribosomes. Moreover, they are encoded by genes that are significantly enriched in functions associated with transcriptional control, implying they may have an important and wide-ranging role in controlling cellular activities. VastDB thus provides an unprecedented resource for investigations of AS function and regulation.

PMID: 28855263 [PubMed - indexed for MEDLINE]



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Structural templating of J-aggregates: Visualizing bis(monoacylglycero)phosphate domains in live cells.

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Structural templating of J-aggregates: Visualizing bis(monoacylglycero)phosphate domains in live cells.

Biochim Biophys Acta. 2017 11;1865(11 Pt B):1687-1695

Authors: Mo GCH, Yip CM

Abstract
Identifying the key structural and dynamical determinants that drive the association of biomolecules, whether in solution, or perhaps more importantly in a membrane environment, has critical implications for our understanding of cellular dynamics, processes, and signaling. With recent advances in high-resolution imaging techniques, from the development of new molecular labels to technical advances in imaging methodologies and platforms, researchers are now reaping the benefits of being able to directly characterize and quantify local dynamics, structures, and conformations in live cells and tissues. These capabilities are providing unique insights into association stoichiometries, interactions, and structures on sub-micron length scales. We previously examined the role of lipid headgroup chemistry and phase state in guiding the formation of pseudoisocyanine (PIC) dye J-aggregates on supported planar bilayers [Langmuir, 25, 10719]. We describe here how these same J-aggregates can report on the in situ formation of organellar membrane domains in live cells. Live cell hyperspectral confocal microscopy using GFP-conjugated GTPase markers of early (Rab5) and late (Rab7) endosomes revealed that the PIC J-aggregates were confined to domains on either the limiting membrane or intralumenal vesicles (ILV) of late endosomes, known to be enriched in the anionic lipid bis(monoacylglycero)phosphate (BMP). Correlated confocal fluorescence - atomic force microscopy performed on endosomal membrane-mimetic supported planar lipid bilayers confirmed BMP-specific templating of the PIC J-aggregates. These data provide strong evidence for the formation of BMP-rich lipid domains during multivesicular body formation and portend the application of structured dye aggregates as markers of cellular membrane domain structure, size, and formation.

PMID: 28844737 [PubMed - indexed for MEDLINE]



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A Broad-Spectrum Inhibitor of CRISPR-Cas9.

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A Broad-Spectrum Inhibitor of CRISPR-Cas9.

Cell. 2017 Sep 07;170(6):1224-1233.e15

Authors: Harrington LB, Doxzen KW, Ma E, Liu JJ, Knott GJ, Edraki A, Garcia B, Amrani N, Chen JS, Cofsky JC, Kranzusch PJ, Sontheimer EJ, Davidson AR, Maxwell KL, Doudna JA

Abstract
CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9. A crystal structure of an AcrIIC1-Cas9 HNH domain complex shows how AcrIIC1 traps Cas9 in a DNA-bound but catalytically inactive state. By contrast, AcrIIC3 blocks activity of a single Cas9 ortholog and induces Cas9 dimerization while preventing binding to the target DNA. These two orthogonal mechanisms allow for separate control of Cas9 target binding and cleavage and suggest applications to allow DNA binding while preventing DNA cutting by Cas9.

PMID: 28844692 [PubMed - indexed for MEDLINE]



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The Parkinson's disease-associated GPR37 receptor interacts with striatal adenosine A2A receptor controlling its cell surface expression and function in vivo.

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The Parkinson's disease-associated GPR37 receptor interacts with striatal adenosine A2A receptor controlling its cell surface expression and function in vivo.

Sci Rep. 2017 Aug 25;7(1):9452

Authors: Morató X, Luján R, López-Cano M, Gandía J, Stagljar I, Watanabe M, Cunha RA, Fernández-Dueñas V, Ciruela F

Abstract
G protein-coupled receptor 37 (GPR37) is an orphan receptor associated to Parkinson's disease (PD) neuropathology. Here, we identified GPR37 as an inhibitor of adenosine A2A receptor (A2AR) cell surface expression and function in vivo. In addition, we showed that GPR37 and A2AR do oligomerize in the striatum. Thus, a close proximity of GPR37 and A2AR at the postsynaptic level of striatal synapses was observed by double-labelling post-embedding immunogold detection. Indeed, the direct receptor-receptor interaction was further substantiated by proximity ligation in situ assay. Interestingly, GPR37 deletion promoted striatal A2AR cell surface expression that correlated well with an increased A2AR agonist-mediated cAMP accumulation, both in primary striatal neurons and nerve terminals. Furthermore, GPR37-/- mice showed enhanced A2AR agonist-induced catalepsy and an increased response to A2AR antagonist-mediated locomotor activity. Overall, these results revealed a key role for GPR37 controlling A2AR biology in the striatum, which may be relevant for PD management.

PMID: 28842709 [PubMed - in process]



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Taxonomically Restricted Genes with Essential Functions Frequently Play Roles in Chromosome Segregation in Caenorhabditis elegans and Saccharomyces cerevisiae.

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Taxonomically Restricted Genes with Essential Functions Frequently Play Roles in Chromosome Segregation in Caenorhabditis elegans and Saccharomyces cerevisiae.

G3 (Bethesda). 2017 Oct 05;7(10):3337-3347

Authors: Verster AJ, Styles EB, Mateo A, Derry WB, Andrews BJ, Fraser AG

Abstract
Genes encoding essential components of core cellular processes are typically highly conserved across eukaryotes. However, a small proportion of essential genes are highly taxonomically restricted; there appear to be no similar genes outside the genomes of highly related species. What are the functions of these poorly characterized taxonomically restricted genes (TRGs)? Systematic screens in Saccharomyces cerevisiae and Caenorhabditis elegans previously identified yeast or nematode TRGs that are essential for viability and we find that these genes share many molecular features, despite having no significant sequence similarity. Specifically, we find that those TRGs with essential phenotypes have an expression profile more similar to highly conserved genes, they have more protein-protein interactions and more protein disorder. Surprisingly, many TRGs play central roles in chromosome segregation; a core eukaryotic process. We thus find that genes that appear to be highly evolutionarily restricted do not necessarily play roles in species-specific biological functions but frequently play essential roles in core eukaryotic processes.

PMID: 28839119 [PubMed - indexed for MEDLINE]



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Genome-Scale Genetic Interactions and Cell Imaging Confirm Cytokinesis as Deleterious to Transient Topoisomerase II Deficiency in Saccharomyces cerevisiae.

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Genome-Scale Genetic Interactions and Cell Imaging Confirm Cytokinesis as Deleterious to Transient Topoisomerase II Deficiency in Saccharomyces cerevisiae.

G3 (Bethesda). 2017 Oct 05;7(10):3379-3391

Authors: Ramos-Pérez C, Ayra-Plasencia J, Matos-Perdomo E, Lisby M, Brown GW, Machín F

Abstract
Topoisomerase II (Top2) is an essential protein that resolves DNA catenations. When Top2 is inactivated, mitotic catastrophe results from massive entanglement of chromosomes. Top2 is also the target of many first-line anticancer drugs, the so-called Top2 poisons. Often, tumors become resistant to these drugs by acquiring hypomorphic mutations in the genes encoding Top2 Here, we have compared the cell cycle and nuclear segregation of two coisogenic Saccharomyces cerevisiae strains carrying top2 thermosensitive alleles that differ in their resistance to Top2 poisons: the broadly-used poison-sensitive top2-4 and the poison-resistant top2-5 Furthermore, we have performed genome-scale synthetic genetic array (SGA) analyses for both alleles under permissive conditions, chronic sublethal Top2 downregulation, and acute, yet transient, Top2 inactivation. We find that slowing down mitotic progression, especially at the time of execution of the mitotic exit network (MEN), protects against Top2 deficiency. In all conditions, genetic protection was stronger in top2-5; this correlated with cell biology experiments in this mutant, whereby we observed destabilization of both chromatin and ultrafine anaphase bridges by execution of MEN and cytokinesis. Interestingly, whereas transient inactivation of the critical MEN driver Cdc15 partly suppressed top2-5 lethality, this was not the case when earlier steps within anaphase were disrupted; i.e., top2-5 cdc14-1 We discuss the basis of this difference and suggest that accelerated progression through mitosis may be a therapeutic strategy to hypersensitize cancer cells carrying hypomorphic mutations in TOP2.

PMID: 28839115 [PubMed - indexed for MEDLINE]



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