We present a liquid chromatography−mass spectrometry (LC−MS) method that capitalizes on the mass-resolving power of the orbitrap to enable sensitive and specific measurement of known and ...unanticipated metabolites in parallel, with a focus on water-soluble species involved in core metabolism. The reversed phase LC method, with a cycle time 25 min, involves a water−methanol gradient on a C18 column with tributylamine as the ion pairing agent. The MS portion involves full scans from 85 to 1000 m/z at 1 Hz and 100 000 resolution in negative ion mode on a stand alone orbitrap (“Exactive”). The median limit of detection, across 80 metabolite standards, was 5 ng/mL with the linear range typically ≥100-fold. For both standards and a cellular extract from Saccharomyces cerevisiae (Baker’s yeast), the median inter-run relative standard deviation in peak intensity was 8%. In yeast exact, we detected 137 known compounds, whose 13C-labeling patterns could also be tracked to probe metabolic flux. In yeast engineered to lack a gene of unknown function (YKL215C), we observed accumulation of an ion of m/z 128.0351, which we subsequently confirmed to be oxoproline, resulting in annotation of YKL215C as an oxoprolinase. These examples demonstrate the suitability of the present method for quantitative metabolomics, fluxomics, and discovery metabolite profiling.
High-throughput cDNA synthesis and sequencing of poly(A)-enriched RNA is rapidly emerging as a technology competing to replace microarrays as a quantitative platform for measuring gene expression.
...Consequently, we compared full length cDNA sequencing to 2-channel gene expression microarrays in the context of measuring differential gene expression. Because of its comparable cost to a gene expression microarray, our study focused on the data obtainable from a single lane of an Illumina 1 G sequencer. We compared sequencing data to a highly replicated microarray experiment profiling two divergent strains of S. cerevisiae.
Using a large number of quantitative PCR (qPCR) assays, more than previous studies, we found that neither technology is decisively better at measuring differential gene expression. Further, we report sequencing results from a diploid hybrid of two strains of S. cerevisiae that indicate full length cDNA sequencing can discover heterozygosity and measure quantitative allele-specific expression simultaneously.
Diet greatly influences gene expression and physiology. In mammals, elucidating the effects and mechanisms of individual nutrients is challenging due to the complexity of both the animal and its ...diet. Here, we used an interspecies systems biology approach with Caenorhabditis elegans and two of its bacterial diets, Escherichia coli and Comamonas aquatica, to identify metabolites that affect the animal’s gene expression and physiology. We identify vitamin B12 as the major dilutable metabolite provided by Comamonas aq. that regulates gene expression, accelerates development, and reduces fertility but does not affect lifespan. We find that vitamin B12 has a dual role in the animal: it affects development and fertility via the methionine/S-Adenosylmethionine (SAM) cycle and breaks down the short-chain fatty acid propionic acid, preventing its toxic buildup. Our interspecies systems biology approach provides a paradigm for understanding complex interactions between diet and physiology.
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•Interspecies systems biology paradigm links genetics to metabolites•Vitamin B12 is produced by the Comamonas, but not the E. coli, diet•Vitamin B12 affects development, fertility, and propionic acid toxicity•Vitamin B12 accelerates development via SAM metabolism
An interspecies systems biology approach identifies vitamin B12, supplied by a diet of Comamonas bacteria, but not the standard E. coli diet, as a key micronutrient affecting C. elegans development and fertility.
Genome-wide gene-expression studies have shown that hundreds of yeast genes are induced or repressed transiently by changes in temperature; many are annotated to stress response on this basis. To ...obtain a genome-scale assessment of which genes are functionally important for innate and/or acquired thermotolerance, we combined the use of a barcoded pool of ∼4,800 nonessential, prototrophic Saccharomyces cerevisiae deletion strains with Illumina-based deep-sequencing technology. As reported in other recent studies that have used deletion mutants to study stress responses, we observed that gene deletions resulting in the highest thermosensitivity generally are not the same as those transcriptionally induced in response to heat stress. Functional analysis of identified genes revealed that metabolism, cellular signaling, and chromatin regulation play roles in regulating thermotolerance and in acquired thermotolerance. However, for most of the genes identified, the molecular mechanism behind this action remains unclear. In fact, a large fraction of identified genes are annotated as having unknown functions, further underscoring our incomplete understanding of the response to heat shock. We suggest that survival after heat shock depends on a small number of genes that function in assessing the metabolic health of the cell and/or regulate its growth in a changing environment.
The mitochondrial inner membrane contains a large protein complex that functions in inner membrane organization and formation of membrane contact sites. The complex was variably named the ...mitochondrial contact site complex, mitochondrial inner membrane organizing system, mitochondrial organizing structure, or Mitofilin/Fcj1 complex. To facilitate future studies, we propose to unify the nomenclature and term the complex "mitochondrial contact site and cristae organizing system" and its subunits Mic10 to Mic60.
Riboneogenesis in Yeast Clasquin, Michelle F.; Melamud, Eugene; Singer, Alexander ...
Cell,
06/2011, Volume:
145, Issue:
6
Journal Article
Peer reviewed
Open access
Glucose is catabolized in yeast via two fundamental routes, glycolysis and the oxidative pentose phosphate pathway, which produces NADPH and the essential nucleotide component ribose-5-phosphate. ...Here, we describe riboneogenesis, a thermodynamically driven pathway that converts glycolytic intermediates into ribose-5-phosphate without production of NADPH. Riboneogenesis begins with synthesis, by the combined action of transketolase and aldolase, of the seven-carbon bisphosphorylated sugar sedoheptulose-1,7-bisphosphate. In the pathway's committed step, sedoheptulose bisphosphate is hydrolyzed to sedoheptulose-7-phosphate by the enzyme sedoheptulose-1,7-bisphosphatase (SHB17), whose activity we identified based on metabolomic analysis of the corresponding knockout strain. The crystal structure of Shb17 in complex with sedoheptulose-1,7-bisphosphate reveals that the substrate binds in the closed furan form in the active site. Sedoheptulose-7-phosphate is ultimately converted by known enzymes of the nonoxidative pentose phosphate pathway to ribose-5-phosphate. Flux through SHB17 increases when ribose demand is high relative to demand for NADPH, including during ribosome biogenesis in metabolically synchronized yeast cells.
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► Riboneogenesis converts three-carbon glycolytic intermediates into ribose ► Sedoheptulose-1,7-bisphosphate is a key intermediate in riboneogenesis in yeast ► A sedoheptulose bisphosphatase, Shb17, thermodynamically drives ribose biosynthesis ► Flux through Shb17 is high when demand for ribose exceeds that for reducing power
Most heritable traits, including many human diseases, are caused by multiple loci. Studies in both humans and model organisms, such as yeast, have failed to detect a large fraction of the loci that ...underlie such complex traits. A lack of statistical power to identify multiple loci with small effects is undoubtedly one of the primary reasons for this problem. We have developed a method in yeast that allows the use of much larger sample sizes than previously possible and hence permits the detection of multiple loci with small effects. The method involves generating very large numbers of progeny from a cross between two Saccharomyces cerevisiae strains and then phenotyping and genotyping pools of these offspring. We applied the method to 17 chemical resistance traits and mitochondrial function, and identified loci for each of these phenotypes. We show that the level of genetic complexity underlying these quantitative traits is highly variable, with some traits influenced by one major locus and others by at least 20 loci. Our results provide an empirical demonstration of the genetic complexity of a number of traits and show that it is possible to identify many of the underlying factors using straightforward techniques. Our method should have broad applications in yeast and can be extended to other organisms.
Systematic analysis of complex genetic interactions Kuzmin, Elena; VanderSluis, Benjamin; Wang, Wen ...
Science (American Association for the Advancement of Science),
2018-Apr-20, 2018-04-20, 20180420, Volume:
360, Issue:
6386
Journal Article
Peer reviewed
Open access
To systematically explore complex genetic interactions, we constructed ~200,000 yeast triple mutants and scored negative trigenic interactions. We selected double-mutant query genes across a broad ...spectrum of biological processes, spanning a range of quantitative features of the global digenic interaction network and tested for a genetic interaction with a third mutation. Trigenic interactions often occurred among functionally related genes, and essential genes were hubs on the trigenic network. Despite their functional enrichment, trigenic interactions tended to link genes in distant bioprocesses and displayed a weaker magnitude than digenic interactions. We estimate that the global trigenic interaction network is ~100 times as large as the global digenic network, highlighting the potential for complex genetic interactions to affect the biology of inheritance, including the genotype-to-phenotype relationship.
Background Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) results in systemic autoimmunity from birth and can be caused by mutations in the transcription factor forkhead box ...P3 (FOXP3). Objective To determine if Foxp3 is required for the generation of IL-10–expressing T regulatory cells. Methods CD4 lymphocytes were isolated from patients with IPEX-like syndromes and activated with antibodies to CD3 and CD46 to generate IL-10–expressing T regulatory cells. Results We describe a patient with clinical manifestations of IPEX that had a normal Foxp3 gene, but who had CD25 deficiency due to autosomal recessive mutations in this gene. This patient exhibited defective IL-10 expression from CD4 lymphocytes, whereas a Foxp3-deficient patient expressed normal levels of IL-10. Conclusion These data show that CD25 deficiency results in an IPEX-like syndrome and suggests that although Foxp3 is not required for normal IL-10 expression by human CD4 lymphocytes, CD25 expression is important. Clinical implications Any patient with features of IPEX but with a normal Foxp3 gene should be screened for mutations in the IL-2 receptor subunit CD25.
Mitochondria are central to many cellular processes including respiration, ion homeostasis, and apoptosis. Using computational predictions combined with traditional quantitative experiments, we have ...identified 100 proteins whose deficiency alters mitochondrial biogenesis and inheritance in Saccharomyces cerevisiae. In addition, we used computational predictions to perform targeted double-mutant analysis detecting another nine genes with synthetic defects in mitochondrial biogenesis. This represents an increase of about 25% over previously known participants. Nearly half of these newly characterized proteins are conserved in mammals, including several orthologs known to be involved in human disease. Mutations in many of these genes demonstrate statistically significant mitochondrial transmission phenotypes more subtle than could be detected by traditional genetic screens or high-throughput techniques, and 47 have not been previously localized to mitochondria. We further characterized a subset of these genes using growth profiling and dual immunofluorescence, which identified genes specifically required for aerobic respiration and an uncharacterized cytoplasmic protein required for normal mitochondrial motility. Our results demonstrate that by leveraging computational analysis to direct quantitative experimental assays, we have characterized mutants with subtle mitochondrial defects whose phenotypes were undetected by high-throughput methods.
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