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Saccharomyces cerevisiae

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https://www.readbyqxmd.com/read/29045784/metal-dependence-of-xylose-isomerase-from-piromyces-sp-e2-explored-by-activity-profiling-and-protein-crystallography
#1
Misun Lee, Henriette J Rozeboom, Paul de Waal, Rene de Jong, Hanna M Dudek, Dick B Janssen
Xylose isomerase from Piromyces sp. E2 (PirXI) can be used for equipping Saccharomyces cerevisiae with the capacity to ferment xylose to ethanol. The biochemical properties and structure of the enzyme have not been described even though its metal content, catalytic parameters and expression level are critical for rapid xylose utilization. We have isolated the enzyme after high-level expression in E. coli, analyzed the metal-dependence of its catalytic properties and solved twelve crystal structures in the presence of different metals, substrates, and substrate analogs...
October 18, 2017: Biochemistry
https://www.readbyqxmd.com/read/29045428/the-role-of-pka-in-the-translational-response-to-heat-stress-in-saccharomyces-cerevisiae
#2
Carla E Barraza, Clara A Solari, Irina Marcovich, Christopher Kershaw, Fiorella Galello, Silvia Rossi, Mark P Ashe, Paula Portela
Cellular responses to stress stem from a variety of different mechanisms, including translation arrest and relocation of the translationally repressed mRNAs to ribonucleoprotein particles like stress granules (SGs) and processing bodies (PBs). Here, we examine the role of PKA in the S. cerevisiae heat shock response. Under mild heat stress Tpk3 aggregates and promotes aggregation of eIF4G, Pab1 and eIF4E, whereas severe heat stress leads to the formation of PBs and SGs that contain both Tpk2 and Tpk3 and a larger 48S translation initiation complex...
2017: PloS One
https://www.readbyqxmd.com/read/29043653/quantitative-analysis-of-dna-damage-signaling-responses-to-chemical-and-genetic-perturbations
#3
Francisco M Bastos de Oliveira, Dongsung Kim, Michael Lanz, Marcus B Smolka
Phosphorylation-mediated signaling is essential for maintenance of the eukaryotic genome. The evolutionarily conserved kinases ATR and ATM sense specific DNA structures generated upon DNA damage or replication stress and mediate an extensive signaling network that impinges upon most nuclear processes. ATR/ATM signaling is highly regulated and can function in a context-dependent manner. Thus, the ability to quantitatively monitor most, if not all, signaling events in this network is essential to investigate the mechanisms by which kinases maintain genome integrity...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29043651/reporter-based-synthetic-genetic-array-analysis-a-functional-genomics-approach-for-investigating-transcript-or-protein-abundance-using-fluorescent-proteins-in-saccharomyces-cerevisiae
#4
Hendrikje Göttert, Mojca Mattiazzi Usaj, Adam P Rosebrock, Brenda J Andrews
Fluorescent reporter genes have long been used to quantify various cell features such as transcript and protein abundance. Here, we describe a method, reporter synthetic genetic array (R-SGA) analysis, which allows for the simultaneous quantification of any fluorescent protein readout in thousands of yeast strains using an automated pipeline. R-SGA combines a fluorescent reporter system with standard SGA analysis and can be used to examine any array-based strain collection available to the yeast community. This protocol describes the R-SGA methodology for screening different arrays of yeast mutants including the deletion collection, a collection of temperature-sensitive strains for the assessment of essential yeast genes and a collection of inducible overexpression strains...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29043650/rewiring-the-budding-yeast-proteome-using-synthetic-physical-interactions
#5
Guðjón Ólafsson, Peter H Thorpe
Artificially tethering two proteins or protein fragments together is a powerful method to query molecular mechanisms. However, this approach typically relies upon a prior understanding of which two proteins, when fused, are most likely to provide a specific function and is therefore not readily amenable to large-scale screening. Here, we describe the Synthetic Physical Interaction (SPI) method to create proteome-wide forced protein associations in the budding yeast Saccharomyces cerevisiae. This method allows thousands of protein-protein associations to be screened for those that affect either normal growth or sensitivity to drugs or specific conditions...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29043649/genome-wide-quantitative-fitness-analysis-qfa-of-yeast-cultures
#6
Eva-Maria Holstein, Conor Lawless, Peter Banks, David Lydall
We provide a detailed protocol for robot-assisted, genome-wide measurement of fitness in the model yeast Saccharomyces cerevisiae using Quantitative Fitness Analysis (QFA). We first describe how we construct thousands of double or triple mutant yeast strains in parallel using Synthetic Genetic Array (SGA) procedures. Strains are inoculated onto solid agar surfaces by liquid spotting followed by repeated photography of agar plates. Growth curves are constructed and the fitness of each strain is estimated. Robot-assisted QFA, can be used to identify genetic interactions and chemical sensitivity/resistance in genome-wide experiments, but QFA can also be used in smaller scale, manual workflows...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29043643/imaging-of-dna-ultrafine-bridges-in-budding-yeast
#7
Oliver Quevedo, Michael Lisby
DNA ultrafine bridges (UFBs) are a type of chromatin-free DNA bridges that connect sister chromatids in anaphase and pose a threat to genome stability. However, little is known about the origin of these structures, and how they are sensed and resolved by the cell. In this chapter, we review tools and methods for studying UFBs by fluorescence microscopy including chemical and genetic approaches to induce UFBs in the model organism Saccharomyces cerevisiae.
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29043641/measuring-dynamic-behavior-of-trinucleotide-repeat-tracts-in-vivo-in-saccharomyces-cerevisiae
#8
Gregory M Williams, Jennifer A Surtees
Trinucleotide repeat (TNR) tracts are inherently unstable during DNA replication, leading to repeat expansions and/or contractions. Expanded tracts are the cause of over 40 neurodegenerative and neuromuscular diseases. In this chapter, we focus on the (CNG)n repeat sequences that, when expanded, lead to Huntington's disease (HD), myotonic dystrophy type 1 (DM1), and a number of other neurodegenerative diseases. We describe a series of in vivo assays, using the model system Saccharomyces cerevisiae, to determine and characterize the dynamic behavior of TNR tracts that are in the early stages of expansion, i...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29043639/methods-to-study-repeat-fragility-and-instability-in-saccharomyces-cerevisiae
#9
Erica J Polleys, Catherine H Freudenreich
Trinucleotide repeats are common in the human genome and can undergo changes in repeat length. Expanded CAG repeats have been linked to over 14 human diseases and are considered hotspots for breakage and genomic rearrangement. Here, we describe two Saccharomyces cerevisiae based assays that evaluate the rate of chromosome breakage that occurs within a repeat tract (fragility), and a PCR-based assay to evaluate tract length changes (instability). The first fragility assay utilizes end-loss and subsequent telomere addition as the main mode of repair of a yeast artificial chromosome (YAC)...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29043636/analysis-of-de-novo-telomere-addition-by-southern-blot
#10
Diego Bonetti, Maria Pia Longhese
Telomere length is maintained in most eukaryotes by the action of a specialized enzyme, the telomerase. However, the complexity of mechanisms regulating telomeric DNA length as well as the heterogeneity in length of each telomere in a population of cells has made it very difficult to understand how telomerase is regulated in vivo. Here, we describe a method developed in Saccharomyces cerevisiae to monitor the addition of telomeric sequences to a single newly generated telomere in vivo. The primary strain consists of a HO endonuclease cleavage site that is placed directly adjacent to an 81-base-pair stretch of telomeric DNA inserted into the ADH4 locus of chromosome VII...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29043635/detection-of-dna-rna-hybrids-in-vivo
#11
María García-Rubio, Sonia I Barroso, Andrés Aguilera
DNA-RNA hybrids form naturally during essential cellular functions such as transcription and replication. However, they may be an important source of genome instability, a hallmark of cancer and genetic diseases. Detection of DNA-RNA hybrids in cells is becoming crucial to understand an increasing number of molecular biology processes in genome dynamics and function and to identify new factors and mechanisms responsible for disease in biomedical research. Here, we describe two different procedures for the reliable detection of DNA-RNA hybrids in the yeast Saccharomyces cerevisiae and in human cells: DNA-RNA Immunoprecipitation (DRIP) and Immunofluorescence...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29043634/mapping-ribonucleotides-incorporated-into-dna-by-hydrolytic-end-sequencing
#12
Clinton D Orebaugh, Scott A Lujan, Adam B Burkholder, Anders R Clausen, Thomas A Kunkel
Ribonucleotides embedded within DNA render the DNA sensitive to the formation of single-stranded breaks under alkali conditions. Here, we describe a next-generation sequencing method called hydrolytic end sequencing (HydEn-seq) to map ribonucleotides inserted into the genome of Saccharomyce cerevisiae strains deficient in ribonucleotide excision repair. We use this method to map several genomic features in wild-type and replicase variant yeast strains.
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29043633/measuring-the-levels-of-ribonucleotides-embedded-in-genomic-dna
#13
Alice Meroni, Giulia M Nava, Sarah Sertic, Paolo Plevani, Marco Muzi-Falconi, Federico Lazzaro
Ribonucleotides (rNTPs) are incorporated into genomic DNA at a relatively high frequency during replication. They have beneficial effects but, if not removed from the chromosomes, increase genomic instability. Here, we describe a fast method to easily estimate the amounts of embedded ribonucleotides into the genome. The protocol described is performed in Saccharomyces cerevisiae and allows us to quantify altered levels of rNMPs due to different mutations in the replicative polymerase ε. However, this protocol can be easily applied to cells derived from any organism...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29043622/alkaline-denaturing-southern-blot-analysis-to-monitor-double-strand-break-processing
#14
Chiara Vittoria Colombo, Luca Menin, Michela Clerici
Generation of 3' single-stranded DNA (ssDNA) tails at the ends of a double-strand break (DSB) is essential to repair the break through accurate homology-mediated repair pathways. Several methods have been developed to measure ssDNA accumulation at a DSB in the budding yeast Saccharomyces cerevisiae. Here, we describe one of these assays, which is based on the inability of restriction enzymes to cleave ssDNA. Digestion of genomic DNA prepared at different time points after DSB generation leads to the formation of ssDNA fragments whose length increases as the 5' strand degradation proceeds beyond restriction sites...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29043616/analyzing-genome-rearrangements-in-saccharomyces-cerevisiae
#15
Anjana Srivatsan, Christopher D Putnam, Richard D Kolodner
Genome rearrangements underlie different human diseases including many cancers. Determining the rates at which genome rearrangements arise and isolating unique, independent genome rearrangements is critical to understanding the genes and pathways that prevent or promote genome rearrangements. Here, we describe quantitative S. cerevisiae genetic assays for measuring the rates of accumulating genome rearrangements including deletions, translocations, and broken chromosomes healed by de novo telomere addition that result in the deletion of two counter-selectable genes, CAN1 and URA3, placed in the nonessential regions of the S...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29043613/the-chromosome-transmission-fidelity-assay-for-measuring-chromosome-loss-in-yeast
#16
Supipi Duffy, Philip Hieter
The budding yeast Saccharomyces cerevisiae has served as an excellent model system for studying highly conserved biological pathways including pathways involved in genome transmission and maintenance. The Chromosome Transmission Fidelity (CTF) colony color assay was developed to assess chromosome instability (CIN) in yeast, by monitoring the loss or gain during cell division of an artificial chromosome fragment carrying a visual marker. The CTF assay monitors changes in chromosome number, allowing the detection of mutants that exhibit increased rates of chromosome nondisjunction or chromosome loss...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29043612/the-a-like-faker-assay-for-measuring-yeast-chromosome-iii-stability
#17
Carolina A Novoa, J Sidney Ang, Peter C Stirling
The ability to rapidly assess chromosome instability (CIN) has enabled profiling of most yeast genes for potential effects on genome stability. The A-like faker (ALF) assay is one of several qualitative and quantitative marker loss assays that indirectly measure loss or conversion of genetic material using a counterselection step. The ALF assay relies on the ability to count spurious mating events that occur upon loss of the MATα locus of haploid Saccharomyces cerevisiae strains. Here, we describe the deployment of the ALF assay for both rapid and simple qualitative, and more in-depth quantitative analysis allowing determination of absolute ALF frequencies...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29043484/the-role-of-yeast-m-6-a-methyltransferase-in-peroxisomal-fatty-acid-oxidation
#18
REVIEW
Pradeep Kumar Yadav, Praveen Kumar Rajvanshi, Ram Rajasekharan
The precise and controlled regulation of gene expression at transcriptional and post-transcriptional levels is crucial for the eukaryotic cell survival and functions. In eukaryotes, more than 100 types of post-transcriptional RNA modifications have been identified. The N(6)-methyladenosine (m(6)A) modification in mRNA is among the most common post-transcriptional RNA modifications known in eukaryotic organisms, and the m(6)A RNA modification can regulate gene expression. The role of yeast m(6)A methyltransferase (Ime4) in meiosis, sporulation, triacylglycerol metabolism, vacuolar morphology, and mitochondrial functions has been reported...
October 17, 2017: Current Genetics
https://www.readbyqxmd.com/read/29043356/cationic-amphiphilic-zn-porphyrin-with-high-antifungal-photodynamic-potency
#19
Sara Moghnie, Artak Tovmasyan, James Craik, Ines Batinic-Haberle, Ludmil Benov
Photodynamic therapy (PDT) is a promising alternative approach particularly attractive for treatment of localized fungal infections. It is based on compounds, photosensitizers (PSs), which when excited with visible light, generate reactive species that ultimately cause cell death. Such species have short lifespans; as a consequence, efficiency and selectivity of the PDT treatment depend mainly on the properties of the PSs. This study is the first to explore the effect of cationic porphyrin-based photosensitizers on Saccharomyces cerevisiae, a member of the fungus kingdom...
October 18, 2017: Photochemical & Photobiological Sciences
https://www.readbyqxmd.com/read/29042445/analysis-of-substrate-specificity-of-trypanosoma-brucei-osts-by-functional-expression-of-domain-swapped-chimeras-in-yeast
#20
Kristina Poljak, Jörg Breitling, Robert Gauss, George Rugarabamu, Mauro Pellanda, Markus Aebi
N-linked protein glycosylation is an essential and highly conserved post-translational modification in eukaryotes. The transfer of a glycan from a lipid-linked oligosaccharide (LLO) donor to the asparagine residue of a nascent polypeptide chain is catalyzed by an oligosaccharyltransferase (OST) in the lumen of the endoplasmic reticulum (ER). Trypanosoma brucei encodes three paralogue single protein OSTs called TbSTT3A, TbSTT3B and TbSTT3C that can functionally complement the Saccharomyces cerevisiae OST, making it an ideal experimental system to study the fundamental properties of OST activity...
October 17, 2017: Journal of Biological Chemistry
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