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Sample records for yeast eukaryotic translation

  1. HIV-1 Replication and the Cellular Eukaryotic Translation Apparatus

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    Santiago Guerrero

    2015-01-01

    Full Text Available Eukaryotic translation is a complex process composed of three main steps: initiation, elongation, and termination. During infections by RNA- and DNA-viruses, the eukaryotic translation machinery is used to assure optimal viral protein synthesis. Human immunodeficiency virus type I (HIV-1 uses several non-canonical pathways to translate its own proteins, such as leaky scanning, frameshifting, shunt, and cap-independent mechanisms. Moreover, HIV-1 modulates the host translation machinery by targeting key translation factors and overcomes different cellular obstacles that affect protein translation. In this review, we describe how HIV-1 proteins target several components of the eukaryotic translation machinery, which consequently improves viral translation and replication.

  2. On the Diversification of the Translation Apparatus across Eukaryotes

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    Greco Hernández

    2012-01-01

    Full Text Available Diversity is one of the most remarkable features of living organisms. Current assessments of eukaryote biodiversity reaches 1.5 million species, but the true figure could be several times that number. Diversity is ingrained in all stages and echelons of life, namely, the occupancy of ecological niches, behavioral patterns, body plans and organismal complexity, as well as metabolic needs and genetics. In this review, we will discuss that diversity also exists in a key biochemical process, translation, across eukaryotes. Translation is a fundamental process for all forms of life, and the basic components and mechanisms of translation in eukaryotes have been largely established upon the study of traditional, so-called model organisms. By using modern genome-wide, high-throughput technologies, recent studies of many nonmodel eukaryotes have unveiled a surprising diversity in the configuration of the translation apparatus across eukaryotes, showing that this apparatus is far from being evolutionarily static. For some of the components of this machinery, functional differences between different species have also been found. The recent research reviewed in this article highlights the molecular and functional diversification the translational machinery has undergone during eukaryotic evolution. A better understanding of all aspects of organismal diversity is key to a more profound knowledge of life.

  3. Novel characteristics of the biological properties of the yeast Saccharomyces cerevisiae eukaryotic initiation factor 2A.

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    Komar, Anton A; Gross, Stephane R; Barth-Baus, Diane; Strachan, Ryan; Hensold, Jack O; Goss Kinzy, Terri; Merrick, William C

    2005-04-22

    Eukaryotic initiation factor 2A (eIF2A) has been shown to direct binding of the initiator methionyl-tRNA (Met-tRNA(i)) to 40 S ribosomal subunits in a codon-dependent manner, in contrast to eIF2, which requires GTP but not the AUG codon to bind initiator tRNA to 40 S subunits. We show here that yeast eIF2A genetically interacts with initiation factor eIF4E, suggesting that both proteins function in the same pathway. The double eIF2A/eIF4E-ts mutant strain displays a severe slow growth phenotype, which correlated with the accumulation of 85% of the double mutant cells arrested at the G(2)/M border. These cells also exhibited a disorganized actin cytoskeleton and elevated actin levels, suggesting that eIF2A might be involved in controlling the expression of genes involved in morphogenic processes. Further insights into eIF2A function were gained from the studies of eIF2A distribution in ribosomal fractions obtained from either an eIF5BDelta (fun12Delta) strain or a eIF3b-ts (prt1-1) strain. It was found that the binding of eIF2A to 40 and 80 S ribosomes was not impaired in either strain. We also found that eIF2A functions as a suppressor of Ure2p internal ribosome entry site-mediated translation in yeast cells. The regulation of expression from the URE2 internal ribosome entry site appears to be through the levels of eIF2A protein, which has been found to be inherently unstable with a half-life of approximately 17 min. It was hypothesized that this instability allows for translational control through the level of eIF2A protein in yeast cells.

  4. MetWAMer: eukaryotic translation initiation site prediction

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    Brendel Volker

    2008-09-01

    Full Text Available Abstract Background Translation initiation site (TIS identification is an important aspect of the gene annotation process, requisite for the accurate delineation of protein sequences from transcript data. We have developed the MetWAMer package for TIS prediction in eukaryotic open reading frames of non-viral origin. MetWAMer can be used as a stand-alone, third-party tool for post-processing gene structure annotations generated by external computational programs and/or pipelines, or directly integrated into gene structure prediction software implementations. Results MetWAMer currently implements five distinct methods for TIS prediction, the most accurate of which is a routine that combines weighted, signal-based translation initiation site scores and the contrast in coding potential of sequences flanking TISs using a perceptron. Also, our program implements clustering capabilities through use of the k-medoids algorithm, thereby enabling cluster-specific TIS parameter utilization. In practice, our static weight array matrix-based indexing method for parameter set lookup can be used with good results in data sets exhibiting moderate levels of 5'-complete coverage. Conclusion We demonstrate that improvements in statistically-based models for TIS prediction can be achieved by taking the class of each potential start-methionine into account pending certain testing conditions, and that our perceptron-based model is suitable for the TIS identification task. MetWAMer represents a well-documented, extensible, and freely available software system that can be readily re-trained for differing target applications and/or extended with existing and novel TIS prediction methods, to support further research efforts in this area.

  5. Origins of robustness in translational control via eukaryotic translation initiation factor (eIF) 2.

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    Khan, Mohammad Farhan; Spurgeon, Sarah; von der Haar, Tobias

    2018-05-14

    Phosphorylation of eukaryotic translation initiation factor 2 (eIF2) is one of the best studied and most widely used means for regulating protein synthesis activity in eukaryotic cells. This pathway regulates protein synthesis in response to stresses, viral infections, and nutrient depletion, among others. We present analyses of an ordinary differential equation-based model of this pathway, which aim to identify its principal robustness-conferring features. Our analyses indicate that robustness is a distributed property, rather than arising from the properties of any one individual pathway species. However, robustness-conferring properties are unevenly distributed between the different species, and we identify a guanine nucleotide dissociation inhibitor (GDI) complex as a species that likely contributes strongly to the robustness of the pathway. Our analyses make further predictions on the dynamic response to different types of kinases that impinge on eIF2. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. Interaction of the RNP1 motif in PRT1 with HCR1 promotes 40S binding of eukaryotic initiation factor 3 in yeast

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    Nielsen, Klaus H; Valásek, Leos; Sykes, Caroah

    2006-01-01

    We found that mutating the RNP1 motif in the predicted RRM domain in yeast eukaryotic initiation factor 3 (eIF3) subunit b/PRT1 (prt1-rnp1) impairs its direct interactions in vitro with both eIF3a/TIF32 and eIF3j/HCR1. The rnp1 mutation in PRT1 confers temperature-sensitive translation initiation...

  7. Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions.

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    Hesketh, Andy; Vergnano, Marta; Wan, Chris; Oliver, Stephen G

    2017-07-25

    We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA) and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR) inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection. IMPORTANCE During infections, pathogenic bacteria can release nucleotides into the cells of their eukaryotic hosts. These nucleotides are recognized as signals that contribute to the initiation of defensive immune responses that help the infected

  8. Neural Network Prediction of Translation Initiation Sites in Eukaryotes: Perspectives for EST and Genome analysis

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    Pedersen, Anders Gorm; Nielsen, Henrik

    1997-01-01

    Translation in eukaryotes does not always start at the first AUG in an mRNA, implying that context information also plays a role.This makes prediction of translation initiation sites a non-trivial task, especially when analysing EST and genome data where the entire mature mRNA sequence is not known...

  9. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

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    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Initiation of translation in bacteria by a structured eukaryotic IRES RNA.

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    Colussi, Timothy M; Costantino, David A; Zhu, Jianyu; Donohue, John Paul; Korostelev, Andrei A; Jaafar, Zane A; Plank, Terra-Dawn M; Noller, Harry F; Kieft, Jeffrey S

    2015-03-05

    The central dogma of gene expression (DNA to RNA to protein) is universal, but in different domains of life there are fundamental mechanistic differences within this pathway. For example, the canonical molecular signals used to initiate protein synthesis in bacteria and eukaryotes are mutually exclusive. However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life. We wanted to explore whether an undiscovered RNA-based signal might be able to use these conserved features, bypassing mechanisms specific to each domain of life, and initiate protein synthesis in both bacteria and eukaryotes. Although structured internal ribosome entry site (IRES) RNAs can manipulate ribosomes to initiate translation in eukaryotic cells, an analogous RNA structure-based mechanism has not been observed in bacteria. Here we report our discovery that a eukaryotic viral IRES can initiate translation in live bacteria. We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resolution, revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by transfer RNAs. Initiation in both bacteria and eukaryotes depends on the structure of the IRES RNA, but in bacteria this RNA uses a different mechanism that includes a form of ribosome repositioning after initial recruitment. This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.

  11. Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions

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    Andy Hesketh

    2017-07-01

    Full Text Available We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP, cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection.

  12. A set of ligation-independent in vitro translation vectors for eukaryotic protein production

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    Endo Yaeta

    2008-03-01

    Full Text Available Abstract Background The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags. Results We designed four ligation independent cloning (LIC vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein. Conclusion Four newly

  13. Present status of DNA repair mechanisms in uv irradiated yeast taken as a model eukaryotic system

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    Moustacchi, E.; Waters, R.; Heude, M.; Chanet, R.

    1975-01-01

    The repair mechanisms of altered DNA are generally less well understood for eukaryotes than they are for prokaryotes and bacteriophages. For mammalian cell lines cultured in vitro the specific labelling of DNA has allowed the biochemical analysis of some of the steps of the repair processes whereas the determination of their genetic controls is, with a few exceptions, obviously difficult. On the other hand, with fungi and more specifically with yeast taken as a model unicellular eukaryotic system, the genetic approach has been extensively explored: radiosensitive mutants are readily detected and genetically analyzed, double and multiple mutants can be constructed and from their responses to irradiation the number of repair pathways involved can be suggested. The lack of thymidine kinase in these organisms has hampered for a certain time the biochemical analysis of repair. However, the recent isolation of yeast strains capable of taking up and incorporating thymidine 5'-monophosphate into their DNA opens new possibilities for the future. In spite of this difficulty, attempts to measure the induction and removal of uv-induced pyrimidine dimers were performed by several groups during the last three years. The two main repair pathways described for E. coli, i.e., the excision-resynthesis and post-replicative recombinational repair pathways, do exist in yeast. The existence of the former pathway is supported not only by indirect evidence but also by biochemical analysis. The rad 1 and rad 2 mutants for instance have been shown to be blocked in the excision of uv-induced pyrimidine dimers. Other loci are epistatic to rad 1 and rad 2 (rad 3 , rad 4 ) and are likely to act on this excision pathway. The genetic control of the mitochondrial response to a uv treatment involves nuclear genes and mitochondrial determinants

  14. Model of cap-dependent translation initiation in sea urchin: a step towards the eukaryotic translation regulation network.

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    Bellé, Robert; Prigent, Sylvain; Siegel, Anne; Cormier, Patrick

    2010-03-01

    The large and rapid increase in the rate of protein synthesis following fertilization of the sea urchin egg has long been a paradigm of translational control, an important component of the regulation of gene expression in cells. This translational up-regulation is linked to physiological changes that occur upon fertilization and is necessary for entry into first cell division cycle. Accumulated knowledge on cap-dependent initiation of translation makes it suited and timely to start integrating the data into a system view of biological functions. Using a programming environment for system biology coupled with model validation (named Biocham), we have built an integrative model for cap-dependent initiation of translation. The model is described by abstract rules. It contains 51 reactions involved in 74 molecular complexes. The model proved to be coherent with existing knowledge by using queries based on computational tree logic (CTL) as well as Boolean simulations. The model could simulate the change in translation occurring at fertilization in the sea urchin model. It could also be coupled with an existing model designed for cell-cycle control. Therefore, the cap-dependent translation initiation model can be considered a first step towards the eukaryotic translation regulation network.

  15. A novel nuclear genetic code alteration in yeasts and the evolution of codon reassignment in eukaryotes.

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    Mühlhausen, Stefanie; Findeisen, Peggy; Plessmann, Uwe; Urlaub, Henning; Kollmar, Martin

    2016-07-01

    The genetic code is the cellular translation table for the conversion of nucleotide sequences into amino acid sequences. Changes to the meaning of sense codons would introduce errors into almost every translated message and are expected to be highly detrimental. However, reassignment of single or multiple codons in mitochondria and nuclear genomes, although extremely rare, demonstrates that the code can evolve. Several models for the mechanism of alteration of nuclear genetic codes have been proposed (including "codon capture," "genome streamlining," and "ambiguous intermediate" theories), but with little resolution. Here, we report a novel sense codon reassignment in Pachysolen tannophilus, a yeast related to the Pichiaceae. By generating proteomics data and using tRNA sequence comparisons, we show that Pachysolen translates CUG codons as alanine and not as the more usual leucine. The Pachysolen tRNACAG is an anticodon-mutated tRNA(Ala) containing all major alanine tRNA recognition sites. The polyphyly of the CUG-decoding tRNAs in yeasts is best explained by a tRNA loss driven codon reassignment mechanism. Loss of the CUG-tRNA in the ancient yeast is followed by gradual decrease of respective codons and subsequent codon capture by tRNAs whose anticodon is not part of the aminoacyl-tRNA synthetase recognition region. Our hypothesis applies to all nuclear genetic code alterations and provides several testable predictions. We anticipate more codon reassignments to be uncovered in existing and upcoming genome projects. © 2016 Mühlhausen et al.; Published by Cold Spring Harbor Laboratory Press.

  16. Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them.

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    Leppek, Kathrin; Das, Rhiju; Barna, Maria

    2018-03-01

    RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5' untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5' UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms.

  17. Reassigning stop codons via translation termination: How a few eukaryotes broke the dogma.

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    Alkalaeva, Elena; Mikhailova, Tatiana

    2017-03-01

    The genetic code determines how amino acids are encoded within mRNA. It is universal among the vast majority of organisms, although several exceptions are known. Variant genetic codes are found in ciliates, mitochondria, and numerous other organisms. All revealed genetic codes (standard and variant) have at least one codon encoding a translation stop signal. However, recently two new genetic codes with a reassignment of all three stop codons were revealed in studies examining the protozoa transcriptomes. Here, we discuss this finding and the recent studies of variant genetic codes in eukaryotes. We consider the possible molecular mechanisms allowing the use of certain codons as sense and stop signals simultaneously. The results obtained by studying these amazing organisms represent a new and exciting insight into the mechanism of stop codon decoding in eukaryotes. Also see the video abstract here. © 2017 WILEY Periodicals, Inc.

  18. Heterologous Expression of Membrane and Soluble Proteins Derepresses GCN4 mRNA Translation in the Yeast Saccharomyces cerevisiae

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    Steffensen, L.; Pedersen, P. A.

    2006-01-01

    -ATPase also induced GCN4 translation. Derepression of GCN4 translation required phosphorylation of eIF-2 , the tRNA binding domain of Gcn2p, and the ribosome-associated proteins Gcn1p and Gcn20p. The increase in Gcn4p density in response to heterologous expression did not induce transcription from the HIS4...... promoter, a traditional Gcn4p target.......This paper describes the first physiological response at the translational level towards heterologous protein production in Saccharomyces cerevisiae. In yeast, the phosphorylation of eukaryotic initiation factor 2 (eIF-2 ) by Gcn2p protein kinase mediates derepression of GCN4 mRNA translation. Gcn4...

  19. Non-AUG translation: a new start for protein synthesis in eukaryotes.

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    Kearse, Michael G; Wilusz, Jeremy E

    2017-09-01

    Although it was long thought that eukaryotic translation almost always initiates at an AUG start codon, recent advancements in ribosome footprint mapping have revealed that non-AUG start codons are used at an astonishing frequency. These non-AUG initiation events are not simply errors but instead are used to generate or regulate proteins with key cellular functions; for example, during development or stress. Misregulation of non-AUG initiation events contributes to multiple human diseases, including cancer and neurodegeneration, and modulation of non-AUG usage may represent a novel therapeutic strategy. It is thus becoming increasingly clear that start codon selection is regulated by many trans -acting initiation factors as well as sequence/structural elements within messenger RNAs and that non-AUG translation has a profound impact on cellular states. © 2017 Kearse and Wilusz; Published by Cold Spring Harbor Laboratory Press.

  20. Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment

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    Fan Ying; Shi Lichi; Ladizhansky, Vladimir; Brown, Leonid S.

    2011-01-01

    Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives, successfully used for producing proteins for solution NMR studies, is yeast expression systems, particularly Pichia pastoris. We report on successful implementation and optimization of isotope labeling protocols, previously used for soluble secreted proteins, to produce homogeneous samples of a eukaryotic seven-transmembrane helical protein, rhodopsin from Leptosphaeria maculans. Even in shake-flask cultures, yields exceeded 5 mg of purified uniformly 13 C, 15 N-labeled protein per liter of culture. The protein was stable (at least several weeks at 5°C) and functionally active upon reconstitution into lipid membranes at high protein-to-lipid ratio required for solid-state NMR. The samples gave high-resolution 13 C and 15 N solid-state magic angle spinning NMR spectra, amenable to a detailed structural analysis. We believe that similar protocols can be adopted for challenging mammalian targets, which often resist characterization by other structural methods.

  1. Speed Controls in Translating Secretory Proteins in Eukaryotes - an Evolutionary Perspective

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    Mahlab, Shelly; Linial, Michal

    2014-01-01

    Protein translation is the most expensive operation in dividing cells from bacteria to humans. Therefore, managing the speed and allocation of resources is subject to tight control. From bacteria to humans, clusters of relatively rare tRNA codons at the N′-terminal of mRNAs have been implicated in attenuating the process of ribosome allocation, and consequently the translation rate in a broad range of organisms. The current interpretation of “slow” tRNA codons does not distinguish between protein translations mediated by free- or endoplasmic reticulum (ER)-bound ribosomes. We demonstrate that proteins translated by free- or ER-bound ribosomes exhibit different overall properties in terms of their translation efficiency and speed in yeast, fly, plant, worm, bovine and human. We note that only secreted or membranous proteins with a Signal peptide (SP) are specified by segments of “slow” tRNA at the N′-terminal, followed by abundant codons that are considered “fast.” Such profiles apply to 3100 proteins of the human proteome that are composed of secreted and signal peptide (SP)-assisted membranous proteins. Remarkably, the bulks of the proteins (12,000), or membranous proteins lacking SP (3400), do not have such a pattern. Alternation of “fast” and “slow” codons was found also in proteins that translocate to mitochondria through transit peptides (TP). The differential clusters of tRNA adapted codons is not restricted to the N′-terminal of transcripts. Specifically, Glycosylphosphatidylinositol (GPI)-anchored proteins are unified by clusters of low adapted tRNAs codons at the C′-termini. Furthermore, selection of amino acids types and specific codons was shown as the driving force which establishes the translation demands for the secretory proteome. We postulate that “hard-coded” signals within the secretory proteome assist the steps of protein maturation and folding. Specifically, “speed control” signals for delaying the translation

  2. Depletion of eIF4G from yeast cells narrows the range of translational efficiencies genome-wide

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    Hinnebusch Alan G

    2011-01-01

    Full Text Available Abstract Background Eukaryotic translation initiation factor 4G (eIF4G is thought to influence the translational efficiencies of cellular mRNAs by its roles in forming an eIF4F-mRNA-PABP mRNP that is competent for attachment of the 43S preinitiation complex, and in scanning through structured 5' UTR sequences. We have tested this hypothesis by determining the effects of genetically depleting eIF4G from yeast cells on global translational efficiencies (TEs, using gene expression microarrays to measure the abundance of mRNA in polysomes relative to total mRNA for ~5900 genes. Results Although depletion of eIF4G is lethal and reduces protein synthesis by ~75%, it had small effects (less than a factor of 1.5 on the relative TE of most genes. Within these limits, however, depleting eIF4G narrowed the range of translational efficiencies genome-wide, with mRNAs of better than average TE being translated relatively worse, and mRNAs with lower than average TE being translated relatively better. Surprisingly, the fraction of mRNAs most dependent on eIF4G display an average 5' UTR length at or below the mean for all yeast genes. Conclusions This finding suggests that eIF4G is more critical for ribosome attachment to mRNAs than for scanning long, structured 5' UTRs. Our results also indicate that eIF4G, and the closed-loop mRNP it assembles with the m7 G cap- and poly(A-binding factors (eIF4E and PABP, is not essential for translation of most (if not all mRNAs but enhances the differentiation of translational efficiencies genome-wide.

  3. Eukaryotic elongation factor 2 controls TNF-α translation in LPS-induced hepatitis

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    González-Terán, Bárbara; Cortés, José R.; Manieri, Elisa; Matesanz, Nuria; Verdugo, ρngeles; Rodríguez, María E.; González-Rodríguez, ρgueda; Valverde, ρngela; Martín, Pilar; Davis, Roger J.; Sabio, Guadalupe

    2012-01-01

    Bacterial LPS (endotoxin) has been implicated in the pathogenesis of acute liver disease through its induction of the proinflammatory cytokine TNF-α. TNF-α is a key determinant of the outcome in a well-established mouse model of acute liver failure during septic shock. One possible mechanism for regulating TNF-α expression is through the control of protein elongation during translation, which would allow rapid cell adaptation to physiological changes. However, the regulation of translational elongation is poorly understood. We found that expression of p38γ/δ MAPK proteins is required for the elongation of nascent TNF-α protein in macrophages. The MKK3/6-p38γ/δ pathway mediated an inhibitory phosphorylation of eukaryotic elongation factor 2 (eEF2) kinase, which in turn promoted eEF2 activation (dephosphorylation) and subsequent TNF-α elongation. These results identify a new signaling pathway that regulates TNF-α production in LPS-induced liver damage and suggest potential cell-specific therapeutic targets for liver diseases in which TNF-α production is involved. PMID:23202732

  4. Characterization of a eukaryotic translation initiation factor 5A homolog from Tamarix androssowii involved in plant abiotic stress tolerance

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    Wang Liuqiang

    2012-07-01

    Full Text Available Abstract Background The eukaryotic translation initiation factor 5A (eIF5A promotes formation of the first peptide bond at the onset of protein synthesis. However, the function of eIF5A in plants is not well understood. Results In this study, we characterized the function of eIF5A (TaeIF5A1 from Tamarix androssowii. The promoter of TaeIF5A1 with 1,486 bp in length was isolated, and the cis-elements in the promoter were identified. A WRKY (TaWRKY and RAV (TaRAV protein can specifically bind to a W-box motif in the promoter of TaeIF5A1 and activate the expression of TaeIF5A1. Furthermore, TaeIF5A1, TaWRKY and TaRAV share very similar expression pattern and are all stress-responsive gene that functions in the abscisic acid (ABA signaling pathway, indicating that they are components of a single regulatory pathway. Transgenic yeast and poplar expressing TaeIF5A1 showed elevated protein levels combined with improved abiotic stresses tolerance. Furthermore, TaeIF5A1-transformed plants exhibited enhanced superoxide dismutase (SOD and peroxidase (POD activities, lower electrolyte leakage and higher chlorophyll content under salt stress. Conclusions These results suggested that TaeIF5A1 is involved in abiotic stress tolerance, and is likely regulated by transcription factors TaWRKY and TaRAV both of which can bind to the W-box motif. In addition, TaeIF5A1 may mediate stress tolerance by increasing protein synthesis, enhancing ROS scavenging by improving SOD and POD activities, and preventing chlorophyll loss and membrane damage. Therefore, eIF5A may play an important role in plant adaptation to changing environmental conditions.

  5. Characterization of a eukaryotic translation initiation factor 5A homolog from Tamarix androssowii involved in plant abiotic stress tolerance.

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    Wang, Liuqiang; Xu, Chenxi; Wang, Chao; Wang, Yucheng

    2012-07-26

    The eukaryotic translation initiation factor 5A (eIF5A) promotes formation of the first peptide bond at the onset of protein synthesis. However, the function of eIF5A in plants is not well understood. In this study, we characterized the function of eIF5A (TaeIF5A1) from Tamarix androssowii. The promoter of TaeIF5A1 with 1,486 bp in length was isolated, and the cis-elements in the promoter were identified. A WRKY (TaWRKY) and RAV (TaRAV) protein can specifically bind to a W-box motif in the promoter of TaeIF5A1 and activate the expression of TaeIF5A1. Furthermore, TaeIF5A1, TaWRKY and TaRAV share very similar expression pattern and are all stress-responsive gene that functions in the abscisic acid (ABA) signaling pathway, indicating that they are components of a single regulatory pathway. Transgenic yeast and poplar expressing TaeIF5A1 showed elevated protein levels combined with improved abiotic stresses tolerance. Furthermore, TaeIF5A1-transformed plants exhibited enhanced superoxide dismutase (SOD) and peroxidase (POD) activities, lower electrolyte leakage and higher chlorophyll content under salt stress. These results suggested that TaeIF5A1 is involved in abiotic stress tolerance, and is likely regulated by transcription factors TaWRKY and TaRAV both of which can bind to the W-box motif. In addition, TaeIF5A1 may mediate stress tolerance by increasing protein synthesis, enhancing ROS scavenging by improving SOD and POD activities, and preventing chlorophyll loss and membrane damage. Therefore, eIF5A may play an important role in plant adaptation to changing environmental conditions.

  6. Yeast 2.0-connecting the dots in the construction of the world's first functional synthetic eukaryotic genome.

    Science.gov (United States)

    Pretorius, I S; Boeke, J D

    2018-06-01

    Historians of the future may well describe 2018 as the year that the world's first functional synthetic eukaryotic genome became a reality. Without the benefit of hindsight, it might be hard to completely grasp the long-term significance of a breakthrough moment in the history of science like this. The role of synthetic biology in the imminent birth of a budding Saccharomyces cerevisiae yeast cell carrying 16 man-made chromosomes causes the world of science to teeter on the threshold of a future-defining scientific frontier. The genome-engineering tools and technologies currently being developed to produce the ultimate yeast genome will irreversibly connect the dots between our improved understanding of the fundamentals of a complex cell containing its DNA in a specialised nucleus and the application of bioengineered eukaryotes designed for advanced biomanufacturing of beneficial products. By joining up the dots between the findings and learnings from the international Synthetic Yeast Genome project (known as the Yeast 2.0 or Sc2.0 project) and concurrent advancements in biodesign tools and smart data-intensive technologies, a future world powered by a thriving bioeconomy seems realistic. This global project demonstrates how a collaborative network of dot connectors-driven by a tinkerer's indomitable curiosity to understand how things work inside a eukaryotic cell-are using cutting-edge biodesign concepts and synthetic biology tools to advance science and to positively frame human futures (i.e. improved quality of life) in a planetary context (i.e. a sustainable environment). Explorations such as this have a rich history of resulting in unexpected discoveries and unanticipated applications for the benefit of people and planet. However, we must learn from past explorations into controversial futuristic sciences and ensure that researchers at the forefront of an emerging science such as synthetic biology remain connected to all stakeholders' concerns about the

  7. The N-terminal region of eukaryotic translation initiation factor 5A signals to nuclear localization of the protein

    International Nuclear Information System (INIS)

    Parreiras-e-Silva, Lucas T.; Gomes, Marcelo D.; Oliveira, Eduardo B.; Costa-Neto, Claudio M.

    2007-01-01

    The eukaryotic translation initiation factor 5A (eIF5A) is a ubiquitous protein of eukaryotic and archaeal organisms which undergoes hypusination, a unique post-translational modification. We have generated a polyclonal antibody against murine eIF5A, which in immunocytochemical assays in B16-F10 cells revealed that the endogenous protein is preferentially localized to the nuclear region. We therefore analyzed possible structural features present in eIF5A proteins that could be responsible for that characteristic. Multiple sequence alignment analysis of eIF5A proteins from different eukaryotic and archaeal organisms showed that the former sequences have an extended N-terminal segment. We have then performed in silico prediction analyses and constructed different truncated forms of murine eIF5A to verify any possible role that the N-terminal extension might have in determining the subcellular localization of the eIF5A in eukaryotic organisms. Our results indicate that the N-terminal extension of the eukaryotic eIF5A contributes in signaling this protein to nuclear localization, despite of bearing no structural similarity with classical nuclear localization signals

  8. Improved Ribosome-Footprint and mRNA Measurements Provide Insights into Dynamics and Regulation of Yeast Translation

    Science.gov (United States)

    2016-02-11

    unlimited. Improved Ribosome-Footprint and mRNA Measurements Provide Insights into Dynamics and Regulation of Yeast Translation The views, opinions and...into Dynamics and Regulation of Yeast Translation Report Title Ribosome-footprint profiling provides genome-wide snapshots of translation, but...tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was

  9. Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms

    OpenAIRE

    Kahvejian, Avak; Svitkin, Yuri V.; Sukarieh, Rami; M'Boutchou, Marie-Noël; Sonenberg, Nahum

    2005-01-01

    Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5′-end of the mRNA to promote the recruitment of the ribosome. Although the 3′ poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for ...

  10. Yeast for virus research

    Science.gov (United States)

    Zhao, Richard Yuqi

    2017-01-01

    Budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are two popular model organisms for virus research. They are natural hosts for viruses as they carry their own indigenous viruses. Both yeasts have been used for studies of plant, animal and human viruses. Many positive sense (+) RNA viruses and some DNA viruses replicate with various levels in yeasts, thus allowing study of those viral activities during viral life cycle. Yeasts are single cell eukaryotic organisms. Hence, many of the fundamental cellular functions such as cell cycle regulation or programed cell death are highly conserved from yeasts to higher eukaryotes. Therefore, they are particularly suited to study the impact of those viral activities on related cellular activities during virus-host interactions. Yeasts present many unique advantages in virus research over high eukaryotes. Yeast cells are easy to maintain in the laboratory with relative short doubling time. They are non-biohazardous, genetically amendable with small genomes that permit genome-wide analysis of virologic and cellular functions. In this review, similarities and differences of these two yeasts are described. Studies of virologic activities such as viral translation, viral replication and genome-wide study of virus-cell interactions in yeasts are highlighted. Impacts of viral proteins on basic cellular functions such as cell cycle regulation and programed cell death are discussed. Potential applications of using yeasts as hosts to carry out functional analysis of small viral genome and to develop high throughput drug screening platform for the discovery of antiviral drugs are presented. PMID:29082230

  11. Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms.

    Science.gov (United States)

    Kahvejian, Avak; Svitkin, Yuri V; Sukarieh, Rami; M'Boutchou, Marie-Noël; Sonenberg, Nahum

    2005-01-01

    Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5'-end of the mRNA to promote the recruitment of the ribosome. Although the 3' poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (approximately 65% vs. approximately 35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5'-end of mRNA.

  12. Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them

    Science.gov (United States)

    Leppek, Kathrin; Das, Rhiju; Barna, Maria

    2017-01-01

    RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5′ untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5′ UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms. PMID:29165424

  13. Diversity of Eukaryotic Translational Initiation Factor eIF4E in Protists.

    Science.gov (United States)

    Jagus, Rosemary; Bachvaroff, Tsvetan R; Joshi, Bhavesh; Place, Allen R

    2012-01-01

    The greatest diversity of eukaryotic species is within the microbial eukaryotes, the protists, with plants and fungi/metazoa representing just two of the estimated seventy five lineages of eukaryotes. Protists are a diverse group characterized by unusual genome features and a wide range of genome sizes from 8.2 Mb in the apicomplexan parasite Babesia bovis to 112,000-220,050 Mb in the dinoflagellate Prorocentrum micans. Protists possess numerous cellular, molecular and biochemical traits not observed in "text-book" model organisms. These features challenge some of the concepts and assumptions about the regulation of gene expression in eukaryotes. Like multicellular eukaryotes, many protists encode multiple eIF4Es, but few functional studies have been undertaken except in parasitic species. An earlier phylogenetic analysis of protist eIF4Es indicated that they cannot be grouped within the three classes that describe eIF4E family members from multicellular organisms. Many more protist sequences are now available from which three clades can be recognized that are distinct from the plant/fungi/metazoan classes. Understanding of the protist eIF4Es will be facilitated as more sequences become available particularly for the under-represented opisthokonts and amoebozoa. Similarly, a better understanding of eIF4Es within each clade will develop as more functional studies of protist eIF4Es are completed.

  14. Ribosome stalling regulates IRES-mediated translation in eukaryotes, a parallel to prokaryotic attenuation

    NARCIS (Netherlands)

    Fernandez, James; Yaman, Ibrahim; Huang, Charles; Liu, Haiyan; Lopez, Alex B.; Komar, Anton A.; Caprara, Mark G.; Merrick, William C.; Snider, Martin D.; Kaufman, Randal J.; Lamers, Wouter H.; Hatzoglou, Maria

    2005-01-01

    It was previously shown that the mRNA for the cat-1 Arg/Lys transporter is translated from an internal ribosome entry site (IRES) that is regulated by cellular stress. Amino acid starvation stimulated cat-1 translation via a mechanism that requires translation of an ORF in the mRNA leader and

  15. AUG is the only initiation codon in eukaryotes

    Energy Technology Data Exchange (ETDEWEB)

    Sherman, F; McKnight, G; Stewart, J W

    1980-01-01

    An analysis of mutants of the yeast Saccharomyces cerevisiae indicates that AUG is the sole codon capable of initiating translation of iso-1-cytochrome c. This result with yeast and the sequence results of numerous eukaryotic genes indicate that AUG is the only initiation codon in eukaryotes; in contrast, results with Escherichia colia and bacteriophages indicate that both AUG and GUG are initiation codons in prokaryotes. The difference can be explained by the lack of the t/sup 6/ A hypermodified nucleoside (N-(9-(..beta..-D-ribofuranosyl)purin-6-ylcarbamoyl)threonine) in prokaryotic initiator tRNA and its presence in eukaryotic initiator tRNA.

  16. The long non-coding RNA GAS5 cooperates with the eukaryotic translation initiation factor 4E to regulate c-Myc translation.

    Directory of Open Access Journals (Sweden)

    Guangzhen Hu

    Full Text Available Long noncoding RNAs (lncRNAs are important regulators of transcription; however, their involvement in protein translation is not well known. Here we explored whether the lncRNA GAS5 is associated with translation initiation machinery and regulates translation. GAS5 was enriched with eukaryotic translation initiation factor-4E (eIF4E in an RNA-immunoprecipitation assay using lymphoma cell lines. We identified two RNA binding motifs within eIF4E protein and the deletion of each motif inhibited the binding of GAS5 with eIF4E. To confirm the role of GAS5 in translation regulation, GAS5 siRNA and in vitro transcribed GAS5 RNA were used to knock down or overexpress GAS5, respectively. GAS5 siRNA had no effect on global protein translation but did specifically increase c-Myc protein level without an effect on c-Myc mRNA. The mechanism of this increase in c-Myc protein was enhanced association of c-Myc mRNA with the polysome without any effect on protein stability. In contrast, overexpression of in vitro transcribed GAS5 RNA suppressed c-Myc protein without affecting c-Myc mRNA. Interestingly, GAS5 was found to be bound with c-Myc mRNA, suggesting that GAS5 regulates c-Myc translation through lncRNA-mRNA interaction. Our findings have uncovered a role of GAS5 lncRNA in translation regulation through its interactions with eIF4E and c-Myc mRNA.

  17. Nutrient control of eukaryote cell growth: a systems biology study in yeast

    Directory of Open Access Journals (Sweden)

    Lilley Kathryn S

    2010-05-01

    Full Text Available Abstract Background To elucidate the biological processes affected by changes in growth rate and nutrient availability, we have performed a comprehensive analysis of the transcriptome, proteome and metabolome responses of chemostat cultures of the yeast, Saccharomyces cerevisiae, growing at a range of growth rates and in four different nutrient-limiting conditions. Results We find significant changes in expression for many genes in each of the four nutrient-limited conditions tested. We also observe several processes that respond differently to changes in growth rate and are specific to each nutrient-limiting condition. These include carbohydrate storage, mitochondrial function, ribosome synthesis, and phosphate transport. Integrating transcriptome data with proteome measurements allows us to identify previously unrecognized examples of post-transcriptional regulation in response to both nutrient and growth-rate signals. Conclusions Our results emphasize the unique properties of carbon metabolism and the carbon substrate, the limitation of which induces significant changes in gene regulation at the transcriptional and post-transcriptional level, as well as altering how many genes respond to growth rate. By comparison, the responses to growth limitation by other nutrients involve a smaller set of genes that participate in specific pathways. See associated commentary http://www.biomedcentral.com/1741-7007/8/62

  18. Mutation of a Nicotiana tabacum L. eukaryotic translation-initiation factor gene reduces susceptibility to a resistance-breaking strain of Potato Virus Y.

    Science.gov (United States)

    Takakura, Yoshimitsu; Udagawa, Hisashi; Shinjo, Akira; Koga, Kazuharu

    2018-04-06

    Eukaryotic translation-initiation factors eIF4E and eIF(iso)4E in plants play key roles in infection by potyviruses and other plant RNA viruses. Mutations in the genes encoding these factors reduce susceptibility to the viruses, and are the basis of several recessive virus-resistance genes widely used in plant breeding. Because virus variants occasionally break such resistance, the molecular basis for this process must be elucidated. Although deletion mutants of eIF4E1-S of tobacco (Nicotiana tabacum L.) resist Potato virus Y (PVY; the type member of the genus Potyvirus), resistance-breaking strains of PVY threaten tobacco production worldwide. Here, we used RNA interference technology to knock down tobacco eIF4E2-S and eIF4E2-T genes or eIF(iso)4E-S and eIF(iso)4E-T genes. Transgenic plants with reduced transcript levels of both eIF(iso)4E-S and eIF(iso)4E-T showed reduced susceptibility to a resistance-breaking PVY strain with a K105E mutation in the viral genome-associated protein (VPg). By screening a population of chemically-induced mutants of eIF(iso)4E-S and eIF(iso)4E-T, we showed that plants with a nonsense mutation in eIF(iso)4E-T, but not eIF(iso)4E-S, showed reduced susceptibility to the resistance-breaking PVY strain. In a yeast two-hybrid assay, VPg of the resistance-breaking strain, but not wild-type PVY, physically interacted with the eIF(iso)4E-T protein. Thus, eIF4E1-S is required for infection by PVY, but eIF(iso)4E-T is required for infection by the resistance-breaking strain. Our study provides the first evidence for the involvement of a host eukaryotic translation-initiation factor in the infection cycle of a resistance-breaking virus strain. The eIF(iso)4E-T mutants will be useful in tobacco breeding to introduce resistance against resistance-breaking PVY strains. This article is protected by copyright. All rights reserved. © 2018 BSPP and John Wiley & Sons Ltd.

  19. Please do not recycle! Translation reinitiation in microbes and higher eukaryotes

    Czech Academy of Sciences Publication Activity Database

    Gunišová, Stanislava; Hronová, Vladislava; Mohammad, Mahabub Pasha; Hinnebusch, A.G.; Valášek, Leoš Shivaya

    2018-01-01

    Roč. 42, č. 2 (2018), s. 165-192 E-ISSN 1574-6976 R&D Projects: GA ČR GA15-10116S EU Projects: Wellcome Trust(GB) 090812/B/09/Z Institutional support: RVO:61388971 Keywords : translation reinitiation * termination-reinitiation * uORF Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology

  20. Extensive proteomic remodeling is induced by eukaryotic translation elongation factor 1Bγ deletion in Aspergillus fumigatus.

    Science.gov (United States)

    O'Keeffe, Grainne; Jöchl, Christoph; Kavanagh, Kevin; Doyle, Sean

    2013-11-01

    The opportunistic pathogen Aspergillus fumigatus is ubiquitous in the environment and predominantly infects immunocompromised patients. The functions of many genes remain unknown despite sequencing of the fungal genome. A putative translation elongation factor 1Bγ (eEF1Bγ, termed elfA; 750 bp) is expressed, and exhibits glutathione S-transferase activity, in A. fumigatus. Here, we demonstrate the role of ElfA in the oxidative stress response, as well as a possible involvement in translation and actin cytoskeleton organization, respectively. Comparative proteomics, in addition to phenotypic analysis, under basal and oxidative stress conditions, demonstrated a role for A. fumigatus elfA in the oxidative stress response. An elfA-deficient strain (A. fumigatus ΔelfA) was significantly more sensitive to the oxidants H2O2, diamide, and 4,4'-dipyridyl disulfide (DPS) than the wild-type. This was further supported with the identification of differentially expressed proteins of the oxidative stress response, including; mitochondrial peroxiredoxin Prx1, molecular chaperone Hsp70 and mitochondrial glycerol-3-phosphate dehydrogenase. Phenotypic analysis also revealed that A. fumigatus ΔelfA was significantly more tolerant to voriconazole than the wild-type. The differential expression of two aminoacyl-tRNA synthetases suggests a role for A. fumigatus elfA in translation, while the identification of actin-bundling protein Sac6 and vacuolar dynamin-like GTPase VpsA link A. fumigatus elfA to the actin cytoskeleton. Overall, this work highlights the diverse roles of A. fumigatus elfA, with respect to translation, oxidative stress and actin cytoskeleton organization. In addition to this, the strategy of combining targeted gene deletion with comparative proteomics for elucidating the role of proteins of unknown function is further revealed. © 2013 The Protein Society.

  1. Inhibition of eukaryotic translation elongation by the antitumor natural product Mycalamide B.

    Science.gov (United States)

    Dang, Yongjun; Schneider-Poetsch, Tilman; Eyler, Daniel E; Jewett, John C; Bhat, Shridhar; Rawal, Viresh H; Green, Rachel; Liu, Jun O

    2011-08-01

    Mycalamide B (MycB) is a marine sponge-derived natural product with potent antitumor activity. Although it has been shown to inhibit protein synthesis, the molecular mechanism of action by MycB remains incompletely understood. We verified the inhibition of translation elongation by in vitro HCV IRES dual luciferase assays, ribosome assembly, and in vivo [(35)S]methinione labeling experiments. Similar to cycloheximide (CHX), MycB inhibits translation elongation through blockade of eEF2-mediated translocation without affecting the eEF1A-mediated loading of tRNA onto the ribosome, AUG recognition, or dipeptide synthesis. Using chemical footprinting, we identified the MycB binding site proximal to the C3993 28S rRNA residue on the large ribosomal subunit. However, there are also subtle, but significant differences in the detailed mechanisms of action of MycB and CHX. First, MycB arrests the ribosome on the mRNA one codon ahead of CHX. Second, MycB specifically blocked tRNA binding to the E-site of the large ribosomal subunit. Moreover, they display different polysome profiles in vivo. Together, these observations shed new light on the mechanism of inhibition of translation elongation by MycB.

  2. Sox2 is translationally activated by eukaryotic initiation factor 4E in human glioma-initiating cells

    Energy Technology Data Exchange (ETDEWEB)

    Ge, Yuqing; Zhou, Fengbiao; Chen, Hong; Cui, Chunhong; Liu, Dan [Key Laboratory of Glycoconjuates Research, Ministry of Public Health and Gene Research Center, Shanghai Medical College of Fudan University, Shanghai 200032 (China); Li, Qiuping [Zhongshan Hospital of Fudan University, Shanghai 200032 (China); Yang, Zhiyuan; Wu, Guoqiang [Key Laboratory of Glycoconjuates Research, Ministry of Public Health and Gene Research Center, Shanghai Medical College of Fudan University, Shanghai 200032 (China); Sun, Shuhui [Key Laboratory of Medical Molecular Virology, Ministry of Education and Health, Shanghai Medical College of Fudan University, Shanghai 200032 (China); Gu, Jianxin [Key Laboratory of Glycoconjuates Research, Ministry of Public Health and Gene Research Center, Shanghai Medical College of Fudan University, Shanghai 200032 (China); Institutes of Biomedical Sciences of Fudan University, Shanghai 200032 (China); Wei, Yuanyan, E-mail: yywei@fudan.edu.cn [Key Laboratory of Glycoconjuates Research, Ministry of Public Health and Gene Research Center, Shanghai Medical College of Fudan University, Shanghai 200032 (China); Jiang, Jianhai, E-mail: jianhaijiang@fudan.edu.cn [Key Laboratory of Glycoconjuates Research, Ministry of Public Health and Gene Research Center, Shanghai Medical College of Fudan University, Shanghai 200032 (China)

    2010-07-09

    Sox2, a master transcription factor, contributes to the generation of induced pluripotent stem cells and plays significant roles in sustaining the self-renewal of neural stem cells and glioma-initiating cells. Understanding the functional differences of Sox2 between glioma-initiating cells and normal neural stem cells would contribute to therapeutic approach for treatment of brain tumors. Here, we first demonstrated that Sox2 could contribute to the self-renewal and proliferation of glioma-initiating cells. The following experiments showed that Sox2 was activated at translational level in a subset of human glioma-initiating cells compared with the normal neural stem cells. Further investigation revealed there was a positive correlation between Sox2 and eukaryotic initiation factor 4E (eIF4E) in glioma tissues. Down-regulation of eIF4E decreased Sox2 protein level without altering its mRNA level in glioma-initiating cells, indicating that Sox2 was activated by eIF4E at translational level. Furthermore, eIF4E was presumed to regulate the expression of Sox2 by its 5' untranslated region (5' UTR) sequence. Our results suggest that the eIF4E-Sox2 axis is a novel mechanism of unregulated self-renewal of glioma-initiating cells, providing a potential therapeutic target for glioma.

  3. Modifying the 5'-Cap for Click Reactions of Eukaryotic mRNA and To Tune Translation Efficiency in Living Cells.

    Science.gov (United States)

    Holstein, Josephin M; Anhäuser, Lea; Rentmeister, Andrea

    2016-08-26

    The 5'-cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5'-cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7-modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N(2) -modified 5'-caps, can be used to tune translation of the respective mRNAs both in vitro and in cells. Appropriate modifications allow subsequent bioorthogonal chemistry, as demonstrated by intracellular live-cell labeling of a target mRNA. The efficient and versatile N7 manipulation of the mRNA cap makes mRNAs amenable to both modulation of their biological function and intracellular labeling, and represents a valuable addition to the chemical biology toolbox. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Sox2 is translationally activated by eukaryotic initiation factor 4E in human glioma-initiating cells

    International Nuclear Information System (INIS)

    Ge, Yuqing; Zhou, Fengbiao; Chen, Hong; Cui, Chunhong; Liu, Dan; Li, Qiuping; Yang, Zhiyuan; Wu, Guoqiang; Sun, Shuhui; Gu, Jianxin; Wei, Yuanyan; Jiang, Jianhai

    2010-01-01

    Sox2, a master transcription factor, contributes to the generation of induced pluripotent stem cells and plays significant roles in sustaining the self-renewal of neural stem cells and glioma-initiating cells. Understanding the functional differences of Sox2 between glioma-initiating cells and normal neural stem cells would contribute to therapeutic approach for treatment of brain tumors. Here, we first demonstrated that Sox2 could contribute to the self-renewal and proliferation of glioma-initiating cells. The following experiments showed that Sox2 was activated at translational level in a subset of human glioma-initiating cells compared with the normal neural stem cells. Further investigation revealed there was a positive correlation between Sox2 and eukaryotic initiation factor 4E (eIF4E) in glioma tissues. Down-regulation of eIF4E decreased Sox2 protein level without altering its mRNA level in glioma-initiating cells, indicating that Sox2 was activated by eIF4E at translational level. Furthermore, eIF4E was presumed to regulate the expression of Sox2 by its 5' untranslated region (5' UTR) sequence. Our results suggest that the eIF4E-Sox2 axis is a novel mechanism of unregulated self-renewal of glioma-initiating cells, providing a potential therapeutic target for glioma.

  5. The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication.

    Science.gov (United States)

    Morais, Ana Ts; Terzian, Ana Cb; Duarte, Danilo Vb; Bronzoni, Roberta Vm; Madrid, Maria Cfs; Gavioli, Arieli F; Gil, Laura Hvg; Oliveira, Amanda G; Zanelli, Cleslei F; Valentini, Sandro R; Rahal, Paula; Nogueira, Mauricio L

    2013-06-22

    Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role

  6. Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast.

    Directory of Open Access Journals (Sweden)

    Jeffrey A Hussmann

    2015-12-01

    Full Text Available Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon. Some experiments have used cycloheximide (CHX to stabilize ribosomes before measuring their positions, and these studies all counterintuitively report a weak negative correlation between the translation rate of a codon and the abundance of its cognate tRNA. In contrast, some experiments performed without CHX report strong positive correlations. To explain this contradiction, we identify unexpected patterns in ribosome density downstream of each type of codon in experiments that use CHX. These patterns are evidence that elongation continues to occur in the presence of CHX but with dramatically altered codon-specific elongation rates. The measured positions of ribosomes in these experiments therefore do not reflect the amounts of time ribosomes spend at each position in vivo. These results suggest that conclusions from experiments in yeast using CHX may need reexamination. In particular, we show that in all such experiments, codons decoded by less abundant tRNAs were in fact being translated more slowly before the addition of CHX disrupted these dynamics.

  7. Eukaryotic translation initiation factor 3 subunit e controls intracellular calcium homeostasis by regulation of cav1.2 surface expression.

    Directory of Open Access Journals (Sweden)

    Pawel Buda

    Full Text Available Inappropriate surface expression of voltage-gated Ca(2+channels (CaV in pancreatic ß-cells may contribute to the development of type 2 diabetes. First, failure to increase intracellular Ca(2+ concentrations at the sites of exocytosis impedes insulin release. Furthermore, excessive Ca(2+ influx may trigger cytotoxic effects. The regulation of surface expression of CaV channels in the pancreatic β-cells remains unknown. Here, we used real-time 3D confocal and TIRFM imaging, immunocytochemistry, cellular fractionation, immunoprecipitation and electrophysiology to study trafficking of L-type CaV1.2 channels upon β-cell stimulation. We found decreased surface expression of CaV1.2 and a corresponding reduction in L-type whole-cell Ca(2+ currents in insulin-secreting INS-1 832/13 cells upon protracted (15-30 min stimulation. This internalization occurs by clathrin-dependent endocytosis and could be prevented by microtubule or dynamin inhibitors. eIF3e (Eukaryotic translation initiation factor 3 subunit E is part of the protein translation initiation complex, but its effect on translation are modest and effects in ion channel trafficking have been suggested. The factor interacted with CaV1.2 and regulated CaV1.2 traffic bidirectionally. eIF3e silencing impaired CaV1.2 internalization, which resulted in an increased intracellular Ca(2+ load upon stimulation. These findings provide a mechanism for regulation of L-type CaV channel surface expression with consequences for β-cell calcium homeostasis, which will affect pancreatic β-cell function and insulin production.

  8. The Jigsaw Puzzle of mRNA Translation Initiation in Eukaryotes: A Decade of Structures Unraveling the Mechanics of the Process.

    Science.gov (United States)

    Hashem, Yaser; Frank, Joachim

    2018-03-01

    Translation initiation in eukaryotes is a highly regulated and rate-limiting process. It results in the assembly and disassembly of numerous transient and intermediate complexes involving over a dozen eukaryotic initiation factors (eIFs). This process culminates in the accommodation of a start codon marking the beginning of an open reading frame at the appropriate ribosomal site. Although this process has been extensively studied by hundreds of groups for nearly half a century, it has been only recently, especially during the last decade, that we have gained deeper insight into the mechanics of the eukaryotic translation initiation process. This advance in knowledge is due in part to the contributions of structural biology, which have shed light on the molecular mechanics underlying the different functions of various eukaryotic initiation factors. In this review, we focus exclusively on the contribution of structural biology to the understanding of the eukaryotic initiation process, a long-standing jigsaw puzzle that is just starting to yield the bigger picture. Expected final online publication date for the Annual Review of Biophysics Volume 47 is May 20, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  9. Eukaryotic translation initiation factor 5A (eIF5A) is essential for HIF-1α activation in hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Tariq, Mohammad [Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Graduate School of Science and Engineering, Saitama University, 645 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan); Ito, Akihiro, E-mail: akihiro-i@riken.jp [Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Japan Agency for Medical Research and Development, AMED-CREST, 1-7-1 Otemachi, Chiyoda-ku, Tokyo, 100-0004 (Japan); Ishfaq, Muhammad; Bradshaw, Elliot [Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Graduate School of Science and Engineering, Saitama University, 645 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan); Yoshida, Minoru [Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Graduate School of Science and Engineering, Saitama University, 645 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan); Japan Agency for Medical Research and Development, AMED-CREST, 1-7-1 Otemachi, Chiyoda-ku, Tokyo, 100-0004 (Japan)

    2016-02-05

    The eukaryotic initiation factor 5A (eIF5A) is an essential protein involved in translation elongation and cell proliferation. eIF5A undergoes several post-translational modifications including hypusination and acetylation. Hypusination is indispensable for the function of eIF5A. On the other hand, the precise function of acetylation remains unknown, but it may render the protein inactive since hypusination blocks acetylation. Here, we report that acetylation of eIF5A increases under hypoxia. During extended hypoxic periods an increase in the level of eIF5A acetylation correlated with a decrease in HIF-1α, suggesting involvement of eIF5A activity in HIF-1α expression under hypoxia. Indeed, suppression of eIF5A by siRNA oligo-mediated knockdown or treatment with GC7, a deoxyhypusine synthase inhibitor, led to significant reduction of HIF-1α activity. Furthermore, knockdown of eIF5A or GC7 treatment reduced tumor spheroid formation with a concomitant decrease in HIF-1α expression. Our results suggest that functional, hypusinated eIF5A is necessary for HIF-1α expression during hypoxia and that eIF5A is an attractive target for cancer therapy. - Highlights: • Hypoxia induces acetylation of eIF5A. • Active eIF5A is necessary for HIF-1α activation in hypoxia. • Active eIF5A is important for tumor spheroid growth.

  10. Eukaryotic translation initiation factor 5A (eIF5A) is essential for HIF-1α activation in hypoxia

    International Nuclear Information System (INIS)

    Tariq, Mohammad; Ito, Akihiro; Ishfaq, Muhammad; Bradshaw, Elliot; Yoshida, Minoru

    2016-01-01

    The eukaryotic initiation factor 5A (eIF5A) is an essential protein involved in translation elongation and cell proliferation. eIF5A undergoes several post-translational modifications including hypusination and acetylation. Hypusination is indispensable for the function of eIF5A. On the other hand, the precise function of acetylation remains unknown, but it may render the protein inactive since hypusination blocks acetylation. Here, we report that acetylation of eIF5A increases under hypoxia. During extended hypoxic periods an increase in the level of eIF5A acetylation correlated with a decrease in HIF-1α, suggesting involvement of eIF5A activity in HIF-1α expression under hypoxia. Indeed, suppression of eIF5A by siRNA oligo-mediated knockdown or treatment with GC7, a deoxyhypusine synthase inhibitor, led to significant reduction of HIF-1α activity. Furthermore, knockdown of eIF5A or GC7 treatment reduced tumor spheroid formation with a concomitant decrease in HIF-1α expression. Our results suggest that functional, hypusinated eIF5A is necessary for HIF-1α expression during hypoxia and that eIF5A is an attractive target for cancer therapy. - Highlights: • Hypoxia induces acetylation of eIF5A. • Active eIF5A is necessary for HIF-1α activation in hypoxia. • Active eIF5A is important for tumor spheroid growth.

  11. Externalization and recognition by macrophages of large subunit of eukaryotic translation initiation factor 3 in apoptotic cells

    International Nuclear Information System (INIS)

    Nakai, Yuji; Shiratsuchi, Akiko; Manaka, Junko; Nakayama, Hiroshi; Takio, Koji; Zhang Jianting; Suganuma, Tatsuo; Nakanishi, Yoshinobu

    2005-01-01

    We previously isolated a monoclonal antibody named PH2 that inhibits phosphatidylserine-mediated phagocytosis of apoptotic cells by macrophages [C. Fujii, A. Shiratsuchi, J. Manaka, S. Yonehara, Y. Nakanishi. Cell Death Differ. 8 (2001) 1113-1122]. We report here the identification of the cognate antigen. A protein bound by PH2 in Western blotting was identified as the 170-kDa subunit of eukaryotic translation initiation factor 3 (eIF3 p170/eIF3a). When eIF3a was expressed in a culture cell line as a protein fused to green fluorescence protein, the fusion protein was detected at the cell surface only after the induction of apoptosis. The same phenomenon was seen when the localization of endogenous eIF3a was determined using anti-eIF3a antibody, and eIF3a seemed to be partially degraded during apoptosis. Furthermore, bacterially expressed N-terminal half of eIF3a fused to glutathione S-transferase bound to the surface of macrophages and inhibited phagocytosis of apoptotic cells by macrophages when it was added to phagocytosis reactions. These results collectively suggest that eIF3a translocates to the cell surface upon apoptosis, probably after partial degradation, and bridges apoptotic cells and macrophages to enhance phagocytosis

  12. The nematode eukaryotic translation initiation factor 4E/G complex works with a trans-spliced leader stem-loop to enable efficient translation of trimethylguanosine-capped RNAs.

    Science.gov (United States)

    Wallace, Adam; Filbin, Megan E; Veo, Bethany; McFarland, Craig; Stepinski, Janusz; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Davis, Richard E

    2010-04-01

    Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5' end through the eIF4F initiation complex binding to the 5' m(7)G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5' end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m(7)G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5' untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs.

  13. The Nematode Eukaryotic Translation Initiation Factor 4E/G Complex Works with a trans-Spliced Leader Stem-Loop To Enable Efficient Translation of Trimethylguanosine-Capped RNAs ▿ †

    Science.gov (United States)

    Wallace, Adam; Filbin, Megan E.; Veo, Bethany; McFarland, Craig; Stepinski, Janusz; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Davis, Richard E.

    2010-01-01

    Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5′ end through the eIF4F initiation complex binding to the 5′ m7G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5′ end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m7G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5′ untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs. PMID:20154140

  14. The control of translational accuracy is a determinant of healthy ageing in yeast.

    Science.gov (United States)

    von der Haar, Tobias; Leadsham, Jane E; Sauvadet, Aimie; Tarrant, Daniel; Adam, Ilectra S; Saromi, Kofo; Laun, Peter; Rinnerthaler, Mark; Breitenbach-Koller, Hannelore; Breitenbach, Michael; Tuite, Mick F; Gourlay, Campbell W

    2017-01-01

    Life requires the maintenance of molecular function in the face of stochastic processes that tend to adversely affect macromolecular integrity. This is particularly relevant during ageing, as many cellular functions decline with age, including growth, mitochondrial function and energy metabolism. Protein synthesis must deliver functional proteins at all times, implying that the effects of protein synthesis errors like amino acid misincorporation and stop-codon read-through must be minimized during ageing. Here we show that loss of translational accuracy accelerates the loss of viability in stationary phase yeast. Since reduced translational accuracy also reduces the folding competence of at least some proteins, we hypothesize that negative interactions between translational errors and age-related protein damage together overwhelm the cellular chaperone network. We further show that multiple cellular signalling networks control basal error rates in yeast cells, including a ROS signal controlled by mitochondrial activity, and the Ras pathway. Together, our findings indicate that signalling pathways regulating growth, protein homeostasis and energy metabolism may jointly safeguard accurate protein synthesis during healthy ageing. © 2017 The Authors.

  15. Crystallization and preliminary crystallographic studies of the W2 domain of Drosophila melanogaster eukaryotic translation initiation factor 5C domain-containing protein

    International Nuclear Information System (INIS)

    Zhao, Hui; Wang, Hong; Liu, Huihui; Teng, Maikun; Li, Xu

    2012-01-01

    The crystallization and preliminary crystallographic studies of the carboxy-terminal domain of D. melanogaster eukaryotic translation initiation factor 5C domain-containing protein are reported. The Drosophila melanogaster eukaryotic translation initiation factor 5C domain-containing protein (ECP) is composed of two independently folded domains which belong to the basic leucine-zipper and W2 domain-containing protein (BZW) family. Based on the sequence similarity between the C-terminal W2 domain of ECP and some eukaryotic translation initiation factors (such as eIF2B∊, eIF4γ, eIF5 etc.), ECP has been speculated to participate in the translation initiation process. Structural information on the C-terminal W2 domain of ECP would be helpful in understanding the specific cellular function of this protein. Here, the W2 domain of ECP was expressed and crystallized. Crystals grown by the hanging-drop vapour-diffusion method diffracted to 2.70 Å resolution and belonged to space group I4, with unit-cell parameters a = b = 81.05, c = 57.44 Å. The Matthews coefficient suggested that there was one molecule per asymmetric unit in the crystal

  16. Translation initiation on mRNAs bound by nuclear cap-binding protein complex CBP80/20 requires interaction between CBP80/20-dependent translation initiation factor and eukaryotic translation initiation factor 3g.

    Science.gov (United States)

    Choe, Junho; Oh, Nara; Park, Sungjin; Lee, Ye Kyung; Song, Ok-Kyu; Locker, Nicolas; Chi, Sung-Gil; Kim, Yoon Ki

    2012-05-25

    In the cytoplasm of mammalian cells, either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF) 4E can direct the initiation of translation. Although the recruitment of ribosomes to mRNAs during eIF4E-dependent translation (ET) is well characterized, the molecular mechanism for CBP80/20-dependent translation (CT) remains obscure. Here, we show that CBP80/20-dependent translation initiation factor (CTIF), which has been shown to be preferentially involved in CT but not ET, specifically interacts with eIF3g, a component of the eIF3 complex involved in ribosome recruitment. By interacting with eIF3g, CTIF serves as an adaptor protein to bridge the CBP80/20 and the eIF3 complex, leading to efficient ribosome recruitment during CT. Accordingly, down-regulation of CTIF using a small interfering RNA causes a redistribution of CBP80 from polysome fractions to subpolysome fractions, without significant consequence to eIF4E distribution. In addition, down-regulation of eIF3g inhibits the efficiency of nonsense-mediated mRNA decay, which is tightly coupled to CT but not to ET. Moreover, the artificial tethering of CTIF to an intercistronic region of dicistronic mRNA results in translation of the downstream cistron in an eIF3-dependent manner. These findings support the idea that CT mechanistically differs from ET.

  17. Different roles of eukaryotic MutS and MutL complexes in repair of small insertion and deletion loops in yeast.

    Directory of Open Access Journals (Sweden)

    Nina V Romanova

    2013-10-01

    Full Text Available DNA mismatch repair greatly increases genome fidelity by recognizing and removing replication errors. In order to understand how this fidelity is maintained, it is important to uncover the relative specificities of the different components of mismatch repair. There are two major mispair recognition complexes in eukaryotes that are homologues of bacterial MutS proteins, MutSα and MutSβ, with MutSα recognizing base-base mismatches and small loop mispairs and MutSβ recognizing larger loop mispairs. Upon recognition of a mispair, the MutS complexes then interact with homologues of the bacterial MutL protein. Loops formed on the primer strand during replication lead to insertion mutations, whereas loops on the template strand lead to deletions. We show here in yeast, using oligonucleotide transformation, that MutSα has a strong bias toward repair of insertion loops, while MutSβ has an even stronger bias toward repair of deletion loops. Our results suggest that this bias in repair is due to the different interactions of the MutS complexes with the MutL complexes. Two mutants of MutLα, pms1-G882E and pms1-H888R, repair deletion mispairs but not insertion mispairs. Moreover, we find that a different MutL complex, MutLγ, is extremely important, but not sufficient, for deletion repair in the presence of either MutLα mutation. MutSβ is present in many eukaryotic organisms, but not in prokaryotes. We suggest that the biased repair of deletion mispairs may reflect a critical eukaryotic function of MutSβ in mismatch repair.

  18. The rice eukaryotic translation initiation factor 3 subunit f (OseIF3f is involved in microgametogenesis

    Directory of Open Access Journals (Sweden)

    Qi eLi

    2016-04-01

    Full Text Available Microgametogenesis is the postmeiotic pollen developmental phase when unicellular microspores develop into mature tricellular pollen. In rice, microgametogenesis can influence grain yields to a great degree because pollen abortion occurs more easily during microgametogenesis than during other stages of pollen development. However, our knowledge of the genes involved in microgametogenesis in rice remains limited. Due to the dependence of pollen development on the regulatory mechanisms of protein expression, we identified the encoding gene of the eukaryotic translation initiation factor 3, subunit f in Oryza sativa (OseIF3f. Immunoprecipitation combined with mass spectrometry confirmed that OseIF3f was a subunit of rice eIF3, which consisted of at least 12 subunits including eIF3a, eIF3b, eIF3c, eIF3d, eIF3e, eIF3f, eIF3g, eIF3h, eIF3i, eIF3k, eIF3l and eIF3m. OseIF3f showed high mRNA levels in immature florets and is highly abundant in developing anthers. Subcellular localization analysis showed that OseIF3f was localized to the cytosol and the endoplasmic reticulum in rice root cells. We further analyzed the biological function of OseIF3f using the double-stranded RNA-mediated interference (RNAi approach. The OseIF3f-RNAi lines grew normally at the vegetative stage but displayed a large reduction in seed production and pollen viability, which is associated with the down-regulation of OseIF3f. Further cytological observations of pollen development revealed that the OseIF3f-RNAi lines showed no obvious abnormalities at the male meiotic stage and the unicellular microspore stage. However, compared to the wild type, OseIF3f-RNAi lines contained a higher percentage of arrested unicellular pollen at the bicellular stage and a higher percentage of arrested unicellular and bicellular pollen, and aborted pollen at the tricellular stage. These results indicate that OseIF3f plays a role in microgametogenesis.

  19. Electrospray mass spectrometry characterization of post-translational modifications of barley alpha-amylase 1 produced in yeast

    DEFF Research Database (Denmark)

    Søgaard, M; Andersen, Jens S.; Roepstorff, P

    1993-01-01

    We have used electrospray mass spectrometry (ESMS) in combination with protein chemistry and genetics to delineate post-translational modifications in yeast of barley alpha-amylase 1 (AMY1), a 45 kD enzyme crucial for production of malt, an important starting material in the manufacture of beer...

  20. Eukaryotic elongation factor 1-beta interacts with the 5' untranslated region of the M gene of Nipah virus to promote mRNA translation.

    Science.gov (United States)

    Uchida, Shotaro; Sato, Hiroki; Yoneda, Misako; Kai, Chieko

    2016-09-01

    Nipah virus belongs to the genus Henipavirus in the family Paramyxoviridae, and its RNA genome is larger than those of other paramyxoviruses because it has long untranslated regions (UTRs) in each gene. However, the functions of these UTRs are not fully understood. In this study, we investigated the functions of the 5' UTRs and found that the 5' UTR of the M gene upregulated the translation of a reporter gene. Using an RNA pull-down assay, we showed that eukaryotic elongation factor 1-beta (EEF1B2) interacts with nucleotides 81-100 of the M 5' UTR and specifically enhances its translation efficiency. Our results suggest that the M 5' UTR promotes the production of M protein and viral budding by recruiting EEF1B2.

  1. Eukaryotic translation initiation factor 2B-beta (eIF2Bβ), a new class of plant virus resistance gene.

    Science.gov (United States)

    Shopan, Jannat; Mou, Haipeng; Zhang, Lili; Zhang, Changtong; Ma, Weiwei; Walsh, John A; Hu, Zhongyuan; Yang, Jinghua; Zhang, Mingfang

    2017-06-01

    Recessive resistances to plant viruses in the Potyvirus genus have been found to be based on mutations in the plant eukaryotic translation initiation factors, eIF4E and eIF4G or their isoforms. Here we report that natural, monogenic recessive resistance to the Potyvirus Turnip mosaic virus (TuMV) has been found in a number of mustard (Brassica juncea) accessions. Bulked segregant analysis and sequencing of resistant and susceptible plant lines indicated the resistance is controlled by a single recessive gene, recessive TuMV resistance 03 (retr03), an allele of the eukaryotic translation initiation factor 2B-beta (eIF2Bβ). Silencing of eIF2Bβ in a TuMV-susceptible mustard plant line and expression of eIF2Bβ from a TuMV-susceptible line in a TuMV-resistant mustard plant line confirmed the new resistance mechanism. A functional copy of a specific allele of eIF2Bβ is required for efficient TuMV infection. eIF2Bβ represents a new class of virus resistance gene conferring resistance to any pathogen. eIF2B acts as a guanine nucleotide exchange factor (GEF) for its GTP-binding protein partner eIF2 via interaction with eIF2·GTP at an early step in translation initiation. Further genotyping indicated that a single non-synonymous substitution (A120G) in the N-terminal region of eIF2Bβ was responsible for the TuMV resistance. A reproducible marker has been developed, facilitating marker-assisted selection for TuMV resistance in B. juncea. Our findings provide a new target for seeking natural resistance to potyviruses and new opportunities for the control of potyviruses using genome editing techniques targeted on eIF2Bβ. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  2. Norovirus translation requires an interaction between the C Terminus of the genome-linked viral protein VPg and eukaryotic translation initiation factor 4G.

    Science.gov (United States)

    Chung, Liliane; Bailey, Dalan; Leen, Eoin N; Emmott, Edward P; Chaudhry, Yasmin; Roberts, Lisa O; Curry, Stephen; Locker, Nicolas; Goodfellow, Ian G

    2014-08-01

    Viruses have evolved a variety of mechanisms to usurp the host cell translation machinery to enable translation of the viral genome in the presence of high levels of cellular mRNAs. Noroviruses, a major cause of gastroenteritis in man, have evolved a mechanism that relies on the interaction of translation initiation factors with the virus-encoded VPg protein covalently linked to the 5' end of the viral RNA. To further characterize this novel mechanism of translation initiation, we have used proteomics to identify the components of the norovirus translation initiation factor complex. This approach revealed that VPg binds directly to the eIF4F complex, with a high affinity interaction occurring between VPg and eIF4G. Mutational analyses indicated that the C-terminal region of VPg is important for the VPg-eIF4G interaction; viruses with mutations that alter or disrupt this interaction are debilitated or non-viable. Our results shed new light on the unusual mechanisms of protein-directed translation initiation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Strong eukaryotic IRESs have weak secondary structure.

    Directory of Open Access Journals (Sweden)

    Xuhua Xia

    Full Text Available BACKGROUND: The objective of this work was to investigate the hypothesis that eukaryotic Internal Ribosome Entry Sites (IRES lack secondary structure and to examine the generality of the hypothesis. METHODOLOGY/PRINCIPAL FINDINGS: IRESs of the yeast and the fruit fly are located in the 5'UTR immediately upstream of the initiation codon. The minimum folding energy (MFE of 60 nt RNA segments immediately upstream of the initiation codons was calculated as a proxy of secondary structure stability. MFE of the reverse complements of these 60 nt segments was also calculated. The relationship between MFE and empirically determined IRES activity was investigated to test the hypothesis that strong IRES activity is associated with weak secondary structure. We show that IRES activity in the yeast and the fruit fly correlates strongly with the structural stability, with highest IRES activity found in RNA segments that exhibit the weakest secondary structure. CONCLUSIONS: We found that a subset of eukaryotic IRESs exhibits very low secondary structure in the 5'-UTR sequences immediately upstream of the initiation codon. The consistency in results between the yeast and the fruit fly suggests a possible shared mechanism of cap-independent translation initiation that relies on an unstructured RNA segment.

  4. Establishment and Application of a High Throughput Screening System Targeting the Interaction between HCV Internal Ribosome Entry Site and Human Eukaryotic Translation Initiation Factor 3

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    Yuying Zhu

    2017-05-01

    Full Text Available Viruses are intracellular obligate parasites and the host cellular machinery is usually recruited for their replication. Human eukaryotic translation initiation factor 3 (eIF3 could be directly recruited by the hepatitis C virus (HCV internal ribosome entry site (IRES to promote the translation of viral proteins. In this study, we establish a fluorescence polarization (FP based high throughput screening (HTS system targeting the interaction between HCV IRES and eIF3. By screening a total of 894 compounds with this HTS system, two compounds (Mucl39526 and NP39 are found to disturb the interaction between HCV IRES and eIF3. And these two compounds are further demonstrated to inhibit the HCV IRES-dependent translation in vitro. Thus, this HTS system is functional to screen the potential HCV replication inhibitors targeting human eIF3, which is helpful to overcome the problem of viral resistance. Surprisingly, one compound HP-3, a kind of oxytocin antagonist, is discovered to significantly enhance the interaction between HCV IRES and eIF3 by this HTS system. HP-3 is demonstrated to directly interact with HCV IRES and promote the HCV IRES-dependent translation both in vitro and in vivo, which strongly suggests that HP-3 has potentials to promote HCV replication. Therefore, this HTS system is also useful to screen the potential HCV replication enhancers, which is meaningful for understanding the viral replication and screening novel antiviral drugs. To our knowledge, this is the first HTS system targeting the interaction between eIF3 and HCV IRES, which could be applied to screen both potential HCV replication inhibitors and enhancers.

  5. Molecular modeling of the human eukaryotic translation initiation factor 5A (eIF5A) based on spectroscopic and computational analyses

    International Nuclear Information System (INIS)

    Costa-Neto, Claudio M.; Parreiras-e-Silva, Lucas T.; Ruller, Roberto; Oliveira, Eduardo B.; Miranda, Antonio; Oliveira, Laerte; Ward, Richard J.

    2006-01-01

    The eukaryotic translation initiation factor 5A (eIF5A) is a protein ubiquitously present in archaea and eukarya, which undergoes a unique two-step post-translational modification called hypusination. Several studies have shown that hypusination is essential for a variety of functional roles for eIF5A, including cell proliferation and synthesis of proteins involved in cell cycle control. Up to now neither a totally selective inhibitor of hypusination nor an inhibitor capable of directly binding to eIF5A has been reported in the literature. The discovery of such an inhibitor might be achieved by computer-aided drug design based on the 3D structure of the human eIF5A. In this study, we present a molecular model for the human eIF5A protein based on the crystal structure of the eIF5A from Leishmania brasiliensis, and compare the modeled conformation of the loop bearing the hypusination site with circular dichroism data obtained with a synthetic peptide of this loop. Furthermore, analysis of amino acid variability between different human eIF5A isoforms revealed peculiar structural characteristics that are of functional relevance

  6. Interaction in vitro between the proteinase of Tomato ringspot virus (genus Nepovirus) and the eukaryotic translation initiation factor iso4E from Arabidopsis thaliana.

    Science.gov (United States)

    Léonard, Simon; Chisholm, Joan; Laliberté, Jean-François; Sanfaçon, Hélène

    2002-08-01

    Eukaryotic initiation factor eIF(iso)4E binds to the cap structure of mRNAs leading to assembly of the translation complex. This factor also interacts with the potyvirus VPg and this interaction has been correlated with virus infectivity. In this study, we show an interaction between eIF(iso)4E and the proteinase (Pro) of a nepovirus (Tomato ringspot virus; ToRSV) in vitro. The ToRSV VPg did not interact with eIF(iso)4E although its presence on the VPg-Pro precursor increased the binding affinity of Pro for the initiation factor. A major determinant of the interaction was mapped to the first 93 residues of Pro. Formation of the complex was inhibited by addition of m(7)GTP (a cap analogue), suggesting that Pro-containing molecules compete with cellular mRNAs for eIF(iso)4E binding. The possible implications of this interaction for translation and/or replication of the virus genome are discussed.

  7. Structural Studies of RNA Helicases Involved in Eukaryotic Pre-mRNA Splicing, Ribosome Biogenesis, and Translation Initiation

    DEFF Research Database (Denmark)

    He, Yangzi

    and ligates the neighbouring exons to generate mature mRNAs. Prp43 is an RNA helicase of the DEAH/RHA family. In yeast, once mRNAs are released, Prp43 catalyzes the disassembly of spliceosomes. The 18S, 5.8S and 25S rRNAs are transcribed as a single polycistronic transcript—the 35S pre......-rRNA. It is nucleolytically cleaved and chemically modified to generate mature rRNAs, which assemble with ribosomal proteins to form the ribosome. Prp43 is required for the processing of the 18S rRNA. Using X-ray crystallography, I determined a high resolution structure of Prp43 bound to ADP, the first structure of a DEAH....../RHA helicase. It defined the conserved structural features of all DEAH/RHA helicases, and unveiled a novel nucleotide binding site. Additionally a preliminary low resolution structure of a ternary complex comprising Prp43, a non-hydrolyzable ATP analogue, and a single-stranded RNA, was obtained. The ribosome...

  8. A universal trend of reduced mRNA stability near the translation-initiation site in prokaryotes and eukaryotes.

    Directory of Open Access Journals (Sweden)

    Wanjun Gu

    2010-02-01

    Full Text Available Recent studies have suggested that the thermodynamic stability of mRNA secondary structure near the start codon can regulate translation efficiency in Escherichia coli, and that translation is more efficient the less stable the secondary structure. We survey the complete genomes of 340 species for signals of reduced mRNA secondary structure near the start codon. Our analysis includes bacteria, archaea, fungi, plants, insects, fishes, birds, and mammals. We find that nearly all species show evidence for reduced mRNA stability near the start codon. The reduction in stability generally increases with increasing genomic GC content. In prokaryotes, the reduction also increases with decreasing optimal growth temperature. Within genomes, there is variation in the stability among genes, and this variation correlates with gene GC content, codon bias, and gene expression level. For birds and mammals, however, we do not find a genome-wide trend of reduced mRNA stability near the start codon. Yet the most GC rich genes in these organisms do show such a signal. We conclude that reduced stability of the mRNA secondary structure near the start codon is a universal feature of all cellular life. We suggest that the origin of this reduction is selection for efficient recognition of the start codon by initiator-tRNA.

  9. Leucine-rich diet alters the eukaryotic translation initiation factors expression in skeletal muscle of tumour-bearing rats

    Directory of Open Access Journals (Sweden)

    Mello Maria

    2007-03-01

    Full Text Available Abstract Background Cancer-cachexia induces a variety of metabolic disorders on protein turnorver, decreasing protein synthesis and increasing protein degradation. Controversly, insulin, other hormones, and branched-chain amino acids, especially leucine, stimulate protein synthesis and modulate the activity of translation initiation factors involved in protein synthesis. Since the tumour effects are more pronounced when associated with pregnancy, ehancing muscle-wasting proteolysis, in this study, the influence of a leucine-rich diet on the protein synthesis caused by cancer were investigated. Methods Pregnant rats with or without Walker 256 tumour were distributed into six groups. During 20 days of experiment, three groups were fed with a control diet: C – pregnant control, W – tumour-bearing, and P – pair-fed, which received the same amount of food as ingested by the W group; three other groups of pregnant rats were fed a leucine-rich diet: L – pregnant leucine, WL – tumour-bearing, and PL – pair-fed, which received the same amount of food as ingested by the WL group. Results The gastrocnemius muscle of WL rats showed increased incorporation of leucine in protein compared to W rats; the leucine-rich diet also prevented the decrease in plasma insulin normally seen in W. The expression of translation initiation factors increased when tumour-bearing rats fed leucine-rich diet, with increase of ~35% for eIF2α and eIF5, ~17% for eIF4E and 20% for eIF4G; the expression of protein kinase S6K1 and protein kinase C was also highly enhanced. Conclusion The results suggest that a leucine-rich diet increased the protein synthesis in skeletal muscle in tumour-bearing rats possibly through the activation of eIF factors and/or the S6kinase pathway.

  10. Leucine-rich diet alters the eukaryotic translation initiation factors expression in skeletal muscle of tumour-bearing rats

    International Nuclear Information System (INIS)

    Ventrucci, Gislaine; Mello, Maria Alice R; Gomes-Marcondes, Maria Cristina C

    2007-01-01

    Cancer-cachexia induces a variety of metabolic disorders on protein turnorver, decreasing protein synthesis and increasing protein degradation. Controversly, insulin, other hormones, and branched-chain amino acids, especially leucine, stimulate protein synthesis and modulate the activity of translation initiation factors involved in protein synthesis. Since the tumour effects are more pronounced when associated with pregnancy, ehancing muscle-wasting proteolysis, in this study, the influence of a leucine-rich diet on the protein synthesis caused by cancer were investigated. Pregnant rats with or without Walker 256 tumour were distributed into six groups. During 20 days of experiment, three groups were fed with a control diet: C – pregnant control, W – tumour-bearing, and P – pair-fed, which received the same amount of food as ingested by the W group; three other groups of pregnant rats were fed a leucine-rich diet: L – pregnant leucine, WL – tumour-bearing, and PL – pair-fed, which received the same amount of food as ingested by the WL group. The gastrocnemius muscle of WL rats showed increased incorporation of leucine in protein compared to W rats; the leucine-rich diet also prevented the decrease in plasma insulin normally seen in W. The expression of translation initiation factors increased when tumour-bearing rats fed leucine-rich diet, with increase of ~35% for eIF2α and eIF5, ~17% for eIF4E and 20% for eIF4G; the expression of protein kinase S6K1 and protein kinase C was also highly enhanced. The results suggest that a leucine-rich diet increased the protein synthesis in skeletal muscle in tumour-bearing rats possibly through the activation of eIF factors and/or the S6kinase pathway

  11. Leucine-rich diet alters the eukaryotic translation initiation factors expression in skeletal muscle of tumour-bearing rats

    Energy Technology Data Exchange (ETDEWEB)

    Ventrucci, Gislaine [Laboratório de Nutrição e Câncer, Departamento de Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, 13083-970, São Paulo (Brazil); Mello, Maria Alice R [Departamento de Fisiologia e Biofísica, Instituto Biociências, Universidade Estadual de São Paulo, UNESP, Rio Claro, 13506-900, São Paulo (Brazil); Gomes-Marcondes, Maria Cristina C [Laboratório de Nutrição e Câncer, Departamento de Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, 13083-970, São Paulo (Brazil)

    2007-03-06

    Cancer-cachexia induces a variety of metabolic disorders on protein turnorver, decreasing protein synthesis and increasing protein degradation. Controversly, insulin, other hormones, and branched-chain amino acids, especially leucine, stimulate protein synthesis and modulate the activity of translation initiation factors involved in protein synthesis. Since the tumour effects are more pronounced when associated with pregnancy, ehancing muscle-wasting proteolysis, in this study, the influence of a leucine-rich diet on the protein synthesis caused by cancer were investigated. Pregnant rats with or without Walker 256 tumour were distributed into six groups. During 20 days of experiment, three groups were fed with a control diet: C – pregnant control, W – tumour-bearing, and P – pair-fed, which received the same amount of food as ingested by the W group; three other groups of pregnant rats were fed a leucine-rich diet: L – pregnant leucine, WL – tumour-bearing, and PL – pair-fed, which received the same amount of food as ingested by the WL group. The gastrocnemius muscle of WL rats showed increased incorporation of leucine in protein compared to W rats; the leucine-rich diet also prevented the decrease in plasma insulin normally seen in W. The expression of translation initiation factors increased when tumour-bearing rats fed leucine-rich diet, with increase of ~35% for eIF2α and eIF5, ~17% for eIF4E and 20% for eIF4G; the expression of protein kinase S6K1 and protein kinase C was also highly enhanced. The results suggest that a leucine-rich diet increased the protein synthesis in skeletal muscle in tumour-bearing rats possibly through the activation of eIF factors and/or the S6kinase pathway.

  12. Targeting eukaryotic translation in mesothelioma cells with an eIF4E-specific antisense oligonucleotide.

    Directory of Open Access Journals (Sweden)

    Blake A Jacobson

    Full Text Available BACKGROUND: Aberrant cap-dependent translation is implicated in tumorigenesis in multiple tumor types including mesothelioma. In this study, disabling the eIF4F complex by targeting eIF4E with eIF4E-specific antisense oligonucleotide (4EASO is assessed as a therapy for mesothelioma. METHODS: Mesothelioma cells were transfected with 4EASO, designed to target eIF4E mRNA, or mismatch-ASO control. Cell survival was measured in mesothelioma treated with 4EASO alone or combined with either gemcitabine or pemetrexed. Levels of eIF4E, ODC, Bcl-2 and β-actin were assessed following treatment. Binding to a synthetic cap-analogue was used to study the strength of eIF4F complex activation following treatment. RESULTS: eIF4E level and the formation of eIF4F cap-complex decreased in response to 4EASO, but not mismatch control ASO, resulting in cleavage of PARP indicating apoptosis. 4EASO treatment resulted in dose dependent decrease in eIF4E levels, which corresponded to cytotoxicity of mesothelioma cells. 4EASO resulted in decreased levels of eIF4E in non-malignant LP9 cells, but this did not correspond to increased cytotoxicity. Proteins thought to be regulated by cap-dependent translation, Bcl-2 and ODC, were decreased upon treatment with 4EASO. Combination therapy of 4EASO with pemetrexed or gemcitabine further reduced cell number. CONCLUSION: 4EASO is a novel drug that causes apoptosis and selectively reduces eIF4E levels, eIF4F complex formation, and proliferation of mesothelioma cells. eIF4E knockdown results in decreased expression of anti-apoptotic and pro-growth proteins and enhances chemosensitivity.

  13. Expression of the Eukaryotic Translation Initiation Factors 4E and 2α in Non-Hodgkin’s Lymphomas

    Science.gov (United States)

    Wang, Songtao; Rosenwald, Igor B.; Hutzler, Michael J.; Pihan, German A.; Savas, Lou; Chen, Jane-Jane; Woda, Bruce A.

    1999-01-01

    Transition of cells from quiescence to proliferation requires an increase in the rate of protein synthesis, which is regulated in part by two key translation initiation factors, 4E and 2α. The expression and activity of both factors are increased transiently when normal resting cells are stimulated to proliferate. They are constitutively elevated in oncogene transformed cultured cells, and overexpression of either initiation factor in rodent cells makes them tumorigenic. In this study we investigate an association between the expression of translation initiation factors and lymphomagenesis. We have analyzed the expression of the protein synthesis initiation factors 4E and 2α by immunohistochemistry in reactive lymph nodes and several types of non-Hodgkin’s lymphoma representing a wide range of clinical behaviors based on the Revised European-American Lymphoma behavioral classification. The study included 7 benign lymph nodes with follicular hyperplasia, 26 indolent lymphomas (6 marginal zone lymphomas, 7 small lymphocytic lymphomas, and 13 follicular lymphomas, grades 1 and 2), 16 moderately aggressive lymphomas (8 mantle cell lymphomas and 8 follicular lymphomas, grade 3), 24 aggressive lymphomas (14 large-B-cell lymphomas and 10 anaplastic large-cell lymphomas), and 15 highly aggressive lymphomas (7 lymphoblastic lymphomas and 8 Burkitt’s lymphomas). Strong expression of initiation factors 4E and 2α was demonstrated in the germinal centers of reactive follicles. Minimal or no expression was seen in the mantle zones and surrounding paracortices, indicating that high expression of initiation factors 4E and 2α is associated with the active proliferation of lymphocytes. Most cases of aggressive and highly aggressive lymphomas showed strong expression of initiation factors 4E and 2α, in contrast to the cases of indolent and moderately aggressive lymphoma, in which their expression was intermediate between the germinal centers and the mantles of reactive

  14. Eukaryotic translation initiation factor 5A induces apoptosis in colon cancer cells and associates with the nucleus in response to tumour necrosis factor α signalling

    International Nuclear Information System (INIS)

    Taylor, Catherine A.; Sun Zhong; Cliche, Dominic O.; Ming, Hong; Eshaque, Bithi; Jin Songmu; Hopkins, Marianne T.; Thai, Boun; Thompson, John E.

    2007-01-01

    Eukaryotic translation initiation factor 5A (eIF5A) is thought to function as a nucleocytoplasmic shuttle protein. There are reports of its involvement in cell proliferation, and more recently it has also been implicated in the regulation of apoptosis. In the present study, we examined the effects of eIF5A over-expression on apoptosis and of siRNA-mediated suppression of eIF5A on expression of the tumour suppressor protein, p53. Over-expression of either eIF5A or a mutant of eIF5A incapable of being hypusinated was found to induce apoptosis in colon carcinoma cells. Our results also indicate that eIF5A is required for expression of p53 following the induction of apoptosis by treatment with Actinomycin D. Depiction of eIF5A localization by indirect immunofluorescence has indicated, for the first time, that the protein is rapidly translocated from the cytoplasm to the nucleus by death receptor activation or following treatment with Actinomycin D. These findings collectively indicate that unhypusinated eIF5A may have pro-apoptotic functions and that eIF5A is rapidly translocated to the nucleus following the induction of apoptotic cell death

  15. Mapping the human translation elongation factor eEF1H complex using the yeast two-hybrid system

    DEFF Research Database (Denmark)

    Mansilla, Francisco; Friis, Irene; Jadidi, Mandana

    2002-01-01

    In eukaryotes, the eukaryotic translation elongation factor eEF1A responsible for transporting amino-acylated tRNA to the ribosome forms a higher-order complex, eEF1H, with its guanine-nucleotide-exchange factor eEF1B. In metazoans, eEF1B consists of three subunits: eEF1B alpha, eEF1B eta and eEF1B...... of in vitro experiments have been proposed for the macromolecular organization of the eEF1H complex. However, these models differ in various aspects. This might be due to the difficulties of handling, particularly the eEF1B beta and eEF1B gamma subunits in vitro. Here, the human eEF1H complex is for the first...... gamma:eEF1B beta, where the last was observed using a three-hybrid approach. Surprisingly, eEF1A2 showed no or only little affinity for the guanine-nucleotide-exchange factors. Truncated versions of the subunits of eEF1B were used to orientate these subunits within the resulting model. The model unit...

  16. Dual Role for Hsc70 in the Biogenesis and Regulation of the Heme-Regulated Kinase of the α Subunit of Eukaryotic Translation Initiation Factor 2

    Science.gov (United States)

    Uma, Sheri; Thulasiraman, Vanitha; Matts, Robert L.

    1999-01-01

    The heme-regulated kinase of the α subunit of eukaryotic initiation factor 2 (HRI) is activated in rabbit reticulocyte lysate (RRL) in response to a number of environmental conditions, including heme deficiency, heat shock, and oxidative stress. Activation of HRI causes an arrest of initiation of protein synthesis. Recently, we have demonstrated that the heat shock cognate protein Hsc70 negatively modulates the activation of HRI in RRL in response to these environmental conditions. Hsc70 is also known to be a critical component of the Hsp90 chaperone machinery in RRL, which plays an obligatory role for HRI to acquire and maintain a conformation that is competent to activate. Using de novo-synthesized HRI in synchronized pulse-chase translations, we have examined the role of Hsc70 in the regulation of HRI biogenesis and activation. Like Hsp90, Hsc70 interacted with nascent HRI and HRI that was matured to a state which was competent to undergo stimulus-induced activation (mature-competent HRI). Interaction of HRI with Hsc70 was required for the transformation of HRI, as the Hsc70 antagonist clofibric acid inhibited the folding of HRI into a mature-competent conformation. Unlike Hsp90, Hsc70 also interacted with transformed HRI. Clofibric acid disrupted the interaction of Hsc70 with transformed HRI that had been matured and transformed in the absence of the drug. Disruption of Hsc70 interaction with transformed HRI in heme-deficient RRL resulted in its hyperactivation. Furthermore, activation of HRI in response to heat shock or denatured proteins also resulted in a similar blockage of Hsc70 interaction with transformed HRI. These results indicate that Hsc70 is required for the folding and transformation of HRI into an active kinase but is subsequently required to negatively attenuate the activation of transformed HRI. PMID:10454533

  17. Over-expression of eukaryotic translation initiation factor 4 gamma 1 correlates with tumor progression and poor prognosis in nasopharyngeal carcinoma

    Directory of Open Access Journals (Sweden)

    Li Xin

    2010-04-01

    Full Text Available Abstract Background The aim of the present study was to analyze the expression of eukaryotic translation initiation factor 4 gamma 1 (EIF4G1 in nasopharyngeal carcinoma (NPC and its correlation with clinicopathologic features, including patients' survival time. Methods Using real-time PCR, we detected the expression of EIF4G1 in normal nasopharyngeal tissues, immortalized nasopharyngeal epithelial cell lines NP69, NPC tissues and cell lines. EIF4G1 protein expression in NPC tissues was examined using immunohistochemistry. Survival analysis was performed using Kaplan-Meier method. The effect of EIF4G1 on cell invasion and tumorigenesis were investigated. Results The expression levels of EIF4G1 mRNA were significantly greater in NPC tissues and cell lines than those in the normal nasopharyngeal tissues and NP69 cells (P EIF4G1 protein was higher in NPC tissues than that in the nasopharyngeal tissues (P EIF4G1 protein in tumors were positively correlated with tumor T classification (P = 0.039, lymph node involvement (N classification, P = 0.008, and the clinical stages (P = 0.003 of NPC patients. Patients with higher EIF4G1 expression had shorter overall survival time (P = 0.019. Multivariate analysis showed that EIF4G1 expression was an independent prognostic indicator for the overall survival of NPC patients. Using shRNA to knock down the expression of EIF4G1 not only markedly inhibited cell cycle progression, proliferation, migration, invasion, and colony formation, but also dramatically suppressed in vivo xenograft tumor growth. Conclusion Our data suggest that EIF4G1 can serve as a biomarker for the prognosis of NPC patients.

  18. Genomics and the making of yeast biodiversity

    NARCIS (Netherlands)

    Hittinger, Chris Todd; Rokas, Antonis; Bai, Feng-Yan; Boekhout, Teun; Gonçalves, Paula; Jeffries, Thomas W; Kominek, Jacek; Lachance, Marc-André; Libkind, Diego; Rosa, Carlos A; Sampaio, José Paulo; Kurtzman, Cletus P

    2015-01-01

    Yeasts are unicellular fungi that do not form fruiting bodies. Although the yeast lifestyle has evolved multiple times, most known species belong to the subphylum Saccharomycotina (syn. Hemiascomycota, hereafter yeasts). This diverse group includes the premier eukaryotic model system, Saccharomyces

  19. Global translational impacts of the loss of the tRNA modification t6A in yeast

    Directory of Open Access Journals (Sweden)

    Patrick C. Thiaville

    2015-12-01

    Full Text Available The universal tRNA modification t6A is found at position 37 of nearly all tRNAs decoding ANN codons. The absence of t6A37 leads to severe growth defects in baker’s yeast, phenotypes similar to those caused by defects in mcm5s2U34 synthesis. Mutants in mcm5s2U34 can be suppressed by overexpression of tRNALysUUU, but we show t6A phenotypes could not be suppressed by expressing any individual ANN decoding tRNA, and t6A and mcm5s2U are not determinants for each other’s formation. Our results suggest that t6A deficiency, like mcm5s2U deficiency, leads to protein folding defects, and show that the absence of t6A led to stress sensitivities (heat, ethanol, salt and sensitivity to TOR pathway inhibitors. Additionally, L-homoserine suppressed the slow growth phenotype seen in t6A-deficient strains, and proteins aggregates and Advanced Glycation End-products (AGEs were increased in the mutants. The global consequences on translation caused by t6A absence were examined by ribosome profiling. Interestingly, the absence of t6A did not lead to global translation defects, but did increase translation initiation at upstream non-AUG codons and increased frame-shifting in specific genes. Analysis of codon occupancy rates suggests that one of the major roles of t6A is to homogenize the process of elongation by slowing the elongation rate at codons decoded by high abundance tRNAs and I34:C3 pairs while increasing the elongation rate of rare tRNAs and G34:U3 pairs. This work reveals that the consequences of t6A absence are complex and multilayered and has set the stage to elucidate the molecular basis of the observed phenotypes.

  20. A Dehydration-Induced Eukaryotic Translation Initiation Factor iso4G Identified in a Slow Wilting Soybean Cultivar Enhances Abiotic Stress Tolerance in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Juan P. Gallino

    2018-03-01

    Full Text Available Water is usually the main limiting factor for soybean productivity worldwide and yet advances in genetic improvement for drought resistance in this crop are still limited. In the present study, we investigated the physiological and molecular responses to drought in two soybean contrasting genotypes, a slow wilting N7001 and a drought sensitive TJS2049 cultivars. Measurements of stomatal conductance, carbon isotope ratios and accumulated dry matter showed that N7001 responds to drought by employing mechanisms resulting in a more efficient water use than TJS2049. To provide an insight into the molecular mechanisms that these cultivars employ to deal with water stress, their early and late transcriptional responses to drought were analyzed by suppression subtractive hybridization. A number of differentially regulated genes from N7001 were identified and their expression pattern was compared between in this genotype and TJS2049. Overall, the data set indicated that N7001 responds to drought earlier than TJ2049 by up-regulating a larger number of genes, most of them encoding proteins with regulatory and signaling functions. The data supports the idea that at least some of the phenotypic differences between slow wilting and drought sensitive plants may rely on the regulation of the level and timing of expression of specific genes. One of the genes that exhibited a marked N7001-specific drought induction profile encoded a eukaryotic translation initiation factor iso4G (GmeIFiso4G-1a. GmeIFiso4G-1a is one of four members of this protein family in soybean, all of them sharing high sequence identity with each other. In silico analysis of GmeIFiso4G-1 promoter sequences suggested a possible functional specialization between distinct family members, which can attain differences at the transcriptional level. Conditional overexpression of GmeIFiso4G-1a in Arabidopsis conferred the transgenic plants increased tolerance to osmotic, salt, drought and low

  1. MicroRNA-9 enhances sensitivity to cetuximab in epithelial phenotype hepatocellular carcinoma cells through regulation of the eukaryotic translation initiation factor 5A-2.

    Science.gov (United States)

    Xue, Fei; Liang, Yuntian; Li, Zhenrong; Liu, Yanhui; Zhang, Hongwei; Wen, Yu; Yan, Lei; Tang, Qiang; Xiao, Erhui; Zhang, Dongyi

    2018-01-01

    Hepatocellular carcinoma (HCC) is one of the most widespread malignant human tumors worldwide. Treatment options include radiotherapy, surgical intervention and chemotherapy; however, drug resistance is an ongoing treatment concern. In the present study, the effects of a microRNA (miR/miRNA), miR-9, on the sensitivity of HCC cell lines to the epidermal growth factor receptor inhibitor, cetuximab, were examined. miR-9 has been proposed to serve a role in tumorigenesis and tumor progression. In the present study, bioinformatics analyses identified the eukaryotic translation initiation factor 5A2 (eIF-5A-2) as a target of miR-9. The expression levels of miR-9 and eIF-5A-2 were examined by reverse transcription-quantitative polymerase chain reaction and HCC cell lines were transfected with miR-9 mimics and inhibitors to determine the effects of the miRNA on cell proliferation and viability. The miR-9 mimic was revealed to significantly increase the sensitivity of epithelial phenotype HCC cells (Hep3B and Huh7) to cetuximab, while the miR-9 inhibitor triggered the opposite effect. There were no significant differences in sensitivity to cetuximab observed in mesenchymal phenotype HCC cells (SNU387 and SNU449). Cells lines displaying high expression levels of eIF-5A-2 were more resistant to cetuximab. Transfection of cells with a miR-9 mimic resulted in downregulation of the expression of eIF-5A-2 mRNA, while an miR-9 inhibitor increased expression. When expression of eIF-5A-2 was knocked down with siRNA, the effects of miR-9 on cetuximab sensitivity were no longer observed. Taken together, these data support a role for miR-9 in enhancing the sensitivity of epithelial phenotype HCC cells to cetuximab through regulation of eIF-5A-2.

  2. The Level of Autoantibodies Targeting Eukaryote Translation Elongation Factor 1 α1 and Ubiquitin-Conjugating Enzyme 2L3 in Nondiabetic Young Adults

    Directory of Open Access Journals (Sweden)

    Eunhee G. Kim

    2016-01-01

    Full Text Available BackgroundThe prevalence of novel type 1 diabetes mellitus (T1DM antibodies targeting eukaryote translation elongation factor 1 alpha 1 autoantibody (EEF1A1-AAb and ubiquitin-conjugating enzyme 2L3 autoantibody (UBE2L3-AAb has been shown to be negatively correlated with age in T1DM subjects. Therefore, we aimed to investigate whether age affects the levels of these two antibodies in nondiabetic subjects.MethodsEEF1A1-AAb and UBE2L3-AAb levels in nondiabetic control subjects (n=150 and T1DM subjects (n=101 in various ranges of age (18 to 69 years were measured using an enzyme-linked immunosorbent assay. The cutoff point for the presence of each autoantibody was determined based on control subjects using the formula: [mean absorbance+3×standard deviation].ResultsIn nondiabetic subjects, there were no significant correlations between age and EEF1A1-AAb and UBE2L3-AAb levels. However, there was wide variation in EEF1A1-AAb and UBE2L3-AAb levels among control subjects <40 years old; the prevalence of both EEF1A1-AAb and UBE2L3-AAb in these subjects was 4.4%. When using cutoff points determined from the control subjects <40 years old, the prevalence of both autoantibodies in T1DM subjects was decreased (EEFA1-AAb, 15.8% to 8.9%; UBE2L3-AAb, 10.9% to 7.9% when compared to the prevalence using the cutoff derived from the totals for control subjects.ConclusionThere was no association between age and EEF1A1-AAb or UBE2L3-AAb levels in nondiabetic subjects. However, the wide variation in EEF1A1-AAb and UBE2L3-AAb levels apparent among the control subjects <40 years old should be taken into consideration when determining the cutoff reference range for the diagnosis of T1DM.

  3. Translation, modification and cellular distribution of two AC4 variants of African cassava mosaic virus in yeast and their pathogenic potential in plants

    Energy Technology Data Exchange (ETDEWEB)

    Hipp, Katharina, E-mail: katharina.hipp@bio.uni-stuttgart.de [University of Stuttgart, Institute of Biomaterials and biomolecular Systems, Department of Molecular Biology and Plant Virology, Pfaffenwaldring 57, 70550 Stuttgart (Germany); Rau, Peter; Schäfer, Benjamin [University of Stuttgart, Institute of Biomaterials and biomolecular Systems, Department of Molecular Biology and Plant Virology, Pfaffenwaldring 57, 70550 Stuttgart (Germany); Pfannstiel, Jens [University of Hohenheim, Mass Spectrometry Core Facility, August-von-Hartmann-Straße 3, 70599 Stuttgart (Germany); Jeske, Holger [University of Stuttgart, Institute of Biomaterials and biomolecular Systems, Department of Molecular Biology and Plant Virology, Pfaffenwaldring 57, 70550 Stuttgart (Germany)

    2016-11-15

    Plant infecting geminiviruses encode a small (A)C4 protein within the open reading frame of the replication-initiator protein. In African cassava mosaic virus, two in-frame start codons may be used for the translation of a longer and a shorter AC4 variant. Both were fused to green fluorescent protein or glutathione-S-transferase genes and expressed in fission yeast. The longer variant accumulated in discrete spots in the cytoplasm, whereas the shorter variant localized to the plasma membrane. A similar expression pattern was found in plants. A myristoylation motif may promote a targeting of the shorter variant to the plasma membrane. Mass spectrometry analysis of the yeast-expressed shorter variant detected the corresponding myristoylation. The biological relevance of the second start codon was confirmed using mutated infectious clones. Whereas mutating the first start codon had no effect on the infectivity in Nicotiana benthamiana plants, the second start codon proved to be essential. -- Highlights: •The ACMV AC4 may be translated from one or the other in-frame start codon. •Both AC4 variants are translated in fission yeast. •The long AC4 protein localizes to the cytoplasm, the short to the plasma membrane. •The short variant is myristoylated in yeast and may promote membrane localization. •Only the shorter AC4 variant has an impact on viral infections in plants.

  4. Translation, modification and cellular distribution of two AC4 variants of African cassava mosaic virus in yeast and their pathogenic potential in plants

    International Nuclear Information System (INIS)

    Hipp, Katharina; Rau, Peter; Schäfer, Benjamin; Pfannstiel, Jens; Jeske, Holger

    2016-01-01

    Plant infecting geminiviruses encode a small (A)C4 protein within the open reading frame of the replication-initiator protein. In African cassava mosaic virus, two in-frame start codons may be used for the translation of a longer and a shorter AC4 variant. Both were fused to green fluorescent protein or glutathione-S-transferase genes and expressed in fission yeast. The longer variant accumulated in discrete spots in the cytoplasm, whereas the shorter variant localized to the plasma membrane. A similar expression pattern was found in plants. A myristoylation motif may promote a targeting of the shorter variant to the plasma membrane. Mass spectrometry analysis of the yeast-expressed shorter variant detected the corresponding myristoylation. The biological relevance of the second start codon was confirmed using mutated infectious clones. Whereas mutating the first start codon had no effect on the infectivity in Nicotiana benthamiana plants, the second start codon proved to be essential. -- Highlights: •The ACMV AC4 may be translated from one or the other in-frame start codon. •Both AC4 variants are translated in fission yeast. •The long AC4 protein localizes to the cytoplasm, the short to the plasma membrane. •The short variant is myristoylated in yeast and may promote membrane localization. •Only the shorter AC4 variant has an impact on viral infections in plants.

  5. Assigning Quantitative Function to Post-Translational Modifications Reveals Multiple Sites of Phosphorylation That Tune Yeast Pheromone Signaling Output

    Energy Technology Data Exchange (ETDEWEB)

    Pincus, David; Ryan, Christopher J.; Smith, Richard D.; Brent, Roger; Resnekov, Orna; Hakimi, Mohamed Ali

    2013-03-12

    Cell signaling systems transmit information by post-­translationally modifying signaling proteins, often via phosphorylation. While thousands of sites of phosphorylation have been identified in proteomic studies, the vast majority of sites have no known function. Assigning functional roles to the catalog of uncharacterized phosphorylation sites is a key research challenge. Here we present a general approach to address this challenge and apply it to a prototypical signaling pathway, the pheromone response pathway in Saccharomyces cerevisiae. The pheromone pathway includes a mitogen activated protein kinase (MAPK) cascade activated by a G-­protein coupled receptor (GPCR). We used mass spectrometry-based proteomics to identify sites whose phosphorylation changed when the system was active, and evolutionary conservation to assign priority to a list of candidate MAPK regulatory sites. We made targeted alterations in those sites, and measured the effects of the mutations on pheromone pathway output in single cells. Our work identified six new sites that quantitatively tuned system output. We developed simple computational models to find system architectures that recapitulated the quantitative phenotypes of the mutants. Our results identify a number of regulated phosphorylation events that contribute to adjust the input-­output relationship of this model eukaryotic signaling system. We believe this combined approach constitutes a general means not only to reveal modification sites required to turn a pathway on and off, but also those required for more subtle quantitative effects that tune pathway output. Our results further suggest that relatively small quantitative influences from individual regulatory phosphorylation events endow signaling systems with plasticity that evolution may exploit to quantitatively tailor signaling outcomes.

  6. Multiple Transceptors for Macro- and Micro-Nutrients Control Diverse Cellular Properties Through the PKA Pathway in Yeast: A Paradigm for the Rapidly Expanding World of Eukaryotic Nutrient Transceptors Up to Those in Human Cells.

    Science.gov (United States)

    Steyfkens, Fenella; Zhang, Zhiqiang; Van Zeebroeck, Griet; Thevelein, Johan M

    2018-01-01

    of receptors from nutrient transporters during evolution. The nutrient-sensing transceptor system in yeast for activation of the PKA pathway has served as a paradigm for similar studies on candidate nutrient transceptors in other eukaryotes and we succinctly discuss the many examples of transceptors that have already been documented in other yeast species, filamentous fungi, plants, and animals, including the examples in human cells.

  7. Multiple Transceptors for Macro- and Micro-Nutrients Control Diverse Cellular Properties Through the PKA Pathway in Yeast: A Paradigm for the Rapidly Expanding World of Eukaryotic Nutrient Transceptors Up to Those in Human Cells

    Directory of Open Access Journals (Sweden)

    Fenella Steyfkens

    2018-03-01

    the development of receptors from nutrient transporters during evolution. The nutrient-sensing transceptor system in yeast for activation of the PKA pathway has served as a paradigm for similar studies on candidate nutrient transceptors in other eukaryotes and we succinctly discuss the many examples of transceptors that have already been documented in other yeast species, filamentous fungi, plants, and animals, including the examples in human cells.

  8. Eukaryotic initiation factor 3 (eIF3) and 5’ mRNA leader sequences as agents of translational regulation in Arabidopsis. Final report

    Energy Technology Data Exchange (ETDEWEB)

    von Arnim, Albrecht G. [Univ. of Tennessee, Knoxville, TN (United States)

    2015-02-04

    Protein synthesis, or translation, consumes a sizable fraction of the cell’s energy budget, estimated at 5% and up to 50% in differentiated and growing cells, respectively. Plants also invest significant energy and biomass to construct and maintain the translation apparatus. Translation is regulated by a variety of external stimuli. Compared to transcriptional control, attributes of translational control include reduced sensitivity to stochastic fluctuation, a finer gauge of control, and more rapid responsiveness to environmental stimuli. Yet, our murky understanding of translational control allows few generalizations. Consequently, translational regulation is underutilized in the context of transgene regulation, although synthetic biologists are now beginning to appropriate RNA-level gene regulation into their regulatory circuits. We also know little about how translational control contributes to the diversity of plant form and function. This project explored how an emerging regulatory mRNA sequence element, upstream open reading frames (uORFs), is integrated with the general translation initiation machinery to permit translational regulation on specific mRNAs.

  9. Repression of mitochondrial translation, respiration and a metabolic cycle-regulated gene, SLF1, by the yeast Pumilio-family protein Puf3p.

    Directory of Open Access Journals (Sweden)

    Marc Chatenay-Lapointe

    Full Text Available Synthesis and assembly of the mitochondrial oxidative phosphorylation (OXPHOS system requires genes located both in the nuclear and mitochondrial genomes, but how gene expression is coordinated between these two compartments is not fully understood. One level of control is through regulated expression mitochondrial ribosomal proteins and other factors required for mitochondrial translation and OXPHOS assembly, which are all products of nuclear genes that are subsequently imported into mitochondria. Interestingly, this cadre of genes in budding yeast has in common a 3'-UTR element that is bound by the Pumilio family protein, Puf3p, and is coordinately regulated under many conditions, including during the yeast metabolic cycle. Multiple functions have been assigned to Puf3p, including promoting mRNA degradation, localizing nucleus-encoded mitochondrial transcripts to the outer mitochondrial membrane, and facilitating mitochondria-cytoskeletal interactions and motility. Here we show that Puf3p has a general repressive effect on mitochondrial OXPHOS abundance, translation, and respiration that does not involve changes in overall mitochondrial biogenesis and largely independent of TORC1-mitochondrial signaling. We also identified the cytoplasmic translation factor Slf1p as yeast metabolic cycle-regulated gene that is repressed by Puf3p at the post-transcriptional level and promotes respiration and extension of yeast chronological life span when over-expressed. Altogether, these results should facilitate future studies on which of the many functions of Puf3p is most relevant for regulating mitochondrial gene expression and the role of nuclear-mitochondrial communication in aging and longevity.

  10. The yeast ADH7 promoter enables gene expression under pronounced translation repression caused by the combined stress of vanillin, furfural, and 5-hydroxymethylfurfural.

    Science.gov (United States)

    Ishida, Yoko; Nguyen, Trinh Thi My; Izawa, Shingo

    2017-06-20

    Lignocellulosic biomass conversion inhibitors such as vanillin, furfural, and 5-hydroxymethylfurfural (HMF) inhibit the growth of and fermentation by Saccharomyces cerevisiae. A high concentration of each fermentation inhibitor represses translation and increases non-translated mRNAs. We previously reported that the mRNAs of ADH7 and BDH2, which encode putative NADPH- and NADH-dependent alcohol dehydrogenases, respectively, were efficiently translated even with translation repression in response to severe vanillin stress. However, the combined effects of these fermentation inhibitors on the expression of ADH7 and BDH2 remain unclear. We herein demonstrated that exposure to a combined stress of vanillin, furfural, and HMF repressed translation. The protein synthesis of Adh7, but not Bdh2 was significantly induced under combined stress conditions, even though the mRNA levels of ADH7 and BDH2 were up-regulated. Additionally, adh7Δ cells were more sensitive to the combined stress than wild-type and bdh2Δ cells. These results suggest that induction of the ADH7 expression plays a role in the tolerance to the combined stress of vanillin, furfural, and HMF. Furthermore, we succeeded in improving yeast tolerance to the combined stress by controlling the expression of ALD6 with the ADH7 promoter. Our results demonstrate that the ADH7 promoter can overcome the pronounced translation repression caused by the combined stress of vanillin, furfural, and HMF, and also suggest a new gene engineering strategy to breed robust and optimized yeasts for bioethanol production from a lignocellulosic biomass. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Yeast synthetic biology for the production of recombinant therapeutic proteins.

    Science.gov (United States)

    Kim, Hyunah; Yoo, Su Jin; Kang, Hyun Ah

    2015-02-01

    The production of recombinant therapeutic proteins is one of the fast-growing areas of molecular medicine and currently plays an important role in treatment of several diseases. Yeasts are unicellular eukaryotic microbial host cells that offer unique advantages in producing biopharmaceutical proteins. Yeasts are capable of robust growth on simple media, readily accommodate genetic modifications, and incorporate typical eukaryotic post-translational modifications. Saccharomyces cerevisiae is a traditional baker's yeast that has been used as a major host for the production of biopharmaceuticals; however, several nonconventional yeast species including Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica have gained increasing attention as alternative hosts for the industrial production of recombinant proteins. In this review, we address the established and emerging genetic tools and host strains suitable for recombinant protein production in various yeast expression systems, particularly focusing on current efforts toward synthetic biology approaches in developing yeast cell factories for the production of therapeutic recombinant proteins. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  12. Structural modelling and phylogenetic analyses of PgeIF4A2 (Eukaryotic translation initiation factor) from Pennisetum glaucum reveal signature motifs with a role in stress tolerance and development.

    Science.gov (United States)

    Agarwal, Aakrati; Mudgil, Yashwanti; Pandey, Saurabh; Fartyal, Dhirendra; Reddy, Malireddy K

    2016-01-01

    Eukaryotic translation initiation factor 4A (eIF4A) is an indispensable component of the translation machinery and also play a role in developmental processes and stress alleviation in plants and animals. Different eIF4A isoforms are present in the cytosol of the cell, namely, eIF4A1, eIF4A2, and eIF4A3 and their expression is tightly regulated in cap-dependent translation. We revealed the structural model of PgeIF4A2 protein using the crystal structure of Homo sapiens eIF4A3 (PDB ID: 2J0S) as template by Modeller 9.12. The resultant PgeIF4A2 model structure was refined by PROCHECK, ProSA, Verify3D and RMSD that showed the model structure is reliable with 77 % amino acid sequence identity with template. Investigation revealed two conserved signatures for ATP-dependent RNA Helicase DEAD-box conserved site (VLDEADEML) and RNA helicase DEAD-box type, Q-motif in sheet-turn-helix and α-helical region respectively. All these conserved motifs are responsible for response during developmental stages and stress tolerance in plants.

  13. Translation initiation factors eIF3 and HCR1 control translation termination and stop codon read-through in yeast cells

    Czech Academy of Sciences Publication Activity Database

    Beznosková, P.; Cuchalová, Lucie; Wagner, S.; Shoemaker, Ch. J.; Gunišová, S.; von der Haar, T.; Valášek, L. S.

    2013-01-01

    Roč. 9, č. 11 (2013), e1003962_1-e1003962_17 ISSN 1553-7404 Institutional support: RVO:61389013 Keywords : translation initiation * translation termination * eIF3 Subject RIV: CD - Macromolecular Chemistry Impact factor: 8.167, year: 2013

  14. The eukaryotic translation initiation factor 3 subunit E binds to classical swine fever virus NS5A and facilitates viral replication.

    Science.gov (United States)

    Liu, Xiaofeng; Wang, Xiaoyu; Wang, Qian; Luo, Mingyang; Guo, Huancheng; Gong, Wenjie; Tu, Changchun; Sun, Jinfu

    2018-02-01

    Classical swine fever virus (CSFV) NS5A protein is a multifunctional protein, playing critical roles in viral RNA replication, translation and assembly. To further explore its functions in viral replication, interaction of NS5A with host factors was assayed using a his-tag "pull down" assay coupled with shotgun LC-MS/MS. Host protein translation initiation factor 3 subunit E was identified as a binding partner of NS5A, and confirmed by co-immunoprecipitation and co-localization analysis. Overexpression of eIF3E markedly enhanced CSFV genomic replication, viral protein expression and production of progeny virus, and downregulation of eIF3E by siRNA significantly decreased viral proliferation in PK-15 cells. Luciferase reporter assay showed an enhancement of translational activity of the internal ribosome entry site of CSFV by eIF3E and a decrease in cellular translation by NS5A. These data indicate that eIF3E plays an important role in CSFV replication, thereby identifying it as a potential target for inhibition of the virus. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Applications of Recombinant DNA Technology in Gastrointestinal Medicine and Hepatology: Basic Paradigms of Molecular Cell Biology. Part C: Protein Synthesis and Post-Translational Processing in Eukaryotic Cells

    Directory of Open Access Journals (Sweden)

    Gary E Wild

    2000-01-01

    Full Text Available The translation of mRNA constitutes the first step in the synthesis of a functional protein. The polypeptide chain is subsequently folded into the appropriate three-dimensional configuration and undergoes a variety of processing steps before being converted into its active form. These processing steps are intimately related to the cellular events that occur in the endoplasmic reticulum and Golgi compartments, and determine the sorting and transport of different proteins to their appropriate destinations within the cell. While the regulation of gene expression occurs primarily at the level of transcription, the expression of many genes can also be controlled at the level of translation. Most proteins can be regulated in response to extracellular signals. In addition, intracellular protein levels can be controlled by differential rates of protein degradation. Thus, the regulation of both the amounts and activities of intracellular proteins ultimately determines all aspects of cell behaviour.

  16. Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes.

    Science.gov (United States)

    Crepin, Thibaut; Shalak, Vyacheslav F; Yaremchuk, Anna D; Vlasenko, Dmytro O; McCarthy, Andrew; Negrutskii, Boris S; Tukalo, Michail A; El'skaya, Anna V

    2014-11-10

    Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon-anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the 'GTP'- and 'GDP'-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Short RNA guides cleavage by eukaryotic RNase III.

    Directory of Open Access Journals (Sweden)

    Bruno Lamontagne

    Full Text Available In eukaryotes, short RNAs guide a variety of enzymatic activities that range from RNA editing to translation repression. It is hypothesized that pre-existing proteins evolved to bind and use guide RNA during evolution. However, the capacity of modern proteins to adopt new RNA guides has never been demonstrated. Here we show that Rnt1p, the yeast orthologue of the bacterial dsRNA-specific RNase III, can bind short RNA transcripts and use them as guides for sequence-specific cleavage. Target cleavage occurred at a constant distance from the Rnt1p binding site, leaving the guide RNA intact for subsequent cleavage. Our results indicate that RNase III may trigger sequence-specific RNA degradation independent of the RNAi machinery, and they open the road for a new generation of precise RNA silencing tools that do not trigger a dsRNA-mediated immune response.

  18. Eukaryotic acquisition of a bacterial operon

    Science.gov (United States)

    The yeast Saccharomyces cerevisiae is one of the champions of basic biomedical research due to its compact eukaryotic genome and ease of experimental manipulation. Despite these immense strengths, its impact on understanding the genetic basis of natural phenotypic variation has been limited by strai...

  19. Binding of DEAD-box helicase Dhh1 to the 5'-untranslated region of ASH1 mRNA represses localized translation of ASH1 in yeast cells.

    Science.gov (United States)

    Zhang, Qianjun; Meng, Xiuhua; Li, Delin; Chen, Shaoyin; Luo, Jianmin; Zhu, Linjie; Singer, Robert H; Gu, Wei

    2017-06-09

    Local translation of specific mRNAs is regulated by dynamic changes in their subcellular localization, and these changes are due to complex mechanisms controlling cytoplasmic mRNA transport. The budding yeast Saccharomyces cerevisiae is well suited to studying these mechanisms because many of its transcripts are transported from the mother cell to the budding daughter cell. Here, we investigated the translational control of ASH1 mRNA after transport and localization. We show that although ASH1 transcripts were translated after they reached the bud tip, some mRNAs were bound by the RNA-binding protein Puf6 and were non-polysomal. We also found that the DEAD-box helicase Dhh1 complexed with the untranslated ASH1 mRNA and Puf6. Loss of Dhh1 affected local translation of ASH1 mRNA and resulted in delocalization of ASH1 transcript in the bud. Forcibly shifting the non-polysomal ASH1 mRNA into polysomes was associated with Dhh1 dissociation. We further demonstrated that Dhh1 is not recruited to ASH1 mRNA co-transcriptionally, suggesting that it could bind to ASH1 mRNA within the cytoplasm. Of note, Dhh1 bound to the 5'-UTR of ASH1 mRNA and inhibited its translation in vitro These results suggest that after localization to the bud tip, a portion of the localized ASH1 mRNA becomes translationally inactive because of binding of Dhh1 and Puf6 to the 5'- and 3'-UTRs of ASH1 mRNA. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Programmed cell death in the leaves of the Arabidopsis spontaneous necrotic spots (sns-D mutant correlates with increased expression of the eukaryotic translation initiation factor eIF4B2

    Directory of Open Access Journals (Sweden)

    Gwenael M.D.J.-M. Gaussand

    2011-04-01

    Full Text Available From a pool of transgenic Arabidopsis (Arabidopsis thaliana plants harboring an activator T-DNA construct, one mutant was identified that developed spontaneous necrotic spots (sns-D on the rosette leaves under aseptic conditions. The sns-D mutation is dominant and homozygous plants are embryo lethal. The mutant produced smaller rosettes with a different number of stomata than the wild-type. DNA fragmentation in the nuclei of cells in the necrotic spots and a significant increase of caspase-3 and caspase-6 like activities in sns-D leaf extracts indicated that the sns-D mutation caused programmed cell death (PCD. The integration of the activator T-DNA caused an increase of the expression level of At1g13020, which encodes the eukaryotic translation initiation factor eIF4B2. The expression level of eIF4B2 was positively correlated with the severity of sns-D mutant phenotype. Overexpression of the eIF4B2 cDNA mimicked phenotypic traits of the sns-D mutant indicating that the sns-D mutant phenotype is indeed caused by activation tagging of eIF4B2. Thus, incorrect regulation of translation initiation may result in PCD.

  1. The eukaryotic translation elongation factor eEF1A2 induces neoplastic properties and mediates tumorigenic effects of ZNF217 in precursor cells of human ovarian carcinomas

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Yu; Wong, Nicholas; Guan, Yinghui; Salamanca, Clara M.; Cheng, Jung Chien; Lee, Jonathan M.; Gray, Joe W.; Auersperg, Nelly

    2008-04-25

    Ovarian epithelial carcinomas (OEC) frequently exhibit amplifications at the 20q13 locus which is the site of several oncogenes, including the eukaryotic elongation factor EEF1A2 and the transcription factor ZNF217. We reported previously that overexpressed ZNF217 induces neoplastic characteristics in precursor cells of OEC. Unexpectedly, ZNF217, which is a transcriptional repressor, enhanced expression of eEF1A2. In this study, array comparative genomic hybridization, single nucleotide polymorphism and Affymetrix analysis of ZNF217-overexpressing cell lines confirmed consistently increased expression of eEF1A2 but not of other oncogenes, and revealed early changes in EEF1A2 gene copy numbers and increased expression at crisis during immortalization. We defined the influence of eEF1A2 overexpression on immortalized ovarian surface epithelial cells, and investigated interrelationships between effects of ZNF217 and eEF1A2 on cellular phenotypes. Lentivirally induced eEF1A2 overexpression caused delayed crisis, apoptosis resistance and increases in serum-independence, saturation densities, and anchorage independence. siRNA to eEF1A2 reversed apoptosis resistance and reduced anchorage independence in eEF1A2-overexpressing lines. Remarkably, siRNA to eEF1A2 was equally efficient in inhibiting both anchorage independence and resistance to apoptosis conferred by ZNF217 overexpression. Our data define neoplastic properties that are caused by eEF1A2 in nontumorigenic ovarian cancer precursor cells, and suggest that eEF1A2 plays a role in mediating ZNF217-induced neoplastic progression.

  2. Biotechnical Microbiology, yeast and bacteria

    DEFF Research Database (Denmark)

    Villadsen, Ingrid Stampe

    1999-01-01

    This section contains the following single lecture notes: Eukaryotic Cell Biology. Kingdom Fungi. Cell Division. Meiosis and Recombination. Genetics of Yeast. Organisation of the Chromosome. Organization and genetics of the mitochondrial Geneme. Regulatio of Gene Expression. Intracellular Compart...

  3. The small molecule '1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate' and its derivatives regulate global protein synthesis by inactivating eukaryotic translation initiation factor 2-alpha.

    Science.gov (United States)

    Hong, Mi-Na; Nam, Ky-Youb; Kim, Kyung Kon; Kim, So-Young; Kim, InKi

    2016-05-01

    By environmental stresses, cells can initiate a signaling pathway in which eukaryotic translation initiation factor 2-alpha (eIF2-α) is involved to regulate the response. Phosphorylation of eIF2-α results in the reduction of overall protein neogenesis, which allows cells to conserve resources and to reprogram energy usage for effective stress control. To investigate the role of eIF2-α in cell stress responses, we conducted a viability-based compound screen under endoplasmic reticulum (ER) stress condition, and identified 1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate (AMC-01) and its derivatives as eIF2-α-inactivating chemical. Molecular characterization of this signaling pathway revealed that AMC-01 induced inactivation of eIF2-α by phosphorylating serine residue 51 in a dose- and time-dependent manner, while the negative control compounds did not affect eIF2-α phosphorylation. In contrast with ER stress induction by thapsigargin, phosphorylation of eIF2-α persisted for the duration of incubation with AMC-01. By pathway analysis, AMC-01 clearly induced the activation of protein kinase RNA-activated (PKR) kinase and nuclear factor-κB (NF-κB), whereas it did not modulate the activity of PERK or heme-regulated inhibitor (HRI). Finally, we could detect a lower protein translation rate in cells incubated with AMC-01, establishing AMC-01 as a potent chemical probe that can regulate eIF2-α activity. We suggest from these data that AMC-01 and its derivative compounds can be used as chemical probes in future studies of the role of eIF2-α in protein synthesis-related cell physiology.

  4. Deep sequencing leads to the identification of eukaryotic translation initiation factor 5A as a key element in Rsv1-mediated lethal systemic hypersensitive response to Soybean mosaic virus infection in soybean.

    Science.gov (United States)

    Chen, Hui; Adam Arsovski, Andrej; Yu, Kangfu; Wang, Aiming

    2017-04-01

    Rsv1, a single dominant resistance locus in soybean, confers extreme resistance to the majority of Soybean mosaic virus (SMV) strains, but is susceptible to the G7 strain. In Rsv1-genotype soybean, G7 infection provokes a lethal systemic hypersensitive response (LSHR), a delayed host defence response. The Rsv1-mediated LSHR signalling pathway remains largely unknown. In this study, we employed a genome-wide investigation to gain an insight into the molecular interplay between SMV G7 and Rsv1-genotype soybean. Small RNA (sRNA), degradome and transcriptome sequencing analyses were used to identify differentially expressed genes (DEGs) and microRNAs (DEMs) in response to G7 infection. A number of DEGs, DEMs and microRNA targets, and the interaction network of DEMs and their target mRNAs responsive to G7 infection, were identified. Knock-down of one of the identified DEGs, the eukaryotic translation initiation factor 5A (eIF5A), diminished the LSHR and enhanced viral accumulation, suggesting the essential role of eIF5A in the G7-induced, Rsv1-mediated LSHR signalling pathway. This work provides an in-depth genome-wide analysis of high-throughput sequencing data, and identifies multiple genes and microRNA signatures that are associated with the Rsv1-mediated LSHR. © 2016 HER MAJESTY THE QUEEN IN RIGHT OF CANADA MOLECULAR PLANT PATHOLOGY © 2016 BSPP AND JOHN WILEY & SONS LTD.

  5. Orthogonal translation components for the in vivo incorporation of unnatural amino acids

    Science.gov (United States)

    Schultz, Peter G.; Xie, Jianming; Zeng, Huaqiang

    2012-07-10

    The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate unnatural amino acids into proteins produced in eubacterial host cells such as E. coli, or in a eukaryotic host such as a yeast cell. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing unnatural amino acids, and translation systems.

  6. Different polyamine pathways from bacteria have replaced eukaryotic spermidine biosynthesis in ciliates Tetrahymena thermophila and Paramecium tetaurelia.

    Science.gov (United States)

    Li, Bin; Kim, Sok Ho; Zhang, Yang; Hanfrey, Colin C; Elliott, Katherine A; Ealick, Steven E; Michael, Anthony J

    2015-09-01

    The polyamine spermidine is absolutely required for growth and cell proliferation in eukaryotes, due to its role in post-translational modification of essential translation elongation factor eIF5A, mediated by deoxyhypusine synthase. We have found that free-living ciliates Tetrahymena and Paramecium lost the eukaryotic genes encoding spermidine biosynthesis: S-adenosylmethionine decarboxylase (AdoMetDC) and spermidine synthase (SpdSyn). In Tetrahymena, they were replaced by a gene encoding a fusion protein of bacterial AdoMetDC and SpdSyn, present as three copies. In Paramecium, a bacterial homospermidine synthase replaced the eukaryotic genes. Individual AdoMetDC-SpdSyn fusion protein paralogues from Tetrahymena exhibit undetectable AdoMetDC activity; however, when two paralogous fusion proteins are mixed, AdoMetDC activity is restored and spermidine is synthesized. Structural modelling indicates a functional active site is reconstituted by sharing critical residues from two defective protomers across the heteromer interface. Paramecium was found to accumulate homospermidine, suggesting it replaces spermidine for growth. To test this concept, a budding yeast spermidine auxotrophic strain was found to grow almost normally with homospermidine instead of spermidine. Biosynthesis of spermidine analogue aminopropylcadaverine, but not exogenously provided norspermidine, correlated with some growth. Finally, we found that diverse single-celled eukaryotic parasites and multicellular metazoan Schistosoma worms have lost the spermidine biosynthetic pathway but retain deoxyhypusine synthase. © 2015 John Wiley & Sons Ltd.

  7. Eukaryotic translation initiator protein 1A isoform, CCS-3, enhances the transcriptional repression of p21CIP1 by proto-oncogene FBI-1 (Pokemon/ZBTB7A).

    Science.gov (United States)

    Choi, Won-Il; Kim, Youngsoo; Kim, Yuri; Yu, Mi-young; Park, Jungeun; Lee, Choong-Eun; Jeon, Bu-Nam; Koh, Dong-In; Hur, Man-Wook

    2009-01-01

    FBI-1, a member of the POK (POZ and Kruppel) family of transcription factors, plays a role in differentiation, oncogenesis, and adipogenesis. eEF1A is a eukaryotic translation elongation factor involved in several cellular processes including embryogenesis, oncogenic transformation, cell proliferation, and cytoskeletal organization. CCS-3, a potential cervical cancer suppressor, is an isoform of eEF1A. We found that eEF1A forms a complex with FBI-1 by co-immunoprecipitation, SDS-PAGE, and MALDI-TOF Mass analysis of the immunoprecipitate. GST fusion protein pull-downs showed that FBI-1 directly interacts with eEF1A and CCS-3 via the zinc finger and POZ-domain of FBI-1. FBI-1 co-localizes with either eEF1A or CCS-3 at the nuclear periplasm. CCS-3 enhances transcriptional repression of the p21CIP1 gene (hereafter referred to as p21) by FBI-1. The POZ-domain of FBI-1 interacts with the co-repressors, SMRT and BCoR. We found that CCS-3 also interacts with the co-repressors independently. The molecular interaction between the co-repressors and CCS-3 at the POZ-domain of FBI-1 appears to enhance FBI-1 mediated transcriptional repression. Our data suggest that CCS-3 may be important in cell differentiation, tumorigenesis, and oncogenesis by interacting with the proto-oncogene FBI-1 and transcriptional co-repressors. Copyright 2009 S. Karger AG, Basel.

  8. Post-Translational Modifications of TRP Channels

    Directory of Open Access Journals (Sweden)

    Olaf Voolstra

    2014-04-01

    Full Text Available Transient receptor potential (TRP channels constitute an ancient family of cation channels that have been found in many eukaryotic organisms from yeast to human. TRP channels exert a multitude of physiological functions ranging from Ca2+ homeostasis in the kidney to pain reception and vision. These channels are activated by a wide range of stimuli and undergo covalent post-translational modifications that affect and modulate their subcellular targeting, their biophysical properties, or channel gating. These modifications include N-linked glycosylation, protein phosphorylation, and covalent attachment of chemicals that reversibly bind to specific cysteine residues. The latter modification represents an unusual activation mechanism of ligand-gated ion channels that is in contrast to the lock-and-key paradigm of receptor activation by its agonists. In this review, we summarize the post-translational modifications identified on TRP channels and, when available, explain their physiological role.

  9. Structure and Mechanism of a Eukaryotic FMN Adenylyltransferase

    OpenAIRE

    Huerta, Carlos; Borek, Dominika; Machius, Mischa; Grishin, Nick V.; Zhang, Hong

    2009-01-01

    Flavin mononucleotide adenylyltransferase (FMNAT) catalyzes the formation of the essential flavocoenzyme FAD and plays an important role in flavocoenzyme homeostasis regulation. By sequence comparison, bacterial and eukaryotic FMNAT enzymes belong to two different protein superfamilies and apparently utilize different set of active site residues to accomplish the same chemistry. Here we report the first structural characterization of a eukaryotic FMNAT from a pathogenic yeast Candida glabrata...

  10. Yeast genome sequencing:

    DEFF Research Database (Denmark)

    Piskur, Jure; Langkjær, Rikke Breinhold

    2004-01-01

    For decades, unicellular yeasts have been general models to help understand the eukaryotic cell and also our own biology. Recently, over a dozen yeast genomes have been sequenced, providing the basis to resolve several complex biological questions. Analysis of the novel sequence data has shown...... of closely related species helps in gene annotation and to answer how many genes there really are within the genomes. Analysis of non-coding regions among closely related species has provided an example of how to determine novel gene regulatory sequences, which were previously difficult to analyse because...... they are short and degenerate and occupy different positions. Comparative genomics helps to understand the origin of yeasts and points out crucial molecular events in yeast evolutionary history, such as whole-genome duplication and horizontal gene transfer(s). In addition, the accumulating sequence data provide...

  11. Autophagy in unicellular eukaryotes

    NARCIS (Netherlands)

    Kiel, J.A.K.W.

    2010-01-01

    Cells need a constant supply of precursors to enable the production of macromolecules to sustain growth and survival. Unlike metazoans, unicellular eukaryotes depend exclusively on the extracellular medium for this supply. When environmental nutrients become depleted, existing cytoplasmic components

  12. Enzymes from Higher Eukaryotes for Industrial Biocatalysis

    Directory of Open Access Journals (Sweden)

    Zhibin Liu

    2004-01-01

    Full Text Available The industrial production of fine chemicals, feed and food ingredients, pharmaceuticals, agrochemicals and their respective intermediates relies on an increasing application of biocatalysis, i.e. on enzyme or whole-cell catalyzed conversions of molecules. Simple procedures for discovery, cloning and over-expression as well as fast growth favour fungi, yeasts and especially bacteria as sources of biocatalysts. Higher eukaryotes also harbour an almost unlimited number of potential biocatalysts, although to date the limited supply of enzymes, the high heterogeneity of enzyme preparations and the hazard of infectious contaminants keep some interesting candidates out of reach for industrial bioprocesses. In the past only a few animal and plant enzymes from agricultural waste materials were employed in food processing. The use of bacterial expression strains or non-conventional yeasts for the heterologous production of efficient eukaryotic enzymes can overcome the bottleneck in enzyme supply and provide sufficient amounts of homogenous enzyme preparations for reliable and economically feasible applications at large scale. Ideal enzymatic processes represent an environmentally friendly, »near-to-completion« conversion of (mostly non-natural substrates to pure products. Recent developments demonstrate the commercial feasibility of large-scale biocatalytic processes employing enzymes from higher eukaryotes (e.g. plants, animals and also their usefulness in some small-scale industrial applications.

  13. Yeast replicative aging: a paradigm for defining conserved longevity interventions

    OpenAIRE

    Wasko, Brian M.; Kaeberlein, Matt

    2013-01-01

    The finite replicative life span of budding yeast mother cells was demonstrated as early as 1959, but the idea that budding yeast could be used to model aging of multicellular eukaryotes did not enter the scientific mainstream until relatively recently. Despite continued skepticism by some, there are now abundant data that several interventions capable of extending yeast replicative life span have a similar effect in multicellular eukaryotes including nematode worms, fruit flies, and rodents....

  14. Constraints and consequences of the emergence of amino acid repeats in eukaryotic proteins.

    Science.gov (United States)

    Chavali, Sreenivas; Chavali, Pavithra L; Chalancon, Guilhem; de Groot, Natalia Sanchez; Gemayel, Rita; Latysheva, Natasha S; Ing-Simmons, Elizabeth; Verstrepen, Kevin J; Balaji, Santhanam; Babu, M Madan

    2017-09-01

    Proteins with amino acid homorepeats have the potential to be detrimental to cells and are often associated with human diseases. Why, then, are homorepeats prevalent in eukaryotic proteomes? In yeast, homorepeats are enriched in proteins that are essential and pleiotropic and that buffer environmental insults. The presence of homorepeats increases the functional versatility of proteins by mediating protein interactions and facilitating spatial organization in a repeat-dependent manner. During evolution, homorepeats are preferentially retained in proteins with stringent proteostasis, which might minimize repeat-associated detrimental effects such as unregulated phase separation and protein aggregation. Their presence facilitates rapid protein divergence through accumulation of amino acid substitutions, which often affect linear motifs and post-translational-modification sites. These substitutions may result in rewiring protein interaction and signaling networks. Thus, homorepeats are distinct modules that are often retained in stringently regulated proteins. Their presence facilitates rapid exploration of the genotype-phenotype landscape of a population, thereby contributing to adaptation and fitness.

  15. Use of prokaryotic transcriptional activators as metabolite biosensors in eukaryotic cells

    DEFF Research Database (Denmark)

    2018-01-01

    The present invention relates to the use of transcriptional activators from prokaryotic organisms for use in eukaryotic cells, such as yeast as sensors of intracellular and extracellular accumulation of a ligand or metabolite specifically activating this transcriptional activator in a eukaryot...

  16. Principles of mRNA transport in yeast.

    Science.gov (United States)

    Heym, Roland Gerhard; Niessing, Dierk

    2012-06-01

    mRNA localization and localized translation is a common mechanism by which cellular asymmetry is achieved. In higher eukaryotes the mRNA transport machinery is required for such diverse processes as stem cell division and neuronal plasticity. Because mRNA localization in metazoans is highly complex, studies at the molecular level have proven to be cumbersome. However, active mRNA transport has also been reported in fungi including Saccharomyces cerevisiae, Ustilago maydis and Candida albicans, in which these events are less difficult to study. Amongst them, budding yeast S. cerevisiae has yielded mechanistic insights that exceed our understanding of other mRNA localization events to date. In contrast to most reviews, we refrain here from summarizing mRNA localization events from different organisms. Instead we give an in-depth account of ASH1 mRNA localization in budding yeast. This approach is particularly suited to providing a more holistic view of the interconnection between the individual steps of mRNA localization, from transcriptional events to cytoplasmic mRNA transport and localized translation. Because of our advanced mechanistic understanding of mRNA localization in yeast, the present review may also be informative for scientists working, for example, on mRNA localization in embryogenesis or in neurons.

  17. Redox characteristics of the eukaryotic cytosol

    DEFF Research Database (Denmark)

    López-Mirabal, H Reynaldo; Winther, Jakob R

    2007-01-01

    The eukaryotic cytoplasm has long been regarded as a cellular compartment in which the reduced state of protein cysteines is largely favored. Under normal conditions, the cytosolic low-molecular weight redox buffer, comprising primarily of glutathione, is highly reducing and reactive oxygen species...... (ROS) and glutathionylated proteins are maintained at very low levels. In the present review, recent progress in the understanding of the cytosolic thiol-disulfide redox metabolism and novel analytical approaches to studying cytosolic redox properties are discussed. We will focus on the yeast model...... organism, Saccharomyces cerevisiae, where the combination of genetic and biochemical approaches has brought us furthest in understanding the mechanisms underlying cellular redox regulation. It has been shown in yeast that, in addition to the enzyme glutathione reductase, other mechanisms may exist...

  18. Eukaryotic Cell Panorama

    Science.gov (United States)

    Goodsell, David S.

    2011-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. This report describes the scientific results that support an illustration of a eukaryotic cell, enlarged by one million times to show the distribution and arrangement of macromolecules. The panoramic cross section includes eight panels that extend…

  19. Eukaryotic cell flattening

    Science.gov (United States)

    Bae, Albert; Westendorf, Christian; Erlenkamper, Christoph; Galland, Edouard; Franck, Carl; Bodenschatz, Eberhard; Beta, Carsten

    2010-03-01

    Eukaryotic cell flattening is valuable for improving microscopic observations, ranging from bright field to total internal reflection fluorescence microscopy. In this talk, we will discuss traditional overlay techniques, and more modern, microfluidic based flattening, which provides a greater level of control. We demonstrate these techniques on the social amoebae Dictyostelium discoideum, comparing the advantages and disadvantages of each method.

  20. Comparative Genomics of Eukaryotes.

    NARCIS (Netherlands)

    Noort, V. van

    2007-01-01

    This thesis focuses on developing comparative genomics methods in eukaryotes, with an emphasis on applications for gene function prediction and regulatory element detection. In the past, methods have been developed to predict functional associations between gene pairs in prokaryotes. The challenge

  1. A Functional, Genome-wide Evaluation of Liposensitive Yeast Identifies the “ARE2 Required for Viability” (ARV1) Gene Product as a Major Component of Eukaryotic Fatty Acid Resistance*

    Science.gov (United States)

    Ruggles, Kelly V.; Garbarino, Jeanne; Liu, Ying; Moon, James; Schneider, Kerry; Henneberry, Annette; Billheimer, Jeff; Millar, John S.; Marchadier, Dawn; Valasek, Mark A.; Joblin-Mills, Aidan; Gulati, Sonia; Munkacsi, Andrew B.; Repa, Joyce J.; Rader, Dan; Sturley, Stephen L.

    2014-01-01

    The toxic subcellular accumulation of lipids predisposes several human metabolic syndromes, including obesity, type 2 diabetes, and some forms of neurodegeneration. To identify pathways that prevent lipid-induced cell death, we performed a genome-wide fatty acid sensitivity screen in Saccharomyces cerevisiae. We identified 167 yeast mutants as sensitive to 0.5 mm palmitoleate, 45% of which define pathways that were conserved in humans. 63 lesions also impacted the status of the lipid droplet; however, this was not correlated to the degree of fatty acid sensitivity. The most liposensitive yeast strain arose due to deletion of the “ARE2 required for viability” (ARV1) gene, encoding an evolutionarily conserved, potential lipid transporter that localizes to the endoplasmic reticulum membrane. Down-regulation of mammalian ARV1 in MIN6 pancreatic β-cells or HEK293 cells resulted in decreased neutral lipid synthesis, increased fatty acid sensitivity, and lipoapoptosis. Conversely, elevated expression of human ARV1 in HEK293 cells or mouse liver significantly increased triglyceride mass and lipid droplet number. The ARV1-induced hepatic triglyceride accumulation was accompanied by up-regulation of DGAT1, a triglyceride synthesis gene, and the fatty acid transporter, CD36. Furthermore, ARV1 was identified as a transcriptional of the protein peroxisome proliferator-activated receptor α (PPARα), a key regulator of lipid homeostasis whose transcriptional targets include DGAT1 and CD36. These results implicate ARV1 as a protective factor in lipotoxic diseases due to modulation of fatty acid metabolism. In conclusion, a lipotoxicity-based genetic screen in a model microorganism has identified 75 human genes that may play key roles in neutral lipid metabolism and disease. PMID:24273168

  2. A functional, genome-wide evaluation of liposensitive yeast identifies the "ARE2 required for viability" (ARV1) gene product as a major component of eukaryotic fatty acid resistance.

    Science.gov (United States)

    Ruggles, Kelly V; Garbarino, Jeanne; Liu, Ying; Moon, James; Schneider, Kerry; Henneberry, Annette; Billheimer, Jeff; Millar, John S; Marchadier, Dawn; Valasek, Mark A; Joblin-Mills, Aidan; Gulati, Sonia; Munkacsi, Andrew B; Repa, Joyce J; Rader, Dan; Sturley, Stephen L

    2014-02-14

    The toxic subcellular accumulation of lipids predisposes several human metabolic syndromes, including obesity, type 2 diabetes, and some forms of neurodegeneration. To identify pathways that prevent lipid-induced cell death, we performed a genome-wide fatty acid sensitivity screen in Saccharomyces cerevisiae. We identified 167 yeast mutants as sensitive to 0.5 mm palmitoleate, 45% of which define pathways that were conserved in humans. 63 lesions also impacted the status of the lipid droplet; however, this was not correlated to the degree of fatty acid sensitivity. The most liposensitive yeast strain arose due to deletion of the "ARE2 required for viability" (ARV1) gene, encoding an evolutionarily conserved, potential lipid transporter that localizes to the endoplasmic reticulum membrane. Down-regulation of mammalian ARV1 in MIN6 pancreatic β-cells or HEK293 cells resulted in decreased neutral lipid synthesis, increased fatty acid sensitivity, and lipoapoptosis. Conversely, elevated expression of human ARV1 in HEK293 cells or mouse liver significantly increased triglyceride mass and lipid droplet number. The ARV1-induced hepatic triglyceride accumulation was accompanied by up-regulation of DGAT1, a triglyceride synthesis gene, and the fatty acid transporter, CD36. Furthermore, ARV1 was identified as a transcriptional of the protein peroxisome proliferator-activated receptor α (PPARα), a key regulator of lipid homeostasis whose transcriptional targets include DGAT1 and CD36. These results implicate ARV1 as a protective factor in lipotoxic diseases due to modulation of fatty acid metabolism. In conclusion, a lipotoxicity-based genetic screen in a model microorganism has identified 75 human genes that may play key roles in neutral lipid metabolism and disease.

  3. Sexual differentiation in fission yeast

    DEFF Research Database (Denmark)

    Egel, R; Nielsen, O; Weilguny, D

    1990-01-01

    The regulation of sexual reproduction in yeast constitutes the highest level of differentiation observed in these unicellular organisms. The various ramifications of this system involve DNA rearrangement, transcriptional control, post-translational modification (such as protein phosphorylation) a......) and receptor/signal processing. A few basic similarities are common to both fission and budding yeasts. The wiring of the regulatory circuitry, however, varies considerably between these divergent yeast groups....

  4. Arsenic and Antimony Transporters in Eukaryotes

    Directory of Open Access Journals (Sweden)

    Ewa Maciaszczyk-Dziubinska

    2012-03-01

    Full Text Available Arsenic and antimony are toxic metalloids, naturally present in the environment and all organisms have developed pathways for their detoxification. The most effective metalloid tolerance systems in eukaryotes include downregulation of metalloid uptake, efflux out of the cell, and complexation with phytochelatin or glutathione followed by sequestration into the vacuole. Understanding of arsenic and antimony transport system is of high importance due to the increasing usage of arsenic-based drugs in the treatment of certain types of cancer and diseases caused by protozoan parasites as well as for the development of bio- and phytoremediation strategies for metalloid polluted areas. However, in contrast to prokaryotes, the knowledge about specific transporters of arsenic and antimony and the mechanisms of metalloid transport in eukaryotes has been very limited for a long time. Here, we review the recent advances in understanding of arsenic and antimony transport pathways in eukaryotes, including a dual role of aquaglyceroporins in uptake and efflux of metalloids, elucidation of arsenic transport mechanism by the yeast Acr3 transporter and its role in arsenic hyperaccumulation in ferns, identification of vacuolar transporters of arsenic-phytochelatin complexes in plants and forms of arsenic substrates recognized by mammalian ABC transporters.

  5. Arsenic and Antimony Transporters in Eukaryotes

    Science.gov (United States)

    Maciaszczyk-Dziubinska, Ewa; Wawrzycka, Donata; Wysocki, Robert

    2012-01-01

    Arsenic and antimony are toxic metalloids, naturally present in the environment and all organisms have developed pathways for their detoxification. The most effective metalloid tolerance systems in eukaryotes include downregulation of metalloid uptake, efflux out of the cell, and complexation with phytochelatin or glutathione followed by sequestration into the vacuole. Understanding of arsenic and antimony transport system is of high importance due to the increasing usage of arsenic-based drugs in the treatment of certain types of cancer and diseases caused by protozoan parasites as well as for the development of bio- and phytoremediation strategies for metalloid polluted areas. However, in contrast to prokaryotes, the knowledge about specific transporters of arsenic and antimony and the mechanisms of metalloid transport in eukaryotes has been very limited for a long time. Here, we review the recent advances in understanding of arsenic and antimony transport pathways in eukaryotes, including a dual role of aquaglyceroporins in uptake and efflux of metalloids, elucidation of arsenic transport mechanism by the yeast Acr3 transporter and its role in arsenic hyperaccumulation in ferns, identification of vacuolar transporters of arsenic-phytochelatin complexes in plants and forms of arsenic substrates recognized by mammalian ABC transporters. PMID:22489166

  6. Protein splicing and its evolution in eukaryotes

    Directory of Open Access Journals (Sweden)

    Starokadomskyy P. L.

    2010-02-01

    Full Text Available Inteins, or protein introns, are parts of protein sequences that are post-translationally excised, their flanking regions (exteins being spliced together. This process was called protein splicing. Originally inteins were found in prokaryotic or unicellular eukaryotic organisms. But the general principles of post-translation protein rearrangement are evolving yielding different post-translation modification of proteins in multicellular organisms. For clarity, these non-intein mediated events call either protein rearrangements or protein editing. The most intriguing example of protein editing is proteasome-mediated splicing of antigens in vertebrates that may play important role in antigen presentation. Other examples of protein rearrangements are maturation of Hg-proteins (critical receptors in embryogenesis as well as maturation of several metabolic enzymes. Despite a lack of experimental data we try to analyze some intriguing examples of protein splicing evolution.

  7. Yeast glycolipid biosurfactants.

    Science.gov (United States)

    Jezierska, Sylwia; Claus, Silke; Van Bogaert, Inge

    2017-10-25

    Various yeasts, both conventional and exotic ones, are known to produce compounds useful to mankind. Ethanol is the most known of these compounds, but more complex molecules such as amphiphilic biosurfactants can also be derived from eukaryotic microorganisms at an industrially and commercially relevant scale. Among them, glycolipids are the most promising, due to their attractive properties and high product titers. Many of these compounds can be considered as secondary metabolites with a specific function for the host. Hence, a dedicated biosynthetic process enables regulation and combines pathways delivering the lipidic moiety and the hydrophilic carbohydrate part of the glycolipid. In this Review, we will discuss the biosynthetic and regulatory aspects of the yeast-derived sophorolipids, mannosylerythritol lipids, and cellobiose lipids, with special emphasis on the relation between glycolipid synthesis and the general lipid metabolism. © 2017 Federation of European Biochemical Societies.

  8. Interaction of tRNA with Eukaryotic Ribosome

    Directory of Open Access Journals (Sweden)

    Dmitri Graifer

    2015-03-01

    Full Text Available This paper is a review of currently available data concerning interactions of tRNAs with the eukaryotic ribosome at various stages of translation. These data include the results obtained by means of cryo-electron microscopy and X-ray crystallography applied to various model ribosomal complexes, site-directed cross-linking with the use of tRNA derivatives bearing chemically or photochemically reactive groups in the CCA-terminal fragment and chemical probing of 28S rRNA in the region of the peptidyl transferase center. Similarities and differences in the interactions of tRNAs with prokaryotic and eukaryotic ribosomes are discussed with concomitant consideration of the extent of resemblance between molecular mechanisms of translation in eukaryotes and bacteria.

  9. Eukaryotic DNA Replication Fork.

    Science.gov (United States)

    Burgers, Peter M J; Kunkel, Thomas A

    2017-06-20

    This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

  10. Transcriptional Waves in the Yeast Cell Cycle

    OpenAIRE

    Oliva, Anna; Rosebrock, Adam; Ferrezuelo, Francisco; Pyne, Saumyadipta; Chen, Haiying; Skiena, Steve; Futcher, Bruce; Leatherwood, Janet

    2005-01-01

    Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast) and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast). The 750 genes with the most significant oscillat...

  11. Predicted RNA Binding Proteins Pes4 and Mip6 Regulate mRNA Levels, Translation, and Localization during Sporulation in Budding Yeast.

    Science.gov (United States)

    Jin, Liang; Zhang, Kai; Sternglanz, Rolf; Neiman, Aaron M

    2017-05-01

    In response to starvation, diploid cells of Saccharomyces cerevisiae undergo meiosis and form haploid spores, a process collectively referred to as sporulation. The differentiation into spores requires extensive changes in gene expression. The transcriptional activator Ndt80 is a central regulator of this process, which controls many genes essential for sporulation. Ndt80 induces ∼300 genes coordinately during meiotic prophase, but different mRNAs within the NDT80 regulon are translated at different times during sporulation. The protein kinase Ime2 and RNA binding protein Rim4 are general regulators of meiotic translational delay, but how differential timing of individual transcripts is achieved was not known. This report describes the characterization of two related NDT80 -induced genes, PES4 and MIP6 , encoding predicted RNA binding proteins. These genes are necessary to regulate the steady-state expression, translational timing, and localization of a set of mRNAs that are transcribed by NDT80 but not translated until the end of meiosis II. Mutations in the predicted RNA binding domains within PES4 alter the stability of target mRNAs. PES4 and MIP6 affect only a small portion of the NDT80 regulon, indicating that they act as modulators of the general Ime2/Rim4 pathway for specific transcripts. Copyright © 2017 American Society for Microbiology.

  12. RNA Export through the NPC in Eukaryotes.

    Science.gov (United States)

    Okamura, Masumi; Inose, Haruko; Masuda, Seiji

    2015-03-20

    In eukaryotic cells, RNAs are transcribed in the nucleus and exported to the cytoplasm through the nuclear pore complex. The RNA molecules that are exported from the nucleus into the cytoplasm include messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), micro RNAs (miRNAs), and viral mRNAs. Each RNA is transported by a specific nuclear export receptor. It is believed that most of the mRNAs are exported by Nxf1 (Mex67 in yeast), whereas rRNAs, snRNAs, and a certain subset of mRNAs are exported in a Crm1/Xpo1-dependent manner. tRNAs and miRNAs are exported by Xpot and Xpo5. However, multiple export receptors are involved in the export of some RNAs, such as 60S ribosomal subunit. In addition to these export receptors, some adapter proteins are required to export RNAs. The RNA export system of eukaryotic cells is also used by several types of RNA virus that depend on the machineries of the host cell in the nucleus for replication of their genome, therefore this review describes the RNA export system of two representative viruses. We also discuss the NPC anchoring-dependent mRNA export factors that directly recruit specific genes to the NPC.

  13. Mitochondrial uncoupling proteins in unicellular eukaryotes.

    Science.gov (United States)

    Jarmuszkiewicz, Wieslawa; Woyda-Ploszczyca, Andrzej; Antos-Krzeminska, Nina; Sluse, Francis E

    2010-01-01

    Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier protein family that are present in the mitochondrial inner membrane and mediate free fatty acid (FFA)-activated, purine nucleotide (PN)-inhibited proton conductance. Since 1999, the presence of UCPs has been demonstrated in some non-photosynthesising unicellular eukaryotes, including amoeboid and parasite protists, as well as in non-fermentative yeast and filamentous fungi. In the mitochondria of these organisms, UCP activity is revealed upon FFA-induced, PN-inhibited stimulation of resting respiration and a decrease in membrane potential, which are accompanied by a decrease in membranous ubiquinone (Q) reduction level. UCPs in unicellular eukaryotes are able to divert energy from oxidative phosphorylation and thus compete for a proton electrochemical gradient with ATP synthase. Our recent work indicates that membranous Q is a metabolic sensor that might utilise its redox state to release the PN inhibition of UCP-mediated mitochondrial uncoupling under conditions of phosphorylation and resting respiration. The action of reduced Q (QH2) could allow higher or complete activation of UCP. As this regulatory feature was demonstrated for microorganism UCPs (A. castellanii UCP), plant and mammalian UCP1 analogues, and UCP1 in brown adipose tissue, the process could involve all UCPs. Here, we discuss the functional connection and physiological role of UCP and alternative oxidase, two main energy-dissipating systems in the plant-type mitochondrial respiratory chain of unicellular eukaryotes, including the control of cellular energy balance as well as preventive action against the production of reactive oxygen species. Copyright © 2009 Elsevier B.V. All rights reserved.

  14. Translation elongation factor 1A facilitates the assembly of the tombusvirus replicase and stimulates minus-strand synthesis.

    OpenAIRE

    Zhenghe Li; Judit Pogany; Steven Tupman; Anthony M Esposito; Terri Goss Kinzy; Peter D Nagy

    2010-01-01

    Replication of plus-strand RNA viruses depends on host factors that are recruited into viral replicase complexes. Previous studies showed that eukaryotic translation elongation factor (eEF1A) is one of the resident host proteins in the highly purified tombusvirus replicase complex. Using a random library of eEF1A mutants, we identified one mutant that decreased and three mutants that increased Tomato bushy stunt virus (TBSV) replication in a yeast model host. Additional in vitro assays with w...

  15. DNA repair and the genetic control of radiosensitivity in yeast

    International Nuclear Information System (INIS)

    Haynes, R.H.

    1975-01-01

    The following topics are discussed: advantages of yeasts for easily manipulated model systems for studies on molecular biology of eukaryotes; induction of x-ray-resistant mutants by radiations and chemicals; genetics of uv-sensitive mutants; loci of genes affecting radiosensitivity; gene interactions in multiple mutants; liquid-holding recovery; mitotic and meiotic recombination; and repair of yeast mitochondrial DNA

  16. Industrial relevance of chromosomal copy number variation in Saccharomyces yeasts

    NARCIS (Netherlands)

    Gorter de Vries, A.R.; Pronk, J.T.; Daran, J.G.

    2017-01-01

    Chromosomal copy number variation (CCNV) plays a key role in evolution and health of eukaryotes. The unicellular yeast Saccharomyces cerevisiae is an important model for studying the generation, physiological impact, and evolutionary significance of CCNV. Fundamental studies of this yeast have

  17. Biomass conversion inhibitors furfural and 5-hydroxymethylfurfural induce formation of messenger RNP granules and attenuate translation activity in Saccharomyces cerevisiae.

    Science.gov (United States)

    Iwaki, Aya; Kawai, Takao; Yamamoto, Yosuke; Izawa, Shingo

    2013-03-01

    Various forms of stress can cause an attenuation of bulk translation activity and the accumulation of nontranslating mRNAs into cytoplasmic messenger RNP (mRNP) granules termed processing bodies (P-bodies) and stress granules (SGs) in eukaryotic cells. Furfural and 5-hydroxymethylfurfural (HMF), derived from lignocellulosic biomass, inhibit yeast growth and fermentation as stressors. Since there is no report regarding their effects on the formation of cytoplasmic mRNP granules, here we investigated whether furfural and HMF cause the assembly of yeast P-bodies and SGs accompanied by translational repression. We found that furfural and HMF cause the attenuation of bulk translation activity and the assembly of cytoplasmic mRNP granules in Saccharomyces cerevisiae. Notably, a combination of furfural and HMF induced the remarkable repression of translation initiation and SG formation. These findings provide new information about the physiological effects of furfural and HMF on yeast cells, and also suggest the potential usefulness of cytoplasmic mRNP granules as a warning sign or index of the deterioration of cellular physiological status in the fermentation of lignocellulosic hydrolysates.

  18. The RNA recognition motif of eukaryotic translation initiation factor 3g (eIF3g) is required for resumption of scanning of posttermination ribosomes for reinitiation on GCN4 and together with eIF3i stimulates linear scanning.

    Science.gov (United States)

    Cuchalová, Lucie; Kouba, Tomás; Herrmannová, Anna; Dányi, István; Chiu, Wen-Ling; Valásek, Leos

    2010-10-01

    Recent reports have begun unraveling the details of various roles of individual eukaryotic translation initiation factor 3 (eIF3) subunits in translation initiation. Here we describe functional characterization of two essential Saccharomyces cerevisiae eIF3 subunits, g/Tif35 and i/Tif34, previously suggested to be dispensable for formation of the 48S preinitiation complexes (PICs) in vitro. A triple-Ala substitution of conserved residues in the RRM of g/Tif35 (g/tif35-KLF) or a single-point mutation in the WD40 repeat 6 of i/Tif34 (i/tif34-Q258R) produces severe growth defects and decreases the rate of translation initiation in vivo without affecting the integrity of eIF3 and formation of the 43S PICs in vivo. Both mutations also diminish induction of GCN4 expression, which occurs upon starvation via reinitiation. Whereas g/tif35-KLF impedes resumption of scanning for downstream reinitiation by 40S ribosomes terminating at upstream open reading frame 1 (uORF1) in the GCN4 mRNA leader, i/tif34-Q258R prevents full GCN4 derepression by impairing the rate of scanning of posttermination 40S ribosomes moving downstream from uORF1. In addition, g/tif35-KLF reduces processivity of scanning through stable secondary structures, and g/Tif35 specifically interacts with Rps3 and Rps20 located near the ribosomal mRNA entry channel. Together these results implicate g/Tif35 and i/Tif34 in stimulation of linear scanning and, specifically in the case of g/Tif35, also in proper regulation of the GCN4 reinitiation mechanism.

  19. Eukaryotic DNA Replicases

    KAUST Repository

    Zaher, Manal S.; Oke, Muse; Hamdan, Samir

    2014-01-01

    The current model of the eukaryotic DNA replication fork includes three replicative DNA polymerases, polymerase α/primase complex (Pol α), polymerase δ (Pol δ), and polymerase ε (Pol ε). The primase synthesizes 8–12 nucleotide RNA primers that are extended by the DNA polymerization activity of Pol α into 30–35 nucleotide RNA-DNA primers. Replication factor C (RFC) opens the polymerase clamp-like processivity factor, proliferating cell nuclear antigen (PCNA), and loads it onto the primer-template. Pol δ utilizes PCNA to mediate highly processive DNA synthesis, while Pol ε has intrinsic high processivity that is modestly stimulated by PCNA. Pol ε replicates the leading strand and Pol δ replicates the lagging strand in a division of labor that is not strict. The three polymerases are comprised of multiple subunits and share unifying features in their large catalytic and B subunits. The remaining subunits are evolutionarily not related and perform diverse functions. The catalytic subunits are members of family B, which are distinguished by their larger sizes due to inserts in their N- and C-terminal regions. The sizes of these inserts vary among the three polymerases, and their functions remain largely unknown. Strikingly, the quaternary structures of Pol α, Pol δ, and Pol ε are arranged similarly. The catalytic subunits adopt a globular structure that is linked via its conserved C-terminal region to the B subunit. The remaining subunits are linked to the catalytic and B subunits in a highly flexible manner.

  20. Eukaryotic DNA Replicases

    KAUST Repository

    Zaher, Manal S.

    2014-11-21

    The current model of the eukaryotic DNA replication fork includes three replicative DNA polymerases, polymerase α/primase complex (Pol α), polymerase δ (Pol δ), and polymerase ε (Pol ε). The primase synthesizes 8–12 nucleotide RNA primers that are extended by the DNA polymerization activity of Pol α into 30–35 nucleotide RNA-DNA primers. Replication factor C (RFC) opens the polymerase clamp-like processivity factor, proliferating cell nuclear antigen (PCNA), and loads it onto the primer-template. Pol δ utilizes PCNA to mediate highly processive DNA synthesis, while Pol ε has intrinsic high processivity that is modestly stimulated by PCNA. Pol ε replicates the leading strand and Pol δ replicates the lagging strand in a division of labor that is not strict. The three polymerases are comprised of multiple subunits and share unifying features in their large catalytic and B subunits. The remaining subunits are evolutionarily not related and perform diverse functions. The catalytic subunits are members of family B, which are distinguished by their larger sizes due to inserts in their N- and C-terminal regions. The sizes of these inserts vary among the three polymerases, and their functions remain largely unknown. Strikingly, the quaternary structures of Pol α, Pol δ, and Pol ε are arranged similarly. The catalytic subunits adopt a globular structure that is linked via its conserved C-terminal region to the B subunit. The remaining subunits are linked to the catalytic and B subunits in a highly flexible manner.

  1. A role for eukaryotic translation initiation factor 2B (eIF2B) in taste memory consolidation and in thermal control establishment during the critical period for sensory development.

    Science.gov (United States)

    Tirosh, Sharon; Elkobi, Alina; Rosenblum, Kobi; Meiri, Noam

    2007-05-01

    All species exhibit critical periods for sensory development, yet very little is known about the molecules involved in the changes in the network wiring that underlies this process. Here the role of transcription regulation of the translation machinery was determined by evaluating the expression of eIF2Bepsilon, an essential component of translation initiation, in both taste-preference development and thermal control establishment in chicks. Analysis of the expression pattern of this gene after passive-avoidance training revealed clear induction of eIF2Bepsilon in both the mesopallium intermediomediale (IMM) and in the striatum mediale (StM). In addition, a correlation was found between the concentration of methylanthranilate (MeA), which was the malaise substrate in the passive-avoidance training procedure, the duration of memory, and the expression level of eIF2Bepsilon. Training chicks on a low concentration of MeA induced short-term memory and low expression level of eIF2Bepsilon, whereas a high concentration of MeA induced long-term memory and a high expression level of eIF2Bepsilon in both the IMM and StM. Furthermore, eIF2Bepsilon-antisense "knock-down" not only reduced the amount of eIF2Bepsilon but also attenuated taste memory formation. In order to determine whether induction of eIF2Bepsilon is a general feature of neuronal plasticity, we checked whether it was induced in other forms of neuronal plasticity, with particular attention to its role in temperature control establishment, which represents hypothalamic-related plasticity. It was established that eIF2Bepsilon-mRNA was induced in the preopotic anterior hypothalamus during heat conditioning. Taken together, these results correlate eIF2Bepsilon with sensory development.

  2. Structure of a yeast 40S-eIF1-eIF1A-eIF3-eIF3j initiation complex.

    Science.gov (United States)

    Aylett, Christopher H S; Boehringer, Daniel; Erzberger, Jan P; Schaefer, Tanja; Ban, Nenad

    2015-03-01

    Eukaryotic translation initiation requires cooperative assembly of a large protein complex at the 40S ribosomal subunit. We have resolved a budding yeast initiation complex by cryo-EM, allowing placement of prior structures of eIF1, eIF1A, eIF3a, eIF3b and eIF3c. Our structure highlights differences in initiation-complex binding to the ribosome compared to that of mammalian eIF3, demonstrates a direct contact between eIF3j and eIF1A and reveals the network of interactions between eIF3 subunits.

  3. Mapping replication origins in yeast chromosomes.

    Science.gov (United States)

    Brewer, B J; Fangman, W L

    1991-07-01

    The replicon hypothesis, first proposed in 1963 by Jacob and Brenner, states that DNA replication is controlled at sites called origins. Replication origins have been well studied in prokaryotes. However, the study of eukaryotic chromosomal origins has lagged behind, because until recently there has been no method for reliably determining the identity and location of origins from eukaryotic chromosomes. Here, we review a technique we developed with the yeast Saccharomyces cerevisiae that allows both the mapping of replication origins and an assessment of their activity. Two-dimensional agarose gel electrophoresis and Southern hybridization with total genomic DNA are used to determine whether a particular restriction fragment acquires the branched structure diagnostic of replication initiation. The technique has been used to localize origins in yeast chromosomes and assess their initiation efficiency. In some cases, origin activation is dependent upon the surrounding context. The technique is also being applied to a variety of eukaryotic organisms.

  4. Heat shock-induced accumulation of translation elongation and termination factors precedes assembly of stress granules in S. cerevisiae.

    Directory of Open Access Journals (Sweden)

    Tomas Grousl

    Full Text Available In response to severe environmental stresses eukaryotic cells shut down translation and accumulate components of the translational machinery in stress granules (SGs. Since they contain mainly mRNA, translation initiation factors and 40S ribosomal subunits, they have been referred to as dominant accumulations of stalled translation preinitiation complexes. Here we present evidence that the robust heat shock-induced SGs of S. cerevisiae also contain translation elongation factors eEF3 (Yef3p and eEF1Bγ2 (Tef4p as well as translation termination factors eRF1 (Sup45p and eRF3 (Sup35p. Despite the presence of the yeast prion protein Sup35 in heat shock-induced SGs, we found out that its prion-like domain is not involved in the SGs assembly. Factors eEF3, eEF1Bγ2 and eRF1 were accumulated and co-localized with Dcp2 foci even upon a milder heat shock at 42°C independently of P-bodies scaffolding proteins. We also show that eEF3 accumulations at 42°C determine sites of the genuine SGs assembly at 46°C. We suggest that identification of translation elongation and termination factors in SGs might help to understand the mechanism of the eIF2α factor phosphorylation-independent repression of translation and SGs assembly.

  5. A Synthetic Biology Framework for Programming Eukaryotic Transcription Functions

    Science.gov (United States)

    Khalil, Ahmad S.; Lu, Timothy K.; Bashor, Caleb J.; Ramirez, Cherie L.; Pyenson, Nora C.; Joung, J. Keith; Collins, James J.

    2013-01-01

    SUMMARY Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks. PMID:22863014

  6. Regulated eukaryotic DNA replication origin firing with purified proteins.

    Science.gov (United States)

    Yeeles, Joseph T P; Deegan, Tom D; Janska, Agnieszka; Early, Anne; Diffley, John F X

    2015-03-26

    Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

  7. A second pathway to degrade pyrimidine nucleic acid precursors in eukaryotes

    DEFF Research Database (Denmark)

    Andersen, Gorm; Bjornberg, Olof; Polakova, Silvia

    2008-01-01

    Pyrimidine bases are the central precursors for RNA and DNA, and their intracellular pools are determined by de novo, salvage and catabolic pathways. In eukaryotes, degradation of uracil has been believed to proceed only via the reduction to dihydrouracil. Using a yeast model, Saccharomyces kluyv...... of the eukaryotic or prokaryotic genes involved in pyrimidine degradation described to date.......Pyrimidine bases are the central precursors for RNA and DNA, and their intracellular pools are determined by de novo, salvage and catabolic pathways. In eukaryotes, degradation of uracil has been believed to proceed only via the reduction to dihydrouracil. Using a yeast model, Saccharomyces......, respectively. The gene products of URC1 and URC4 are highly conserved proteins with so far unknown functions and they are present in a variety of prokaryotes and fungi. In bacteria and in some fungi, URC1 and URC4 are linked on the genome together with the gene for uracil phosphoribosyltransferase (URC6). Urc1...

  8. Functions and structures of eukaryotic recombination proteins

    International Nuclear Information System (INIS)

    Ogawa, Tomoko

    1994-01-01

    We have found that Rad51 and RecA Proteins form strikingly similar structures together with dsDNA and ATP. Their right handed helical nucleoprotein filaments extend the B-form DNA double helixes to 1.5 times in length and wind the helix. The similarity and uniqueness of their structures must reflect functional homologies between these proteins. Therefore, it is highly probable that similar recombination proteins are present in various organisms of different evolutional states. We have succeeded to clone RAD51 genes from human, mouse, chicken and fission yeast genes, and found that the homologues are widely distributed in eukaryotes. The HsRad51 and MmRad51 or ChRad51 proteins consist of 339 amino acids differing only by 4 or 12 amino acids, respectively, and highly homologous to both yeast proteins, but less so to Dmcl. All of these proteins are homologous to the region from residues 33 to 240 of RecA which was named ''homologous core. The homologous core is likely to be responsible for functions common for all of them, such as the formation of helical nucleoprotein filament that is considered to be involved in homologous pairing in the recombination reaction. The mouse gene is transcribed at a high level in thymus, spleen, testis, and ovary, at lower level in brain and at a further lower level in some other tissues. It is transcribed efficiently in recombination active tissues. A clear functional difference of Rad51 homologues from RecA was suggested by the failure of heterologous genes to complement the deficiency of Scrad51 mutants. This failure seems to reflect the absence of a compatible partner, such as ScRad52 protein in the case of ScRad51 protein, between different species. Thus, these discoveries play a role of the starting point to understand the fundamental gene targeting in mammalian cells and in gene therapy. (J.P.N.)

  9. A general strategy to construct small molecule biosensors in eukaryotes.

    Science.gov (United States)

    Feng, Justin; Jester, Benjamin W; Tinberg, Christine E; Mandell, Daniel J; Antunes, Mauricio S; Chari, Raj; Morey, Kevin J; Rios, Xavier; Medford, June I; Church, George M; Fields, Stanley; Baker, David

    2015-12-29

    Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.

  10. PSI1 is responsible for the stearic acid enrichment that is characteristic of phosphatidylinositol in yeast

    NARCIS (Netherlands)

    Le Guédard, Marina; Bessoule, Jean-Jacques; Boyer, Valérie; Ayciriex, Sophie; Velours, Gisèle; Kulik, Willem; Ejsing, Christer S.; Shevchenko, Andrej; Coulon, Denis; Lessire, René; Testet, Eric

    2009-01-01

    In yeast, both phosphatidylinositol and phosphatidylserine are synthesized from cytidine diphosphate-diacylglycerol. Because, as in other eukaryotes, phosphatidylinositol contains more saturated fatty acids than phosphatidylserine (and other phospholipids), it has been hypothesized that either

  11. Eukaryotic translation initiation factor 5A of wheat: Identification ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-05-18

    . The available literature indi- cated that the expression of eIF5A was temporal and spatial difference and suppressing eIF5A activation causes pleiotropic effects. Transcript analysis reveals that two tobacco eIF5A genes ...

  12. Eukaryotic translation initiation factor 5A of wheat: Identification ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-05-18

    May 18, 2009 ... Alignment of the amino acid sequence of eIF5A from wheat, tomato, potato, rice, Arabidopsis, maize, human, mouse, rabbit and C. elegan. The protein sequence shown in the diagrams are listed in the GenBank database under the following accession number: AteIF5A-1 (AAD39281), CeeIF5A-1 (P34563), ...

  13. Functional heterologous protein expression by genetically engineered probiotic yeast Saccharomyces boulardii.

    Directory of Open Access Journals (Sweden)

    Lauren E Hudson

    Full Text Available Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.

  14. Functional heterologous protein expression by genetically engineered probiotic yeast Saccharomyces boulardii.

    Science.gov (United States)

    Hudson, Lauren E; Fasken, Milo B; McDermott, Courtney D; McBride, Shonna M; Kuiper, Emily G; Guiliano, David B; Corbett, Anita H; Lamb, Tracey J

    2014-01-01

    Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.

  15. Gene expression dynamics in the oxidative stress response of fission yeast

    DEFF Research Database (Denmark)

    Papadakis, Emmanouil

    Changes in the environment continuously challenge living organisms during their lifetime. A cell’s survival depends on its ability to coordinate a rapid and successful stress response when exposed to acute doses of damaging agents. Oxidative stress caused by an excess of reactive oxygen species......, especially using model organisms. The fission yeast Schizosaccharomyces pombe is a unicellular eukaryotic organism that possesses genome features and molecular pathways that are highly conserved in humans. Moreover, the limited redundancy of its genome make S. pombe well suited for phenotypic studies...... (HP, 0.5 mM). The applied experimental design allowed us to measure both the activation and recovery phases of the response at a sufficiently high time resolution to model transcription and translation dynamics. Absolute expression levels (copies per cell) and time-resolved expression profiles for 4...

  16. The fission yeast MTREC and EJC orthologs ensure the maturation of meiotic transcripts during meiosis.

    Science.gov (United States)

    Marayati, Bahjat Fadi; Hoskins, Victoria; Boger, Robert W; Tucker, James F; Fishman, Emily S; Bray, Andrew S; Zhang, Ke

    2016-09-01

    Meiosis is a highly regulated process by which genetic information is transmitted through sexual reproduction. It encompasses unique mechanisms that do not occur in vegetative cells, producing a distinct, well-regulated meiotic transcriptome. During vegetative growth, many meiotic genes are constitutively transcribed, but most of the resulting mRNAs are rapidly eliminated by the Mmi1-MTREC (Mtl1-Red1 core) complex. While Mmi1-MTREC targets premature meiotic RNAs for degradation by the nuclear 3'-5' exoribonuclease exosome during mitotic growth, its role in meiotic gene expression during meiosis is not known. Here, we report that Red5, an essential MTREC component, interacts with pFal1, an ortholog of eukaryotic translation initiation factor eIF4aIII in the fission yeast Schizosaccharomyces pombe In mammals, together with MAGO (Mnh1), Rnps1, and Y14, elF4AIII (pFal1) forms the core of the exon junction complex (EJC), which is essential for transcriptional surveillance and localization of mature mRNAs. In fission yeast, two EJC orthologs, pFal1 and Mnh1, are functionally connected with MTREC, specifically in the process of meiotic gene expression during meiosis. Although pFal1 interacts with Mnh1, Y14, and Rnps1, its association with Mnh1 is not disrupted upon loss of Y14 or Rnps1. Mutations of Red1, Red5, pFal1, or Mnh1 produce severe meiotic defects; the abundance of meiotic transcripts during meiosis decreases; and mRNA maturation processes such as splicing are impaired. Since studying meiosis in mammalian germline cells is difficult, our findings in fission yeast may help to define the general mechanisms involved in accurate meiotic gene expression in higher eukaryotes. © 2016 Marayati et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  17. Translation Theory 'Translated'

    DEFF Research Database (Denmark)

    Wæraas, Arild; Nielsen, Jeppe

    2016-01-01

    Translation theory has proved to be a versatile analytical lens used by scholars working from different traditions. On the basis of a systematic literature review, this study adds to our understanding of the ‘translations’ of translation theory by identifying the distinguishing features of the most...... common theoretical approaches to translation within the organization and management discipline: actor-network theory, knowledge-based theory, and Scandinavian institutionalism. Although each of these approaches already has borne much fruit in research, the literature is diverse and somewhat fragmented......, but also overlapping. We discuss the ways in which the three versions of translation theory may be combined and enrich each other so as to inform future research, thereby offering a more complete understanding of translation in and across organizational settings....

  18. Defect in the GTPase activating protein (GAP) function of eIF5 causes repression of GCN4 translation.

    Science.gov (United States)

    Antony A, Charles; Alone, Pankaj V

    2017-05-13

    In eukaryotes, the eIF5 protein plays an important role in translation start site selection by providing the GAP (GTPase activating protein) function. However, in yeast translation initiation fidelity defective eIF5 G31R mutant causes preferential utilization of UUG as initiation codon and is termed as Suppressor of initiation codon (Sui - ) phenotype due to its hyper GTPase activity. The eIF5 G31R mutant dominantly represses GCN4 expression and confers sensitivity to 3-Amino-1,2,4-Trizole (3AT) induced starvation. The down-regulation of the GCN4 expression (Gcn - phenotype) in the eIF5 G31R mutant was not because of leaky scanning defects; rather was due to the utilization of upUUG initiation codons at the 5' regulatory region present between uORF1 and the main GCN4 ORF. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Gonococcal attachment to eukaryotic cells

    International Nuclear Information System (INIS)

    James, J.F.; Lammel, C.J.; Draper, D.L.; Brown, D.A.; Sweet, R.L.; Brooks, G.F.

    1983-01-01

    The attachment of Neisseria gonorrhoeae to eukaryotic cells grown in tissue culture was analyzed by use of light and electron microscopy and by labeling of the bacteria with [ 3 H]- and [ 14 C]adenine. Isogenic piliated and nonpiliated N. gonorrhoeae from opaque and transparent colonies were studied. The results of light microscopy studies showed that the gonococci attached to cells of human origin, including Flow 2000, HeLa 229, and HEp 2. Studies using radiolabeled gonococci gave comparable results. Piliated N. gonorrhoeae usually attached in larger numbers than nonpiliated organisms, and those from opaque colonies attached more often than isogenic variants from transparent colonies. Day-to-day variation in rate of attachment was observed. Scanning electron microscopy studies showed the gonococcal attachment to be specific for microvilli of the host cells. It is concluded that more N. gonorrhoeae from opaque colonies, as compared with isogenic variants from transparent colonies, attach to eukaryotic cells grown in tissue culture

  20. In vivo unnatural amino acid expression in the methylotrophic yeast Pichia pastoris

    Energy Technology Data Exchange (ETDEWEB)

    Young, Travis; Schultz, Peter G.

    2017-08-15

    The invention provides orthogonal translation systems for the production of polypeptides comprising unnatural amino acids in methylotrophic yeast such as Pichia pastoris. Methods for producing polypeptides comprising unnatural amino acids in methylotrophic yeast such as Pichia pastoris are also provided.

  1. In vivo unnatural amino acid expression in the methylotrophic yeast Pichia pastoris

    Science.gov (United States)

    Young, Travis [San Diego, CA; Schultz, Peter G [La Jolla, CA

    2014-02-11

    The invention provides orthogonal translation systems for the production of polypeptides comprising unnatural amino acids in methyltrophic yeast such as Pichia pastoris. Methods for producing polypeptides comprising unnatural amino acids in methyltrophic yeast such as Pichia pastoris are also provided.

  2. Defensins: antifungal lessons from eukaryotes

    Directory of Open Access Journals (Sweden)

    Patrícia M. Silva

    2014-03-01

    Full Text Available Over the last years, antimicrobial peptides (AMPs have been the focus of intense research towards the finding of a viable alternative to current antifungal drugs. Defensins are one of the major families of AMPs and the most represented among all eukaryotic groups, providing an important first line of host defense against pathogenic microorganisms. Several of these cysteine-stabilized peptides present a relevant effect against fungi. Defensins are the AMPs with the broader distribution across all eukaryotic kingdoms, namely, Fungi, Plantæ and Animalia, and were recently shown to have an ancestor in a bacterial organism. As a part of the host defense, defensins act as an important vehicle of information between innate and adaptive immune system and have a role in immunomodulation. This multidimensionality represents a powerful host shield, hard for microorganisms to overcome using single approach resistance strategies. Pathogenic fungi resistance to conventional antimycotic drugs is becoming a major problem. Defensins, as other AMPs, have shown to be an effective alternative to the current antimycotic therapies, demonstrating potential as novel therapeutic agents or drug leads. In this review, we summarize the current knowledge on some eukaryotic defensins with antifungal action. An overview of the main targets in the fungal cell and the mechanism of action of these AMPs (namely, the selectivity for some fungal membrane components are presented. Additionally, recent works on antifungal defensins structure, activity and citotoxicity are also reviewed.

  3. Tandem affinity purification of histones, coupled to mass spectrometry, identifies associated proteins and new sites of post-translational modification in Saccharomyces cerevisiae.

    Science.gov (United States)

    Valero, M Luz; Sendra, Ramon; Pamblanco, Mercè

    2016-03-16

    Histones and their post-translational modifications contribute to regulating fundamental biological processes in all eukaryotic cells. We have applied a conventional tandem affinity purification strategy to histones H3 and H4 of the yeast Saccharomyces cerevisiae. Mass spectrometry analysis of the co-purified proteins revealed multiple associated proteins, including core histones, which indicates that tagged histones may be incorporated to the nucleosome particle. Among the many other co-isolated proteins there are histone chaperones, elements of chromatin remodeling, of nucleosome assembly/disassembly, and of histone modification complexes. The histone chaperone Rtt106p, two members of chromatin assembly FACT complex and Psh1p, an ubiquitin ligase, were the most abundant proteins obtained with both H3-TAP and H4-TAP, regardless of the cell extraction medium stringency. Our mass spectrometry analyses have also revealed numerous novel post-translational modifications, including 30 new chemical modifications in histones, mainly by ubiquitination. We have discovered not only new sites of ubiquitination but that, besides lysine, also serine and threonine residues are targets of ubiquitination on yeast histones. Our results show the standard tandem affinity purification procedure is suitable for application to yeast histones, in order to isolate and characterize histone-binding proteins and post-translational modifications, avoiding the bias caused by histone purification from a chromatin-enriched fraction. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Plasticity and diversity of tRNA anticodon determinants of substrate recognition by eukaryotic A37 isopentenyltransferases

    OpenAIRE

    Lamichhane, Tek N.; Blewett, Nathan H.; Maraia, Richard J.

    2011-01-01

    Transfer RNAs are subject to a wide variety of modifications. Once such modification is N6-(isopentenyl)adenosine. This paper examines the substrate specificity of modifying enzymes from budding and fission yeast. Complex patterns of substrate determinants are uncovered. These determinants differ between the budding and fission yeast in enzymes. This study demonstrates previously unappreciated molecular plasticity and biological diversity of the tRNA-isopentenyltransferase system in eukaryotes.

  5. Death of a dogma: eukaryotic mRNAs can code for more than one protein.

    Science.gov (United States)

    Mouilleron, Hélène; Delcourt, Vivian; Roucou, Xavier

    2016-01-08

    mRNAs carry the genetic information that is translated by ribosomes. The traditional view of a mature eukaryotic mRNA is a molecule with three main regions, the 5' UTR, the protein coding open reading frame (ORF) or coding sequence (CDS), and the 3' UTR. This concept assumes that ribosomes translate one ORF only, generally the longest one, and produce one protein. As a result, in the early days of genomics and bioinformatics, one CDS was associated with each protein-coding gene. This fundamental concept of a single CDS is being challenged by increasing experimental evidence indicating that annotated proteins are not the only proteins translated from mRNAs. In particular, mass spectrometry (MS)-based proteomics and ribosome profiling have detected productive translation of alternative open reading frames. In several cases, the alternative and annotated proteins interact. Thus, the expression of two or more proteins translated from the same mRNA may offer a mechanism to ensure the co-expression of proteins which have functional interactions. Translational mechanisms already described in eukaryotic cells indicate that the cellular machinery is able to translate different CDSs from a single viral or cellular mRNA. In addition to summarizing data showing that the protein coding potential of eukaryotic mRNAs has been underestimated, this review aims to challenge the single translated CDS dogma. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Yeast Genomics for Bread, Beer, Biology, Bucks and Breath

    Science.gov (United States)

    Sakharkar, Kishore R.; Sakharkar, Meena K.

    The rapid advances and scale up of projects in DNA sequencing dur ing the past two decades have produced complete genome sequences of several eukaryotic species. The versatile genetic malleability of the yeast, and the high degree of conservation between its cellular processes and those of human cells have made it a model of choice for pioneering research in molecular and cell biology. The complete sequence of yeast genome has proven to be extremely useful as a reference towards the sequences of human and for providing systems to explore key gene functions. Yeast has been a ‘legendary model’ for new technologies and gaining new biological insights into basic biological sciences and biotechnology. This chapter describes the awesome power of yeast genetics, genomics and proteomics in understanding of biological function. The applications of yeast as a screening tool to the field of drug discovery and development are highlighted and the traditional importance of yeast for bakers and brewers is discussed.

  7. Genomics and the making of yeast biodiversity.

    Science.gov (United States)

    Hittinger, Chris Todd; Rokas, Antonis; Bai, Feng-Yan; Boekhout, Teun; Gonçalves, Paula; Jeffries, Thomas W; Kominek, Jacek; Lachance, Marc-André; Libkind, Diego; Rosa, Carlos A; Sampaio, José Paulo; Kurtzman, Cletus P

    2015-12-01

    Yeasts are unicellular fungi that do not form fruiting bodies. Although the yeast lifestyle has evolved multiple times, most known species belong to the subphylum Saccharomycotina (syn. Hemiascomycota, hereafter yeasts). This diverse group includes the premier eukaryotic model system, Saccharomyces cerevisiae; the common human commensal and opportunistic pathogen, Candida albicans; and over 1000 other known species (with more continuing to be discovered). Yeasts are found in every biome and continent and are more genetically diverse than angiosperms or chordates. Ease of culture, simple life cycles, and small genomes (∼10-20Mbp) have made yeasts exceptional models for molecular genetics, biotechnology, and evolutionary genomics. Here we discuss recent developments in understanding the genomic underpinnings of the making of yeast biodiversity, comparing and contrasting natural and human-associated evolutionary processes. Only a tiny fraction of yeast biodiversity and metabolic capabilities has been tapped by industry and science. Expanding the taxonomic breadth of deep genomic investigations will further illuminate how genome function evolves to encode their diverse metabolisms and ecologies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Chronological aging-induced apoptosis in yeast

    OpenAIRE

    Fabrizio, Paola; Longo, Valter D.

    2008-01-01

    Saccharomyces cerevisiae is the simplest among the major eukaryotic model organisms for aging and diseases. Longevity in the chronological life span paradigm is measured as the mean and maximum survival period of populations of non-dividing yeast. This paradigm has been used successfully to identify several life-regulatory genes and three evolutionary conserved pro-aging pathways. More recently, Schizosaccharomyces pombe has been shown to age chronologically in a manner that resembles that of...

  9. An isoform of eukaryotic initiation factor 4E from Chrysanthemum morifolium interacts with Chrysanthemum virus B coat protein.

    Directory of Open Access Journals (Sweden)

    Aiping Song

    Full Text Available BACKGROUND: Eukaryotic translation initiation factor 4E (eIF4E plays an important role in plant virus infection as well as the regulation of gene translation. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe the isolation of a cDNA encoding CmeIF(iso4E (GenBank accession no. JQ904592, an isoform of eIF4E from chrysanthemum, using RACE PCR. We used the CmeIF(iso4E cDNA for expression profiling and to analyze the interaction between CmeIF(iso4E and the Chrysanthemum virus B coat protein (CVBCP. Multiple sequence alignment and phylogenetic tree analysis showed that the sequence similarity of CmeIF(iso4E with other reported plant eIF(iso4E sequences varied between 69.12% and 89.18%, indicating that CmeIF(iso4E belongs to the eIF(iso4E subfamily of the eIF4E family. CmeIF(iso4E was present in all chrysanthemum organs, but was particularly abundant in the roots and flowers. Confocal microscopy showed that a transiently transfected CmeIF(iso4E-GFP fusion protein distributed throughout the whole cell in onion epidermis cells. A yeast two hybrid assay showed CVBCP interacted with CmeIF(iso4E but not with CmeIF4E. BiFC assay further demonstrated the interaction between CmeIF(iso4E and CVBCP. Luminescence assay showed that CVBCP increased the RLU of Luc-CVB, suggesting CVBCP might participate in the translation of viral proteins. CONCLUSIONS/SIGNIFICANCE: These results inferred that CmeIF(iso4E as the cap-binding subunit eIF(iso4F may be involved in Chrysanthemum Virus B infection in chrysanthemum through its interaction with CVBCP in spatial.

  10. [Mitochondria inheritance in yeast saccharomyces cerevisiae].

    Science.gov (United States)

    Fizikova, A Iu

    2011-01-01

    The review is devoted to the main mechanisms of mitochondria inheritance in yeast Saccharonmyces cerevisiae. The genetic mechanisms of functionally active mitochondria inheritance in eukaryotic cells is one of the most relevant in modem researches. A great number of genetic diseases are associated with mitochondria dysfunction. Plasticity of eukaryotic cell metabolism according to the environmental changes is ensured by adequate mitochondria functioning by means of ATP synthesis coordination, reactive oxygen species accumulation, apoptosis regulation and is an important factor of cell adaptation to stress. Mitochondria participation in important for cell vitality processes masters the presence of accurate mechanisms of mitochondria functions regulation according to environment fluctuations. The mechanisms of mitochondria division and distribution are highly conserved. Baker yeast S. cerevisiae is an ideal model object for mitochondria researches due to energetic metabolism lability, ability to switch over respiration to fermentation, and petite-positive phenotype. Correction of metabolism according to the environmental changes is necessary for cell vitality. The influence of respiratory, carbon, amino acid and phosphate metabolism on mitochondria functions was shown. As far as the mechanisms that stabilize functions of mitochondria and mtDNA are highly conserve, we can project yeast regularities on higher eukaryotes systems. This makes it possible to approximate understanding the etiology and pathogenesis of a great number of human diseases.

  11. Graph theoretic analysis of protein interaction networks of eukaryotes

    Science.gov (United States)

    Goh, K.-I.; Kahng, B.; Kim, D.

    2005-11-01

    Owing to the recent progress in high-throughput experimental techniques, the datasets of large-scale protein interactions of prototypical multicellular species, the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster, have been assayed. The datasets are obtained mainly by using the yeast hybrid method, which contains false-positive and false-negative simultaneously. Accordingly, while it is desirable to test such datasets through further wet experiments, here we invoke recent developed network theory to test such high-throughput datasets in a simple way. Based on the fact that the key biological processes indispensable to maintaining life are conserved across eukaryotic species, and the comparison of structural properties of the protein interaction networks (PINs) of the two species with those of the yeast PIN, we find that while the worm and yeast PIN datasets exhibit similar structural properties, the current fly dataset, though most comprehensively screened ever, does not reflect generic structural properties correctly as it is. The modularity is suppressed and the connectivity correlation is lacking. Addition of interologs to the current fly dataset increases the modularity and enhances the occurrence of triangular motifs as well. The connectivity correlation function of the fly, however, remains distinct under such interolog additions, for which we present a possible scenario through an in silico modeling.

  12. Molecular characterization of factors involved in regulation of archaeal translation

    NARCIS (Netherlands)

    Blombach, F.

    2010-01-01

    The three domains of life – Bacteria, Archaea, and Eukaryotes – can be easily distinguished based on how the genetic information is processed during transcription, translation, and (DNA) replication. Generally, Eukaryotes turned out to employ machineries for these processes that are in their essence

  13. Understanding Translation

    DEFF Research Database (Denmark)

    Schjoldager, Anne Gram; Gottlieb, Henrik; Klitgård, Ida

    Understanding Translation is designed as a textbook for courses on the theory and practice of translation in general and of particular types of translation - such as interpreting, screen translation and literary translation. The aim of the book is to help you gain an in-depth understanding...... of the phenomenon of translation and to provide you with a conceptual framework for the analysis of various aspects of professional translation. Intended readers are students of translation and languages, but the book will also be relevant for others who are interested in the theory and practice of translation...... - translators, language teachers, translation users and literary, TV and film critics, for instance. Discussions focus on translation between Danish and English....

  14. Translation Techniques

    OpenAIRE

    Marcia Pinheiro

    2015-01-01

    In this paper, we discuss three translation techniques: literal, cultural, and artistic. Literal translation is a well-known technique, which means that it is quite easy to find sources on the topic. Cultural and artistic translation may be new terms. Whilst cultural translation focuses on matching contexts, artistic translation focuses on matching reactions. Because literal translation matches only words, it is not hard to find situations in which we should not use this technique.  Because a...

  15. Positive selection for unpreferred codon usage in eukaryotic genomes

    Directory of Open Access Journals (Sweden)

    Galagan James E

    2007-07-01

    Full Text Available Abstract Background Natural selection has traditionally been understood as a force responsible for pushing genes to states of higher translational efficiency, whereas lower translational efficiency has been explained by neutral mutation and genetic drift. We looked for evidence of directional selection resulting in increased unpreferred codon usage (and presumably reduced translational efficiency in three divergent clusters of eukaryotic genomes using a simple optimal-codon-based metric (Kp/Ku. Results Here we show that for some genes natural selection is indeed responsible for causing accelerated unpreferred codon substitution, and document the scope of this selection. In Cryptococcus and to a lesser extent Drosophila, we find many genes showing a statistically significant signal of selection for unpreferred codon usage in one or more lineages. We did not find evidence for this type of selection in Saccharomyces. The signal of positive selection observed from unpreferred synonymous codon substitutions is coincident in Cryptococcus and Drosophila with the distribution of upstream open reading frames (uORFs, another genic feature known to reduce translational efficiency. Functional enrichment analysis of genes exhibiting low Kp/Ku ratios reveals that genes in regulatory roles are particularly subject to this type of selection. Conclusion Through genome-wide scans, we find recent selection for unpreferred codon usage at approximately 1% of genetic loci in a Cryptococcus and several genes in Drosophila. Unpreferred codons can impede translation efficiency, and we find that genes with translation-impeding uORFs are enriched for this selection signal. We find that regulatory genes are particularly likely to be subject to selection for unpreferred codon usage. Given that expression noise can propagate through regulatory cascades, and that low translational efficiency can reduce expression noise, this finding supports the hypothesis that translational

  16. Biomedical applications of yeast- a patent view, part one: yeasts as workhorses for the production of therapeutics and vaccines.

    Science.gov (United States)

    Roohvand, Farzin; Shokri, Mehdi; Abdollahpour-Alitappeh, Meghdad; Ehsani, Parastoo

    2017-08-01

    Yeasts, as Eukaryotes, offer unique features for ease of growth and genetic manipulation possibilities, making it an exceptional microbial host. Areas covered: This review provides general and patent-oriented insights into production of biopharmaceuticals by yeasts. Patents, wherever possible, were correlated to the original or review articles. The review describes applications of major GRAS (generally regarded as safe) yeasts for the production of therapeutic proteins and subunit vaccines; additionally, immunomodulatory properties of yeast cell wall components were reviewed for use of whole yeast cells as a new vaccine platform. The second part of the review will discuss yeast- humanization strategies and innovative applications. Expert opinion: Biomedical applications of yeasts were initiated by utilization of Saccharomyces cerevisiae, for production of leavened (fermented) products, and advanced to serve to produce biopharmaceuticals. Higher biomass production and expression/secretion yields, more similarity of glycosylation patterns to mammals and possibility of host-improvement strategies through application of synthetic biology might enhance selection of Pichia pastoris (instead of S. cerevisiae) as a host for production of biopharmaceutical in future. Immunomodulatory properties of yeast cell wall β-glucans and possibility of intracellular expression of heterologous pathogen/tumor antigens in yeast cells have expanded their application as a new platform, 'Whole Yeast Vaccines'.

  17. Yeast diversity and native vigor for flavor phenotypes.

    Science.gov (United States)

    Carrau, Francisco; Gaggero, Carina; Aguilar, Pablo S

    2015-03-01

    Saccharomyces cerevisiae, the yeast used widely for beer, bread, cider, and wine production, is the most resourceful eukaryotic model used for genetic engineering. A typical concern about using engineered yeasts for food production might be negative consumer perception of genetically modified organisms. However, we believe the true pitfall of using genetically modified yeasts is their limited capacity to either refine or improve the sensory properties of fermented foods under real production conditions. Alternatively, yeast diversity screening to improve the aroma and flavors could offer groundbreaking opportunities in food biotechnology. We propose a 'Yeast Flavor Diversity Screening' strategy which integrates knowledge from sensory analysis and natural whole-genome evolution with information about flavor metabolic networks and their regulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Noise minimization in eukaryotic gene expression.

    Directory of Open Access Journals (Sweden)

    Hunter B Fraser

    2004-06-01

    Full Text Available All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or "noise." Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.

  19. Noise minimization in eukaryotic gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Fraser, Hunter B.; Hirsh, Aaron E.; Giaever, Guri; Kumm, Jochen; Eisen, Michael B.

    2004-01-15

    All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or noise. Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.

  20. Noise minimization in eukaryotic gene expression

    International Nuclear Information System (INIS)

    Fraser, Hunter B.; Hirsh, Aaron E.; Giaever, Guri; Kumm, Jochen; Eisen, Michael B.

    2004-01-01

    All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or noise. Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection

  1. Mechanisms of iron sensing and regulation in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Martínez-Pastor, María Teresa; Perea-García, Ana; Puig, Sergi

    2017-04-01

    Iron is a redox active element that functions as an essential cofactor in multiple metabolic pathways, including respiration, DNA synthesis and translation. While indispensable for eukaryotic life, excess iron can lead to oxidative damage of macromolecules. Therefore, living organisms have developed sophisticated strategies to optimally regulate iron acquisition, storage and utilization in response to fluctuations in environmental iron bioavailability. In the yeast Saccharomyces cerevisiae, transcription factors Aft1/Aft2 and Yap5 regulate iron metabolism in response to low and high iron levels, respectively. In addition to producing and assembling iron cofactors, mitochondrial iron-sulfur (Fe/S) cluster biogenesis has emerged as a central player in iron sensing. A mitochondrial signal derived from Fe/S synthesis is exported and converted into an Fe/S cluster that interacts directly with Aft1/Aft2 and Yap5 proteins to regulate their transcriptional function. Various conserved proteins, such as ABC mitochondrial transporter Atm1 and, for Aft1/Aft2, monothiol glutaredoxins Grx3 and Grx4 are implicated in this iron-signaling pathway. The analysis of a wide range of S. cerevisiae strains of different geographical origins and sources has shown that yeast strains adapted to high iron display growth defects under iron-deficient conditions, and highlighted connections that exist in the response to both opposite conditions. Changes in iron accumulation and gene expression profiles suggest differences in the regulation of iron homeostasis genes.

  2. Introns Protect Eukaryotic Genomes from Transcription-Associated Genetic Instability.

    Science.gov (United States)

    Bonnet, Amandine; Grosso, Ana R; Elkaoutari, Abdessamad; Coleno, Emeline; Presle, Adrien; Sridhara, Sreerama C; Janbon, Guilhem; Géli, Vincent; de Almeida, Sérgio F; Palancade, Benoit

    2017-08-17

    Transcription is a source of genetic instability that can notably result from the formation of genotoxic DNA:RNA hybrids, or R-loops, between the nascent mRNA and its template. Here we report an unexpected function for introns in counteracting R-loop accumulation in eukaryotic genomes. Deletion of endogenous introns increases R-loop formation, while insertion of an intron into an intronless gene suppresses R-loop accumulation and its deleterious impact on transcription and recombination in yeast. Recruitment of the spliceosome onto the mRNA, but not splicing per se, is shown to be critical to attenuate R-loop formation and transcription-associated genetic instability. Genome-wide analyses in a number of distant species differing in their intron content, including human, further revealed that intron-containing genes and the intron-richest genomes are best protected against R-loop accumulation and subsequent genetic instability. Our results thereby provide a possible rationale for the conservation of introns throughout the eukaryotic lineage. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Challenges in Whole-Genome Annotation of Pyrosequenced Eukaryotic Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Kuo, Alan; Grigoriev, Igor

    2009-04-17

    Pyrosequencing technologies such as 454/Roche and Solexa/Illumina vastly lower the cost of nucleotide sequencing compared to the traditional Sanger method, and thus promise to greatly expand the number of sequenced eukaryotic genomes. However, the new technologies also bring new challenges such as shorter reads and new kinds and higher rates of sequencing errors, which complicate genome assembly and gene prediction. At JGI we are deploying 454 technology for the sequencing and assembly of ever-larger eukaryotic genomes. Here we describe our first whole-genome annotation of a purely 454-sequenced fungal genome that is larger than a yeast (>30 Mbp). The pezizomycotine (filamentous ascomycote) Aspergillus carbonarius belongs to the Aspergillus section Nigri species complex, members of which are significant as platforms for bioenergy and bioindustrial technology, as members of soil microbial communities and players in the global carbon cycle, and as agricultural toxigens. Application of a modified version of the standard JGI Annotation Pipeline has so far predicted ~;;10k genes. ~;;12percent of these preliminary annotations suffer a potential frameshift error, which is somewhat higher than the ~;;9percent rate in the Sanger-sequenced and conventionally assembled and annotated genome of fellow Aspergillus section Nigri member A. niger. Also,>90percent of A. niger genes have potential homologs in the A. carbonarius preliminary annotation. Weconclude, and with further annotation and comparative analysis expect to confirm, that 454 sequencing strategies provide a promising substrate for annotation of modestly sized eukaryotic genomes. We will also present results of annotation of a number of other pyrosequenced fungal genomes of bioenergy interest.

  4. Budding yeast for budding geneticists: a primer on the Saccharomyces cerevisiae model system.

    Science.gov (United States)

    Duina, Andrea A; Miller, Mary E; Keeney, Jill B

    2014-05-01

    The budding yeast Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of eukaryotic cell biology. This Primer article presents a brief historical perspective on the emergence of this organism as a premier experimental system over the course of the past century. An overview of the central features of the S. cerevisiae genome, including the nature of its genetic elements and general organization, is also provided. Some of the most common experimental tools and resources available to yeast geneticists are presented in a way designed to engage and challenge undergraduate and graduate students eager to learn more about the experimental amenability of budding yeast. Finally, a discussion of several major discoveries derived from yeast studies highlights the far-reaching impact that the yeast system has had and will continue to have on our understanding of a variety of cellular processes relevant to all eukaryotes, including humans.

  5. Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Ito, Keisuke; Sugawara, Taishi; Shiroishi, Mitsunori; Tokuda, Natsuko; Kurokawa, Azusa; Misaka, Takumi; Makyio, Hisayoshi; Yurugi-Kobayashi, Takami; Shimamura, Tatsuro; Nomura, Norimichi; Murata, Takeshi; Abe, Keiko; Iwata, So

    2008-01-01

    Crystallization of eukaryotic membrane proteins is a challenging, iterative process. The protein of interest is often modified in an attempt to improve crystallization and diffraction results. To accelerate this process, we took advantage of a GFP-fusion yeast expression system that uses PCR to direct homologous recombination and gene cloning. We explored the possibility of employing more than one PCR fragment to introduce various mutations in a single step, and found that when up to five PCR fragments were co-transformed into yeast, the recombination frequency was maintained as the number of fragments was increased. All transformants expressed the model membrane protein, while the resulting plasmid from each clone contained the designed mutations only. Thus, we have demonstrated a technique allowing the expression of mutant membrane proteins within 5 days, combining a GFP-fusion expression system and yeast homologous recombination

  6. Translational Creativity

    DEFF Research Database (Denmark)

    Nielsen, Sandro

    2010-01-01

    A long-established approach to legal translation focuses on terminological equivalence making translators strictly follow the words of source texts. Recent research suggests that there is room for some creativity allowing translators to deviate from the source texts. However, little attention...... is given to genre conventions in source texts and the ways in which they can best be translated. I propose that translators of statutes with an informative function in expert-to-expert communication may be allowed limited translational creativity when translating specific types of genre convention....... This creativity is a result of translators adopting either a source-language or a target-language oriented strategy and is limited by the pragmatic principle of co-operation. Examples of translation options are provided illustrating the different results in target texts. The use of a target-language oriented...

  7. Yeast signaling pathways in the oxidative stress response

    Energy Technology Data Exchange (ETDEWEB)

    Ikner, Aminah [Section of Microbiology, Division of Biological Sciences, University of California, Davis, CA 95616 (United States); Shiozaki, Kazuhiro [Section of Microbiology, Division of Biological Sciences, University of California, Davis, CA 95616 (United States)]. E-mail: kshiozaki@ucdavis.edu

    2005-01-06

    Oxidative stress that generates the reactive oxygen species (ROS) is one of the major causes of DNA damage and mutations. The 'DNA damage checkpoint' that arrests cell cycle and repairs damaged DNA has been a focus of recent studies, and the genetically amenable model systems provided by yeasts have been playing a leading role in the eukaryotic checkpoint research. However, means to eliminate ROS are likely to be as important as the DNA repair mechanisms in order to suppress mutations in the chromosomal DNA, and yeasts also serve as excellent models to understand how eukaryotes combat oxidative stress. In this article, we present an overview of the signaling pathways that sense oxidative stress and induce expression of various anti-oxidant genes in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe and the pathogenic yeast Candida albicans. Three conserved signaling modules have been identified in the oxidative stress response of these diverse yeast species: the stress-responsive MAP kinase cascade, the multistep phosphorelay and the AP-1-like transcription factor. The structure and function of these signaling modules are discussed.

  8. Yeast signaling pathways in the oxidative stress response

    International Nuclear Information System (INIS)

    Ikner, Aminah; Shiozaki, Kazuhiro

    2005-01-01

    Oxidative stress that generates the reactive oxygen species (ROS) is one of the major causes of DNA damage and mutations. The 'DNA damage checkpoint' that arrests cell cycle and repairs damaged DNA has been a focus of recent studies, and the genetically amenable model systems provided by yeasts have been playing a leading role in the eukaryotic checkpoint research. However, means to eliminate ROS are likely to be as important as the DNA repair mechanisms in order to suppress mutations in the chromosomal DNA, and yeasts also serve as excellent models to understand how eukaryotes combat oxidative stress. In this article, we present an overview of the signaling pathways that sense oxidative stress and induce expression of various anti-oxidant genes in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe and the pathogenic yeast Candida albicans. Three conserved signaling modules have been identified in the oxidative stress response of these diverse yeast species: the stress-responsive MAP kinase cascade, the multistep phosphorelay and the AP-1-like transcription factor. The structure and function of these signaling modules are discussed

  9. Machine Translation

    Indian Academy of Sciences (India)

    Research Mt System Example: The 'Janus' Translating Phone Project. The Janus ... based on laptops, and simultaneous translation of two speakers in a dialogue. For more ..... The current focus in MT research is on using machine learning.

  10. How natural a kind is "eukaryote?".

    Science.gov (United States)

    Doolittle, W Ford

    2014-06-02

    Systematics balances uneasily between realism and nominalism, uncommitted as to whether biological taxa are discoveries or inventions. If the former, they might be taken as natural kinds. I briefly review some philosophers' concepts of natural kinds and then argue that several of these apply well enough to "eukaryote." Although there are some sticky issues around genomic chimerism and when eukaryotes first appeared, if we allow for degrees in the naturalness of kinds, existing eukaryotes rank highly, higher than prokaryotes. Most biologists feel this intuitively: All I attempt to do here is provide some conceptual justification. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  11. Yeast metabolic engineering--targeting sterol metabolism and terpenoid formation.

    Science.gov (United States)

    Wriessnegger, Tamara; Pichler, Harald

    2013-07-01

    Terpenoids comprise various structures conferring versatile functions to eukaryotes, for example in the form of prenyl-anchors they attach proteins to membranes. The physiology of eukaryotic membranes is fine-tuned by another terpenoid class, namely sterols. Evidence is accumulating that numerous membrane proteins require specific sterol structural features for function. Moreover, sterols are intermediates in the synthesis of steroids serving as hormones in higher eukaryotes. Like steroids many compounds of the terpenoid family do not contribute to membrane architecture, but serve as signalling, protective or attractant/repellent molecules. Particularly plants have developed a plenitude of terpenoid biosynthetic routes branching off early in the sterol biosynthesis pathway and, thereby, forming one of the largest groups of naturally occurring organic compounds. Many of these aromatic and volatile molecules are interesting for industrial application ranging from foods to pharmaceuticals. Combining the fortunate situation that sterol biosynthesis is highly conserved in eukaryotes with the amenability of yeasts to genetic and metabolic engineering, basically all naturally occurring terpenoids might be produced involving yeasts. Such engineered yeasts are useful for the study of biological functions and molecular interactions of terpenoids as well as for the large-scale production of high-value compounds, which are unavailable in sufficient amounts from natural sources due to their low abundance. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

    Energy Technology Data Exchange (ETDEWEB)

    Tsuji, Toshikazu; Kawai-Noma, Shigeko [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Pack, Chan-Gi [Cellular Informatics Laboratory, RIKEN Advanced Science Institute, Wako-shi, Saitama 351-0198 (Japan); Terajima, Hideki [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Yajima, Junichiro; Nishizaka, Takayuki [Department of Physics, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588 (Japan); Kinjo, Masataka [Laboratory of Molecular Cell Dynamics, Graduate School of Life Sciences, Hokkaido University, Sapporo 001-0021 (Japan); Taguchi, Hideki, E-mail: taguchi@bio.titech.ac.jp [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan)

    2011-02-25

    Research highlights: {yields} We develop a method to track a quantum dot-conjugated protein in yeast cells. {yields} We incorporate the conjugated quantum dot proteins into yeast spheroplasts. {yields} We track the motions by conventional or 3D tracking microscopy. -- Abstract: Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.

  13. Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

    International Nuclear Information System (INIS)

    Tsuji, Toshikazu; Kawai-Noma, Shigeko; Pack, Chan-Gi; Terajima, Hideki; Yajima, Junichiro; Nishizaka, Takayuki; Kinjo, Masataka; Taguchi, Hideki

    2011-01-01

    Research highlights: → We develop a method to track a quantum dot-conjugated protein in yeast cells. → We incorporate the conjugated quantum dot proteins into yeast spheroplasts. → We track the motions by conventional or 3D tracking microscopy. -- Abstract: Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.

  14. Prions in yeast

    OpenAIRE

    Bezdíčka, Martin

    2013-01-01

    The thesis describes yeast prions and their biological effects on yeast in general. It defines the basic characteristics of yeast prions, that distinguish prions from other proteins. The thesis introduces various possibilities of prion formation, and propagation as well as specific types of yeast prions, including various functions of most studied types of prions. The thesis also focuses on chaperones that affect the state of yeast prions in cells. Lastly, the thesis indicates similarities be...

  15. Lariat capping as a tool to manipulate the 5' end of individual yeast mRNA species in vivo

    DEFF Research Database (Denmark)

    Krogh, Nicolai; Pietschmann, Max; Schmid, Manfred

    2017-01-01

    The 5' cap structure of eukaryotic mRNA is critical for its processing, transport, translation, and stability. The many functions of the cap and the fact that most, if not all, mRNA carries the same type of cap makes it difficult to analyze cap function in vivo at individual steps of gene...... in the budding yeast Saccharomyces cerevisiae presumably without cofactors and that lariat capping occurs cotranscriptionally. The lariat-capped reporter mRNA is efficiently exported to the cytoplasm where it is found to be oligoadenylated and evenly distributed. Both the oligoadenylated form and a lariat......-capped mRNA with a templated poly(A) tail translates poorly, underlining the critical importance of the m(7)G cap in translation. Finally, the lariat-capped RNA exhibits a threefold longer half-life compared to its m(7)G-capped counterpart, consistent with a key role for the m(7)G cap in mRNA turnover. Our...

  16. Machine translation

    Energy Technology Data Exchange (ETDEWEB)

    Nagao, M

    1982-04-01

    Each language has its own structure. In translating one language into another one, language attributes and grammatical interpretation must be defined in an unambiguous form. In order to parse a sentence, it is necessary to recognize its structure. A so-called context-free grammar can help in this respect for machine translation and machine-aided translation. Problems to be solved in studying machine translation are taken up in the paper, which discusses subjects for semantics and for syntactic analysis and translation software. 14 references.

  17. Arabinogalactan proteins have deep roots in eukaryotes

    DEFF Research Database (Denmark)

    Hervé, Cécile; Siméon, Amandine; Jam, Murielle

    2016-01-01

    Arabinogalactan proteins (AGPs) are highly glycosylated, hydroxyproline-rich proteins found at the cell surface of plants, where they play key roles in developmental processes. Brown algae are marine, multicellular, photosynthetic eukaryotes. They belong to the phylum Stramenopiles, which...

  18. Mechanisms by Which Different Functional States of Mitochondria Define Yeast Longevity

    Science.gov (United States)

    Beach, Adam; Leonov, Anna; Arlia-Ciommo, Anthony; Svistkova, Veronika; Lutchman, Vicky; Titorenko, Vladimir I.

    2015-01-01

    Mitochondrial functionality is vital to organismal physiology. A body of evidence supports the notion that an age-related progressive decline in mitochondrial function is a hallmark of cellular and organismal aging in evolutionarily distant eukaryotes. Studies of the baker’s yeast Saccharomyces cerevisiae, a unicellular eukaryote, have led to discoveries of genes, signaling pathways and chemical compounds that modulate longevity-defining cellular processes in eukaryotic organisms across phyla. These studies have provided deep insights into mechanistic links that exist between different traits of mitochondrial functionality and cellular aging. The molecular mechanisms underlying the essential role of mitochondria as signaling organelles in yeast aging have begun to emerge. In this review, we discuss recent progress in understanding mechanisms by which different functional states of mitochondria define yeast longevity, outline the most important unanswered questions and suggest directions for future research. PMID:25768339

  19. Mechanisms by Which Different Functional States of Mitochondria Define Yeast Longevity

    Directory of Open Access Journals (Sweden)

    Adam Beach

    2015-03-01

    Full Text Available Mitochondrial functionality is vital to organismal physiology. A body of evidence supports the notion that an age-related progressive decline in mitochondrial function is a hallmark of cellular and organismal aging in evolutionarily distant eukaryotes. Studies of the baker’s yeast Saccharomyces cerevisiae, a unicellular eukaryote, have led to discoveries of genes, signaling pathways and chemical compounds that modulate longevity-defining cellular processes in eukaryotic organisms across phyla. These studies have provided deep insights into mechanistic links that exist between different traits of mitochondrial functionality and cellular aging. The molecular mechanisms underlying the essential role of mitochondria as signaling organelles in yeast aging have begun to emerge. In this review, we discuss recent progress in understanding mechanisms by which different functional states of mitochondria define yeast longevity, outline the most important unanswered questions and suggest directions for future research.

  20. Transfer of DNA from Bacteria to Eukaryotes

    Directory of Open Access Journals (Sweden)

    Benoît Lacroix

    2016-07-01

    Full Text Available Historically, the members of the Agrobacterium genus have been considered the only bacterial species naturally able to transfer and integrate DNA into the genomes of their eukaryotic hosts. Yet, increasing evidence suggests that this ability to genetically transform eukaryotic host cells might be more widespread in the bacterial world. Indeed, analyses of accumulating genomic data reveal cases of horizontal gene transfer from bacteria to eukaryotes and suggest that it represents a significant force in adaptive evolution of eukaryotic species. Specifically, recent reports indicate that bacteria other than Agrobacterium, such as Bartonella henselae (a zoonotic pathogen, Rhizobium etli (a plant-symbiotic bacterium related to Agrobacterium, or even Escherichia coli, have the ability to genetically transform their host cells under laboratory conditions. This DNA transfer relies on type IV secretion systems (T4SSs, the molecular machines that transport macromolecules during conjugative plasmid transfer and also during transport of proteins and/or DNA to the eukaryotic recipient cells. In this review article, we explore the extent of possible transfer of genetic information from bacteria to eukaryotic cells as well as the evolutionary implications and potential applications of this transfer.

  1. PfeIK1, a eukaryotic initiation factor 2α kinase of the human malaria parasite Plasmodium falciparum, regulates stress-response to amino-acid starvation

    Directory of Open Access Journals (Sweden)

    Ranford-Cartwright Lisa

    2009-05-01

    Full Text Available Abstract Background Post-transcriptional control of gene expression is suspected to play an important role in malaria parasites. In yeast and metazoans, part of the stress response is mediated through phosphorylation of eukaryotic translation initiation factor 2α (eIF2α, which results in the selective translation of mRNAs encoding stress-response proteins. Methods The impact of starvation on the phosphorylation state of PfeIF2α was examined. Bioinformatic methods were used to identify plasmodial eIF2α kinases. The activity of one of these, PfeIK1, was investigated using recombinant protein with non-physiological substrates and recombinant PfeIF2α. Reverse genetic techniques were used to disrupt the pfeik1 gene. Results The data demonstrate that the Plasmodium falciparum eIF2α orthologue is phosphorylated in response to starvation, and provide bioinformatic evidence for the presence of three eIF2α kinases in P. falciparum, only one of which (PfPK4 had been described previously. Evidence is provided that one of the novel eIF2α kinases, PfeIK1, is able to phosphorylate the P. falciparum eIF2α orthologue in vitro. PfeIK1 is not required for asexual or sexual development of the parasite, as shown by the ability of pfeik1- parasites to develop into sporozoites. However, eIF2α phosphorylation in response to starvation is abolished in pfeik1- asexual parasites Conclusion This study strongly suggests that a mechanism for versatile regulation of translation by several kinases with a similar catalytic domain but distinct regulatory domains, is conserved in P. falciparum.

  2. Industrial Relevance of Chromosomal Copy Number Variation in Saccharomyces Yeasts

    Science.gov (United States)

    Gorter de Vries, Arthur R.; Pronk, Jack T.

    2017-01-01

    ABSTRACT Chromosomal copy number variation (CCNV) plays a key role in evolution and health of eukaryotes. The unicellular yeast Saccharomyces cerevisiae is an important model for studying the generation, physiological impact, and evolutionary significance of CCNV. Fundamental studies of this yeast have contributed to an extensive set of methods for analyzing and introducing CCNV. Moreover, these studies provided insight into the balance between negative and positive impacts of CCNV in evolutionary contexts. A growing body of evidence indicates that CCNV not only frequently occurs in industrial strains of Saccharomyces yeasts but also is a key contributor to the diversity of industrially relevant traits. This notion is further supported by the frequent involvement of CCNV in industrially relevant traits acquired during evolutionary engineering. This review describes recent developments in genome sequencing and genome editing techniques and discusses how these offer opportunities to unravel contributions of CCNV in industrial Saccharomyces strains as well as to rationally engineer yeast chromosomal copy numbers and karyotypes. PMID:28341679

  3. mRNA processing in yeast

    International Nuclear Information System (INIS)

    Stevens, A.

    1982-01-01

    Investigations in this laboratory center on basic enzymatic reactions of RNA. Still undefined are reactions involved in the conversion of precursors of mRA (pre-mRNA) to mRNA in eukaryotes. The pre-mRNA is called heterogeneous nuclear RNA and is 2 to 6 times larger than mRNA. The conversion, called splicing, involves a removal of internal sequences called introns by endoribonuclease action followed by a rejoining of the 3'- and 5'-end fragments, called exons, by ligating activity. It has not been possible yet to study the enzymes involved in vitro. Also undefined are reactions involved in the turnover or discarding of certain of the pre-mRNA molecules. Yeast is a simple eukaryote and may be expected to have the same, but perhaps simpler, processing reactions as the higher eukaryotes. Two enzymes involved in the processing of pre-mRNA and mRNA in yeast are under investigation. Both enzymes have been partially purified from ribonucleoprotein particles of yeast. The first is a unique decapping enzyme which cleaves [ 3 H]m 7 Gppp [ 14 C]RNA-poly (A) of yeast, yielding [ 3 H]m 7 GDP and is suggested by the finding that the diphosphate product, m 7 GpppA(G), and UDP-glucose are not hydrolyzed. The second enzyme is an endoribonuclease which converts both the [ 3 H] and [ 14 C] labels of [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) from an oligo(dT)-cellulose bound form to an unbound, acid-insoluble form. Results show that the stimulation involves an interaction of the labeled RNA with the small nuclear RNA. The inhibition of the enzyme by ethidium bromide and its stimulation by small nuclear RNA suggest that it may be a processing ribonuclease, requiring specific double-stranded features in its substrate. The characterization of the unique decapping enzyme and endoribonuclease may help to understand reactions involved in the processing of pre-mRNA and mRNA in eukaryotes

  4. Convergent use of RhoGAP toxins by eukaryotic parasites and bacterial pathogens.

    Directory of Open Access Journals (Sweden)

    Dominique Colinet

    2007-12-01

    Full Text Available Inactivation of host Rho GTPases is a widespread strategy employed by bacterial pathogens to manipulate mammalian cellular functions and avoid immune defenses. Some bacterial toxins mimic eukaryotic Rho GTPase-activating proteins (GAPs to inactivate mammalian GTPases, probably as a result of evolutionary convergence. An intriguing question remains whether eukaryotic pathogens or parasites may use endogenous GAPs as immune-suppressive toxins to target the same key genes as bacterial pathogens. Interestingly, a RhoGAP domain-containing protein, LbGAP, was recently characterized from the parasitoid wasp Leptopilina boulardi, and shown to protect parasitoid eggs from the immune response of Drosophila host larvae. We demonstrate here that LbGAP has structural characteristics of eukaryotic RhoGAPs but that it acts similarly to bacterial RhoGAP toxins in mammals. First, we show by immunocytochemistry that LbGAP enters Drosophila immune cells, plasmatocytes and lamellocytes, and that morphological changes in lamellocytes are correlated with the quantity of LbGAP they contain. Demonstration that LbGAP displays a GAP activity and specifically interacts with the active, GTP-bound form of the two Drosophila Rho GTPases Rac1 and Rac2, both required for successful encapsulation of Leptopilina eggs, was then achieved using biochemical tests, yeast two-hybrid analysis, and GST pull-down assays. In addition, we show that the overall structure of LbGAP is similar to that of eukaryotic RhoGAP domains, and we identify distinct residues involved in its interaction with Rac GTPases. Altogether, these results show that eukaryotic parasites can use endogenous RhoGAPs as virulence factors and that despite their differences in sequence and structure, eukaryotic and bacterial RhoGAP toxins are similarly used to target the same immune pathways in insects and mammals.

  5. Translating India

    CERN Document Server

    Kothari, Rita

    2014-01-01

    The cultural universe of urban, English-speaking middle class in India shows signs of growing inclusiveness as far as English is concerned. This phenomenon manifests itself in increasing forms of bilingualism (combination of English and one Indian language) in everyday forms of speech - advertisement jingles, bilingual movies, signboards, and of course conversations. It is also evident in the startling prominence of Indian Writing in English and somewhat less visibly, but steadily rising, activity of English translation from Indian languages. Since the eighties this has led to a frenetic activity around English translation in India's academic and literary circles. Kothari makes this very current phenomenon her chief concern in Translating India.   The study covers aspects such as the production, reception and marketability of English translation. Through an unusually multi-disciplinary approach, this study situates English translation in India amidst local and global debates on translation, representation an...

  6. Translating Inclusion

    DEFF Research Database (Denmark)

    Fallov, Mia Arp; Birk, Rasmus

    2018-01-01

    The purpose of this paper is to explore how practices of translation shape particular paths of inclusion for people living in marginalized residential areas in Denmark. Inclusion, we argue, is not an end-state, but rather something which must be constantly performed. Active citizenship, today......, is not merely a question of participation, but of learning to become active in all spheres of life. The paper draws on empirical examples from a multi-sited field work in 6 different sites of local community work in Denmark, to demonstrate how different dimensions of translation are involved in shaping active...... citizenship. We propose the following different dimensions of translation: translating authority, translating language, translating social problems. The paper takes its theoretical point of departure from assemblage urbanism, arguing that cities are heterogeneous assemblages of socio-material interactions...

  7. Enzymes of yeast polyphosphate metabolism: structure, enzymology and biological roles.

    Science.gov (United States)

    Gerasimaitė, Rūta; Mayer, Andreas

    2016-02-01

    Inorganic polyphosphate (polyP) is found in all living organisms. The known polyP functions in eukaryotes range from osmoregulation and virulence in parasitic protozoa to modulating blood coagulation, inflammation, bone mineralization and cellular signalling in mammals. However mechanisms of regulation and even the identity of involved proteins in many cases remain obscure. Most of the insights obtained so far stem from studies in the yeast Saccharomyces cerevisiae. Here, we provide a short overview of the properties and functions of known yeast polyP metabolism enzymes and discuss future directions for polyP research. © 2016 Authors; published by Portland Press Limited.

  8. Vaginal yeast infection

    Science.gov (United States)

    Yeast infection - vagina; Vaginal candidiasis; Monilial vaginitis ... Most women have a vaginal yeast infection at some time. Candida albicans is a common type of fungus. It is often found in small amounts ...

  9. Atypical mitochondrial inheritance patterns in eukaryotes.

    Science.gov (United States)

    Breton, Sophie; Stewart, Donald T

    2015-10-01

    Mitochondrial DNA (mtDNA) is predominantly maternally inherited in eukaryotes. Diverse molecular mechanisms underlying the phenomenon of strict maternal inheritance (SMI) of mtDNA have been described, but the evolutionary forces responsible for its predominance in eukaryotes remain to be elucidated. Exceptions to SMI have been reported in diverse eukaryotic taxa, leading to the prediction that several distinct molecular mechanisms controlling mtDNA transmission are present among the eukaryotes. We propose that these mechanisms will be better understood by studying the deviations from the predominating pattern of SMI. This minireview summarizes studies on eukaryote species with unusual or rare mitochondrial inheritance patterns, i.e., other than the predominant SMI pattern, such as maternal inheritance of stable heteroplasmy, paternal leakage of mtDNA, biparental and strictly paternal inheritance, and doubly uniparental inheritance of mtDNA. The potential genes and mechanisms involved in controlling mitochondrial inheritance in these organisms are discussed. The linkage between mitochondrial inheritance and sex determination is also discussed, given that the atypical systems of mtDNA inheritance examined in this minireview are frequently found in organisms with uncommon sexual systems such as gynodioecy, monoecy, or andromonoecy. The potential of deviations from SMI for facilitating a better understanding of a number of fundamental questions in biology, such as the evolution of mtDNA inheritance, the coevolution of nuclear and mitochondrial genomes, and, perhaps, the role of mitochondria in sex determination, is considerable.

  10. Comparative genomics and evolution of eukaryotic phospholipidbiosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Lykidis, Athanasios

    2006-12-01

    Phospholipid biosynthetic enzymes produce diverse molecular structures and are often present in multiple forms encoded by different genes. This work utilizes comparative genomics and phylogenetics for exploring the distribution, structure and evolution of phospholipid biosynthetic genes and pathways in 26 eukaryotic genomes. Although the basic structure of the pathways was formed early in eukaryotic evolution, the emerging picture indicates that individual enzyme families followed unique evolutionary courses. For example, choline and ethanolamine kinases and cytidylyltransferases emerged in ancestral eukaryotes, whereas, multiple forms of the corresponding phosphatidyltransferases evolved mainly in a lineage specific manner. Furthermore, several unicellular eukaryotes maintain bacterial-type enzymes and reactions for the synthesis of phosphatidylglycerol and cardiolipin. Also, base-exchange phosphatidylserine synthases are widespread and ancestral enzymes. The multiplicity of phospholipid biosynthetic enzymes has been largely generated by gene expansion in a lineage specific manner. Thus, these observations suggest that phospholipid biosynthesis has been an actively evolving system. Finally, comparative genomic analysis indicates the existence of novel phosphatidyltransferases and provides a candidate for the uncharacterized eukaryotic phosphatidylglycerol phosphate phosphatase.

  11. Compositional translation

    NARCIS (Netherlands)

    Appelo, Lisette; Janssen, Theo; Jong, de F.M.G.; Landsbergen, S.P.J.

    1994-01-01

    This book provides an in-depth review of machine translation by discussing in detail a particular method, called compositional translation, and a particular system, Rosetta, which is based on this method. The Rosetta project is a unique combination of fundamental research and large-scale

  12. Communities of microbial eukaryotes in the mammalian gut within the context of environmental eukaryotic diversity

    Energy Technology Data Exchange (ETDEWEB)

    Parfrey, Laura Wegener; Walters, William A.; Lauber, Christian L.; Clemente, Jose C.; Berg-Lyons, Donna; Teiling, Clotilde; Kodira, Chinnappa; Mohiuddin, Mohammed; Brunelle, Julie; Driscoll, Mark; Fierer, Noah; Gilbert, Jack A.; Knight, Rob

    2014-06-19

    Eukaryotic microbes (protists) residing in the vertebrate gut influence host health and disease, but their diversity and distribution in healthy hosts is poorly understood. Protists found in the gut are typically considered parasites, but many are commensal and some are beneficial. Further, the hygiene hypothesis predicts that association with our co-evolved microbial symbionts may be important to overall health. It is therefore imperative that we understand the normal diversity of our eukaryotic gut microbiota to test for such effects and avoid eliminating commensal organisms. We assembled a dataset of healthy individuals from two populations, one with traditional, agrarian lifestyles and a second with modern, westernized lifestyles, and characterized the human eukaryotic microbiota via high-throughput sequencing. To place the human gut microbiota within a broader context our dataset also includes gut samples from diverse mammals and samples from other aquatic and terrestrial environments. We curated the SILVA ribosomal database to reflect current knowledge of eukaryotic taxonomy and employ it as a phylogenetic framework to compare eukaryotic diversity across environment. We show that adults from the non-western population harbor a diverse community of protists, and diversity in the human gut is comparable to that in other mammals. However, the eukaryotic microbiota of the western population appears depauperate. The distribution of symbionts found in mammals reflects both host phylogeny and diet. Eukaryotic microbiota in the gut are less diverse and more patchily distributed than bacteria. More broadly, we show that eukaryotic communities in the gut are less diverse than in aquatic and terrestrial habitats, and few taxa are shared across habitat types, and diversity patterns of eukaryotes are correlated with those observed for bacteria. These results outline the distribution and diversity of microbial eukaryotic communities in the mammalian gut and across

  13. Translational Repression in Malaria Sporozoites

    Science.gov (United States)

    Turque, Oliver; Tsao, Tiffany; Li, Thomas; Zhang, Min

    2016-01-01

    Malaria is a mosquito-borne infectious disease of humans and other animals. It is caused by the parasitic protozoan, Plasmodium. Sporozoites, the infectious form of malaria parasites, are quiescent when they remain in the salivary glands of the Anopheles mosquito until transmission into a mammalian host. Metamorphosis of the dormant sporozoite to its active form in the liver stage requires transcriptional and translational regulations. Here, we summarize recent advances in the translational repression of gene expression in the malaria sporozoite. In sporozoites, many mRNAs that are required for liver stage development are translationally repressed. Phosphorylation of eukaryotic Initiation Factor 2α (eIF2α) leads to a global translational repression in sporozoites. The eIF2α kinase, known as Upregulated in Infectious Sporozoite 1 (UIS1), is dominant in the sporozoite. The eIF2α phosphatase, UIS2, is translationally repressed by the Pumilio protein Puf2. This translational repression is alleviated when sporozoites are delivered into the mammalian host. PMID:28357358

  14. Translational repression in malaria sporozoites

    Directory of Open Access Journals (Sweden)

    Oliver Turque

    2016-04-01

    Full Text Available Malaria is a mosquito-borne infectious disease of humans and other animals. It is caused by the parasitic protozoan, Plasmodium. Sporozoites, the infectious form of malaria parasites, are quiescent when they remain in the salivary glands of the Anopheles mosquito until transmission into a mammalian host. Metamorphosis of the dormant sporozoite to its active form in the liver stage requires transcriptional and translational regulations. Here, we summarize recent advances in the translational repression of gene expression in the malaria sporozoite. In sporozoites, many mRNAs that are required for liver stage development are translationally repressed. Phosphorylation of eukaryotic Initiation Factor 2α (eIF2α leads to a global translational repression in sporozoites. The eIF2α kinase, known as Upregulated in Infectious Sporozoite 1 (UIS1, is dominant in the sporozoite. The eIF2α phosphatase, UIS2, is translationally repressed by the Pumilio protein Puf2. This translational repression is alleviated when sporozoites are delivered into the mammalian host.

  15. Eukaryotic Cell Cycle as a Test Case for Modeling Cellular Regulation in a Collaborative Problem-Solving Environment

    Science.gov (United States)

    2007-03-01

    computer models of cell cycle regulation in a variety of organisms, including yeast cells, amphibian embryos, bacterial cells and human cells. These...and meiosis ), but they do not nullify the central role played by irreversible, alternating START and FINISH transitions in the cell cycle. 32...AFRL-IF-RS-TR-2007-69 Final Technical Report March 2007 EUKARYOTIC CELL CYCLE AS A TEST CASE FOR MODELING CELLULAR REGULATION IN A

  16. Eukaryotes first: how could that be?

    Science.gov (United States)

    Mariscal, Carlos; Doolittle, W Ford

    2015-09-26

    In the half century since the formulation of the prokaryote : eukaryote dichotomy, many authors have proposed that the former evolved from something resembling the latter, in defiance of common (and possibly common sense) views. In such 'eukaryotes first' (EF) scenarios, the last universal common ancestor is imagined to have possessed significantly many of the complex characteristics of contemporary eukaryotes, as relics of an earlier 'progenotic' period or RNA world. Bacteria and Archaea thus must have lost these complex features secondarily, through 'streamlining'. If the canonical three-domain tree in which Archaea and Eukarya are sisters is accepted, EF entails that Bacteria and Archaea are convergently prokaryotic. We ask what this means and how it might be tested. © 2015 The Author(s).

  17. Implication of Ca2+ in the Regulation of Replicative Life Span of Budding Yeast*

    OpenAIRE

    Tsubakiyama, Ryohei; Mizunuma, Masaki; Gengyo, Anri; Yamamoto, Josuke; Kume, Kazunori; Miyakawa, Tokichi; Hirata, Dai

    2011-01-01

    In eukaryotic cells, Ca2+-triggered signaling pathways are used to regulate a wide variety of cellular processes. Calcineurin, a highly conserved Ca2+/calmodulin-dependent protein phosphatase, plays key roles in the regulation of diverse biological processes in organisms ranging from yeast to humans. We isolated a mutant of the SIR3 gene, implicated in the regulation of life span, as a suppressor of the Ca2+ sensitivity of zds1Δ cells in the budding yeast Saccharomyces cerevisiae. Therefore, ...

  18. Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry

    DEFF Research Database (Denmark)

    Ejsing, Christer S.; Sampaio, Julio L; Surendranath, Vineeth

    2009-01-01

    95% coverage of the yeast lipidome achieved with 125-fold improvement in sensitivity compared with previous approaches. Comparative lipidomics demonstrated that growth temperature and defects in lipid biosynthesis induce ripple effects throughout the molecular composition of the yeast lipidome....... This work serves as a resource for molecular characterization of eukaryotic lipidomes, and establishes shotgun lipidomics as a powerful platform for complementing biochemical studies and other systems-level approaches....

  19. Bacterial toxin-antitoxin gene system as containment control in yeast cells

    DEFF Research Database (Denmark)

    Kristoffersen, P.; Jensen, G. B.; Gerdes, K.

    2000-01-01

    The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae, Expression of the relE gene was highly toxic to yeast cells. However, expression...... fermentation processes in which the escape of genetically modified cells would be considered highly risky....

  20. Inhibition of Ribosome Recruitment Induces Stress Granule Formation Independently of Eukaryotic Initiation Factor 2α Phosphorylation

    OpenAIRE

    Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J.; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed; Pelletier, Jerry

    2006-01-01

    Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2α phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity...

  1. Expression of the lysostaphin gene of Staphylococcus simulans in a eukaryotic system.

    OpenAIRE

    Williamson, C M; Bramley, A J; Lax, A J

    1994-01-01

    The lysostaphin gene of Staphylococcus simulans was cloned into Escherichia coli. The 5' end of the gene was modified to include a eukaryotic start codon, the Kozak expression start site consensus sequence, and an enzyme site to facilitate manipulation of the gene. Transcription of the modified gene in vitro yielded an RNA transcript which, when added to a rabbit reticulocyte cell-free translation system, directed the synthesis of several products. The largest product, migrating at approximat...

  2. Fungal Meiosis and Parasexual Reproduction – Lessons from Pathogenic Yeast

    OpenAIRE

    Sherwood, Racquel K.; Bennett, Richard J.

    2009-01-01

    Meiosis is an integral part of sexual reproduction in eukaryotic species. It performs the dual functions of halving the genetic content in the cell, as well as increasing genetic diversity by promoting recombination between chromosome homologs. Despite extensive studies of meiosis in model yeast, it is now apparent that both the regulation of meiosis and the machinery mediating recombination has significantly diverged, even between closely related species. To highlight this, we discuss new st...

  3. Freedom and Responsibility in Synthetic Genomics: The Synthetic Yeast Project

    OpenAIRE

    Sliva, Anna; Yang, Huanming; Boeke, Jef D.; Mathews, Debra J. H.

    2015-01-01

    First introduced in 2011, the Synthetic Yeast Genome (Sc2.0) Project is a large international synthetic genomics project that will culminate in the first eukaryotic cell (Saccharomyces cerevisiae) with a fully synthetic genome. With collaborators from across the globe and from a range of institutions spanning from do-it-yourself biology (DIYbio) to commercial enterprises, it is important that all scientists working on this project are cognizant of the ethical and policy issues associated with...

  4. Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise; Snoek, Tim; Kildegaard, Kanchana Rueksomtawin

    2016-01-01

    ,cis-muconic acid at different levels, and found that reporter gene output correlated with production. The transplantation of prokaryotic transcriptional activators into the eukaryotic chassis illustrates the potential of a hitherto untapped biosensor resource useful for biotechnological applications....... real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily...

  5. Scheffersomyces cryptocercus: a new xylose-fermenting yeast associated with the gut of wood roaches and new combinations in the Sugiyamaella yeast clade.

    Science.gov (United States)

    Urbina, Hector; Frank, Robert; Blackwell, Meredith

    2013-01-01

    The gut of wood-feeding insects is a microhabitat for a specialized community of microbes, including bacteria and several groups of eukaryotes such as nematodes, parabasalids and fungi. The characterization of gut yeast communities from a variety of insects has shown that certain yeasts often are associated with the insects. The gut of wood-feeding insects is rich in ascomycete yeasts and in particular xylose-fermenting (X-F) and assimilating yeasts have been consistently present in the gut of lignicolous insects. The objective of this study was the characterization of the yeast flora from the gut of the wood roach Cryptocercus sp. (Blattodea: Cryptocercidae). Five wood roaches were collected along the Appalachian Trail near the border between Tennessee and North Carolina, USA. We isolated 18 yeast strains from the wood roaches identified as Sugiyamaella paludigena and Sugiyamaella lignohabitans, xylose-assimilating yeasts, and Scheffersomyces cryptocercus (NRRL Y-48824(T) = CBS 12658) a new species of X-F yeast. The presence of X-F and certain non X-F yeasts in the gut of the subsocial wood roach Cryptocercus sp. extends the previous findings of associations between certain ascomycete yeasts and lignicolous insects. New combinations were made for 13 asexual members of the Sugiyamaella clade.

  6. Reproduction, symbiosis, and the eukaryotic cell

    Science.gov (United States)

    Godfrey-Smith, Peter

    2015-01-01

    This paper develops a conceptual framework for addressing questions about reproduction, individuality, and the units of selection in symbiotic associations, with special attention to the origin of the eukaryotic cell. Three kinds of reproduction are distinguished, and a possible evolutionary sequence giving rise to a mitochondrion-containing eukaryotic cell from an endosymbiotic partnership is analyzed as a series of transitions between each of the three forms of reproduction. The sequence of changes seen in this “egalitarian” evolutionary transition is compared with those that apply in “fraternal” transitions, such as the evolution of multicellularity in animals. PMID:26286983

  7. Modelling the CDK-dependent transcription cycle in fission yeast.

    Science.gov (United States)

    Sansó, Miriam; Fisher, Robert P

    2013-12-01

    CDKs (cyclin-dependent kinases) ensure directionality and fidelity of the eukaryotic cell division cycle. In a similar fashion, the transcription cycle is governed by a conserved subfamily of CDKs that phosphorylate Pol II (RNA polymerase II) and other substrates. A genetic model organism, the fission yeast Schizosaccharomyces pombe, has yielded robust models of cell-cycle control, applicable to higher eukaryotes. From a similar approach combining classical and chemical genetics, fundamental principles of transcriptional regulation by CDKs are now emerging. In the present paper, we review the current knowledge of each transcriptional CDK with respect to its substrate specificity, function in transcription and effects on chromatin modifications, highlighting the important roles of CDKs in ensuring quantity and quality control over gene expression in eukaryotes.

  8. Unicellular eukaryotes as models in cell and molecular biology: critical appraisal of their past and future value.

    Science.gov (United States)

    Simon, Martin; Plattner, Helmut

    2014-01-01

    Unicellular eukaryotes have been appreciated as model systems for the analysis of crucial questions in cell and molecular biology. This includes Dictyostelium (chemotaxis, amoeboid movement, phagocytosis), Tetrahymena (telomere structure, telomerase function), Paramecium (variant surface antigens, exocytosis, phagocytosis cycle) or both ciliates (ciliary beat regulation, surface pattern formation), Chlamydomonas (flagellar biogenesis and beat), and yeast (S. cerevisiae) for innumerable aspects. Nowadays many problems may be tackled with "higher" eukaryotic/metazoan cells for which full genomic information as well as domain databases, etc., were available long before protozoa. Established molecular tools, commercial antibodies, and established pharmacology are additional advantages available for higher eukaryotic cells. Moreover, an increasing number of inherited genetic disturbances in humans have become elucidated and can serve as new models. Among lower eukaryotes, yeast will remain a standard model because of its peculiarities, including its reduced genome and availability in the haploid form. But do protists still have a future as models? This touches not only the basic understanding of biology but also practical aspects of research, such as fund raising. As we try to scrutinize, due to specific advantages some protozoa should and will remain favorable models for analyzing novel genes or specific aspects of cell structure and function. Outstanding examples are epigenetic phenomena-a field of rising interest. © 2014 Elsevier Inc. All rights reserved.

  9. Independent Evolution of Winner Traits without Whole Genome Duplication in Dekkera Yeasts.

    Directory of Open Access Journals (Sweden)

    Yi-Cheng Guo

    Full Text Available Dekkera yeasts have often been considered as alternative sources of ethanol production that could compete with S. cerevisiae. The two lineages of yeasts independently evolved traits that include high glucose and ethanol tolerance, aerobic fermentation, and a rapid ethanol fermentation rate. The Saccharomyces yeasts attained these traits mainly through whole genome duplication approximately 100 million years ago (Mya. However, the Dekkera yeasts, which were separated from S. cerevisiae approximately 200 Mya, did not undergo whole genome duplication (WGD but still occupy a niche similar to S. cerevisiae. Upon analysis of two Dekkera yeasts and five closely related non-WGD yeasts, we found that a massive loss of cis-regulatory elements occurred in an ancestor of the Dekkera yeasts, which led to improved mitochondrial functions similar to the S. cerevisiae yeasts. The evolutionary analysis indicated that genes involved in the transcription and translation process exhibited faster evolution in the Dekkera yeasts. We detected 90 positively selected genes, suggesting that the Dekkera yeasts evolved an efficient translation system to facilitate adaptive evolution. Moreover, we identified that 12 vacuolar H+-ATPase (V-ATPase function genes that were under positive selection, which assists in developing tolerance to high alcohol and high sugar stress. We also revealed that the enzyme PGK1 is responsible for the increased rate of glycolysis in the Dekkera yeasts. These results provide important insights to understand the independent adaptive evolution of the Dekkera yeasts and provide tools for genetic modification promoting industrial usage.

  10. Binary translation using peephole translation rules

    Science.gov (United States)

    Bansal, Sorav; Aiken, Alex

    2010-05-04

    An efficient binary translator uses peephole translation rules to directly translate executable code from one instruction set to another. In a preferred embodiment, the translation rules are generated using superoptimization techniques that enable the translator to automatically learn translation rules for translating code from the source to target instruction set architecture.

  11. The origin of the eukaryotic cell

    Science.gov (United States)

    Hartman, H.

    1984-01-01

    The endosymbiotic hypothesis for the origin of the eukaryotic cell has been applied to the origin of the mitochondria and chloroplasts. However as has been pointed out by Mereschowsky in 1905, it should also be applied to the nucleus as well. If the nucleus, mitochondria and chloroplasts are endosymbionts, then it is likely that the organism that did the engulfing was not a DNA-based organism. In fact, it is useful to postulate that this organism was a primitive RNA-based organism. This hypothesis would explain the preponderance of RNA viruses found in eukaryotic cells. The centriole and basal body do not have a double membrane or DNA. Like all MTOCs (microtubule organising centres), they have a structural or morphic RNA implicated in their formation. This would argue for their origin in the early RNA-based organism rather than in an endosymbiotic event involving bacteria. Finally, the eukaryotic cell uses RNA in ways quite unlike bacteria, thus pointing to a greater emphasis of RNA in both control and structure in the cell. The origin of the eukaryotic cell may tell us why it rather than its prokaryotic relative evolved into the metazoans who are reading this paper.

  12. NMR comparison of prokaryotic and eukaryotic cytochromes c

    International Nuclear Information System (INIS)

    Chau, Meihing; Cai, Meng Li; Timkovich, R.

    1990-01-01

    1 H NMR spectroscopy has been used to examine ferrocytochrome c-551 from Pseudomonas aeruginosa (ATCC 19429) over the pH range 3.5-10.6 and the temperature range 4-60 degree C. Resonance assignments are proposed for main-chain and side-chain protons. Comparison of results for cytochrome c-551 to recently assigned spectra for horse cytochrome c and mutants of yeast iso-1 cytochrome reveals some unique resonances with unusual chemical shifts in all cytochromes that may serve as markers for the heme region. Results for cytochrome c-551 indicate that in the smaller prokaryotic cytochrome, all benzoid side chains are rapidly flipping on the NMR time scale. In contrast, in eukaryotic cytochromes there are some rings flipping slowly on the NMR time scale. The ferrocytochrome c-551 undergoes a transition linked to pH with a pK around 7. The pH behavior of assigned resonances provides evidence that the site of protonation is the inner or buried 17-propionic acid heme substituent (IUPAC-IUB porphyrin nomenclature). Conformational heterogeneity has been observed for segments near the inner heme propionate substituent

  13. Precision translator

    Science.gov (United States)

    Reedy, Robert P.; Crawford, Daniel W.

    1984-01-01

    A precision translator for focusing a beam of light on the end of a glass fiber which includes two turning fork-like members rigidly connected to each other. These members have two prongs each with its separation adjusted by a screw, thereby adjusting the orthogonal positioning of a glass fiber attached to one of the members. This translator is made of simple parts with capability to keep adjustment even in condition of rough handling.

  14. Anaerobic energy metabolism in unicellular photosynthetic eukaryotes.

    Science.gov (United States)

    Atteia, Ariane; van Lis, Robert; Tielens, Aloysius G M; Martin, William F

    2013-02-01

    Anaerobic metabolic pathways allow unicellular organisms to tolerate or colonize anoxic environments. Over the past ten years, genome sequencing projects have brought a new light on the extent of anaerobic metabolism in eukaryotes. A surprising development has been that free-living unicellular algae capable of photoautotrophic lifestyle are, in terms of their enzymatic repertoire, among the best equipped eukaryotes known when it comes to anaerobic energy metabolism. Some of these algae are marine organisms, common in the oceans, others are more typically soil inhabitants. All these species are important from the ecological (O(2)/CO(2) budget), biotechnological, and evolutionary perspectives. In the unicellular algae surveyed here, mixed-acid type fermentations are widespread while anaerobic respiration, which is more typical of eukaryotic heterotrophs, appears to be rare. The presence of a core anaerobic metabolism among the algae provides insights into its evolutionary origin, which traces to the eukaryote common ancestor. The predicted fermentative enzymes often exhibit an amino acid extension at the N-terminus, suggesting that these proteins might be compartmentalized in the cell, likely in the chloroplast or the mitochondrion. The green algae Chlamydomonas reinhardtii and Chlorella NC64 have the most extended set of fermentative enzymes reported so far. Among the eukaryotes with secondary plastids, the diatom Thalassiosira pseudonana has the most pronounced anaerobic capabilities as yet. From the standpoints of genomic, transcriptomic, and biochemical studies, anaerobic energy metabolism in C. reinhardtii remains the best characterized among photosynthetic protists. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Translational Control in Plasmodium and Toxoplasma Parasites

    Science.gov (United States)

    Joyce, Bradley R.; Sullivan, William J.; Nussenzweig, Victor

    2013-01-01

    The life cycles of apicomplexan parasites such as Plasmodium spp. and Toxoplasma gondii are complex, consisting of proliferative and latent stages within multiple hosts. Dramatic transformations take place during the cycles, and they demand precise control of gene expression at all levels, including translation. This review focuses on the mechanisms that regulate translational control in Plasmodium and Toxoplasma, with a particular emphasis on the phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). Phosphorylation of eIF2α (eIF2α∼P) is a conserved mechanism that eukaryotic cells use to repress global protein synthesis while enhancing gene-specific translation of a subset of mRNAs. Elevated levels of eIF2α∼P have been observed during latent stages in both Toxoplasma and Plasmodium, indicating that translational control plays a role in maintaining dormancy. Parasite-specific eIF2α kinases and phosphatases are also required for proper developmental transitions and adaptation to cellular stresses encountered during the life cycle. Identification of small-molecule inhibitors of apicomplexan eIF2α kinases may selectively interfere with parasite translational control and lead to the development of new therapies to treat malaria and toxoplasmosis. PMID:23243065

  16. Translational control in Plasmodium and toxoplasma parasites.

    Science.gov (United States)

    Zhang, Min; Joyce, Bradley R; Sullivan, William J; Nussenzweig, Victor

    2013-02-01

    The life cycles of apicomplexan parasites such as Plasmodium spp. and Toxoplasma gondii are complex, consisting of proliferative and latent stages within multiple hosts. Dramatic transformations take place during the cycles, and they demand precise control of gene expression at all levels, including translation. This review focuses on the mechanisms that regulate translational control in Plasmodium and Toxoplasma, with a particular emphasis on the phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). Phosphorylation of eIF2α (eIF2α∼P) is a conserved mechanism that eukaryotic cells use to repress global protein synthesis while enhancing gene-specific translation of a subset of mRNAs. Elevated levels of eIF2α∼P have been observed during latent stages in both Toxoplasma and Plasmodium, indicating that translational control plays a role in maintaining dormancy. Parasite-specific eIF2α kinases and phosphatases are also required for proper developmental transitions and adaptation to cellular stresses encountered during the life cycle. Identification of small-molecule inhibitors of apicomplexan eIF2α kinases may selectively interfere with parasite translational control and lead to the development of new therapies to treat malaria and toxoplasmosis.

  17. A high-throughput method for quantifying metabolically active yeast cells

    DEFF Research Database (Denmark)

    Nandy, Subir Kumar; Knudsen, Peter Boldsen; Rosenkjær, Alexander

    2015-01-01

    By redesigning the established methylene blue reduction test for bacteria and yeast, we present a cheap and efficient methodology for quantitative physiology of eukaryotic cells applicable for high-throughput systems. Validation of themethod in fermenters and highthroughput systems proved....... The drop in metabolic activity associated with the diauxic shift in yeast proved more pronounced for the MBRT-derived curve compared with OD curves, consistent with a dramatic shift in the ratio between live and dead cells at this metabolic event. This method provides a tool with numerous applications, e.......g. characterizing the death phase of stationary phase cultures, or in drug screens with pathogenic yeasts....

  18. The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

    NARCIS (Netherlands)

    Joosten, V.; Lokman, C.; Hondel, C.A.M.J.J. van den; Punt, P.J.

    2003-01-01

    In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications

  19. Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast

    DEFF Research Database (Denmark)

    Halim, Adnan; Larsen, Ida Signe Bohse; Neubert, Patrick

    2015-01-01

    developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-linked mannose (O-Man) glycoproteome and used this to identify a pleothora of O-Man glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied...... the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and in doing so, we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic, and mitochondrial proteins in S. cerevisiae and S. pombe. Such O-Man glycoproteins were not found in our analysis of human cell lines....... However, the type of yeast O-Man nucleocytoplasmic proteins and the localization of identified O-Man residues mirror that of the O-GlcNAc glycoproteome found in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery...

  20. Yeasts from Different Habitats and Their Potential as Biocontrol Agents

    Directory of Open Access Journals (Sweden)

    Julia Pretscher

    2018-04-01

    Full Text Available Ever since plant diseases began causing losses in viticulture, the control of phytopathogenic fungi has become of vital interest for winemakers. The occurrence of novel pests, fungicide resistance, and changed consumer expectations have led to an enormous demand for novel plant protection strategies. As part of integrated protection measures, antagonistic microorganisms have been investigated to a large extent. Such microorganisms can be applied not only in conventional, but also in organic farming as biological control agents (BCA. Particularly, yeasts were found to be interesting candidates for the development of BCA. Many of these eukaryotic microorganisms are found as part of the phylloplane microflora. In this study, we assessed a set of 38 yeast isolates from different habitats, including the guts of termites, for inhibitory effects against some phytopathogenic fungi that have received less attention in earlier studies. The majority of yeasts were found to interfere with fungi infecting grapevine (Eutypa lata, Botrytis cinerea, and Roesleria subterranea, stone fruits (Monilinia fructicola, or rice (Magnaporte oryzae, as well in vitro and in model experiment on fruits. Although most yeast strains secreted glycoside hydrolases and proteases, attempts to demonstrate direct antagonistic activities of lytic enzymes failed. However, in culture filtrates of the termite yeast Papiliotrema odontotermitis OO5, a low molecular thermostable antagonistic factor was detected. Iron depletion as a BCA mechanism was confirmed for strains of Metschnikowia pulcherrima but not for other yeasts.

  1. Translation termination in pyrrolysine-utilizing archaea.

    Science.gov (United States)

    Alkalaeva, Elena; Eliseev, Boris; Ambrogelly, Alexandre; Vlasov, Peter; Kondrashov, Fyodor A; Gundllapalli, Sharath; Frolova, Lyudmila; Söll, Dieter; Kisselev, Lev

    2009-11-03

    Although some data link archaeal and eukaryotic translation, the overall mechanism of protein synthesis in archaea remains largely obscure. Both archaeal (aRF1) and eukaryotic (eRF1) single release factors recognize all three stop codons. The archaeal genus Methanosarcinaceae contains two aRF1 homologs, and also uses the UAG stop to encode the 22nd amino acid, pyrrolysine. Here we provide an analysis of the last stage of archaeal translation in pyrrolysine-utilizing species. We demonstrated that only one of two Methanosarcina barkeri aRF1 homologs possesses activity and recognizes all three stop codons. The second aRF1 homolog may have another unknown function. The mechanism of pyrrolysine incorporation in the Methanosarcinaceae is discussed.

  2. Protein N-myristoylation in Escherichia coli: Reconstitution of a eukaryotic protein modification in bacteria

    International Nuclear Information System (INIS)

    Duronio, R.J.; Jackson-Machelski, E.; Heuckeroth, R.O.; Gordon, J.I.; Olins, P.O.; Devine, C.S.; Yonemoto, W.; Slice, L.W.; Taylor, S.S.

    1990-01-01

    Protein N-myristoylation refers to the covalent attachment of a myristoyl group (C14:0), via amide linkage, to the NH 2 -terminal glycine residue of certain cellular and viral proteins. Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes this cotranslational modification. The authors have developed a system for studying the substrate requirements and biological effects of protein N-myristoylation as well as NMT structure-activity relationships. Expression of the yeast NMT1 gene in Escherichia coli, a bacterium that has no endogenous NMT activity, results in production of the intact 53-kDa NMT polypeptide as well as a truncated polypeptide derived from proteolytic removal of its NH 2 -terminal 39 amino acids. By using a dual plasmid system, N-myristoylation of a mammalian protein was reconstituted in E. coli by simultaneous expression of the yeast NMT1 gene and a murine cDNA encoding the catalytic (C) subunit of cAMP-dependent protein kinase (PK-A). A major advantage of the bacterial system over eukaryotic systems is the absence of endogenous NMT and substrates, providing a more straightforward way of preparing myristoylated, analog-substituted, and nonmyristoylated forms of a given protein for comparison of their structural and functional properties. The experimental system may prove useful for recapitulating other eukaryotic protein modifications in E. coli so that structure-activity relationships of modifying enzymes and their substrates can be more readily assessed

  3. Machine Translation and Other Translation Technologies.

    Science.gov (United States)

    Melby, Alan

    1996-01-01

    Examines the application of linguistic theory to machine translation and translator tools, discusses the use of machine translation and translator tools in the real world of translation, and addresses the impact of translation technology on conceptions of language and other issues. Findings indicate that the human mind is flexible and linguistic…

  4. Symbiosis and the origin of eukaryotic motility

    Science.gov (United States)

    Margulis, L.; Hinkle, G.

    1991-01-01

    Ongoing work to test the hypothesis of the origin of eukaryotic cell organelles by microbial symbioses is discussed. Because of the widespread acceptance of the serial endosymbiotic theory (SET) of the origin of plastids and mitochondria, the idea of the symbiotic origin of the centrioles and axonemes for spirochete bacteria motility symbiosis was tested. Intracellular microtubular systems are purported to derive from symbiotic associations between ancestral eukaryotic cells and motile bacteria. Four lines of approach to this problem are being pursued: (1) cloning the gene of a tubulin-like protein discovered in Spirocheata bajacaliforniesis; (2) seeking axoneme proteins in spirochets by antibody cross-reaction; (3) attempting to cultivate larger, free-living spirochetes; and (4) studying in detail spirochetes (e.g., Cristispira) symbiotic with marine animals. Other aspects of the investigation are presented.

  5. Towards New Antifolates Targeting Eukaryotic Opportunistic Infections

    Energy Technology Data Exchange (ETDEWEB)

    Liu, J.; Bolstad, D; Bolstad, E; Wright, D; Anderson, A

    2009-01-01

    Trimethoprim, an antifolate commonly prescribed in combination with sulfamethoxazole, potently inhibits several prokaryotic species of dihydrofolate reductase (DHFR). However, several eukaryotic pathogenic organisms are resistant to trimethoprim, preventing its effective use as a therapeutic for those infections. We have been building a program to reengineer trimethoprim to more potently and selectively inhibit eukaryotic species of DHFR as a viable strategy for new drug discovery targeting several opportunistic pathogens. We have developed a series of compounds that exhibit potent and selective inhibition of DHFR from the parasitic protozoa Cryptosporidium and Toxoplasma as well as the fungus Candida glabrata. A comparison of the structures of DHFR from the fungal species Candida glabrata and Pneumocystis suggests that the compounds may also potently inhibit Pneumocystis DHFR.

  6. Inorganic phosphate uptake in unicellular eukaryotes.

    Science.gov (United States)

    Dick, Claudia F; Dos-Santos, André L A; Meyer-Fernandes, José R

    2014-07-01

    Inorganic phosphate (Pi) is an essential nutrient for all organisms. The route of Pi utilization begins with Pi transport across the plasma membrane. Here, we analyzed the gene sequences and compared the biochemical profiles, including kinetic and modulator parameters, of Pi transporters in unicellular eukaryotes. The objective of this review is to evaluate the recent findings regarding Pi uptake mechanisms in microorganisms, such as the fungi Neurospora crassa and Saccharomyces cerevisiae and the parasite protozoans Trypanosoma cruzi, Trypanosoma rangeli, Leishmania infantum and Plasmodium falciparum. Pi uptake is the key step of Pi homeostasis and in the subsequent signaling event in eukaryotic microorganisms. Biochemical and structural studies are important for clarifying mechanisms of Pi homeostasis, as well as Pi sensor and downstream pathways, and raise possibilities for future studies in this field. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Proteomic analysis of post-translational modifications

    DEFF Research Database (Denmark)

    Mann, Matthias; Jensen, Ole N

    2003-01-01

    Post-translational modifications modulate the activity of most eukaryote proteins. Analysis of these modifications presents formidable challenges but their determination generates indispensable insight into biological function. Strategies developed to characterize individual proteins are now...... systematically applied to protein populations. The combination of function- or structure-based purification of modified 'subproteomes', such as phosphorylated proteins or modified membrane proteins, with mass spectrometry is proving particularly successful. To map modification sites in molecular detail, novel...

  8. Evolution of an intricate J-protein network driving protein disaggregation in eukaryotes.

    Science.gov (United States)

    Nillegoda, Nadinath B; Stank, Antonia; Malinverni, Duccio; Alberts, Niels; Szlachcic, Anna; Barducci, Alessandro; De Los Rios, Paolo; Wade, Rebecca C; Bukau, Bernd

    2017-05-15

    Hsp70 participates in a broad spectrum of protein folding processes extending from nascent chain folding to protein disaggregation. This versatility in function is achieved through a diverse family of J-protein cochaperones that select substrates for Hsp70. Substrate selection is further tuned by transient complexation between different classes of J-proteins, which expands the range of protein aggregates targeted by metazoan Hsp70 for disaggregation. We assessed the prevalence and evolutionary conservation of J-protein complexation and cooperation in disaggregation. We find the emergence of a eukaryote-specific signature for interclass complexation of canonical J-proteins. Consistently, complexes exist in yeast and human cells, but not in bacteria, and correlate with cooperative action in disaggregation in vitro. Signature alterations exclude some J-proteins from networking, which ensures correct J-protein pairing, functional network integrity and J-protein specialization. This fundamental change in J-protein biology during the prokaryote-to-eukaryote transition allows for increased fine-tuning and broadening of Hsp70 function in eukaryotes.

  9. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

    Directory of Open Access Journals (Sweden)

    Chew Chieng Yeo

    2016-02-01

    Full Text Available Toxin-antitoxin (TA systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  10. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

    Science.gov (United States)

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-01-01

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies. PMID:26907343

  11. Active role of a human genomic insert in replication of a yeast artificial chromosome.

    Science.gov (United States)

    van Brabant, A J; Fangman, W L; Brewer, B J

    1999-06-01

    Yeast artificial chromosomes (YACs) are a common tool for cloning eukaryotic DNA. The manner by which large pieces of foreign DNA are assimilated by yeast cells into a functional chromosome is poorly understood, as is the reason why some of them are stably maintained and some are not. We examined the replication of a stable YAC containing a 240-kb insert of DNA from the human T-cell receptor beta locus. The human insert contains multiple sites that serve as origins of replication. The activity of these origins appears to require the yeast ARS consensus sequence and, as with yeast origins, additional flanking sequences. In addition, the origins in the human insert exhibit a spacing, a range of activation efficiencies, and a variation in times of activation during S phase similar to those found for normal yeast chromosomes. We propose that an appropriate combination of replication origin density, activation times, and initiation efficiencies is necessary for the successful maintenance of YAC inserts.

  12. Comparative evolutionary analysis of protein complexes in E. coli and yeast

    Directory of Open Access Journals (Sweden)

    Ranea Juan AG

    2010-02-01

    Full Text Available Abstract Background Proteins do not act in isolation; they frequently act together in protein complexes to carry out concerted cellular functions. The evolution of complexes is poorly understood, especially in organisms other than yeast, where little experimental data has been available. Results We generated accurate, high coverage datasets of protein complexes for E. coli and yeast in order to study differences in the evolution of complexes between these two species. We show that substantial differences exist in how complexes have evolved between these organisms. A previously proposed model of complex evolution identified complexes with cores of interacting homologues. We support findings of the relative importance of this mode of evolution in yeast, but find that it is much less common in E. coli. Additionally it is shown that those homologues which do cluster in complexes are involved in eukaryote-specific functions. Furthermore we identify correlated pairs of non-homologous domains which occur in multiple protein complexes. These were identified in both yeast and E. coli and we present evidence that these too may represent complex cores in yeast but not those of E. coli. Conclusions Our results suggest that there are differences in the way protein complexes have evolved in E. coli and yeast. Whereas some yeast complexes have evolved by recruiting paralogues, this is not apparent in E. coli. Furthermore, such complexes are involved in eukaryotic-specific functions. This implies that the increase in gene family sizes seen in eukaryotes in part reflects multiple family members being used within complexes. However, in general, in both E. coli and yeast, homologous domains are used in different complexes.

  13. How MCM loading and spreading specify eukaryotic DNA replication initiation sites [version 1; referees: 4 approved

    Directory of Open Access Journals (Sweden)

    Olivier Hyrien

    2016-08-01

    Full Text Available DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs, the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC, they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes.

  14. How MCM loading and spreading specify eukaryotic DNA replication initiation sites.

    Science.gov (United States)

    Hyrien, Olivier

    2016-01-01

    DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb) non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs), the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC), they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes.

  15. Translation Competence

    DEFF Research Database (Denmark)

    Vandepitte, Sonia; Mousten, Birthe; Maylath, Bruce

    2014-01-01

    After Kiraly (2000) introduced the collaborative form of translation in classrooms, Pavlovic (2007), Kenny (2008), and Huertas Barros (2011) provided empirical evidence that testifies to the impact of collaborative learning. This chapter sets out to describe the collaborative forms of learning at...

  16. Translating Harbourscapes

    DEFF Research Database (Denmark)

    Diedrich, Lisa Babette

    -specific design are proposed for all actors involved in harbour transformation. The study ends with an invitation to further investigate translation as a powerful metaphor for the way existing qualities of a site can be transformed, rather than erased or rewritten, and to explore how this metaphor can foster new...

  17. Word translation entropy in translation

    DEFF Research Database (Denmark)

    Schaeffer, Moritz; Dragsted, Barbara; Hvelplund, Kristian Tangsgaard

    2016-01-01

    This study reports on an investigation into the relationship between the number of translation alternatives for a single word and eye movements on the source text. In addition, the effect of word order differences between source and target text on eye movements on the source text is studied....... In particular, the current study investigates the effect of these variables on early and late eye movement measures. Early eye movement measures are indicative of processes that are more automatic while late measures are more indicative of conscious processing. Most studies that found evidence of target...... language activation during source text reading in translation, i.e. co-activation of the two linguistic systems, employed late eye movement measures or reaction times. The current study therefore aims to investigate if and to what extent earlier eye movement measures in reading for translation show...

  18. The fission yeast spindle orientation checkpoint: a model that generates tension?

    Science.gov (United States)

    Gachet, Yannick; Reyes, Céline; Goldstone, Sherilyn; Tournier, Sylvie

    2006-10-15

    In all eukaryotes, the alignment of the mitotic spindle with the axis of cell polarity is essential for accurate chromosome segregation as well as for the establishment of cell fate, and thus morphogenesis, during development. Studies in invertebrates, higher eukaryotes and yeast suggest that astral microtubules interact with the cell cortex to position the spindle. These microtubules are thought to impose pushing or pulling forces on the spindle poles to affect the rotation or movement of the spindle. In the fission yeast model, where cell division is symmetrical, spindle rotation is dependent on the interaction of astral microtubules with the cortical actin cytoskeleton. In these cells, a bub1-dependent mitotic checkpoint, the spindle orientation checkpoint (SOC), is activated when the spindles fail to align with the cell polarity axis. In this paper we review the mechanism that orientates the spindle during mitosis in fission yeast, and discuss the consequences of misorientation on metaphase progression. Copyright 2006 John Wiley & Sons, Ltd.

  19. Yeast as a Heterologous Model System to Uncover Type III Effector Function.

    Directory of Open Access Journals (Sweden)

    Crina Popa

    2016-02-01

    Full Text Available Type III effectors (T3E are key virulence proteins that are injected by bacterial pathogens inside the cells of their host to subvert cellular processes and contribute to disease. The budding yeast Saccharomyces cerevisiae represents an important heterologous system for the functional characterisation of T3E proteins in a eukaryotic environment. Importantly, yeast contains eukaryotic processes with low redundancy and are devoid of immunity mechanisms that counteract T3Es and mask their function. Expression in yeast of effectors from both plant and animal pathogens that perturb conserved cellular processes often resulted in robust phenotypes that were exploited to elucidate effector functions, biochemical properties, and host targets. The genetic tractability of yeast and its amenability for high-throughput functional studies contributed to the success of this system that, in recent years, has been used to study over 100 effectors. Here, we provide a critical view on this body of work and describe advantages and limitations inherent to the use of yeast in T3E research. "Favourite" targets of T3Es in yeast are cytoskeleton components and small GTPases of the Rho family. We describe how mitogen-activated protein kinase (MAPK signalling, vesicle trafficking, membrane structures, and programmed cell death are also often altered by T3Es in yeast and how this reflects their function in the natural host. We describe how effector structure-function studies and analysis of candidate targeted processes or pathways can be carried out in yeast. We critically analyse technologies that have been used in yeast to assign biochemical functions to T3Es, including transcriptomics and proteomics, as well as suppressor, gain-of-function, or synthetic lethality screens. We also describe how yeast can be used to select for molecules that block T3E function in search of new antibacterial drugs with medical applications. Finally, we provide our opinion on the limitations

  20. Sparse "1"3C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins

    International Nuclear Information System (INIS)

    Liu, Jing; Liu, Chang; Fan, Ying; Munro, Rachel A.; Ladizhansky, Vladimir; Brown, Leonid S.; Wang, Shenlin

    2016-01-01

    We demonstrate a novel sparse "1"3C labelling approach for methylotrophic yeast P. pastoris expression system, towards solid-state NMR studies of eukaryotic membrane proteins. The labelling scheme was achieved by co-utilizing natural abundance methanol and specifically "1"3C labelled glycerol as carbon sources in the expression medium. This strategy improves the spectral resolution by 1.5 fold, displays site-specific labelling patterns, and has advantages for collecting long-range distance restraints for structure determination of large eukaryotic membrane proteins by solid-state NMR.

  1. Recovery of soil unicellular eukaryotes: an efficiency and activity analysis on the single cell level.

    Science.gov (United States)

    Lentendu, Guillaume; Hübschmann, Thomas; Müller, Susann; Dunker, Susanne; Buscot, François; Wilhelm, Christian

    2013-12-01

    Eukaryotic unicellular organisms are an important part of the soil microbial community, but they are often neglected in soil functional microbial diversity analysis, principally due to the absence of specific investigation methods in the special soil environment. In this study we used a method based on high-density centrifugation to specifically isolate intact algal and yeast cells, with the aim to analyze them with flow cytometry and sort them for further molecular analysis such as deep sequencing. Recovery efficiency was tested at low abundance levels that fit those in natural environments (10(4) to 10(6) cells per g soil). Five algae and five yeast morphospecies isolated from soil were used for the testing. Recovery efficiency was between 1.5 to 43.16% and 2 to 30.2%, respectively, and was dependent on soil type for three of the algae. Control treatments without soil showed that the majority of cells were lost due to the method itself (58% and 55.8% respectively). However, the cell extraction technique did not much compromise cell vitality because a fluorescein di-acetate assay indicated high viability percentages (73.3% and 97.2% of cells, respectively). The low abundant algae and yeast morphospecies recovered from soil were cytometrically analyzed and sorted. Following, their DNA was isolated and amplified using specific primers. The developed workflow enables isolation and enrichment of intact autotrophic and heterotrophic soil unicellular eukaryotes from natural environments for subsequent application of deep sequencing technologies. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Yeast-expressed human membrane protein aquaporin-1 yields excellent resolution of solid-state MAS NMR spectra

    International Nuclear Information System (INIS)

    Emami, Sanaz; Fan Ying; Munro, Rachel; Ladizhansky, Vladimir; Brown, Leonid S.

    2013-01-01

    One of the biggest challenges in solid-state NMR studies of membrane proteins is to obtain a homogeneous natively folded sample giving high spectral resolution sufficient for structural studies. Eukaryotic membrane proteins are especially difficult and expensive targets in this respect. Methylotrophic yeast Pichia pastoris is a reliable producer of eukaryotic membrane proteins for crystallography and a promising economical source of isotopically labeled proteins for NMR. We show that eukaryotic membrane protein human aquaporin 1 can be doubly ( 13 C/ 15 N) isotopically labeled in this system and functionally reconstituted into phospholipids, giving excellent resolution of solid-state magic angle spinning NMR spectra.

  3. Patterns of prokaryotic lateral gene transfers affecting parasitic microbial eukaryotes

    DEFF Research Database (Denmark)

    Alsmark, Cecilia; Foster, Peter G; Sicheritz-Pontén, Thomas

    2013-01-01

    BACKGROUND: The influence of lateral gene transfer on gene origins and biology in eukaryotes is poorly understood compared with those of prokaryotes. A number of independent investigations focusing on specific genes, individual genomes, or specific functional categories from various eukaryotes have...... approach to systematically investigate lateral gene transfer affecting the proteomes of thirteen, mainly parasitic, microbial eukaryotes, representing four of the six eukaryotic super-groups. All of the genomes investigated have been significantly affected by prokaryote-to-eukaryote lateral gene transfers...... indicated that lateral gene transfer does indeed affect eukaryotic genomes. However, the lack of common methodology and criteria in these studies makes it difficult to assess the general importance and influence of lateral gene transfer on eukaryotic genome evolution. RESULTS: We used a phylogenomic...

  4. Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells

    International Nuclear Information System (INIS)

    Takahashi, Hideo; Shimada, Ichio

    2010-01-01

    The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins are not functionally expressed in E. coli, due to problems related to disulfide bond formation, post-translational modifications, and folding. In such cases, other expression systems are required for producing proteins for biomolecular NMR analyses. In this paper, we review the recent advances in expression systems for isotopically labeled heterologous proteins, utilizing non-E. coli prokaryotic and eukaryotic cells.

  5. Synonymous codon ordering: a subtle but prevalent strategy of bacteria to improve translational efficiency.

    Directory of Open Access Journals (Sweden)

    Zhu-Qing Shao

    Full Text Available BACKGROUND: In yeast coding sequences, once a particular codon has been used, subsequent occurrence of the same amino acid tends to use codons sharing the same tRNA. Such a phenomenon of co-tRNA codons pairing bias (CTCPB is also found in some other eukaryotes but it is not known whether it occurs in prokaryotes. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we focused on a total of 773 bacterial genomes to investigate their synonymous codon pairing preferences. After calculating the actual frequencies of synonymous codon pairs and comparing them with their expected values, we detected an obvious pairing bias towards identical codon pairs. This seems consistent with the previously reported CTCPB phenomenon, since identical codons are certainly read by the same tRNA. However, among co-tRNA but non-identical codon pairs, only 22 were often found overrepresented, suggesting that many co-tRNA codons actually do not preferentially pair together in prokaryotes. Therefore, the previously reported co-tRNA codons pairing rule needs to be more rigorously defined. The affinity differences between a tRNA anticodon and its readable codons should be taken into account. Moreover, both within-gene-shuffling tests and phylogenetic analyses support the idea that translational selection played an important role in shaping the observed synonymous codon pairing pattern in prokaryotes. CONCLUSIONS: Overall, a high level of synonymous codon pairing bias was detected in 73% investigated bacterial species, suggesting the synonymous codon ordering strategy has been prevalently adopted by prokaryotes to improve their translational efficiencies. The findings in this study also provide important clues to better understand the complex dynamics of translational process.

  6. Recent advances in the genome-wide study of DNA replication origins in yeast

    Science.gov (United States)

    Peng, Chong; Luo, Hao; Zhang, Xi; Gao, Feng

    2015-01-01

    DNA replication, one of the central events in the cell cycle, is the basis of biological inheritance. In order to be duplicated, a DNA double helix must be opened at defined sites, which are called DNA replication origins (ORIs). Unlike in bacteria, where replication initiates from a single replication origin, multiple origins are utilized in the eukaryotic genomes. Among them, the ORIs in budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have been best characterized. In recent years, advances in DNA microarray and next-generation sequencing technologies have increased the number of yeast species involved in ORIs research dramatically. The ORIs in some non-conventional yeast species such as Kluyveromyces lactis and Pichia pastoris have also been genome-widely identified. Relevant databases of replication origins in yeast were constructed, then the comparative genomic analysis can be carried out. Here, we review several experimental approaches that have been used to map replication origins in yeast and some of the available web resources related to yeast ORIs. We also discuss the sequence characteristics and chromosome structures of ORIs in the four yeast species, which can be utilized to improve yeast replication origins prediction. PMID:25745419

  7. Translational genomics

    Directory of Open Access Journals (Sweden)

    Martin Kussmann

    2014-09-01

    Full Text Available The term “Translational Genomics” reflects both title and mission of this new journal. “Translational” has traditionally been understood as “applied research” or “development”, different from or even opposed to “basic research”. Recent scientific and societal developments have triggered a re-assessment of the connotation that “translational” and “basic” are either/or activities: translational research nowadays aims at feeding the best science into applications and solutions for human society. We therefore argue here basic science to be challenged and leveraged for its relevance to human health and societal benefits. This more recent approach and attitude are catalyzed by four trends or developments: evidence-based solutions; large-scale, high dimensional data; consumer/patient empowerment; and systems-level understanding.

  8. Beyond Translation

    DEFF Research Database (Denmark)

    Olwig, Mette Fog

    2013-01-01

    This article contributes to the growing scholarship on local development practitioners by re-examining conceptualizations of practitioners as ‘brokers’ strategically translating between ‘travelling’ (development institution) rationalities and ‘placed’ (recipient area) rationalities in relation...... and practice spurred by new challenges deriving from climate change anxiety, the study shows how local practitioners often make local activities fit into travelling development rationalities as a matter of habit, rather than as a conscious strategy. They may therefore cease to ‘translate’ between different...... rationalities. This is shown to have important implications for theory, research and practice concerning disaster risk reduction and climate change adaptation in which such translation is often expected....

  9. Revising Translations

    DEFF Research Database (Denmark)

    Rasmussen, Kirsten Wølch; Schjoldager, Anne

    2011-01-01

    The paper explains the theoretical background and findings of an empirical study of revision policies, using Denmark as a case in point. After an overview of important definitions, types and parameters, the paper explains the methods and data gathered from a questionnaire survey and an interview...... survey. Results clearly show that most translation companies regard both unilingual and comparative revisions as essential components of professional quality assurance. Data indicate that revision is rarely fully comparative, as the preferred procedure seems to be a unilingual revision followed by a more...... or less comparative rereading. Though questionnaire data seem to indicate that translation companies use linguistic correctness and presentation as the only revision parameters, interview data reveal that textual and communicative aspects are also considered. Generally speaking, revision is not carried...

  10. Prokaryotes versus Eukaryotes: Who is hosting whom?

    Directory of Open Access Journals (Sweden)

    Guillermo eTellez

    2014-10-01

    Full Text Available Microorganisms represent the largest component of biodiversity in our world. For millions of years, prokaryotic microorganisms have functioned as a major selective force shaping eukaryotic evolution. Microbes that live inside and on animals outnumber the animals’ actual somatic and germ cells by an estimated 10-fold. Collectively, the intestinal microbiome represents a ‘forgotten organ’, functioning as an organ inside another that can execute many physiological responsibilities. The nature of primitive eukaryotes was drastically changed due to the association with symbiotic prokaryotes facilitating mutual coevolution of host and microbe. Phytophagous insects have long been used to test theories of evolutionary diversification; moreover, the diversification of a number of phytophagous insect lineages has been linked to mutualisms with microbes. From termites and honey bees to ruminants and mammals, depending on novel biochemistries provided by the prokaryotic microbiome, the association helps to metabolize several nutrients that the host cannot digest and converting these into useful end products (such as short chain fatty acids, a process which has huge impact on the biology and homeostasis of metazoans. More importantly, in a direct and/or indirect way, the intestinal microbiota influences the assembly of gut-associated lymphoid tissue, helps to educate immune system, affects the integrity of the intestinal mucosal barrier, modulates proliferation and differentiation of its epithelial lineages, regulates angiogenesis, and modifies the activity of enteric as well as the central nervous system,. Despite these important effects, the mechanisms by which the gut microbial community influences the host’s biology remains almost entirely unknown. Our aim here is to encourage empirical inquiry into the relationship between mutualism and evolutionary diversification between prokaryotes and eukaryotes which encourage us to postulate: Who is

  11. Yeast expression proteomics by high-resolution mass spectrometry

    DEFF Research Database (Denmark)

    Walther, Tobias C; Olsen, Jesper Velgaard; Mann, Matthias

    2010-01-01

    -translational controls contribute majorly to regulation of protein abundance, for example in heat shock stress response. The development of new sample preparation methods, high-resolution mass spectrometry and novel bioinfomatic tools close this gap and allow the global quantitation of the yeast proteome under different...

  12. Footprinting analysis of interactions between the largest eukaryotic RNase P/MRP protein Pop1 and RNase P/MRP RNA components.

    Science.gov (United States)

    Fagerlund, Robert D; Perederina, Anna; Berezin, Igor; Krasilnikov, Andrey S

    2015-09-01

    Ribonuclease (RNase) P and RNase MRP are closely related catalytic ribonucleoproteins involved in the metabolism of a wide range of RNA molecules, including tRNA, rRNA, and some mRNAs. The catalytic RNA component of eukaryotic RNase P retains the core elements of the bacterial RNase P ribozyme; however, the peripheral RNA elements responsible for the stabilization of the global architecture are largely absent in the eukaryotic enzyme. At the same time, the protein makeup of eukaryotic RNase P is considerably more complex than that of the bacterial RNase P. RNase MRP, an essential and ubiquitous eukaryotic enzyme, has a structural organization resembling that of eukaryotic RNase P, and the two enzymes share most of their protein components. Here, we present the results of the analysis of interactions between the largest protein component of yeast RNases P/MRP, Pop1, and the RNA moieties of the enzymes, discuss structural implications of the results, and suggest that Pop1 plays the role of a scaffold for the stabilization of the global architecture of eukaryotic RNase P RNA, substituting for the network of RNA-RNA tertiary interactions that maintain the global RNA structure in bacterial RNase P. © 2015 Fagerlund et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  13. Mutant power: using mutant allele collections for yeast functional genomics.

    Science.gov (United States)

    Norman, Kaitlyn L; Kumar, Anuj

    2016-03-01

    The budding yeast has long served as a model eukaryote for the functional genomic analysis of highly conserved signaling pathways, cellular processes and mechanisms underlying human disease. The collection of reagents available for genomics in yeast is extensive, encompassing a growing diversity of mutant collections beyond gene deletion sets in the standard wild-type S288C genetic background. We review here three main types of mutant allele collections: transposon mutagen collections, essential gene collections and overexpression libraries. Each collection provides unique and identifiable alleles that can be utilized in genome-wide, high-throughput studies. These genomic reagents are particularly informative in identifying synthetic phenotypes and functions associated with essential genes, including those modeled most effectively in complex genetic backgrounds. Several examples of genomic studies in filamentous/pseudohyphal backgrounds are provided here to illustrate this point. Additionally, the limitations of each approach are examined. Collectively, these mutant allele collections in Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans promise insights toward an advanced understanding of eukaryotic molecular and cellular biology. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  14. A review on sustainable yeast biotechnological processes and applications

    DEFF Research Database (Denmark)

    Nandy, Subir Kumar; Srivastava, R. K.

    2018-01-01

    Yeast is very well known eukaryotic organism for its remarkable biodiversity and extensive industrial applications. Saccharomyces cerevisiae is one of the most widely used microorganisms in biotechnology with successful applications in the biochemical production. Biological conversion with the fo......Yeast is very well known eukaryotic organism for its remarkable biodiversity and extensive industrial applications. Saccharomyces cerevisiae is one of the most widely used microorganisms in biotechnology with successful applications in the biochemical production. Biological conversion...... with the focus on the different utilization of renewable feedstocks into fuels and chemicals has been intensively investigated due to increasing concerns on sustainability issues worldwide. Compared with its counterparts, Saccharomyces cerevisiae, the baker's yeast, is more industrially relevant due to known...... genetic and physiological background, the availability of a large collection of genetic tools, the compatibility of high-density and large-scale fermentation, and optimize the pathway for variety of products. Therefore, S. cerevisiae is one of the most popular cell factories and has been successfully used...

  15. Consistent mutational paths predict eukaryotic thermostability

    Directory of Open Access Journals (Sweden)

    van Noort Vera

    2013-01-01

    Full Text Available Abstract Background Proteomes of thermophilic prokaryotes have been instrumental in structural biology and successfully exploited in biotechnology, however many proteins required for eukaryotic cell function are absent from bacteria or archaea. With Chaetomium thermophilum, Thielavia terrestris and Thielavia heterothallica three genome sequences of thermophilic eukaryotes have been published. Results Studying the genomes and proteomes of these thermophilic fungi, we found common strategies of thermal adaptation across the different kingdoms of Life, including amino acid biases and a reduced genome size. A phylogenetics-guided comparison of thermophilic proteomes with those of other, mesophilic Sordariomycetes revealed consistent amino acid substitutions associated to thermophily that were also present in an independent lineage of thermophilic fungi. The most consistent pattern is the substitution of lysine by arginine, which we could find in almost all lineages but has not been extensively used in protein stability engineering. By exploiting mutational paths towards the thermophiles, we could predict particular amino acid residues in individual proteins that contribute to thermostability and validated some of them experimentally. By determining the three-dimensional structure of an exemplar protein from C. thermophilum (Arx1, we could also characterise the molecular consequences of some of these mutations. Conclusions The comparative analysis of these three genomes not only enhances our understanding of the evolution of thermophily, but also provides new ways to engineer protein stability.

  16. Animal vaccines based on orally presented yeast recombinants.

    Science.gov (United States)

    Shin, Min-Kyoung; Yoo, Han Sang

    2013-09-13

    In veterinary vaccinology, the oral route of administration is an attractive alternative compared to the commonly used parenteral route. Yeasts have a number of properties that make them potential live delivery systems for oral vaccination purposes such as their high expression levels, their GRAS status, adjuvant properties, and post-translational modification possibilities. Consequently, yeasts have been employed for the expression of heterologous genes and for the production of therapeutic proteins. Yeast-based vaccines are reviewed with regard to their ability to express and produce antigens from pathogens for veterinary use. Many of these vaccines have been shown to elicit protective immune responses following oral immunization in animals. Ultimately, yeast-based oral vaccines may offer a potential opportunity for the development of novel ideal vaccines in veterinary medicine. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. An SVD-based comparison of nine whole eukaryotic genomes supports a coelomate rather than ecdysozoan lineage

    Directory of Open Access Journals (Sweden)

    Stuart Gary W

    2004-12-01

    Full Text Available Abstract Background Eukaryotic whole genome sequences are accumulating at an impressive rate. Effective methods for comparing multiple whole eukaryotic genomes on a large scale are needed. Most attempted solutions involve the production of large scale alignments, and many of these require a high stringency pre-screen for putative orthologs in order to reduce the effective size of the dataset and provide a reasonably high but unknown fraction of correctly aligned homologous sites for comparison. As an alternative, highly efficient methods that do not require the pre-alignment of operationally defined orthologs are also being explored. Results A non-alignment method based on the Singular Value Decomposition (SVD was used to compare the predicted protein complement of nine whole eukaryotic genomes ranging from yeast to man. This analysis resulted in the simultaneous identification and definition of a large number of well conserved motifs and gene families, and produced a species tree supporting one of two conflicting hypotheses of metazoan relationships. Conclusions Our SVD-based analysis of the entire protein complement of nine whole eukaryotic genomes suggests that highly conserved motifs and gene families can be identified and effectively compared in a single coherent definition space for the easy extraction of gene and species trees. While this occurs without the explicit definition of orthologs or homologous sites, the analysis can provide a basis for these definitions.

  18. Nitrile Metabolizing Yeasts

    Science.gov (United States)

    Bhalla, Tek Chand; Sharma, Monica; Sharma, Nitya Nand

    Nitriles and amides are widely distributed in the biotic and abiotic components of our ecosystem. Nitrile form an important group of organic compounds which find their applications in the synthesis of a large number of compounds used as/in pharmaceutical, cosmetics, plastics, dyes, etc>. Nitriles are mainly hydro-lyzed to corresponding amide/acid in organic chemistry. Industrial and agricultural activities have also lead to release of nitriles and amides into the environment and some of them pose threat to human health. Biocatalysis and biotransformations are increasingly replacing chemical routes of synthesis in organic chemistry as a part of ‘green chemistry’. Nitrile metabolizing organisms or enzymes thus has assumed greater significance in all these years to convert nitriles to amides/ acids. The nitrile metabolizing enzymes are widely present in bacteria, fungi and yeasts. Yeasts metabolize nitriles through nitrilase and/or nitrile hydratase and amidase enzymes. Only few yeasts have been reported to possess aldoxime dehydratase. More than sixty nitrile metabolizing yeast strains have been hither to isolated from cyanide treatment bioreactor, fermented foods and soil. Most of the yeasts contain nitrile hydratase-amidase system for metabolizing nitriles. Transformations of nitriles to amides/acids have been carried out with free and immobilized yeast cells. The nitrilases of Torulopsis candida>and Exophiala oligosperma>R1 are enantioselec-tive and regiospecific respectively. Geotrichum>sp. JR1 grows in the presence of 2M acetonitrile and may have potential for application in bioremediation of nitrile contaminated soil/water. The nitrilase of E. oligosperma>R1 being active at low pH (3-6) has shown promise for the hydroxy acids. Immobilized yeast cells hydrolyze some additional nitriles in comparison to free cells. It is expected that more focus in future will be on purification, characterization, cloning, expression and immobilization of nitrile metabolizing

  19. Interplay of noncoding RNAs, mRNAs, and proteins during the growth of eukaryotic cells

    International Nuclear Information System (INIS)

    Zhdanov, V. P.

    2010-01-01

    Numerous biological functions of noncoding RNAs (ncRNAs) in eukaryotic cells are based primarily on their ability to pair with target mRNAs and then either to prevent translation or to result in rapid degradation of the mRNA-ncRNA complex. Using a general model describing this scenario, we show that ncRNAs may help to maintain constant mRNA and protein concentrations during the growth of cells. The possibility of observation of this effect on the global scale is briefly discussed.

  20. Regulation of eukaryotic elongation factor 1 alpha (eEF1A) by dynamic lysine methylation

    DEFF Research Database (Denmark)

    Jakobsson, Magnus E; Małecki, Jędrzej; Falnes, Pål Ø

    2018-01-01

    Lysine methylation is a frequent post-translational protein modification, which has been intensively studied in the case of histone proteins. Lysine methylations are also found on many non-histone proteins, and one prominent example is eukaryotic elongation factor 1 alpha (eEF1A). Besides its...... essential role in the protein synthesis machinery, a number of non-canonical functions have also been described for eEF1A, such as regulation of the actin cytoskeleton and the promotion of viral replication. The functional significance of the extensive lysine methylations on eEF1A, as well as the identity...

  1. The Genome of Naegleria gruberi Illuminates Early Eukaryotic Versatility

    Energy Technology Data Exchange (ETDEWEB)

    Fritz-Laylin, Lillian K.; Prochnik, Simon E.; Ginger, Michael L.; Dacks, Joel; Carpenter, Meredith L.; Field, Mark C.; Kuo, Alan; Paredez, Alex; Chapman, Jarrod; Pham, Jonathan; Shu, Shengqiang; Neupane, Rochak; Cipriano, Michael; Mancuso, Joel; Tu, Hank; Salamov, Asaf; Lindquist, Erika; Shapiro, Harris; Lucas, Susan; Grigoriev, Igor V.; Cande, W. Zacheus; Fulton, Chandler; Rokhsar, Daniel S.; Dawson, Scott C.

    2010-03-01

    Genome sequences of diverse free-living protists are essential for understanding eukaryotic evolution and molecular and cell biology. The free-living amoeboflagellate Naegleria gruberi belongs to a varied and ubiquitous protist clade (Heterolobosea) that diverged from other eukaryotic lineages over a billion years ago. Analysis of the 15,727 protein-coding genes encoded by Naegleria's 41 Mb nuclear genome indicates a capacity for both aerobic respiration and anaerobic metabolism with concomitant hydrogen production, with fundamental implications for the evolution of organelle metabolism. The Naegleria genome facilitates substantially broader phylogenomic comparisons of free-living eukaryotes than previously possible, allowing us to identify thousands of genes likely present in the pan-eukaryotic ancestor, with 40% likely eukaryotic inventions. Moreover, we construct a comprehensive catalog of amoeboid-motility genes. The Naegleria genome, analyzed in the context of other protists, reveals a remarkably complex ancestral eukaryote with a rich repertoire of cytoskeletal, sexual, signaling, and metabolic modules.

  2. 酵母细胞表面展示技术%Recent Advances in Yeast Cell-Surface Display Technology

    Institute of Scientific and Technical Information of China (English)

    王佳堃; 孙中远; 刘建新

    2011-01-01

    酵母细胞表达体系具备较为完善的蛋白质翻译后修饰和分泌的机制.以酵母为基础的细胞表面展示技术是一项新兴的真核蛋白展示技术,已成功应用于蛋白质识别、蛋白质的固定化和定向进化研究,成为了蛋白质工程研究的重要工具.根据与酵母细胞壁的外源目的蛋白融合部位的不同,酿酒酵母表面展示系统主要分为凝集素系统和絮凝素系统2大系统.将该技术引入动物营养研究领域,有望通过诱导宿主产生原虫或甲烷菌的抗体,对瘤胃微生态进行调控,从而以减少甲烷排放;生产高活性的全细胞催化剂,以提高反刍动物和单胃动物对纤维物质的利用效率.为此,本文就该技术的原理、特点和应用领域进行了综述.%Yeast cell has a perfect system for protein post-translational modification and secretion. The cell surface display technology based on yeast is a novel eukaryotic protein display technology. It has been successfully used for protein recognition, immobilization and remediation, and becomes an important tool for the study of protein engineering. Yeast cell surface display technology is divided into two systems, which are agglutinin and flocculin, according to the different insertion sites of the yeast cell wall for target heterologous proteins. If introduced to animal nutrition research, this technology will be a promising strategy to reduce the methane production by the regulation on ruminal microbial ecology, which is inducing of production of antibodies for protozoa and methanogen in host; highly active whole cell biocatalysts will be produced by increasing fiber utilization in ruminant and monogastric animal. Therefore, mechanism, characteristic and application of the technology are reviewed in this paper.

  3. The flavoprotein Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells

    Energy Technology Data Exchange (ETDEWEB)

    Nishimura, Akira; Kawahara, Nobuhiro [Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192 (Japan); Takagi, Hiroshi, E-mail: hiro@bs.naist.jp [Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192 (Japan)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer NO is produced from L-arginine in response to elevated temperature in yeast. Black-Right-Pointing-Pointer Tah18 was first identified as the yeast protein involved in NO synthesis. Black-Right-Pointing-Pointer Tah18-dependent NO synthesis confers tolerance to high-temperature on yeast cells. -- Abstract: Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. In the unicellular eukaryote yeast, NO may be involved in stress response pathways, but its role is poorly understood due to the lack of mammalian NO synthase (NOS) orthologues. Previously, we have proposed the oxidative stress-induced L-arginine synthesis and its physiological role under stress conditions in yeast Saccharomyces cerevisiae. Here, our experimental results indicated that increased conversion of L-proline into L-arginine led to NO production in response to elevated temperature. We also showed that the flavoprotein Tah18, which was previously reported to transfer electrons to the Fe-S cluster protein Dre2, was involved in NO synthesis in yeast. Gene knockdown analysis demonstrated that Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells. As it appears that such a unique cell protection mechanism is specific to yeasts and fungi, it represents a promising target for antifungal activity.

  4. Recent advances in the genome-wide study of DNA replication origins in yeast

    Directory of Open Access Journals (Sweden)

    Chong ePeng

    2015-02-01

    Full Text Available DNA replication, one of the central events in the cell cycle, is the basis of biological inheritance. In order to be duplicated, a DNA double helix must be opened at defined sites, which are called DNA replication origins (ORIs. Unlike in bacteria, where replication initiates from a single replication origin, multiple origins are utilized in the eukaryotic genome. Among them, the ORIs in budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have been best characterized. In recent years, advances in DNA microarray and next-generation sequencing technologies have increased the number of yeast species involved in ORIs research dramatically. The ORIs in some nonconventional yeast species such as Kluyveromyces lactis and Pichia pastoris have also been genome-widely identified. Relevant databases of replication origins in yeast were constructed, then the comparative genomic analysis can be carried out. Here, we review several experimental approaches that have been used to map replication origins in yeast and some of the available web resources related to yeast ORIs. We also discuss the sequence characteristics and chromosome structures of ORIs in the four yeast species, which can be utilized to improve the replication origins prediction.

  5. The flavoprotein Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells

    International Nuclear Information System (INIS)

    Nishimura, Akira; Kawahara, Nobuhiro; Takagi, Hiroshi

    2013-01-01

    Highlights: ► NO is produced from L-arginine in response to elevated temperature in yeast. ► Tah18 was first identified as the yeast protein involved in NO synthesis. ► Tah18-dependent NO synthesis confers tolerance to high-temperature on yeast cells. -- Abstract: Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. In the unicellular eukaryote yeast, NO may be involved in stress response pathways, but its role is poorly understood due to the lack of mammalian NO synthase (NOS) orthologues. Previously, we have proposed the oxidative stress-induced L-arginine synthesis and its physiological role under stress conditions in yeast Saccharomyces cerevisiae. Here, our experimental results indicated that increased conversion of L-proline into L-arginine led to NO production in response to elevated temperature. We also showed that the flavoprotein Tah18, which was previously reported to transfer electrons to the Fe–S cluster protein Dre2, was involved in NO synthesis in yeast. Gene knockdown analysis demonstrated that Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells. As it appears that such a unique cell protection mechanism is specific to yeasts and fungi, it represents a promising target for antifungal activity.

  6. Crystallization and preliminary X-ray analysis of beta-alanine synthase from the yeast Saccharomyces kluyveri

    DEFF Research Database (Denmark)

    Dobritzsch, D.; Gojkovic, Zoran; Andersen, Birgit

    2003-01-01

    In eukaryotes and some bacteria, the third step of reductive pyrimidine catabolism is catalyzed by beta-alanine synthase (EC 3.5.1.6). Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri were obtained using sodium citrate as a precipitant. The crystals belong to space group P2...

  7. Reconstruction of the yeast Snf1 kinase regulatory network reveals its role as a global energy regulator

    DEFF Research Database (Denmark)

    Usaite, Renata; Jewett, Michael Christopher; Soberano de Oliveira, Ana Paula

    2009-01-01

    Highly conserved among eukaryotic cells, the AMP-activated kinase (AMPK) is a central regulator of carbon metabolism. To map the complete network of interactions around AMPK in yeast (Snf1) and to evaluate the role of its regulatory subunit Snf4, we measured global mRNA, protein and metabolite...

  8. Ultrastructural diversity between centrioles of eukaryotes.

    Science.gov (United States)

    Gupta, Akshari; Kitagawa, Daiju

    2018-02-16

    Several decades of centriole research have revealed the beautiful symmetry present in these microtubule-based organelles, which are required to form centrosomes, cilia, and flagella in many eukaryotes. Centriole architecture is largely conserved across most organisms, however, individual centriolar features such as the central cartwheel or microtubule walls exhibit considerable variability when examined with finer resolution. Here, we review the ultrastructural characteristics of centrioles in commonly studied organisms, highlighting the subtle and not-so-subtle differences between specific structural components of these centrioles. Additionally, we survey some non-canonical centriole structures that have been discovered in various species, from the coaxial bicentrioles of protists and lower land plants to the giant irregular centrioles of the fungus gnat Sciara. Finally, we speculate on the functional significance of these differences between centrioles, and the contribution of individual structural elements such as the cartwheel or microtubules towards the stability of centrioles.Centriole structure, cartwheel, triplet microtubules, SAS-6, centrosome.

  9. Bacterial proteins pinpoint a single eukaryotic root

    Czech Academy of Sciences Publication Activity Database

    Derelle, R.; Torruella, G.; Klimeš, V.; Brinkmann, H.; Kim, E.; Vlček, Čestmír; Lang, B.F.; Eliáš, M.

    2015-01-01

    Roč. 112, č. 7 (2015), E693-E699 ISSN 0027-8424 R&D Projects: GA ČR GA13-24983S Grant - others:GA MŠk(CZ) ED2.1.00/03.0100; Howard Hughes Medical Institute International Early Career Scientist Program(US) 55007424; Spanish Ministry of Economy and Competitiveness, European Molecular Biology Organization Young Investigator Program(ES) BFU2012-31329; Spanish Ministry of Economy and Competitiveness, "Centro de Excelencia Severo Ochoa" - European Regional Development Fund(ES) Sev-2012-0208, BES-2013-064004 Institutional support: RVO:68378050 Keywords : eukaryote phylogeny * phylogenomics * Opimoda * Diphoda * LECA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 9.423, year: 2015

  10. Translating democracy

    DEFF Research Database (Denmark)

    Doerr, Nicole

    2012-01-01

    Linguistic barriers may pose problems for politicians trying to communicate delicate decisions to a European-wide public, as well as for citizens wishing to protest at the European level. In this article I present a counter-intuitive position on the language question, one that explores how...... Forum (ESF). I compare deliberative practices in the multilingual ESF preparatory meetings with those in monolingual national Social Forum meetings in three Western European countries. My comparison shows that multilingualism does not reduce the inclusivity of democratic deliberation as compared...... in institutionalized habits and norms of deliberation. Addressing democratic theorists, my findings suggest that translation could be a way to think about difference not as a hindrance but as a resource for democracy in linguistically heterogeneous societies and public spaces, without presupposing a shared language...

  11. Translator's preface.

    Science.gov (United States)

    Lamiell, James T

    2013-08-01

    Presents a preface from James T. Lamiell, who translates Wilhelm Wundt's Psychology's Struggle for Existence (Die Psychologie im Kampf ums Dasein), in which Wundt advised against the impending divorce of psychology from philosophy, into English. Lamiell comments that more than a decade into the 21st century, it appears that very few psychologists have any interest at all in work at the interface of psychology and philosophy. He notes that one clear indication of this is that the Society for Theoretical and Philosophical Psychology, which is Division 24 of the American Psychological Association (APA), remains one of the smallest of the APA's nearly 60 divisions. (PsycINFO Database Record (c) 2013 APA, all rights reserved).

  12. Effect of baculovirus infection on the mRNA and protein levels of the Spodoptera frugiperda eukaryotic initiation factor 4E

    NARCIS (Netherlands)

    Oers, van M.M.; Veken, van der L.T.J.N.; Vlak, J.M.; Thomas, A.A.M.

    2001-01-01

    The cDNA sequence of eukaryotic translation initiation factor eIF4E was derived from a Spodoptera frugiperda cDNA library. Eight tryptophan residues, typical for eIF4E, are strictly conserved in the encoded 210 amino acid protein. A polyclonal antiserum detected a 26 kDa protein in lepidopteran cell

  13. Mapping Translation Technology Research in Translation Studies

    DEFF Research Database (Denmark)

    Schjoldager, Anne; Christensen, Tina Paulsen; Flanagan, Marian

    2017-01-01

    section aims to improve this situation by presenting new and innovative research papers that reflect on recent technological advances and their impact on the translation profession and translators from a diversity of perspectives and using a variety of methods. In Section 2, we present translation......Due to the growing uptake of translation technology in the language industry and its documented impact on the translation profession, translation students and scholars need in-depth and empirically founded knowledge of the nature and influences of translation technology (e.g. Christensen....../Schjoldager 2010, 2011; Christensen 2011). Unfortunately, the increasing professional use of translation technology has not been mirrored within translation studies (TS) by a similar increase in research projects on translation technology (Munday 2009: 15; O’Hagan 2013; Doherty 2016: 952). The current thematic...

  14. Horizontal gene transfer promoted evolution of the ability to propagate under anaerobic conditions in yeasts

    DEFF Research Database (Denmark)

    Gojkovic, Zoran; Knecht, Wolfgang; Warneboldt, J.

    2004-01-01

    The ability to propagate under anaerobic conditions is an essential and unique trait of brewer's or baker's yeast (Saccharomyces cervisiae). To understand the evolution of facultative anaerobiosis we studied the dependence of de novo pyrimidine biosynthesis, more precisely the fourth enzymic...... activity catalysed by dihydroorotate dehydrogenase (DHODase), on the enzymes of the respiratory chain in several yeast species. While the majority of yeasts possess a mitochondrial DHODase, Saccharomyces cerevisiae has a cytoplasmatic enzyme, whose activity is independent of the presence of oxygen. From....... We show that these two S. kluyveri enzymes, and their coding genes, differ in their dependence on the presence of oxygen. Only the cytoplasmic DHODase promotes growth in the absence of oxygen. Apparently a Saccharomyces yeast progenitor which had a eukaryotic-like mitochondrial DHODase acquired...

  15. Initiation at closely spaced replication origins in a yeast chromosome.

    Science.gov (United States)

    Brewer, B J; Fangman, W L

    1993-12-10

    Replication of eukaryotic chromosomes involves initiation at origins spaced an average of 50 to 100 kilobase pairs. In yeast, potential origins can be recognized as autonomous replication sequences (ARSs) that allow maintenance of plasmids. However, there are more ARS elements than active chromosomal origins. The possibility was examined that close spacing of ARSs can lead to inactive origins. Two ARSs located 6.5 kilobase pairs apart can indeed interfere with each other. Replication is initiated from one or the other ARS with equal probability, but rarely (< 5%) from both ARSs on the same DNA molecule.

  16. Tus-Ter as a tool to study site-specific DNA replication perturbation in eukaryotes

    DEFF Research Database (Denmark)

    Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

    2014-01-01

    The high-affinity binding of the Tus protein to specific 21-bp sequences, called Ter, causes site-specific, and polar, DNA replication fork arrest in E coli. The Tus-Ter complex serves to coordinate DNA replication with chromosome segregation in this organism. A number of recent and ongoing studies...... have demonstrated that Tus-Ter can be used as a heterologous tool to generate site-specific perturbation of DNA replication when reconstituted in eukaryotes. Here, we review these recent findings and explore the molecular mechanism by which Tus-Ter mediates replication fork (RF) arrest in the budding...... yeast, S. cerevisiae. We propose that Tus-Ter is a versatile, genetically tractable, and regulatable RF blocking system that can be utilized for disrupting DNA replication in a diverse range of host cells....

  17. Tus-Ter as a tool to study site-specific DNA replication perturbation in eukaryotes.

    Science.gov (United States)

    Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

    2014-01-01

    The high-affinity binding of the Tus protein to specific 21-bp sequences, called Ter, causes site-specific, and polar, DNA replication fork arrest in E coli. The Tus-Ter complex serves to coordinate DNA replication with chromosome segregation in this organism. A number of recent and ongoing studies have demonstrated that Tus-Ter can be used as a heterologous tool to generate site-specific perturbation of DNA replication when reconstituted in eukaryotes. Here, we review these recent findings and explore the molecular mechanism by which Tus-Ter mediates replication fork (RF) arrest in the budding yeast, S. cerevisiae. We propose that Tus-Ter is a versatile, genetically tractable, and regulatable RF blocking system that can be utilized for disrupting DNA replication in a diverse range of host cells.

  18. Architecture of eukaryotic mRNA 3′-end processing machinery

    Science.gov (United States)

    Hill, Chris H.; Easter, Ashley D.; Emsley, Paul; Degliesposti, Gianluca; Gordiyenko, Yuliya; Santhanam, Balaji; Wolf, Jana; Wiederhold, Katrin; Dornan, Gillian L.; Skehel, Mark; Robinson, Carol V.; Passmore, Lori A.

    2018-01-01

    Newly transcribed eukaryotic precursor messenger RNAs (pre-mRNAs) are processed at their 3′ ends by the ~1-megadalton multiprotein cleavage and polyadenylation factor (CPF). CPF cleaves pre-mRNAs, adds a polyadenylate tail, and triggers transcription termination, but it is unclear how its various enzymes are coordinated and assembled. Here, we show that the nuclease, polymerase, and phosphatase activities of yeast CPF are organized into three modules. Using electron cryomicroscopy, we determined a 3.5-angstrom-resolution structure of the ~200-kilodalton polymerase module. This revealed four β propellers, in an assembly markedly similar to those of other protein complexes that bind nucleic acid. Combined with in vitro reconstitution experiments, our data show that the polymerase module brings together factors required for specific and efficient polyadenylation, to help coordinate mRNA 3′-end processing. PMID:29074584

  19. The COG database: an updated version includes eukaryotes

    Directory of Open Access Journals (Sweden)

    Sverdlov Alexander V

    2003-09-01

    Full Text Available Abstract Background The availability of multiple, essentially complete genome sequences of prokaryotes and eukaryotes spurred both the demand and the opportunity for the construction of an evolutionary classification of genes from these genomes. Such a classification system based on orthologous relationships between genes appears to be a natural framework for comparative genomics and should facilitate both functional annotation of genomes and large-scale evolutionary studies. Results We describe here a major update of the previously developed system for delineation of Clusters of Orthologous Groups of proteins (COGs from the sequenced genomes of prokaryotes and unicellular eukaryotes and the construction of clusters of predicted orthologs for 7 eukaryotic genomes, which we named KOGs after eukaryotic orthologous groups. The COG collection currently consists of 138,458 proteins, which form 4873 COGs and comprise 75% of the 185,505 (predicted proteins encoded in 66 genomes of unicellular organisms. The eukaryotic orthologous groups (KOGs include proteins from 7 eukaryotic genomes: three animals (the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster and Homo sapiens, one plant, Arabidopsis thaliana, two fungi (Saccharomyces cerevisiae and Schizosaccharomyces pombe, and the intracellular microsporidian parasite Encephalitozoon cuniculi. The current KOG set consists of 4852 clusters of orthologs, which include 59,838 proteins, or ~54% of the analyzed eukaryotic 110,655 gene products. Compared to the coverage of the prokaryotic genomes with COGs, a considerably smaller fraction of eukaryotic genes could be included into the KOGs; addition of new eukaryotic genomes is expected to result in substantial increase in the coverage of eukaryotic genomes with KOGs. Examination of the phyletic patterns of KOGs reveals a conserved core represented in all analyzed species and consisting of ~20% of the KOG set. This conserved portion of the

  20. Regulation of oxidative enzyme activity and eukaryotic elongation factor 2 in human skeletal muscle: influence of gender and exercise

    DEFF Research Database (Denmark)

    Roepstorff, Carsten; Schjerling, P.; Vistisen, Bodil

    2005-01-01

    AIM: To investigate gender-related differences in the responses of oxidative enzymes and eukaryotic elongation factor-2 (eEF2) to exercise. METHODS: The influence of exercise (90 min, 60%VO(2peak)) on citrate synthase (CS) and beta-hydroxyacyl-CoA dehydrogenase (HAD) activity and mRNA content...... expression and phosphorylation were unaffected by training status (NS). CONCLUSION: Basal transcriptional, translational, and/or post-translational control of CS and HAD seems to be gender-dependent. Also, gender differences in translation and/or post-translational protein modification of CS occur during...... not differ between females and males (NS). In females only, CS activity was enhanced (P differ between UT and ET but, nevertheless, CS activity was 56% higher in ET than in UT volunteers (P

  1. How does yeast respond to pressure?

    Directory of Open Access Journals (Sweden)

    Fernandes P.M.B.

    2005-01-01

    Full Text Available The brewing and baking yeast Saccharomyces cerevisiae has been used as a model for stress response studies of eukaryotic cells. In this review we focus on the effect of high hydrostatic pressure (HHP on S. cerevisiae. HHP exerts a broad effect on yeast cells characteristic of common stresses, mainly associated with protein alteration and lipid bilayer phase transition. Like most stresses, pressure induces cell cycle arrest. Below 50 MPa (500 atm yeast cell morphology is unaffected whereas above 220 MPa wild-type cells are killed. S. cerevisiae cells can acquire barotolerance if they are pretreated with a sublethal stress due to temperature, ethanol, hydrogen peroxide, or pressure. Nevertheless, pressure only leads to protection against severe stress if, after pressure pretreatment, the cells are also re-incubated at room pressure. We attribute this effect to the inhibition of the protein synthesis apparatus under HHP. The global genome expression analysis of S. cerevisiae cells submitted to HHP revealed a stress response profile. The majority of the up-regulated genes are involved in stress defense and carbohydrate metabolism while most repressed genes belong to the cell cycle progression and protein synthesis categories. However, the signaling pathway involved in the pressure response is still to be elucidated. Nitric oxide, a signaling molecule involved in the regulation of a large number of cellular functions, confers baroprotection. Furthermore, S. cerevisiae cells in the early exponential phase submitted to 50-MPa pressure show induction of the expression level of the nitric oxide synthase inducible isoform. As pressure becomes an important biotechnological tool, studies concerning this kind of stress in microorganisms are imperative.

  2. Synthetic yeast based cell factories for vanillin-glucoside production

    DEFF Research Database (Denmark)

    Strucko, Tomas

    and controlled expression/overexpression of genes of interest. De novo biosynthetic pathway for vanillin-β-glucoside production was employed as a model system for several case studies in this project. In order to construct yeast cell factories fulfilling current demands of industrial biotechnology, methods......The yeast Saccharomyces cerevisiae is well a characterized microorganism and widely used as eukaryotic model organism as well as a key cell factory for bioproduction of various products. The latter comprise a large variety of scientifically and industrially relevant products such as low-value bulk...... chemicals and biofuels, food additives, high-value chemicals and recombinant proteins. Despite the recent achievements in the fields of systems biology and metabolic engineering together with availability of broad genetic engineering toolbox, the full potential of S. cerevisiae as a cell factory is not yet...

  3. DNA to DNA transcription might exist in eukaryotic cells

    OpenAIRE

    Li, Gao-De

    2016-01-01

    Till now, in biological sciences, the term, transcription, mainly refers to DNA to RNA transcription. But our recently published experimental findings obtained from Plasmodium falciparum strongly suggest the existence of DNA to DNA transcription in the genome of eukaryotic cells, which could shed some light on the functions of certain noncoding DNA in the human and other eukaryotic genomes.

  4. Expression and function of the human estrogen receptor in yeast

    International Nuclear Information System (INIS)

    White, J.H.; Metzger, D.; Chambon, P.

    1988-01-01

    Gene expression in eukaryotes is regulated at many levels. Moreover, there is increasing evidence that the basic control mechanisms of transcription initiation have been conserved across the range of eukaryotes from yeast to man. In vertebrates, the nuclear receptors, whose activity is dependent on the binding of specific ligands, stimulate transcription by interacting with specific cis-acting sequences and display all of the hallmarks of inducible enhancer factors. Alignment of their amino acid sequences indicates that they are composed of a series of conserved domains. The domain structure of the human estrogen receptor (hER) is typical of receptor proteins. Region C, containing two putative zinc fingers, comprises the DNA-binding domain responsible for specific recognition of estrogen response elements (ERE). Region E contains the hormone-binding domain and domain(s) responsible for transcription activation. A mutant of the hER, called HE15, which lacks the hormone-binding domain, binds DNA in vivo and in vitro but activates transcription only poorly in a constitutive manner in vivo in HeLa cells. A series of studies have demonstrated that the hormone- and DNA-binding domains of the nuclear receptors function independently. Chimeric proteins consisting of the DNA-binding domain of yeast GAL4 coupled to the hormone-binding domains of either the hER or glucocorticoid receptor element (GRE) will stimulate transcription in HeLa cells when bound to a UAS. Taken together, these results demonstrate that the hER and other nuclear receptors, as well as GAL4 and GCN4 proteins of yeast, consist of discrete and separable DNA-binding and transcription-activation functions. To investigate these striking parallels further, the authors have expressed the hER in the yeast Saccharomyces cerevisiae and have analyzed its hormone- and DNA-binding properties in vitro and its ability to stimulate transcription in vivo

  5. Structural basis for the initiation of eukaryotic transcription-coupled DNA repair.

    Science.gov (United States)

    Xu, Jun; Lahiri, Indrajit; Wang, Wei; Wier, Adam; Cianfrocco, Michael A; Chong, Jenny; Hare, Alissa A; Dervan, Peter B; DiMaio, Frank; Leschziner, Andres E; Wang, Dong

    2017-11-30

    Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block translocation of RNA polymerase II (Pol II). Cockayne syndrome group B (CSB, also known as ERCC6) protein in humans (or its yeast orthologues, Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe) is among the first proteins to be recruited to the lesion-arrested Pol II during the initiation of eukaryotic TCR. Mutations in CSB are associated with the autosomal-recessive neurological disorder Cockayne syndrome, which is characterized by progeriod features, growth failure and photosensitivity. The molecular mechanism of eukaryotic TCR initiation remains unclear, with several long-standing unanswered questions. How cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II, the role of CSB in TCR initiation, and how CSB interacts with the arrested Pol II complex are all unknown. The lack of structures of CSB or the Pol II-CSB complex has hindered our ability to address these questions. Here we report the structure of the S. cerevisiae Pol II-Rad26 complex solved by cryo-electron microscopy. The structure reveals that Rad26 binds to the DNA upstream of Pol II, where it markedly alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes the forward movement of Pol II, and elucidate key roles for Rad26 in both TCR and transcription elongation.

  6. Morphological and ecological complexity in early eukaryotic ecosystems.

    Science.gov (United States)

    Javaux, E J; Knoll, A H; Walter, M R

    2001-07-05

    Molecular phylogeny and biogeochemistry indicate that eukaryotes differentiated early in Earth history. Sequence comparisons of small-subunit ribosomal RNA genes suggest a deep evolutionary divergence of Eukarya and Archaea; C27-C29 steranes (derived from sterols synthesized by eukaryotes) and strong depletion of 13C (a biogeochemical signature of methanogenic Archaea) in 2,700 Myr old kerogens independently place a minimum age on this split. Steranes, large spheroidal microfossils, and rare macrofossils of possible eukaryotic origin occur in Palaeoproterozoic rocks. Until now, however, evidence for morphological and taxonomic diversification within the domain has generally been restricted to very late Mesoproterozoic and Neoproterozoic successions. Here we show that the cytoskeletal and ecological prerequisites for eukaryotic diversification were already established in eukaryotic microorganisms fossilized nearly 1,500 Myr ago in shales of the early Mesoproterozoic Roper Group in northern Australia.

  7. Purification and biochemical characterisation of the yeast ABC transporter Pdr11p

    DEFF Research Database (Denmark)

    Laub, Katrine Rude

    Sterols constitute an essential lipid class in eukaryotic membranes where intracellular distributions are highly regulated. In the yeast Saccharomyces cerevisiae sterol uptake has been attributed to the two plasma membrane-localised ATP-binding cassette (ABC) transporters, Aus1p and Pdr11p...... of the yeast ABC transporter Pdr11p. This includes optimising its overexpression utilising the galactose induction system in S. cerevisiae, screening for the best detergent to extract the protein from the membrane, and establishing purification and reconstitution protocols. By providing a purification...

  8. MAP kinase pathways in the yeast Saccharomyces cerevisiae

    Science.gov (United States)

    Gustin, M. C.; Albertyn, J.; Alexander, M.; Davenport, K.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.

  9. Industrial Relevance of Chromosomal Copy Number Variation in Saccharomyces Yeasts.

    Science.gov (United States)

    Gorter de Vries, Arthur R; Pronk, Jack T; Daran, Jean-Marc G

    2017-06-01

    Chromosomal copy number variation (CCNV) plays a key role in evolution and health of eukaryotes. The unicellular yeast Saccharomyces cerevisiae is an important model for studying the generation, physiological impact, and evolutionary significance of CCNV. Fundamental studies of this yeast have contributed to an extensive set of methods for analyzing and introducing CCNV. Moreover, these studies provided insight into the balance between negative and positive impacts of CCNV in evolutionary contexts. A growing body of evidence indicates that CCNV not only frequently occurs in industrial strains of Saccharomyces yeasts but also is a key contributor to the diversity of industrially relevant traits. This notion is further supported by the frequent involvement of CCNV in industrially relevant traits acquired during evolutionary engineering. This review describes recent developments in genome sequencing and genome editing techniques and discusses how these offer opportunities to unravel contributions of CCNV in industrial Saccharomyce s strains as well as to rationally engineer yeast chromosomal copy numbers and karyotypes. Copyright © 2017 Gorter de Vries et al.

  10. Genetics of Yeasts

    Science.gov (United States)

    Querol, Amparo; Fernández-Espinar, M. Teresa; Belloch, Carmela

    The use of yeasts in biotechnology processes dates back to ancient days. Before 7000 BC, beer was produced in Sumeria. Wine was made in Assyria in 3500 BC, and ancient Rome had over 250 bakeries, which were making leavened bread by 100 BC. And milk has been made into Kefyr and Koumiss in Asia for many centuries (Demain, Phaff, & Kurtzman, 1999). However, the importance of yeast in the food and beverage industries was only realized about 1860, when their role in food manufacturing became evident.

  11. L-arabinose fermenting yeast

    Science.gov (United States)

    Zhang, Min; Singh, Arjun; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric; Suominen, Pirkko

    2010-12-07

    An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. Methods of producing ethanol include utilizing these modified yeast strains. ##STR00001##

  12. Genomic impact of eukaryotic transposable elements.

    Science.gov (United States)

    Arkhipova, Irina R; Batzer, Mark A; Brosius, Juergen; Feschotte, Cédric; Moran, John V; Schmitz, Jürgen; Jurka, Jerzy

    2012-11-21

    The third international conference on the genomic impact of eukaryotic transposable elements (TEs) was held 24 to 28 February 2012 at the Asilomar Conference Center, Pacific Grove, CA, USA. Sponsored in part by the National Institutes of Health grant 5 P41 LM006252, the goal of the conference was to bring together researchers from around the world who study the impact and mechanisms of TEs using multiple computational and experimental approaches. The meeting drew close to 170 attendees and included invited floor presentations on the biology of TEs and their genomic impact, as well as numerous talks contributed by young scientists. The workshop talks were devoted to computational analysis of TEs with additional time for discussion of unresolved issues. Also, there was ample opportunity for poster presentations and informal evening discussions. The success of the meeting reflects the important role of Repbase in comparative genomic studies, and emphasizes the need for close interactions between experimental and computational biologists in the years to come.

  13. Genomic impact of eukaryotic transposable elements

    Directory of Open Access Journals (Sweden)

    Arkhipova Irina R

    2012-11-01

    Full Text Available Abstract The third international conference on the genomic impact of eukaryotic transposable elements (TEs was held 24 to 28 February 2012 at the Asilomar Conference Center, Pacific Grove, CA, USA. Sponsored in part by the National Institutes of Health grant 5 P41 LM006252, the goal of the conference was to bring together researchers from around the world who study the impact and mechanisms of TEs using multiple computational and experimental approaches. The meeting drew close to 170 attendees and included invited floor presentations on the biology of TEs and their genomic impact, as well as numerous talks contributed by young scientists. The workshop talks were devoted to computational analysis of TEs with additional time for discussion of unresolved issues. Also, there was ample opportunity for poster presentations and informal evening discussions. The success of the meeting reflects the important role of Repbase in comparative genomic studies, and emphasizes the need for close interactions between experimental and computational biologists in the years to come.

  14. Do lipids shape the eukaryotic cell cycle?

    Science.gov (United States)

    Furse, Samuel; Shearman, Gemma C

    2018-01-01

    Successful passage through the cell cycle presents a number of structural challenges to the cell. Inceptive studies carried out in the last five years have produced clear evidence of modulations in the lipid profile (sometimes referred to as the lipidome) of eukaryotes as a function of the cell cycle. This mounting body of evidence indicates that lipids play key roles in the structural transformations seen across the cycle. The accumulation of this evidence coincides with a revolution in our understanding of how lipid composition regulates a plethora of biological processes ranging from protein activity through to cellular signalling and membrane compartmentalisation. In this review, we discuss evidence from biological, chemical and physical studies of the lipid fraction across the cell cycle that demonstrate that lipids are well-developed cellular components at the heart of the biological machinery responsible for managing progress through the cell cycle. Furthermore, we discuss the mechanisms by which this careful control is exercised. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  15. Kinetics of gene and chromosome mutations induced by UV-C in yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Koltovaya, N.; Kokoreva, A.; Senchenko, D.; Shvaneva, N.; Zhuchkina, N.

    2017-01-01

    The systematic study of the kinetics of UV-induced gene and structural mutations in eukaryotic cells was carried out on the basis of model yeast S. cerevisiae. A variety of genetic assays (all types of base pair substitutions, frameshifts, forward mutations canl, chromosomal and plasmid rearrangements) in haploid strains were used. Yeast cells were treated by UV-C light of fluence of energy up to 200 J/m"2. The kinetics of the induced gene and structural mutations is represented by a linear-quadratic and exponential functions. The slope of curves in log-log plots was not constant, had the value 2-4 and depended on the interval of doses. It was suggested that it is the superposition and dynamics of different pathways form the mutagenic responses of eukaryotic cells to UV-C light that cause the high-order curves. [ru

  16. Origins and evolution of viruses of eukaryotes: The ultimate modularity

    International Nuclear Information System (INIS)

    Koonin, Eugene V.; Dolja, Valerian V.; Krupovic, Mart

    2015-01-01

    Viruses and other selfish genetic elements are dominant entities in the biosphere, with respect to both physical abundance and genetic diversity. Various selfish elements parasitize on all cellular life forms. The relative abundances of different classes of viruses are dramatically different between prokaryotes and eukaryotes. In prokaryotes, the great majority of viruses possess double-stranded (ds) DNA genomes, with a substantial minority of single-stranded (ss) DNA viruses and only limited presence of RNA viruses. In contrast, in eukaryotes, RNA viruses account for the majority of the virome diversity although ssDNA and dsDNA viruses are common as well. Phylogenomic analysis yields tangible clues for the origins of major classes of eukaryotic viruses and in particular their likely roots in prokaryotes. Specifically, the ancestral genome of positive-strand RNA viruses of eukaryotes might have been assembled de novo from genes derived from prokaryotic retroelements and bacteria although a primordial origin of this class of viruses cannot be ruled out. Different groups of double-stranded RNA viruses derive either from dsRNA bacteriophages or from positive-strand RNA viruses. The eukaryotic ssDNA viruses apparently evolved via a fusion of genes from prokaryotic rolling circle-replicating plasmids and positive-strand RNA viruses. Different families of eukaryotic dsDNA viruses appear to have originated from specific groups of bacteriophages on at least two independent occasions. Polintons, the largest known eukaryotic transposons, predicted to also form virus particles, most likely, were the evolutionary intermediates between bacterial tectiviruses and several groups of eukaryotic dsDNA viruses including the proposed order “Megavirales” that unites diverse families of large and giant viruses. Strikingly, evolution of all classes of eukaryotic viruses appears to have involved fusion between structural and replicative gene modules derived from different sources

  17. Origins and evolution of viruses of eukaryotes: The ultimate modularity

    Energy Technology Data Exchange (ETDEWEB)

    Koonin, Eugene V., E-mail: koonin@ncbi.nlm.nih.gov [National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894 (United States); Dolja, Valerian V., E-mail: doljav@science.oregonstate.edu [Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331 (United States); Krupovic, Mart, E-mail: krupovic@pasteur.fr [Institut Pasteur, Unité Biologie Moléculaire du Gène chez les Extrêmophiles, Department of Microbiology, Paris 75015 (France)

    2015-05-15

    Viruses and other selfish genetic elements are dominant entities in the biosphere, with respect to both physical abundance and genetic diversity. Various selfish elements parasitize on all cellular life forms. The relative abundances of different classes of viruses are dramatically different between prokaryotes and eukaryotes. In prokaryotes, the great majority of viruses possess double-stranded (ds) DNA genomes, with a substantial minority of single-stranded (ss) DNA viruses and only limited presence of RNA viruses. In contrast, in eukaryotes, RNA viruses account for the majority of the virome diversity although ssDNA and dsDNA viruses are common as well. Phylogenomic analysis yields tangible clues for the origins of major classes of eukaryotic viruses and in particular their likely roots in prokaryotes. Specifically, the ancestral genome of positive-strand RNA viruses of eukaryotes might have been assembled de novo from genes derived from prokaryotic retroelements and bacteria although a primordial origin of this class of viruses cannot be ruled out. Different groups of double-stranded RNA viruses derive either from dsRNA bacteriophages or from positive-strand RNA viruses. The eukaryotic ssDNA viruses apparently evolved via a fusion of genes from prokaryotic rolling circle-replicating plasmids and positive-strand RNA viruses. Different families of eukaryotic dsDNA viruses appear to have originated from specific groups of bacteriophages on at least two independent occasions. Polintons, the largest known eukaryotic transposons, predicted to also form virus particles, most likely, were the evolutionary intermediates between bacterial tectiviruses and several groups of eukaryotic dsDNA viruses including the proposed order “Megavirales” that unites diverse families of large and giant viruses. Strikingly, evolution of all classes of eukaryotic viruses appears to have involved fusion between structural and replicative gene modules derived from different sources

  18. Redox-dependent conformational changes in eukaryotic cytochromes revealed by paramagnetic NMR spectroscopy

    International Nuclear Information System (INIS)

    Volkov, Alexander N.; Vanwetswinkel, Sophie; Van de Water, Karen; Nuland, Nico A. J. van

    2012-01-01

    Cytochrome c (Cc) is a soluble electron carrier protein, transferring reducing equivalents between Cc reductase and Cc oxidase in eukaryotes. In this work, we assessed the structural differences between reduced and oxidized Cc in solution by paramagnetic NMR spectroscopy. First, we have obtained nearly-complete backbone NMR resonance assignments for iso-1-yeast Cc and horse Cc in both oxidation states. These were further used to derive pseudocontact shifts (PCSs) arising from the paramagnetic haem group. Then, an extensive dataset comprising over 450 measured PCSs and high-resolution X-ray and solution NMR structures of both proteins were used to define the anisotropic magnetic susceptibility tensor, Δχ. For most nuclei, the PCSs back-calculated from the Δχ tensor are in excellent agreement with the experimental PCS values. However, several contiguous stretches—clustered around G41, N52, and A81—exhibit large deviations both in yeast and horse Cc. This behaviour is indicative of redox-dependent structural changes, the extent of which is likely conserved in the protein family. We propose that the observed discrepancies arise from the changes in protein dynamics and discuss possible functional implications.

  19. Redox-dependent conformational changes in eukaryotic cytochromes revealed by paramagnetic NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Volkov, Alexander N.; Vanwetswinkel, Sophie; Van de Water, Karen; Nuland, Nico A. J. van, E-mail: nvnuland@vub.ac.be [Vrije Universiteit Brussel, Jean Jeener NMR Centre, Structural Biology Brussels (Belgium)

    2012-03-15

    Cytochrome c (Cc) is a soluble electron carrier protein, transferring reducing equivalents between Cc reductase and Cc oxidase in eukaryotes. In this work, we assessed the structural differences between reduced and oxidized Cc in solution by paramagnetic NMR spectroscopy. First, we have obtained nearly-complete backbone NMR resonance assignments for iso-1-yeast Cc and horse Cc in both oxidation states. These were further used to derive pseudocontact shifts (PCSs) arising from the paramagnetic haem group. Then, an extensive dataset comprising over 450 measured PCSs and high-resolution X-ray and solution NMR structures of both proteins were used to define the anisotropic magnetic susceptibility tensor, {Delta}{chi}. For most nuclei, the PCSs back-calculated from the {Delta}{chi} tensor are in excellent agreement with the experimental PCS values. However, several contiguous stretches-clustered around G41, N52, and A81-exhibit large deviations both in yeast and horse Cc. This behaviour is indicative of redox-dependent structural changes, the extent of which is likely conserved in the protein family. We propose that the observed discrepancies arise from the changes in protein dynamics and discuss possible functional implications.

  20. Inhibition of Ribosome Recruitment Induces Stress Granule Formation Independently of Eukaryotic Initiation Factor 2α Phosphorylation

    Science.gov (United States)

    Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J.; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed

    2006-01-01

    Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2α phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2α phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2α to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2α phosphorylation-dependent and -independent pathways that target translation initiation. PMID:16870703

  1. Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation.

    Science.gov (United States)

    Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed; Pelletier, Jerry

    2006-10-01

    Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2alpha phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2alpha phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2alpha to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2alpha phosphorylation-dependent and -independent pathways that target translation initiation.

  2. Yeast Infection during Pregnancy

    Science.gov (United States)

    ... disrupt the pH balance of the vagina. Common yeast infection symptoms include vaginal itching and a white, thick discharge that looks ... and Prevention. http://www.cdc.gov/std/tg2015/candidiasis.htm. Accessed Aug. 27, ... Vagina, Cervix, Toxic Shock Syndrome, Endometritis, and Salpingitis. In: ...

  3. Polysome Profile Analysis - Yeast

    Czech Academy of Sciences Publication Activity Database

    Pospíšek, M.; Valášek, Leoš Shivaya

    2013-01-01

    Roč. 530, č. 2013 (2013), s. 173-181 ISSN 0076-6879 Institutional support: RVO:61388971 Keywords : grow yeast cultures * polysome profile analysis * sucrose density gradient centrifugation Subject RIV: CE - Biochemistry Impact factor: 2.194, year: 2013

  4. Interactions between subunits of Saccharomyces cerevisiae RNase MRP support a conserved eukaryotic RNase P/MRP architecture.

    Science.gov (United States)

    Aspinall, Tanya V; Gordon, James M B; Bennett, Hayley J; Karahalios, Panagiotis; Bukowski, John-Paul; Walker, Scott C; Engelke, David R; Avis, Johanna M

    2007-01-01

    Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve our understanding of subunit roles and enzyme architecture, we have examined protein-protein and protein-RNA interactions in vitro, complementing existing yeast two-hybrid data. In total, 31 direct protein-protein interactions were identified, each protein interacting with at least three others. Furthermore, seven proteins self-interact, four strongly, pointing to subunit multiplicity in the holoenzyme. Six protein subunits interact directly with MRP RNA and four with pre-rRNA. A comparative analysis with existing data for the yeast and human RNase P/MRP systems enables confident identification of Pop1p, Pop4p and Rpp1p as subunits that lie at the enzyme core, with probable addition of Pop5p and Pop3p. Rmp1p is confirmed as an integral subunit, presumably associating preferentially with RNase MRP, rather than RNase P, via interactions with Snm1p and MRP RNA. Snm1p and Rmp1p may act together to assist enzyme specificity, though roles in substrate binding are also indicated for Pop4p and Pop6p. The results provide further evidence of a conserved eukaryotic RNase P/MRP architecture and provide a strong basis for studies of enzyme assembly and subunit function.

  5. Genome-reconstruction for eukaryotes from complex natural microbial communities.

    Science.gov (United States)

    West, Patrick T; Probst, Alexander J; Grigoriev, Igor V; Thomas, Brian C; Banfield, Jillian F

    2018-04-01

    Microbial eukaryotes are integral components of natural microbial communities, and their inclusion is critical for many ecosystem studies, yet the majority of published metagenome analyses ignore eukaryotes. In order to include eukaryotes in environmental studies, we propose a method to recover eukaryotic genomes from complex metagenomic samples. A key step for genome recovery is separation of eukaryotic and prokaryotic fragments. We developed a k -mer-based strategy, EukRep, for eukaryotic sequence identification and applied it to environmental samples to show that it enables genome recovery, genome completeness evaluation, and prediction of metabolic potential. We used this approach to test the effect of addition of organic carbon on a geyser-associated microbial community and detected a substantial change of the community metabolism, with selection against almost all candidate phyla bacteria and archaea and for eukaryotes. Near complete genomes were reconstructed for three fungi placed within the Eurotiomycetes and an arthropod. While carbon fixation and sulfur oxidation were important functions in the geyser community prior to carbon addition, the organic carbon-impacted community showed enrichment for secreted proteases, secreted lipases, cellulose targeting CAZymes, and methanol oxidation. We demonstrate the broader utility of EukRep by reconstructing and evaluating relatively high-quality fungal, protist, and rotifer genomes from complex environmental samples. This approach opens the way for cultivation-independent analyses of whole microbial communities. © 2018 West et al.; Published by Cold Spring Harbor Laboratory Press.

  6. Potential of industrial biotechnology with cyanobacteria and eukaryotic microalgae.

    Science.gov (United States)

    Wijffels, René H; Kruse, Olaf; Hellingwerf, Klaas J

    2013-06-01

    Both cyanobacteria and eukaryotic microalgae are promising organisms for sustainable production of bulk products such as food, feed, materials, chemicals and fuels. In this review we will summarize the potential and current biotechnological developments. Cyanobacteria are promising host organisms for the production of small molecules that can be secreted such as ethanol, butanol, fatty acids and other organic acids. Eukaryotic microalgae are interesting for products for which cellular storage is important such as proteins, lipids, starch and alkanes. For the development of new and promising lines of production, strains of both cyanobacteria and eukaryotic microalgae have to be improved. Transformation systems have been much better developed in cyanobacteria. However, several products would be preferably produced with eukaryotic microalgae. In the case of cyanobacteria a synthetic-systems biology approach has a great potential to exploit cyanobacteria as cell factories. For eukaryotic microalgae transformation systems need to be further developed. A promising strategy is transformation of heterologous (prokaryotic and eukaryotic) genes in established eukaryotic hosts such as Chlamydomonas reinhardtii. Experimental outdoor pilots under containment for the production of genetically modified cyanobacteria and microalgae are in progress. For full scale production risks of release of genetically modified organisms need to be assessed. Copyright © 2013. Published by Elsevier Ltd.

  7. Mapping yeast origins of replication via single-stranded DNA detection.

    Science.gov (United States)

    Feng, Wenyi; Raghuraman, M K; Brewer, Bonita J

    2007-02-01

    Studies in th Saccharomyces cerevisiae have provided a framework for understanding how eukaryotic cells replicate their chromosomal DNA to ensure faithful transmission of genetic information to their daughter cells. In particular, S. cerevisiae is the first eukaryote to have its origins of replication mapped on a genomic scale, by three independent groups using three different microarray-based approaches. Here we describe a new technique of origin mapping via detection of single-stranded DNA in yeast. This method not only identified the majority of previously discovered origins, but also detected new ones. We have also shown that this technique can identify origins in Schizosaccharomyces pombe, illustrating the utility of this method for origin mapping in other eukaryotes.

  8. L-arabinose fermenting yeast

    Science.gov (United States)

    Zhang, Min; Singh, Arjun; Suominen, Pirkko; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric

    2013-02-12

    An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. A yeast strain engineered to metabolize arabinose through a novel pathway is also disclosed. Methods of producing ethanol include utilizing these modified yeast strains.

  9. Functional and Biochemical Characterization of Human Eukaryotic Translation Initiation Factor 3 in Living Cells

    Czech Academy of Sciences Publication Activity Database

    Wagner, Susan; Herrmannová, Anna; Malík, Radek; Peclinovská, L.; Valášek, Leoš Shivaya

    2014-01-01

    Roč. 34, č. 16 (2014), s. 3041-3052 ISSN 0270-7306 R&D Projects: GA ČR GAP305/10/0335 Institutional support: RVO:61388971 ; RVO:68378050 Keywords : MESSENGER-RNA RECRUITMENT * FACTOR EIF3 * IN-VIVO Subject RIV: CE - Biochemistry Impact factor: 4.777, year: 2014

  10. Cross-species complementation of bacterial- and eukaryotic-type cardiolipin synthases

    Directory of Open Access Journals (Sweden)

    Petra Gottier

    2017-11-01

    Full Text Available The glycerophospholipid cardiolipin is a unique constituent of bacterial and mitochondrial membranes. It is involved in forming and stabilizing high molecular mass membrane protein complexes and in maintaining membrane architecture. Absence of cardiolipin leads to reduced efficiency of the electron transport chain, decreased membrane potential, and, ultimately, impaired respiratory metabolism. For the protozoan parasite Trypanosoma brucei cardiolipin synthesis is essential for survival, indicating that the enzymes involved in cardiolipin production represent potential drug targets. T. brucei cardiolipin synthase (TbCLS is unique as it belongs to the family of phospholipases D (PLD, harboring a prokaryotic-type cardiolipin synthase (CLS active site domain. In contrast, most other eukaryotic CLS, including the yeast ortholog ScCrd1, are members of the CDP-alcohol phosphatidyl­ transferase family. To study if these mechanistically distinct CLS enzymes are able to catalyze cardiolipin production in a cell that normally expresses a different type of CLS, we expressed TbCLS and ScCrd1 in CLS-deficient yeast and trypanosome strains, respectively. Our results show that TbCLS complemented cardiolipin production in CRD1 knockout yeast and partly restored wild-type colony forming capability under stress conditions. Remarkably, CL remodeling appeared to be impaired in the transgenic construct, suggesting that CL production and remodeling are tightly coupled processes that may require a clustering of the involved proteins into specific CL-synthesizing domains. In contrast, no complementation was observed by heterologous expression of ScCrd1 in conditional TbCLS knockout trypanosomes, despite proper mitochondrial targeting of the protein.

  11. Eukaryote-wide sequence analysis of mitochondrial β-barrel outer membrane proteins

    Directory of Open Access Journals (Sweden)

    Fujita Naoya

    2011-01-01

    Full Text Available Abstract Background The outer membranes of mitochondria are thought to be homologous to the outer membranes of Gram negative bacteria, which contain 100's of distinct families of β-barrel membrane proteins (BOMPs often forming channels for transport of nutrients or drugs. However, only four families of mitochondrial BOMPs (MBOMPs have been confirmed to date. Although estimates as high as 100 have been made in the past, the number of yet undiscovered MBOMPs is an open question. Fortunately, the recent discovery of a membrane integration signal (the β-signal for MBOMPs gave us an opportunity to look for undiscovered MBOMPs. Results We present the results of a comprehensive survey of eukaryotic protein sequences intended to identify new MBOMPs. Our search employs recent results on β-signals as well as structural information and a novel BOMP predictor trained on both bacterial and mitochondrial BOMPs. Our principal finding is circumstantial evidence suggesting that few MBOMPs remain to be discovered, if one assumes that, like known MBOMPs, novel MBOMPs will be monomeric and β-signal dependent. In addition to this, our analysis of MBOMP homologs reveals some exceptions to the current model of the β-signal, but confirms its consistent presence in the C-terminal region of MBOMP proteins. We also report a β-signal independent search for MBOMPs against the yeast and Arabidopsis proteomes. We find no good candidates MBOMPs in yeast but the Arabidopsis results are less conclusive. Conclusions Our results suggest there are no remaining MBOMPs left to discover in yeast; and if one assumes all MBOMPs are β-signal dependent, few MBOMP families remain undiscovered in any sequenced organism.

  12. Highly variable rates of genome rearrangements between hemiascomycetous yeast lineages.

    Directory of Open Access Journals (Sweden)

    2006-03-01

    Full Text Available Hemiascomycete yeasts cover an evolutionary span comparable to that of the entire phylum of chordates. Since this group currently contains the largest number of complete genome sequences it presents unique opportunities to understand the evolution of genome organization in eukaryotes. We inferred rates of genome instability on all branches of a phylogenetic tree for 11 species and calculated species-specific rates of genome rearrangements. We characterized all inversion events that occurred within synteny blocks between six representatives of the different lineages. We show that the rates of macro- and microrearrangements of gene order are correlated within individual lineages but are highly variable across different lineages. The most unstable genomes correspond to the pathogenic yeasts Candida albicans and Candida glabrata. Chromosomal maps have been intensively shuffled by numerous interchromosomal rearrangements, even between species that have retained a very high physical fraction of their genomes within small synteny blocks. Despite this intensive reshuffling of gene positions, essential genes, which cluster in low recombination regions in the genome of Saccharomyces cerevisiae, tend to remain syntenic during evolution. This work reveals that the high plasticity of eukaryotic genomes results from rearrangement rates that vary between lineages but also at different evolutionary times of a given lineage.

  13. A Three-Dimensional Model of the Yeast Genome

    Science.gov (United States)

    Noble, William; Duan, Zhi-Jun; Andronescu, Mirela; Schutz, Kevin; McIlwain, Sean; Kim, Yoo Jung; Lee, Choli; Shendure, Jay; Fields, Stanley; Blau, C. Anthony

    Layered on top of information conveyed by DNA sequence and chromatin are higher order structures that encompass portions of chromosomes, entire chromosomes, and even whole genomes. Interphase chromosomes are not positioned randomly within the nucleus, but instead adopt preferred conformations. Disparate DNA elements co-localize into functionally defined aggregates or factories for transcription and DNA replication. In budding yeast, Drosophila and many other eukaryotes, chromosomes adopt a Rabl configuration, with arms extending from centromeres adjacent to the spindle pole body to telomeres that abut the nuclear envelope. Nonetheless, the topologies and spatial relationships of chromosomes remain poorly understood. Here we developed a method to globally capture intra- and inter-chromosomal interactions, and applied it to generate a map at kilobase resolution of the haploid genome of Saccharomyces cerevisiae. The map recapitulates known features of genome organization, thereby validating the method, and identifies new features. Extensive regional and higher order folding of individual chromosomes is observed. Chromosome XII exhibits a striking conformation that implicates the nucleolus as a formidable barrier to interaction between DNA sequences at either end. Inter-chromosomal contacts are anchored by centromeres and include interactions among transfer RNA genes, among origins of early DNA replication and among sites where chromosomal breakpoints occur. Finally, we constructed a three-dimensional model of the yeast genome. Our findings provide a glimpse of the interface between the form and function of a eukaryotic genome.

  14. Translational Control of Cell Division by Elongator

    Directory of Open Access Journals (Sweden)

    Fanelie Bauer

    2012-05-01

    Full Text Available Elongator is required for the synthesis of the mcm5s2 modification found on tRNAs recognizing AA-ending codons. In order to obtain a global picture of the role of Elongator in translation, we used reverse protein arrays to screen the fission yeast proteome for translation defects. Unexpectedly, this revealed that Elongator inactivation mainly affected three specific functional groups including proteins implicated in cell division. The absence of Elongator results in a delay in mitosis onset and cytokinesis defects. We demonstrate that the kinase Cdr2, which is a central regulator of mitosis and cytokinesis, is under translational control by Elongator due to the Lysine codon usage bias of the cdr2 coding sequence. These findings uncover a mechanism by which the codon usage, coupled to tRNA modifications, fundamentally contributes to gene expression and cellular functions.

  15. David and Goliath: chemical perturbation of eukaryotes by bacteria.

    Science.gov (United States)

    Ho, Louis K; Nodwell, Justin R

    2016-03-01

    Environmental microbes produce biologically active small molecules that have been mined extensively as antibiotics and a smaller number of drugs that act on eukaryotic cells. It is known that there are additional bioactives to be discovered from this source. While the discovery of new antibiotics is challenged by the frequent discovery of known compounds, we contend that the eukaryote-active compounds may be less saturated. Indeed, despite there being far fewer eukaryotic-active natural products these molecules interact with a far richer diversity of molecular and cellular targets.

  16. Yeast ecology of Kombucha fermentation.

    Science.gov (United States)

    Teoh, Ai Leng; Heard, Gillian; Cox, Julian

    2004-09-01

    Kombucha is a traditional fermentation of sweetened tea, involving a symbiosis of yeast species and acetic acid bacteria. Despite reports of different yeast species being associated with the fermentation, little is known of the quantitative ecology of yeasts in Kombucha. Using oxytetracycline-supplemented malt extract agar, yeasts were isolated from four commercially available Kombucha products and identified using conventional biochemical and physiological tests. During the fermentation of each of the four products, yeasts were enumerated from both the cellulosic pellicle and liquor of the Kombucha. The number and diversity of species varied between products, but included Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii and Zygosaccharomyces bailii. While these yeast species are known to occur in Kombucha, the enumeration of each species present throughout fermentation of each of the four Kombucha cultures demonstrated for the first time the dynamic nature of the yeast ecology. Kombucha fermentation is, in general, initiated by osmotolerant species, succeeded and ultimately dominated by acid-tolerant species.

  17. Transcript specificity in yeast pre-mRNA splicing revealed by mutations in core spliceosomal components.

    Directory of Open Access Journals (Sweden)

    Jeffrey A Pleiss

    2007-04-01

    Full Text Available Appropriate expression of most eukaryotic genes requires the removal of introns from their pre-messenger RNAs (pre-mRNAs, a process catalyzed by the spliceosome. In higher eukaryotes a large family of auxiliary factors known as SR proteins can improve the splicing efficiency of transcripts containing suboptimal splice sites by interacting with distinct sequences present in those pre-mRNAs. The yeast Saccharomyces cerevisiae lacks functional equivalents of most of these factors; thus, it has been unclear whether the spliceosome could effectively distinguish among transcripts. To address this question, we have used a microarray-based approach to examine the effects of mutations in 18 highly conserved core components of the spliceosomal machinery. The kinetic profiles reveal clear differences in the splicing defects of particular pre-mRNA substrates. Most notably, the behaviors of ribosomal protein gene transcripts are generally distinct from other intron-containing transcripts in response to several spliceosomal mutations. However, dramatically different behaviors can be seen for some pairs of transcripts encoding ribosomal protein gene paralogs, suggesting that the spliceosome can readily distinguish between otherwise highly similar pre-mRNAs. The ability of the spliceosome to distinguish among its different substrates may therefore offer an important opportunity for yeast to regulate gene expression in a transcript-dependent fashion. Given the high level of conservation of core spliceosomal components across eukaryotes, we expect that these results will significantly impact our understanding of how regulated splicing is controlled in higher eukaryotes as well.

  18. Measuring strand discontinuity-directed mismatch repair in yeast Saccharomyces cerevisiae by cell-free nuclear extracts.

    Science.gov (United States)

    Yuan, Fenghua; Lai, Fangfang; Gu, Liya; Zhou, Wen; El Hokayem, Jimmy; Zhang, Yanbin

    2009-05-01

    Mismatch repair corrects biosynthetic errors generated during DNA replication, whose deficiency causes a mutator phenotype and directly underlies hereditary non-polyposis colorectal cancer and sporadic cancers. Because of remarkably high conservation of the mismatch repair machinery between the budding yeast (Saccharomyces cerevisiae) and humans, the study of mismatch repair in yeast has provided tremendous insights into the mechanisms of this repair pathway in humans. In addition, yeast cells possess an unbeatable advantage over human cells in terms of the easy genetic manipulation, the availability of whole genome deletion strains, and the relatively low cost for setting up the system. Although many components of eukaryotic mismatch repair have been identified, it remains unclear if additional factors, such as DNA helicase(s) and redundant nuclease(s) besides EXO1, participate in eukaryotic mismatch repair. To facilitate the discovery of novel mismatch repair factors, we developed a straightforward in vitro cell-free repair system. Here, we describe the practical protocols for preparation of yeast cell-free nuclear extracts and DNA mismatch substrates, and the in vitro mismatch repair assay. The validity of the cell-free system was confirmed by the mismatch repair deficient yeast strain (Deltamsh2) and the complementation assay with purified yeast MSH2-MSH6.

  19. Mapping Translation Technology Research in Translation Studies

    DEFF Research Database (Denmark)

    Schjoldager, Anne; Christensen, Tina Paulsen; Flanagan, Marian

    2017-01-01

    /Schjoldager 2010, 2011; Christensen 2011). Unfortunately, the increasing professional use of translation technology has not been mirrored within translation studies (TS) by a similar increase in research projects on translation technology (Munday 2009: 15; O’Hagan 2013; Doherty 2016: 952). The current thematic...... section aims to improve this situation by presenting new and innovative research papers that reflect on recent technological advances and their impact on the translation profession and translators from a diversity of perspectives and using a variety of methods. In Section 2, we present translation...... technology research as a subdiscipline of TS, and we define and discuss some basic concepts and models of the field that we use in the rest of the paper. Based on a small-scale study of papers published in TS journals between 2006 and 2016, Section 3 attempts to map relevant developments of translation...

  20. Phosphorylation and cellular function of the human Rpa2 N-terminus in the budding yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Ghospurkar, Padmaja L; Wilson, Timothy M; Liu, Shengqin; Herauf, Anna; Steffes, Jenna; Mueller, Erica N; Oakley, Gregory G; Haring, Stuart J

    2015-02-01

    Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3-4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Synthetic genome engineering forging new frontiers for wine yeast.

    Science.gov (United States)

    Pretorius, Isak S

    2017-02-01

    Over the past 15 years, the seismic shifts caused by the convergence of biomolecular, chemical, physical, mathematical, and computational sciences alongside cutting-edge developments in information technology and engineering have erupted into a new field of scientific endeavor dubbed Synthetic Biology. Recent rapid advances in high-throughput DNA sequencing and DNA synthesis techniques are enabling the design and construction of new biological parts (genes), devices (gene networks) and modules (biosynthetic pathways), and the redesign of biological systems (cells and organisms) for useful purposes. In 2014, the budding yeast Saccharomyces cerevisiae became the first eukaryotic cell to be equipped with a fully functional synthetic chromosome. This was achieved following the synthesis of the first viral (poliovirus in 2002 and bacteriophage Phi-X174 in 2003) and bacterial (Mycoplasma genitalium in 2008 and Mycoplasma mycoides in 2010) genomes, and less than two decades after revealing the full genome sequence of a laboratory (S288c in 1996) and wine (AWRI1631 in 2008) yeast strain. A large international project - the Synthetic Yeast Genome (Sc2.0) Project - is now underway to synthesize all 16 chromosomes (∼12 Mb carrying ∼6000 genes) of the sequenced S288c laboratory strain by 2018. If successful, S. cerevisiae will become the first eukaryote to cross the horizon of in silico design of complex cells through de novo synthesis, reshuffling, and editing of genomes. In the meantime, yeasts are being used as cell factories for the semi-synthetic production of high-value compounds, such as the potent antimalarial artemisinin, and food ingredients, such as resveratrol, vanillin, stevia, nootkatone, and saffron. As a continuum of previously genetically engineered industrially important yeast strains, precision genome engineering is bound to also impact the study and development of wine yeast strains supercharged with synthetic DNA. The first taste of what the future

  2. Potential of industrial biotechnology with cyanobacteria and eukaryotic microalgae.

    NARCIS (Netherlands)

    Wijffels, R.H.; Kruse, O.; Hellingwerf, K.J.

    2013-01-01

    Both cyanobacteria and eukaryotic microalgae are promising organisms for sustainable production of bulk products such as food, feed, materials, chemicals and fuels. In this review we will summarize the potential and current biotechnological developments. Cyanobacteria are promising host organisms

  3. Conservation and Variability of Meiosis Across the Eukaryotes.

    Science.gov (United States)

    Loidl, Josef

    2016-11-23

    Comparisons among a variety of eukaryotes have revealed considerable variability in the structures and processes involved in their meiosis. Nevertheless, conventional forms of meiosis occur in all major groups of eukaryotes, including early-branching protists. This finding confirms that meiosis originated in the common ancestor of all eukaryotes and suggests that primordial meiosis may have had many characteristics in common with conventional extant meiosis. However, it is possible that the synaptonemal complex and the delicate crossover control related to its presence were later acquisitions. Later still, modifications to meiotic processes occurred within different groups of eukaryotes. Better knowledge on the spectrum of derived and uncommon forms of meiosis will improve our understanding of many still mysterious aspects of the meiotic process and help to explain the evolutionary basis of functional adaptations to the meiotic program.

  4. DNA mismatch repair and its many roles in eukaryotic cells

    DEFF Research Database (Denmark)

    Liu, Dekang; Keijzers, Guido; Rasmussen, Lene Juel

    2017-01-01

    in the clinic, and as a biomarker of cancer susceptibility in animal model systems. Prokaryotic MMR is well-characterized at the molecular and mechanistic level; however, MMR is considerably more complex in eukaryotic cells than in prokaryotic cells, and in recent years, it has become evident that MMR plays...... novel roles in eukaryotic cells, several of which are not yet well-defined or understood. Many MMR-deficient human cancer cells lack mutations in known human MMR genes, which strongly suggests that essential eukaryotic MMR components/cofactors remain unidentified and uncharacterized. Furthermore......, the mechanism by which the eukaryotic MMR machinery discriminates between the parental (template) and the daughter (nascent) DNA strand is incompletely understood and how cells choose between the EXO1-dependent and the EXO1–independent subpathways of MMR is not known. This review summarizes recent literature...

  5. Nitrate storage and dissimilatory nitrate reduction by eukaryotic microbes

    DEFF Research Database (Denmark)

    Kamp, Anja; Høgslund, Signe; Risgaard-Petersen, Nils

    2015-01-01

    The microbial nitrogen cycle is one of the most complex and environmentally important element cycles on Earth and has long been thought to be mediated exclusively by prokaryotic microbes. Rather recently, it was discovered that certain eukaryotic microbes are able to store nitrate intracellularly......, suggesting that eukaryotes may rival prokaryotes in terms of dissimilatory nitrate reduction. Finally, this review article sketches some evolutionary perspectives of eukaryotic nitrate metabolism and identifies open questions that need to be addressed in future investigations....... and use it for dissimilatory nitrate reduction in the absence of oxygen. The paradigm shift that this entailed is ecologically significant because the eukaryotes in question comprise global players like diatoms, foraminifers, and fungi. This review article provides an unprecedented overview of nitrate...

  6. Massive expansion of the calpain gene family in unicellular eukaryotes

    Directory of Open Access Journals (Sweden)

    Zhao Sen

    2012-09-01

    Full Text Available Abstract Background Calpains are Ca2+-dependent cysteine proteases that participate in a range of crucial cellular processes. Dysfunction of these enzymes may cause, for instance, life-threatening diseases in humans, the loss of sex determination in nematodes and embryo lethality in plants. Although the calpain family is well characterized in animal and plant model organisms, there is a great lack of knowledge about these genes in unicellular eukaryote species (i.e. protists. Here, we study the distribution and evolution of calpain genes in a wide range of eukaryote genomes from major branches in the tree of life. Results Our investigations reveal 24 types of protein domains that are combined with the calpain-specific catalytic domain CysPc. In total we identify 41 different calpain domain architectures, 28 of these domain combinations have not been previously described. Based on our phylogenetic inferences, we propose that at least four calpain variants were established in the early evolution of eukaryotes, most likely before the radiation of all the major supergroups of eukaryotes. Many domains associated with eukaryotic calpain genes can be found among eubacteria or archaebacteria but never in combination with the CysPc domain. Conclusions The analyses presented here show that ancient modules present in prokaryotes, and a few de novo eukaryote domains, have been assembled into many novel domain combinations along the evolutionary history of eukaryotes. Some of the new calpain genes show a narrow distribution in a few branches in the tree of life, likely representing lineage-specific innovations. Hence, the functionally important classical calpain genes found among humans and vertebrates make up only a tiny fraction of the calpain family. In fact, a massive expansion of the calpain family occurred by domain shuffling among unicellular eukaryotes and contributed to a wealth of functionally different genes.

  7. Massive expansion of the calpain gene family in unicellular eukaryotes.

    Science.gov (United States)

    Zhao, Sen; Liang, Zhe; Demko, Viktor; Wilson, Robert; Johansen, Wenche; Olsen, Odd-Arne; Shalchian-Tabrizi, Kamran

    2012-09-29

    Calpains are Ca2+-dependent cysteine proteases that participate in a range of crucial cellular processes. Dysfunction of these enzymes may cause, for instance, life-threatening diseases in humans, the loss of sex determination in nematodes and embryo lethality in plants. Although the calpain family is well characterized in animal and plant model organisms, there is a great lack of knowledge about these genes in unicellular eukaryote species (i.e. protists). Here, we study the distribution and evolution of calpain genes in a wide range of eukaryote genomes from major branches in the tree of life. Our investigations reveal 24 types of protein domains that are combined with the calpain-specific catalytic domain CysPc. In total we identify 41 different calpain domain architectures, 28 of these domain combinations have not been previously described. Based on our phylogenetic inferences, we propose that at least four calpain variants were established in the early evolution of eukaryotes, most likely before the radiation of all the major supergroups of eukaryotes. Many domains associated with eukaryotic calpain genes can be found among eubacteria or archaebacteria but never in combination with the CysPc domain. The analyses presented here show that ancient modules present in prokaryotes, and a few de novo eukaryote domains, have been assembled into many novel domain combinations along the evolutionary history of eukaryotes. Some of the new calpain genes show a narrow distribution in a few branches in the tree of life, likely representing lineage-specific innovations. Hence, the functionally important classical calpain genes found among humans and vertebrates make up only a tiny fraction of the calpain family. In fact, a massive expansion of the calpain family occurred by domain shuffling among unicellular eukaryotes and contributed to a wealth of functionally different genes.

  8. Repair of DNA DSB in higher eukaryotes

    International Nuclear Information System (INIS)

    Wang, H.; Perrault, A.R.; Takeda, Y.; Iliakis, G.

    2003-01-01

    Cells of higher eukaryotes process within minutes double strand breaks (DSBs) in their genome using a NHEJ apparatus that engages DNA-PKcs, Ku, DNA ligase IV, XRCC4, and other as of yet unidentified factors. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DNA DSBs using an alternative pathway operating with slower kinetics. This alternative pathway is active in mutants deficient in genes of the RAD52 epistasis group. We proposed, therefore, that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK- dependent (D-NHEJ) pathway, rather than homology directed repair of DSBs. We studied the role of Ku and DNA-PKcs in the coordination of these pathways using as a model end joining of restriction endonuclease linearized plasmid DNA in whole cell extracts. Efficient error-free endjoining observed in such in-vitro reactions is strongly inhibited by anti-Ku antibodies. The inhibition requires DNA-PKcs, despite that fact that Ku efficiently binds DNA ends in the presence of antibodies, or in the absence of DNA-PKcs. Strong inhibition of DNA endjoining is also mediated by wortmannin, an inhibitor of DNA-PKcs, in the presence but not in the absence of Ku, and this inhibition can be rescued by pre-incubating the reaction with double stranded oligonucleotides. The results are compatible with a role of Ku in directing endjoining to a DNA-PK dependent pathway, mediated by efficient end binding and productive interactions with DNA-PKcs. On the other hand, efficient end joining is observed in extracts of cells lacking DNA-PKcs, as well as in Ku-depleted extracts sugggesting the operation of alternative pathways. Extracts depleted of Ku and DNA-PKcs rejoin blunt ends, as well as homologous ends with 3' or 5' protruding single strands with similar efficiency, but addition of Ku suppresses joining of blunt ends and homologous ends with 3' overhangs. We propose that the

  9. Genetically engineered yeast

    DEFF Research Database (Denmark)

    2014-01-01

    A genetically modified Saccharomyces cerevisiae comprising an active fermentation pathway producing 3-HP expresses an exogenous gene expressing the aminotransferase YhxA from Bacillus cereus AH1272 catalysing a transamination reaction between beta-alanine and pyruvate to produce malonate semialde......A genetically modified Saccharomyces cerevisiae comprising an active fermentation pathway producing 3-HP expresses an exogenous gene expressing the aminotransferase YhxA from Bacillus cereus AH1272 catalysing a transamination reaction between beta-alanine and pyruvate to produce malonate...... semialdehyde. The yeast may also express a 3-hydroxyisobutyrate dehydrogenase (HIBADH) and a 3-hydroxypropanoate dehydrogenase (3-HPDH) and aspartate 1-decarboxylase. Additionally the yeast may express pyruvate carboxylase and aspartate aminotransferase....

  10. Single Cell Genomics and Transcriptomics for Unicellular Eukaryotes

    Energy Technology Data Exchange (ETDEWEB)

    Ciobanu, Doina; Clum, Alicia; Singh, Vasanth; Salamov, Asaf; Han, James; Copeland, Alex; Grigoriev, Igor; James, Timothy; Singer, Steven; Woyke, Tanja; Malmstrom, Rex; Cheng, Jan-Fang

    2014-03-14

    Despite their small size, unicellular eukaryotes have complex genomes with a high degree of plasticity that allow them to adapt quickly to environmental changes. Unicellular eukaryotes live with prokaryotes and higher eukaryotes, frequently in symbiotic or parasitic niches. To this day their contribution to the dynamics of the environmental communities remains to be understood. Unfortunately, the vast majority of eukaryotic microorganisms are either uncultured or unculturable, making genome sequencing impossible using traditional approaches. We have developed an approach to isolate unicellular eukaryotes of interest from environmental samples, and to sequence and analyze their genomes and transcriptomes. We have tested our methods with six species: an uncharacterized protist from cellulose-enriched compost identified as Platyophrya, a close relative of P. vorax; the fungus Metschnikowia bicuspidate, a parasite of water flea Daphnia; the mycoparasitic fungi Piptocephalis cylindrospora, a parasite of Cokeromyces and Mucor; Caulochytrium protosteloides, a parasite of Sordaria; Rozella allomycis, a parasite of the water mold Allomyces; and the microalgae Chlamydomonas reinhardtii. Here, we present the four components of our approach: pre-sequencing methods, sequence analysis for single cell genome assembly, sequence analysis of single cell transcriptomes, and genome annotation. This technology has the potential to uncover the complexity of single cell eukaryotes and their role in the environmental samples.

  11. An Evolutionary Framework for Understanding the Origin of Eukaryotes

    Directory of Open Access Journals (Sweden)

    Neil W. Blackstone

    2016-04-01

    Full Text Available Two major obstacles hinder the application of evolutionary theory to the origin of eukaryotes. The first is more apparent than real—the endosymbiosis that led to the mitochondrion is often described as “non-Darwinian” because it deviates from the incremental evolution championed by the modern synthesis. Nevertheless, endosymbiosis can be accommodated by a multi-level generalization of evolutionary theory, which Darwin himself pioneered. The second obstacle is more serious—all of the major features of eukaryotes were likely present in the last eukaryotic common ancestor thus rendering comparative methods ineffective. In addition to a multi-level theory, the development of rigorous, sequence-based phylogenetic and comparative methods represents the greatest achievement of modern evolutionary theory. Nevertheless, the rapid evolution of major features in the eukaryotic stem group requires the consideration of an alternative framework. Such a framework, based on the contingent nature of these evolutionary events, is developed and illustrated with three examples: the putative intron proliferation leading to the nucleus and the cell cycle; conflict and cooperation in the origin of eukaryotic bioenergetics; and the inter-relationship between aerobic metabolism, sterol synthesis, membranes, and sex. The modern synthesis thus provides sufficient scope to develop an evolutionary framework to understand the origin of eukaryotes.

  12. Compositional patterns in the genomes of unicellular eukaryotes.

    Science.gov (United States)

    Costantini, Maria; Alvarez-Valin, Fernando; Costantini, Susan; Cammarano, Rosalia; Bernardi, Giorgio

    2013-11-05

    The genomes of multicellular eukaryotes are compartmentalized in mosaics of isochores, large and fairly homogeneous stretches of DNA that belong to a small number of families characterized by different average GC levels, by different gene concentration (that increase with GC), different chromatin structures, different replication timing in the cell cycle, and other different properties. A question raised by these basic results concerns how far back in evolution the compartmentalized organization of the eukaryotic genomes arose. In the present work we approached this problem by studying the compositional organization of the genomes from the unicellular eukaryotes for which full sequences are available, the sample used being representative. The average GC levels of the genomes from unicellular eukaryotes cover an extremely wide range (19%-60% GC) and the compositional patterns of individual genomes are extremely different but all genomes tested show a compositional compartmentalization. The average GC range of the genomes of unicellular eukaryotes is very broad (as broad as that of prokaryotes) and individual compositional patterns cover a very broad range from very narrow to very complex. Both features are not surprising for organisms that are very far from each other both in terms of phylogenetic distances and of environmental life conditions. Most importantly, all genomes tested, a representative sample of all supergroups of unicellular eukaryotes, are compositionally compartmentalized, a major difference with prokaryotes.

  13. Cell signaling, post-translational protein modifications and NMR spectroscopy

    International Nuclear Information System (INIS)

    Theillet, Francois-Xavier; Smet-Nocca, Caroline; Liokatis, Stamatios; Thongwichian, Rossukon; Kosten, Jonas; Yoon, Mi-Kyung; Kriwacki, Richard W.; Landrieu, Isabelle; Lippens, Guy; Selenko, Philipp

    2012-01-01

    Post-translationally modified proteins make up the majority of the proteome and establish, to a large part, the impressive level of functional diversity in higher, multi-cellular organisms. Most eukaryotic post-translational protein modifications (PTMs) denote reversible, covalent additions of small chemical entities such as phosphate-, acyl-, alkyl- and glycosyl-groups onto selected subsets of modifiable amino acids. In turn, these modifications induce highly specific changes in the chemical environments of individual protein residues, which are readily detected by high-resolution NMR spectroscopy. In the following, we provide a concise compendium of NMR characteristics of the main types of eukaryotic PTMs: serine, threonine, tyrosine and histidine phosphorylation, lysine acetylation, lysine and arginine methylation, and serine, threonine O-glycosylation. We further delineate the previously uncharacterized NMR properties of lysine propionylation, butyrylation, succinylation, malonylation and crotonylation, which, altogether, define an initial reference frame for comprehensive PTM studies by high-resolution NMR spectroscopy.

  14. Tapping into yeast diversity.

    Science.gov (United States)

    Fay, Justin C

    2012-11-01

    Domesticated organisms demonstrate our capacity to influence wild species but also provide us with the opportunity to understand rapid evolution in the context of substantially altered environments and novel selective pressures. Recent advances in genetics and genomics have brought unprecedented insights into the domestication of many organisms and have opened new avenues for further improvements to be made. Yet, our ability to engineer biological systems is not without limits; genetic manipulation is often quite difficult. The budding yeast, Saccharomyces cerevisiae, is not only one of the most powerful model organisms, but is also the premier producer of fermented foods and beverages around the globe. As a model system, it entertains a hefty workforce dedicated to deciphering its genome and the function it encodes at a rich mechanistic level. As a producer, it is used to make leavened bread, and dozens of different alcoholic beverages, such as beer and wine. Yet, applying the awesome power of yeast genetics to understanding its origins and evolution requires some knowledge of its wild ancestors and the environments from which they were derived. A number of surprisingly diverse lineages of S. cerevisiae from both primeval and secondary forests in China have been discovered by Wang and his colleagues. These lineages substantially expand our knowledge of wild yeast diversity and will be a boon to elucidating the ecology, evolution and domestication of this academic and industrial workhorse.

  15. Mechanisms of uv mutagenesis in yeast

    International Nuclear Information System (INIS)

    Lawrence, C.W.; Christensen, R.; Schwartz, A.

    1982-01-01

    The uv mutagenesis in yeast depends on the function of the RAD6 locus, a gene that is also responsible for a substantial fraction of wild-type resistance, suggesting that this eukaryote may possess a misrepair mechanism analogous to that proposed for Escherichia coli. The molecular mechanism responsible for RAD6 repair or recovery is not yet known, but it is different from either excision or recombination-dependent repair, processes carried out by the other two main repair pathways in yeast. RAD6-dependent mutagenesis has been found to have the following characteristics. It is associated at best with only a small fraction of RAD6-dependent repair, the majority of the sensitivity of rad6 mutants being due to their lack of nonmutagenic repair. SRS2 metabolic suppressors restore a substantial fraction of uv resistance to rad6 mutants but do not restore their uv mutability. Strains containing mutations at loci (rev, umr) that are probably more directly involved in mutagenesis are only mildly sensitive, and there is a poor correlation between their sensitivity and mutational deficiency. The uv mutagenesis appears to require a large number of gene functions, perhaps ten or more. Where examined in detail, these genes have been found to be concerned in the production of only a specific range of mutational events, not all of them. Mating experiments have shown that a substantial fraction, probably 40% or more, of uv-induced mutations are untargeted, that is, occur in lesion-free regions of DNA. The uv irradiation, therefore, produces a general reduction in the normally high fidelity with which DNA is replicated on undamaged templates. It does not appear to be necessary for the causal lesion to be present in the same chromosome as the mutation it induces. The reduction in fidelity may be the consequence of the production of a diffusible factor in uv-irradiated cells, but definite evidence supporting this proposal has not yet been obtained

  16. On Various Negative Translations

    Directory of Open Access Journals (Sweden)

    Gilda Ferreira

    2011-01-01

    Full Text Available Several proof translations of classical mathematics into intuitionistic mathematics have been proposed in the literature over the past century. These are normally referred to as negative translations or double-negation translations. Among those, the most commonly cited are translations due to Kolmogorov, Godel, Gentzen, Kuroda and Krivine (in chronological order. In this paper we propose a framework for explaining how these different translations are related to each other. More precisely, we define a notion of a (modular simplification starting from Kolmogorov translation, which leads to a partial order between different negative translations. In this derived ordering, Kuroda and Krivine are minimal elements. Two new minimal translations are introduced, with Godel and Gentzen translations sitting in between Kolmogorov and one of these new translations.

  17. Gender issues in translation

    OpenAIRE

    ERGASHEVA G.I.

    2015-01-01

    The following research is done regarding gender in translation dealing specifically with the issue of the translators’ gender identity and its effect on their translations, as well as on how gender itself is translated and produced. We will try to clarify what gender is, how gender manifests itself in the system of language, and what problems translators encounter when translating or producing gender-related materials

  18. Cultural Context and Translation

    Institute of Scientific and Technical Information of China (English)

    张敏

    2009-01-01

    cultural context plays an important role in translation. Because translation is a cross-culture activity, the culture context that influ-ences translating is consisted of both the culture contexts of source language and target language. This article firstly analyzes the concept of context and cultural context, then according to the procedure of translating classifies cultural context into two stages and talks about how they respectively influence translating.

  19. Evolutionary Conservation and Diversification of the Translation Initiation Apparatus in Trypanosomatids

    Directory of Open Access Journals (Sweden)

    Alexandra Zinoviev

    2012-01-01

    Full Text Available Trypanosomatids are ancient eukaryotic parasites that migrate between insect vectors and mammalian hosts, causing a range of diseases in humans and domestic animals. Trypanosomatids feature a multitude of unusual molecular features, including polycistronic transcription and subsequent processing by trans-splicing and polyadenylation. Regulation of protein coding genes is posttranscriptional and thus, translation regulation is fundamental for activating the developmental program of gene expression. The spliced-leader RNA is attached to all mRNAs. It contains an unusual hypermethylated cap-4 structure in its 5 end. The cap-binding complex, eIF4F, has gone through evolutionary changes in accordance with the requirement to bind cap-4. The eIF4F components in trypanosomatids are highly diverged from their orthologs in higher eukaryotes, and their potential functions are discussed. The cap-binding activity in all eukaryotes is a target for regulation and plays a similar role in trypanosomatids. Recent studies revealed a novel eIF4E-interacting protein, involved in directing stage-specific and stress-induced translation pathways. Translation regulation during stress also follows unusual regulatory cues, as the increased translation of Hsp83 following heat stress is driven by a defined element in the 3 UTR, unlike higher eukaryotes. Overall, the environmental switches experienced by trypanosomatids during their life cycle seem to affect their translational machinery in unique ways.

  20. Rec8p, a meiotic recombination and sister chromatid cohesion phosphoprotein of the Rad21p family conserved from fision yeast to humans.

    NARCIS (Netherlands)

    S. Parisi; M.J. McKay (Michael); M. Molnar; M.A. Thompson (Anne); P.J. van der Spek (Peter); E. van Drunen-Schoenmaker; R. Kanaar (Roland); E. Lehmann; J.H.J. Hoeijmakers (Jan); J. Kohli

    1999-01-01

    textabstractOur work and that of others defined mitosis-specific (Rad21 subfamily) and meiosis-specific (Rec8 subfamily) proteins involved in sister chromatid cohesion in several eukaryotes, including humans. Mutation of the fission yeast Schizosaccharomyces pombe rec8 gene was previously shown to

  1. A Western Blot-based Investigation of the Yeast Secretory Pathway Designed for an Intermediate-Level Undergraduate Cell Biology Laboratory

    Science.gov (United States)

    Hood-DeGrenier, Jennifer K.

    2008-01-01

    The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in…

  2. Translation-coupling systems

    Science.gov (United States)

    Pfleger, Brian; Mendez-Perez, Daniel

    2013-11-05

    Disclosed are systems and methods for coupling translation of a target gene to a detectable response gene. A version of the invention includes a translation-coupling cassette. The translation-coupling cassette includes a target gene, a response gene, a response-gene translation control element, and a secondary structure-forming sequence that reversibly forms a secondary structure masking the response-gene translation control element. Masking of the response-gene translation control element inhibits translation of the response gene. Full translation of the target gene results in unfolding of the secondary structure and consequent translation of the response gene. Translation of the target gene is determined by detecting presence of the response-gene protein product. The invention further includes RNA transcripts of the translation-coupling cassettes, vectors comprising the translation-coupling cassettes, hosts comprising the translation-coupling cassettes, methods of using the translation-coupling cassettes, and gene products produced with the translation-coupling cassettes.

  3. Studies of the expression of human poly(ADP-ribose) polymerase-1 in Saccharomyces cerevisiae and identification of PARP-1 substrates by yeast proteome microarray screening.

    Science.gov (United States)

    Tao, Zhihua; Gao, Peng; Liu, Hung-Wen

    2009-12-15

    Poly(ADP-ribosyl)ation of various nuclear proteins catalyzed by a family of NAD(+)-dependent enzymes, poly(ADP-ribose) polymerases (PARPs), is an important posttranslational modification reaction. PARP activity has been demonstrated in all types of eukaryotic cells with the exception of yeast, in which the expression of human PARP-1 was shown to lead to retarded cell growth. We investigated the yeast growth inhibition caused by human PARP-1 expression in Saccharomyces cerevisiae. Flow cytometry analysis reveals that PARP-1-expressing yeast cells accumulate in the G(2)/M stage of the cell cycle. Confocal microscopy analysis shows that human PARP-1 is distributed throughout the nucleus of yeast cells but is enriched in the nucleolus. Utilizing yeast proteome microarray screening, we identified 33 putative PARP-1 substrates, six of which are known to be involved in ribosome biogenesis. The poly(ADP-ribosyl)ation of three of these yeast proteins, together with two human homologues, was confirmed by an in vitro PARP-1 assay. Finally, a polysome profile analysis using sucrose gradient ultracentrifugation demonstrated that the ribosome levels in yeast cells expressing PARP-1 are lower than those in control yeast cells. Overall, our data suggest that human PARP-1 may affect ribosome biogenesis by modifying certain nucleolar proteins in yeast. The artificial PARP-1 pathway in yeast may be used as a simple platform to identify substrates and verify function of this important enzyme.

  4. Electrochemical Glucose Biosensor Based on Glucose Oxidase Displayed on Yeast Surface.

    Science.gov (United States)

    Wang, Hongwei; Lang, Qiaolin; Liang, Bo; Liu, Aihua

    2015-01-01

    The conventional enzyme-based biosensor requires chemical or physical immobilization of purified enzymes on electrode surface, which often results in loss of enzyme activity and/or fractions immobilized over time. It is also costly. A major advantage of yeast surface display is that it enables the direct utilization of whole cell catalysts with eukaryote-produced proteins being displayed on the cell surface, providing an economic alternative to traditional production of purified enzymes. Herein, we describe the details of the display of glucose oxidase (GOx) on yeast cell surface and its application in the development of electrochemical glucose sensor. In order to achieve a direct electrochemistry of GOx, the entire cell catalyst (yeast-GOx) was immobilized together with multiwalled carbon nanotubes on the electrode, which allowed sensitive and selective glucose detection.

  5. Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

    DEFF Research Database (Denmark)

    Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L.

    2015-01-01

    There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing...... interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV...... mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant a-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330...

  6. Genome sequence of the oleaginous yeast Rhodotorula toruloides strain CGMCC 2.1609

    Directory of Open Access Journals (Sweden)

    Christine Sambles

    2017-09-01

    Full Text Available Most eukaryotic oleaginous species are yeasts and among them the basidiomycete red yeast, Rhodotorula (Rhodosporidium toruloides (Pucciniomycotina is known to produce high quantities of lipids when grown in nitrogen-limiting media, and has potential for biodiesel production. The genome of the CGMCC 2.1609 strain of this oleaginous red yeast was sequenced using a hybrid of Roche 454 and Illumina technology generating 13× coverage. The de novo assembly was carried out using MIRA and scaffolded using MAQ and BAMBUS. The sequencing and assembly resulted in 365 scaffolds with total genome size of 33.4 Mb. The complete genome sequence of this strain was deposited in GenBank and the accession number is LKER00000000. The annotation is available on Figshare (doi:10.6084/m9.figshare.4754251.

  7. Non-Genetic Engineering Approaches for Isolating and Generating Novel Yeasts for Industrial Applications

    Science.gov (United States)

    Chambers, P. J.; Bellon, J. R.; Schmidt, S. A.; Varela, C.; Pretorius, I. S.

    Generating novel yeast strains for industrial applications should be quite straightforward; after all, research into the genetics, biochemistry and physiology of Baker's Yeast, Saccharomyces cerevisiae, has paved the way for many advances in the modern biological sciences. We probably know more about this humble eukaryote than any other, and it is the most tractable of organisms for manipulation using modern genetic engineering approaches. In many countries, however, there are restrictions on the use of genetically-modified organisms (GMOs), particularly in foods and beverages, and the level of consumer acceptance of GMOs is, at best, variable. Thus, many researchers working with industrial yeasts use genetic engineering techniques primarily as research tools, and strain development continues to rely on non-GM technologies. This chapter explores the non-GM tools and strategies available to such researchers.

  8. Modifying chemotherapy response by targeted inhibition of eukaryotic initiation factor 4A

    International Nuclear Information System (INIS)

    Cencic, R; Robert, F; Galicia-Vázquez, G; Malina, A; Ravindar, K; Somaiah, R; Pierre, P; Tanaka, J; Deslongchamps, P; Pelletier, J

    2013-01-01

    Translation is regulated predominantly at the initiation phase by several signal transduction pathways that are often usurped in human cancers, including the PI3K/Akt/mTOR axis. mTOR exerts unique administration over translation by regulating assembly of eukaryotic initiation factor (eIF) 4F, a heterotrimeric complex responsible for recruiting 40S ribosomes (and associated factors) to mRNA 5′ cap structures. Hence, there is much interest in targeted therapies that block eIF4F activity to assess the consequences on tumor cell growth and chemotherapy response. We report here that hippuristanol (Hipp), a translation initiation inhibitor that selectively inhibits the eIF4F RNA helicase subunit, eIF4A, resensitizes Eμ-Myc lymphomas to DNA damaging agents, including those that overexpress eIF4E—a modifier of rapamycin responsiveness. As Mcl-1 levels are significantly affected by Hipp, combining its use with the Bcl-2 family inhibitor, ABT-737, leads to a potent synergistic response in triggering cell death in mouse and human lymphoma and leukemia cells. Suppression of eIF4AI using RNA interference also synergized with ABT-737 in murine lymphomas, highlighting eIF4AI as a therapeutic target for modulating tumor cell response to chemotherapy

  9. Genome and metabolic engineering in non-conventional yeasts: Current advances and applications

    Directory of Open Access Journals (Sweden)

    Ann-Kathrin Löbs

    2017-09-01

    Full Text Available Microbial production of chemicals and proteins from biomass-derived and waste sugar streams is a rapidly growing area of research and development. While the model yeast Saccharomyces cerevisiae is an excellent host for the conversion of glucose to ethanol, production of other chemicals from alternative substrates often requires extensive strain engineering. To avoid complex and intensive engineering of S. cerevisiae, other yeasts are often selected as hosts for bioprocessing based on their natural capacity to produce a desired product: for example, the efficient production and secretion of proteins, lipids, and primary metabolites that have value as commodity chemicals. Even when using yeasts with beneficial native phenotypes, metabolic engineering to increase yield, titer, and production rate is essential. The non-conventional yeasts Kluyveromyces lactis, K. marxianus, Scheffersomyces stipitis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris have been developed as eukaryotic hosts because of their desirable phenotypes, including thermotolerance, assimilation of diverse carbon sources, and high protein secretion. However, advanced metabolic engineering in these yeasts has been limited. This review outlines the challenges of using non-conventional yeasts for strain and pathway engineering, and discusses the developed solutions to these problems and the resulting applications in industrial biotechnology.

  10. Genome and metabolic engineering in non-conventional yeasts: Current advances and applications.

    Science.gov (United States)

    Löbs, Ann-Kathrin; Schwartz, Cory; Wheeldon, Ian

    2017-09-01

    Microbial production of chemicals and proteins from biomass-derived and waste sugar streams is a rapidly growing area of research and development. While the model yeast Saccharomyces cerevisia e is an excellent host for the conversion of glucose to ethanol, production of other chemicals from alternative substrates often requires extensive strain engineering. To avoid complex and intensive engineering of S. cerevisiae, other yeasts are often selected as hosts for bioprocessing based on their natural capacity to produce a desired product: for example, the efficient production and secretion of proteins, lipids, and primary metabolites that have value as commodity chemicals. Even when using yeasts with beneficial native phenotypes, metabolic engineering to increase yield, titer, and production rate is essential. The non-conventional yeasts Kluyveromyces lactis, K. marxianus, Scheffersomyces stipitis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris have been developed as eukaryotic hosts because of their desirable phenotypes, including thermotolerance, assimilation of diverse carbon sources, and high protein secretion. However, advanced metabolic engineering in these yeasts has been limited. This review outlines the challenges of using non-conventional yeasts for strain and pathway engineering, and discusses the developed solutions to these problems and the resulting applications in industrial biotechnology.

  11. Network Thermodynamic Curation of Human and Yeast Genome-Scale Metabolic Models

    Science.gov (United States)

    Martínez, Verónica S.; Quek, Lake-Ee; Nielsen, Lars K.

    2014-01-01

    Genome-scale models are used for an ever-widening range of applications. Although there has been much focus on specifying the stoichiometric matrix, the predictive power of genome-scale models equally depends on reaction directions. Two-thirds of reactions in the two eukaryotic reconstructions Homo sapiens Recon 1 and Yeast 5 are specified as irreversible. However, these specifications are mainly based on biochemical textbooks or on their similarity to other organisms and are rarely underpinned by detailed thermodynamic analysis. In this study, a to our knowledge new workflow combining network-embedded thermodynamic and flux variability analysis was used to evaluate existing irreversibility constraints in Recon 1 and Yeast 5 and to identify new ones. A total of 27 and 16 new irreversible reactions were identified in Recon 1 and Yeast 5, respectively, whereas only four reactions were found with directions incorrectly specified against thermodynamics (three in Yeast 5 and one in Recon 1). The workflow further identified for both models several isolated internal loops that require further curation. The framework also highlighted the need for substrate channeling (in human) and ATP hydrolysis (in yeast) for the essential reaction catalyzed by phosphoribosylaminoimidazole carboxylase in purine metabolism. Finally, the framework highlighted differences in proline metabolism between yeast (cytosolic anabolism and mitochondrial catabolism) and humans (exclusively mitochondrial metabolism). We conclude that network-embedded thermodynamics facilitates the specification and validation of irreversibility constraints in compartmentalized metabolic models, at the same time providing further insight into network properties. PMID:25028891

  12. Writing Through: Practising Translation

    Directory of Open Access Journals (Sweden)

    Joel Scott

    2010-05-01

    Full Text Available This essay exists as a segment in a line of study and writing practice that moves between a critical theory analysis of translation studies conceptions of language, and the practical questions of what those ideas might mean for contemporary translation and writing practice. Although the underlying preoccupation of this essay, and my more general line of inquiry, is translation studies and practice, in many ways translation is merely a way into a discussion on language. For this essay, translation is the threshold of language. But the two trails of the discussion never manage to elude each other, and these concatenations have informed two experimental translation methods, referred to here as Live Translations and Series Translations. Following the essay are a number of poems in translation, all of which come from Blanco Nuclear by the contemporary Spanish poet, Esteban Pujals Gesalí. The first group, the Live Translations consist of transcriptions I made from audio recordings read in a public setting, in which the texts were translated in situ, either off the page of original Spanish-language poems, or through a process very much like that carried out by simultaneous translators, for which readings of the poems were played back to me through headphones at varying speeds to be translated before the audience. The translations collected are imperfect renderings, attesting to a moment in language practice rather than language objects. The second method involves an iterative translation process, by which three versions of any one poem are rendered, with varying levels of fluency, fidelity and servility. All three translations are presented one after the other as a series, with no version asserting itself as the primary translation. These examples, as well as the translation methods themselves, are intended as preliminary experiments within an endlessly divergent continuum of potential methods and translations, and not as a complete representation of

  13. Entropy analysis in yeast DNA

    International Nuclear Information System (INIS)

    Kim, Jongkwang; Kim, Sowun; Lee, Kunsang; Kwon, Younghun

    2009-01-01

    In this article, we investigate the language structure in yeast 16 chromosomes. In order to find it, we use the entropy analysis for codons (or amino acids) of yeast 16 chromosomes, developed in analysis of natural language by Montemurro et al. From the analysis, we can see that there exists a language structure in codons (or amino acids) of yeast 16 chromosomes. Also we find that the grammar structure of amino acids of yeast 16 chromosomes has a deep relationship with secondary structure of protein.

  14. Sharing the cell's bounty - organelle inheritance in yeast.

    Science.gov (United States)

    Knoblach, Barbara; Rachubinski, Richard A

    2015-02-15

    Eukaryotic cells replicate and partition their organelles between the mother cell and the daughter cell at cytokinesis. Polarized cells, notably the budding yeast Saccharomyces cerevisiae, are well suited for the study of organelle inheritance, as they facilitate an experimental dissection of organelle transport and retention processes. Much progress has been made in defining the molecular players involved in organelle partitioning in yeast. Each organelle uses a distinct set of factors - motor, anchor and adaptor proteins - that ensures its inheritance by future generations of cells. We propose that all organelles, regardless of origin or copy number, are partitioned by the same fundamental mechanism involving division and segregation. Thus, the mother cell keeps, and the daughter cell receives, their fair and equitable share of organelles. This mechanism of partitioning moreover facilitates the segregation of organelle fragments that are not functionally equivalent. In this Commentary, we describe how this principle of organelle population control affects peroxisomes and other organelles, and outline its implications for yeast life span and rejuvenation. © 2015. Published by The Company of Biologists Ltd.

  15. Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA–protein interactions

    Science.gov (United States)

    Khanova, Elena; Esakova, Olga; Perederina, Anna; Berezin, Igor; Krasilnikov, Andrey S.

    2012-01-01

    Eukaryotic ribonuclease (RNase) P and RNase MRP are closely related ribonucleoprotein complexes involved in the metabolism of various RNA molecules including tRNA, rRNA, and some mRNAs. While evolutionarily related to bacterial RNase P, eukaryotic enzymes of the RNase P/MRP family are much more complex. Saccharomyces cerevisiae RNase P consists of a catalytic RNA component and nine essential proteins; yeast RNase MRP has an RNA component resembling that in RNase P and 10 essential proteins, most of which are shared with RNase P. The structural organizations of eukaryotic RNases P/MRP are not clear. Here we present the results of RNA–protein UV crosslinking studies performed on RNase P and RNase MRP holoenzymes isolated from yeast. The results indicate locations of specific protein-binding sites in the RNA components of RNase P and RNase MRP and shed light on the structural organizations of these large ribonucleoprotein complexes. PMID:22332141

  16. Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA-protein interactions.

    Science.gov (United States)

    Khanova, Elena; Esakova, Olga; Perederina, Anna; Berezin, Igor; Krasilnikov, Andrey S

    2012-04-01

    Eukaryotic ribonuclease (RNase) P and RNase MRP are closely related ribonucleoprotein complexes involved in the metabolism of various RNA molecules including tRNA, rRNA, and some mRNAs. While evolutionarily related to bacterial RNase P, eukaryotic enzymes of the RNase P/MRP family are much more complex. Saccharomyces cerevisiae RNase P consists of a catalytic RNA component and nine essential proteins; yeast RNase MRP has an RNA component resembling that in RNase P and 10 essential proteins, most of which are shared with RNase P. The structural organizations of eukaryotic RNases P/MRP are not clear. Here we present the results of RNA-protein UV crosslinking studies performed on RNase P and RNase MRP holoenzymes isolated from yeast. The results indicate locations of specific protein-binding sites in the RNA components of RNase P and RNase MRP and shed light on the structural organizations of these large ribonucleoprotein complexes.

  17. Eukaryotic tRNAs fingerprint invertebrates vis-à-vis vertebrates.

    Science.gov (United States)

    Mitra, Sanga; Das, Pijush; Samadder, Arpa; Das, Smarajit; Betai, Rupal; Chakrabarti, Jayprokas

    2015-01-01

    During translation, aminoacyl-tRNA synthetases recognize the identities of the tRNAs to charge them with their respective amino acids. The conserved identities of 58,244 eukaryotic tRNAs of 24 invertebrates and 45 vertebrates in genomic tRNA database were analyzed and their novel features extracted. The internal promoter sequences, namely, A-Box and B-Box, were investigated and evidence gathered that the intervention of optional nucleotides at 17a and 17b correlated with the optimal length of the A-Box. The presence of canonical transcription terminator sequences at the immediate vicinity of tRNA genes was ventured. Even though non-canonical introns had been reported in red alga, green alga, and nucleomorph so far, fairly motivating evidence of their existence emerged in tRNA genes of other eukaryotes. Non-canonical introns were seen to interfere with the internal promoters in two cases, questioning their transcription fidelity. In a first of its kind, phylogenetic constructs based on tRNA molecules delineated and built the trees of the vast and diverse invertebrates and vertebrates. Finally, two tRNA models representing the invertebrates and the vertebrates were drawn, by isolating the dominant consensus in the positional fluctuations of nucleotide compositions.

  18. Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia

    Energy Technology Data Exchange (ETDEWEB)

    Larson, Eric T.; Kim, Jessica E.; Napuli, Alberto J.; Verlinde, Christophe L. M. J.; Fan, Erkang; Zucker, Frank H.; Van Voorhis, Wesley C.; Buckner, Frederick S.; Hol, Wim G. J.; Merritt, Ethan A., E-mail: merritt@u.washington.edu [Medical Structural Genomics of Pathogenic Protozoa, (United States); University of Washington, Seattle, WA 98195 (United States)

    2012-09-01

    The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.

  19. Molecular architecture of the yeast Mediator complex

    Science.gov (United States)

    Robinson, Philip J; Trnka, Michael J; Pellarin, Riccardo; Greenberg, Charles H; Bushnell, David A; Davis, Ralph; Burlingame, Alma L; Sali, Andrej; Kornberg, Roger D

    2015-01-01

    The 21-subunit Mediator complex transduces regulatory information from enhancers to promoters, and performs an essential role in the initiation of transcription in all eukaryotes. Structural information on two-thirds of the complex has been limited to coarse subunit mapping onto 2-D images from electron micrographs. We have performed chemical cross-linking and mass spectrometry, and combined the results with information from X-ray crystallography, homology modeling, and cryo-electron microscopy by an integrative modeling approach to determine a 3-D model of the entire Mediator complex. The approach is validated by the use of X-ray crystal structures as internal controls and by consistency with previous results from electron microscopy and yeast two-hybrid screens. The model shows the locations and orientations of all Mediator subunits, as well as subunit interfaces and some secondary structural elements. Segments of 20–40 amino acid residues are placed with an average precision of 20 Å. The model reveals roles of individual subunits in the organization of the complex. DOI: http://dx.doi.org/10.7554/eLife.08719.001 PMID:26402457

  20. Crystallization and preliminary X-ray analysis of eukaryotic initiation factor 4E from Pisum sativum

    International Nuclear Information System (INIS)

    Ashby, Jamie A.; Stevenson, Clare E. M.; Maule, Andrew J.; Lawson, David M.

    2009-01-01

    Crystals of N-terminally truncated eIF4E from pea were obtained and X-ray data were recorded in-house to a resolution of 2.2 Å. Crystals of an N-terminally truncated 20 kDa fragment of Pisum sativum eIF4E (ΔN-eIF4E) were grown by vapour diffusion. X-ray data were recorded to a resolution of 2.2 Å from a single crystal in-house. Indexing was consistent with primitive monoclinic symmetry and solvent-content estimations suggested that between four and nine copies of the eIF4E fragment were possible per crystallographic asymmetric unit. eIF4E is an essential component of the eukaryotic translation machinery and recent studies have shown that point mutations of plant eIF4Es can confer resistance to potyvirus infection

  1. Comparative genomics of phylogenetically diverse unicellular eukaryotes provide new insights into the genetic basis for the evolution of the programmed cell death machinery.

    Science.gov (United States)

    Nedelcu, Aurora M

    2009-03-01

    Programmed cell death (PCD) represents a significant component of normal growth and development in multicellular organisms. Recently, PCD-like processes have been reported in single-celled eukaryotes, implying that some components of the PCD machinery existed early in eukaryotic evolution. This study provides a comparative analysis of PCD-related sequences across more than 50 unicellular genera from four eukaryotic supergroups: Unikonts, Excavata, Chromalveolata, and Plantae. A complex set of PCD-related sequences that correspond to domains or proteins associated with all main functional classes--from ligands and receptors to executors of PCD--was found in many unicellular lineages. Several PCD domains and proteins previously thought to be restricted to animals or land plants are also present in unicellular species. Noteworthy, the yeast, Saccharomyces cerevisiae--used as an experimental model system for PCD research, has a rather reduced set of PCD-related sequences relative to other unicellular species. The phylogenetic distribution of the PCD-related sequences identified in unicellular lineages suggests that the genetic basis for the evolution of the complex PCD machinery present in extant multicellular lineages has been established early in the evolution of eukaryotes. The shaping of the PCD machinery in multicellular lineages involved the duplication, co-option, recruitment, and shuffling of domains already present in their unicellular ancestors.

  2. Why Translation Is Difficult

    DEFF Research Database (Denmark)

    Carl, Michael; Schaeffer, Moritz Jonas

    2017-01-01

    The paper develops a definition of translation literality that is based on the syntactic and semantic similarity of the source and the target texts. We provide theoretical and empirical evidence that absolute literal translations are easy to produce. Based on a multilingual corpus of alternative...... translations we investigate the effects of cross-lingual syntactic and semantic distance on translation production times and find that non-literality makes from-scratch translation and post-editing difficult. We show that statistical machine translation systems encounter even more difficulties with non-literality....

  3. Elucidating the composition and conservation of the autophagy pathway in photosynthetic eukaryotes

    Science.gov (United States)

    Shemi, Adva; Ben-Dor, Shifra; Vardi, Assaf

    2015-01-01

    Aquatic photosynthetic eukaryotes represent highly diverse groups (green, red, and chromalveolate algae) derived from multiple endosymbiosis events, covering a wide spectrum of the tree of life. They are responsible for about 50% of the global photosynthesis and serve as the foundation for oceanic and fresh water food webs. Although the ecophysiology and molecular ecology of some algal species are extensively studied, some basic aspects of algal cell biology are still underexplored. The recent wealth of genomic resources from algae has opened new frontiers to decipher the role of cell signaling pathways and their function in an ecological and biotechnological context. Here, we took a bioinformatic approach to explore the distribution and conservation of TOR and autophagy-related (ATG) proteins (Atg in yeast) in diverse algal groups. Our genomic analysis demonstrates conservation of TOR and ATG proteins in green algae. In contrast, in all 5 available red algal genomes, we could not detect the sequences that encode for any of the 17 core ATG proteins examined, albeit TOR and its interacting proteins are conserved. This intriguing data suggests that the autophagy pathway is not conserved in red algae as it is in the entire eukaryote domain. In contrast, chromalveolates, despite being derived from the red-plastid lineage, retain and express ATG genes, which raises a fundamental question regarding the acquisition of ATG genes during algal evolution. Among chromalveolates, Emiliania huxleyi (Haptophyta), a bloom-forming coccolithophore, possesses the most complete set of ATG genes, and may serve as a model organism to study autophagy in marine protists with great ecological significance. PMID:25915714

  4. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

    Science.gov (United States)

    Klionsky, Daniel J.; Abeliovich, Hagai; Agostinis, Patrizia; Agrawal, Devendra K.; Aliev, Gjumrakch; Askew, David S.; Baba, Misuzu; Baehrecke, Eric H.; Bahr, Ben A.; Ballabio, Andrea; Bamber, Bruce A.; Bassham, Diane C.; Bergamini, Ettore; Bi, Xiaoning; Biard-Piechaczyk, Martine; Blum, Janice S.; Bredesen, Dale E.; Brodsky, Jeffrey L.; Brumell, John H.; Brunk, Ulf T.; Bursch, Wilfried; Camougrand, Nadine; Cebollero, Eduardo; Cecconi, Francesco; Chen, Yingyu; Chin, Lih-Shen; Choi, Augustine; Chu, Charleen T.; Chung, Jongkyeong; Clarke, Peter G.H.; Clark, Robert S.B.; Clarke, Steven G.; Clavé, Corinne; Cleveland, John L.; Codogno, Patrice; Colombo, María I.; Coto-Montes, Ana; Cregg, James M.; Cuervo, Ana Maria; Debnath, Jayanta; Demarchi, Francesca; Dennis, Patrick B.; Dennis, Phillip A.; Deretic, Vojo; Devenish, Rodney J.; Di Sano, Federica; Dice, J. Fred; DiFiglia, Marian; Dinesh-Kumar, Savithramma; Distelhorst, Clark W.; Djavaheri-Mergny, Mojgan; Dorsey, Frank C.; Dröge, Wulf; Dron, Michel; Dunn, William A.; Duszenko, Michael; Eissa, N. Tony; Elazar, Zvulun; Esclatine, Audrey; Eskelinen, Eeva-Liisa; Fésüs, László; Finley, Kim D.; Fuentes, José M.; Fueyo, Juan; Fujisaki, Kozo; Galliot, Brigitte; Gao, Fen-Biao; Gewirtz, David A.; Gibson, Spencer B.; Gohla, Antje; Goldberg, Alfred L.; Gonzalez, Ramon; González-Estévez, Cristina; Gorski, Sharon; Gottlieb, Roberta A.; Häussinger, Dieter; He, You-Wen; Heidenreich, Kim; Hill, Joseph A.; Høyer-Hansen, Maria; Hu, Xun; Huang, Wei-Pang; Iwasaki, Akiko; Jäättelä, Marja; Jackson, William T.; Jiang, Xuejun; Jin, Shengkan; Johansen, Terje; Jung, Jae U.; Kadowaki, Motoni; Kang, Chanhee; Kelekar, Ameeta; Kessel, David H.; Kiel, Jan A.K.W.; Kim, Hong Pyo; Kimchi, Adi; Kinsella, Timothy J.; Kiselyov, Kirill; Kitamoto, Katsuhiko; Knecht, Erwin; Komatsu, Masaaki; Kominami, Eiki; Kondo, Seiji; Kovács, Attila L.; Kroemer, Guido; Kuan, Chia-Yi; Kumar, Rakesh; Kundu, Mondira; Landry, Jacques; Laporte, Marianne; Le, Weidong; Lei, Huan-Yao; Lenardo, Michael J.; Levine, Beth; Lieberman, Andrew; Lim, Kah-Leong; Lin, Fu-Cheng; Liou, Willisa; Liu, Leroy F.; Lopez-Berestein, Gabriel; López-Otín, Carlos; Lu, Bo; Macleod, Kay F.; Malorni, Walter; Martinet, Wim; Matsuoka, Ken; Mautner, Josef; Meijer, Alfred J.; Meléndez, Alicia; Michels, Paul; Miotto, Giovanni; Mistiaen, Wilhelm P.; Mizushima, Noboru; Mograbi, Baharia; Monastyrska, Iryna; Moore, Michael N.; Moreira, Paula I.; Moriyasu, Yuji; Motyl, Tomasz; Münz, Christian; Murphy, Leon O.; Naqvi, Naweed I.; Neufeld, Thomas P.; Nishino, Ichizo; Nixon, Ralph A.; Noda, Takeshi; Nürnberg, Bernd; Ogawa, Michinaga; Oleinick, Nancy L.; Olsen, Laura J.; Ozpolat, Bulent; Paglin, Shoshana; Palmer, Glen E.; Papassideri, Issidora; Parkes, Miles; Perlmutter, David H.; Perry, George; Piacentini, Mauro; Pinkas-Kramarski, Ronit; Prescott, Mark; Proikas-Cezanne, Tassula; Raben, Nina; Rami, Abdelhaq; Reggiori, Fulvio; Rohrer, Bärbel; Rubinsztein, David C.; Ryan, Kevin M.; Sadoshima, Junichi; Sakagami, Hiroshi; Sakai, Yasuyoshi; Sandri, Marco; Sasakawa, Chihiro; Sass, Miklós; Schneider, Claudio; Seglen, Per O.; Seleverstov, Oleksandr; Settleman, Jeffrey; Shacka, John J.; Shapiro, Irving M.; Sibirny, Andrei; Silva-Zacarin, Elaine C.M.; Simon, Hans-Uwe; Simone, Cristiano; Simonsen, Anne; Smith, Mark A.; Spanel-Borowski, Katharina; Srinivas, Vickram; Steeves, Meredith; Stenmark, Harald; Stromhaug, Per E.; Subauste, Carlos S.; Sugimoto, Seiichiro; Sulzer, David; Suzuki, Toshihiko; Swanson, Michele S.; Tabas, Ira; Takeshita, Fumihiko; Talbot, Nicholas J.; Tallóczy, Zsolt; Tanaka, Keiji; Tanaka, Kozo; Tanida, Isei; Taylor, Graham S.; Taylor, J. Paul; Terman, Alexei; Tettamanti, Gianluca; Thompson, Craig B.; Thumm, Michael; Tolkovsky, Aviva M.; Tooze, Sharon A.; Truant, Ray; Tumanovska, Lesya V.; Uchiyama, Yasuo; Ueno, Takashi; Uzcátegui, Néstor L.; van der Klei, Ida; Vaquero, Eva C.; Vellai, Tibor; Vogel, Michael W.; Wang, Hong-Gang; Webster, Paul; Wiley, John W.; Xi, Zhijun; Xiao, Gutian; Yahalom, Joachim; Yang, Jin-Ming; Yap, George; Yin, Xiao-Ming; Yoshimori, Tamotsu; Yu, Li; Yue, Zhenyu; Yuzaki, Michisuke; Zabirnyk, Olga; Zheng, Xiaoxiang; Zhu, Xiongwei; Deter, Russell L.

    2009-01-01

    Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response. PMID:18188003

  5. Current awareness on yeast.

    Science.gov (United States)

    2002-02-01

    In order to keep subscribers up-to-date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly-published material on yeasts. Each bibliography is divided into 10 sections. 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (3 weeks journals - search completed 5th. Dec. 2001)

  6. Archaeal “Dark Matter” and the Origin of Eukaryotes

    Science.gov (United States)

    Williams, Tom A.; Embley, T. Martin

    2014-01-01

    Current hypotheses about the history of cellular life are mainly based on analyses of cultivated organisms, but these represent only a small fraction of extant biodiversity. The sequencing of new environmental lineages therefore provides an opportunity to test, revise, or reject existing ideas about the tree of life and the origin of eukaryotes. According to the textbook three domains hypothesis, the eukaryotes emerge as the sister group to a monophyletic Archaea. However, recent analyses incorporating better phylogenetic models and an improved sampling of the archaeal domain have generally supported the competing eocyte hypothesis, in which core genes of eukaryotic cells originated from within the Archaea, with important implications for eukaryogenesis. Given this trend, it was surprising that a recent analysis incorporating new genomes from uncultivated Archaea recovered a strongly supported three domains tree. Here, we show that this result was due in part to the use of a poorly fitting phylogenetic model and also to the inclusion by an automated pipeline of genes of putative bacterial origin rather than nucleocytosolic versions for some of the eukaryotes analyzed. When these issues were resolved, analyses including the new archaeal lineages placed core eukaryotic genes within the Archaea. These results are consistent with a number of recent studies in which improved archaeal sampling and better phylogenetic models agree in supporting the eocyte tree over the three domains hypothesis. PMID:24532674

  7. Eukaryotic systematics: a user's guide for cell biologists and parasitologists.

    Science.gov (United States)

    Walker, Giselle; Dorrell, Richard G; Schlacht, Alexander; Dacks, Joel B

    2011-11-01

    Single-celled parasites like Entamoeba, Trypanosoma, Phytophthora and Plasmodium wreak untold havoc on human habitat and health. Understanding the position of the various protistan pathogens in the larger context of eukaryotic diversity informs our study of how these parasites operate on a cellular level, as well as how they have evolved. Here, we review the literature that has brought our understanding of eukaryotic relationships from an idea of parasites as primitive cells to a crystallized view of diversity that encompasses 6 major divisions, or supergroups, of eukaryotes. We provide an updated taxonomic scheme (for 2011), based on extensive genomic, ultrastructural and phylogenetic evidence, with three differing levels of taxonomic detail for ease of referencing and accessibility (see supplementary material at Cambridge Journals On-line). Two of the most pressing issues in cellular evolution, the root of the eukaryotic tree and the evolution of photosynthesis in complex algae, are also discussed along with ideas about what the new generation of genome sequencing technologies may contribute to the field of eukaryotic systematics. We hope that, armed with this user's guide, cell biologists and parasitologists will be encouraged about taking an increasingly evolutionary point of view in the battle against parasites representing real dangers to our livelihoods and lives.

  8. Evolution of DNA replication protein complexes in eukaryotes and Archaea.

    Directory of Open Access Journals (Sweden)

    Nicholas Chia

    Full Text Available BACKGROUND: The replication of DNA in Archaea and eukaryotes requires several ancillary complexes, including proliferating cell nuclear antigen (PCNA, replication factor C (RFC, and the minichromosome maintenance (MCM complex. Bacterial DNA replication utilizes comparable proteins, but these are distantly related phylogenetically to their archaeal and eukaryotic counterparts at best. METHODOLOGY/PRINCIPAL FINDINGS: While the structures of each of the complexes do not differ significantly between the archaeal and eukaryotic versions thereof, the evolutionary dynamic in the two cases does. The number of subunits in each complex is constant across all taxa. However, they vary subtly with regard to composition. In some taxa the subunits are all identical in sequence, while in others some are homologous rather than identical. In the case of eukaryotes, there is no phylogenetic variation in the makeup of each complex-all appear to derive from a common eukaryotic ancestor. This is not the case in Archaea, where the relationship between the subunits within each complex varies taxon-to-taxon. We have performed a detailed phylogenetic analysis of these relationships in order to better understand the gene duplications and divergences that gave rise to the homologous subunits in Archaea. CONCLUSION/SIGNIFICANCE: This domain level difference in evolution suggests that different forces have driven the evolution of DNA replication proteins in each of these two domains. In addition, the phylogenies of all three gene families support the distinctiveness of the proposed archaeal phylum Thaumarchaeota.

  9. The Eukaryotic Cell Originated in the Integration and Redistribution of Hyperstructures from Communities of Prokaryotic Cells Based on Molecular Complementarity

    Directory of Open Access Journals (Sweden)

    Vic Norris

    2009-06-01

    Full Text Available In the “ecosystems-first” approach to the origins of life, networks of non-covalent assemblies of molecules (composomes, rather than individual protocells, evolved under the constraints of molecular complementarity. Composomes evolved into the hyperstructures of modern bacteria. We extend the ecosystems-first approach to explain the origin of eukaryotic cells through the integration of mixed populations of bacteria. We suggest that mutualism and symbiosis resulted in cellular mergers entailing the loss of redundant hyperstructures, the uncoupling of transcription and translation, and the emergence of introns and multiple chromosomes. Molecular complementarity also facilitated integration of bacterial hyperstructures to perform cytoskeletal and movement functions.

  10. Aging yeast gain a competitive advantage on non-optimal carbon sources.

    Science.gov (United States)

    Frenk, Stephen; Pizza, Grazia; Walker, Rachael V; Houseley, Jonathan

    2017-06-01

    Animals, plants and fungi undergo an aging process with remarkable physiological and molecular similarities, suggesting that aging has long been a fact of life for eukaryotes and one to which our unicellular ancestors were subject. Key biochemical pathways that impact longevity evolved prior to multicellularity, and the interactions between these pathways and the aging process therefore emerged in ancient single-celled eukaryotes. Nevertheless, we do not fully understand how aging impacts the fitness of unicellular organisms, and whether such cells gain a benefit from modulating rather than simply suppressing the aging process. We hypothesized that age-related loss of fitness in single-celled eukaryotes may be counterbalanced, partly or wholly, by a transition from a specialist to a generalist life-history strategy that enhances adaptability to other environments. We tested this hypothesis in budding yeast using competition assays and found that while young cells are more successful in glucose, highly aged cells outcompete young cells on other carbon sources such as galactose. This occurs because aged yeast divide faster than young cells in galactose, reversing the normal association between age and fitness. The impact of aging on single-celled organisms is therefore complex and may be regulated in ways that anticipate changing nutrient availability. We propose that pathways connecting nutrient availability with aging arose in unicellular eukaryotes to capitalize on age-linked diversity in growth strategy and that individual cells in higher eukaryotes may similarly diversify during aging to the detriment of the organism as a whole. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  11. Analysis of Schizosaccharomyces pombe mediator reveals a set of essential subunits conserved between yeast and metazoan cells

    DEFF Research Database (Denmark)

    Spåhr, H; Samuelsen, C O; Baraznenok, V

    2001-01-01

    . cerevisiae share an essential protein module, which associates with nonessential speciesspecific subunits. In support of this view, sequence analysis of the conserved yeast Mediator components Med4 and Med8 reveals sequence homology to the metazoan Mediator components Trap36 and Arc32. Therefore, 8 of 10...... essential genes conserved between S. pombe and S. cerevisiae also have a metazoan homolog, indicating that an evolutionary conserved Mediator core is present in all eukaryotic cells. Our data suggest a closer functional relationship between yeast and metazoan Mediator than previously anticipated....

  12. Evolutionary conservation of TORC1 components, TOR, Raptor, and LST8, between rice and yeast.

    Science.gov (United States)

    Maegawa, Kentaro; Takii, Rumi; Ushimaru, Takashi; Kozaki, Akiko

    2015-10-01

    Target of rapamycin (TOR) is a conserved eukaryotic serine/threonine kinase that functions as a central controller of cell growth. TOR protein is structurally defined by the presence several conserved domains such as the HEAT repeat, focal adhesion target (FAT), FKBP12/rapamycin binding (FRB), kinase, and FATC domains starting from the N-terminus. In most eukaryotes, TOR forms two distinct physical and functional complexes, which are termed as TOR complex 1 (TORC1) and TORC2. However, plants contain only TORC1 components, i.e., TOR, Raptor, and LST8. In this study, we analyzed the gene structure and functions of TORC components in rice to understand the properties of the TOR complex in plants. Comparison of the locations of introns in these genes among rice and other eukaryotes showed that they were well conserved among plants except for Chlamydomonas. Moreover, the intron positions in the coding sequence of human Raptor and LST8 were closer to those of plants than of fly or nematode. Complementation tests of rice TOR (OsTOR) components in yeast showed that although OsTOR did not complement yeast tor mutants, chimeric TOR, which consisted of the HEAT repeat and FAT domain from yeast and other regions from rice, rescued the tor mutants, indicating that the HEAT repeat and FAT domains are important for species-specific signaling. OsRaptor perfectly complemented a kog1 (yeast Raptor homolog) mutant, and OsLST8 partially complemented an lst8 mutant. Together, these data suggest the importance of the N-terminal region of the TOR, HEAT, and FAT domains for functional diversification of the TOR complex.

  13. Inheritance of the yeast mitochondrial genome

    DEFF Research Database (Denmark)

    Piskur, Jure

    1994-01-01

    Mitochondrion, extrachromosomal genetics, intergenic sequences, genome size, mitochondrial DNA, petite mutation, yeast......Mitochondrion, extrachromosomal genetics, intergenic sequences, genome size, mitochondrial DNA, petite mutation, yeast...

  14. Yeasts preservation: alternatives for lyophilisation

    NARCIS (Netherlands)

    Nyanga, L.K.; Nout, M.J.R.; Smid, E.J.; Boekhout, T.; Zwietering, M.H.

    2012-01-01

    The aim of the study was to compare the effect of two low-cost, low technology traditional methods for drying starter cultures with standard lyophilisation. Lyophilised yeast cultures and yeast cultures preserved in dry rice cakes and dry plant fibre strands were examined for viable cell counts

  15. Determinants of translation ambiguity

    Science.gov (United States)

    Degani, Tamar; Prior, Anat; Eddington, Chelsea M.; Arêas da Luz Fontes, Ana B.; Tokowicz, Natasha

    2016-01-01

    Ambiguity in translation is highly prevalent, and has consequences for second-language learning and for bilingual lexical processing. To better understand this phenomenon, the current study compared the determinants of translation ambiguity across four sets of translation norms from English to Spanish, Dutch, German and Hebrew. The number of translations an English word received was correlated across these different languages, and was also correlated with the number of senses the word has in English, demonstrating that translation ambiguity is partially determined by within-language semantic ambiguity. For semantically-ambiguous English words, the probability of the different translations in Spanish and Hebrew was predicted by the meaning-dominance structure in English, beyond the influence of other lexical and semantic factors, for bilinguals translating from their L1, and translating from their L2. These findings are consistent with models postulating direct access to meaning from L2 words for moderately-proficient bilinguals. PMID:27882188

  16. Translation in ESL Classes

    Directory of Open Access Journals (Sweden)

    Nagy Imola Katalin

    2015-12-01

    Full Text Available The problem of translation in foreign language classes cannot be dealt with unless we attempt to make an overview of what translation meant for language teaching in different periods of language pedagogy. From the translation-oriented grammar-translation method through the complete ban on translation and mother tongue during the times of the audio-lingual approaches, we have come today to reconsider the role and status of translation in ESL classes. This article attempts to advocate for translation as a useful ESL class activity, which can completely fulfil the requirements of communicativeness. We also attempt to identify some activities and games, which rely on translation in some books published in the 1990s and the 2000s.

  17. Translation and Quality Management

    DEFF Research Database (Denmark)

    Petersen, Margrethe

    1996-01-01

    theory which would seem likely to be of interest in this connection and section 2. gives a linguist's introduction to the part of the area of quality management which I consider relevant for present purposes. Section 3. is devoted to the case study of a small translation firm which has been certified......The aim of this article is to consider the issue of quality in translation. Specifically, the question under consideration is whether quality assurance in relation to translation is feasible and, if so, what some of the implications for translation theory, translation practice and the teaching...... of translation would be. To provide a backdrop against which the issue may be discussed, I present an overview of the two areas which seem most likely to hold potential answers, viz., that of translation theory and that of quality management. Section 1. gives a brief outline of some contributions to translation...

  18. Production of Food Grade Yeasts

    Directory of Open Access Journals (Sweden)

    Argyro Bekatorou

    2006-01-01

    Full Text Available Yeasts have been known to humans for thousands of years as they have been used in traditional fermentation processes like wine, beer and bread making. Today, yeasts are also used as alternative sources of high nutritional value proteins, enzymes and vitamins, and have numerous applications in the health food industry as food additives, conditioners and flavouring agents, for the production of microbiology media and extracts, as well as livestock feeds. Modern scientific advances allow the isolation, construction and industrial production of new yeast strains to satisfy the specific demands of the food industry. Types of commercial food grade yeasts, industrial production processes and raw materials are highlighted. Aspects of yeast metabolism, with respect to carbohydrate utilization, nutritional aspects and recent research advances are also discussed.

  19. Evolutionary History of Ascomyceteous Yeasts

    Energy Technology Data Exchange (ETDEWEB)

    Haridas, Sajeet; Riley, Robert; Salamov, Asaf; Goker, Markus; Klenk, Hans-Peter; Kurtzman, Cletus P.; Blackwell, Meredith; Grigoriev, Igor; Jeffries, Thomas W.

    2014-06-06

    Yeasts are important for many industrial and biotechnological processes and show remarkable diversity despite morphological similarities. We have sequenced the genomes of 16 ascomycete yeasts of taxonomic and industrial importance including members of Saccharomycotina and Taphrinomycotina. A comparison of these with several other previously published yeast genomes have added increased confidence to the phylogenetic positions of previously poorly placed species including Saitoella complicata, Babjeviella inositovora and Metschnikowia bicuspidata. Phylogenetic analysis also showed that yeasts with alternative nuclear codon usage where CUG encodes serine instead of leucine are monophyletic within the Saccharomycotina. Most of the yeasts have compact genomes with a large fraction of single exon genes with Lipomyces starkeyi and the previously published Pneumocystis jirovecii being notable exceptions. Intron analysis suggests that early diverging species have more introns. We also observed a large number of unclassified lineage specific non-simple repeats in these genomes.

  20. Genomic signatures of adaptation to wine biological ageing conditions in biofilm-forming flor yeasts.

    Science.gov (United States)

    Coi, A L; Bigey, F; Mallet, S; Marsit, S; Zara, G; Gladieux, P; Galeote, V; Budroni, M; Dequin, S; Legras, J L

    2017-04-01

    The molecular and evolutionary processes underlying fungal domestication remain largely unknown despite the importance of fungi to bioindustry and for comparative adaptation genomics in eukaryotes. Wine fermentation and biological ageing are performed by strains of S. cerevisiae with, respectively, pelagic fermentative growth on glucose and biofilm aerobic growth utilizing ethanol. Here, we use environmental samples of wine and flor yeasts to investigate the genomic basis of yeast adaptation to contrasted anthropogenic environments. Phylogenetic inference and population structure analysis based on single nucleotide polymorphisms revealed a group of flor yeasts separated from wine yeasts. A combination of methods revealed several highly differentiated regions between wine and flor yeasts, and analyses using codon-substitution models for detecting molecular adaptation identified sites under positive selection in the high-affinity transporter gene ZRT1. The cross-population composite likelihood ratio revealed selective sweeps at three regions, including in the hexose transporter gene HXT7, the yapsin gene YPS6 and the membrane protein coding gene MTS27. Our analyses also revealed that the biological ageing environment has led to the accumulation of numerous mutations in proteins from several networks, including Flo11 regulation and divalent metal transport. Together, our findings suggest that the tuning of FLO11 expression and zinc transport networks are a distinctive feature of the genetic changes underlying the domestication of flor yeasts. Our study highlights the multiplicity of genomic changes underlying yeast adaptation to man-made habitats and reveals that flor/wine yeast lineage can serve as a useful model for studying the genomics of adaptive divergence. © 2017 John Wiley & Sons Ltd.

  1. Codon usage regulates protein structure and function by affecting translation elongation speed in Drosophila cells.

    Science.gov (United States)

    Zhao, Fangzhou; Yu, Chien-Hung; Liu, Yi

    2017-08-21

    Codon usage biases are found in all eukaryotic and prokaryotic genomes and have been proposed to regulate different aspects of translation process. Codon optimality has been shown to regulate translation elongation speed in fungal systems, but its effect on translation elongation speed in animal systems is not clear. In this study, we used a Drosophila cell-free translation system to directly compare the velocity of mRNA translation elongation. Our results demonstrate that optimal synonymous codons speed up translation elongation while non-optimal codons slow down translation. In addition, codon usage regulates ribosome movement and stalling on mRNA during translation. Finally, we show that codon usage affects protein structure and function in vitro and in Drosophila cells. Together, these results suggest that the effect of codon usage on translation elongation speed is a conserved mechanism from fungi to animals that can affect protein folding in eukaryotic organisms. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Searching for eukaryotic life preserved in Antarctic permafrost

    DEFF Research Database (Denmark)

    Zucconi, L.; Selbmann, L.; Buzzini, P.

    2012-01-01

    Fungi and yeasts isolated in pure culture from Antarctic permafrost collected at different depths in the McMurdo Dry Valleys were identified with cultural, physiological and molecular methods. Fungi belonged to the genera Penicillium, Eurotium, Cladosporium, Alternaria, Engyodonthium, Aureobasidium...

  3. Memetics and Translation Studies

    OpenAIRE

    Andrew, Chesterman

    2000-01-01

    Translation Studies is a branch of memetics. This is a claim, a hypothesis. More specifically, it is an interpretive hypothesis: I claim that Translation Studies can be thus interpreted, and that this is a useful thing to do because it offers a new and beneficial way of understanding translation.

  4. Sound Effects in Translation

    DEFF Research Database (Denmark)

    Mees, Inger M.; Dragsted, Barbara; Gorm Hansen, Inge

    2015-01-01

    ), Translog was employed to measure task times. The quality of the products was assessed by three experienced translators, and the number and types of misrecognitions were identified by a phonetician. Results indicate that SR translation provides a potentially useful supplement to written translation...

  5. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes.

    Science.gov (United States)

    Riechmann, J L; Heard, J; Martin, G; Reuber, L; Jiang, C; Keddie, J; Adam, L; Pineda, O; Ratcliffe, O J; Samaha, R R; Creelman, R; Pilgrim, M; Broun, P; Zhang, J Z; Ghandehari, D; Sherman, B K; Yu, G

    2000-12-15

    The completion of the Arabidopsis thaliana genome sequence allows a comparative analysis of transcriptional regulators across the three eukaryotic kingdoms. Arabidopsis dedicates over 5% of its genome to code for more than 1500 transcription factors, about 45% of which are from families specific to plants. Arabidopsis transcription factors that belong to families common to all eukaryotes do not share significant similarity with those of the other kingdoms beyond the conserved DNA binding domains, many of which have been arranged in combinations specific to each lineage. The genome-wide comparison reveals the evolutionary generation of diversity in the regulation of transcription.

  6. Eukaryotic ribosome display with in situ DNA recovery.

    Science.gov (United States)

    He, Mingyue; Edwards, Bryan M; Kastelic, Damjana; Taussig, Michael J

    2012-01-01

    Ribosome display is a cell-free display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic ribosome display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the ribosome.

  7. Characterization of prokaryotic and eukaryotic promoters usinghidden Markov models

    DEFF Research Database (Denmark)

    Pedersen, Anders Gorm; Baldi, Pierre; Brunak, Søren

    1996-01-01

    In this paper we utilize hidden Markov models (HMMs) and information theory to analyze prokaryotic and eukaryotic promoters. We perform this analysis with special emphasis on the fact that promoters are divided into a number of different classes, depending on which polymerase-associated factors...... that bind to them. We find that HMMs trained on such subclasses of Escherichia coli promoters (specifically, the so-called sigma-70 and sigma-54 classes) give an excellent classification of unknown promoters with respect to sigma-class. HMMs trained on eukaryotic sequences from human genes also model nicely...

  8. Characterization of prokaryotic and eukaryotic promoters using hidden Markov models

    DEFF Research Database (Denmark)

    Pedersen, Anders Gorm; Baldi, P.; Chauvin, Y.

    1996-01-01

    In this paper we utilize hidden Markov models (HMMs) and information theory to analyze prokaryotic and eukaryotic promoters. We perform this analysis with special emphasis on the fact that promoters are divided into a number of different classes, depending on which polymerase-associated factors...... that bind to them. We find that HMMs trained on such subclasses of Escherichia coli promoters (specifically, the so-called sigma 70 and sigma 54 classes) give an excellent classification of unknown promoters with respect to sigma-class. HMMs trained on eukaryotic sequences from human genes also model nicely...

  9. Identification of candidate new cancer susceptibility genes using yeast genomics

    International Nuclear Information System (INIS)

    Brown, M.; Brown, J.A.; Game, J.C.

    2003-01-01

    A large proportion of cancer susceptibility syndromes are the result of mutations in genes in DNA repair or in cell-cycle checkpoints in response to DNA damage, such as ataxia telangiectasia (AT), Fanconi's anemia (FA), Bloom's syndrome (BS), Nijmegen breakage syndrome (NBS), and xeroderma pigmentosum (XP). Mutations in these genes often cause gross chromosomal instability leading to an increased mutation rate of all genes including those directly responsible for cancer. We have proposed that because the orthologs of these genes in budding yeast, S. cerevisiae, confer protection against killing by DNA damaging agents it should be possible to identify new cancer susceptibility genes by identifying yeast genes whose deletion causes sensitivity to DNA damage. We therefore screened the recently completed collection of individual gene deletion mutants to identify genes that affect sensitivity to DNA-damaging agents. Screening for sensitivity in this obtained up to now with the F98 glioma model othe fact that each deleted gene is replaced by a cassette containing two molecular 'barcodes', or 20-mers, that uniquely identify the strain when DNA from a pool of strains is hybridized to an oligonucleotide array containing the complementary sequences of the barcodes. We performed the screen with UV, IR, H 2 0 2 and other DNA damaging agents. In addition to identifying genes already known to confer resistance to DNA damaging agents we have identified, and individually confirmed, several genes not previously associated with resistance. Several of these are of unknown function. We have also examined the chromosomal stability of selected strains and found that IR sensitive strains often but not always exhibit genomic instability. We are presently constructing a yeast artificial chromosome to globally interrogate all the genes in the deletion pool for their involvement in genomic stability. This work shows that budding yeast is a valuable eukaryotic model organism to identify

  10. Fusel Alcohols Regulate Translation Initiation by Inhibiting eIF2B to Reduce Ternary Complex in a Mechanism That May Involve Altering the Integrity and Dynamics of the eIF2B Body

    Science.gov (United States)

    Taylor, Eleanor J.; Campbell, Susan G.; Griffiths, Christian D.; Reid, Peter J.; Slaven, John W.; Harrison, Richard J.; Sims, Paul F.G.; Pavitt, Graham D.; Delneri, Daniela

    2010-01-01

    Recycling of eIF2-GDP to the GTP-bound form constitutes a core essential, regulated step in eukaryotic translation. This reaction is mediated by eIF2B, a heteropentameric factor with important links to human disease. eIF2 in the GTP-bound form binds to methionyl initiator tRNA to form a ternary complex, and the levels of this ternary complex can be a critical determinant of the rate of protein synthesis. Here we show that eIF2B serves as the target for translation inhibition by various fusel alcohols in yeast. Fusel alcohols are endpoint metabolites from amino acid catabolism, which signal nitrogen scarcity. We show that the inhibition of eIF2B leads to reduced ternary complex levels and that different eIF2B subunit mutants alter fusel alcohol sensitivity. A DNA tiling array strategy was developed that overcame difficulties in the identification of these mutants where the phenotypic distinctions were too subtle for classical complementation cloning. Fusel alcohols also lead to eIF2α dephosphorylation in a Sit4p-dependent manner. In yeast, eIF2B occupies a large cytoplasmic body where guanine nucleotide exchange on eIF2 can occur and be regulated. Fusel alcohols impact on both the movement and dynamics of this 2B body. Overall, these results confirm that the guanine nucleotide exchange factor, eIF2B, is targeted by fusel alcohols. Moreover, they highlight a potential connection between the movement or integrity of the 2B body and eIF2B regulation. PMID:20444979

  11. The Temple Translator's Workstation Project

    National Research Council Canada - National Science Library

    Vanni, Michelle; Zajac, Remi

    1996-01-01

    .... The Temple Translator's Workstation is incorporated into a Tipster document management architecture and it allows both translator/analysts and monolingual analysts to use the machine- translation...

  12. Distinct gene number-genome size relationships for eukaryotes and non-eukaryotes: gene content estimation for dinoflagellate genomes.

    Directory of Open Access Journals (Sweden)

    Yubo Hou

    Full Text Available The ability to predict gene content is highly desirable for characterization of not-yet sequenced genomes like those of dinoflagellates. Using data from completely sequenced and annotated genomes from phylogenetically diverse lineages, we investigated the relationship between gene content and genome size using regression analyses. Distinct relationships between log(10-transformed protein-coding gene number (Y' versus log(10-transformed genome size (X', genome size in kbp were found for eukaryotes and non-eukaryotes. Eukaryotes best fit a logarithmic model, Y' = ln(-46.200+22.678X', whereas non-eukaryotes a linear model, Y' = 0.045+0.977X', both with high significance (p0.91. Total gene number shows similar trends in both groups to their respective protein coding regressions. The distinct correlations reflect lower and decreasing gene-coding percentages as genome size increases in eukaryotes (82%-1% compared to higher and relatively stable percentages in prokaryotes and viruses (97%-47%. The eukaryotic regression models project that the smallest dinoflagellate genome (3x10(6 kbp contains 38,188 protein-coding (40,086 total genes and the largest (245x10(6 kbp 87,688 protein-coding (92,013 total genes, corresponding to 1.8% and 0.05% gene-coding percentages. These estimates do not likely represent extraordinarily high functional diversity of the encoded proteome but rather highly redundant genomes as evidenced by high gene copy numbers documented for various dinoflagellate species.

  13. Sound Effects in Translation

    DEFF Research Database (Denmark)

    Mees, Inger M.; Dragsted, Barbara; Gorm Hansen, Inge

    2013-01-01

    On the basis of a pilot study using speech recognition (SR) software, this paper attempts to illustrate the benefits of adopting an interdisciplinary approach in translator training. It shows how the collaboration between phoneticians, translators and interpreters can (1) advance research, (2) have......), Translog was employed to measure task times. The quality of the products was assessed by three experienced translators, and the number and types of misrecognitions were identified by a phonetician. Results indicate that SR translation provides a potentially useful supplement to written translation...

  14. Lost in translation

    DEFF Research Database (Denmark)

    Hedegaard, Steffen; Simonsen, Jakob Grue

    2011-01-01

    of translated texts. Our results suggest (i) that frame-based classifiers are usable for author attribution of both translated and untranslated texts; (ii) that framebased classifiers generally perform worse than the baseline classifiers for untranslated texts, but (iii) perform as well as, or superior...... to the baseline classifiers on translated texts; (iv) that—contrary to current belief—naïve classifiers based on lexical markers may perform tolerably on translated texts if the combination of author and translator is present in the training set of a classifier....

  15. Speaking your Translation

    DEFF Research Database (Denmark)

    Dragsted, Barbara; Mees, Inger M.; Gorm Hansen, Inge

    2011-01-01

    In this article we discuss the translation processes and products of 14 MA students who produced translations from Danish (L1) into English (L2) under different working conditions: (1) written translation, (2) sight translation, and (3) sight translation with a speech recognition (SR) tool. Audio......, since students were dictating in their L2, we looked into the number and types of error that occurred when using the SR software. Items that were misrecognised by the program could be divided into three categories: homophones, hesitations, and incorrectly pronounced words. Well over fifty per cent...

  16. Accumulation of long-chain bases in yeast promotes their conversion to a long-chain base vinyl ether

    OpenAIRE

    Martínez-Montañés, Fernando; Lone, Museer A.; Hsu, Fong-Fu; Schneiter3, Roger

    2017-01-01

    Long-chain bases (LCBs) are the precursors to ceramide and sphingolipids in eukaryotic cells. They are formed by the action of serine palmitoyl-CoA transferase (SPT), a complex of integral membrane proteins located in the endoplasmic reticulum. SPT activity is negatively regulated by Orm proteins to prevent the toxic overaccumulation of LCBs. Here we show that overaccumulation of LCBs in yeast results in their conversion to a hitherto undescribed LCB derivative, an LCB vinyl ether. The ...

  17. Cdc7-Dbf4 Regulates NDT80 Transcription as Well as Reductional Segregation during Budding Yeast Meiosis

    OpenAIRE

    Lo, Hsiao-Chi; Wan, Lihong; Rosebrock, Adam; Futcher, Bruce; Hollingsworth, Nancy M.

    2008-01-01

    In budding yeast, as in other eukaryotes, the Cdc7 protein kinase is important for initiation of DNA synthesis in vegetative cells. In addition, Cdc7 has crucial meiotic functions: it facilitates premeiotic DNA replication, and it is essential for the initiation of recombination. This work uses a chemical genetic approach to demonstrate that Cdc7 kinase has additional roles in meiosis. First, Cdc7 allows expression of NDT80, a meiosis-specific transcriptional activator required for the induct...

  18. Lost in translation?

    DEFF Research Database (Denmark)

    Granas, Anne Gerd; Nørgaard, Lotte Stig; Sporrong, Sofia Kälvemark

    2014-01-01

    OBJECTIVE: The "Beliefs about Medicines Questionnaire" (BMQ) assess balance of necessity and concern of medicines. The BMQ has been translated from English to many languages. However, the original meaning of statements, such as "My medicine is a mystery to me", may be lost in translation. The aim...... of this study is to compare three Scandinavian translations of the BMQ. (1) How reliable are the translations? (2) Are they still valid after translation? METHODS: Translated Norwegian, Swedish and Danish versions of the BMQ were scrutinized by three native Scandinavian researchers. Linguistic differences...... and ambiguities in the 5-point Likert scale and the BMQ statements were compared. RESULTS: In the Scandinavian translations, the Likert scale expanded beyond the original version at one endpoint (Swedish) or both endpoints (Danish). In the BMQ statements, discrepancies ranged from smaller inaccuracies toward...

  19. What is a translator?

    Directory of Open Access Journals (Sweden)

    Martha Pulido

    2016-08-01

    Full Text Available I copied the title from Foucault’s text, "Qu'est-ce qu'un auteur" in Dits et écrits [1969], Paris, Gallimard, 1994, that I read in French, then in English in Donald F. Bouchard’s and Sherry Simon’s translation, and finally in Spanish in Yturbe Corina’s translation, and applied for the translator some of the analysis that Foucault presents to define the author. Foucault suggests that if we cannot define an author, at least we can see where their function is reflected. My purpose in this paper is to present those surfaces where the function of the translator is reflected or where it can be revealed, and to analyse the categories that could lead us to the elaboration of a suitable definition of a Translator. I dare already give a compound noun for the translator: Translator-Function.

  20. What is a translator?

    Directory of Open Access Journals (Sweden)

    Martha Martha Pulido

    2016-05-01

    Full Text Available I copied the title from Foucault’s text, "Qu'est-ce qu'un auteur" in Dits et écrits [1969], Paris, Gallimard, 1994, that I read in French, then in English in Donald F. Bouchard’s and Sherry Simon’s translation, and finally in Spanish in Yturbe Corina’s translation, and applied for the translator some of the analysis that Foucault presents to define the author. Foucault suggests that if we cannot define an author, at least we can see where their function is reflected. My purpose in this paper is to present those surfaces where the function of the translator is reflected or where it can be revealed, and to analyse the categories that could lead us to the elaboration of a suitable definition of a Translator. I dare already give a compound noun for the translator: Translator-Function.

  1. Genetic study on yeast

    International Nuclear Information System (INIS)

    Mortimer, R.K.

    1981-01-01

    Research during the past year has moved ahead on several fronts. A major compilation of all the genetic mapping data for the yeast Saccharomyces cerevisiae has been completed. The map describes the location of over 300 genes on 17 chromosomes. A report on this work will appear in Microbiological Reviews in December 1980. Recombinant DNA procedures have been introduced into the experiments and RAD52 (one of the genes involved in recombination and repair damage), has been successfully cloned. This clone will be used to determine the gene product. Diploid cells homozygous for RAD52 have exceptionally high frequencies of mitotic loss of chromosomes. This loss is stimulated by ionizing radiation. This effect is a very significant finding. The effect has also been seen with certain other RAD mutants

  2. Lager Yeast Comes of Age

    Science.gov (United States)

    2014-01-01

    Alcoholic fermentations have accompanied human civilizations throughout our history. Lager yeasts have a several-century-long tradition of providing fresh beer with clean taste. The yeast strains used for lager beer fermentation have long been recognized as hybrids between two Saccharomyces species. We summarize the initial findings on this hybrid nature, the genomics/transcriptomics of lager yeasts, and established targets of strain improvements. Next-generation sequencing has provided fast access to yeast genomes. Its use in population genomics has uncovered many more hybridization events within Saccharomyces species, so that lager yeast hybrids are no longer the exception from the rule. These findings have led us to propose network evolution within Saccharomyces species. This “web of life” recognizes the ability of closely related species to exchange DNA and thus drain from a combined gene pool rather than be limited to a gene pool restricted by speciation. Within the domesticated lager yeasts, two groups, the Saaz and Frohberg groups, can be distinguished based on fermentation characteristics. Recent evidence suggests that these groups share an evolutionary history. We thus propose to refer to the Saaz group as Saccharomyces carlsbergensis and to the Frohberg group as Saccharomyces pastorianus based on their distinct genomes. New insight into the hybrid nature of lager yeast will provide novel directions for future strain improvement. PMID:25084862

  3. Interaction Between Yeasts and Zinc

    Science.gov (United States)

    Nicola, Raffaele De; Walker, Graeme

    Zinc is an essential trace element in biological systems. For example, it acts as a cellular membrane stabiliser, plays a critical role in gene expression and genome modification and activates nearly 300 enzymes, including alcohol dehydrogenase. The present chapter will be focused on the influence of zinc on cell physiology of industrial yeast strains of Saccharomyces cerevisiae, with special regard to the uptake and subsequent utilisation of this metal. Zinc uptake by yeast is metabolism-dependent, with most of the available zinc translocated very quickly into the vacuole. At cell division, zinc is distributed from mother to daughter cells and this effectively lowers the individual cellular zinc concentration, which may become zinc depleted at the onset of the fermentation. Zinc influences yeast fermentative performance and examples will be provided relating to brewing and wine fermentations. Industrial yeasts are subjected to several stresses that may impair fermentation performance. Such stresses may also impact on yeast cell zinc homeostasis. This chapter will discuss the practical implications for the correct management of zinc bioavailability for yeast-based biotechnologies aimed at improving yeast growth, viability, fermentation performance and resistance to environmental stresses

  4. An algorithm for detecting eukaryotic sequences in metagenomic ...

    Indian Academy of Sciences (India)

    species but also from accidental contamination from the genome of eukaryotic host cells. The latter scenario generally occurs in the case of host-associated metagenomes, e.g. microbes living in human gut. In such cases, one needs to identify and remove contaminating host DNA sequences, since the latter sequences will ...

  5. Potential of industrial biotechnology with cyanobacteria and eukaryotic microalgae

    NARCIS (Netherlands)

    Wijffels, R.H.; Kruse, O.; Hellingwerf, K.J.

    2013-01-01

    Both cyanobacteria and eukaryotic microalgae are promising organisms for sustainable production of bulk products such as food, feed, materials, chemicals and fuels. In this review we will summarize the potential and current biotechnological developments.Cyanobacteria are promising host organisms for

  6. Geminin: a major DNA replication safeguard in higher eukaryotes

    DEFF Research Database (Denmark)

    Melixetian, Marina; Helin, Kristian

    2004-01-01

    Eukaryotes have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. These mechanisms prevent relicensing of origins of replication after initiation of DNA replication in S phase until the end of mitosis. Most of our knowledge of mechanisms controlling prereplication...

  7. Eu-Detect: An algorithm for detecting eukaryotic sequences in ...

    Indian Academy of Sciences (India)

    Supplementary figure 1. Plots depicting the classification accuracy of Eu-Detect with various combinations of. 'cumulative sequence count' (40K, 50K, 60K, 70K, 80K) and 'coverage threshold' (20%, 30%, 40%, 50%, 60%, 70%,. 80%). While blue bars represent Eu-Detect's average classification accuracy with eukaryotic ...

  8. Uncoupling of Sister Replisomes during Eukaryotic DNA Replication

    NARCIS (Netherlands)

    Yardimci, Hasan; Loveland, Anna B.; Habuchi, Satoshi; van Oijen, Antoine M.; Walter, Johannes C.

    2010-01-01

    The duplication of eukaryotic genomes involves the replication of DNA from multiple origins of replication. In S phase, two sister replisomes assemble at each active origin, and they replicate DNA in opposite directions. Little is known about the functional relationship between sister replisomes.

  9. Apple Can Act as Anti-Aging on Yeast Cells

    Directory of Open Access Journals (Sweden)

    Vanessa Palermo

    2012-01-01

    Full Text Available In recent years, epidemiological and biochemical studies have shown that eating apples is associated with reduction of occurrence of cancer, degenerative, and cardiovascular diseases. This association is often attributed to the presence of antioxidants such as ascorbic acid (vitamin C and polyphenols. The substances that hinder the presence of free radicals are also able to protect cells from aging. In our laboratory we used yeast, a unicellular eukaryotic organism, to determine in vivo efficacy of entire apples and their components, such as flesh, skin and polyphenolic fraction, to influence aging and oxidative stress. Our results indicate that all the apple components increase lifespan, with the best result given by the whole fruit, indicating a cooperative role of all apple components.

  10. Nuclear fusion and genome encounter during yeast zygote formation.

    Science.gov (United States)

    Tartakoff, Alan Michael; Jaiswal, Purnima

    2009-06-01

    When haploid cells of Saccharomyces cerevisiae are crossed, parental nuclei congress and fuse with each other. To investigate underlying mechanisms, we have developed assays that evaluate the impact of drugs and mutations. Nuclear congression is inhibited by drugs that perturb the actin and tubulin cytoskeletons. Nuclear envelope (NE) fusion consists of at least five steps in which preliminary modifications are followed by controlled flux of first outer and then inner membrane proteins, all before visible dilation of the waist of the nucleus or coalescence of the parental spindle pole bodies. Flux of nuclear pore complexes occurs after dilation. Karyogamy requires both the Sec18p/NSF ATPase and ER/NE luminal homeostasis. After fusion, chromosome tethering keeps tagged parental genomes separate from each other. The process of NE fusion and evidence of genome independence in yeast provide a prototype for understanding related events in higher eukaryotes.

  11. Homology of yeast photoreactivating gene fragment with human genomic digests

    International Nuclear Information System (INIS)

    Meechan, P.J.; Milam, K.M.; Cleaver, J.E.

    1984-01-01

    Enzymatic photoreactivation of UV-induced DNA lesions has been demonstrated for a variety of prokaryotic and eukaryotic organisms. Its presence in placental mammals, however, has not been clearly established. The authors attempted to resolve this question by assaying for the presence (or absence) of sequences in human DNA complimentary to a fragment of the photoreactivating gene from S. cerevisiae that has recently been cloned. In another study, DNA from human, chick E. coli and yeast cells was digested with either HindIII of BglII, electrophoresed on a 0.5% agarose gel, transferred (Southern blot) to a nylon membrane and probed for homology against a Sau3A restriction fragment from S. cerevisiae that compliments phr/sup -/ cells. Hybridization to human DNA digests was observed only under relatively non-stringent conditions indicating the gene is not conserved in placental mammals. These results are correlated with current literature data concerning photoreactivating enzymes

  12. Uniting sex and eukaryote origins in an emerging oxygenic world.

    Science.gov (United States)

    Gross, Jeferson; Bhattacharya, Debashish

    2010-08-23

    Theories about eukaryote origins (eukaryogenesis) need to provide unified explanations for the emergence of diverse complex features that define this lineage. Models that propose a prokaryote-to-eukaryote transition are gridlocked between the opposing "phagocytosis first" and "mitochondria as seed" paradigms, neither of which fully explain the origins of eukaryote cell complexity. Sex (outcrossing with meiosis) is an example of an elaborate trait not yet satisfactorily addressed in theories about eukaryogenesis. The ancestral nature of meiosis and its dependence on eukaryote cell biology suggest that the emergence of sex and eukaryogenesis were simultaneous and synergic and may be explained by a common selective pressure. We propose that a local rise in oxygen levels, due to cyanobacterial photosynthesis in ancient Archean microenvironments, was highly toxic to the surrounding biota. This selective pressure drove the transformation of an archaeal (archaebacterial) lineage into the first eukaryotes. Key is that oxygen might have acted in synergy with environmental stresses such as ultraviolet (UV) radiation and/or desiccation that resulted in the accumulation of reactive oxygen species (ROS). The emergence of eukaryote features such as the endomembrane system and acquisition of the mitochondrion are posited as strategies to cope with a metabolic crisis in the cell plasma membrane and the accumulation of ROS, respectively. Selective pressure for efficient repair of ROS/UV-damaged DNA drove the evolution of sex, which required cell-cell fusions, cytoskeleton-mediated chromosome movement, and emergence of the nuclear envelope. Our model implies that evolution of sex and eukaryogenesis were inseparable processes. Several types of data can be used to test our hypothesis. These include paleontological predictions, simulation of ancient oxygenic microenvironments, and cell biological experiments with Archaea exposed to ROS and UV stresses. Studies of archaeal conjugation

  13. Patterns of intron gain and conservation in eukaryotic genes

    Directory of Open Access Journals (Sweden)

    Wolf Yuri I

    2007-10-01

    Full Text Available Abstract Background: The presence of introns in protein-coding genes is a universal feature of eukaryotic genome organization, and the genes of multicellular eukaryotes, typically, contain multiple introns, a substantial fraction of which share position in distant taxa, such as plants and animals. Depending on the methods and data sets used, researchers have reached opposite conclusions on the causes of the high fraction of shared introns in orthologous genes from distant eukaryotes. Some studies conclude that shared intron positions reflect, almost entirely, a remarkable evolutionary conservation, whereas others attribute it to parallel gain of introns. To resolve these contradictions, it is crucial to analyze the evolution of introns by using a model that minimally relies on arbitrary assumptions. Results: We developed a probabilistic model of evolution that allows for variability of intron gain and loss rates over branches of the phylogenetic tree, individual genes, and individual sites. Applying this model to an extended set of conserved eukaryotic genes, we find that parallel gain, on average, accounts for only ~8% of the shared intron positions. However, the distribution of parallel gains over the phylogenetic tree of eukaryotes is highly non-uniform. There are, practically, no parallel gains in closely related lineages, whereas for distant lineages, such as animals and plants, parallel gains appear to contribute up to 20% of the shared intron positions. In accord with these findings, we estimated that ancestral introns have a high probability to be retained in extant genomes, and conversely, that a substantial fraction of extant introns have retained their positions since the early stages of eukaryotic evolution. In addition, the density of sites that are available for intron insertion is estimated to be, approximately, one in seven basepairs. Conclusion: We obtained robust estimates of the contribution of parallel gain to the observed

  14. Calcium homeostasis and signaling in fungi and their relevance for pathogenicity of yeasts and filamentous fungi

    Directory of Open Access Journals (Sweden)

    Renata Tisi

    2016-09-01

    Full Text Available Though fungi show peculiarities in the purposes and specific traits of calcium signaling pathways, the general scheme and the most important players are well conserved if compared to higher eukaryotes. This provides a powerful opportunity either to investigate shared features using yeast as a model or to exploit fungal specificities as potential targets for antifungal therapies. The sequenced genomes from yeast Saccharomyces cerevisiae, Schizosaccharomyces pombe and the filamentous fungus Neurospora crassa were already published more than ten years ago. More recently the genome sequences of filamentous fungi of Aspergillus genus, some of which threatening pathogens, and dimorphic fungi Ustilago maydis were published, giving the chance to identify several proteins involved in calcium signaling based on their homology to yeast or mammalian counterparts. Nonetheless, unidentified calcium transporters are still present in these organisms which await to be molecularly characterized. Despite the relative simplicity in yeast calcium machinery and the availability of sophisticated molecular tools, in the last years, a number of new actors have been identified, albeit not yet fully characterized. This review will try to describe the state of the art in calcium channels and calcium signaling knowledge in yeast, with particular attention to the relevance of this knowledge with respect to pathological fungi.

  15. Implication of Ca2+ in the regulation of replicative life span of budding yeast.

    Science.gov (United States)

    Tsubakiyama, Ryohei; Mizunuma, Masaki; Gengyo, Anri; Yamamoto, Josuke; Kume, Kazunori; Miyakawa, Tokichi; Hirata, Dai

    2011-08-19

    In eukaryotic cells, Ca(2+)-triggered signaling pathways are used to regulate a wide variety of cellular processes. Calcineurin, a highly conserved Ca(2+)/calmodulin-dependent protein phosphatase, plays key roles in the regulation of diverse biological processes in organisms ranging from yeast to humans. We isolated a mutant of the SIR3 gene, implicated in the regulation of life span, as a suppressor of the Ca(2+) sensitivity of zds1Δ cells in the budding yeast Saccharomyces cerevisiae. Therefore, we investigated a relationship between Ca(2+) signaling and life span in yeast. Here we show that Ca(2+) affected the replicative life span (RLS) of yeast. Increased external and intracellular Ca(2+) levels caused a reduction in their RLS. Consistently, the increase in calcineurin activity by either the zds1 deletion or the constitutively activated calcineurin reduced RLS. Indeed, the shortened RLS of zds1Δ cells was suppressed by the calcineurin deletion. Further, the calcineurin deletion per se promoted aging without impairing the gene silencing typically observed in short-lived sir mutants, indicating that calcineurin plays an important role in a regulation of RLS even under normal growth condition. Thus, our results indicate that Ca(2+) homeostasis/Ca(2+) signaling are required to regulate longevity in budding yeast.

  16. Assessing phagotrophy in the mixotrophic ciliate Paramecium bursaria using GFP-expressing yeast cells.

    Science.gov (United States)

    Miura, Takashi; Moriya, Hisao; Iwai, Sosuke

    2017-07-03

    We used cells of the yeast Saccharomyces cerevisiae expressing green fluorescent protein (GFP) as fluorescently labelled prey to assess the phagocytic activities of the mixotrophic ciliate Paramecium bursaria, which harbours symbiotic Chlorella-like algae. Because of different fluorescence spectra of GFP and algal chlorophyll, ingested GFP-expressing yeast cells can be distinguished from endosymbiotic algal cells and directly counted in individual P. bursaria cells using fluorescence microscopy. By using GFP-expressing yeast cells, we found that P. bursaria altered ingestion activities under different physiological conditions, such as different growth phases or the presence/absence of endosymbionts. Use of GFP-expressing yeast cells allowed us to estimate the digestion rates of live prey of the ciliate. In contrast to the ingestion activities, the digestion rate within food vacuoles was not affected by the presence of endosymbionts, consistent with previous findings that food and perialgal vacuoles are spatially and functionally separated in P. bursaria. Thus, GFP-expressing yeast may provide a valuable tool to assess both ingestion and digestion activities of ciliates that feed on eukaryotic organisms. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Structural differences between yeast and mammalian microtubules revealed by cryo-EM

    Energy Technology Data Exchange (ETDEWEB)

    Howes, Stuart C. [Univ. of California, Berkeley, CA (United States). Biophysics Graduate Group; Geyer, Elisabeth A. [Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biophysics; Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biochemistry; LaFrance, Benjamin [Univ. of California, Berkeley, CA (United States). Molecular and Cell Biology Graduate Program; Zhang, Rui [Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Kellogg, Elizabeth H. [Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Westermann, Stefan [Univ. of Duisburg-Essen, Essen (Germany). Dept. of Molecular Genetics, Center for Medical Biotechnology; Rice, Luke M. [Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biophysics; Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biochemistry; Nogales, Eva [Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Univ. of California, Berkeley, CA (United States). Dept. of Molecular Biology and California Inst. for Quantitative Biosciences; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division

    2017-06-26

    Microtubules are polymers of αβ-tubulin heterodimers essential for all eukaryotes. Despite sequence conservation, there are significant structural differences between microtubules assembled in vitro from mammalian or budding yeast tubulin. Yeast MTs were not observed to undergo compaction at the interdimer interface as seen for mammalian microtubules upon GTP hydrolysis. Lack of compaction might reflect slower GTP hydrolysis or a different degree of allosteric coupling in the lattice. The microtubule plus end–tracking protein Bim1 binds yeast microtubules both between αβ-tubulin heterodimers, as seen for other organisms, and within tubulin dimers, but binds mammalian tubulin only at interdimer contacts. At the concentrations used in cryo-electron microscopy, Bim1 causes the compaction of yeast microtubules and induces their rapid disassembly. In conclusion, our studies demonstrate structural differences between yeast and mammalian microtubules that likely underlie their differing polymerization dynamics. These differences may reflect adaptations to the demands of different cell size or range of physiological growth temperatures.

  18. The cauliflower Orange gene enhances petiole elongation by suppressing expression of eukaryotic release factor 1.

    Science.gov (United States)

    Zhou, Xiangjun; Sun, Tian-Hu; Wang, Ning; Ling, Hong-Qing; Lu, Shan; Li, Li

    2011-04-01

    The cauliflower (Brassica oleracea var. botrytis) Orange (Or) gene affects plant growth and development in addition to conferring β-carotene accumulation. This study was undertaken to investigate the molecular basis for the effects of the Or gene mutation in on plant growth. The OR protein was found to interact with cauliflower and Arabidopsis eukaryotic release factor 1-2 (eRF1-2), a member of the eRF1 family, by yeast two-hybrid analysis and by bimolecular fluorescence complementation (BiFC) assay. Concomitantly, the Or mutant showed reduced expression of the BoeRF1 family genes. Transgenic cauliflower plants with suppressed expression of BoeRF1-2 and BoeRF1-3 were generated by RNA interference. Like the Or mutant, the BoeRF1 RNAi lines showed increased elongation of the leaf petiole. This long-petiole phenotype was largely caused by enhanced cell elongation, which resulted from increased cell length and elevated expression of genes involved in cell-wall loosening. These findings demonstrate that the cauliflower Or gene controls petiole elongation by suppressing the expression of eRF1 genes, and provide new insights into the molecular mechanism of leaf petiole regulation. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

  19. Functional conservation of coenzyme Q biosynthetic genes among yeasts, plants, and humans.

    Directory of Open Access Journals (Sweden)

    Kazuhiro Hayashi

    Full Text Available Coenzyme Q (CoQ is an essential factor for aerobic growth and oxidative phosphorylation in the electron transport system. The biosynthetic pathway for CoQ has been proposed mainly from biochemical and genetic analyses of Escherichia coli and Saccharomyces cerevisiae; however, the biosynthetic pathway in higher eukaryotes has been explored in only a limited number of studies. We previously reported the roles of several genes involved in CoQ synthesis in the fission yeast Schizosaccharomyces pombe. Here, we expand these findings by identifying ten genes (dps1, dlp1, ppt1, and coq3-9 that are required for CoQ synthesis. CoQ10-deficient S. pombe coq deletion strains were generated and characterized. All mutant fission yeast strains were sensitive to oxidative stress, produced a large amount of sulfide, required an antioxidant to grow on minimal medium, and did not survive at the stationary phase. To compare the biosynthetic pathway of CoQ in fission yeast with that in higher eukaryotes, the ability of CoQ biosynthetic genes from humans and plants (Arabidopsis thaliana to functionally complement the S. pombe coq deletion strains was determined. With the exception of COQ9, expression of all other human and plant COQ genes recovered CoQ10 production by the fission yeast coq deletion strains, although the addition of a mitochondrial targeting sequence was required for human COQ3 and COQ7, as well as A. thaliana COQ6. In summary, this study describes the functional conservation of CoQ biosynthetic genes between yeasts, humans, and plants.

  20. Yeasts preservation: alternatives for lyophilisation

    OpenAIRE

    Nyanga, Loveness K.; Nout, Martinus J. R.; Smid, Eddy J.; Boekhout, Teun; Zwietering, Marcel H.

    2012-01-01

    The aim of the study was to compare the effect of two low-cost, low technology traditional methods for drying starter cultures with standard lyophilisation. Lyophilised yeast cultures and yeast cultures preserved in dry rice cakes and dry plant fibre strands were examined for viable cell counts during 6 months storage at 4 and 25 °C. None of the yeast cultures showed a significant loss in viable cell count during 6 months of storage at 4 °C upon lyophilisation and preservation in dry rice cak...

  1. [Distiller Yeasts Producing Antibacterial Peptides].

    Science.gov (United States)

    Klyachko, E V; Morozkina, E V; Zaitchik, B Ts; Benevolensky, S V

    2015-01-01

    A new method of controlling lactic acid bacteria contamination was developed with the use of recombinant Saccharomyces cerevisiae strains producing antibacterial peptides. Genes encoding the antibacterial peptides pediocin and plantaricin with codons preferable for S. cerevisiae were synthesized, and a system was constructed for their secretory expression. Recombinant S. cerevisiae strains producing antibacterial peptides effectively inhibit the growth of Lactobacillus sakei, Pediacoccus pentasaceus, Pediacoccus acidilactici, etc. The application of distiller yeasts producing antibacterial peptides enhances the ethanol yield in cases of bacterial contamination. Recombinant yeasts producing the antibacterial peptides pediocin and plantaricin can successfully substitute the available industrial yeast strains upon ethanol production.

  2. Yeast synthetic biology toolbox and applications for biofuel production.

    Science.gov (United States)

    Tsai, Ching-Sung; Kwak, Suryang; Turner, Timothy L; Jin, Yong-Su

    2015-02-01

    Yeasts are efficient biofuel producers with numerous advantages outcompeting bacterial counterparts. While most synthetic biology tools have been developed and customized for bacteria especially for Escherichia coli, yeast synthetic biological tools have been exploited for improving yeast to produce fuels and chemicals from renewable biomass. Here we review the current status of synthetic biological tools and their applications for biofuel production, focusing on the model strain Saccharomyces cerevisiae We describe assembly techniques that have been developed for constructing genes, pathways, and genomes in yeast. Moreover, we discuss synthetic parts for allowing precise control of gene expression at both transcriptional and translational levels. Applications of these synthetic biological approaches have led to identification of effective gene targets that are responsible for desirable traits, such as cellulosic sugar utilization, advanced biofuel production, and enhanced tolerance against toxic products for biofuel production from renewable biomass. Although an array of synthetic biology tools and devices are available, we observed some gaps existing in tool development to achieve industrial utilization. Looking forward, future tool development should focus on industrial cultivation conditions utilizing industrial strains. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  3. Regulation of eukaryotic initiation factor 4AII by MyoD during murine myogenic cell differentiation.

    Directory of Open Access Journals (Sweden)

    Gabriela Galicia-Vázquez

    Full Text Available Gene expression during muscle cell differentiation is tightly regulated at multiple levels, including translation initiation. The PI3K/mTOR signalling pathway exerts control over protein synthesis by regulating assembly of eukaryotic initiation factor (eIF 4F, a heterotrimeric complex that stimulates recruitment of ribosomes to mRNA templates. One of the subunits of eIF4F, eIF4A, supplies essential helicase function during this phase of translation. The presence of two cellular eIF4A isoforms, eIF4AI and eIF4AII, has long thought to impart equivalent functions to eIF4F. However, recent experiments have alluded to distinct activities between them. Herein, we characterize distinct regulatory mechanisms between the eIF4A isoforms during muscle cell differentiation. We find that eIF4AI levels decrease during differentiation whereas eIF4AII levels increase during myofiber formation in a MyoD-dependent manner. This study characterizes a previously undefined mechanism for eIF4AII regulation in differentiation and highlights functional differences between eIF4AI and eIF4AII. Finally, RNAi-mediated alterations in eIF4AI and eIF4AII levels indicate that the myogenic process can tolerate short term reductions in eIF4AI or eIF4AII levels, but not both.

  4. Efficient Production of γ-GABA Using Recombinant E. coli Expressing Glutamate Decarboxylase (GAD) Derived from Eukaryote Saccharomyces cerevisiae.

    Science.gov (United States)

    Xiong, Qiang; Xu, Zheng; Xu, Lu; Yao, Zhong; Li, Sha; Xu, Hong

    2017-12-01

    γ-Aminobutyric acid (γ-GABA) is a non-proteinogenic amino acid, which acts as a major regulator in the central nervous system. Glutamate decarboxylase (namely GAD, EC 4.1.1.15) is known to be an ideal enzyme for γ-GABA production using L-glutamic acid as substrate. In this study, we cloned and expressed GAD gene from eukaryote Saccharomyces cerevisiae (ScGAD) in E. coli BL21(DE3). This enzyme was further purified and its optimal reaction temperature and pH were 37 °C and pH 4.2, respectively. The cofactor of ScGAD was verified to be either pyridoxal 5'-phosphate (PLP) or pyridoxal hydrochloride. The optimal concentration of either cofactor was 50 mg/L. The optimal medium for E. coli-ScGAD cultivation and expression were 10 g/L lactose, 5 g/L glycerol, 20 g/L yeast extract, and 10 g/L sodium chloride, resulting in an activity of 55 U/mL medium, three times higher than that of using Luria-Bertani (LB) medium. The maximal concentration of γ-GABA was 245 g/L whereas L-glutamic acid was near completely converted. These findings provided us a good example for bio-production of γ-GABA using recombinant E. coli expressing a GAD enzyme derived from eukaryote.

  5. Discourse Analysis in Translator Training

    OpenAIRE

    Gülfidan Ayvaz

    2015-01-01

    Translator training enables students to gain experience in both linguistic parameters and translation practice. Discourse Analysis is one of the strategies that lead to a better translation process and quality in translation. In that regard, this study aims to present DA as a translation strategy for translation practice and a useful tool for translator training. The relationship between DA and Translator Training is not widely studied. Therefore this study aims to define DA and how it can be...

  6. Yeast strains and methods of use thereof

    OpenAIRE

    Goddard, Matthew Robert; Gardner, Richard Clague; Anfang, Nicole

    2013-01-01

    The present invention relates to yeast strains and, in particular, to yeast stains for use in fermentation processes. The invention also relates to methods of fermentation using the yeast strains of the invention either alone or in combination with other yeast strains. The invention thither relates to methods for the selection of yeast strains suitable for fermentation cultures by screening for various metabolic products and the use of specific nutrient sources.

  7. Silibinin inhibits translation initiation: implications for anticancer therapy.

    Science.gov (United States)

    Lin, Chen-Ju; Sukarieh, Rami; Pelletier, Jerry

    2009-06-01

    Silibinin is a nontoxic flavonoid reported to have anticancer properties. In this study, we show that silibinin exhibits antiproliferative activity on MCF-7 breast cancer cells. Exposure to silibinin leads to a concentration-dependent decrease in global protein synthesis associated with reduced levels of eukaryotic initiation factor 4F complex. Moreover, polysome profile analysis of silibinin-treated cells shows a decrease in polysome content and translation of cyclin D1 mRNA. Silibinin exerts its effects on translation initiation by inhibiting the mammalian target of rapamycin signaling pathway by acting upstream of TSC2. Our results show that silibinin blocks mammalian target of rapamycin signaling with a concomitant reduction in translation initiation, thus providing a possible molecular mechanism of how silibinin can inhibit growth of transformed cells.

  8. Struggling with Translations

    DEFF Research Database (Denmark)

    Obed Madsen, Søren

    This paper shows empirical how actors have difficulties with translating strategy texts. The paper uses four cases as different examples of what happens, and what might be difficult, when actors translate organizational texts. In order to explore this, it draws on a translation training method from...... translation theory. The study shows that for those who have produced the text, it is difficult to translate a strategy where they have to change the words so others who don’t understand the language in the text can understand it. It also shows that for those who haven’t been a part of the production, it very...... challenge the notion that actors understand all texts and that managers per se can translate a text....

  9. Identification of SUMO conjugation sites in the budding yeast proteome

    Directory of Open Access Journals (Sweden)

    Miguel Esteras

    2017-10-01

    Full Text Available Post-translational modification by the small ubiquitin-like modifier (SUMO is an important mechanism regulating protein function. Identification of SUMO conjugation sites on substrates is a challenging task. Here we employed a proteomic method to map SUMO acceptor lysines in budding yeast proteins. We report the identification of 257 lysine residues where SUMO is potentially attached. Amongst the hits, we identified already known SUMO substrates and sites, confirming the success of the approach. In addition, we tested several of the novel substrates using SUMO immunoprecipitation analysis and confirmed that the SUMO acceptor lysines identified in these proteins are indeed bona fide SUMOylation sites. We believe that the collection of SUMO sites presented here is an important resource for future functional studies of SUMOylation in yeast.

  10. Dietary live yeast alters metabolic profiles, protein biosynthesis and thermal stress tolerance of Drosophila melanogaster.

    Science.gov (United States)

    Colinet, Hervé; Renault, David

    2014-04-01

    The impact of nutritional factors on insect's life-history traits such as reproduction and lifespan has been excessively examined; however, nutritional determinant of insect's thermal tolerance has not received a lot of attention. Dietary live yeast represents a prominent source of proteins and amino acids for laboratory-reared drosophilids. In this study, Drosophila melanogaster adults were fed on diets supplemented or not with live yeast. We hypothesized that manipulating nutritional conditions through live yeast supplementation would translate into altered physiology and stress tolerance. We verified how live yeast supplementation affected body mass characteristics, total lipids and proteins, metabolic profiles and cold tolerance (acute and chronic stress). Females fed with live yeast had increased body mass and contained more lipids and proteins. Using GC/MS profiling, we found distinct metabolic fingerprints according to nutritional conditions. Metabolite pathway enrichment analysis corroborated that live yeast supplementation was associated with amino acid and protein biosyntheses. The cold assays revealed that the presence of dietary live yeast greatly promoted cold tolerance. Hence, this study conclusively demonstrates a significant interaction between nutritional conditions and thermal tolerance. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Structural investigations of yeast mannans

    International Nuclear Information System (INIS)

    Rademacher, K.H.

    1983-01-01

    Cell wall mannans were isolated from 8 different Candida species and separated in oligosaccharides by partial acetolysis. After gel chromatography specific acetolysis patterns were obtained. The 13 C NMR spectra of mannans and oligosaccharides were recorded. Signals at delta = 93.1 - 105.4 were assigned to certain chemical structures. Both the spectral patterns and the acetolysis patterns of the yeast mannans can be used for the discrimination of related yeasts. (author)

  12. Translational ecology for hydrogeology.

    Science.gov (United States)

    Schlesinger, William H

    2013-01-01

    Translational ecology--a special discipline aimed to improve the accessibility of science to policy makers--will help hydrogeologists contribute to the solution of pressing environmental problems. Patterned after translational medicine, translational ecology is a partnership to ensure that the right science gets done in a timely fashion, so that it can be communicated to those who need it. © 2013, National Ground Water Association.

  13. Oral yeast colonization throughout pregnancy.

    Science.gov (United States)

    Rio, R; Simões-Silva, L; Garro, S; Silva, M-J; Azevedo, Á; Sampaio-Maia, B

    2017-03-01

    Recent studies suggest that placenta may harbour a unique microbiome that may have origin in maternal oral microbiome. Although the major physiological and hormonal adjustments observed in pregnant women lead to biochemical and microbiological modifications of the oral environment, very few studies evaluated the changes suffered by the oral microbiota throughout pregnancy. So, the aim of our study was to evaluate oral yeast colonization throughout pregnancy and to compare it with non-pregnant women. The oral yeast colonization was assessed in saliva of 30 pregnant and non-pregnant women longitudinally over a 6-months period. Demographic information was collected, a non-invasive intra-oral examination was performed and saliva flow and pH were determined. Pregnant and non-pregnant groups were similar regarding age and level of education. Saliva flow rate did not differ, but saliva pH was lower in pregnant than in non-pregnant women. Oral yeast prevalence was higher in pregnant than in non-pregnant women, either in the first or in the third trimester, but did not attain statistical significance. In individuals colonized with yeast, the total yeast quantification (Log10CFU/mL) increase from the 1st to the 3rd trimester in pregnant women, but not in non-pregnant women. Pregnancy may favour oral yeast growth that may be associated with an acidic oral environment.

  14. Biotechnological Applications of Dimorphic Yeasts

    Science.gov (United States)

    Doiphode, N.; Joshi, C.; Ghormade, V.; Deshpande, M. V.

    The dimorphic yeasts have the equilibrium between spherical growth (budding) and polarized (hyphal or pseudohyphal tip elongation) which can be triggered by change in the environmental conditions. The reversible growth phenomenon has made dimorphic yeasts as an useful model to understand fungal evolution and fungal differentiation, in general. In nature dimorphism is clearly evident in plant and animal fungal pathogens, which survive and most importantly proliferate in the respective hosts. However, number of organisms with no known pathogenic behaviour also show such a transition, which can be exploited for the technological applications due to their different biochemical make up under different morphologies. For instance, chitin and chitosan production using dimorphic Saccharomyces, Mucor, Rhizopus and Benjaminiella, oil degradation and biotransformation with yeast-form of Yarrowia species, bioremediation of organic pollutants, exopolysac-charide production by yeast-phase of Aureobasidium pullulans, to name a few. Myrothecium verrucaria can be used for seed dressing in its yeast form and it produces a mycolytic enzyme complex in its hyphal-form for the biocontrol of fungal pathogens, while Beauveria bassiana and other entomopathogens kill the insect pest by producing yeast- like cells in the insect body. The form-specific expression of protease, chitinase, lipase, ornithine decarboxylase, glutamate dehydrogenases, etc. make Benjaminiella poitrasii, Basidiobolus sp., and Mucor rouxii strains important in bioremediation, nanobiotechnology, fungal evolution and other areas.

  15. Translation and Intertextuality

    Directory of Open Access Journals (Sweden)

    Mohammad Rahimi

    2015-09-01

    Full Text Available This study is intends to describe and Presents a new theory of translation based on the "Intertextuality" unlike the Translation theories that presented to date, what all are based on the principle of "Equivalence". Our theory is based on the examples of Arabic poetry translated into Persian poetry. The major findings of this study show that the Intertextuality can serve as a link between the original text and the target. it can also interact with other texts is the translation result in the target language, Whtich is the book of poetic eloquence is addressed and was mentioned Literary robbery.

  16. Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks

    Science.gov (United States)

    Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

    2016-05-01

    RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks.

  17. Yeast ribosomal proteins

    International Nuclear Information System (INIS)

    Otaka, E.; Kobata, K.

    1978-01-01

    The cytoplasmic 80s ribosomal proteins from the cells of yeast Saccharomyces cerevisiae were analyzed by SDS two-dimensional polyacrylamide gel electrophoresis. Seventyfour proteins were identified and consecutively numbered from 1 to 74. Upon oxidation of the 80s proteins with performic acid, ten proteins (no. 15, 20, 35, 40, 44, 46, 49, 51, 54 and 55) were dislocated on the gel without change of the total number of protein spots. Five proteins (no. 8, 14, 16, 36 and 74) were phosphorylated in vivo as seen in 32 P-labelling experiments. The large and small subunits separated in low magnesium medium were analyzed by the above gel electrophoresis. At least forty-five and twenty-eight proteins were assumed to be in the large and small subunits, respectively. All proteins found in the 80s ribosomes, except for no. 3, were detected in either subunit without appearance of new spots. The acidic protein no. 3 seems to be lost during subunit dissociation. (orig.) [de

  18. Metabolic regulation of yeast

    Science.gov (United States)

    Fiechter, A.

    1982-12-01

    Metabolic regulation which is based on endogeneous and exogeneous process variables which may act constantly or time dependently on the living cell is discussed. The observed phenomena of the regulation are the result of physical, chemical, and biological parameters. These parameters are identified. Ethanol is accumulated as an intermediate product and the synthesis of biomass is reduced. This regulatory effect of glucose is used for the aerobic production of ethanol. Very high production rates are thereby obtained. Understanding of the regulation mechanism of the glucose effect has improved. In addition to catabolite repression, several other mechanisms of enzyme regulation have been described, that are mostly governed by exogeneous factors. Glucose also affects the control of respiration in a third class of yeasts which are unable to make use of ethanol as a substrate for growth. This is due to the lack of any anaplerotic activity. As a consequence, diauxic growth behavior is reduced to a one-stage growth with a drastically reduced cell yield. The pulse chemostat technique, a systematic approach for medium design is developed and medium supplements that are essential for metabolic control are identified.

  19. Nonselective enrichment for yeast adenine mutants by flow cytometry

    Science.gov (United States)

    Bruschi, C. V.; Chuba, P. J.

    1988-01-01

    The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.

  20. Long-read sequencing data analysis for yeasts.

    Science.gov (United States)

    Yue, Jia-Xing; Liti, Gianni

    2018-06-01

    Long-read sequencing technologies have become increasingly popular due to their strengths in resolving complex genomic regions. As a leading model organism with small genome size and great biotechnological importance, the budding yeast Saccharomyces cerevisiae has many isolates currently being sequenced with long reads. However, analyzing long-read sequencing data to produce high-quality genome assembly and annotation remains challenging. Here, we present a modular computational framework named long-read sequencing data analysis for yeasts (LRSDAY), the first one-stop solution that streamlines this process. Starting from the raw sequencing reads, LRSDAY can produce chromosome-level genome assembly and comprehensive genome annotation in a highly automated manner with minimal manual intervention, which is not possible using any alternative tool available to date. The annotated genomic features include centromeres, protein-coding genes, tRNAs, transposable elements (TEs), and telomere-associated elements. Although tailored for S. cerevisiae, we designed LRSDAY to be highly modular and customizable, making it adaptable to virtually any eukaryotic organism. When applying LRSDAY to an S. cerevisiae strain, it takes ∼41 h to generate a complete and well-annotated genome from ∼100× Pacific Biosciences (PacBio) running the basic workflow with four threads. Basic experience working within the Linux command-line environment is recommended for carrying out the analysis using LRSDAY.

  1. Yeast Acid Phosphatases and Phytases: Production, Characterization and Commercial Prospects

    Science.gov (United States)

    Kaur, Parvinder; Satyanarayana, T.

    The element phosphorus is critical to all life forms as it forms the basic component of nucleic acids and ATP and has a number of indispensable biochemical roles. Unlike C or N, the biogeochemical cycling of phosphorus is very slow, and thus making it the growth-limiting element in most soils and aquatic systems. Phosphohydrolases (e.g. acid phosphatases and phytases) are enzymes that break the C-O-P ester bonds and provide available inorganic phosphorus from various inassimilable organic forms of phosphorus like phytates. These enzymes are of significant value in effectively combating phosphorus pollution. Although phytases and acid phosphatases are produced by various plants, animals and micro organisms, microbial sources are more promising for the production on a commercial scale. Yeasts being the simplest eukaryotes are ideal candidates for phytase and phos-phatase research due to their mostly non-pathogenic and GRAS status. They have not, however, been utilized to their full potential. This chapter focuses attention on the present state of knowledge on the production, characterization and potential commercial prospects of yeast phytases and acid phosphatases.

  2. Anionic lipids and the maintenance of membrane electrostatics in eukaryotes.

    Science.gov (United States)

    Platre, Matthieu Pierre; Jaillais, Yvon

    2017-02-01

    A wide range of signaling processes occurs at the cell surface through the reversible association of proteins from the cytosol to the plasma membrane. Some low abundant lipids are enriched at the membrane of specific compartments and thereby contribute to the identity of cell organelles by acting as biochemical landmarks. Lipids also influence membrane biophysical properties, which emerge as an important feature in specifying cellular territories. Such parameters are crucial for signal transduction and include lipid packing, membrane curvature and electrostatics. In particular, membrane electrostatics specifies the identity of the plasma membrane inner leaflet. Membrane surface charges are carried by anionic phospholipids, however the exact nature of the lipid(s) that powers the plasma membrane electrostatic field varies among eukaryotes and has been hotly debated during the last decade. Herein, we discuss the role of anionic lipids in setting up plasma membrane electrostatics and we compare similarities and differences that were found in different eukaryotic cells.

  3. The MCM Helicase Motor of the Eukaryotic Replisome.

    Science.gov (United States)

    Abid Ali, Ferdos; Costa, Alessandro

    2016-05-08

    The MCM motor of the CMG helicase powers ahead of the eukaryotic replication machinery to unwind DNA, in a process that requires ATP hydrolysis. The reconstitution of DNA replication in vitro has established the succession of events that lead to replication origin activation by the MCM and recent studies have started to elucidate the structural basis of duplex DNA unwinding. Despite the exciting progress, how the MCM translocates on DNA remains a matter of debate. Copyright © 2016. Published by Elsevier Ltd.

  4. Biological Influence of Deuterium on Procariotic and Eukaryotic Cells

    OpenAIRE

    Oleg Mosin; Ignat Ignatov

    2014-01-01

    Biologic influence of deuterium (D) on cells of various taxonomic groups of prokaryotic and eukaryotic microorganisms realizing methylotrophic, chemoheterotrophic, photo-organotrophic, and photosynthetic ways of assimilation of carbon substrates are investigated at growth on media with heavy water (D2О). The method of step by step adaptation technique of cells to D2О was developed, consisting in plating of cells on 2 % agarose nutrient media containing increasing gradient of concentration of ...

  5. An Evolutionary Framework for Understanding the Origin of Eukaryotes

    OpenAIRE

    Neil W. Blackstone

    2016-01-01

    Two major obstacles hinder the application of evolutionary theory to the origin of eukaryotes. The first is more apparent than real?the endosymbiosis that led to the mitochondrion is often described as ?non-Darwinian? because it deviates from the incremental evolution championed by the modern synthesis. Nevertheless, endosymbiosis can be accommodated by a multi-level generalization of evolutionary theory, which Darwin himself pioneered. The second obstacle is more serious?all of the major fea...

  6. Gram-Negative Bacterial Sensors for Eukaryotic Signal Molecules

    Directory of Open Access Journals (Sweden)

    Olivier Lesouhaitier

    2009-09-01

    Full Text Available Ample evidence exists showing that eukaryotic signal molecules synthesized and released by the host can activate the virulence of opportunistic pathogens. The sensitivity of prokaryotes to host signal molecules requires the presence of bacterial sensors. These prokaryotic sensors, or receptors, have a double function: stereospecific recognition in a complex environment and transduction of the message in order to initiate bacterial physiological modifications. As messengers are generally unable to freely cross the bacterial membrane, they require either the presence of sensors anchored in the membrane or transporters allowing direct recognition inside the bacterial cytoplasm. Since the discovery of quorum sensing, it was established that the production of virulence factors by bacteria is tightly growth-phase regulated. It is now obvious that expression of bacterial virulence is also controlled by detection of the eukaryotic messengers released in the micro-environment as endocrine or neuro-endocrine modulators. In the presence of host physiological stress many eukaryotic factors are released and detected by Gram-negative bacteria which in return rapidly adapt their physiology. For instance, Pseudomonas aeruginosa can bind elements of the host immune system such as interferon-γ and dynorphin and then through quorum sensing circuitry enhance its virulence. Escherichia coli sensitivity to the neurohormones of the catecholamines family appears relayed by a recently identified bacterial adrenergic receptor. In the present review, we will describe the mechanisms by which various eukaryotic signal molecules produced by host may activate Gram-negative bacteria virulence. Particular attention will be paid to Pseudomonas, a genus whose representative species, P. aeruginosa, is a common opportunistic pathogen. The discussion will be particularly focused on the pivotal role played by these new types of pathogen sensors from the sensing to the transduction

  7. Replication and Transcription of Eukaryotic DNA in Esherichia coli

    Science.gov (United States)

    Morrow, John F.; Cohen, Stanley N.; Chang, Annie C. Y.; Boyer, Herbert W.; Goodman, Howard M.; Helling, Robert B.

    1974-01-01

    Fragments of amplified Xenopus laevis DNA, coding for 18S and 28S ribosomal RNA and generated by EcoRI restriction endonuclease, have been linked in vitro to the bacterial plasmid pSC101; and the recombinant molecular species have been introduced into E. coli by transformation. These recombinant plasmids, containing both eukaryotic and prokaryotic DNA, replicate stably in E. coli. RNA isolated from E. coli minicells harboring the plasmids hybridizes to amplified X. laevis rDNA. Images PMID:4600264

  8. Extreme Diversity of Diplonemid Eukaryotes in the Ocean

    Czech Academy of Sciences Publication Activity Database

    Flegontova, Olga; Flegontov, Pavel; Malviya, S.; Audic, S.; Wincker, P.; de Vargas, C.; Bowler, C.; Lukeš, Julius; Horák, Aleš

    2016-01-01

    Roč. 26, č. 22 (2016), s. 3060-3065 ISSN 0960-9822 R&D Projects: GA ČR GPP506/12/P931; GA ČR(CZ) GA14-23986S Institutional support: RVO:60077344 Keywords : virus-sized particles * microbial eukaryotes * sea-floor * phytoplankton * communities * euglenozoa * dispersal * ecosystem Subject RIV: EG - Zoology Impact factor: 8.851, year: 2016

  9. Effects of background fluid on the efficiency of inactivating yeast with non-thermal atmospheric pressure plasma.

    Directory of Open Access Journals (Sweden)

    Young-Hyo Ryu

    Full Text Available Non-thermal plasma at atmospheric pressure has been actively applied to sterilization. However, its efficiency for inactivating microorganisms often varies depending on microbial species and environments surrounding the microorganisms. We investigated the influence of environmental factors (surrounding media on the efficiency of microbial inactivation by plasma using an eukaryotic model microbe, Saccharomyces cerevisiae, to elucidate the mechanisms for differential efficiency of sterilization by plasma. Yeast cells treated with plasma in water showed the most severe damage in viability and cell morphology as well as damage to membrane lipids, and genomic DNA. Cells in saline were less damaged compared to those in water, and those in YPD (Yeast extract, Peptone, Dextrose were least impaired. HOG1 mitogen activated protein kinase was activated in cells exposed to plasma in water and saline. Inactivation of yeast cells in water and saline was due to the acidification of the solutions by plasma, but higher survival of yeast cells treated in saline may have resulted from the additional effect related to salt strength. Levels of hydroxyl radical (OH· produced by plasma were the highest in water and the lowest in YPD. This may have resulted in differential inactivation of yeast cells in water, saline, and YPD by plasma. Taken together, our data suggest that the surrounding media (environment can crucially affect the outcomes of yeast cell plasma treatment because plasma modulates vital properties of media, and the toxic nature of plasma can also be altered by the surrounding media.

  10. Lost in Translation?

    NARCIS (Netherlands)

    Jonkers, Peter

    2017-01-01

    Translating sacred scriptures is not only a praxis that is crucial for the fruitful, i.e. non-distorted and unbiased dialogue between different religious traditions, but also raises some fundamental theoretical questions when it comes to translating the sacred texts of the religious other or

  11. Translating VDM to Alloy

    DEFF Research Database (Denmark)

    Lausdahl, Kenneth

    2013-01-01

    specifications. However, to take advantage of the automated analysis of Alloy, the model-oriented VDM specifications must be translated into a constraint-based Alloy specifications. We describe how a sub- set of VDM can be translated into Alloy and how assertions can be expressed in VDM and checked by the Alloy...

  12. Students' Differentiated Translation Processes

    Science.gov (United States)

    Bossé, Michael J.; Adu-Gyamfi, Kwaku; Chandler, Kayla

    2014-01-01

    Understanding how students translate between mathematical representations is of both practical and theoretical importance. This study examined students' processes in their generation of symbolic and graphic representations of given polynomial functions. The purpose was to investigate how students perform these translations. The result of the study…

  13. Creativity, Culture and Translation

    Science.gov (United States)

    Babaee, Siamak; Wan Yahya, Wan Roselezam; Babaee, Ruzbeh

    2014-01-01

    Some scholars (Bassnett-McGuire, Catford, Brislin) suggest that a good piece of translation should be a strict reflection of the style of the original text while some others (Gui, Newmark, Wilss) consider the original text untranslatable unless it is reproduced. Opposing views by different critics suggest that translation is still a challenging…

  14. Translation as (Global) Writing

    Science.gov (United States)

    Horner, Bruce; Tetreault, Laura

    2016-01-01

    This article explores translation as a useful point of departure and framework for taking a translingual approach to writing engaging globalization. Globalization and the knowledge economy are putting renewed emphasis on translation as a key site of contest between a dominant language ideology of monolingualism aligned with fast capitalist…

  15. Measuring Translation Literality

    DEFF Research Database (Denmark)

    Carl, Michael; Schaeffer, Moritz

    2017-01-01

    Tirkkonen-Condit (2005: 407–408) argues that “It looks as if literal translation is [the result of] a default rendering procedure”. As a corollary, more literal translations should be easier to process, and less literal ones should be associated with more cognitive effort. In order to assess this...

  16. Text Coherence in Translation

    Science.gov (United States)

    Zheng, Yanping

    2009-01-01

    In the thesis a coherent text is defined as a continuity of senses of the outcome of combining concepts and relations into a network composed of knowledge space centered around main topics. And the author maintains that in order to obtain the coherence of a target language text from a source text during the process of translation, a translator can…

  17. TRANSLATING SERVICE TECHNICAL PROSE

    African Journals Online (AJOL)

    language. The Application of Technical Service. Prose. To form a good idea of the appl ication .... cost lives. In this particular domain, translators must have a sound technical ... These semantic ... another language and often, in doing so, changing its meaning. The words ..... He will hand out tasks to each translator and after.

  18. Stimulating translational research

    DEFF Research Database (Denmark)

    Bentires-Alj, Mohamed; Rajan, Abinaya; van Harten, Wim

    2015-01-01

    Translational research leaves no-one indifferent and everyone expects a particular benefit. We as EU-LIFE (www.eu-life.eu), an alliance of 13 research institutes in European life sciences, would like to share our experience in an attempt to identify measures to promote translational research with...... without undermining basic exploratory research and academic freedom....

  19. Translation, Quality and Growth

    DEFF Research Database (Denmark)

    Petersen, Margrethe

    The paper investigates the feasibility and some of the possible consequences of applying quality management to translation. It first gives an introduction to two different schools of translation and to (total) quality management. It then examines whether quality management may, in theory...

  20. Translation, Interpreting and Lexicography

    DEFF Research Database (Denmark)

    Dam, Helle Vrønning; Tarp, Sven

    2018-01-01

    in the sense that their practice fields are typically ‘about something else’. Translators may, for example, be called upon to translate medical texts, and interpreters may be assigned to work on medical speeches. Similarly, practical lexicography may produce medical dictionaries. In this perspective, the three...

  1. Translation between cultures

    Directory of Open Access Journals (Sweden)

    Henrique de Oliveira Lee

    2016-05-01

    Full Text Available This article will question the pertinence of understanding interculturality in terms of translation between cultures. I shall study this hypothesis in two ways : 1 / the cosmopolitan horizon, which the idea of translation may implicate ; 2 / the critique of the premises of unique origin and homogeneity of cultures which this hypothesis makes possible.

  2. Idioms and Back Translation

    Science.gov (United States)

    Griffin, Frank

    2004-01-01

    The challenges of intercultural communication are an integral part of many undergraduate business communication courses. Marketing gaffes clearly illustrate the pitfalls of translation and underscore the importance of a knowledge of the culture with which one is attempting to communicate. A good way to approach the topic of translation pitfalls in…

  3. Pleiotropic functions of the yeast Greatwall-family protein kinase Rim15p: a novel target for the control of alcoholic fermentation.

    Science.gov (United States)

    Watanabe, Daisuke; Takagi, Hiroshi

    2017-06-01

    Rim15p, a Greatwall-family protein kinase in yeast Saccharomyces cerevisiae, is required for cellular nutrient responses, such as the entry into quiescence and the induction of meiosis and sporulation. In higher eukaryotes, the orthologous gene products are commonly involved in the cell cycle G 2 /M transition. How are these pleiotropic functions generated from a single family of protein kinases? Recent advances in both research fields have identified the conserved Greatwall-mediated signaling pathway and a variety of downstream target molecules. In addition, our studies of S. cerevisiae sake yeast strains revealed that Rim15p also plays a significant role in the control of alcoholic fermentation. Despite an extensive history of research on glycolysis and alcoholic fermentation, there has been no critical clue to artificial modification of fermentation performance of yeast cells. Our finding of an in vivo metabolic regulatory mechanism is expected to provide a major breakthrough in yeast breeding technologies for fermentation applications.

  4. diArk – a resource for eukaryotic genome research

    Directory of Open Access Journals (Sweden)

    Kollmar Martin

    2007-04-01

    Full Text Available Abstract Background The number of completed eukaryotic genome sequences and cDNA projects has increased exponentially in the past few years although most of them have not been published yet. In addition, many microarray analyses yielded thousands of sequenced EST and cDNA clones. For the researcher interested in single gene analyses (from a phylogenetic, a structural biology or other perspective it is therefore important to have up-to-date knowledge about the various resources providing primary data. Description The database is built around 3 central tables: species, sequencing projects and publications. The species table contains commonly and alternatively used scientific names, common names and the complete taxonomic information. For projects the sequence type and links to species project web-sites and species homepages are stored. All publications are linked to projects. The web-interface provides comprehensive search modules with detailed options and three different views of the selected data. We have especially focused on developing an elaborate taxonomic tree search tool that allows the user to instantaneously identify e.g. the closest relative to the organism of interest. Conclusion We have developed a database, called diArk, to store, organize, and present the most relevant information about completed genome projects and EST/cDNA data from eukaryotes. Currently, diArk provides information about 415 eukaryotes, 823 sequencing projects, and 248 publications.

  5. Biotransformation of arsenic by a Yellowstone thermoacidophilic eukaryotic alga.

    Science.gov (United States)

    Qin, Jie; Lehr, Corinne R; Yuan, Chungang; Le, X Chris; McDermott, Timothy R; Rosen, Barry P

    2009-03-31

    Arsenic is the most common toxic substance in the environment, ranking first on the Superfund list of hazardous substances. It is introduced primarily from geochemical sources and is acted on biologically, creating an arsenic biogeocycle. Geothermal environments are known for their elevated arsenic content and thus provide an excellent setting in which to study microbial redox transformations of arsenic. To date, most studies of microbial communities in geothermal environments have focused on Bacteria and Archaea, with little attention to eukaryotic microorganisms. Here, we show the potential of an extremophilic eukaryotic alga of the order Cyanidiales to influence arsenic cycling at elevated temperatures. Cyanidioschyzon sp. isolate 5508 oxidized arsenite [As(III)] to arsenate [As(V)], reduced As(V) to As(III), and methylated As(III) to form trimethylarsine oxide (TMAO) and dimethylarsenate [DMAs(V)]. Two arsenic methyltransferase genes, CmarsM7 and CmarsM8, were cloned from this organism and demonstrated to confer resistance to As(III) in an arsenite hypersensitive strain of Escherichia coli. The 2 recombinant CmArsMs were purified and shown to transform As(III) into monomethylarsenite, DMAs(V), TMAO, and trimethylarsine gas, with a T(opt) of 60-70 degrees C. These studies illustrate the importance of eukaryotic microorganisms to the biogeochemical cycling of arsenic in geothermal systems, offer a molecular explanation for how these algae tolerate arsenic in their environment, and provide the characterization of algal methyltransferases.

  6. Ubiquitination dynamics in the early-branching eukaryote Giardia intestinalis

    Science.gov (United States)

    Niño, Carlos A; Chaparro, Jenny; Soffientini, Paolo; Polo, Simona; Wasserman, Moises

    2013-01-01

    Ubiquitination is a highly dynamic and versatile posttranslational modification that regulates protein function, stability, and interactions. To investigate the roles of ubiquitination in a primitive eukaryotic lineage, we utilized the early-branching eukaryote Giardia intestinalis. Using a combination of biochemical, immunofluorescence-based, and proteomics approaches, we assessed the ubiquitination status during the process of differentiation in Giardia. We observed that different types of ubiquitin modifications present specific cellular and temporal distribution throughout the Giardia life cycle from trophozoites to cyst maturation. Ubiquitin signal was detected in the wall of mature cysts, and enzymes implicated in cyst wall biogenesis were identified as substrates for ubiquitination. Interestingly, inhibition of proteasome activity did not affect trophozoite replication and differentiation, while it caused a decrease in cyst viability, arguing for proteasome involvement in cyst wall maturation. Using a proteomics approach, we identified around 200 high-confidence ubiquitinated candidates that vary their ubiquitination status during differentiation. Our results indicate that ubiquitination is critical for several cellular processes in this primitive eukaryote. PMID:23613346

  7. Gene Transfer in Eukaryotic Cells Using Activated Dendrimers

    Science.gov (United States)

    Dennig, Jörg

    Gene transfer into eukaryotic cells plays an important role in cell biology. Over the last 30 years a number of transfection methods have been developed to mediate gene transfer into eukaryotic cells. Classical methods include co-precipitation of DNA with calcium phosphate, charge-dependent precipitation of DNA with DEAE-dextran, electroporation of nucleic acids, and formation of transfection complexes between DNA and cationic liposomes. Gene transfer technologies based on activated PAMAM-dendrimers provide another class of transfection reagents. PAMAM-dendrimers are highly branched, spherical molecules. Activation of newly synthesized dendrimers involves hydrolytic removal of some of the branches, and results in a molecule with a higher degree of flexibility. Activated dendrimers assemble DNA into compact structures via charge interactions. Activated dendrimer - DNA complexes bind to the cell membrane of eukaryotic cells, and are transported into the cell by non-specific endocytosis. A structural model of the activated dendrimer - DNA complex and a potential mechanism for its uptake into cells will be discussed.

  8. Enzymes involved in organellar DNA replication in photosynthetic eukaryotes.

    Science.gov (United States)

    Moriyama, Takashi; Sato, Naoki

    2014-01-01

    Plastids and mitochondria possess their own genomes. Although the replication mechanisms of these organellar genomes remain unclear in photosynthetic eukaryotes, several organelle-localized enzymes related to genome replication, including DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, primer removal enzyme, and several DNA recombination-related enzymes, have been identified. In the reference Eudicot plant Arabidopsis thaliana, the replication-related enzymes of plastids and mitochondria are similar because many of them are dual targeted to both organelles, whereas in the red alga Cyanidioschyzon merolae, plastids and mitochondria contain different replication machinery components. The enzymes involved in organellar genome replication in green plants and red algae were derived from different origins, including proteobacterial, cyanobacterial, and eukaryotic lineages. In the present review, we summarize the available data for enzymes related to organellar genome replication in green plants and red algae. In addition, based on the type and distribution of replication enzymes in photosynthetic eukaryotes, we discuss the transitional history of replication enzymes in the organelles of plants.

  9. Mechanisms and regulation of DNA replication initiation in eukaryotes.

    Science.gov (United States)

    Parker, Matthew W; Botchan, Michael R; Berger, James M

    2017-04-01

    Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation - from selecting replication start sites to replicative helicase loading and activation - and describe how these events are often distinctly regulated across different eukaryotic model organisms.

  10. Translational selection in human: More pronounced in housekeeping genes

    KAUST Repository

    Ma, Lina

    2014-07-10

    Background: Translational selection is a ubiquitous and significant mechanism to regulate protein expression in prokaryotes and unicellular eukaryotes. Recent evidence has shown that translational selection is weakly operative in highly expressed genes in human and other vertebrates. However, it remains unclear whether translational selection acts differentially on human genes depending on their expression patterns.Results: Here we report that human housekeeping (HK) genes that are strictly defined as genes that are expressed ubiquitously and consistently in most or all tissues, are under stronger translational selection.Conclusions: These observations clearly show that translational selection is also closely associated with expression pattern. Our results suggest that human HK genes are more efficiently and/or accurately translated into proteins, which will inevitably open up a new understanding of HK genes and the regulation of gene expression.Reviewers: This article was reviewed by Yuan Yuan, Baylor College of Medicine; Han Liang, University of Texas MD Anderson Cancer Center (nominated by Dr Laura Landweber) Eugene Koonin, NCBI, NLM, NIH, United States of America Sandor Pongor, International Centre for Genetic Engineering and biotechnology (ICGEB), Italy. © 2014 Ma et al.; licensee BioMed Central Ltd.

  11. Genetic structure and evolution of the Vps25 family, a yeast ESCRT-II component

    Directory of Open Access Journals (Sweden)

    Slater Ruth

    2006-08-01

    Full Text Available Abstract Background Vps25p is the product of yeast gene VPS25 and is found in an endosomal sorting complex required for transport (ESCRT-II, along with Vps22p and Vps36p. This complex is essential for sorting of ubiquitinated biosynthetic and endosomal cargoes into endosomes. Results We found that VPS25 is a highly conserved and widely expressed eukaryotic gene, with single orthologs in chromalveolate, excavate, amoebozoan, plant, fungal and metazoan species. Two paralogs were found in Trichomonas vaginalis. An ortholog was strikingly absent from the Encephalitozoon cuniculi genome. Intron positions were analyzed in VPS25 from 36 species. We found evidence for five ancestral VPS25 introns, intron loss, and single instances of intron gain (a Paramecium species and intron slippage (Theileria species. Processed pseudogenes were identified in four mammalian genomes, with a notable absence in the mouse genome. Two retropseudogenes were found in the chimpanzee genome, one more recently inserted, and one evolving from a common primate ancestor. The amino acid sequences of 119 Vps25 orthologs are aligned, compared with the known secondary structure of yeast Vps25p, and used to carry out phylogenetic analysis. Residues in two amino-terminal PPXY motifs (motif I and II, involved in dimerization of Vps25p and interaction with Vps22p and Vps36p, were closely, but not absolutely conserved. Specifically, motif I was absent in Vps25 homologs of chromalveolates, euglenozoa, and diplomonads. A highly conserved carboxy-terminal lysine was identified, which suggests Vps25 is ubiquitinated. Arginine-83 of yeast Vps25p involved in Vps22p interaction was highly, but not absolutely, conserved. Human tissue expression analysis showed universal expression. Conclusion We have identified 119 orthologs of yeast Vps25p. Expression of mammalian VPS25 in a wide range of tissues, and the presence in a broad range of eukaryotic species, indicates a basic role in eukaryotic cell

  12. Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains.

    Science.gov (United States)

    Walker, Sarah E; Zhou, Fujun; Mitchell, Sarah F; Larson, Victoria S; Valasek, Leos; Hinnebusch, Alan G; Lorsch, Jon R

    2013-02-01

    Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B's domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome's mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment.

  13. Translation Ambiguity but Not Word Class Predicts Translation Performance

    Science.gov (United States)

    Prior, Anat; Kroll, Judith F.; Macwhinney, Brian

    2013-01-01

    We investigated the influence of word class and translation ambiguity on cross-linguistic representation and processing. Bilingual speakers of English and Spanish performed translation production and translation recognition tasks on nouns and verbs in both languages. Words either had a single translation or more than one translation. Translation…

  14. Examining English-German Translation Ambiguity Using Primed Translation Recognition

    Science.gov (United States)

    Eddington, Chelsea M.; Tokowicz, Natasha

    2013-01-01

    Many words have more than one translation across languages. Such "translation-ambiguous" words are translated more slowly and less accurately than their unambiguous counterparts. We examine the extent to which word context and translation dominance influence the processing of translation-ambiguous words. We further examine how these factors…

  15. Yeast Flocculation—Sedimentation and Flotation

    Directory of Open Access Journals (Sweden)

    Graham G. Stewart

    2018-04-01

    Full Text Available Unlike most fermentation alcohol beverage production processes, brewers recycle their yeast. This is achieved by employing a yeast culture’s: flocculation, adhesion, sedimentation, flotation, and cropping characteristics. As a consequence of yeast recycling, the quality of the cropped yeast culture’s characteristics is critical. However, the other major function of brewer’s yeast is to metabolise wort into ethanol, carbon dioxide, glycerol, and other fermentation products, many of which contribute to beer’s overall flavour characteristics. This review will only focus on brewer’s yeast flocculation characteristics.

  16. Modulation of intracellular protein degradation by SSB1-SIS1 chaperon system in yeast S. cerevisiae.

    Science.gov (United States)

    Ohba, M

    1997-06-09

    In prokaryotes, DnaK-DnaJ chaperon is involved in the protein degradation catalyzed by proteases La and ClpA/B complex as shown in E. coli. To extend this into eukaryotic cells, we examined the effects of hsp70 genes, SSA1 and SSB1, and DnaJ genes, SIS1 and YDJ1, on the growth of proteasome subunit mutants of the yeast S. cerevisiae. The results identified SSB1 and SIS1 as a pair of chaperon genes specifically involved in efficient protein turnover in the yeast, whose overexpression suppressed the growth defects caused by the proteasome mutations. Moreover, a single amino acid substitution in the putative peptide-binding site of SSB1 protein profoundly enhanced the suppression activity, indicating that the activity is mediated by the peptide-binding activity of this chaperon. Thus SSB1, with its partner DnaJ, SIS1, modulates the efficiency of protein turnover through its chaperon activity.

  17. Speciation driven by hybridization and chromosomal plasticity in a wild yeast.

    Science.gov (United States)

    Leducq, Jean-Baptiste; Nielly-Thibault, Lou; Charron, Guillaume; Eberlein, Chris; Verta, Jukka-Pekka; Samani, Pedram; Sylvester, Kayla; Hittinger, Chris Todd; Bell, Graham; Landry, Christian R

    2016-01-11

    Hybridization is recognized as a powerful mechanism of speciation and a driving force in generating biodiversity. However, only few multicellular species, limited to a handful of plants and animals, have been shown to fulfil all the criteria of homoploid hybrid speciation. This lack of evidence could lead to the interpretation that speciation by hybridization has a limited role in eukaryotes, particularly in single-celled organisms. Laboratory experiments have revealed that fungi such as budding yeasts can rapidly develop reproductive isolation and novel phenotypes through hybridization, showing that in principle homoploid speciation could occur in nature. Here, we report a case of homoploid hybrid speciation in natural populations of the budding yeast Saccharomyces paradoxus inhabiting the North American forests. We show that the rapid evolution of chromosome architecture and an ecological context that led to secondary contact between nascent species drove the formation of an incipient hybrid species with a potentially unique ecological niche.

  18. Enrichment of Circular Code Motifs in the Genes of the Yeast Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Christian J. Michel

    2017-12-01

    Full Text Available A set X of 20 trinucleotides has been found to have the highest average occurrence in the reading frame, compared to the two shifted frames, of genes of bacteria, archaea, eukaryotes, plasmids and viruses. This set X has an interesting mathematical property, since X is a maximal C 3 self-complementary trinucleotide circular code. Furthermore, any motif obtained from this circular code X has the capacity to retrieve, maintain and synchronize the original (reading frame. Since 1996, the theory of circular codes in genes has mainly been developed by analysing the properties of the 20 trinucleotides of X , using combinatorics and statistical approaches. For the first time, we test this theory by analysing the X motifs, i.e., motifs from the circular code X , in the complete genome of the yeast Saccharomyces cerevisiae. Several properties of X motifs are identified by basic statistics (at the frequency level, and evaluated by comparison to R motifs, i.e., random motifs generated from 30 different random codes R . We first show that the frequency of X motifs is significantly greater than that of R motifs in the genome of S. cerevisiae. We then verify that no significant difference is observed between the frequencies of X and R motifs in the non-coding regions of S. cerevisiae, but that the occurrence number of X motifs is significantly higher than R motifs in the genes (protein-coding regions. This property is true for all cardinalities of X motifs (from 4 to 20 and for all 16 chromosomes. We further investigate the distribution of X motifs in the three frames of S. cerevisiae genes and show that they occur more frequently in the reading frame, regardless of their cardinality or their length. Finally, the ratio of X genes, i.e., genes with at least one X motif, to non- X genes, in the set of verified genes is significantly different to that observed in the set of putative or dubious genes with no experimental evidence. These results, taken together

  19. Enrichment of Circular Code Motifs in the Genes of the Yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Michel, Christian J; Ngoune, Viviane Nguefack; Poch, Olivier; Ripp, Raymond; Thompson, Julie D

    2017-12-03

    A set X of 20 trinucleotides has been found to have the highest average occurrence in the reading frame, compared to the two shifted frames, of genes of bacteria, archaea, eukaryotes, plasmids and viruses. This set X has an interesting mathematical property, since X is a maximal C3 self-complementary trinucleotide circular code. Furthermore, any motif obtained from this circular code X has the capacity to retrieve, maintain and synchronize the original (reading) frame. Since 1996, the theory of circular codes in genes has mainly been developed by analysing the properties of the 20 trinucleotides of X, using combinatorics and statistical approaches. For the first time, we test this theory by analysing the X motifs, i.e., motifs from the circular code X, in the complete genome of the yeast Saccharomyces cerevisiae . Several properties of X motifs are identified by basic statistics (at the frequency level), and evaluated by comparison to R motifs, i.e., random motifs generated from 30 different random codes R. We first show that the frequency of X motifs is significantly greater than that of R motifs in the genome of S. cerevisiae . We then verify that no significant difference is observed between the frequencies of X and R motifs in the non-coding regions of S. cerevisiae , but that the occurrence number of X motifs is significantly higher than R motifs in the genes (protein-coding regions). This property is true for all cardinalities of X motifs (from 4 to 20) and for all 16 chromosomes. We further investigate the distribution of X motifs in the three frames of S. cerevisiae genes and show that they occur more frequently in the reading frame, regardless of their cardinality or their length. Finally, the ratio of X genes, i.e., genes with at least one X motif, to non-X genes, in the set of verified genes is significantly different to that observed in the set of putative or dubious genes with no experimental evidence. These results, taken together, represent the first

  20. Combining independent de novo assemblies optimizes the coding transcriptome for nonconventional model eukaryotic organisms.

    Science.gov (United States)

    Cerveau, Nicolas; Jackson, Daniel J

    2016-12-09

    Next-generation sequencing (NGS) technologies are arguably the most revolutionary technical development to join the list of tools available to molecular biologists since PCR. For researchers working with nonconventional model organisms one major problem with the currently dominant NGS platform (Illumina) stems from the obligatory fragmentation of nucleic acid material that occurs prior to sequencing during library preparation. This step creates a significant bioinformatic challenge for accurate de novo assembly of novel transcriptome data. This challenge becomes apparent when a variety of modern assembly tools (of which there is no shortage) are applied to the same raw NGS dataset. With the same assembly parameters these tools can generate markedly different assembly outputs. In this study we present an approach that generates an optimized consensus de novo assembly of eukaryotic coding transcriptomes. This approach does not represent a new assembler, rather it combines the outputs of a variety of established assembly packages, and removes redundancy via a series of clustering steps. We test and validate our approach using Illumina datasets from six phylogenetically diverse eukaryotes (three metazoans, two plants and a yeast) and two simulated datasets derived from metazoan reference genome annotations. All of these datasets were assembled using three currently popular assembly packages (CLC, Trinity and IDBA-tran). In addition, we experimentally demonstrate that transcripts unique to one particular assembly package are likely to be bioinformatic artefacts. For all eight datasets our pipeline generates more concise transcriptomes that in fact possess more unique annotatable protein domains than any of the three individual assemblers we employed. Another measure of assembly completeness (using the purpose built BUSCO databases) also confirmed that our approach yields more information. Our approach yields coding transcriptome assemblies that are more likely to be