Academic literature on the topic 'Yeast cell factory'
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Journal articles on the topic "Yeast cell factory"
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Spier, R. E. "Yeast as a Cell Factory." Enzyme and Microbial Technology 26, no. 9-10 (June 2000): 639. http://dx.doi.org/10.1016/s0141-0229(00)00223-4.
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Nielsen, Jens. "Yeast Systems Biology: Model Organism and Cell Factory." Biotechnology Journal 14, no. 9 (May 20, 2019): 1800421. http://dx.doi.org/10.1002/biot.201800421.
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dos Santos, Sandra C., and Isabel Sá-Correia. "Yeast toxicogenomics: lessons from a eukaryotic cell model and cell factory." Current Opinion in Biotechnology 33 (June 2015): 183–91. http://dx.doi.org/10.1016/j.copbio.2015.03.001.
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van Dijk, Ralf, Klaas Nico Faber, Jan A. K. W. Kiel, Marten Veenhuis, and Ida van der Klei. "The methylotrophic yeast Hansenula polymorpha: a versatile cell factory." Enzyme and Microbial Technology 26, no. 9-10 (June 2000): 793–800. http://dx.doi.org/10.1016/s0141-0229(00)00173-3.
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Eliasson Lantz, Anna, Songsak Wattanachaisaereekul, Michael Lynge Nielsen, and Jens Nielsen. "Towards a yeast cell factory platform for polyketide production." Journal of Biotechnology 131, no. 2 (September 2007): S199. http://dx.doi.org/10.1016/j.jbiotec.2007.07.355.
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Kampranis, Sotirios C., and Antonios M. Makris. "DEVELOPING A YEAST CELL FACTORY FOR THE PRODUCTION OF TERPENOIDS." Computational and Structural Biotechnology Journal 3, no. 4 (October 2012): e201210006. http://dx.doi.org/10.5936/csbj.201210006.
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Rustiaty, Banon. "OPTIMALISASI SEL Saccharomyces cerevisiae UNTUK MENINGKATKAN PRODUKTIVITAS DAN EFISIENSI INDUSTRI ETANOL [Optimization of Saccharomyces cerevisiae Cell to Increase Productivity and Efficiency of Ethanol Industry]." Jurnal Teknologi & Industri Hasil Pertanian 23, no. 2 (September 18, 2018): 97. http://dx.doi.org/10.23960/jtihp.v23i2.97-102.
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The development of bioethanol as fuel substitution is believed to overcome the potency of the world energy crisis including Indonesia. The bioethanol development can be done by increasing the production capacity of the existing bioethanol factory plant by improving yeast culture for enhancing the performance of the fermentation process. This study was aimed at obtaining a method of optimizing the ability of Saccharomyces cerevisiae fermentation that can be applied by the alcohol industry in Indonesia for increasing factory productivity, thereby reducing the cost of producing alcohol. In this study, the adaptation of Saccharomyces cerevisiae Watei and Saccharomyces cerevisiae Hakken I were adopted in environment condition with high ethanol content up to 13%. The results showed that the yeast was able to grow in environments with high ethanol content with higher specific growth rate and larger cell size than those within the original yeast. This condition showed that adapted strains can overcome stress caused by high ethanol. These results promise the good performance yeasts with ability in growing and performing metabolic activities in high alcohol-containing environment conditions
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Park, Jongbeom, In Jung Kim, and Soo Rin Kim. "Nonconventional Yeasts Engineered Using the CRISPR-Cas System as Emerging Microbial Cell Factories." Fermentation 8, no. 11 (November 19, 2022): 656. http://dx.doi.org/10.3390/fermentation8110656.
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Because the petroleum-based chemical synthesis of industrial products causes serious environmental and societal issues, biotechnological production using microorganisms is an alternative approach to achieve a more sustainable economy. In particular, the yeast Saccharomyces cerevisiae is widely used as a microbial cell factory to produce biofuels and valuable biomaterials. However, product profiles are often restricted due to the Crabtree-positive nature of S. cerevisiae, and ethanol production from lignocellulose is possibly enhanced by developing alternative stress-resistant microbial platforms. With desirable metabolic pathways and regulation in addition to strong resistance to diverse stress factors, nonconventional yeasts (NCY) may be considered an alternative microbial platform for industrial uses. Irrespective of their high industrial value, the lack of genetic information and useful gene editing tools makes it challenging to develop metabolic engineering-guided scaled-up applications using yeasts. The recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system is a powerful gene editing tool for NCYs. This review describes the current status of and recent advances in promising NCYs in terms of industrial and biotechnological applications, highlighting CRISPR-Cas9 system-based metabolic engineering strategies. This will serve as a basis for the development of novel yeast applications.
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Porro, Danilo, and Paola Branduardi. "Yeast cell factory: fishing for the best one or engineering it?" Microbial Cell Factories 8, no. 1 (2009): 51. http://dx.doi.org/10.1186/1475-2859-8-51.
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Prado, Angelica Rodriguez, Kanchana Kildegaard, Mingji Li, Irina Borodina, and Jens Nielsen. "Development of a yeast cell factory for production of aromatic products." New Biotechnology 31 (July 2014): S130. http://dx.doi.org/10.1016/j.nbt.2014.05.1934.
Full textDissertations / Theses on the topic "Yeast cell factory"
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MAESTRONI, LETIZIA. "TACKLING THE CHALLENGE OF BIO-BASED PRODUCTIONS BY LEVERAGING THE POTENTIAL OF YEAST BIODIVERSITY AND SYNTHETIC BIOLOGY." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2023. https://hdl.handle.net/10281/402374.
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Il ruolo principale delle biotecnologie industriali è quello di fornire soluzioni innovative per alcune delle più grandi sfide del mondo. Nonostante il potenziale e le tecniche innovative applicate, i processi microbiologici bio-based necessitano ancora di ulteriori studi per diventare pervasivi e quindi sostituire i processi di produzione tradizionali.
Per rendere i processi microbici economicamente fattibili e rispettosi dell'ambiente, uno dei fattori chiave risiede nella scelta della biomassa di partenza. In una logica di bioeconomia circolare, i sottoprodotti e le biomasse residue devono essere considerati come materie prime di partenza del processo. L'uso di queste biomasse non solleva questioni etiche e allo stesso tempo è economicamente vantaggioso e orientato all'ambiente. La maggior parte di queste biomasse residue sono residui agricoli e forestali, una famiglia di biomasse caratterizzate da una struttura lignocellulosica. Il problema legato al loro utilizzo nelle bioraffinerie a base microbica è quello di trovare un pretrattamento efficiente per convertirli in zuccheri fermentabili e altri nutrienti, riducendo al minimo il rilascio di inibitori della crescita microbica.
Parlando di bioraffinerie microbiche, ci sono due aspetti principali da tenere a mente durante la progettazione del processo: la biomassa di partenza e l'ospite microbico. L’host finale può essere scelto seguendo due approcci complementari: i) sfruttare la biodiversità microbica già presente in natura, scegliendo l'ospite finale in base alle sue caratteristiche innate, particolarmente vantaggiose in uno specifico processo produttivo; ii) lavorare su una cell factory già nota, customizzandola secondo le necessità.
Nel Capitolo 2 è stata valutata una specifica classe di lieviti non convenzionali, denominata lieviti oleaginosi, per ottenere oli microbici (SCOs) per la produzione di biodiesel a partire da scarti dell'industria della barbabietola da zucchero. Lipomyces starkeyi è stato selezionato come cell factory per la conversione della polpa di barbabietola da zucchero e della melassa di barbabietola da zucchero per massimizzare l'accumulo di SCOs. Con questo esempio applicativo abbiamo dimostrato la possibilità di sfruttare microrganismi non convenzionali per ottenere bio-carburanti più sostenibili.
D'altra parte, la scelta di Saccharomyces cerevisiae come ospite finale ha il grande vantaggio di sfruttare l'ampia conoscenza che lo circonda, compreso l’enorme numero di approcci di biologia sintetica per disegnarlo nella forma finale necessaria.
Nel Capitolo 3 presento una nuova combinazione di approcci di biologia sintetica per accelerare le procedure di ingegnerizzazione, consentendo l’over-espressione e lo studio di vie biosintetiche eterologhe sempre più complesse. Inoltre, mostro l'applicazione di questo nuovo kit di strumenti alla produzione di un metabolita secondario di pianta. Nel capitolo 4 descrivo la progettazione di un nuovo vettore per migliorare le procedure di editing del genoma in S. cerevisiae. Anche in questo secondo progetto l'obiettivo finale è stato quello di velocizzare le fasi di progettazione e costruzione e le procedure di laboratorio, standardizzandole il più possibile per semplificare una parte del lavoro e lasciare più spazio alle fasi successive di test & learn. Nel Capitolo 5 propongo il concetto di co-localizzazione spaziale degli enzimi come campo d'avanguardia nella biologia sintetica per massimizzare il flusso di carbonio verso il prodotto di interesse, sfruttando l'uso di scaffold proteici sintetici e domini di interazione sintetici.
La tesi qui presentata vuole porsi come esempio pratico di come le biotecnologie industriali possano essere utilizzate come potente strumento nella difficile transizione da una società basata sul petrolio e una più sostenibile.The role of industrial biotechnology is to provide game-changing solutions for some of the world’s greatest challenges. From climate change to alternative energy sources and to sustainable productions, industrial biotechnology is fighting to find new sustainable solutions. Despite the promising potential and the innovative techniques applied, bio-based biological processes still need further studies for becoming pervasive and therefore substituting the traditional processes of production. To make microbial processes economically feasible and environmentally friendly, one of the key factors resides in the choice of the starting biomass. In a logic of circular bioeconomy, by-products and residual biomasses have to be considered as starting feedstocks of the process. The use of these biomasses does not raise ethical issues and at the same time is economically advantageous and environment oriented. Indeed, they do not compete with the food industry, as they are usually production waste. Most of these residual biomasses are agricultural and forest residues, a family of biomasses characterised by a lignocellulosic structure. The problem related to their use in microbial-based biorefineries is to find an efficient pretreatment to convert them into fermentable sugars and other nutrients, while reducing to a minimum the release of inhibitors of microbial growth. Talking about microbial-based biorefinery as a substitute to petrol-based refinery, there are two main topics to keep in mind during the process design: the starting biomass and the microbial host. The chassis which will be involved in the final production process can be chosen following two complementary approaches: i) exploiting microbial biodiversity already present in nature by picking the final host depending on its innate characteristics, particularly advantageous in a specific production process; ii) working on a well-known cell factory by customising it as needed. In this thesis both principles were followed. In Chapter 2 a specific class of non-conventional yeasts, named oleaginous yeasts, was evaluated to obtain single cell oils (SCOs) for biodiesel production starting from wastes of the sugar beet industry. Lipomyces starkeyi was selected as cell factory for the conversion of sugar beet pulp and sugar beet molasses to maximise SCOs accumulation. With this applicative example we showed the possibility to take advantage of non-conventional microorganisms to achieve a more sustainable way to produce fuels. On the other hand, choosing Saccharomyces cerevisiae as final host has the major advantage of exploiting the wide knowledge around it, starting from its genome and physiology, and arriving at the tremendous number of synthetic biology approaches to engineer it and manipulate it in the desired final form. In Chapter 3 I introduce a novel toolkit: a new combination of synthetic biology approaches to accelerate the engineering procedures allowing the overexpression and the study of more and more complex biosynthetic heterologous pathways. Moreover, I show the application of this novel toolkit to the production of a selected plant secondary metabolite. In Chapter 4 I describe the design of a new vector to improve genome editing procedures in S. cerevisiae. Even in this second project the final goal was to speed up the design and build stages and laboratory procedures, standardising them as much as possible to simplify one part of scientists' work, to leave more space to the subsequent phases of testing and learning. In Chapter 5 I propose the concept of enzyme spatial co-localisation as a forefront field in synthetic biology to maximise the carbon flux toward the product of interest, exploiting the use of protein synthetic scaffolds and synthetic interaction domains. The presented thesis wants to pose itself as a practical example on how industrial biotechnology can be used as a powerful tool in the difficult transition to a more sustainable society.
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Nomura, Teruyuki. "Factors affecting yeast cell viability." Thesis, Heriot-Watt University, 1986. http://hdl.handle.net/10399/1061.
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Brown, Steven Richard. "A design of experiments approach for engineering carbon metabolism in the yeast Saccharomyces cerevisiae." Thesis, University of Exeter, 2016. http://hdl.handle.net/10871/26158.
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The proven ability to ferment Saccharomyces cerevisiae on a large scale presents an attractive target for producing chemicals and fuels from sustainable sources. Efficient and predominant carbon flux through to ethanol is a significant engineering issue in the development of this yeast as a multi-product cell chassis used in biorefineries. In order to evaluate diversion of carbon flux away from ethanol, combinatorial deletions were investigated in genes encoding the six isozymes of alcohol dehydrogenase (ADH), which catalyse the terminal step in ethanol production. The scarless, dominant and counter- selectable amdSYM gene deletion method was optimised for generation of a combinatorial ADH knockout library in an industrially relevant strain of S. cerevisiae. Current understanding of the individual ADH genes fails to fully evaluate genotype-by-genotype and genotype-by-environment interactions: rather, further research of such a complex biological process requires a multivariate mathematical modelling approach. Application of such an approach using the Design of Experiments (DoE) methodology is appraised here as essential for detailed empirical evaluation of complex systems. DoE provided empirical evidence that in S. cerevisiae: i) the ADH2 gene is not associated with producing ethanol under anaerobic culture conditions in combination with 25 g l-1 glucose substrate concentrations; ii) ADH4 is associated with increased ethanol production when the cell is confronted with a zinc-limited [1 μM] environment; and iii) ADH5 is linked with the production of ethanol, predominantly at pH 4.5. A successful metabolic engineering strategy is detailed which increases the product portfolio of S. cerevisiae, currently used for large-scale production of bioethanol. Heterologous expression of the cytochrome P450 fatty acid peroxygenase from Jeotgalicoccus sp., OleTJE, fused to the RhFRED reductase from Rhodococcus sp. NCIMB 978 converted free fatty acid precursors to C13, C15 and C17 alkenes (3.81 ng μl-1 total alkene concentration).
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Okolo, Bartholomew Ndubuisi. "Alcohol tolerance in yeast : on factors influencing the inhibitory and toxic effects of alcohols on distilling yeast." Thesis, University of Strathclyde, 1986. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=24267.
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An investigation of the factors influencing the inhibitory and toxic effects of ethanol and higher alcohols, byproducts of alcoholic fermentation, on yeast, is presented. The relative potency of alcohols was found to correlate strongly with the carbon chain-length or molecular size and the lipid solubility of the respective alcohols. Higher alcohols act synergistically with each other and with ethanol in causing cell death of suspensions of non-growing Saccharomyces cerevisiae. The presence of higher alcohols in fermented broth, even at low concentrations, and other by-products of alcoholic fermentation, could explain the higher potency of ethanol produced during fermentation compared to added ethanol. The kinetics of uptake of labelled ethanol supplied at different concentrations gave no evidence of enzymic involvement in the ethanol uptake process. The rate of release of labelled ethanol by cells fed labelled glucose paralled the rate of p14sC-C0b2s release. This does not support the view that ethanol accumulates within the cells to higher concentrations than occur in the medium. Supplementation of a basal synthetic medium with various nutrients did not confer additional survival capacity on yeast against the adverse effects of alcohol. Osmotic pressure did not influence alcohol toxicity below 10% (w/v) sorbitol equivalent of osmotic pressure. Alcohol toxicity is not influenced by hydrogen ion concentration (pH) over a range of pH 5.3 to 3.5.
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Pia, Chen-Chun. "Analysis of GINS and other replication factors in the fission yeast cell cycle." Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.504447.
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Hughes, Marcus Daniel. "The M-factor pheromone from the fission yeast Schizosaccharomyces pombe : investigation into its proteolysis." Thesis, University of Warwick, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342552.
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Meeker, Timothy J. "Live Yeast Cell Derivative leads to rapid phosphorylation of Epidermal Growth Factor Receptor." University of Cincinnati / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1337888734.
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Groß, Annett. "Genetically Tailored Yeast Strains for Cell-based Biosensors in White Biotechnology." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-83341.
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This work was performed in the framework of two application-oriented research projects that focus on the generation and evaluation of fluorescent Saccharomyces (S.) cerevisiae-based sensor and reporter cells for white biotechnology as well as the extension of the conventional single-cell/single-construct principle of ordinary yeast biosensor approaches. Numerous products are currently generated by biotechnological processes which require continuous and precise process control and monitoring. These demands are only partially met by physical or physiochemical sensors since they measure parameters off-line or use surrogate parameters that consequently provide only indirect information about the actual process performance. Biosensors, in particular whole cell-based biosensors, have the unique potential to near-line and long-term monitor parameters such as nutrient availability during fermentation processes. Moreover, they allow for the assessment of an analyte’s biological relevance.
Prototype yeast sensor and reporter strains derived from common laboratory strains were transformed with multicopy expression plasmids that mediate constitutive or inducible expression of a fluorescence reporter gene. Performance of these cells was examined by various qualitative and quantitative detection methods – representative of putative transducer technologies. Analyses were performed on the population level by microplate reader-based fluorometry and Western blot as well as on the single-cell level by fluorescence microscopy and flow cytometry. ‘Signature’ promoters that are activated or repressed during particular nutrient-limited growth conditions were selected in order to generate yeast nutrient sensor strains for monitoring the biological availability of nitrogen, phosphorus or sulphur. For each category, at least one promoter mediating at least threefold changed green fluorescence levels between sensor cells in non-limited and nutrient-limited conditions was identified. Sensor strains were evaluated in detail regarding sensitivity, analyte selectivity and the ability to restore basic fluorescence after shift from nutrient-limited to non-limited conditions (regeneration). The applicability for bioprocess monitoring purposes was tested by growth of yeast nutrient sensor cells in microalgae media and supernatants. Despite successful proof of principle, numerous challenges still need to be solved to realise prospective implementation in this field of white biotechnology.
The major drawback of plasmid-borne detection constructs is a high fluorescence variance between individual cells. By generation of a nitrogen sensor strain with a genome-integrated detection construct, uniform expression on the single-cell level and simultaneous maintenance of basic properties (ability of fluorescence induction/regeneration and lack of cross-reactivity) was achieved. However, due to the singular detection construct per cell, significantly weaker overall fluorescence was observed. The traditional single-cell/single-construct approach was expanded upon in two ways. Firstly, a practical dual-colour sensor strain was created by simultaneous, constitutive expression of a red fluorescence reporter gene in green fluorescent nitrogen sensor cells.
Secondly, an innovative cellular communication and signal amplification system inspired by the natural S. cerevisiae pheromone system and mating response was established successfully. It features the yeast pheromone alpha-factor as a trigger and alpha-factor-responsive reporter cells which express a fluorescence reporter gene from the pheromone-inducible FIG1 promoter as an output signal. The system was functional both with synthetic and cell-secreted alpha-factor, provided that recombinant cells were deleted for the alpha-factor protease Bar1p. Integration of amplifier cells which secrete alpha-factor in response to stimulation with the pheromone itself could increase the system\'s sensitivity further. Signal amplification was demonstrated for phosphorus sensor cells as a proof of concept. Therefore, the alpha-factor-based cellular communication and signal amplification system might be useful in applications that suffer from poor signal yield. Due to its modular design, the system could be applied in basically any cell-based biosensor or sensor-actor system.
Immobilisation of the generated sensor and reporter cells in transparent natural polymers can be beneficial considering biosensor fabrication. Functionality of sensor and reporter cells in calcium-alginate beads or nano-printed arrays was successfully demonstrated. For the latter setup, fluorescence scanning and software-assisted fluorescence quantification was applied as a new detection method. In an experiment using an agarose-based two-compartment setup proposed by Jahn, 2011, properties of the alpha-factor-based cellular communication and signal amplification system after immobilisation were tested. These studies provide an initial experimental basis for an appropriate geometry of miniaturised immobilisation matrices with fluorescent yeast sensor and reporter cells in prospective biosensor designs.
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Tippins, T. A. "Various factors which affect the response of yeast cells to environmental mutagens." Thesis, Swansea University, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.639246.
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This project investigates the factors which may affect the response of yeast cells to potential mutagens and thus to optimise their response. The problem was approached from four main angles as follows: i) permeability - rendering the cells more permeable either by pre-treatment with selected chemicals or by selecting clones with cell wall defects; ii) repair capacity - preventing adequate repair of damaged DNA either by post-treatment with repair inhibitors or by using strains with defective repair genes; iii) genetic background - looking at reversion in the same gene but in a different genetic background or different genes in the same background; iv) treatment conditions - treating cells in buffer or broth, with or without exogenous activation, at 28 C or 37 C. The general conclusions which may be drawn from these studies are: a) most chemical mutagens are able to enter the yeast cells in sufficient quantities to cause damage to the DNA without pre-treating the cells to increase their permeability; b) the repair capacity of a cell is a very important factor in its response to a mutagen and if this capacity is greatly impaired, then the chances of survival of the cell after treatment with a mutagen are greatly reduced; c) the genetic background of a cell and the marker under consideration can affect the response of the cell to a mutagen; d) the conditions under which yeast cells are exposed to mutagens affect both the response of the yeast cells and the effectiveness of the mutagen itself. As for optimising the response of the yeast cells to mutagens this can only be done by gathering together all the information already known about the compound under study, and any related compounds, and analysing this data to discover what treatment conditions should be used and possibly what test.
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Awrey, Donald E. "Structural and functional analysis of the yeast general transcript elongation factor, TFIIS." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0009/NQ30069.pdf.
Full textBook chapters on the topic "Yeast cell factory"
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Mittal, Milky, Adya Varshney, Nimisha Singh, Ashok Saini, and Indra Mani. "Yeast Cell Factory for Production of Biomolecules." In Biomanufacturing for Sustainable Production of Biomolecules, 211–51. Singapore: Springer Nature Singapore, 2023. http://dx.doi.org/10.1007/978-981-19-7911-8_11.
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Godinho, Cláudia P., and Isabel Sá-Correia. "Physiological Genomics of Multistress Resistance in the Yeast Cell Model and Factory: Focus on MDR/MXR Transporters." In Yeasts in Biotechnology and Human Health, 1–35. Cham: Springer International Publishing, 2019. http://dx.doi.org/10.1007/978-3-030-13035-0_1.
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Branduardi, Paola, Liliane Barroso, Laura Dato, Edward J. Louis, and Danilo Porro. "Molecular Tools for Leveraging the Potential of the Acid-Tolerant Yeast Zygosaccharomyces bailii as Cell Factory." In Methods in Molecular Biology, 179–204. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2399-2_11.
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Branduardi, Paola, Laura Dato, and Danilo Porro. "Molecular Tools and Protocols for Engineering the Acid-Tolerant Yeast Zygosaccharomyces bailii as a Potential Cell Factory." In Methods in Molecular Biology, 63–85. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-0563-8_4.
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Kang, Hyun Ah, Hubert G. Schwelberger, and John W. B. Hershey. "Effect of Initiation Factor eIF-5A Depletion on Cell Proliferation and Protein Synthesis." In Protein Synthesis and Targeting in Yeast, 123–29. Berlin, Heidelberg: Springer Berlin Heidelberg, 1993. http://dx.doi.org/10.1007/978-3-642-84921-3_12.
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Wu, Wei-Sheng. "A Computational Method for Identifying Yeast Cell Cycle Transcription Factors." In Methods in Molecular Biology, 209–19. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-2957-3_12.
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Tanaka, Takaaki, and Kazuhiro Nakanishi. "Factors Affecting the Performance of Crossflow Filtration of Yeast Cell Suspension." In Developments in Food Engineering, 653–55. Boston, MA: Springer US, 1994. http://dx.doi.org/10.1007/978-1-4615-2674-2_211.
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Oborská-Oplová, Michaela, Ute Fischer, Martin Altvater, and Vikram Govind Panse. "Eukaryotic Ribosome assembly and Nucleocytoplasmic Transport." In Ribosome Biogenesis, 99–126. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2501-9_7.
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AbstractThe process of eukaryotic ribosome assembly stretches across the nucleolus, the nucleoplasm and the cytoplasm, and therefore relies on efficient nucleocytoplasmic transport. In yeast, the import machinery delivers ~140,000 ribosomal proteins every minute to the nucleus for ribosome assembly. At the same time, the export machinery facilitates translocation of ~2000 pre-ribosomal particles every minute through ~200 nuclear pore complexes (NPC) into the cytoplasm. Eukaryotic ribosome assembly also requires >200 conserved assembly factors, which transiently associate with pre-ribosomal particles. Their site(s) of action on maturing pre-ribosomes are beginning to be elucidated. In this chapter, we outline protocols that enable rapid biochemical isolation of pre-ribosomal particles for single particle cryo-electron microscopy (cryo-EM) and in vitro reconstitution of nuclear transport processes. We discuss cell-biological and genetic approaches to investigate how the ribosome assembly and the nucleocytoplasmic transport machineries collaborate to produce functional ribosomes.
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Brahma, Sandipan, and Steven Henikoff. "CUT&RUN Profiling of the Budding Yeast Epigenome." In Methods in Molecular Biology, 129–47. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2257-5_9.
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AbstractMapping the epigenome is key to describe the relationship between chromatin landscapes and the control of DNA-based cellular processes such as transcription. Cleavage under targets and release using nuclease (CUT&RUN) is an in situ chromatin profiling strategy in which controlled cleavage by antibody-targeted Micrococcal Nuclease solubilizes specific protein-DNA complexes for paired-end DNA sequencing. When applied to budding yeast, CUT&RUN profiling yields precise genome-wide maps of histone modifications, histone variants, transcription factors, and ATP-dependent chromatin remodelers, while avoiding cross-linking and solubilization issues associated with the most commonly used chromatin profiling technique Chromatin Immunoprecipitation (ChIP). Furthermore, targeted chromatin complexes cleanly released by CUT&RUN can be used as input for a subsequent native immunoprecipitation step (CUT&RUN.ChIP) to simultaneously map two epitopes in single molecules genome-wide. The intrinsically low background and high resolution of CUT&RUN and CUT&RUN.ChIP allows for identification of transient genomic features such as dynamic nucleosome-remodeling intermediates. Starting from cells, one can perform CUT&RUN or CUT&RUN.ChIP and obtain purified DNA for sequencing library preparation in 2 days.
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Chernoff, Yury O., Susan M. Uptain, and Susan L. Lindquist. "Analysis of prion factors in yeast." In Guide to Yeast Genetics and Molecular and Cell Biology Part C, 499–538. Elsevier, 2002. http://dx.doi.org/10.1016/s0076-6879(02)51867-x.
Full textConference papers on the topic "Yeast cell factory"
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Nielsen, Jens. "Yeast as a Platform Cell Factory in Future Biorefineries." In 14th Asia Pacific Confederation of Chemical Engineering Congress. Singapore: Research Publishing Services, 2012. http://dx.doi.org/10.3850/978-981-07-1445-1_803.
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Riyanti, Eny Ida, and Edy Listanto. "Understanding yeast tolerance as cell factory for bioethanol production from lignocellulosic biomass." In THE SECOND INTERNATIONAL CONFERENCE ON GENETIC RESOURCES AND BIOTECHNOLOGY: Harnessing Technology for Conservation and Sustainable Use of Genetic Resources for Food and Agriculture. AIP Publishing, 2022. http://dx.doi.org/10.1063/5.0075157.
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Wu, Wei-Sheng. "Identification of yeast cell cycle transcription factors using dynamic system model." In 2010 IEEE Workshop On Signal Processing Systems (SiPS). IEEE, 2010. http://dx.doi.org/10.1109/sips.2010.5624789.
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Lee, S., Q. Chen, and R. S. Amano. "Non-Heating Sterilization Method by High Pressure Carbon Dioxide." In ASME 2003 International Design Engineering Technical Conferences and Computers and Information in Engineering Conference. ASMEDC, 2003. http://dx.doi.org/10.1115/detc2003/cie-48249.
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The compressed CO2 was supplied from the CO2 cylinder to the steel vessel containing yeast-water mixture until the pressure inside the steel vessel reached a desired pressure level. The steel vessel was kept at the prescribed pressure level during the treatment period. After the treatment, the pressure inside the vessel was released to the atmospheric pressure within the evacuation time by controlling the relief control valve. The sterilization effect was evaluated by vial counting the treated sample taken from the steel vessel. Several experiments have been performed varying each factor such as the total treatment time, the evacuation time and the applied pressure. As a result, it was verified that these factors are strongly related to the sterilization effect. In addition, it was observed that increase of the treatment time can enhance the concentration rate of CO2 in the mixture. Furthermore, it was concluded that CO2 penetrated into each yeast cell was rapidly expanded by fast depressurizing. Also, the experimental results showed that when treatment time is longer, the pressure becomes higher, and the evacuation time is shorter, thus, a better sterilizing effect was obtained. Contrary to the results above, both the treatment time and the evacuation time rarely affected sterilization effect under supercritical condition. By adopting Supercritical condition, a desirable result could be achieved with shorter treatment and longer evacuation time. Thus, as long as supercritical condition is adopted for test, either fast evacuation time or long treatment time is not essential any more.
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Lee, S., and R. S. Amano. "Design of Brewery Pasteurization Method by High Pressure Carbon Dioxide." In ASME 2005 International Design Engineering Technical Conferences and Computers and Information in Engineering Conference. ASMEDC, 2005. http://dx.doi.org/10.1115/detc2005-84925.
Full textAbstract:
The compressed CO2 was supplied from the CO2 cylinder to the steel vessel containing yeast-water mixture until the pressure inside the steel vessel reached a desired pressure level. The steel vessel was kept at the prescribed pressure level during the treatment period. After the treatment, the pressure inside the vessel was released to the atmospheric pressure within the evacuation time by controlling the relief control valve. The sterilization effect was evaluated by vial counting the treated sample taken from the steel vessel. Several experiments have been performed varying each factor such as the total treatment time, the evacuation time and the applied pressure. As a result, it was verified that these factors are strongly related to the sterilization effect. In addition, it was observed that increase of the treatment time could enhance the concentration rate of CO2 in the mixture. Furthermore, it was concluded that fast depressurizing rapidly expanded CO2 penetrated into each yeast cell. Also, the experimental results showed that when treatment time is longer, the pressure becomes higher, and the evacuation time is shorter, thus, a better sterilizing effect was obtained. Contrary to the results above, both the treatment time and the evacuation time rarely affected sterilization effect under supercritical condition. By adopting Supercritical condition, a desirable result could be achieved with shorter treatment and longer evacuation time. Thus, as long as supercritical condition is adopted for test, either fast evacuation time or long treatment time is not essential any more.
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He, Dong, Dao Zhou, and Yanhong Zhou. "Identifying synergistic transcriptional factors involved in the yeast cell cycle using Microarray and ChIP-chip data." In 2006 Fifth International Conference on Grid and Cooperative Computing Workshops. IEEE, 2006. http://dx.doi.org/10.1109/gccw.2006.54.
Full text
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Nudurupati, Sai Chaitanya, Pushpendra Singh, and Nadine Aubry. "Effect of Frequency and Electrode Configuration on Yeast Cells Subjected to Traveling Electric Fields." In ASME 2006 2nd Joint U.S.-European Fluids Engineering Summer Meeting Collocated With the 14th International Conference on Nuclear Engineering. ASMEDC, 2006. http://dx.doi.org/10.1115/fedsm2006-98448.
Full textAbstract:
There is great interest in trapping and manipulating small sized particles such as biological, glass, polymer and carbonaceous particles suspended in a liquid. One way to trap such micro/nano sized particles is by means of a microfluidic chamber equipped with electrodes at the bottom and thus generating conventional dielectrophoresis based on an electric field of spatially varying magnitude. In this work, we explore the use of traveling wave dielectrophoresis induced by an electric field of spatially varying phase, which offers both particle capturing/separation and transport capabilities (without having to pump the fluid itself). Particles are subjected to electrostatic and hydrodynamic forces and torques that are computed solving the full equations of motion for both the fluid and the particles without any modeling (from first principles) and using a finite element scheme based on the Distributed Lagrange Multiplier (DLM) method. We consider two typical microfluidic channels (MEMS devices) with electrodes embedded in the bottom wall. It is found that the motion and destination of the particles strongly depend on the frequency dependent complex Clausius-Mossotti factor (the mismatch between the particles and fluid electric properties), and that the hydrodynamic and electrostatic particle-particle interactions play a crucial role on the particles dynamics. These conclusions are demonstrated on model particles having the properties of yeast cells.
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Freund, M., J.-P. Cazenave, M.-L. Wiesel, C. Roitsch, N. Riehl-Bellon, G. Loison, Y. E. Lemoine, S. Brown, and M. Courtney. "RECOMBINANT HIRUDIN INHIBITS EXPERIMENTAL VENOUS THROMBOSIS INDUCED BY INJECTION OF TISSUE FACTOR AND STASIS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643917.
Full textAbstract:
Hirudin (HIR), a polypeptide of 65 aminoacids, is the most potent natural inhibitor of coagulation by forming rapidly a very stable and specific non covalent 1:1 complex with α-thrombin, independent of antithrombin III. Although natural HIR has in vivo anticoagulant and antithrombotic properties, its limited availability for large scale purification has prevented further clinical testing and potential use; this can now be solved by recombinant DNA technology. We have previously reported the cloning and expression of a cDNA encoding one variant (called HV-2) of Hirudo medicinalis HIR (Proc. Natl. Acad. Sci. USA. 1986, 83, 1084-1088). The main factors responsible for venous thrombosis are stasis and thrombin generation secondary to tissue factor liberation from vascular cells and monocytes by injury, endotoxin, interleukin-1 or cachectin and the subsequent activation and circulation of activated clotting factors. We have studied the antithrombotic properties of recombinant HIR, HV-2, in a rat experiemental model of venous thrombosis. HV-2 was expressed in yeast, extracted from culture supernatant and purified by HPLC. Pure HV-2 had an isoleucine NH2-terminus and a specific activity of 13000 ATU/mg.30 male Wistar rats (225-300g) were anesthetized with pentobarbital. At time t (0 min) an i.v. (penis) injection of 0.4 ml of saline or HV-2 (2000 to 8000 ATU/kg) was given, followed at t (5min) by 25 mg/kg tissue factor (Thromboplastin C, Dade) i.v. ; 10 s later stasis of the exposed vena cava between 2 sutures 0.7 cm apart and at t (15 min) removal, blotting, fixation and weighing of the thrombus. Linear regression analysis showed a correlation (r=0.99) between the dose of HV-2 and thrombus weight and a calculated IC50 = 3000 ATU/kg. Total inhibition of thrombus formation was seen after injection of 6000 ATU/kg HV-2 and lasted up to 15 min of circulation, HV-2 being completely eliminated from blood in 60 min and accumulated in the kidneys as shown by gamma imaging with 131I-HV-2. In conclusion, the recombinant HIR HV-2 is a potent immediate antithrombin which inhibits venous thrombosis induced by tissue factor and stasis.
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Liu, Hanli, Yutao Zhang, Mika Kimura, and Britton Chance. "Theoretical and Experimental Investigations on Solute-Induced Changes in Optical Properties in Living Tissues." In Biomedical Optical Spectroscopy and Diagnostics. Washington, D.C.: Optica Publishing Group, 2006. http://dx.doi.org/10.1364/bosd.1996.cm3.
Full textAbstract:
A number of recent studies have focused on the possibility of using the NIR techniques to monitor a change of glucose concentration in tissue [1,2]. The basis of the method rests on the fact that a change of refractive index in the extracellular fluid due to the presence of additional glucose causes a small change in the overall scattering property of the tissue that could be detected by the NIR techniques. Chance et al [ 3] show that in lipid and yeast cell suspensions, an increase in concentration of a general solute, such as sugars and electrolytes, gives rise to a decrease in scattering factor of the suspension. These results are in good agreement with those given in Refs. 1 and 2. However, in the tissue measurement performed on a perfused rat liver, the results obtained by adding mannitol (or glucose) to the perfusate of the perfused liver displayed a behavior in contrast to those in the lipid suspensions [3] and can not be well explained by the change of only refractive index. In order to employ the NIR techniques for a broad use in noninvasive physiological monitoring, we wish to show in this paper the solute-induced correlation between optical properties in tissue and its refractive index as well as its osmolarity.
Reports on the topic "Yeast cell factory"
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Hodges, Thomas K., and David Gidoni. Regulated Expression of Yeast FLP Recombinase in Plant Cells. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7574341.bard.
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Research activities in both our laboratories were directed toward development of control of the FLP/frt recombination system for plants. As described in the text of the research proposal, the US lab has been engaged in developing regulatory strategies such as tissue-specific promoters and the steroid-inducible activation of the FLP enzyme while the main research activities in Israel have been directed toward the development and testing of a copper-regulated expression of flp recombinase in tobacco (this is an example of a promoter activation by metal ions). The Israeli lab hat additionally completed experiments of previous studies regarding factors affecting the efficiency of recombinase activity using both a gain-of-function assay (excisional-activation of a gusA marker) and loss of function assay (excision of a rolC marker) in tobacco. Site-specific recombinase systems, in particular the FLP/frt and R/RS systems of yeast and the Cre/lox system of bacteriophage P1, have become an essential component of targeted genetic transformation procedures both in animal and plant organisms. To provide more flexibility in transgene excisions by the recombinase systems as well as gene targeting, and to widen possible applications, the development of controlled or regulated recombination systems is highly desirable and was therefore the subject of this research proposal. There are a few possible mechanisms to regulate expression of a recombinase system. They include: 1) control of the recombination system by having the target sites (e.g. frt) in one plant and the flp recombinase gene in another, and bringing the two together by cross fertilization. 2) regulation of promoter activities by external stimuli such as temperature, chemicals, metal ions, etc. 3) regulation of promoter activities by internal signals, i.e. cell- or tissue-specific, or developmental regulation. 4) regulation of enzyme activity by providing cofactors essential for biochemical reactions to take place such as steroid molecules in conjunction with a steroid ligand-binding protein (domains). During the course of this research our major emphasis have been focused toward studying the feasibility of hybrid seed production in Arabidopsis, using FLP/frt. Male-sterility was induced using the antisence of a pollen- and tapetum-specific gene, bcp1, isolated from Arabidopsis. The sterility inducing gene was flanked by frt sites. Upon cross pollination of flowers of male-sterile plants with pollen from FLP-containing plants, viable seeds were produced, and the progeny hybrid plants developed normally. The major achievement from this work is the first demonstration of using a site-specific recombinase to restore fertility in male-sterile plants (see attached paper, Luo et al., Plant J 2000; 23:423-430). The implication from this finding is that site-specific recombination systems can be applied in crop plants as a useful alternative method for hybrid seed production.
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Droby, Samir, Michael Wisniewski, Martin Goldway, Wojciech Janisiewicz, and Charles Wilson. Enhancement of Postharvest Biocontrol Activity of the Yeast Candida oleophila by Overexpression of Lytic Enzymes. United States Department of Agriculture, November 2003. http://dx.doi.org/10.32747/2003.7586481.bard.
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Enhancing the activity of biocontrol agents could be the most important factor in their success in controlling fruit disease and their ultimate acceptance in commercial disease management. Direct manipulation of a biocontrol agent resulting in enhancement of diseases control could be achieved by using recent advances in molecular biology techniques. The objectives of this project were to isolate genes from yeast species that were used as postharvest biocontrol agents against postharvest diseases and to determine their role in biocontrol efficacy. The emphasis was to be placed on the yeast, Candida oleophila, which was jointly discovered and developed in our laboratories, and commercialized as the product, Aspire. The general plan was to develop a transformation system for C . oleophila and either knockout or overexpress particular genes of interest. Additionally, biochemical characterization of the lytic peptides was conducted in the wild-type and transgenic isolates. In addition to developing a better understanding of the mode of action of the yeast biocontrol agents, it was also our intent to demonstrate the feasibility of enhancing biocontrol activity via genetic enhancement of yeast with genes known to code for proteins with antimicrobial activity. Major achievements are: 1) Characterization of extracellular lytic enzymes produced by the yeast biocontrol agent Candida oleophila; 2) Development of a transformation system for Candida oleophila; 3) Cloning and analysis of C.oleophila glucanase gene; 4) Overexpression of and knockout of C. oleophila glucanase gene and evaluating its role in the biocontrol activity of C. oleophila; 5) Characterization of defensin gene and its expression in the yeast Pichiapastoris; 6) Cloning and Analysis of Chitinase and Adhesin Genes; 7) Characterization of the rnase secreted by C . oleophila and its inhibitory activity against P. digitatum. This project has resulted in information that enhanced our understanding of the mode of action of the yeast C . oleophila. This was important step towards enhancing the biocontrol activity of the yeast. Fungal cell wall enzymes produced by the yeast antagonist were characterized. Different substrates were identified to enhance there production in vitro. Exo-b-1, 3 glucanase, chitinase and protease production was stimulated by the presence of cell-wall fragments of Penicillium digitatum in the growing medium, in addition to glucose. A transformation system developed was used to study the role of lytic enzymes in the biocontrol activity of the yeast antagonist and was essential for genetic manipulation of C . oleqphila. After cloning and characterization of the exo-glucanase gene from the yeast, the transformation system was efficiently used to study the role of the enzyme in the biocontrol activity by over-expressing or knocking out the activity of the enzyme. At the last phase of the research (still ongoing) the transformation system is being used to study the role of chitinase gene in the mode of action. Knockout and over expression experiments are underway.
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Nelson, Nathan, and Randy Schekman. Functional Biogenesis of V-ATPase in the Vacuolar System of Plants and Fungi. United States Department of Agriculture, September 1996. http://dx.doi.org/10.32747/1996.7574342.bard.
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The vacuolar H+-ATPase (V-ATPase) is one of the most fundamental enzymes in nature. It pumps protons into the vacuolar system of eukaryotic cells and provides the energy for numerous transport systems. Through our BARD grant we discovered a novel family of membrane chaperones that modulate the amount of membrane proteins. We also elucidated the mechanism by which assembly factors guide the membrane sector of V-ATPase from the endoplasmic reticulum to the Golgi apparatus. The major goal of the research was to understand the mechanism of action and biogenesis of V-ATPase in higher plants and fungi. The fundamental question of the extent of acidification in organelles of the vacuolar system was addressed by studying the V-ATPase of lemon fruit, constructing lemon cDNAs libraries and study their expression in mutant yeast cells. The biogenesis of the enzyme and its function in the Golgi apparatus was studied in yeast utilizing a gallery of secretory mutants available in our laboratories. One of the goals of this project is to determine biochemically and genetically how V-ATPase is assembled into the different membranes of a wide variety of organelles and what is the mechanism of its action.The results of this project advanced out knowledge along these lines.
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Elroy-Stein, Orna, and Dmitry Belostotsky. Mechanism of Internal Initiation of Translation in Plants. United States Department of Agriculture, December 2010. http://dx.doi.org/10.32747/2010.7696518.bard.
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Original objectives Elucidation of PABP's role in crTMV148 IRES function in-vitro using wheat germ extract and krebs-2 cells extract. Fully achieved. Elucidation of PABP's role in crTMV148 IRES function in-vivo in Arabidopsis. Characterization of the physical interactions of PABP and other potential ITAFs with crTMV148 IRES. Partly achieved. To conduct search for additional ITAFs using different approaches and evaluate the candidates. Partly achieved. Background of the topic The power of internal translation via the activity of internal ribosomal entry site (IRES) elements allow coordinated synthesis of multiple gene products from a single transcription unit, and thereby enables to bypass the need for sequential transformation with multiple independent transgenes. The key goal of this project was to identify and analyze the IRES-trans-acting factors (ITAFs) that mediate the activity of a crucifer-infecting tobamovirus (crTMV148) IRES. The remarkable conservation of the IRES activity across the phylogenetic spectrum (yeast, plants and animals) strongly suggests that key ITAFs that mediate its activity are themselves highly conserved. Thus, crTMV148 IRES offers opportunity for elucidation of the fundamental mechanisms underlying internal translation in higher plants in order to enable its rational manipulation for the purpose of agricultural biotechnology. Major conclusions and achievements. - CrTMV IRES requires PABP for maximal activity. This conclusion was achieved by PABP depletion and reconstitution of wheat germ- and Krebs2-derived in-vitro translation assays using Arabidopsis-derived PABP2, 3, 5, 8 and yeast Pab1p. - Mutations in the internal polypurine tract of the IRES decrease the high-affinity binding of all phylogenetically divergent PABPs derived from Arabidopsis and yeast in electro mobility gel shift assays. - Mutations in the internal polypurine tract decrease IRES activity in-vivo. - The 3'-poly(A) tail enhances crTMV148 IRES activity more efficiently in the absence of 5'-methylated cap. - In-vivo assembled RNPs containing proteins specifically associated with the IRES were purified from HEK293 cells using the RNA Affinity in Tandem (RAT) approach followed by their identification by mass spectroscopy. - This study yielded a list of potential protein candidates that may serve as ITAFs of crTMV148 IRES activity, among them are a/b tubulin, a/g actin, GAPDH, enolase 1, ribonuclease/angiogenin inhibitor 1, 26S proteasome subunit p45, rpSA, eEF1Bδ, and proteasome b5 subunit. Implications, both scientific and agriculture. The fact that the 3'-poly(A) tail enhances crTMV148 IRES activity more efficiently in the absence of 5'-methylated cap suggests a potential joint interaction between PABP, the IRES sequence and the 3'-poly(A). This has an important scientific implication related to IRES function in general.
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Sessa, Guido, and Gregory Martin. Role of GRAS Transcription Factors in Tomato Disease Resistance and Basal Defense. United States Department of Agriculture, 2005. http://dx.doi.org/10.32747/2005.7696520.bard.
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The research problem: Bacterial spot and bacterial speck diseases of tomato are causedby strains of Xanthomonas campestris pv. vesicatoria (Xcv) and Pseudomonas syringae pv.tomato (Pst), respectively. These bacteria colonize aerial parts of the plant and causesignificant losses in tomato production worldwide. Protection against Xcv and Pst bycultural practices or chemical control has been unsuccessful and there are only limitedsources of genetic resistance to these pathogens. In previous research supported in part byBARD IS-3237-01, we extensively characterized changes in tomato gene expression uponthe onset of spot and speck disease resistance. A remarkable finding of these studies wasthe inducibility in tomato leaves by both Xcv and Pst strains of genes encodingtranscriptional activator of the GRAS family, which has not been previously linked todisease resistance. Goals: Central goals of this research were to investigate the role of GRAS genes in tomatoinnate immunity and to assess their potential use for disease control.Specific objectives were to: 1. Identify GRAS genes that are induced in tomato during thedefense response and analyze their role in disease resistance by loss-of-function experiments.2. Overexpress GRAS genes in tomato and characterize plants for possible broad-spectrumresistance. 3. Identify genes whose transcription is regulated by GRAS family. Our main achievements during this research program are in three major areas:1. Identification of tomato GRAS family members induced in defense responses andanalysis of their role in disease resistance. Genes encoding tomato GRAS family memberswere retrieved from databases and analyzed for their inducibility by Pst avirulent bacteria.Real-time RT-PCR analysis revealed that six SlGRAS transcripts are induced during theonset of disease resistance to Pst. Further expression analysis of two selected GRAS genesshowed that they accumulate in tomato plants in response to different avirulent bacteria orto the fungal elicitor EIX. In addition, eight SlGRAS genes, including the Pst-induciblefamily members, were induced by mechanical stress in part in a jasmonic acid-dependentmanner. Remarkably, SlGRAS6 gene was found to be required for tomato resistance to Pstin virus-induced gene silencing (VIGS) experiments.2. Molecular analysis of pathogen-induced GRAS transcriptional activators. In aheterologous yeast system, Pst-inducible GRAS genes were shown to have the ability toactivate transcription in agreement with their putative function of transcription factors. Inaddition, deletion analysis demonstrated that short sequences at the amino-terminus ofSlGRAS2, SlGRAS4 and SlGRAS6 are sufficient for transcriptional activation. Finally,defense-related SlGRAS proteins were found to localize to the cell nucleus. 3. Disease resistance and expression profiles of transgenic plants overexpressing SlGRASgenes. Transgenic plants overexpressing SlGRAS3 or SlGRAS6 were generated. Diseasesusceptibility tests revealed that these plants are not more resistant to Pst than wild-typeplants. Gene expression profiles of the overexpressing plants identified putative direct orindirect target genes regulated by SlGRAS3 and SlGRAS6. Scientific and agricultural significance: Our research activities established a novel linkbetween the GRAS family of transcription factors, plant disease resistance and mechanicalstress response. SlGRAS6 was found to be required for disease resistance to Pstsuggesting that this and possibly other GRAS family members are involved in thetranscriptional reprogramming that takes place during the onset of disease resistance.Their nuclear localization and transcriptional activation ability support their proposed roleas transcription factors or co-activators. However, the potential of utilizing GRAS familymembers for the improvement of plant disease resistance in agriculture has yet to bedemonstrated.
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Ohad, Nir, and Robert Fischer. Regulation of Fertilization-Independent Endosperm Development by Polycomb Proteins. United States Department of Agriculture, January 2004. http://dx.doi.org/10.32747/2004.7695869.bard.
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Arabidopsis mutants that we have isolated, encode for fertilization-independent endosperm (fie), fertilization-independent seed2 (fis2) and medea (mea) genes, act in the female gametophyte and allow endosperm to develop without fertilization when mutated. We cloned the FIE and MEA genes and showed that they encode WD and SET domain polycomb (Pc G) proteins, respectively. Homologous proteins of FIE and MEA in other organisms are known to regulate gene transcription by modulating chromatin structure. Based on our results, we proposed a model whereby both FIE and MEA interact to suppress transcription of regulatory genes. These genes are transcribed only at proper developmental stages, as in the central cell of the female gametophyte after fertilization, thus activating endosperm development. To test our model, the following questions were addressed: What is the Composition and Function of the Polycomb Complex? Molecular, biochemical, genetic and genomic approaches were offered to identify members of the complex, analyze their interactions, and understand their function. What is the Temporal and Spatial Pattern of Polycomb Proteins Accumulation? The use of transgenic plants expressing tagged FIE and MEA polypeptides as well as specific antibodies were proposed to localize the endogenous polycomb complex. How is Polycomb Protein Activity Controlled? To understand the molecular mechanism controlling the accumulation of FIE protein, transgenic plants as well as molecular approaches were proposed to determine whether FIE is regulated at the translational or posttranslational levels. The objectives of our research program have been accomplished and the results obtained exceeded our expectation. Our results reveal that fie and mea mutations cause parent-of-origin effects on seed development by distinct mechanisms (Publication 1). Moreover our data show that FIE has additional functions besides controlling the development of the female gametophyte. Using transgenic lines in which FIE was not expressed or the protein level was reduced during different developmental stages enabled us for the first time to explore FIE function during sporophyte development (Publication 2 and 3). Our results are consistent with the hypothesis that FIE, a single copy gene in the Arabidopsis genome, represses multiple developmental pathways (i.e., endosperm, embryogenesis, shot formation and flowering). Furthermore, we identified FIE target genes, including key transcription factors known to promote flowering (AG and LFY) as well as shoot and leaf formation (KNAT1) (Publication 2 and 3), thus demonstrating that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Using the Yeast two hybrid system and pull-down assays we demonstrated that FIE protein interact with MEA via the N-terminal region (Publication 1). Moreover, CURLY LEAF protein, an additional member of the SET domain family interacts with FIE as well. The overlapping expression patterns of FIE, with ether MEA or CLF and their common mutant phenotypes, demonstrate the versatility of FIE function. FIE association with different SET domain polycomb proteins, results in differential regulation of gene expression throughout the plant life cycle (Publication 3). In vitro interaction assays we have recently performed demonstrated that FIE interacts with the cell cycle regulatory component Retinobalsoma protein (pRb) (Publication 4). These results illuminate the potential mechanism by which FIE may restrain embryo sac central cell division, at least partly, through interaction with, and suppression of pRb-regulated genes. The results of this program generated new information about the initiation of reproductive development and expanded our understanding of how PcG proteins regulate developmental programs along the plant life cycle. The tools and information obtained in this program will lead to novel strategies which will allow to mange crop plants and to increase crop production.
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Chamovitz, Daniel, and Albrecht Von Arnim. Translational regulation and light signal transduction in plants: the link between eIF3 and the COP9 signalosome. United States Department of Agriculture, November 2006. http://dx.doi.org/10.32747/2006.7696515.bard.
Full textAbstract:
The COP9 signalosome (CSN) is an eight-subunit protein complex that is highly conserved among eukaryotes. Genetic analysis of the signalosome in the plant model species Arabidopsis thaliana has shown that the signalosome is a repressor of light dependent seedling development as mutant Arabidopsis seedlings that lack this complex develop in complete darkness as if exposed to light. These mutant plants die following the seedling stage, even when exposed to light, indicating that the COP9 signalosome also has a central role in the regulation of normal photomorphogenic development. The biochemical mode of action of the signalosome and its position in eukaryotic cell signaling pathways is a matter of controversy and ongoing investigation, and recent results place the CSN at the juncture of kinase signaling pathways and ubiquitin-mediated protein degradation. We have shown that one of the many CSN functions may relate to the regulation of translation through the interaction of the CSN with its related complex, eukaryotic initiation factor (eIF3). While we have established a physical connection between eIF3 subunits and CSN subunits, the physiological and developmental significance of this interaction is still unknown. In an effort to understand the biochemical activity of the signalosome, and its role in regulating translation, we originally proposed to dissect the contribution of "h" subunit of eIF3 (eIF3h) along the following specific aims: (i) Isolation and phenotypic characterization of an Arabidopsis loss-of-function allele for eIF3h from insertional mutagenesis libraries; (ii) Creation of designed gain and loss of function alleles for eIF3h on the basis of its nucleocytoplasmic distribution and its yeast-two-hybrid interactions with other eIF3 and signalosome partner proteins; (iii) Determining the contribution of eIF3h and its interaction with the signalosome by expressing specific mutants of eIF3h in the eIF3h- loss-of function background. During the course of the research, these goals were modified to include examining the genetic interaction between csn and eif3h mutations. More importantly, we extended our effort toward the genetic analysis of mutations in the eIF3e subunit, which also interacts with the CSN. Through the course of this research program we have made several critical scientific discoveries, all concerned with the apparent diametrically opposed roles of eIF3h and eIF3e. We showed that: 1) While eIF3e is essential for growth and development, eIF3h is not essential for growth or basal translation; 2) While eIF3e has a negative role in translational regulation, eIF3h is positively required for efficient translation of transcripts with complex 5' UTR sequences; 3) Over-accumulation of eIF3e and loss-of-function of eIF3h both lead to cop phenotypes in dark-grown seedlings. These results were published in one publication (Kim et al., Plant Cell 2004) and in a second manuscript currently in revision for Embo J. Are results have led to a paradigm shift in translation research – eIF3 is now viewed in all systems as a dynamic entity that contains regulatory subuits that affect translational efficiency. In the long-term agronomic outlook, the proposed research has implications that may be far reaching. Many important plant processes, including developmental and physiological responses to light, abiotic stress, photosynthate, and hormones operate in part by modulating protein translation [23, 24, 40, 75]. Translational regulation is slowly coming of age as a mechanism for regulating foreign gene expression in plants, beginning with translational enhancers [84, 85] and more recently, coordinating the expression of multiple transgenes using internal ribosome entry sites. Our contribution to understanding the molecular mode of action of a protein complex as fundamental as eIF3 is likely to lead to advances that will be applicable in the foreseeable future.