Готові списки джерел за темами / Transient and stable expression

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Добірка наукової літератури з теми "Transient and stable expression"

Автор: Grafiati

Опубліковано: 11 березня 2023

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Статті в журналах з теми "Transient and stable expression"

Chen, Ridong, Soon Seog Jeong, Jun Luo, and Maria Borovilos. "20 Transient and Stable Expression of Authentic Human Proteins." Cytokine 39, no. 1 (July 2007): 6. http://dx.doi.org/10.1016/j.cyto.2007.07.025.

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Peffley, E. B., M. Hart, Z. Xiang, Z. Hiang, A. El-Sharif, N. Dong, and G. C. Phillips. "TRANSIENT EXPRESSION OF GUS IN ONION TRANSFORMANTS." HortScience 31, no. 5 (September 1996): 759b—759. http://dx.doi.org/10.21273/hortsci.31.5.759b.

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Particle bombardment using the Bio-Rad PDS-1000/HE system is being investigated as a means to develop a stable transformation system for the bulb onion. Donor materials for bombardments included sterile meristems and radicals from newly germinated seeds, callus of Allium cepa `TG1015', `Sunlite', and `Buffalo', A. fistulosum `Heshiko', and suspension-cultured cell lines with confirmed regeneration capability, as well as regenerated plants of an F1 interspecific hybrid. Transient expression assays using the B-glucuronidase (GUS) reporter gene system were used to optimize the conditions for transformation. Various promoters combined with the GUS coding sequence were tested. Results indicate genotype specificity for promoter expression. Some tissue continued to exhibit GUS activity after several months in culture, indicating potential for achieving stable transformation of onion.

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Chen, Jun, Marc R. Lake, Reza S. Sabet, Wende Niforatos, Steve D. Pratt, Steven C. Cassar, Jing Xu, et al. "Utility of Large-Scale Transiently Transfected Cells for Cell-Based High-Throughput Screens to Identify Transient Receptor Potential Channel A1 (TRPA1) Antagonists." Journal of Biomolecular Screening 12, no. 1 (November 12, 2006): 61–69. http://dx.doi.org/10.1177/1087057106295220.

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Despite increasing use of cell-based assays in high-throughput screening (HTS) and lead optimization, one challenge is the adequate supply of high-quality cells expressing the target of interest. To this end, cell lines stably expressing targets are often established, maintained, and scaled up by cell culture. These steps require large investments of time and resources. Moreover, significant variability invariably occurs in cell yield, viability, expression levels, and target activities. In particular, stable expression of targets such as transient receptor potential A1 (TRPA1) causes toxicity, cell line degeneration, and loss of functional activity. Therefore, in an effort to identify TRPA1 antagonists, the authors used large-scale transiently transfected (LSTT) cells, enabling rapid establishment of assays suitable for HTS. LSTT cells, which could- be stored frozen for a long period of time (e.g., at least 42 weeks), retained TRPA1 protein expression and could be easily revived to produce robust and consistent signals in calcium influx and electrophysiological assays. Using cells from a single transfection, a chemical library of 700,000 compounds was screened, and TRPA1 antagonists were identified. The use of LSTT circumvented issues associated with stable TRPA1 expression, increased flexibility and consistency, and greatly reduced labor and cost. This approach will also be applicable to other pharmaceutical targets.

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Ju, Xiaoli, Meijia Ren, Keping Chen, and Qiang Wang. "Overexpression of c-Myc enhances recombinant protein production in High Five cells after baculovirus infection." Zeitschrift für Naturforschung C 73, no. 3-4 (February 23, 2018): 147–51. http://dx.doi.org/10.1515/znc-2017-0076.

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AbstractDue to their numerous advantages, baculovirus expression vector systems (BEVS) have been widely used to express recombinant proteins for different purposes. Different strategies have been adopted to increase recombinant protein production. In this study, we transiently or stably expressed mousec-Mycin High Five cells using a commercial pIB/V5 vector. Under the control of theOpIE2promoter, this vector could enhance recombinant protein production. We found that transient expression ofc-Mycin High Five cells improved recombinant protein production. Furthermore, we established two stable cell lines, High Five-c-Myc #1 and High Five-c-Myc #2, that stably expressed mousec-Myc. We further found that the expression level of the recombinant protein was increased in these stable cell lines compared to control cell lines. These data indicate that overexpressingc-Mycin cells is a promising way to improve recombinant protein production in BEVS.

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Liew, Chee-Gee, Jonathan S. Draper, James Walsh, Harry Moore, and Peter W. Andrews. "Transient and Stable Transgene Expression in Human Embryonic Stem Cells." Stem Cells 25, no. 6 (June 2007): 1521–28. http://dx.doi.org/10.1634/stemcells.2006-0634.

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Liew, Chee-Gee, Jonathan S. Draper, James Walsh, Harry Moore, and Peter W. Andrews. "Transient and Stable Transgene Expression in Human Embryonic Stem Cells." Stem Cells 26, no. 4 (April 2008): 1094. http://dx.doi.org/10.1634/stemcells.2006-0634erratum.

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Mortimer, Cara L., Benjamin Dugdale, and James L. Dale. "Updates in inducible transgene expression using viral vectors: from transient to stable expression." Current Opinion in Biotechnology 32 (April 2015): 85–92. http://dx.doi.org/10.1016/j.copbio.2014.11.009.

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Stelzer, Christoph, and Yaakov Benenson. "Precise determination of input-output mapping for multimodal gene circuits using data from transient transfection." PLOS Computational Biology 16, no. 11 (November 30, 2020): e1008389. http://dx.doi.org/10.1371/journal.pcbi.1008389.

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The mapping of molecular inputs to their molecular outputs (input/output, I/O mapping) is an important characteristic of gene circuits, both natural and synthetic. Experimental determination of such mappings for synthetic circuits is best performed using stably integrated genetic constructs. In mammalian cells, stable integration of complex circuits is a time-consuming process that hampers rapid characterization of multiple circuit variants. On the other hand, transient transfection is quick. However, it is an extremely noisy process and it is unclear whether the obtained data have any relevance to the input/output mapping of a circuit obtained in the case of a stable integration. Here we describe a data processing workflow, Peakfinder algorithm for flow cytometry data (PFAFF), that allows extracting precise input/output mapping from single-cell protein expression data gathered by flow cytometry after a transient transfection. The workflow builds on the numerically-proven observation that the multivariate modes of input and output expression of multi-channel flow cytometry datasets, pre-binned by the expression level of an independent transfection reporter gene, harbor cells with circuit gene copy numbers distributions that depend deterministically on the properties of a bin. We validate our method by simulating flow cytometry data for seven multi-node circuit architectures, including a complex bi-modal circuit, under stable integration and transient transfection scenarios. The workflow applied to the simulated transient transfection data results in similar conclusions to those reached with simulated stable integration data. This indicates that the input/output mapping derived from transient transfection data using our method is an excellent approximation of the ground truth. Thus, the method allows to determine input/output mapping of complex gene network using noisy transient transfection data.

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Bailly, A., G. Spath, V. Bender, and M. C. Weiss. "Phenotypic effects of the forced expression of HNF4 and HNF1alpha are conditioned by properties of the recipient cell." Journal of Cell Science 111, no. 16 (August 15, 1998): 2411–21. http://dx.doi.org/10.1242/jcs.111.16.2411.

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Tagged versions of HNF4 or HNF1alpha cDNAs in expression vectors have been introduced by transient and stable transfection into three cell lines of hepatic origin that all fail to express these two liver-enriched transcription factors and hepatic functions. C2 and H5 cells are dedifferentiated rat hepatoma variants and WIF12-E cells are human fibroblast-rat hepatoma hybrids with a reduced complement of human chromosomes. Transfectants were analyzed for the expression state of the endogenous genes coding for these transcription factors and for hepatic functions. Each cell line showed a different response to the forced expression of the transcription factors. In C2 cells, no measurable effect was observed, either upon transitory or stable expression. H5 cells reexpressed the endogenous HNF4 gene only upon transient HNF1alpha transfection, and the endogenous HNF1alpha gene only in stable HNF4 transfectants. WIF12-E cells responded to the forced transient or stable expression of either HNF1alpha or HNF4 by cross-activation of the corresponding endogenous gene. In addition, the stable transfectants reexpress HNF3alpha and C/EBPalpha, as well as all of the hepatic functions examined. Hybrid cells similar to WIF12-E had previously been observed to show pleiotropic reexpression of the hepatic phenotype in parallel with loss of human chromosome 2. For the stable WIF12-E transfectants, it was verified that reexpression of the hepatic phenotype was not due to loss of human chromosome 2. The demonstration of reciprocal cross-regulation between HNF4 and HNF1alpha in transient as well as stable transfectants implies that direct effects are involved.

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Alexander, L., H. Lee, M. Rosenzweig, J. U. Jung, and R. C. Desrosiers. "EGFP-Containing Vector System that Facilitates Stable and Transient Expression Assays." BioTechniques 23, no. 1 (July 1997): 64–66. http://dx.doi.org/10.2144/97231bm12.

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Дисертації з теми "Transient and stable expression"

Godfrey, Charlotte. "Investigation of translational reprogramming during transient and stable expression of monoclonal antibodies in Chinese hamster ovary cells." Thesis, University of Kent, 2017. https://kar.kent.ac.uk/65774/.

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Translational reprogramming and mRNA translation e ciency greatly in uence global protein synthesis, cell proliferation and growth; important parameters in de ning recombinant protein expression yields. Polysome pro ling is a widely-used technique to analyse mRNA transla- tion and its e ciency that provides a snapshot of ribosomes loaded on mRNA transcripts at any particular time. A higher number of polysomes present on a given mRNA suggests that the mRNA is being more heavily translated than those mRNAs with few ribosomes. Fur- ther, a large pool of sub-polysomes (40S, 60S and 80S) compared to polysomes in a sample suggests low translational activity. Here, polysome pro ling has been applied to investigate translational reprogramming in multiple recombinant monoclonal antibody (mAb)-producing Chinese hamster ovary (CHO) cell lines, and to determine how reprogramming re ects the ability of such cells to proliferate and make recombinant proteins in stable and transient mAb expression systems, in batch and fed-batch culture mode. The impact of culture temperature on the polysome pro le and hence on reprogramming was also investigated in transient studies. Polysome pro ling revealed reprogramming di ered between recombinant cell lines. Those with the highest global translational e ciency generally had the fastest cell speci c growth rates, although total ribosome capacity did not directly relate to those with the fastest growth rates or mAb productivities. This suggests it is the ability to utilise available machinery that determines protein synthetic capacity. Recombinant cell lines with higher cell speci c produc- tivities generally maintained a higher polysome to monosome (P:M) ratio during stationary phase and had elevated recombinant mRNA copy numbers localised to translationally active heavy polysomes. In transient systems, the P:M ratio was maintained longer at reduced tem- perature cultivation and related to higher mAb yields being obtained. A number of endogenous transcripts were found to be more or less abundant on polysomes at di erent times of culture, indicative of changes in the cellular requirements of the encoded proteins. Such transcripts could be potential cell engineering targets to help tune the needs of the cell to the demands of a culture process or recombinant protein, or alternatively their untranslated regions harnessed to help preferentially load target mRNAs onto ribosomes. When upstream open reading frames (uORFs) or alternative translation start sites were engineered into recombinant transcripts a range of mAb expressions were observed allowing the tuning of mAb expression, including improvement over a standard untranslated region used industrially as a control. The ndings described in this thesis therefore reveal insights into the mechanisms involved in translational regulation and reprogramming in CHO cells during bioprocessing. These can be utilised for further improvement via targeted cell engineering strategies, cell line screen- ing approaches or modi cation of recombinant transcripts for enhanced industrial host and recombinant cell lines.

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Schmitz, Christian. "Transient and stable expression of the human papilloma virus type 16 (HPV-16) early protein 2 (E2) in human keratinocytes." Thesis, University of York, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326421.

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Ferrari, Ilse Fernanda. "Caracterização de promotores de expressão especifica de cana-de-açucar (Saccharum ssp.) em sistema modelo Micro-Tom (Solanum lycopersicum L)." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/64/64133/tde-18012013-145236/.

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O emprego de novas tecnologias, como a transgenia, associadas ao melhoramento convencional de cana-de-açúcar apresenta grande potencial no combate a pragas e desenvolvimento de novas variedades. Entretanto, a falta de especificidade na expressão temporal e/ou local dos genes introduzidos tem se mostrado um fator limitante para o sucesso de produtos derivados da transgenia. Os promotores das metalotioneínas, por exemplo, podem representar uma alternativa ao uso de promotores constitutivos, particularmente, aqueles de metalotioneínas do tipo 1 (MT1) por apresentarem níveis elevados de expressão, em diferentes tecidos/órgãos e serem responsivos a estresses bióticos e abióticos. O uso de promotores sintéticos, contendo apenas elementos-cis, como GCG-like, W boxes e JERE, tem sido relevante por induzirem a expressão gênica local em resposta ao ataque de agentes bióticos. Este trabalho teve por objetivo a caracterização funcional de promotores de genes de metalotioneínas de cana-de-açúcar e o uso de elementos regulatórios sintéticos e, em paralelo, avaliou-se o uso de promotores sintéticos no controle da expressão gênica em cana-de-açúcar. Após as análises de expressão transiente, verificou-se que o promotor SoMT1b apresentou atividade GUS e GFP em epitélios de cebola e os promotores sintéticos, 4X Wbox, 4X GCC-like, 4X JERE e 4xW 4xS-box, e o promotor SoMT1b foram capazes de dirigir a expressão do gene repórter uidA (GUS) em calos embriogênicos de cana-de-açúcar. Em análise de expressão estável, o promotor SoMT1b foi capaz de dirigir a expressão do gene GUS para os frutos e sementes de tomate \'Micro-Tom\', mas não foi responsivo a estresse por herbivoria, cádmio e cobre. Também foi realizada a transformação de plantas de cana-de-açúcar, as quais ainda estão sendo analisadas. Os resultados obtidos demonstram a funcionalidade do promotor SoMT1b
New technologies, like genetic transformation, associated with conventional breeding of sugarcane have a large potential in developing new varieties tolerant to biotic and abiotic stress. However, the lack of specificity in the spatial and/or temporal expression of the introduced genes has been a limited factor for the success of products derived from transgeny. Metallothionein promoters, for instance, can represent an alternative to the use of constitutive promoters, particularly those from metallothionein type 1 (MT1) because they present high expression levels in different tissues / organs and are responsive to biotic and abiotic stress. Another alternative is the use of synthetic promoters which contain only cis elements, as GCG-like, W-box and JERE, which induces local gene expression in response to pathogen attack. In this work, we aimed to make the functional characterization of sugarcane metallothionein promoters and synthetic regulatory elements, in parallel, we evaluated the use of synthetic promoters in the control of gene expression in sugarcane. After transient expression analyzis, it was found that SoMT1b promoter was able to control the expression of the reporter genes to GUS and GFP in onion epithelium and GUS in sugarcane embryogenic calli. Additionally, synthetic promoters 4X Wbox, 4X GCC-like, 4X JERE and 4xW 4xS-box were able to direct the expression of the gene uidA (GUS) in embryogenic calli of sugarcane. In stable transformation analysis, SoMT1b promoter was capable of directing uidA expression in fruits and seeds of tomato cv. \'Micro-Tom\', but it was not responsive to herbivory, cadmium and copper stress. It was also carried out the transformation of sugarcane plants with the construction containing the SoMT1b promoter, but these are still being analyzed. The results demonstrate the functionality of the SoMT1b promoter in sugarcane and tomato

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Kinauer, Markus. "Transient and Stable Terminal Imido Complexes of Iridium." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2019. http://hdl.handle.net/21.11130/00-1735-0000-0003-C14D-D.

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Addison, Charlene Janet. "Microtubule-associated protein 2 (MAP2) expression in transiently and stably transfected P19 embryonal carcinoma cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq26296.pdf.

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Reed, James. "Transient expression for engineering triterpenoid diversity in plants." Thesis, University of East Anglia, 2016. https://ueaeprints.uea.ac.uk/67059/.

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The triterpenes are one of the largest and most structurally diverse classes of plant specialised metabolites, with important commercial, agronomical and medical potential. However the structural complexity and low abundance of these compounds in nature presents significant challenges for exploiting this diversity. In recent years, advances in our understanding of triterpene biosynthesis in various plant species have greatly facilitated the ability to produce these compounds in other hosts. Plant-based host systems hold great promise because they may provide substrates, cofactors and subcellular compartmentalisation mechanisms facilitating engineering production of complex triterpenoids. Agroinfiltration is a rapid and scalable process that enables transient expression of biosynthetic genes in amenable plants such as Nicotiana benthamiana. This technique holds great promise for selective production and modification of triterpenes, but studies in this area have been limited. In this PhD thesis, transient expression is used to produce and oxidise triterpenes in N. benthamiana. Triterpene yields are improved through expression of rate-limiting genes in the mevalonate pathway (Chapter 3). An enzymatic ‘toolkit’ is established from a collection of previously characterised triterpene biosynthetic genes, allowing production of milligram quantities of a diverse array of simple and oxidised triterpenes in N. benthamiana. Furthermore, combinatorial biosynthesis is utilised to engineer production of novel oxidised triterpenes in planta (Chapter 4). A set of oxidised derivatives of β-amyrin are purified and evaluated for antiproliferative and anti-inflammatory activity in human cell lines, yielding insights into structural features underlying these activities in humans (Chapter 5). Finally, N. benthamiana is used for functional screening of cytochrome P450s implicated in the biosynthesis of agronomically important antifungal triterpene glycosides (avenacins) in oat, leading to identification of new P450s required for disease resistance (Chapter 6). This work highlights the utility of N. benthamiana as triterpene engineering platform and provides a basis for future studies exploring triterpene structure-activity relationships.

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Rahman, Md Mahbubar, Jay Shockey, and Aruna Kilaru. "Characterization of Select Avocado Acyltransferases by Transient Expression." Digital Commons @ East Tennessee State University, 2018. https://dc.etsu.edu/etsu-works/4814.

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Carter, Emma. "EPR investigation of stable and transient oxygen centred radicals over polycristalline titanium dioxide." Thesis, Cardiff University, 2007. http://orca.cf.ac.uk/56178/.

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Electron Paramagnetic Resonance Spectroscopy (EPR) has been used to identify and characterise the nature of several oxygen centred radicals over the surface of noncrystalline TiC2. The observed radicals exhibit varying stabilities over the different polymorphs of TiO2 samples studied, namely mixed-phase P25, pure phase anatase and a pure rutile material. Paramagnetic Ti centres were formed by thermal treatment of Ti02 under vacuum. Following the addition of oxygen, the superoxide anion (O2") was formed and exhibited a distribution of sites on the surface. In particular, O2" preferentially stabilises at oxygen vacancies on the surface. However, the degree of site occupancy was found to be temperature dependent. Pronounced activity for vacancy stabilised anions under the influence of thermal, photochemical and chemical treatment was identified compared to non vacancy sites. Co-adsorption of a series of aliphatic and aromatic ketones and oxygen over P25, followed by low temperature (77K) UV irradiation led to the formation of a series of unstable (transient) alkylperoxy and peroxyacyl radicals. The sequential reaction of acetone with surface adsorbed superoxide anions was also found to result in the formation of an jacetone-02 (a) surface complex which was unstable at temperatures above 250K. Thermally produced Ti centres reacted with acetone to form an unstable organic intermediate. This species subsequently dissociated and underwent further reaction with oxygen to generate peroxy and peroxacyl radicals. The identities of these oxygen centred radicals were confirmed using isotopically enriched O2. Finally, results are presented on the work involved in the development of an Ultra-High Vacuum EPR spectrometer for investigation of paramagnetic species on single crystal oxide surfaces. Samples of Cu(acac)2 supported on quartz and TiC2 (l 10) were examined by EPR and XPS. The two techniques were combined to identify paramagnetic centres on the single crystals and to provide proof of concept in the operation of the spectrometer.

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Lakshmi, Priya Saikumar. "Stable expression of tuberculosis vaccine antigen in lettuce chloroplasts." Master's thesis, University of Central Florida, 2011. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4780.

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Tuberculosis (TB) is caused by Mycobacterium tuberculosis and is one of the leading reasons of death by an infectious bacterial pathogen. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, a potential candidate antigen, ESAT-6 (6 kDa early secretory antigenic target) was fused with cholera toxin B subunit (CTB). Transplastomic lettuce plants were generated expressing these fusion proteins. Site-specific transgene integration into the chloroplast genome was confirmed by polymerase chain reaction and Southern blot analysis. In transplastomic leaves, expression levels of fusion protein (CTB-ESAT6) varied depending upon the developmental stage and time of leaf harvest with highest-level of accumulation in mature leaves harvested at 6PM. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Lyophilization increased CTB-ESAT6 protein content per gram of leaf material by 22 fold. Western blot analysis of lyophilized lettuce leaves showed that the CTB-ESAT6 fusion protein was stable and can be stored for prolonged period at RT. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of ESAT-6 antigen. GM-1 binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to interact with GM1 ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens fused to CTB in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB vaccine with potential for long term storage at room temperature.
ID: 031001453; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Title from PDF title page (viewed July 3, 2013).; Thesis (M.S.)--University of Central Florida, 2011.; Includes bibliographical references (p. 42-46).
M.S.
Masters
Molecular Biology and Micro
Medicine
Biotechnology

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Powers, Sara Lawrence. "Expression and characterization of an extremely stable tetrameric hyperthermophilic protein." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file 3.68 Mb., 189 p, 2006. http://wwwlib.umi.com/dissertations/fullcit/3220724.

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Книги з теми "Transient and stable expression"

Yaghi, Farhan F. Transient and stable expression of human gbsgalactosidase and protective protein in COS-1 and CHO cells. Ottawa: National Library of Canada, 1993.

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1948-, Al-Rubeai Mohamed, ed. Transient expression. Dordrecht: Kluwer Academic Publishers, 2000.

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C, Park K., Pelerin-Dubois Y, and United States. National Aeronautics and Space Administration., eds. An unconditional stable staggered algorithm for transient finite element analysis of coupled thermoelastic problems. [Washington, DC]: National Aeronautics and Space Administration, 1991.

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M, Diner Alex, and United States. Forest Service. Southern Research Station., eds. Transient expression of GUS in bombarded embryogenic longleaf, loblolly, and eastern white pine. Asheville, NC: U.S. Dept. of Agriculture, Forest Service, Southern Research Station, 1999.

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Mulholland, Stephanie. A study of passive smoking and the expression of placental-like heat stable alkaline phosphatase activity in serum. [S.l: The Author], 1993.

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Grant, Oliver H. A study of passive smoking and the expression of placental-like serum heat stable alkaline phosphatase (HSAP) activity. [s.l: The Author], 1990.

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Al-Rubeai, Mohamed. Cell Engineering: Transient Expression. Springer, 2012.

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Al-Rubeai, Mohamed. Cell Engineering: Transient Expression. Springer, 2012.

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Update on Production of Recombinant Therapeutic Protein: Transient Gene Expression. Smithers Rapra Technology, 2013.

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Al-Rubeai, Mohamed. Cell Engineering - Transient Expression (Cell Engineering Volume 2) (Cell Engineering). Springer, 2000.

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Частини книг з теми "Transient and stable expression"

Garabagi, Freydoun, Michael D. McLean, and J. Christopher Hall. "Transient and Stable Expression of Antibodies in Nicotiana Species." In Antibody Engineering, 389–408. Totowa, NJ: Humana Press, 2012. http://dx.doi.org/10.1007/978-1-61779-974-7_23.

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Fromm, Michael, Theodore M. Klein, Stephen A. Goff, Brad Roth, Fionnuala Morrish, and Charles Armstrong. "Transient Expression and Stable Transformation of Maize Using Microprojectiles." In Plant Molecular Biology 2, 219–24. Boston, MA: Springer US, 1991. http://dx.doi.org/10.1007/978-1-4615-3304-7_23.

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Nehlsen, Kristina, Sandra Broll, Raju Kandimalla, Niels Heinz, Markus Heine, Stefanie Binius, Axel Schambach, and Jürgen Bode. "Replicating Minicircles: Overcoming the Limitations of Transient and Stable Expression Systems." In Minicircle and Miniplasmid DNA Vectors, 115–63. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2013. http://dx.doi.org/10.1002/9783527670420.ch8.

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Spiegel, Holger, Stefan Schillberg, and Greta Nölke. "Production of Recombinant Proteins by Agrobacterium-Mediated Transient Expression." In Recombinant Proteins in Plants, 89–102. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2241-4_6.

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AbstractThe agroinfiltration of plant tissue is a robust method that allows the rapid and transient expression of recombinant proteins. Using wild-type plants as biomass, agroinfiltration exploits the ability of plants to synthesize even complex multimeric proteins that require oxidative folding and/or post-translational modifications, while avoiding the expensive and time-consuming creation of stably transformed plant lines. Here we describe a generic method for the transient expression of recombinant proteins in Nicotiana benthamiana at the small to medium laboratory scale, including appropriate binary vectors, the design and cloning of expression constructs, the transformation, selection, and cultivation of recombinant Agrobacterium tumefaciens, the infiltration of plants using a syringe or vacuum device, and finally the extraction of recombinant proteins from plant tissues.

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Séguin, Armand, Denis Lachance, and Pierre J. Charest. "Transient gene expression and stable genetic transformation into conifer tissues by microprojectile bombardment." In Plant Tissue Culture Manual, 19–64. Dordrecht: Springer Netherlands, 1996. http://dx.doi.org/10.1007/978-94-009-0181-0_2.

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Séguin, Armand, Denis Lachance, and Pierre J. Charest. "Transient gene expression and stable genetic transformation into conifer tissues by microprojectile bombardment." In Plant Tissue Culture Manual, 417–62. Dordrecht: Springer Netherlands, 1996. http://dx.doi.org/10.1007/978-94-009-0103-2_24.

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Baichwal, Vijay R., and Bill Sugden. "Vectors for Gene Transfer Derived from Animal DNA Viruses: Transient and Stable Expression of Transferred Genes." In Gene Transfer, 117–48. Boston, MA: Springer US, 1986. http://dx.doi.org/10.1007/978-1-4684-5167-2_5.

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Chiou, Henry C., Sanjay Vasu, Chao Yan Liu, Isabel Cisneros, Meredith B. Jones, and Jonathan F. Zmuda. "Scalable Transient Protein Expression." In Animal Cell Biotechnology, 35–55. Totowa, NJ: Humana Press, 2013. http://dx.doi.org/10.1007/978-1-62703-733-4_4.

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Jostock, Thomas, and Hans-Peter Knopf. "Mammalian Stable Expression of Biotherapeutics." In Methods in Molecular Biology, 227–38. Totowa, NJ: Humana Press, 2012. http://dx.doi.org/10.1007/978-1-61779-921-1_15.

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Schillberg, Stefan, Sabine Zimmermann, Dirk Priifer, Detlef Schuman, and Rainer Fischer. "Transient Gene Expression in Plant Protoplasts." In Recombinant Proteins from Plants, 165–75. Totowa, NJ: Humana Press, 1998. http://dx.doi.org/10.1007/978-1-60327-260-5_13.

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Тези доповідей конференцій з теми "Transient and stable expression"

Couture, Oliver, Eric Lombardi, Kendra Davis, Emily Hays, and Nalini Chandar. "Abstract 5438: Gene expression profiles resulting from stable and transient loss of p53 mirrors its role in tissue differentiation." In Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC. American Association for Cancer Research, 2013. http://dx.doi.org/10.1158/1538-7445.am2013-5438.

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Gralnick, H. R., L. M. Magurder, K. Hansmann, M. Vail, G. Marti, R. McEver, and S. Williams. "THE SURFACE EXPRESSION OF ALPHA GRANULE PROTEINS FOLLOWING THROMBIN STIMULATION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643859.

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We have studied the platelet glycoproteins (GP) GPIb and the GPIIb/IIIa and the expression of alpha granule proteins (AGP) on the platelet (P) surface following thrombin (T) stimulation. The platelets were separated from plasma proteins on a arabinogalactan gradient. The P were stimulated with purified alpha T 0.1u/108p. Either monospecific polyclonal or murine monoclonal antibodies were used to detect the P glycoprotein and AGP. The platelets were analyzed on an EPICS V Flow Cytometer. Resting P had small amounts of AGP (2-8%) present on their surface. Within 1-3 min. after T stimulation significantly increased amounts of PF4 (26%) vWf (8%) Ig (10%) and the 140 kD alpha granule membrane (70%) were present on the P surface. The peak expression of all the AGP occurred within 5 mins. The 140 kD activation protein remained stable over 3-60 mins, in contrast the PF4 and the vWf expression peaked at 5 mins. and then decreased to near baseline levels. The GPIb and GPIIb/IIIa showed different patterns after activation. The GPIb intensity and number of positive cells decreased over time, while the GPIIb/ IIIa increased in flourescent intensity and the number of positive cells. These studies indicate that T stimulation of AGP on the P surface. vWf and P4 have a transient appearance on the P surface while Ig and the 140 kD activation protein both appear to become stable components of the P plasma membrane. This technique of detecting platelet activation is a specific, sensitive, and rapid method.

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Kaufman, Randal J., David G. Bole, and Andrew J. Dorner. "THE INFLUENCE OF N-LINKED GLYCOSYLATION AND BINDING PROTEIN (BiP) ASSOCIATION IN THE SECRETION EFFICIENCY OF COMPLEX GLYCOPROTEINS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644016.

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We have studied the role of Binding Protein (BiP) or glucose regulated protein, GRP 78) in the processing and secretion of factor VIII (fVIII), von Willebrand factor (vWF), and tissue plasminogen activator(tPA) expressed in Chinese hamster ovary cell lines.fVIII is a 300 kDa protein which has a heavily glycosylated internal domain containing 20 clustered potential N-linked glycosylation sites.A significant proportion of the expressed fVIII is bound to BiP in the endoplasmic reticulum (ER) in a stable complex andnever secreted. Deletion of the heavily glycosylatedregion results in a lesser degree of association with BiP and increased secretion. Tunicamycin treatmentof cells producing the deleted form of fVIII resultsin stable association of the unglycosylated fVIII with BiP and inhibition of efficient secretion. vWF contains 17 potential N-linked glycosylation sites which are scattered throughout the molecule. vWF is transiently associated with BiP in the ER, demonstrating that CHO cells are competent to saecrete a complex glycoprotein. tPA, which contains 3 utilized N-linked glycosylation sites, exhibits low level association with BiP and is efficiently secreted. Disruptionof normal N-linked glycosylation of tPA, by site directed mutagenesis of the 3 Asn residues to Gin residues or by tunicamycin treatment of the tPA expressing CHO cells, results in reduced levels of secretion and increased association with BiP. This effect is enhanced by high levels of expression. The findings suggest that occupancy of glycosylation sites may effect protein folding and alter secretion efficiency by influencing the extent and stability of association with BiP.

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Hassini, Amine, and Mihai Arghir. "A Simplified and Consistent Nonlinear Transient Analysis Method for Gas Bearing: Extension to Flexible Rotors." In ASME Turbo Expo 2014: Turbine Technical Conference and Exposition. American Society of Mechanical Engineers, 2014. http://dx.doi.org/10.1115/gt2014-25955.

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A simplified, new method for evaluating the nonlinear fluid forces in air bearings was recently proposed in [1]. The method is based on approximating the frequency dependent linearized dynamic coefficients at several eccentricities, by second order rational functions. A set of ordinary differential equations is then obtained using the inverse of Laplace Transform linking the fluid forces components to the rotor displacements. Coupling these equations with the equations of motion of the rotor lead to a system of ordinary differential equations where displacements and velocities of the rotor and the fluid forces come as unknowns. The numerical results stemming from the proposed approach showed good agreement with the results obtained by solving the full nonlinear transient Reynolds equation coupled to the equation of motion of a point mass rotor. However the method [1] requires a special treatment to ensure continuity of the values of the fluid forces and their first derivatives. More recently, the same authors [2] showed the benefits of imposing the same set of stable poles to the rational functions approximating the impedances. These constrains simplified the expressions of the fluid forces and avoided the introduction of false poles. The method in [2] was applied in the frame of the small perturbation analysis for calculating Campbell and stability diagrams. This approach enhances also the consistency of the fluid forces approximated with the same set of poles because they become naturally continuous over the whole bearing clearance while their increments were not. The present paper shows how easily the new formulation may be applied to compute the nonlinear response of systems with multiple degrees of freedom such as a flexible rotor supported by two air bearings.

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Kuruoglu, Ercan E., Diego Salas, and Diego Pablo Ruiz. "Microarray Gene Expression and Stable Laws." In 2007 IEEE 15th Signal Processing and Communications Applications. IEEE, 2007. http://dx.doi.org/10.1109/siu.2007.4298832.

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Bian, Liming, Robert L. Mauck, and Jason A. Burdick. "Dynamic Compressive Loading and Crosslinking Density Influence the Chondrogenic and Hypertrophic Differentiation of Human Mesenchymal Stem Cells Seeded in Hyaluronic Acid Hydrogels." In ASME 2012 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2012. http://dx.doi.org/10.1115/sbc2012-80048.

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While hyaluronic acid (HA) hydrogels provide a stable 3D environment that is conducive to the chondrogenesis of mesenchymal stem cells (MSCs) in the presence of growth factors [1], the neocartilage that is formed remains inferior to native tissue, even after long culture durations. Additionally, MSCs eventually transit into a hypertrophic phenotype after chondrogenic induction, resulting in the calcification of the ECM after ectopic transplantation [2]. From a material design perspective, variation in the HA hydrogel scaffold crosslinking density via changes in the HA macromer concentration can influence chondrogenesis of MSCs and neocartilage formation [3]. Recent studies have also demonstrated that dynamic compression enhances the expression of chondrogenic markers and cartilage matrix synthesis by MSCs encapsulated in various hydrogels, including agarose [4], alginate [5] and fibrin [6]. Furthermore, mechanical signals also regulate growth plate and articular cartilage chondrocyte hypertrophy via the IHH-PTHrP (India hedgehog, Parathyroid hormone-related protein) pathway [7]. In contrast to biologically inert scaffold materials, HA is capable of interacting with cells (including MSCs) via cell surface receptors (CD44, CD54, and CD168) [8; 9]. Therefore the objectives of this study were to (i) evaluate the effects of both hydrogel crosslinking and dynamic compressive loading on (i) chondrogenesis and cartilage matrix production/distribution of human MSCs encapsulated in HA gels and (ii) hypertrophic differentiation of human MSCs using an in vitro MSC hypertrophy model [10].

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Sindarovska, Yana. "Efficient transient gene expression in basil plants." In ASPB PLANT BIOLOGY 2020. USA: ASPB, 2020. http://dx.doi.org/10.46678/pb.20.1050098.

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Chlis, N. K., E. S. Bei, K. Moirogiorgou, and M. Zervakis. "Extracting reliable gene expression signatures through Stable Bootstrap Validation." In 2015 37th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC). IEEE, 2015. http://dx.doi.org/10.1109/embc.2015.7319384.

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Mak, Terrence W. K., Pascal Van Hentenryck, and Ian A. Hiskens. "A nonlinear optimization model for transient stable line switching." In 2017 American Control Conference (ACC). IEEE, 2017. http://dx.doi.org/10.23919/acc.2017.7963260.

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Shams, Sheyda, Ali Ghafoorzadeh Yazdi, and Masoud Movahhedi. "Unconditionally stable vector-based meshless method for transient electromagnetic analysis." In 2016 24th Iranian Conference on Electrical Engineering (ICEE). IEEE, 2016. http://dx.doi.org/10.1109/iraniancee.2016.7585673.

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Звіти організацій з теми "Transient and stable expression"

Aly, Radi, and John I. Yoder. Development of resistant crop plants to parasitic weeds based on trans-specific gene silencing. United States Department of Agriculture, January 2013. http://dx.doi.org/10.32747/2013.7598146.bard.

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Broomrapes (Orobanche/Phelipanchespp.) are holo parasitic plants that subsist on the roots of a variety of agricultural crops and cause severe losses to the yield quality and quantity. Effective methods for controlling parasitic weeds are scarce, with only a few known cases of genetic resistance. In the current study, we proposed an improved strategy for the control of parasitic weeds based on trans-specific gene-silencing of three parasite genes at once. We used two strategies to express dsRNA containing selected sequences of three Phelipancheaegyptiacagenes PaACS, PaM6PR and PaPrx1 (pma): transient expression using Tobacco rattle virus (TRV:pma) as a virus-induced gene-silencing (VIGS) vector and stable expression in transgenic tomato Solanumlycopersicum(Mill.) plants harboring a hairpin construct (pBINPLUS35:pma). siRNA-mediated transgene-silencing (20–24 nt) was detected in the host plants. Our results demonstrate that the quantities of PaACSand PaM6PR transcripts from P. aegyptiacatubercles grown on transgenic tomato or on Tobacco rattle virus-infected Nicotianabenthamianaplants were significantly reduced. However, only partial reductions in the quantity of PaPrx1 transcripts were observed in the parasite tubercles grown on tomato and on N. benthamianaplants. Concomitant with the suppression of the target genes, there were significant decreases in the number and weight of the parasite tubercles that grew on the host plants, in both the transient and the stable experimental systems. The results of the work carried out using both strategies point to the movement of mobile exogenous siRNA from the host to the parasite, leading to the impaired expression of essential parasite target genes. In light of the importance of parasitic weeds to world agriculture and the difficulty of obtaining resistance by conventional methods, we assume that genetic resistance based on the silencing of key metabolic genes in the parasite is now feasible. BARD Report - Project4622 Page 2 of 60

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Meir, Shimon, Michael S. Reid, Cai-Zhong Jiang, Amnon Lers, and Sonia Philosoph-Hadas. Molecular Studies of Postharvest Leaf and Flower Senescence. United States Department of Agriculture, January 2011. http://dx.doi.org/10.32747/2011.7592657.bard.

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Original objectives: To understand the regulation of abscission by exploring the nature of changes of auxin-related gene expression in tomato (Lycopersicon esculatumMill) abscission zones (AZs) following organ removal, and by analyzing the function of these genes. Our specific goals were: 1) To complete the microarray analyses in tomato flower and leaf AZs, for identifying genes whose expression changes early in response to auxin depletion; 2) To examine, using virus-induced gene silencing (VIGS), the effect of silencing target genes on ethylene sensitivity and abscission competence of the leaf and flower AZs; 3) To isolate and characterize promoters from AZ-specific genes to be used in functional analysis; 4) To generate stable transgenic tomato plants with selected genes silenced with RNAi, under the control of an AZ-specific promoter, for further characterization of their abscission phenotypes. Background: Abscission, the separation of organs from the parent plant, results in postharvest quality loss in many ornamentals and other fresh produce. The process is initiated by changes in the auxin gradient across the AZ, and is triggered by ethylene. Although changes in gene expression have been correlated with the ethylene-mediated execution of abscission, there is almost no information on the initiation of the abscission process, as the AZ becomes sensitized to ethylene. The present project was focused on elucidating these early molecular regulatory events, in order to gain a better control of the abscission process for agricultural manipulations. Major conclusions, solutions, achievements: Microarray analyses, using the Affymetrix Tomato GeneChip®, revealed changes in expression, occurring early in abscission, of many genes with possible regulatory functions. These included a range of auxin- and ethylene-related transcription factors (TFs), other TFs that are transiently induced just after flower removal, and a set of novel AZ-specific genes. We also identified four different defense-related genes, including: Cysteine-type endopeptidase, α- DOX1, WIN2, and SDF2, that are newly-associated with the late stage of the abscission process. This supports the activation of different defense responses and strategies at the late abscission stages, which may enable efficient protection of the exposed tissue toward different environmental stresses. To facilitate functional studies we implemented an efficient VIGS system in tomato, and isolated two abscission-specific promoters (pTAPG1 and pTAPG4) for gene silencing in stable transformation. Using the VIGS system we could demonstrate the importance of TAPGs in abscission of tomato leaf petioles, and evaluated the importance of more than 45 genes in abscission. Among them we identified few critical genes involved in leaf and flower abscission. These included: PTRP-F1, PRP, TKN4, KNOTTED-like homeobox TF, KD1, and KNOX-like homeodomain protein genes, the silencing of which caused a striking retardation of pedicel abscission, and ERF1, ERF4, Clavata-like3 protein, Sucrose transporter protein, and IAA10 genes, the silencing of which delayed petiole abscission. The importance of PRPand KD1 genes in abscission was confirmed also by antisense–silencing using pTAPG4. Experiments testing the effects of RNAi silencing of few other genes are still in progress, The analysis of the microarray results of flower and leaf AZs allowed us to establish a clear sequence of events occurring during acquisition of tissue sensitivity to ethylene, and to confirm our hypothesis that acquisition of ethylene sensitivity in the AZ is associated with altered expression of auxin-regulated genes in both AZs. Implication, both scientific and agricultural: Our studies had provided new insights into the regulation of the abscission process, and shaded light on the molecular mechanisms that drive the acquisition of abscission competence in the AZ. We pointed out some critical genes involved in regulation of abscission, and further expanded our knowledge of auxin-ethylene cross talk during the abscission process. This permits the development of novel techniques for manipulating abscission, and thereby improving the postharvest performance of ornamentals and other crops.

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Liu, Zhanjiang John, Rex Dunham, and Boaz Moav. Developmental and Evaluation of Advanced Expression Vectors with Both Enhanced Integration and Stable Expression for Transgenic Farmed Fish. United States Department of Agriculture, December 2001. http://dx.doi.org/10.32747/2001.7585196.bard.

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The objectives of the project were to develop expression vectors using the Sleeping Beauty transposon technology and the genetic border elements to provide both enhanced integration rate and stable transgene expression, and to evaluate the application of such vectors in farmed fish such as catfish and carp. The panel recommended adding the objective of evaluating the endogenous transposable elements, particularly in catfish, in order to evaluate the applicability of the expression vectors while reduc1ng efforts in real production of transgenic fish considering the focus of the project was to develop the vector and evaluation of its applicability, not producing transgenic fish. Efficient production of transgenic farmed fish is hindered by two major problems: mosaicism due to delayed integration after single-cell stage, and silencing of transgene expression. In this project, we proposed to combat these problems by coupling the Sleeping Beauty transposon technology that can enhance integration rate and the border elements that can insulate transgene from position effect. Our major objective was to develop a new generation of expression vector that contains both of these elements. We have developed expression vectors containing both the Sleeping Beauty transposon signals, inverted repeats and direct repeats (IR and DR, respectively), and the border elements, scs and scs'. Growth hormone minigene has been cloned into this vector for applications of such vectors in growth enhancement. Luc reporter gene has been also cloned into this vector cascades for relative easy evaluation of transgene expression. Transgenic fish have been produced using these expression vectors in both catfish (US) and carp (Israel). Much effort was also devoted to evaluation of the endogenous transposable elements in catfish as recommended by the BARD grant panel. Multiple families of Tcl-like transposons were identified from catfish. Surprisingly, many Tc I-related transcripts were identified. Among these transcripts, both the sense and antisense transcripts were present. Some of the transcripts may be useful for development of novel transposase-based technology for aquaculture applications in the future. This project has both scientific and aquaculture implications. First, to develop expression vectors containing both IR/DR and scs/scs' repeated elements have been reported being extremely technically difficult due to excision of the repeated sequences by the E. coli host during cloning processes. We have successfully constructed this advanced vector that contained very complex cascades for both gene integration and gene regulation. We have produced transgenic fish using such vectors. This advanced expression vector should be useful for production of transgenic fish. By simply replacing the growth hormone gene, any gene of interest can be readily inserted in this vector. Thus this vector should provide technological possibility for early integration and stable expression of any economically important genes in aquaculture. We have also evaluated the applications of the Sleeping Beauty-based vectors in terms of the impact of gene size and found that the size of trans gene drastically affects transposition. The system will be only useful for transferring genes smaller than 5.6 kb. We have also identified novel transposase-related transcripts that may be useful for the development of novel transposase-based technologies for general scientific research and for aquaculture applications.

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Diner, Alex M., Allan Zipf, Rufina Ward, Yinghua Huang, and George Brown. Transient expression of GUS in bombarded embryogenic longleaf, loblolly, and eastern white pine. Asheville, NC: U.S. Department of Agriculture, Forest Service, Southern Research Station, 1999. http://dx.doi.org/10.2737/srs-rn-007.

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Elbaum, Michael, and Peter J. Christie. Type IV Secretion System of Agrobacterium tumefaciens: Components and Structures. United States Department of Agriculture, March 2013. http://dx.doi.org/10.32747/2013.7699848.bard.

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Objectives: The overall goal of the project was to build an ultrastructural model of the Agrobacterium tumefaciens type IV secretion system (T4SS) based on electron microscopy, genetics, and immunolocalization of its components. There were four original aims: Aim 1: Define the contributions of contact-dependent and -independent plant signals to formation of novel morphological changes at the A. tumefaciens polar membrane. Aim 2: Genetic basis for morphological changes at the A. tumefaciens polar membrane. Aim 3: Immuno-localization of VirB proteins Aim 4: Structural definition of the substrate translocation route. There were no major revisions to the aims, and the work focused on the above questions. Background: Agrobacterium presents a unique example of inter-kingdom gene transfer. The process involves cell to cell transfer of both protein and DNA substrates via a contact-dependent mechanism akin to bacterial conjugation. Transfer is mediated by a T4SS. Intensive study of the Agrobacterium T4SS has made it an archetypal model for the genetics and biochemistry. The channel is assembled from eleven protein components encoded on the B operon in the virulence region of the tumor-inducing plasmid, plus an additional coupling protein, VirD4. During the course of our project two structural studies were published presenting X-ray crystallography and three-dimensional reconstruction from electron microscopy of a core complex of the channel assembled in vitro from homologous proteins of E. coli, representing VirB7, VirB9, and VirB10. Another study was published claiming that the secretion channels in Agrobacterium appear on helical arrays around the membrane perimeter and along the entire length of the bacterium. Helical arrangements in bacterial membranes have since fallen from favor however, and that finding was partially retracted in a second publication. Overall, the localization of the T4SS within the bacterial membranes remains enigmatic in the literature, and we believe that our results from this project make a significant advance. Summary of achievements : We found that polar inflations and other membrane disturbances relate to the activation conditions rather than to virulence protein expression. Activation requires low pH and nutrient-poor medium. These stress conditions are also reflected in DNA condensation to varying degrees. Nonetheless, they must be considered in modeling the T4SS as they represent the relevant conditions for its expression and activity. We identified the T4SS core component VirB7 at native expression levels using state of the art super-resolution light microscopy. This marker of the secretion system was found almost exclusively at the cell poles, and typically one pole. Immuno-electron microscopy identified the protein at the inner membrane, rather than at bridges across the inner and outer membranes. This suggests a rare or transient assembly of the secretion-competent channel, or alternatively a two-step secretion involving an intermediate step in the periplasmic space. We followed the expression of the major secreted effector, VirE2. This is a single-stranded DNA binding protein that forms a capsid around the transferred oligonucleotide, adapting the bacterial conjugation to the eukaryotic host. We found that over-expressed VirE2 forms filamentous complexes in the bacterial cytoplasm that could be observed both by conventional fluorescence microscopy and by correlative electron cryo-tomography. Using a non-retentive mutant we observed secretion of VirE2 from bacterial poles. We labeled the secreted substrates in vivo in order detect their secretion and appearance in the plant cells. However the low transfer efficiency and significant background signal have so far hampered this approach.

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Zhao, Bingyu, Saul Burdman, Ronald Walcott, and Gregory E. Welbaum. Control of Bacterial Fruit Blotch of Cucurbits Using the Maize Non-Host Disease Resistance Gene Rxo1. United States Department of Agriculture, September 2013. http://dx.doi.org/10.32747/2013.7699843.bard.

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The specific objectives of this BARD proposal were: (1) To determine whether Rxol can recognize AacavrRxo1 to trigger BFB disease resistance in stable transgenic watermelon plants. (2) To determine the distribution of Aac-avrRxo1 in a global population of Aae and to characterize the biological function of Aac-avrRxo1. (3) To characterize other TIS effectors of Aae and to identify plant R gene(s) that can recognize conserved TIS effectors of this pathogen. Background to the topic: Bacterial fruit blotch (BFB) of cucurbits, caused by Acidovorax avenae subsp. citrulli (Aae), is a devastating disease that affects watermelon (Citrullus lanatus) and melon (Cucumis melo) production worldwide, including both Israel and USA. Two major groups of Aae strains have been classified based on their virulence on host plants, genetics and biochemical properties. Thus far, no effective resistance genes have been identified from cucurbit germplasm. In this project, we assessed the applicability of a non-host disease resistance gene, Rxol, to control BFB in watermelon. We also tried to identify Aae type III secreted (TIS) effectors that can be used as molecular probes to identify novel disease resistance genes in both cucurbits and Nieotianatabaeum. Major conclusions, solutions, achievements: We generated five independent transgenic watermelon (cv. Sugar Babay) plants expressing the Rxol gene. The transgenic plants were evaluated with Aae strains AAC001 and M6 under growth chamber conditions. All transgenic plants were found to be susceptible to both Aae strains. It is possible that watermelon is missing other signaling components that are required for Rxol-mediated disease resistance. In order to screen for novel BFB resistance genes, we inoculated two Aae strains on 60 Nieotiana species. Our disease assay revealed Nicotiana tabaeum is completely resistant to Aae, while its wild relative N. benthamiana is susceptible to Aae. We further demonstrated that Nieotiana benthamiana can be used as a surrogate host for studying the mechanisms of pathogenesis of Aae. We cloned 11 TIS effector genes including the avrRxolhomologues from the genomes of 22 Aae strains collected worldwide. Sequencing analysis revealed that functional avrRxol is conserved in group" but not group I Aae strains. Three effector genes- Aave_1548, Aave_2166 and Aave_2708- possessed the ability to trigger an HR response in N. tabacum when they were transiently expressed by Agrobaeterium. We conclude that N. tabacum carries at least three different non-host resistance genes that can specifically recognize AaeTIS effectors to trigger non-host resistance. Screening 522 cucurbits genotypes with two Aae strains led us to identify two germplasm (P1536473 and P1273650) that are partially resistant to Aae. Interestingly, transient expression of the TIS effector, Aave_1548, in the two germplasms also triggered HR-Iike cell death, which suggests the two lines may carry disease resistance genes that can recognize Aave_1548. Importantly, we also demonstrated that this effector contributes to the virulence of the bacterium in susceptible plants. Therefore, R genes that recognize effector Aave1548 have great potential for breeding for BFB resistance. To better understand the genome diversity of Aae strains, we generated a draft genome sequence of the Israeli Aae strain, M6 (Group I) using Iliumina technology. Comparative analysis of whole genomes of AAC001, and M6 allowed us to identify several effectors genes that differentiate groups I and II. Implications, both scientific and agricultural: The diversity of TIS effectors in group I and II strains of Aae suggests that a subset of effectors could contribute to the host range of group I and II Aae strains. Analysis of these key effectors in a larger Aae population may allow us to predict which cucurbit hosts may be at risk to BFB. Additionally, isolation of tobacco and cucurbit Rgenes that can recognize Aae type III effectors may offer new genetic resources for controlling BFB.

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Ashley, Richard, and Randal J. Verbrugge. The Intermittent Phillips Curve: Finding a Stable (But Persistence-Dependent) Phillips Curve Model Specification. Federal Reserve Bank of Cleveland, February 2023. http://dx.doi.org/10.26509/frbc-wp-201909r2.

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We establish that the Phillips curve is persistence-dependent: inflation responds differently to persistent versus moderately persistent (or versus transient) fluctuations in the unemployment rate gap. This persistence-dependent relationship appears to align with business-cycle stages and is thus consistent with existing theory. Previous work fails to model this dependence, thereby finding numerous "inflation puzzles" – e.g., missing inflation/disinflation – noted in the literature. Our specification eliminates these puzzles; for example, the Phillips curve has not weakened, nor was inflation "stubbornly low" in 2019. The model's coefficients are stable, and it provides accurate conditional recursive forecasts through the Great Recession. There are important monetary policy implications.

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Dunham, Rex A., Boaz Moav, Thomas Chen, and Benzion Cavari. Expression and Inheritance of Growth Hormone Gene Constructs and Selective Breeding of Transgenic Farmed Fish. United States Department of Agriculture, August 1994. http://dx.doi.org/10.32747/1994.7568774.bard.

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Objectives: To accomplish stable expression, inheritance of transgenes and growth improvement in transgenic channel catfish, Ictalurus punctatus, and common carp, Cyprinus carpio, containing growth hormone (GH) genes, develop transgenic fish with all fish constructs, determine the relationships between copy number, expression and growth, determine the combined affect of selective breeding and gene transfer and assess environmental risk of transgenic fish. To develop mechanisms of triploidization for transgenic carp. Results: Performance of transgenic channel catfish was made uniform by selection. Growth of channel catfish and common carp was improved 40-50% more by combining gene transfer of GH genes with selection and crossbreeding than with either selection of crossbreeding. Growth improvement of transgenic catfish was not strongly correlated with copy number and expression levels. Progress was made in producting triploid transgenic common carp. Insertion of salmonid GH gene did not alter reproductive performance in channel catfish. Transgenic channel catfish grew no faster than controls when they had to forage on natural food and transgenic individuals were slightly more vulnerable to predation indicating that fitness of transgenic individuals in natural conditions is less than or equal to non-transgenic channel catfish. Contribution to Agriculture: These experiments are the first to demonstrate that transgenic fish can increase aquaculture production in the aquaculture production in the aquaculture environment. This research also demonstrated that maximum benefit of gene transfer in farmed fish is attained when combined with traditional selective breeding.

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Barkan, Alice, and Zach Adam. The Role of Proteases in Regulating Gene Expression and Assembly Processes in the Chloroplast. United States Department of Agriculture, January 2003. http://dx.doi.org/10.32747/2003.7695852.bard.

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Chloroplasts house many biochemical processes that are essential for plant viability. Foremost, among these is photosynthesis, which requires the protein-rich thylakoid membrane system. The activation of chloroplast genes encoding thylakoid membrane proteins and the targeting and assembly of these proteins together with their nuclear-encoded partners are essential for the elaboration of the thylakoid membrane. Several nuclear-encoded proteins that regulate chloroplast gene expression and that mediate the targeting of proteins to the thylakoid membrane have been identified in recent years, and many more remain to be discovered. The abundance of such proteins is critical and is likely to be determined to a significant extent by their stability, which in turn, is influenced by chloroplast protease activities. The primary goal of this project was to link specific proteases to specific substrates, and in particular, to specific regulatory and assembly proteins. We proposed a two-pronged approach, involving genetic analysis of the consequences of the mutational loss of chloroplast proteases, and biochemical analysis of the degradation pathways of specific proteins that have been shown to control chloroplast gene expression. Our initial bioinformatic analysis of chloroplast proteases allowed us to identify the set of pro teases that is targeted to the chloroplast. We used that information to recover three Arabidopsis mutants with T - DNA insertions in specific chloroplast protease genes. We carried out the first analysis of the stability of a regulator of chloroplast gene expression (CRS2), and found that the protein is much less stable than are typical components of the photosynthetic apparatus. Genetic reagents and analytical methods were developed that have set the stage for a rapid advancement of our understanding of chloroplast proteolysis. The results obtained may be useful for manipulating the expression of transgenes in the chloroplast and for engineering plants whose photosynthetic activity is optimized under harsh environmental conditions.

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