Relevant bibliographies by topics / In vivo inducible promoter

Academic literature on the topic 'In vivo inducible promoter'

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Published: 4 June 2021
Last updated: 25 April 2022

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Journal articles on the topic "In vivo inducible promoter"

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Dunstan, Sarah J., Cameron P. Simmons, and Richard A. Strugnell. "Use of In Vivo-Regulated Promoters To Deliver Antigens from Attenuated Salmonella enterica var. Typhimurium." Infection and Immunity 67, no. 10 (October 1, 1999): 5133–41. http://dx.doi.org/10.1128/iai.67.10.5133-5141.1999.

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ABSTRACT This study describes the construction and analysis of three in vivo-inducible promoter expression plasmids, containing pnirB, ppagC, and pkatG, for the delivery of foreign antigens in the ΔaroAD mutant ofSalmonella enterica var. Typhimurium (hereafter referred to as S. typhimurium). The reporter genes encoding β-galactosidase and firefly luciferase were used to assess the comparative levels of promoter activity in S. typhimuriumin vitro in response to different induction stimuli and in vivo in immunized mice. It was determined that the ppagC construct directed the expression of more β-galactosidase and luciferase inS. typhimurium than the pnirB and pkatG constructs, both in vitro and in vivo. The gene encoding the C fragment of tetanus toxin was expressed in thearoAD mutant of S. typhimurium (BRD509) under the control of the three promoters. Mice orally immunized with attenuated S. typhimurium expressing C fragment under control of the pagC promoter [BRD509(pKK/ppagC/C frag)] mounted the highest tetanus toxoid-specific serum antibody response. Levels of luciferase expression in vivo and C-fragment expression in vitro from thepagC promoter appeared to be equivalent to if not lower than the levels of expression detected with the constitutivetrc promoter. However, mice immunized with BRD509(pKK/ppagC/C frag) induced significantly higher levels of tetanus toxoid-specific antibody than BRD509(pKK/C frag)-immunized mice, suggesting that the specific location of foreign antigen expression may be important for immunogenicity. Mutagenesis of the ribosome binding sites (RBS) in the three promoter/C fragment expression plasmids was also performed. Despite optimization of the RBS in the three different promoter elements, the expression levels in vivo and overall immunogenicity of C fragment when delivered to mice by attenuated S. typhimurium were not affected. These studies suggest that in vivo-inducible promoters may give rise to enhanced immunogenicity and increase the efficacy of S. typhimuriumas a vaccine vector.
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Bateman, B. T., N. P. Donegan, T. M. Jarry, M. Palma, and A. L. Cheung. "Evaluation of a Tetracycline-Inducible Promoter inStaphylococcus aureus In Vitro and In Vivo and Its Application in Demonstrating the Role of sigB in Microcolony Formation." Infection and Immunity 69, no. 12 (December 1, 2001): 7851–57. http://dx.doi.org/10.1128/iai.69.12.7851-7857.2001.

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ABSTRACT An inducible promoter system provides a powerful tool for studying the genetic basis for virulence. A variety of inducible systems have been used in other organisms, including pXyl-xylR-inducible promoter, the pSpac-lacI system, and the arabinose-inducible PBAD promoter, but each of these systems has limitations in its application to Staphylococcus aureus. In this study, we demonstrated the efficacy of a tetracycline-inducible promoter system in inducing gene expression in S. aureus in vitro and inside epithelial cells as well as in an animal model of infection. Using the xyl/tetOpromoter::gfp uvr fusion carried on a shuttle plasmid, we demonstrated that dose-dependant tetracycline induction, as measured by bacterial fluorescence, occurred in each of the above environments while basal activation under noninduced conditions remained low. To ascertain how the system can be used to elucidate the genetic basis of a pathogenic phenotype, we cloned thesigB gene downstream of the inducible promoter. Induction of SigB expression led to dose-dependent attachment of the tested strain to polystyrene microtiter wells. Additionally, bacterial microcolony formation, an event preceding mature biofilm formation, also increased with tetracycline induction of SigB.
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LaPointe, Margot C., Xiao-Ping Yang, Oscar A. Carretero, and Quan He. "Left ventricular targeting of reporter gene expression in vivo by human BNP promoter in an adenoviral vector." American Journal of Physiology-Heart and Circulatory Physiology 283, no. 4 (October 1, 2002): H1439—H1445. http://dx.doi.org/10.1152/ajpheart.01090.2001.

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To selectively introduce genes into the mouse myocardium, we used a recombinant adenovirus encoding a transgene composed of a cardiac-specific promoter [the proximal human brain natriuretic peptide (hBNP) promoter] coupled to a luciferase reporter gene (Ad.hBNPLuc). Activity in vitro and in vivo was compared with Ad.CMVLuc, which contained the cytomegalovirus (CMV) enhancer/promoter. We tested cell-specific and inducible regulation of Ad.hBNPLuc in vitro. Expression was higher in neonatal cardiac myocytes than in a fibroblast cell line and was induced by interleukin-1β, phenylephrine, and isoproterenol in myocytes. For in vivo experiments, Ad.hBNPLuc, Ad.CMVLuc, or vehicle was injected into the left ventricular (LV) free wall of the mouse heart. In Ad.hBNPLuc-injected mice, luciferase activity was only detected in the heart. In contrast, Ad.CMVLuc-injected mice had detectable luciferase activity in all tissues examined. Our studies indicate that 1) the cardiac-specific hBNP promoter and direct cardiac injection limit expression of the transgene to the LV free wall; and 2) transgene expression in vitro is inducible and cardiac myocyte specific. Thus the use of the proximal hBNP promoter in recombinant adenoviral vectors may allow cardiac-specific and inducible expression of therapeutic genes in vivo and prevent some of the side effects of systemic adenovirus administration.
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Xiao, Yong, Takeo Kuwata, Tomoyuki Miura, Masanori Hayami, and Hisatoshi Shida. "Dox-Dependent SIVmac with Tetracycline-Inducible Promoter in the U3 Promoter Region." Virology 269, no. 2 (April 2000): 268–75. http://dx.doi.org/10.1006/viro.2000.0213.

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He, Quan, Ding Wang, Xiao-Ping Yang, Oscar A. Carretero, and Margot C. LaPointe. "Inducible regulation of human brain natriuretic peptide promoter in transgenic mice." American Journal of Physiology-Heart and Circulatory Physiology 280, no. 1 (January 1, 2001): H368—H376. http://dx.doi.org/10.1152/ajpheart.2001.280.1.h368.

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Studies have shown that brain natriuretic peptide (BNP) gene expression is rapidly induced in the infarcted heart and that plasma BNP levels reflect the degree of left ventricular dysfunction. Our previous in vitro work using transiently transfected neonatal rat cardiac myocytes has shown that the human BNP (hBNP) promoter, in particular a region extending from −127 to −40 relative to the start site of transcription, is more active in cardiac myocytes than in fibroblasts. To study tissue-specific and transcriptional regulation of the hBNP gene in vivo, we generated transgenic mice containing the proximal hBNP promoter (−408 to +100) coupled to a luciferase reporter gene. In four lines of transgenic mice, luciferase activity was ∼33- to 100-fold higher in the heart than in other tissues, including the whole brain. To test whether the transgene responded to a pathophysiological stimulus, we induced infarction by coronary artery ligation. Luciferase activity was fivefold higher in the infarcted region of the left ventricle at 48 h than in sham-operated animals and remained elevated for 4 wk. Endogenous BNP mRNA was similarly increased in the infarcted hearts of a separate group of mice. We conclude that 1) the proximal 408-bp region of the hBNP promoter confers cardiac-specific expression and 2) myocardial infarction activates the proximal hBNP promoter in vivo. These data suggest that we have a valid model for the study of basal and inducible regulation of the hBNP gene in vivo.
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Breton, Marc, Evelyne Sagné, Sybille Duret, Laure Béven, Christine Citti, and Joël Renaudin. "First report of a tetracycline-inducible gene expression system for mollicutes." Microbiology 156, no. 1 (January 1, 2010): 198–205. http://dx.doi.org/10.1099/mic.0.034074-0.

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Inducible promoter systems are powerful tools for studying gene function in prokaryotes but have never been shown to function in mollicutes. In this study we evaluated the efficacy of the tetracycline-inducible promoter Pxyl/tetO2 from Bacillus subtilis in controlling gene expression in two mollicutes, the plant pathogen Spiroplasma citri and the animal pathogen Mycoplasma agalactiae. An S. citri plasmid carrying the spiralin gene under the control of the xyl/tetO2 tetracycline-inducible promoter and the TetR repressor gene under the control of a constitutive spiroplasmal promoter was introduced into the spiralin-less S. citri mutant GII3-9a3. In the absence of tetracycline, expression of TetR almost completely abolished expression of spiralin from the xyl/tetO2 promoter. Adding tetracycline (>50 ng ml−1) to the medium induced high-level expression of spiralin. Interestingly, inducible expression of spiralin was also detected in vivo: in S. citri-infected leafhoppers fed on tetracycline-containing medium and in S. citri-infected plants watered with tetracycline. A similar construct was introduced into the M. agalactiae chromosome through transposition. Tetracycline-induced expression of spiralin proved the TetR-Pxyl/tetO2 system to be functional in the ruminant pathogen, suggesting that this tetracycline-inducible promoter system might be of general use in mollicutes.
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Charron, J., H. Richard-Foy, D. S. Berard, G. L. Hager, and J. Drouin. "Independent glucocorticoid induction and repression of two contiguous responsive genes." Molecular and Cellular Biology 9, no. 7 (July 1989): 3127–31. http://dx.doi.org/10.1128/mcb.9.7.3127.

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Specific DNA sequence elements which contain binding sites for the glucocorticoid receptor mediate the action of glucocorticoid hormones on gene transcription. In glucocorticoid-inducible genes, these glucocorticoid-responsive elements behave as hormone-inducible enhancers of transcription. We have taken advantage of the bovine papillomavirus (BPV) system to test the stringency of glucocorticoid regulation of transcription. BPV episomes were constructed to contain two hormone-regulated transcription units in close proximity; one transcription unit is under control of a glucocorticoid-inducible promoter (mouse mammary tumor virus) while the other is under control of a glucocorticoid-inhibited promoter (pro-opiomelanocortin). Glucocorticoids independently regulated transcription of the two physically linked transcription units, irrespective of their relative orientation and of their proximity on the BPV episomes. This result contrasts with the so-called position-independent activity of enhancers and suggests that the multicomponent organization of eucaryotic promoters restricts the action of hormone-responsive regulatory elements to a specific transcription unit, thus accounting for the stringency of hormonal regulation observed in vivo.
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Charron, J., H. Richard-Foy, D. S. Berard, G. L. Hager, and J. Drouin. "Independent glucocorticoid induction and repression of two contiguous responsive genes." Molecular and Cellular Biology 9, no. 7 (July 1989): 3127–31. http://dx.doi.org/10.1128/mcb.9.7.3127-3131.1989.

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Specific DNA sequence elements which contain binding sites for the glucocorticoid receptor mediate the action of glucocorticoid hormones on gene transcription. In glucocorticoid-inducible genes, these glucocorticoid-responsive elements behave as hormone-inducible enhancers of transcription. We have taken advantage of the bovine papillomavirus (BPV) system to test the stringency of glucocorticoid regulation of transcription. BPV episomes were constructed to contain two hormone-regulated transcription units in close proximity; one transcription unit is under control of a glucocorticoid-inducible promoter (mouse mammary tumor virus) while the other is under control of a glucocorticoid-inhibited promoter (pro-opiomelanocortin). Glucocorticoids independently regulated transcription of the two physically linked transcription units, irrespective of their relative orientation and of their proximity on the BPV episomes. This result contrasts with the so-called position-independent activity of enhancers and suggests that the multicomponent organization of eucaryotic promoters restricts the action of hormone-responsive regulatory elements to a specific transcription unit, thus accounting for the stringency of hormonal regulation observed in vivo.
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Piskurich, Janet F., Michael W. Linhoff, Ying Wang, and Jenny P. Y. Ting. "Two Distinct Gamma Interferon-Inducible Promoters of the Major Histocompatibility Complex Class II Transactivator Gene Are Differentially Regulated by STAT1, Interferon Regulatory Factor 1, and Transforming Growth Factor β." Molecular and Cellular Biology 19, no. 1 (January 1, 1999): 431–40. http://dx.doi.org/10.1128/mcb.19.1.431.

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ABSTRACT The major histocompatibility complex (MHC) class II transactivator (CIITA) is the master regulatory factor required for appropriate expression of class II MHC genes. Understanding the expression of CIITA is key to understanding the regulation of class II MHC genes. This report describes the independent regulation of two distinct CIITA promoters by cytokines with opposing functions, gamma interferon (IFN-γ) and transforming growth factor β (TGF-β). A functional analysis of deletion mutations of the upstream promoter (promoter III) identified an IFN-γ-responsive region located approximately 5 kb from the transcriptional start site. An in vivo DNase I hypersensitivity analysis detected a hypersensitive site in this area which supports the relevance of this region. When the downstream promoter (promoter IV) was studied by in vivo genomic footprinting, IFN-γ-induced changes at putative binding sites for STAT1, interferon regulatory factor 1 (IRF-1), and E-box proteins were seen. Gel shift and supershift analyses for IRF-1 confirmed the in vivo footprint results. The role of the IFN-γ-inducible transcription factor STAT1 was examined functionally. Although both promoters were controlled by STAT1, promoter-specific regulation was exhibited. The IFN-γ response of promoter III was completely dependent on STAT1 and not IRF-1, while promoter IV was partially activated by IRF-1 in the total absence of STAT1 expression. While both promoters were affected by TGF-β, activation of promoter III by IFN-γ was more severely diminished by TGF-β treatment. The differential control of CIITA promoters by TGF-β, IRF-1, and STAT1 may be important in refining regulation of class II MHC genes in different cell types and under different stimulatory conditions.
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Ko, H. P., S. T. Okino, Q. Ma, and J. P. Whitlock. "Transactivation domains facilitate promoter occupancy for the dioxin-inducible CYP1A1 gene in vivo." Molecular and Cellular Biology 17, no. 7 (July 1997): 3497–507. http://dx.doi.org/10.1128/mcb.17.7.3497.

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We have studied the transcriptional regulation of the dioxin-inducible mouse CYP1A1 gene in its native chromosomal setting. We analyzed the ability of aromatic hydrocarbon receptor (AhR) mutants and AhR chimeras to restore dioxin responsiveness to the CYP1A1 gene in AhR-defective mouse hepatoma cells. Our data reveal that transactivation domains in AhR's C-terminal half mediate occupancy of the nuclear factor 1 site and TATA box for the CYP1A1 promoter in vivo. Transactivation domains of VP16 and AhR nuclear translocator, but not Sp1, can substitute for AhR's C-terminal half in facilitating protein binding at the promoter. Our data also reveal an apparent linear relationship between promoter occupancy and CYP1A1 gene expression in chromatin. These findings provide new insights into the in vivo mechanism of transcriptional activation for an interesting mammalian gene.
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Dissertations / Theses on the topic "In vivo inducible promoter"

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Saxena, Manvendra, and s3031657@student rmit edu au. "Utilising salmonella to deliver heterologous vaccine antigen." RMIT University. Applied Sciences, 2007. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20080522.095907.

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Live attenuated Salmonella vectors provide a unique alternative in terms of antigen presentation by acting as a vector for heterologous antigens. The efficiency of any live bacterial vector rests with its ability to present sufficient foreign antigen to the human or animal immune system to initiate the desirable protective immune response. Salmonella vectors encoding heterologous protective antigens can elicit the relevant immune responses, be it humoral, mucosal or cell-mediated. STM-1 is a Salmonella mutant developed by RMIT, harbours a mutation in the aroA gene that renders it attenuated, and is a well characterised vaccine strain currently in use to protect livestock against Salmonella infection. In previous work in this laboratory, STM1 was shown to be capable of eliciting immune responses in mice to plasmid-borne antigens. In this study STM-1 was analysed for its ability to vector the model antigen chicken ovalbumin and test antigen C. jejuni major outer membrane protein using in vivo inducible promoters such as pagC and nirB from the plasmid location. The determination of the architecture around the lesion in STM-1 also allowed the development of constructs expressing heterologous antigen from the chromosome. The induction of immune responses, both humoral and cell mediated, was analysed. Another issue addressed in this study was effect of pre-existing immune responses in the animal host against the vector or related strains and the effects on generation of immune responses against the subsequently vectored antigen. Humoral and cellular immune responses to vectored ovalbumin and C. jejuni Momp antigens were observed following vaccination with STM-1, when antigens were expressed from either the plasmid or chromosomal location. Up-regulation of immune responses, both humoral and cell mediated, was observed against the vectored antigens in animals which were pre-exposed to either the bacterial vector or related strains. These results indicate that STM-1 has the potential to be used as a vector to deliver heterologous vaccine antigens from a single copy gene in the field. Lastly, the results from this study indicate that pre-existing immune responses against the bacterial vector or a related strain do in fact enhance both humoral and T cell responses against the heterologous antigen.
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Pinel, Karine. "Imagerie in vivo du contrôle de l’inhibition génique et de l’électroporation d’ARN." Thesis, Bordeaux 2, 2012. http://www.theses.fr/2012BOR22004/document.

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Ces travaux de thèse d’imagerie moléculaire et translationnelle proposent, sur des modèles murins, deux approches innovantes pour les thérapies géniques. La plupart des cancers sont associés à des dérégulations de l’expression génique et certains gènes sont surexprimés. L’utilisation de microARN (miARN) permet d’envisager une réduction de l’expression d’un gène spécifique mais il est nécessaire de limiter cette inhibition au tissu pathologique. L’utilisation des promoteurs thermo-inductibles couplés à un dépôt local de chaleur autorise un contrôle spatial et temporel de l’expression génique in vivo. Notre projet a été de coupler le contrôle spatio-temporel et l’inhibition d’un gène cible. A cette fin, un miARN synthétique a été placé sous contrôle du promoteur thermo-inductible Hsp70B pour induire l’inhibition d’un gène d’imagerie (luciférase firefly) surexprimé dans une tumeur. L’étude a été menée in vitro sur des lignées cellulaires génétiquement modifiées puis in vivo sur un modèle de xénogreffes chez la souris grâce au suivi en imagerie optique de bioluminescence (BLI). Nos résultats montrent la faisabilité d’induire transitoirement l’inhibition génique au sein d’une tumeur. L’induction est modulable par la température. Cette stratégie peut être couplée à des méthodes couramment utilisées en clinique et ouvre des perspectives thérapeutiques intéressantes. Notre travail de thèse s’intéresse également à l’utilisation d’ARN comme molécule thérapeutique pour la thérapie génique. L’électroporation intra-dermique d’ARN codant pour la luciférase permet de suivre et de quantifier in vivo par BLI l’expression génique. Plusieurs types d’ARN ont été utilisés pour comparer les efficacités respectives des différentes voies traductionnelles. Notre travail démontre que les ARN permettent l’expression transitoire, sans risque d’insertion génomique, d’un gène in vivo. Nous montrons ainsi tout le potentiel de l’utilisation des ARN en thérapie génique
The present thesis work in molecular and translational imaging establishes two innovative approaches for gene therapy in mouse models. Abnormal regulation of gene expression is the hallmark of cancer, and some of them are overexpressed. MicroRNA (miRNA) can be used as tools to reduce specific gene expression but requires inhibition to be limited to the pathological tissue. Thermo-inducibles promoters associated with local hyperthermia allow for spatial and temporal control of gene expression in vivo. The goal of the present study was to achieve gene inhibition with spatio-temporal control of miRNA expression to inhibit a target gene. In our strategy, a synthetic miRNA was placed under transcriptional control of the heat-inducible promoter Hsp70B to induce inhibition of the imaging reporter gene firefly luciferase overexpressed in a tumor. The study was conducted both in vitro using genetically modified cells lines and in vivo using a xenograft model in mice monitored by optical bioluminescence imaging (BLI). Our data show the feasibility of transient induction and heat-modulation of gene inhibition within a tumor. This strategy can be performed with currently clinically available methods and thus, offers interesting therapeutics prospects. Our work also includes a study on RNA as therapeutic vector for gene therapy. The intradermic electroporation of RNA encoding the imaging reporter gene firefly luciferase allows to monitor and quantify gene expression by BLI in vivo. Several types of RNA have been used to investigate efficiency of the different translational mechanisms. Our data clearly demonstrate that RNA allows for transient gene expression in vivo without any risk of insertion into the target cell’s genome. Altogether, our data highlight the potential use of RNA in gene therapy
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Yan, Shao-feng. "Development of an inducible promoter system in Leishmania donovani /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/9306.

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Ali, N. A. "Investigations into the regulation of the thiostrepton inducible promoter of Streptomyces lividans." Thesis, Swansea University, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.635755.

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The thiostrepton inducible P-tipA promoter of Streptomyces is widely used as a tool for gene cloning and over expression studies. This thesis describes the experiments carried out to investigate the regulation of P-tipA in S. lividans 1326 in order to improve expression of the promoter. Regulation of the promoter was studied using three different reporter genes, aphII, luxAB and egfp in both plasmid borne and chromosomally integrative forms. The promoter was found to be induced by soya flour as well as thiostrepton (Tsr). Expression of the promoter was determined to be growth-phase development and sensitive to extracellular osmolarity. The expression of the promoter was increased and prolonged in high osmolarity media in the presence and absence of Tsr. The transcriptional activator protein TipAL, which is required for thiostrepton induction was also shown to be required for induction by soya flour and increased extracellular osmolarity. P-tipA has several features, which suggest that it may be responsive to DNA superhelicity. These include a sub-optimally spaced promoter, which is contained within a palindromic sequence with the potential to form a cruciform secondary structure in vivo. The possible role of DNA superhelicity in the regulation of P-tipA was analysed using the gyrase inhibitors, novobiocin and coumermycin. These had little effect on P-tipA expression in the presence of high extracellular osmolarity. The promoter was also exploited for the production of transposon Tn1798, which carries an inducible outward reading P-tipA. The purpose of this transposon is to produce conditional lethal mutations for the study of essential genes. The construction and application of this transposon is described.
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Gross, Tiffany Lauren. "Aedes aegypti Heat Shock 70 Genes and their Inducible Promoters." Diss., Virginia Tech, 2011. http://hdl.handle.net/10919/28305.

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Aedes aegypti is an important vector of the viruses that cause dengue fever, dengue hemorrhagic fever, and yellow fever. In depth genetic studies of vector species have been made possible due to the availability of genome sequences and techniques for producing stably transformed mosquitoes. These resources have also contributed to the establishment of new genetics-based approaches to the control of vector borne disease. Genetic studies of Ae. aegypti have benefited from the ability to drive targeted transgene expression, however a ubiquitous inducible promoter has not been identified in this mosquito. The Drosophila melanogaster heat shock 70 promoter has been shown to drive inducible expression in heterologous systems; however, DmHsp70 possesses significant basal activity in Aedes aegypti. This study characterized the sequence and expression of the heat shock 70 genes of Aedes aegypti. AaHsp70 genes were found to be organized in two clusters, each comprised of three divergent pairs. AaHsp70 genes exhibited robust expression upon heat shock in larvae, pupae, and adults as well as in heads, salivary glands, midguts and ovaries. Genomic regions upstream of AaHsp70 genes were found to drive heat-inducible expression of a reporter in both cell and embryo assays. Deletion analysis of AaHsp70-derived promoters yielded two ~1.5 kb genomic fragments that maintained robust heat inducibility in these systems. Aedes aegypti were transformed with AaHsp70-luciferase gene cassettes using the transposable element Mos1. AaHsp70-luciferase transcripts accumulated specifically after heat shock, and displayed a pattern of rapid induction and decay similar to endogenous AaHsp70 genes. Heat-induced expression of luciferase was observed in transgenic larvae, pupae and adults as well as heads, midguts and ovaries but not salivary glands, with levels varying between transgenic strains. The effect of heat shock on the endogenous RNAi pathway as well as the effect of blood feeding on the expression of AaHsp70 genes was investigated, though reproducible results could not be obtained using the assays employed. In conclusion, the heat shock 70 gene family of Aedes aegypti was identified and characterized. The AaHsp70 promoters described could be valuable for gene function studies as well as for the precise timing of the expression of anti-pathogen molecules.
Ph. D.
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Oduor, Okoth Richard. "Functional Analysis of the Novel Stress- Inducible XVPSAP promoter isolated from Xerophya Viscosa." Doctoral thesis, University of Cape Town, 2009. http://hdl.handle.net/11427/4314.

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Winston, Eugenia Michele. "The Utilization of the Hmg2 Inducible Promoter to Genetically Engineer Parasite Resistance in Tobacco." Diss., Virginia Tech, 2003. http://hdl.handle.net/10919/27200.

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The cyst nematode, Globodera tabacum tabacum Behrens, and the parasitic angiosperm, Egyptian broomrape, Orobanche aegyptiaca Pers., are obligate root parasites that cause severe yield and quality loss of many important crop hosts. Although these represent two diverse classes of parasites, they have significant similarities in the modes of parasitism and complex interactions with their hosts. Conventional control methods have had limited success in controlling these parasites. The overall objective of this research was to engineer resistance to the cyst nematode and Egyptian broomrape by expressing genes encoding parasite specific toxins under the control of parasite-responsive promoters using tobacco (Nicotiana tabacum L. cv. Xanthi). For nematode resistance, an anti-feeding strategy was employed utilizing the tomato proteinase inhibitor I (PI-I) gene as a nematode specific toxin. Transgenic tobacco plants were generated that expressed genes encoding an intracellarly retained or secreted form of tomato PI-I under the control of the nematode-inducible promoter, derived from tomato (Lycopersicon esculentum L.) Hmg2 gene. Our goals were to determine the effectiveness of local PI-I expression on nematode resistance and to determine if intracellular or extracellular PI-I deposition enhances resistance. Two constructs were generated that contained either the coding region of the tomato PI-I gene, lacking the signal sequence (EM1), or the coding region of PI-I including the signal sequence (EM2), fused to the nematode-responsive Hmg2 promoter. Transgenic PI-I plants were inoculated with G. t. tabacum cysts and evaluated for nematode interactions. Our results suggest that local expression of intercellular of PI-I significantly reduced cyst production when compared to the nontransformed controls. For broomrape resistance, a well characterized R/avr gene pair, the tobacco N resistance gene and the tobacco mosaic virus replicase (TMV) gene, was utilized to create novel gene-for-gene resistance via a N gene-mediated hypersensitive response (HR) to limit broomrape parasitism. The bean (Phaselous vulgaris L.) chalcone synthase 8 (CHS8) promoter has been characterized as a broomrapeâ responsive promoter. We introduced the CHS8:TMV replicase gene construct into tobacco plants that contains an endogenous N gene. Transgenic tobacco plants were inoculated with O. aegyptiaca seeds and monitored for parasite attachment and development. The expression of the TMV replicase leads to a significant reduction in broomrape parasitism. These genetic engineering strategies show promise in enhancing resistance to these destructive parasites.
Ph. D.
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Hartman, Andrea H. "Use of an Inducible Promoter to Characterize Type IV Pili Homologues in Clostridium perfringens." Thesis, Virginia Tech, 2012. http://hdl.handle.net/10919/76874.

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Researchers of Clostridium perfringens, a Gram-positive anaerobic pathogen, were lacking a tightlyregulated, inducible promoter system in their genetic toolbox. We constructed a lactose-inducible plasmid-based system utilizing the transcriptional regulator, BgaR. Using the E. coli reporter GusA, we characterized its induction in three different strains of C. perfringens. We then used a newly-developed mutation system to create in-frame deletion mutants in three genes with homology to Type IV pilins, and we used the promoter system described above to complement the mutants. We analyzed each pilin for localization and expression, as well as tested each of the mutants for various phenotypes frequently associated with type IV pili (TFP) and type II secretion systems. PilA2, PilA3, and PilA4 localized to the poles of the cells. PilA2 was expressed in the wildtype when C. perfringens was grown on agar plates, and the PilA3 mutant lacked a von Willebrand factor A domain-containing protein in its secretome. We used our promoter system to express GFP-tagged versions of the TFP ATPase homologues and view them in cells growing on surfaces. We saw that PilB1 and PilB2 co-localized nearly all of the time, while a portion of PilT was independent of the PilB proteins. PilT appeared necessary for the localization of PilB, and it localized independently of TFP proteins in Bacillus subtilis. PilT's typical localization in Bacillus subtilis was disrupted when the GTPase and polymerization activity of cell division protein FtsZ was blocked, suggesting that PilT associates with cell division proteins.
Master of Science
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Modica, Teresa Maria Elisa. "A mouse model to study inducible oncogene cooperation in vivo." Thesis, Open University, 2012. http://oro.open.ac.uk/54234/.

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The current model for cancer development envisions cells under going a series of genetic mutations and/or alterations which result in their inability to respond normally to intracellular and extracellular signals that control proliferation, differentiation and death. The number of required genetic alterations varies for different types of cancer and it is likely that further changes occur during its progression to increased malignancy. Thus, cancer is not a static disease but during the development and progression of tumour, multiple changes occur in two kinds of genes: oncogenes and tumour suppressor genes. Oncogene-products can be classified as growth factors, growth factor receptors, Ras oncoproteins, cytoplasmic protein kinases, transcription factors, anti-apoptotic proteins. In particular, the ras oncogene family includes three members: N-ras, K-ras, H-ras. In non-transformed cells, Ras protein, belonging to G-protein family, transduces growth signals from external to the internal environment. In fact, when activated, Ras exchanges GDP with GTP and this allosteric change allows binding of Ras effector molecules and transduction of signalling cascades. Ras activity is required for cell cycle progression. In cancer it has been observed that this oncogene is constitutively activated by mutations and induces the cell to enter into cell-cycle also in the absence of growth signals. Among the transcription factors, a gene involved in many tumours is myc. This transcription factor plays a key role in cell proliferation as its target proteins include many positive regulators of the cell-cycle. In tumour cells the protein product of this oncogene is overexpressed. The cooperationb etweenm ultiple oncogenesa nd/orl oss of tumour suppressors from different functional classes is necessary for transformation to proceed. In fact, it was observed that, although overexpression of a single oncogene does not transform wild-type mouse embryonic fibroblasts, combinations of myc and H-rasVAL12, can induce cellular transformation and the cells expressing both oncogenes displayeda markedp roliferative advantage. In thyroid, neoplastic transformation generates several different histotypes of tumours, ranging from poorly aggressive and well-differentiated, to highly malignant and undifferentiated anaplastic cancers. The aim of my thesis was to study the tumorigenesis induced by oncogenes and the oncogene cooperation in vivo during the gradual passage from a poorly aggressive to a much more aggressive tumour. To this end a mouse model expressing the two oncogenes H-rasVAL12 and c-myc (referred as ras and myc) in a tissue-specific as well as in a conditional manner was generated. For this purpose, the coding sequences of the two oncogenes were fused in a bicistronic construct and an IRES (Internal Ribosome Entry Sequence or Site) was inserted between them, to ensure the expression of the second oncogene. The construct was inserted under the control of the promoter of the ubiquitously expressed genes ROSA26 and Eeflal. In order to express these oncogenes in a tissue-specific manner, the transcription of the two oncogenesis prevented by a STOP sequence flanked by two LoxP sites. Such a STOP sequence can be removed by Cre recombinase protein. The transgenic mice were crossed with mice expressing Cre in a tissue-specific manner. Two strains of transgenic mice expressing Cre in thyroid cells were used: 1. transgenic mice for TgCre, in which Cre is expressed under the control of Tg promoter after the development of the thyroid; 2. Pax8Cre, in which the Cre sequence is inserted in the Pax8 locus and is expressed during the early stages of the thyroid development. In such a manner the oncogenes were expressed only in thyroid cells, but were still inactive. In particular, ras was fused to the mutated ligand binding domain of the estradiol receptor that is sensitive to tamoxifen and not to endogenous estradiol; while mycwas fused to the mutated ligand binding domain of the progesterone receptor (hPR891) that is sensitive to RU486 and not to endogenous progesterone. With these fused oncogenes it is possible to activate only Ras (with tamoxifen) or only Myc (with RU486) or both (providing both tamoxifen and RU486). Moreover the activity of two oncogenes might be used to immortalize mouse cell lines in culture.
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Giesel, Christian. "Transformation of tobacco with a lupin chitinase gene under control of a stress inducible promoter." Diss., Pretoria : [s.n.], 2007. http://upetd.up.ac.za/thesis/available/etd-07082008-135301.

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Books on the topic "In vivo inducible promoter"

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Karaskov, Elizabeta. In vivo characterization of ordered factor recruitment at CIITA inducible promoter IV. Ottawa: National Library of Canada, 2003.

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Zhang, Xiao-Qun. Functional Studies on the Pdgfra Gene Promoter & Effects of Autocrine Pdgf-A Stimulation in Vivo. Uppsala Universitet, 2001.

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Book chapters on the topic "In vivo inducible promoter"

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Hakamata, Yoji, and Eiji Kobayashi. "Inducible and Conditional Promoter Systems to Generate Transgenic Animals." In Methods in Molecular Biology, 71–79. Totowa, NJ: Humana Press, 2009. http://dx.doi.org/10.1007/978-1-60327-389-3_5.

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Thomson, Bennett, Emmanuelle Graciet, and Frank Wellmer. "Inducible Promoter Systems for Gene Perturbation Experiments in Arabidopsis." In Methods in Molecular Biology, 15–25. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7125-1_2.

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Civenni, Gianluca. "Targeting Promoter-Associated Noncoding RNA In Vivo." In Methods in Molecular Biology, 259–70. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-6716-2_15.

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van der Kop, Dianne A. M., Frans N. J. Droog, Bert J. van der Zaal, and Paul J. J. Hooykaas. "Expression of an auxin-inducible promoter of tobacco in Arabidopsis thaliana." In Plant Hormone Signal Perception and Transduction, 15–22. Dordrecht: Springer Netherlands, 1996. http://dx.doi.org/10.1007/978-94-009-0131-5_3.

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Delhove, Juliette M. K. M., Rajvinder Karda, Kate E. Hawkins, Lorna M. FitzPatrick, Simon N. Waddington, and Tristan R. McKay. "Bioluminescence Monitoring of Promoter Activity In Vitro and In Vivo." In Methods in Molecular Biology, 49–64. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7223-4_5.

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Senf, Sarah M., and Andrew R. Judge. "Determination of Gene Promoter Activity in Skeletal Muscles In Vivo." In Methods in Molecular Biology, 461–72. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-343-1_27.

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Ohtsuru, Akira, Vera Braiden, Yu Cao, Mitsuo Kosaka, and Shunichi Yamashita. "Cancer Gene Therapy in Conjunction with Hyperthermia Under the Control of Heat-Inducible Promoter." In Thermotherapy for Neoplasia, Inflammation, and Pain, 464–70. Tokyo: Springer Japan, 2001. http://dx.doi.org/10.1007/978-4-431-67035-3_53.

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Mazzoletti, Marco, and Gemma Texidó. "In Vivo Target Validation by Inducible RNAi in Human Xenograft Mouse Models." In Target Identification and Validation in Drug Discovery, 325–37. Totowa, NJ: Humana Press, 2013. http://dx.doi.org/10.1007/978-1-62703-311-4_20.

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Yoon, I. S., D. H. Park, H. Mori, B. G. Kang, and H. Imaseki. "Characterization of the Promoter of the Mung Bean Auxin-Inducible ACC Synthase Gene, Vr-ACS6." In Biology and Biotechnology of the Plant Hormone Ethylene II, 21–27. Dordrecht: Springer Netherlands, 1999. http://dx.doi.org/10.1007/978-94-011-4453-7_4.

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Escalante-Alcalde, Diana, and Luis Covarrubias. "Transitory Transgenic Analysis as an In Vivo System to Study Promoter Regulatory Elements." In Microinjection and Transgenesis, 413–25. Berlin, Heidelberg: Springer Berlin Heidelberg, 1998. http://dx.doi.org/10.1007/978-3-642-80343-7_22.

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Conference papers on the topic "In vivo inducible promoter"

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Perrino, Giansimone, and Diego di Bernardo. "Modelling, simulation and control of single cell expression dynamics of the galactose-inducible promoter in yeast." In 2016 IEEE 55th Conference on Decision and Control (CDC). IEEE, 2016. http://dx.doi.org/10.1109/cdc.2016.7798772.

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Chubukova, O. V., Z. R. Vershinina, R. T. Matnyazov, and Al Kh Baymiev. "Using nod genes control system to create rhizospheric microorganisms with regulated gene expression." In 2nd International Scientific Conference "Plants and Microbes: the Future of Biotechnology". PLAMIC2020 Organizing committee, 2020. http://dx.doi.org/10.28983/plamic2020.054.

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Inducible vector containing the full-sized nodD gene and the promoter region of the nod-box under the control of which was cloned the gfp gene was constructed. Modified bacteria R. galegae in which the synthesis of GFP protein was activated by plant flavonoids were obtained.
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YU, YONG A., SHELLEY CALTHARP, and ALADAR A. SZALAY. "INDUCIBLE GENE EXPRESSION IN VIVO USING A RENILLA LUCIFERASE – GFP FUSION CONSTRUCT." In Proceedings of the 11th International Symposium. WORLD SCIENTIFIC, 2001. http://dx.doi.org/10.1142/9789812811158_0113.

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Salam, Muhammad T., Hyang-Min Byun, Carrie V. Breton, Xinhui Wang, Kimberly D. Siegmund, and Frank D. Gilliland. "Genetic And Epigenetic Variations In Inducible Nitric Oxide Synthase Promoter Region, Particulate Pollution And Exhaled Nitric Oxide In Children." In American Thoracic Society 2011 International Conference, May 13-18, 2011 • Denver Colorado. American Thoracic Society, 2011. http://dx.doi.org/10.1164/ajrccm-conference.2011.183.1_meetingabstracts.a5680.

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Harrison, Tyler, Robert J. Paproski, and Roger J. Zemp. "In vivo imaging of inducible tyrosinase gene expression with an ultrasound array-based photoacoustic system." In SPIE BiOS, edited by Alexander A. Oraevsky and Lihong V. Wang. SPIE, 2012. http://dx.doi.org/10.1117/12.908987.

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Spalluto, C. Mirella, Akul Singhania, Christopher H. Woelk, Tilman Sanchez-Elsner, Karl J. Staples, and Tom M. A. Wilkinson. "IFNγ influences bronchial epithelial anti-viral immune responses via inducible epigenetic control of histone methylation of the RIG-I promoter." In Annual Congress 2015. European Respiratory Society, 2015. http://dx.doi.org/10.1183/13993003.congress-2015.oa1782.

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Zelko, Igor N., Marcus W. Stepp, and Rodney J. Folz. "Live In-Vivo Imaging Of Extracellular Superoxide Dismutase (EC-SOD) Promoter Activity In Transgenic Mice." In American Thoracic Society 2012 International Conference, May 18-23, 2012 • San Francisco, California. American Thoracic Society, 2012. http://dx.doi.org/10.1164/ajrccm-conference.2012.185.1_meetingabstracts.a1275.

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Mdoe, Victoria N., and Christopher M. Evans. "Requirement For Epithelial Derived Hypoxia Inducible Factor-1 Alpha(HIF-1±) In Allergic Lung Inflammation In Vivo." In American Thoracic Society 2012 International Conference, May 18-23, 2012 • San Francisco, California. American Thoracic Society, 2012. http://dx.doi.org/10.1164/ajrccm-conference.2012.185.1_meetingabstracts.a4295.

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Blackburn, James, Robert Ng, Daniel Roden, Jianmin Wu, and Richard J. Epstein. "Abstract LB-85: Damage-inducible intragenic demethylation activates transcription of an alternative intronic promoter in TP53-wildtype but not mutant human cells and tumors." In Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA. American Association for Cancer Research, 2014. http://dx.doi.org/10.1158/1538-7445.am2014-lb-85.

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Fu, X., M. Shea, NC Biswal, T. Mitchell, M. Giuliano, NA Healy, KL Meerbrey, et al. "P4-01-03: Establishment and Characterization of an Endocrine Resistance Model In Vitro and In Vivo by Inducible PTEN Knockdown." In Abstracts: Thirty-Fourth Annual CTRC‐AACR San Antonio Breast Cancer Symposium‐‐ Dec 6‐10, 2011; San Antonio, TX. American Association for Cancer Research, 2011. http://dx.doi.org/10.1158/0008-5472.sabcs11-p4-01-03.

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Reports on the topic "In vivo inducible promoter"

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Daniels, C. J. Structure and regulation of an archaebacterial promoter: An in vivo study. Progress report. Office of Scientific and Technical Information (OSTI), December 1993. http://dx.doi.org/10.2172/10169665.

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Dougherty, Shona. Isolation and Functional Characterization of Prostate Tumor-Specific Hypoxia-Inducible Promoter/Enhancer Elements for Use in Gene Therapy. Fort Belvoir, VA: Defense Technical Information Center, June 2001. http://dx.doi.org/10.21236/ada413596.

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Aly, Radi, James H. Westwood, and Carole L. Cramer. Novel Approach to Parasitic Weed Control Based on Inducible Expression of Cecropin in Transgenic Plants. United States Department of Agriculture, May 2003. http://dx.doi.org/10.32747/2003.7586467.bard.

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Our overall goal was to engineer crop plants with enhanced resistance to Orobanche (broomrape) based on the inducible expression of sarcotoxin-like peptide (SLP). A secondary objective was to localize small proteins such as SLP in the host-parasite union in order to begin characterizing the mechanism of SLP toxicity to Orobanche. We have successfully accomplished both of these objectives and have demonstrated that transgenic tobacco plants expressing SLP under control of the HMG2 promoter show enhanced resistance to O. aegyptiaca and O. ramosa . Furthermore, we have shown that proteins much larger than the SLP move into Orobanche tubercles from the host root via either symplastic or apoplastic routes. This project was initiated with the finding that enhanced resistance to Orobanche could be conferred on tobacco, potato, and tomato by expression of SLP (Sarcotoxin IA is a 40-residue peptide produced as an antibiotic by the flesh fly, Sarcophaga peregrina ) under the control of a low-level, root-specific promoter. To improve the level of resistance, we linked the SLP gene to the promoter from HMG2, which is strongly inducible by Orobanche as it parasitizes the host. The resulting transgenic plants express SLP and show increased resistance to Orobanche. Resistance in this case is manifested by increased growth and yield of the host in the presence of the parasite as compared to non-transgenic plants, and decreased parasite growth. The mechanism of resistance appears to operate post-attachment as the parasite tubercles attached to the transgenic root plants turned necrotic and failed to develop normally. Studies examining the movement of GFP (approximately 6X the size of SLP) produced in tobacco roots showed accumulation of green fluorescence in tubercles growing on transformed plants but not in those growing on wild-type plants. This accumulation occurs regardless of whether the GFP is targeted to the cytoplasm (translocated symplastically) or the apoplastic space (translocated in xylem). Plants expressing SLP appear normal as compared to non-transgenic plants in the absence of Orobanche, so there is no obvious unintended impact on the host plant from SLP expression. This project required the creation of several gene constructs and generation of many transformed plant lines in order to address the research questions. The specific objectives of the project were to: 1. Make gene constructs fusing Orobanche-inducible promoter sequences to either the sarcotoxin-like peptide (SLP) gene or the GFP reporter gene. 2. Create transgenic plants containing gene constructs. 3. Characterize patterns of transgene expression and host-to-parasite movement of gene products in tobacco ( Nicotiana tabacum L.) and Arabidopsis thaliana (L.). 4. Characterize response of transgenic potato ( Solanum tuberosum L.) and tomato ( Lycopersicon esculentum Mill .) to Orobanche in lab, greenhouse, and field. Objectives 1 and 2 were largely accomplished during the first year during Dr. Aly's sabbatical visit to Virginia Tech. Transforming and analyzing plants with all the constructs has taken longer than expected, so efforts have concentrated on the most important constructs. Work on objective 4 has been delayed pending the final results of analysis on tobacco and Arabidopsis transgenic plants. The implications of this work are profound, because the Orobanche spp. is an extremely destructive weed that is not controlled effectively by traditional cultural or herbicidal weed control strategies. This is the first example of engineering resistance to parasitic weeds and represents a unique mode of action for selective control of these weeds. This research highlights the possibility of using this technique for resistance to other parasitic species and demonstrates the feasibility of developing other novel strategies for engineering resistance to parasitic weeds.
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Nordeen, Steven. Exploiting Polytene Chromosomes to Identify Transcription CoFactors and Complexes Recruited to an Estrogen Responsive Promoter in Vivo. Fort Belvoir, VA: Defense Technical Information Center, June 2003. http://dx.doi.org/10.21236/ada417949.

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Daniels, C. J. Structure and regulation of an archaebacterial promoter: An in vivo study. Progress report, August 1, 1991--March 31, 1993. Office of Scientific and Technical Information (OSTI), June 1993. http://dx.doi.org/10.2172/10159724.

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Lers, Amnon, E. Lomaniec, S. Burd, A. Khalchitski, L. Canetti, and Pamela J. Green. Analysis of Senescence Inducible Ribonuclease in Tomato: Gene Regulation and Function. United States Department of Agriculture, February 2000. http://dx.doi.org/10.32747/2000.7570563.bard.

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Natural leaf senescence has a negative influence on yield. Postharvest induced senescence contributes to the losses of quality in flowers, foliage, and vegetables. Strategies designed to control the senescence process in crop plants could therefore have great applied significance. Senescence is regulated by differential gene expression yet, functional characterization of the genes specifically induced and study of their expression control, is still in its infancy. Study of senescence-specific genes is required to allow identification of regulatory elements participating in senescence-induced expression and thus provide insights into the genetic regulation of senescence. A main feature of senescence is the hydrolysis of macromolecules by hydrolases of various types such as RNases and proteases. This study was aimed a analysis of senescence-inducible RNases in tomato with the following objectives: Isolation of senescence-inducible RNase cDNA clones; Expression analyses of RNase genes during senescence; Identification of sequences required for senescence-induced gene expression; Functional analyses of senescence-inducible RNases. We narrowed our aims somewhat to focus on the first three objectives because the budget we were awarded was reduced from that requested. We have expanded our research for identification senescence-related RNase/nuclease activities as we thought it will direct us to new RNase/nuclease genes. We have also carried out research in Arabidopsis and parsley, which enabled us to draw mire general conclusions. We completed the first and second objectives and have made considerable progress on the remaining two. We have defined growth conditions suitable for this research and defined the physiological and biochemical parameters characteristic to the advance of leaf senescence. In tomato and arabidopsis we have focused on natural leaf senescence. Parsley was used mainly for study of postharvest senescence in detached leaves. We have identified a 41-kD a tomato nuclease, LeNUCI, specifically induced during senescence which can degrade both RNA and DNA. This activity could be induced by ethylene in young leaves and was subjected to detailed analysis, which enabled its classification as Nuclease I enzyme. LeNUCI may be involved in nucleic acid metabolism during tomato leaf senescence. In parsley senescing leaves we identified 2 main senescence-related nuclease activities of 41 and 39-kDa. These activities were induced in both naturally or artificially senescing leaves, could degrade both DNA and RNA and were very similar in their characteristics to the LeNUCI. Two senescence-induced RNase cDNAs were cloned from tomato. One RNase cDNA was identical to the tomato LX RNase while the second corresponded to the LE RNase. Both were demonstrated before to be induced following phosphate starvation of tomato cell culture but nothing was known about their expression or function in plants. LX gene expression was much more senescence specific and ethylene could activate it in detached young leaves. LE gene expression, which could be transiently induced by wounding, appeared to be activated by abscisic acid. We suggest that the LX RNase has a role in RNA catabolism in the final stage of senescence, and LE may be a defense-related protein. Transgenic plants were generated for altering LX gene expression. No major visible alterations in the phenotype were observed so far. Detailed analysis of senescence in these plants is performed currently. The LX promoter was cloned and its analysis is performed currently for identification of senescence-specific regulatory elements. In Arabidopsis we have identified and characterized a senescence-associated nuclease 1 gene, BFN1, which is highly expressed during leaf and stem senescence. BFN1, is the first example of a senescence- associated gene encoding a nuclease I enzyme as well as the first nuclease I cloned and characterized from Arabidopsis. Our progress should provide excellent tools for the continued analysis of regulation and function of senescence-inducible ribonucleases and nucleases in plants. The cloned genes can be used in reverse genetic approaches, already initiated, which can yield a more direct evidence for the function of these enzymes. Another contribution of this research will be in respect to the molecular mechanism, which controls senescence. We had already initiated in this project and will continue to identify and characterize regulatory elements involved in senescence-specific expression of the genes isolated in this work.
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Shapira, Roni, Judith Grizzle, Nachman Paster, Mark Pines, and Chamindrani Mendis-Handagama. Novel Approach to Mycotoxin Detoxification in Farm Animals Using Probiotics Added to Feed Stuffs. United States Department of Agriculture, May 2010. http://dx.doi.org/10.32747/2010.7592115.bard.

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T-2 toxin, a toxic product belongs to the trichothecene mycotoxins, attracts major interest because of its severe detrimental effects on the health of human and farm animals. The occurrence of trichothecenes contamination is global and they are very resistant to physical or chemical detoxification techniques. Trichothecenes are absorbed in the small intestine into the blood stream. The hypothesis of this project was to develop a protecting system using probiotic bacteria that will express trichothecene 3-O-acetyltransferase (Tri101) that convert T-2 to a less toxic intermediate to reduce ingested levels in-situ. The major obstacle that we had faced during the project is the absence of stable and efficient expression vectors in probiotics. Most of the project period was invested to screen and isolate strong promoter to express high amounts of the detoxify enzyme on one hand and to stabilize the expression vector on the other hand. In order to estimate the detoxification capacity of the isolated promoters we had developed two very sensitive bioassays.The first system was based on Saccharomyces cerevisiae cells expressing the green fluorescent protein (GFP). Human liver cells proliferation was used as the second bioassay system.Using both systems we were able to prove actual detoxification on living cells by probiotic bacteria expressing Tri101. The first step was the isolation of already discovered strong promoters from lactic acid bacteria, cloning them downstream the Tri101 gene and transformed vectors to E. coli, a lactic acid bacteria strain Lactococcuslactis MG1363, and a probiotic strain of Lactobacillus casei. All plasmid constructs transformed to L. casei were unstable. The promoter designated lacA found to be the most efficient in reducing T-2 from the growth media of E. coli and L. lactis. A prompter library was generated from L. casei in order to isolate authentic probiotic promoters. Seven promoters were isolated, cloned downstream Tri101, transformed to bacteria and their detoxification capability was compared. One of those prompters, designated P201 showed a relatively high efficiency in detoxification. Sequence analysis of the promoter region of P201 and another promoter, P41, revealed the consensus region recognized by the sigma factor. We further attempted to isolate an inducible, strong promoter by comparing the protein profiles of L. casei grown in the presence of 0.3% bile salt (mimicking intestine conditions). Six spots that were consistently overexpressed in the presence of bile salts were isolated and identified. Their promoter reigns are now under investigation and characterization.
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Yaron, Zvi, Abigail Elizur, Martin Schreibman, and Yonathan Zohar. Advancing Puberty in the Black Carp (Mylopharyngodon piceus) and the Striped Bass (Morone saxatilis). United States Department of Agriculture, January 2000. http://dx.doi.org/10.32747/2000.7695841.bard.

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Both the genes and cDNA sequences encoding the b-subunits of black carp LH and FSH were isolated, cloned and sequenced. Sequence analysis of the bcFSHb and LHb5'flanking regions revealed that the promoter region of both genes contains canonical TATA sequences, 30 bp and 17 bp upstream of the transcription start site of FSHb and LHb genes, respectively. In addition, they include several sequences of cis-acting motifs, required for inducible and tissue-specific transcriptional regulation: the gonadotropin-specific element (GSE), GnRH responsive element (GRE), half sites of estrogen and androgen response elements, cAMP response element, and AP1. Several methods have been employed by the Israeli team to purify the recombinant b subunits (EtOH precipitation, gel filtration and lentil lectin). While the final objective to produce pure recombinantGtH subunits has not yet been achieved, we have covered much ground towards this goal. The black carp ovary showed a gradual increase in both mass and oocyte diameter. First postvitellogenic oocytes were found in 5 yr old fish. At this age, the testes already contained spermatozoa. The circulating LH levels increased from 0.5 ng/ml in 4 yr old fish to >5ng/ml in 5 yr old fish. In vivo challenge experiments in black carp showed the initial LH response of the pituitary to GnRH in 4 yr old fish. The response was further augmented in 5 yr old fish. The increase in estradiol level in response to gonadotropic stimulation was first noted in 4 yr old fish but this response was much stronger in the following year. In vivo experiments on the FSHb and LHb mRNA levels in response to GnRH were carried out on common carp as a model for synchronom spawning cyprinids. These experiments showed the prevalence of FSHP in maturing fish while LHP mRNA was prevalent in mature fish, especially in females. The gonadal fat-pad was found to originate from the retroperitoneal mesoderm and not from the genital ridge, thus differing from that reported in certain amphibians This tissue possibly serves as the major source of sex steroids in the immature black carp. However, such a function is taken over by the developing gonads in 4 yr old fish. In the striped bass, we described the ontogeny of the neuro-endocrine parameters along the brain-pituitary-gonadal axis during the first four years of life, throughout gonadal development and the onset of puberty. We also described the responsiveness of the reproductive axis to long-term hormonal manipulations at various stages of gonadal development. Most males reached complete sexual maturity during the first year of life. Puberty was initiated during the third year of life in most females, but this first reproductive cycle did not lead to the acquisition of full sexual maturity. This finding indicates that more than one reproductive cycle may be required before adulthood is reached. Out of the three native GnRHs present in striped bass, only sbGnRH and cGnRH II increased concomitantly with the progress of gonadal development and the onset of puberty. This finding, together with data on GtH synthesis and release, suggests that while sbGnRH and cGnRH II may be involved in the regulation of puberty in striped bass, these neuropeptides are not limiting factors to the onset of puberty. Plasma LH levels remained low in all fish, suggesting that LH plays only a minor role in early gonadal development. This hypothesis was further supported by the finding that experimentally elevated plasma LH levels did not result in the induction of complete ovarian and testicular development. The acquisition of complete puberty in 4 yr old females was associated with a rise in the mRNA levels of all GtH subunit genes, including a 218-fold increase in the mRNA levels of bFSH. mRNA levels of the a and PLH subunits increased only 11- and 8-fold, respectively. Although data on plasma FSH levels are unavailable, the dramatic increase in bFSH mRNA suggests a pivotal role for this hormone in regulating the onset and completion of puberty in striped bass. The hormonal regulation of the onset of puberty and of GtH synthesis and release was studied by chronic administration of testosterone (T) and/or an analog of gonadotropin-releasing hormone (G). Sustained administration of T+G increased the mRNA levels of the PLH subunit to the values characteristic of sexually mature fish, and also increased the plasma levels of LH. However, these changes did not result in the acceleration of sexual maturation. The mRNA levels of the bFSH subunit were slightly stimulated, but remained about 1/10 of the values characteristic of sexually mature fish. It is concluded that the stimulation of FSH gene expression and release does not lead to the acceleration of sexual maturity, and that the failure to sufficiently stimulate the bFSH subunit gene expression may underlie the inability of the treatments to advance sexual maturity. Consequently, FSH is suggested to be the key hormone to the initiation and completion of puberty in striped bass. Future efforts to induce precocious puberty in striped bass should focus on understanding the regulation of FSH synthesis and release and on developing technologies to induce these processes. Definite formulation of hormonal manipulation to advance puberty in the striped bass and the black carp seems to be premature at this stage. However, the project has already yielded a great number of experimental tools of DNA technology, slow-release systems and endocrine information on the process of puberty. These systems and certain protocols have been already utilized successfully to advance maturation in other fish (e.g. grey mullet) and will form a base for further study on fish puberty.
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Zhou, Ting, Roni Shapira, Peter Pauls, Nachman Paster, and Mark Pines. Biological Detoxification of the Mycotoxin Deoxynivalenol (DON) to Improve Safety of Animal Feed and Food. United States Department of Agriculture, July 2010. http://dx.doi.org/10.32747/2010.7613885.bard.

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The trichothecene deoxynivalenol (DON, vomitoxin), one of the most common mycotoxin contaminants of grains, is produced by members of the Fusarium genus. DON poses a health risk to consumers and impairs livestock performance because it causes feed refusal, nausea, vomiting, diarrhea, hemolytic effects and cellular injury. The occurrence of trichothecenes contamination is global and they are very resistant to physical or chemical detoxification techniques. Trichothecenes are absorbed in the small intestine into the blood stream. The overall objective of this project was to develop a protecting system using probiotic bacteria that will express trichothecene 3-O-acetyltransferase (Tri101) that convert T-2 to a less toxic intermediate to reduce ingested levels in-situ. The major obstacle that we had faced during the project is the absence of stable and efficient expression vectors in probiotics. Most of the project period was invested to screen and isolate strong promoter to express high amounts of the detoxify enzyme on one hand and to stabilize the expression vector on the other hand. In order to estimate the detoxification capacity of the isolated promoters we had developed two very sensitive bioassays.The first system was based on Saccharomyces cerevisiae cells expressing the green fluorescent protein (GFP). Human liver cells proliferation was used as the second bioassay system.Using both systems we were able to prove actual detoxification on living cells by probiotic bacteria expressing Tri101. The first step was the isolation of already discovered strong promoters from lactic acid bacteria, cloning them downstream the Tri101 gene and transformed vectors to E. coli, a lactic acid bacteria strain Lactococcuslactis MG1363, and a probiotic strain of Lactobacillus casei. All plasmid constructs transformed to L. casei were unstable. The promoter designated lacA found to be the most efficient in reducing T-2 from the growth media of E. coli and L. lactis. A prompter library was generated from L. casei in order to isolate authentic probiotic promoters. Seven promoters were isolated, cloned downstream Tri101, transformed to bacteria and their detoxification capability was compared. One of those prompters, designated P201 showed a relatively high efficiency in detoxification. Sequence analysis of the promoter region of P201 and another promoter, P41, revealed the consensus region recognized by the sigma factor. We further attempted to isolate an inducible, strong promoter by comparing the protein profiles of L. casei grown in the presence of 0.3% bile salt (mimicking intestine conditions). Six spots that were consistently overexpressed in the presence of bile salts were isolated and identified. Their promoter reigns are now under investigation and characterization.
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10

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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