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Inducible Promoter (inducible + promoter)
Selected AbstractsInterconversion of intra- and extra-chromosomal sites of gene amplification by modulation of gene expression and DNA methylationJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2007Noriaki Shimizu Abstract We previously showed that plasmids containing a mammalian replication initiation region and a matrix attachment region were efficiently amplified to few thousand copies per cell, and that they formed extrachromosomal double minutes (DMs) or chromosomal homogeneously staining regions (HSRs). In these structures, the plasmid sequence was arranged as a tandem repeats, and we suggested a mechanism of plasmid amplification. Since amplification was very efficient, easy, and convenient, it might be adapted to a novel method for protein production. In the current study, we found that gene expression from the tandem plasmid repeat was suppressed. We identified several strategies to overcome this suppression, including: (1) use of higher concentrations of antibiotic during cell selection; (2) treatment of cells with agents that influence DNA methylation (5-azacytidine) or histone acetylation (butyrate); (3) co-amplification of an insulator sequence; and (4) co-amplification of sequences that encode a transcriptional activator. Expression from the plasmid repeat was always higher at DMs compared to HSRs. We found that continuous activation of a plasmid-encoded inducible promoter prevented the generation of long HSRs, and favored amplification at DMs. Consistent with this finding, there was a synergistic effect of transcriptional activation and inhibition of DNA methylation on the fragmentation of long HSRs and the generation of DMs and short HSRs. Our results indicate that both transcriptional activation and DNA methylation regulate the interconversion between extra- and intra-chromosomal gene amplification. These results have important implications for both protein production technology, and the generation of chromosomal abnormalities found in human cancer cells. J. Cell. Biochem. 102: 515,529, 2007. © 2007 Wiley-Liss, Inc. [source] Inducible and constitutive expression using new plasmid and integrative expression vectors for Thermus sp.LETTERS IN APPLIED MICROBIOLOGY, Issue 6 2001K.J. Kayser Aims: To develop molecular tools and examine inducible and constitutive gene expression in Thermus thermophilus. Methods and Results: Two plasmid promoter probe vectors and an integrative promoter probe vector were constructed using a promoterless thermostable kanamycin nucleotidyltransferase (KmR) cassette. Three expression vectors were constructed based on a constitutive promoter J17, that functions in both Thermus and Escherichia coli. An inducible expression vector was constructed using the heat-shock inducible promoter (70 to 85°C) from the dnaK gene of T. flavus, and the malate dehydrogenase gene (mdh) from T. flavus was cloned and expressed in both E. coli and T. thermophilus HB27. Conclusions: This report describes the construction and use of improved promoter probe and expression vectors for use in Thermus species. The mdh gene can be used as a high temperature (85°C) reporter gene for Thermus sp. The dnaK promoter is thermo-inducible. Significance and Impact of the Study: The expression vectors and molecular tools described here are significant improvements over previously reported vectors for Thermus sp. The mdh gene and the thermo-inducible dnaK promoter will facilitate high temperature studies employing Thermus species. [source] SigB, an RNA polymerase sigma factor required for osmoprotection and proper differentiation of Streptomyces coelicolorMOLECULAR MICROBIOLOGY, Issue 1 2001You-Hee Cho A gene (sigB) encoding an alternative sigma factor ,B in Streptomyces coelicolor A3(2) was isolated and characterized. It encodes a polypeptide of 281 amino acids (31 546 Da) and is highly homologous to Bacillus subtilis,B. The sigB coding region is preceded by four open reading frames (ORFs): dpsA, orfA, rsbB and rsbA in sequential order. RNA analyses revealed that rsbB, rsbA and sigB constitute an operon (sigB operon). Transcripts were produced constitutively from a promoter (sigBp2) upstream of the rsbB coding region, contributing to the basal level expression of ,B protein. An inducible promoter (sigBp1) resembling the catB promoter (catBp) was located between the rsbA and sigB coding regions. Transcripts from sigBp1 dramatically increased as cells differentiated on solid media, at the stationary phase in liquid media or by osmotic stresses similar to the behaviour of catBp transcripts. Both catBp and sigBp1 promoters were recognized specifically by ,B -containing RNA polymerase in vitro. Disruption of the sigB gene abolished not only the differentiation-associated expression but also the osmotic induction of the catB gene, indicating that catBp is under the control of ,B. The sigB mutant exhibited a similar phenotype to the catB mutant, being sensitive to hyperosmolarity, blocked in forming aerial mycelium and with skewed antibiotic production. Therefore, we conclude that ,B ensures the proper differentiation and osmoprotection of S. coelicolor cells, primarily via regulation of the expression of catalase B. [source] Genome-wide analysis of the general stress response in Bacillus subtilisMOLECULAR MICROBIOLOGY, Issue 4 2001Chester W. Price Bacteria respond to diverse growth-limiting stresses by producing a large set of general stress proteins. In Bacillus subtilis and related Gram-positive pathogens, this response is governed by the ,B transcription factor. To establish the range of cellular functions associated with the general stress response, we compared the transcriptional profiles of wild and mutant strains under conditions that induce ,B activity. Macroarrays representing more than 3900 annotated reading frames of the B. subtilis genome were hybridized to 33P-labelled cDNA populations derived from (i) wild-type and sigB mutant strains that had been subjected to ethanol stress; and (ii) a strain in which ,B expression was controlled by an inducible promoter. On the basis of their significant ,B -dependent expression in three independent experiments, we identified 127 genes as prime candidates for members of the ,B regulon. Of these genes, 30 were known previously or inferred to be ,B dependent by other means. To assist in the analysis of the 97 new genes, we constructed hidden Markov models (HMM) that identified possible ,B recognition sequences preceding 21 of them. To test the HMM and to provide an independent validation of the hybridization experiments, we mapped the ,B -dependent messages for seven representative genes. For all seven, the 5, end of the message lay near typical ,B recognition sequences, and these had been predicted correctly by the HMM for five of the seven examples. Lastly, all 127 gene products were assigned to functional groups by considering their similarity to known proteins. Notably, products with a direct protective function were in the minority. Instead, the general stress response increased relative message levels for known or predicted regulatory proteins, for transporters controlling solute influx and efflux, including potential drug efflux pumps, and for products implicated in carbon metabolism, envelope function and macromolecular turnover. [source] CtsR controls class III heat shock gene expression in the human pathogen Listeria monocytogenesMOLECULAR MICROBIOLOGY, Issue 4 2000Shamila Nair Stress proteins play an important role in virulence, yet little is known about the regulation of stress response in pathogens. In the facultative intracellular pathogen Listeria monocytogenes, the Clp ATPases, including ClpC, ClpP and ClpE, are required for stress survival and intracellular growth. The first gene of the clpC operon of L. monocytogenes encodes a homologue of the Bacillus subtilis CtsR repressor of stress response genes. An L. monocytogenes ctsR -deleted mutant displayed enhanced survival under stress conditions (growth in the presence of 2% NaCl or at 42°C), but its level of virulence in the mouse was not affected. The virulence of a wild-type strain constitutively expressing CtsR is significantly attenuated, presumably because of repression of the stress response. Regulation of the L. monocytogenes clpC, clpP and clpE genes was investigated using transcriptional fusions in B. subtilis as a host. The L. monocytogenes ctsR gene was placed under the control of an inducible promoter, and regulation by CtsR and heat shock was demonstrated in vivo in B. subtilis. The purified CtsR protein of L. monocytogenes binds specifically to the clpC, clpP and clpE regulatory regions, and the extent of the CtsR binding sites was defined by DNase I footprinting. Our results demonstrate that this human pathogen possesses a CtsR regulon controlling class III heat shock genes, strikingly similar to that of the saprophyte B. subtilis. This is the first description of a stress response regulatory gene in a pathogen. [source] Overexpression of rice isoflavone reductase-like gene (OsIRL) confers tolerance to reactive oxygen speciesPHYSIOLOGIA PLANTARUM, Issue 1 2010Sang Gon Kim Isoflavone reductase is an enzyme involved in isoflavonoid biosynthesis in plants. However, rice isoflavone reductase-like gene (OsIRL, accession no. AY071920) has not been unraveled so far. Here, we have characterized its behavior in response to oxidizing agents. Using Northern and Western blot analyses, the OsIRL gene and protein were shown to be down-regulated in young seedling roots treated with reduced glutathione (GSH) and diphenyleneiodonium (DPI), known quenchers of reactive oxygen species (ROS). The OsIRL transcript level in rice suspension-cultured cells was also found to be induced by oxidants such as hydrogen peroxide (H2O2), ferric chloride (FeCl3), methyl viologen (MV) and glucose/glucose oxidase (G/GO), but down-regulated when co-treated with GSH. Furthermore, to investigate whether overexpression of OsIRL in transgenic rice plants promotes resistance to ROS, we generated transgenic rice lines overexpressing the OsIRL gene under an abscisic acid (ABA) inducible promoter. Results showed that the OsIRL transgenic rice line activated by ABA treatment was tolerant against MV and G/GO-induced stress in rice leave and suspension-cultured cells. Our results strongly suggest the involvement of OsIRL in homeostasis of ROS. [source] Over-expression of a Populus peroxisomal ascorbate peroxidase (PpAPX) gene in tobacco plants enhances stress tolerancePLANT BREEDING, Issue 4 2009Y-J. Li Abstract Ascorbate peroxidase (APX) plays an important role in the metabolism of hydrogen peroxide in higher plants. We studied the effect of over-expressing a Populus peroxisomal ascorbate peroxidase (PpAPX) gene under the control of the cauliflower mosaic virus 35S promoter or the rd29 promoter in transgenic tobacco. High levels of PpAPX gene expression were observed in 35S-PpAPX transgenic plants, with a 50% increase in APX activity. The constitutive expression of PpAPX in the tobacco exhibited no morphological abnormalities, while significantly increased root growth was observed in transgenic plants, when compared to control plants. Several independently transformed lines were propagated and evaluated for resistance to methyl viologen (MV), drought and salt stress. Visual assessment of transgenic and control lines exposed to MV (50 or 100 ,mol) confirmed that over-expression of APX minimized leaf damage. APX activity was nearly 80% higher in the leaves of transgenic plants in response to drought or salt stresses. Moreover, the transgenic tobacco also showed significantly improved drought resistance and salt tolerance at the vegetative stage. RNA blot analysis indicated that the PpAPX transcript level was very low under normal growing conditions in rd29Ap-PpAPX plants, but clearly increased under drought stress. Our results show that PpAPX does not play a significant role under normal growing conditions, but did ameliorate oxidative injury under abiotic stress. The Ad29 promoter should be used as an inducible promoter in transgenic works. [source] A role for protein kinase CK2 in plant development: evidence obtained using a dominant-negative mutantTHE PLANT JOURNAL, Issue 1 2008Jordi Moreno-Romero Summary Protein kinase CK2 is an evolutionary conserved Ser/Thr phosphotransferase composed of two distinct subunits, , (catalytic) and , (regulatory), that combine to form a tetrameric complex. Plant genomes contain multiple genes for each subunit, the expression of which gives rise to different active holoenzymes. In order to study the effects of loss of function of CK2 on plant development, we have undertaken a dominant-negative mutant approach. We generated an inactive catalytic subunit by site-directed mutagenesis of an essential lysine residue. The mutated open reading frame was cloned downstream of an inducible promoter, and stably transformed Arabidopsis thaliana plants and tobacco BY2 cells were isolated. Continuous expression of the CK2 kinase-inactive subunit did not prevent seed germination, but seedlings exhibited a strong phenotype, affecting chloroplast development, cotyledon expansion, and root and shoot growth. Prolonged induction of the transgene was lethal. Moreover, dark-germinated seedlings exhibited an apparent de-etiolated phenotype that was not caused by disruption of the light-signalling pathways. Short-term induction of the CK2 kinase-inactive subunit allowed plant survival, but root growth and lateral root formation were significantly affected. The expression pattern of CYCB1;1::GFP in the root meristems of mutant plants demonstrated an important decrease of mitotic activity, and expression of the CK2 kinase-inactive subunit in stably transformed BY2 cells provoked perturbation of the G1/S and G2 phases of the cell cycle. Our results are consistent with a model in which CK2 plays a key role in cell division and cell expansion, with compelling effects on Arabidopsis development. [source] Monitoring of Recombinant Protein Production Using Bioluminescence in a Semiautomated Fermentation ProcessBIOTECHNOLOGY PROGRESS, Issue 4 2003I. Trezzani On-line optimization of fermentation processes can be greatly aided by the availability of information on the physiological state of the cell. The goal of our "BioLux" research project was to design a recombinant cell capable of intracellular monitoring of product synthesis and to use it as part of an automated fermentation system. A recombinant plasmid was constructed containing an inducible promoter that controls the gene coding for a model protein and the genes necessary for bioluminescence. The cells were cultured in microfermenters equipped with an on-line turbidity sensor and a specially designed on-line light sensor capable of continuous measurement of bioluminescence. Initial studies were done under simple culture conditions, and a linear correlation between luminescence and protein production was obtained. Such specially designed recombinant bioluminescent cells can potentially be applied for model-based inference of intracellular product formation, as well as for optimization and control of recombinant fermentation processes. [source] Controlled and localized genetic manipulation in the brainJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 2 2006Rachel Aronoff Abstract Brain structure and function are determined in part through experience and in part through our inherited genes. A powerful approach for unravelling the balance between activity-dependent neuronal plasticity and genetic programs is to directly manipulate the genome. Such molecular genetic studies have been greatly aided by the remarkable progress of large-scale genome sequencing efforts. Sophisticated mouse genetic manipulations allow targeted point-mutations, deletions and additions to the mouse genome. These can be regulated through inducible promoters expressing in genetically specified neuronal cell types. However, despite significant progress it remains difficult to target specific brain regions through transgenesis alone. Recent work suggests that transduction vectors, like lentiviruses and adeno-associated viruses, may provide suitable additional tools for localized and controlled genetic manipulation. Furthermore, studies with such vectors may aid the development of human genetic therapies for brain diseases. [source] A new inducible adenoviral expression system that responds to inflammatory stimuli in vivoTHE JOURNAL OF GENE MEDICINE, Issue 12 2006Gang Cai Abstract Background Gene transfer using inducible promoters, which control expression of transgenic proteins in response to physiological conditions, may have significant advantages. In this study, we tried to achieve an inducible adenoviral expression system for physiologically responsive gene therapy of autoimmune or inflammatory diseases. Methods A luciferase reporter vector with a hybrid promoter containing the human IL-1, enhancer region (,3690 to , 2720) and the human CIITA promoter IV (,399 to + 2) was constructed. A replication-deficient adenovirus was engineered with luciferase controlled by the IL1,/CIITApIV promoter (Ad-IL1,/CIITApIV-Luc). The reporter vector or adenovirus was transfected to C57Bl/6 myeloid dendritic cells (DCs), RAW264.7, and Hep G2 to study the in vitro characteristics of this hybrid promoter. An inflammation model was prepared by injecting lipopolysaccharide (LPS) into Balb/c mice intraperitoneally (i.p.), and infected with Ad-IL1,/CIITApIV-Luc or Ad-CMV-Luc to study the in vivo characteristics of the IL1,/CIITApIV promoter. Results The IL1,/CIITApIV hybrid promoter has pronounced promoter activity, broad-range responsiveness to cytokines or LPS, and can be rechallenged after first induction. In the inflammation model, IL1,/CIITApIV could drive hepatic luciferase expression increasedly rapidly after LPS challenge and in a LPS dose-dependent manner. Conclusions Using the IL1,/CIITApIV hybrid promoter in gene transfer vectors may make it possible to produce transgenic proteins in vivo in direct relationship with the intensity and duration of an individual's status. By providing endogenously controlled production of transgenic proteins, this approach might limit the severity of autoimmune or inflammatory response without interfering with the beneficial components of host defense and immunity. Copyright © 2006 John Wiley & Sons, Ltd. [source] Regulation of transgene expressionACTA OPHTHALMOLOGICA, Issue 2009P KOCH Purpose Regulation of the transgene expression in the targeted cells is of course of major importance when using gene therapy. Actually, we have a huge range of possibilities to regulate gene expression. Methods There are two main classes of promoters: constitutive and inducible promoters. Amongst constitutive promoters, we have two sub-forms: non-tissue and tissue specific promoters. The lasts allows us to better target the tissue or cells in which we want to express our gene of interest. On the other hand, inducible promoters have been widely developed recently and allow us to obtain a regulated expression, depending on different factors. Very recently, disease specific inducible promoters emerged for a more precise regulation. Results We will together examine more precisely the different possibilities offered by gene regulation in Gene Therapy. Thereafter, we will more specifically describe usable promoters in ocular inflammation. Finally we will examine the effects of some inflammatory, disease specific, promoters. Conclusion Regulation of transgene expression is one of the fundaments of efficient gene transfer. Recent developments actually allow us to play within the targeted cell(s) to obtain an expression in specific conditions. [source] Gene transfer of disease regulated promoters during experimental autoimmune uveitisACTA OPHTHALMOLOGICA, Issue 2009V ELMALEH Purpose Adeno-associated virus (AAV) vectors have been successfully used to transfer immunosuppressive genes into the retina to prevent experimental uveitis development. Transgene expression is classically regulated by constitutive or tetracycline inducible promoters. It might be more advantageous that the control of transgene expression depends on the pathological process itself. Inflammation activates transcription factors acting on promoters containing short responsive sequences, responding, for example to nuclear factor kappa B (NF,B-RE). These responsive elements can be used to generate disease regulated promoters. Methods An AAV vector with the GFP gene under the control of a NF-kB-RE containing promoter will be injected subretinally in C57Bl6 mice. Autoimmune uveitis will be induced by adoptive transfer of IRBP specific lymphocytes. Animals will be sacrificed at different time points. GFP expression will be analysed by immunofluorescence. VCAM1, MHC II and CD45 will be analysed by immunofluorescence and used to monitor the level of retinal inflammation. Results One week after disease induction, GFP expression was found in eyes injected with this new vector. Milder GFP expression was also found in mice who did not received adoptive transfer. This background was increased a J14. Conclusion Our preliminary results suggest that disease driven GFP expression can be obtained by the use of AAV vectors containing disease regulated promoters. We still need some more times to improve our model. In the future, we plan to replace the GFP gene by an immunosuppressive gene and test if the system can be use to treat experimental uveitis. [source] | |||||||||||||