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Auswahl der wissenschaftlichen Literatur zum Thema „Plasmide eucaryote“
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Zeitschriftenartikel zum Thema "Plasmide eucaryote"
1
Vasudevachari, M. B., V. Natarajan und N. P. Salzman. „Cotransfection with adenovirus DNA enhances transcription from linear DNA containing eucaryotic promoters“. Molecular and Cellular Biology 7, Nr. 3 (März 1987): 1063–69. http://dx.doi.org/10.1128/mcb.7.3.1063-1069.1987.
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Linear DNAs, containing a copy of the adenovirus serotype 2 (Ad2) inverted terminal repeat sequence at each end, replicate in 293 cells when cotransfected with Ad2 DNA (Hay et al., J. Mol. Biol. 175:493-510, 1984). We have linked either the Ad2 IVa2 promoter (IVa2) or major late promoter (MLP) to the chloramphenicol acetyltransferase gene and inserted this DNA into such a plasmid (pARKR) between its two inverted terminal repeats. These recombinant plasmids were linearized and then used to transfect 293 cells in the presence or absence of Ad2 helper DNA. Synthesis of IVa2 and MLP RNAs, and production of chloramphenicol acetyltransferase was increased dramatically when the Ad2 DNA was included. However, unlike the patterns of temporal regulation which are seen during a cycle of virus replication when these genes are contained within the virion, there was no obvious difference in the timing of RNA synthesis from plasmid IVa2 or MLP after cotransfection. When linearized plasmids containing IVa2 and MLP sequences but lacking inverted terminal repeats at their ends (replication deficient plasmids) were used for transfection, an increase in RNA synthesis from IVa2 or MLP was also observed and similarly required cotransfection with Ad2 DNA. When HeLa cells, which do not constitutively express the adenovirus E1a gene, were cotransfected with linearized plasmids and adenovirus DNA that lacks the E1a region (H5dl312), a stimulation of transcription was also observed, although it was less than the level observed with wild-type DNA. The results of the present study demonstrate that an early gene product(s) besides E1a functions in trans to regulate transcription.
2
Vasudevachari, M. B., V. Natarajan und N. P. Salzman. „Cotransfection with adenovirus DNA enhances transcription from linear DNA containing eucaryotic promoters.“ Molecular and Cellular Biology 7, Nr. 3 (März 1987): 1063–69. http://dx.doi.org/10.1128/mcb.7.3.1063.
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Linear DNAs, containing a copy of the adenovirus serotype 2 (Ad2) inverted terminal repeat sequence at each end, replicate in 293 cells when cotransfected with Ad2 DNA (Hay et al., J. Mol. Biol. 175:493-510, 1984). We have linked either the Ad2 IVa2 promoter (IVa2) or major late promoter (MLP) to the chloramphenicol acetyltransferase gene and inserted this DNA into such a plasmid (pARKR) between its two inverted terminal repeats. These recombinant plasmids were linearized and then used to transfect 293 cells in the presence or absence of Ad2 helper DNA. Synthesis of IVa2 and MLP RNAs, and production of chloramphenicol acetyltransferase was increased dramatically when the Ad2 DNA was included. However, unlike the patterns of temporal regulation which are seen during a cycle of virus replication when these genes are contained within the virion, there was no obvious difference in the timing of RNA synthesis from plasmid IVa2 or MLP after cotransfection. When linearized plasmids containing IVa2 and MLP sequences but lacking inverted terminal repeats at their ends (replication deficient plasmids) were used for transfection, an increase in RNA synthesis from IVa2 or MLP was also observed and similarly required cotransfection with Ad2 DNA. When HeLa cells, which do not constitutively express the adenovirus E1a gene, were cotransfected with linearized plasmids and adenovirus DNA that lacks the E1a region (H5dl312), a stimulation of transcription was also observed, although it was less than the level observed with wild-type DNA. The results of the present study demonstrate that an early gene product(s) besides E1a functions in trans to regulate transcription.
3
Kaufman, R. J., M. V. Davies, V. K. Pathak und J. W. Hershey. „The phosphorylation state of eucaryotic initiation factor 2 alters translational efficiency of specific mRNAs“. Molecular and Cellular Biology 9, Nr. 3 (März 1989): 946–58. http://dx.doi.org/10.1128/mcb.9.3.946-958.1989.
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Phosphorylation of the alpha subunit of the eucaryotic translation initiation factor (eIF-2 alpha) by the double-stranded RNA-activated inhibitor (DAI) kinase correlates with inhibition of translation initiation. The importance of eIF-2 alpha phosphorylation in regulating translation was studied by expression of specific mutants of eIF-2 alpha in COS-1 cells. DNA transfection of certain plasmids could activate DAI kinase and result in poor translation of plasmid-derived mRNAs. In these cases, translation of the plasmid-derived mRNAs was improved by the presence of DAI kinase inhibitors or by the presence of a nonphosphorylatable mutant (serine to alanine) of eIF-2 alpha. The improved translation mediated by expression of the nonphosphorylatable eIF-2 alpha mutant was specific to plasmid-derived mRNA and did not affect global mRNA translation. Expression of a serine-to-aspartic acid mutant eIF-2 alpha, created to mimic the phosphorylated serine, inhibited translation of the mRNAs derived from the transfected plasmid. These results substantiate the hypothesis that DAI kinase activation reduces translation initiation through phosphorylation of eIF-2 alpha and reinforce the importance of phosphorylation of eIF-2 alpha as a way to control initiation of translation in intact cells.
4
Kaufman, R. J., M. V. Davies, V. K. Pathak und J. W. Hershey. „The phosphorylation state of eucaryotic initiation factor 2 alters translational efficiency of specific mRNAs.“ Molecular and Cellular Biology 9, Nr. 3 (März 1989): 946–58. http://dx.doi.org/10.1128/mcb.9.3.946.
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Phosphorylation of the alpha subunit of the eucaryotic translation initiation factor (eIF-2 alpha) by the double-stranded RNA-activated inhibitor (DAI) kinase correlates with inhibition of translation initiation. The importance of eIF-2 alpha phosphorylation in regulating translation was studied by expression of specific mutants of eIF-2 alpha in COS-1 cells. DNA transfection of certain plasmids could activate DAI kinase and result in poor translation of plasmid-derived mRNAs. In these cases, translation of the plasmid-derived mRNAs was improved by the presence of DAI kinase inhibitors or by the presence of a nonphosphorylatable mutant (serine to alanine) of eIF-2 alpha. The improved translation mediated by expression of the nonphosphorylatable eIF-2 alpha mutant was specific to plasmid-derived mRNA and did not affect global mRNA translation. Expression of a serine-to-aspartic acid mutant eIF-2 alpha, created to mimic the phosphorylated serine, inhibited translation of the mRNAs derived from the transfected plasmid. These results substantiate the hypothesis that DAI kinase activation reduces translation initiation through phosphorylation of eIF-2 alpha and reinforce the importance of phosphorylation of eIF-2 alpha as a way to control initiation of translation in intact cells.
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Alwine, J. C. „Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins“. Molecular and Cellular Biology 5, Nr. 5 (Mai 1985): 1034–42. http://dx.doi.org/10.1128/mcb.5.5.1034-1042.1985.
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The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed.
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Alwine, J. C. „Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins.“ Molecular and Cellular Biology 5, Nr. 5 (Mai 1985): 1034–42. http://dx.doi.org/10.1128/mcb.5.5.1034.
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The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed.
7
Chen, XM, SH Zhou, J. Fan, MJ Hu, SQ Wang und YS Yu. „Construction and Identification of an Antisense Glucose Transporter-1 Plasmid“. Journal of International Medical Research 36, Nr. 5 (Oktober 2008): 1001–7. http://dx.doi.org/10.1177/147323000803600517.
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Our aim was to construct a pcDNA3.1(+) eucaryotic expression system vector containing the antisense glucose transporter-1 ( Glut-1) gene. Total RNA was isolated from human Hep-2 laryngeal carcinoma cells, and the Glut-1 and antisense Glut-1 sequences were amplified by polymerase chain reaction. Expression plasmids containing the sense and antisense cDNA were constructed using the pcDNA3.1(+) vector. The resulting sense and antisense vectors, pcDNA3.1(+)-Glut-1 and pcDNA3.1(+)-antiGlut-1, respectively, were examined by restriction analysis and DNA sequencing. The pcDNA3.1(+)-antiGlut-1 was subsequently transfected into Hep-2 cells. Anti Glut-1 mRNA expression was detected, indicating the successful construction of an antisense Glut-1 plasmid capable of transfecting Hep-2 laryngeal carcinoma cells. These data provide a firm basis for additional studies using the plasmid pcDNA3.1(+)-antiGlut-1 to determine its therapeutic potential for the treatment of laryngeal carcinoma.
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Losos, Jan K., David H. Evans und Ann M. Verrinder Gibbins. „Targeted modification of the complete chicken lysozyme gene by poxvirus-mediated recombination“. Biochemistry and Cell Biology 83, Nr. 2 (01.04.2005): 230–38. http://dx.doi.org/10.1139/o05-025.
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We have developed a novel ex vivo system for the rapid one-step targeted modification of large eucaryotic DNA sequences. The highly recombinant environment resulting from infection of rabbit cornea cells with the Shope fibroma virus was exploited to mediate precise modifications of the complete chicken lysozyme gene domain (21.5 kb). Homologous recombination was designed to occur between target DNA (containing the complete lysozyme gene domain) maintained in a λ bacteriophage vector and modified targeting DNA maintained in a plasmid. The targeting plasmids were designed to transfer exogenous sequences (for example, β-galactosidase α-complement, green fluorescent protein, and hydrophobic tail coding sequences) to specific sites within the lysozyme gene domain. Cotransfection of the target phage and a targeting plasmid into Shope fibroma virus infected cells resulted in the poxvirus-mediated transfer of the modified sequences from plasmid to phage. Phage DNA (recombinant and nonrecombinant) was then harvested from the total cellular DNA by packaging into λ phage particles and correct recombinants were identified. Four different gene-targeting pairings were carried out, and from 3% to 11% of the recovered phages were recombinant. Using this poxvirus-mediated targeting system, four different regions of the chicken lysozyme gene domain have been modified precisely by our research group overall with a variety of inserts (6–971 bp), deletions (584–3000 bp), and replacements. We have never failed to obtain the desired recombinant. Poxvirus-mediated recombination thus constitutes a routine, rapid, and remarkably efficient genetic engineering system for the precise modification of large eucaryotic gene domains when compared with traditional practices.Key words: chicken lysozyme, gene targeting, homologous recombination, poxvirus, avian bioreactor.
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Price, Brian M., Adriane L. Liner, Sukjoon Park, Stephen H. Leppla, Alfred Mateczun und Darrell R. Galloway. „Protection against Anthrax Lethal Toxin Challenge by Genetic Immunization with a Plasmid Encoding the Lethal Factor Protein“. Infection and Immunity 69, Nr. 7 (01.07.2001): 4509–15. http://dx.doi.org/10.1128/iai.69.7.4509-4515.2001.
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ABSTRACT The ability of genetic vaccination to protect against a lethal challenge of anthrax toxin was evaluated. BALB/c mice were immunized via gene gun inoculation with eucaryotic expression vector plasmids encoding either a fragment of the protective antigen (PA) or a fragment of lethal factor (LF). Plasmid pCLF4 contains the N-terminal region (amino acids [aa] 10 to 254) of Bacillus anthracis LF cloned into the pCI expression plasmid. Plasmid pCPA contains a biologically active portion (aa 175 to 764) ofB. anthracis PA cloned into the pCI expression vector. One-micrometer-diameter gold particles were coated with plasmid pCLF4 or pCPA or a 1:1 mixture of both and injected into mice via gene gun (1 μg of plasmid DNA/injection) three times at 2-week intervals. Sera were collected and analyzed for antibody titer as well as antibody isotype. Significantly, titers of antibody to both PA and LF from mice immunized with the combination of pCPA and pCLF4 were four to five times greater than titers from mice immunized with either gene alone. Two weeks following the third and final plasmid DNA boost, all mice were challenged with 5 50% lethal doses of lethal toxin (PA plus LF) injected intravenously into the tail vein. All mice immunized with pCLF4, pCPA, or the combination of both survived the challenge, whereas all unimmunized mice did not survive. These results demonstrate that DNA-based immunization alone can provide protection against a lethal toxin challenge and that DNA immunization against the LF antigen alone provides complete protection.
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Chakrabarti, S., und M. M. Seidman. „Intramolecular recombination between transfected repeated sequences in mammalian cells is nonconservative“. Molecular and Cellular Biology 6, Nr. 7 (Juli 1986): 2520–26. http://dx.doi.org/10.1128/mcb.6.7.2520-2526.1986.
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When plasmids carrying a fragmented gene with segments present as direct repeats are introduced into mammalian cells, recombination or gene conversion between the repeated sequences can reconstruct the gene. Intramolecular recombination leads to the deletion of the intervening sequences and the loss of one copy of the repeat. This process is known to be stimulated by double-strand breaks. Two current models for recombination in eucaryotic cells propose that the reaction is initiated by double-strand breaks, but differ in their predictions as to the fate of the intervening sequences. One model suggests that these sequences are always lost, while the other indicates that the reaction will be conservative as a function of the position of the double-strand break. We have constructed a plasmid in which two overlapping portions of the simian virus 40 early region, which contains the origin and T-antigen gene, are present as direct repeats separated by sequences containing a plasmid with a simian virus 40 origin of replication. Recombination across the repeated segments could produce a plasmid with an origin of replication and/or a plasmid with a gene for a functional T-antigen which would drive the replication of both. Introduction of this construction into African green monkey kidney cells, without coinfection, establishes a condition in which the products of the recombination or gene conversion can be interpreted unambiguously. We find that the majority of the reconstruction reactions are nonconservative.
Dissertationen zum Thema "Plasmide eucaryote"
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Quintart, Anne. „Mise au point d'un test de réparation des cassures double-brin de l'ADN, application au déficit immunitaire T(-) B(-) caractérisé par un défaut de recombinaison V(D)J avec radiosensibilité accrue“. Paris 5, 2001. http://www.theses.fr/2001PA05P019.
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Girard, Fabien. „Tethering of molecular parasites on inactive chromatin in eukaryote nucleus“. Electronic Thesis or Diss., Sorbonne université, 2023. http://www.theses.fr/2023SORUS661.
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Les plasmides naturels sont courants chez les procaryotes, mais peu ont été documentés chez les eucaryotes. Le plasmide naturel 2µ présent dans la levure bourgeonnante Saccharomyces cerevisiae est l'un des mieux caractérisés. Cet élément génétique très stable coexiste avec son hôte depuis des millions d'années, ségrégeant efficacement à chaque division cellulaire par un mécanisme qui reste mal compris. En utilisant la ligature de proximité (Hi-C, Micro-C) pour cartographier les contacts entre le plasmide 2µ et les chromosomes de levure dans des dizaines de conditions biologiques différentes, nous avons constaté que le plasmide 2µ se fixe préférentiellement sur des régions à faible activité transcriptionnelle, correspondant souvent à de longs gènes inactifs. Les acteurs communs de la structure des chromosomes, tels que les membres des complexes de maintenance structurale des chromosomes (SMC), ne sont pas impliqués dans ces contacts qui dépendent plutôt d'un signal nucléosomique associé à une déplétion de l'ARN Pol II. Ces contacts sont stables tout au long du cycle cellulaire et peuvent être établis en quelques minutes. Cette stratégie peut aussi être trouvée dans d'autres types de molécules d'ADN et d'autres espèces que S. cerevisiae, comme le suggère le schéma de liaison du plasmide naturel le long des régions silencieuses des chromosomes de Dictyostelium discoideumNatural plasmids are common in prokaryotes but few have been documented in eukaryotes. The natural 2µ plasmid present in budding yeast Saccharomyces cerevisiae is one of the most well characterized. This highly stable genetic element coexists with its host for millions of years, efficiently segregating at each cell division through a mechanism that remains poorly understood. Using proximity ligation (Hi-C, MicroC) to map the contacts between the 2µ and yeast chromosomes under dozens of different biological conditions, we found that the plasmid tether preferentially on regions with low transcriptional activity, often corresponding to long inactive genes, throughout the cell cycle. Common players in chromosome structure such as members of the structural maintenance of chromosome complexes (SMC) are not involved in these contacts, and depend instead on a nucleosomal signal associated with a depletion of RNA Pol II. These contacts are highly stable, and can be established within minutes. Our data show that the plasmid segregates by binding to transcriptionally silent regions of the host chromosomes. This strategy may concern other types of DNA molecules and species beyond S. cerevisiae, as suggested by the binding pattern of the natural Ddp5 plasmid along Dictyostelium discoideum chromosomes’ silent regions
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Gatignol, Anne. „Expression de genes plasmidiques de resistance a la phleomycine chez les eucaryotes“. Toulouse 3, 1987. http://www.theses.fr/1987TOU30257.
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Baudry, Bernadette. „Séquences plasmidiques responsables de l'entrée de Shigella flexneri dans les cellules eucaryotes“. Paris 11, 1988. http://www.theses.fr/1988PA112035.
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Le pouvoir pathogène de Shigella flexneri est dû à la capacité qu'ont ces bactéries de pénétrer et de se multiplier dans les cellules épithéliales de la muqueuse colique humaine. Des études génétiques ont montré que plusieurs loci chromosomiques et un plasmide de 220 kilobases (kb) étaient nécessaires à la pathogénicité. Bien que les gènes chromosomiques soient indispensables à l'expression d’un phénotype complet d'invasion, il a été montré que le plasmide de virulence portait à lui seul toute l'information nécessaire à l'étape d'entrée dans les cellules HeLa. Dans le but d'identifier les gènes responsables de cette étape d'entrée de Shigella dans les cellules, nous avons cloné des fragments du plasmide de virulence d'une souche de S. Flexneri sérotype 5. Plusieurs plasmides recombinants contenant une séquence commune de 37 kb sont apparus capables de promouvoir l'entrée des bactéries dans les cellules. Afin de localiser les gènes de virulence sur cette séquence, l'un des plasmides recombinants a été mutagénisé à l'aide du transposon Tn. 5. Huit insertions, entraînant une inhibition ou une altération du processus d'entrée, ont défini cinq loci répartis sur une région de 20 kb. Des études préliminaires sur l'expression des protéines par les clones recombinants avaient montré que parmi les polypeptides exprimés, quatre étaient reconnus par les sérums de singes convalescents de shigellose. Ces quatre polypeptides constituent les antigènes protéiques majeurs qui provoquent une réponse humorale durant l'infection des hommes et des singes par toutes les espèces de Shigella. Parmi les huit mutants d’insertion ayant une capacité d'entrée altérée, trois présentaient des modifications de l'expression de ces polypeptides. C'est sur la région de 7 kb codant pour ces antigènes, a (78 kilodaltons, kD), b (62 kD), c (43 kD), et d (38 kD), et pour un polypeptide de 21 kD, non exprimé par un des mutants d'insertion, que nous nous sommes concentrés. Les gènes codant pour les quatre polypeptides ont été localisés. L'analyse des résultats a conduit à la proposition d'un modèle d'opéron avec une alternative au niveau de la protéine de 21 kD qui, soit ferait partie de l'opéron, soit en serait un activateur. LpaB et ipaC, les gènes correspondant aux deux antigènes majeurs b et c, ont été clonés et leur séquence nucléotidique déterminée. Le bas pourcentage en G+C et l'utilisation différente des codons par rapport à E. Coli suggèrent une origine non entérobactérienne des gènes de virulence. L'ensemble de ce travail a établi les bases génétiques et moléculaires qui permettront l'étude plus approfondie du processus d'entrée de Shigella dans les cellules eucaryotes.
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Debuchy, Robert. „Mise au point d'un systeme de transformation du champignon filamenteux podospora anserina et recherche de sequences susceptibles d'assurer l'autonomie de replication“. Paris 6, 1987. http://www.theses.fr/1987PA066103.
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Dupou, Laurence. „Contribution a l'etude de la dynamique et de la distribution laterale des lipides dans les membranes plasmiques de cellules eucaryotes“. Toulouse 3, 1987. http://www.theses.fr/1987TOU30043.
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Le, Guyader Laurent. „Utilisation de sondes pyréniques in vivo pour caractériser l'état de phase global de la membrane plasmique de cellules eucaryotes : application à la détection de la liaison d'agonistes au récepteur "delta" opioïde murin“. Toulouse 3, 2007. http://thesesups.ups-tlse.fr/53/.
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Le développement d'outils de criblage de nouvelles molécules capables d'induire une réponse cellulaire identique à celle d'un ligand (agoniste, antagoniste. . . ) est un enjeu important pour la recherche pharmaceutique. Les principales cibles utilisées aujourd'hui sont les récepteurs trans-membranaires couplés aux protéines G (RCPG) dont l'activation déclenche une cascade de réactions intracellulaires (signalisation). L'objectif de ce travail de thèse est la détection de l'initiation de la signalisation cellulaire par spectrométrie de fluorescence pour le récepteur ??opioïde murin (mDOR) qui induit un changement conformationnel responsable d'un épaississement de sa partie transmembranaire lors de la signalisation, au niveau de la membrane plasmique de cellules eucaryotes in vivo. Ce changement est supposé générer des modifications de phase lipidique de la membrane plasmique lors de cette interaction RCPG/agoniste, avec des redistributions locales de certains lipides comme le cholestérol, conduisant à la génération d'une phase liquide-ordonnée (lo). Pour cela deux nouvelles sondes fluorescentes membranaires de la famille des Pyrènecholestérol ont été synthétisées, le 3?-hydroxy-pregn-5-ène-21-(1-méthylpyrényl)-20-one 1 et le 3?-hydroxy-pregn-5-ène-21-(1-méthylpyrényl)-20-méthylidène 2. Dans un premier temps la comparaison du comportement de ces sondes vis-à-vis du cholestérol a été analysée dans différentes membranes modèles phospholipidiques. Les données de microcalorimétrie et de RMN-2H indiquent que ces composés induisent des perturbations des lipides comparables à celle du cholestérol. Les données d'absorption, combinées à la propriété hypochromique du chromophore pyrène ont permis de mettre en évidence un comportement auto-associatif du cholestérol dépendant du degré de saturation des chaînes acyles. De plus ces sondes sont plus idéalement miscibles dans les phases lo. Dans un deuxième temps la sensibilité du spectre d'émission de fluorescence à la polarité de l'environnement a été vérifiée et mise à profit dans des membranes modèles pour discriminer les membranes en phase ld de celles en phase lo (où la polarité du coeur de la membrane est plus faible). Enfin, l'incorporation de la sonde 2 dans la membrane plasmique de cellules vivantes CHO qui sur-expriment mDOR, a permit le suivi dans le temps, par spectrofluorimétrie de la perturbation de phase induite par un agonisteThe development of tools for the screening of new receptors’ agonists is a major issue for pharmaceutical research. Currently, the main targets used are the G-protein coupled receptor of the plasma membrane which the activation trigger the signaling pathways involved in many cell functions. The goal of this thesis is the detection, using a spectrofluorometry approach in vivo, of the signaling pathway ignition by the mouse delta opioid receptor (mDOR). DOR is assumed to generate a relocalisation of some lipids (cholesterol) while agonist binding leading to the formation of a liquid-ordered phase (lo). Thus two fluorescent probes have been synthesized, which the 3beta-hydroxy-pregn-5-ene-21-(1-methylpyrenyl)-20-methylidene. Data from calorimetry and RMN-2H studies show that this probe induces the same lipid membranes disturbance than cholesterol. Absorption data of the probe allowed the assessment of a self-association process of cholesterol in model membranes, which is as a function of the acyl-chains saturation degree of phospholipids. Then the polarity sensitive property of the probe has been used to distinguish between ld and lo phases in model membranes. Finally, we incorporated the probes in CHO cells over-expressing mDOR to monitor, by fluorescence spectroscopy, the mean phase state change of the plasma membrane triggered by a mDOR agonist. This is the first step of a new screening approach
Bücher zum Thema "Plasmide eucaryote"
1
Plasmids of Eukaryotes. Springer Verlag, 1986.
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