Articles de revues : « Eukaryotic plasmid »

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Jankowski, Jacek M., Eva Walczyk et Gordon H. Dixon. « Functional prokaryotic gene control signals within a eukaryotic rainbow trout protamine promoter ». Bioscience Reports 5, n^o 6 (1 juin 1985) : 453–61. http://dx.doi.org/10.1007/bf01116942.

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Following the construction of a series of pSV2-cat derived plasmids containing the chloramphenicol acetyltransferase (CAT) gene under the control of a eukaryotic trout protamine promoter, it was noted thatEscherichia coli, transformed with these plasmids, developed resistance to chloramphenicol (CM). This result suggested that the eukaryotic trout protamine promoter possessed significant prokaryotic promoter activity. Modification of the trout protamine promoter region by removing the region containing the eukaryotic Goldberg-Hogness box in the plasmid p525-cat increased the expression of the CAT gene almost to the wild-type level and conferred strong CM resistance. Sequence comparisons of the plasmid series indicate that prokaryotic promoter elements are present in the trout protamine promoter and that their similarity to the prokaryotic promoter consensus sequences and the distance between the two elements is more favourable in p525-cat, the plasmid which conlers the greatest CM resistance.

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Møller-Jensen, Jakob, et Kenn Gerdes. « Plasmid segregation : spatial awareness at the molecular level ». Journal of Cell Biology 179, n^o 5 (26 novembre 2007) : 813–15. http://dx.doi.org/10.1083/jcb.200710192.

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In bacteria, low-copy number plasmids ensure their stable inheritance by partition loci (par), which actively distribute plasmid replicates to each side of the cell division plane. Using time-lapse fluorescence microscopic tracking of segregating plasmid molecules, a new study provides novel insight into the workings of the par system from Escherichia coli plasmid R1. Despite its relative simplicity, the plasmid partition spindle shares characteristics with the mitotic machinery of eukaryotic cells.

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Soler, Nicolas, Marie Gaudin, Evelyne Marguet et Patrick Forterre. « Plasmids, viruses and virus-like membrane vesicles from Thermococcales ». Biochemical Society Transactions 39, n^o 1 (19 janvier 2011) : 36–44. http://dx.doi.org/10.1042/bst0390036.

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Several families of plasmids and viruses (PVs) have now been described in hyperthermophilic archaea of the order Thermococcales. One family of plasmids replicates by the rolling circle mechanism, whereas most other PVs probably replicate by the θ mode. PVs from Thermococcales encode novel families of DNA replication proteins that have only detectable homologues in other archaeal PVs. PVs from different families share a common gene pool and co-evolve with their hosts. Most Thermococcales also produce virus-like membrane vesicles similar to eukaryotic microparticles (ectosomes). Some membrane vesicles of Thermococcus nautilus harbour the plasmid pTN1, suggesting that vesicles can be involved in plasmid transfer between species.

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Vernis, Laurence, Marion Chasles, Philippe Pasero, Andrée Lepingle, Claude Gaillardin et Philippe Fournier. « Short DNA Fragments without Sequence Similarity Are Initiation Sites for Replication in the Chromosome of the YeastYarrowia lipolytica ». Molecular Biology of the Cell 10, n^o 3 (mars 1999) : 757–69. http://dx.doi.org/10.1091/mbc.10.3.757.

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We have previously shown that both a centromere (CEN) and a replication origin are necessary for plasmid maintenance in the yeastYarrowia lipolytica ( Vernis et al., 1997 ). Because of this requirement, only a small number of centromere-proximal replication origins have been isolated fromYarrowia. We used a CEN-based plasmid to obtain noncentromeric origins, and several new fragments, some unique and some repetitive sequences, were isolated. Some of them were analyzed by two-dimensional gel electrophoresis and correspond to actual sites of initiation (ORI) on the chromosome. We observed that a 125-bp fragment is sufficient for a functionalORI on plasmid, and that chromosomal origins moved to ectopic sites on the chromosome continue to act as initiation sites. These Yarrowia origins share an 8-bp motif, which is not essential for origin function on plasmids. The Yarrowiaorigins do not display any obvious common structural features, like bent DNA or DNA unwinding elements, generally present at or near eukaryotic replication origins. Y. lipolytica origins thus share features of those in the unicellular Saccharomyces cerevisiae and in multicellular eukaryotes: they are discrete and short genetic elements without sequence similarity.

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Capozzo, Alejandra V. E., Virginia Pistone Creydt, Graciela Dran, Gabriela Fernández, Sonia Gómez, Leticia V. Bentancor, Carolina Rubel, Cristina Ibarra, Martín Isturiz et Marina S. Palermo. « Development of DNA Vaccines against Hemolytic-Uremic Syndrome in a Murine Model ». Infection and Immunity 71, n^o 7 (juillet 2003) : 3971–78. http://dx.doi.org/10.1128/iai.71.7.3971-3978.2003.

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ABSTRACT Shiga toxin type 2 (Stx2) produced by Escherichia coli O:157H7 can cause hemolytic-uremic syndrome in children, a disease for which there is neither a vaccine nor an effective treatment. This toxin consists of an enzymatically active A subunit and a pentameric B subunit responsible for the toxin binding to host cells, and also found to be immunogenic in rabbits. In this study we developed eukaryotic plasmids expressing the B subunit gene of Stx2 (pStx2B) and the B subunit plus the gene coding for the A subunit with an active-site deletion (pStx2ΔA). Transfection of eukaryotic cells with these plasmids produced proteins of the expected molecular weight which reacted with specific monoclonal antibodies. Newborn and adult BALB/c mice immunized with two intramuscular injections of each plasmid, either alone or together with the same vector expressing the granulocyte and monocyte colony-stimulating factor (pGM-CSF), elicited a specific Th1-biased humoral response. The effect of pGM-CSF as an adjuvant plasmid was particularly notable in newborn mice and in pStx2B-vaccinated adult mice. Stx2-neutralizing activity, evaluated in vitro on VERO cell monolayers, correlated with in vivo protection. This is the first report using plasmids to induce a neutralizing humoral immune response against the Stx2.

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Liu, Binbo, Shengwu Liu, Xueju Qu et Junyan Liu. « Construction of a eukaryotic expression system for granulysin and its protective effect in mice infected with Mycobacterium tuberculosis ». Journal of Medical Microbiology 55, n^o 10 (1 octobre 2006) : 1389–93. http://dx.doi.org/10.1099/jmm.0.46706-0.

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A full-length cDNA of granulysin was inserted into the pcDNA3.1(−) vector to construct a eukaryotic expression plasmid for granulysin. The recombinant plasmids were injected intramuscularly into mice infected with Mycobacterium tuberculosis to evaluate the protective effect of granulysin. Granulysin significantly decreased the weight index (WI) of the spleen, reduced the numbers of viable bacteria in lung and spleen, and reduced the lesions of lung tissue in granulysin-rDNA-immunized mice compared with those of control group mice. In vitro, the serum of the recombinant-plasmid-immunized mice inhibited the viability of M. tuberculosis by the physical disruption of cell membranes. Therefore, granulysin has a therapeutic effect against M. tuberculosis.

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Xiao, Shan, Yanping Wang, Yuwen Ma, Jue Liu, Can’e Tang, Aiping Deng et Chunxiang Fang. « Dimethylation of eEF1A at Lysine 55 Plays a Key Role in the Regulation of eEF1A2 on Malignant Cell Functions of Acute Myeloid Leukemia ». Technology in Cancer Research & ; Treatment 19 (1 janvier 2020) : 153303382091429. http://dx.doi.org/10.1177/1533033820914295.

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Objective: This study aimed to explore whether eukaryotic translation elongation factor 1 alpha 2 affected cell proliferation, migration, and apoptosis via regulating the dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 in acute myeloid leukemia. Methods: The expressions of eukaryotic translation elongation factor 1 alpha 2 and dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 in acute myeloid leukemia cell lines and human normal bone marrow mononuclear cells (as control) were assessed. Control CRISPR-Cas9 lentivirus, eukaryotic translation elongation factor 1 alpha 2 knockout CRISPR-Cas9 lentivirus, vector plasmid, eukaryotic translation elongation factor 1 alpha 2 wild type overexpression plasmid, and eukaryotic translation elongation factor 1 alpha 2 with a K55R substitution overexpression plasmid were transfected into AML-193 and Kasumi-1 cells combined or alone, and were accordingly divided into 4 groups (Sgcontrol + vector group, SgeEF1A2 + vector group, SgeEF1A2 + eEF1A2WT group, and SgeEFIA2 + eEF1A2K55R group). Results: Eukaryotic translation elongation factor 1 alpha 2 and dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 expressions were higher in AML-193, Kasumi-1, and KG-1 cell lines compared to the control. In AML-193 and Kasumi-1 cells, the knockout and compensated experiments revealed that eukaryotic translation elongation factor 1 alpha 2 promoted cell proliferation and migration but repressed apoptosis. Additionally, the knockout of eukaryotic translation elongation factor 1 alpha 2 decreased dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 expression, meanwhile, eukaryotic translation elongation factor 1 alpha 2 wild type overexpression enhanced while eukaryotic translation elongation factor 1 alpha 2 with a K55R substitution overexpression did not influence the dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 expression. Furthermore, eukaryotic translation elongation factor 1 alpha 2 wild type overexpression promoted cell proliferation, enhanced migration, and decreased apoptosis, but eukaryotic translation elongation factor 1 alpha 2 with a K55R substitution overexpression did not influence these cellular functions in AML-193 and Kasumi-1 cells, suggesting the implication of dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 in eukaryotic translation elongation factor 1 alpha 2 mediated oncogenesis of acute myeloid leukemia. Conclusion: Eukaryotic translation elongation factor 1 alpha 2 and its dimethylated product may serve as therapeutic targets, and these findings may provide support for exploring novel strategies in acute myeloid leukemia treatment.

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Ma, Chien-Hui, Deepanshu Kumar, Makkuni Jayaram, Santanu K. Ghosh et Vishwanath R. Iyer. « The selfish yeast plasmid exploits a SWI/SNF-type chromatin remodeling complex for hitchhiking on chromosomes and ensuring high-fidelity propagation ». PLOS Genetics 19, n^o 10 (9 octobre 2023) : e1010986. http://dx.doi.org/10.1371/journal.pgen.1010986.

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Extra-chromosomal selfish DNA elements can evade the risk of being lost at every generation by behaving as chromosome appendages, thereby ensuring high fidelity segregation and stable persistence in host cell populations. The yeast 2-micron plasmid and episomes of the mammalian gammaherpes and papilloma viruses that tether to chromosomes and segregate by hitchhiking on them exemplify this strategy. We document for the first time the utilization of a SWI/SNF-type chromatin remodeling complex as a conduit for chromosome association by a selfish element. One principal mechanism for chromosome tethering by the 2-micron plasmid is the bridging interaction of the plasmid partitioning proteins (Rep1 and Rep2) with the yeast RSC2 complex and the plasmid partitioning locus STB. We substantiate this model by multiple lines of evidence derived from genomics, cell biology and interaction analyses. We describe a Rep-STB bypass system in which a plasmid engineered to non-covalently associate with the RSC complex mimics segregation by chromosome hitchhiking. Given the ubiquitous prevalence of SWI/SNF family chromatin remodeling complexes among eukaryotes, it is likely that the 2-micron plasmid paradigm or analogous ones will be encountered among other eukaryotic selfish elements.

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Luo, Ben-yan, Xiang-ming Chen, Min Tang, Feng Chen et Zhi Chen. « Construction of a eukaryotic expression plasmid of Humanin ». Journal of Zhejiang University SCIENCE 6B, n^o 1 (janvier 2005) : 11–13. http://dx.doi.org/10.1631/jzus.2005.b0011.

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Bao, G. Y., K. Y. Lu, S. F. Cui et L. Xu. « DKK1 eukaryotic expression plasmid and expression product identification ». Genetics and Molecular Research 14, n^o 2 (2015) : 6312–18. http://dx.doi.org/10.4238/2015.june.11.5.

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WARDAL, EWA, EWA SADOWY et WALERIA HRYNIEWICZ. « Complex Nature of Enterococcal Pheromone-Responsive Plasmids ». Polish Journal of Microbiology 59, n^o 2 (2010) : 79–87. http://dx.doi.org/10.33073/pjm-2010-012.

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Pheromone-responsive plasmids constitute a unique group of approximately 20 plasmids identified, as yet, only among enterococcal species. Several of their representatives, e.g. pAD1, pCF10, pPD1 and pAM373 have been extensively studied. These plasmids possess a sophisticated conjugation mechanism based on response to sex pheromones--small peptides produced by plasmid-free recipient cells. Detailed analysis of regulation and function of the pheromone response process revealed its great complexity and dual role--in plasmid conjugation and modulation of enterococcal virulence. Among other functional modules identified in pheromone plasmids, the stabilization/partition systems play a crucial role in stable maintenance of the plasmid molecule in host bacteria. Among them, the par locus of pAD1 is one of the exceptional RNA addiction systems. Pheromone-responsive plasmids contribute also to enterococcal phenotype being an important vehicle of antibiotic resistance in this genus. Both types of acquired vancomycin resistance determinants, vanA and vanB, as well many other resistant phenotypes, were found to be located on these plasmids. They also encode two basic agents of enterococcal virulence, i.e. aggregation substance (AS) and cytolysin. AS participates in mating-pair formation during conjugation but can also facilitate the adherence ofenterococci to human tissues during infection. The second protein, cytolysin, displays hemolytic activity and helps to invade eukaryotic cells. There are still many aspects of the nature of pheromone plasmids that remain unclear and more detailed studies are needed to understand their uniqueness and complexity.

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Block, Gregory J., Christopher H. Eskiw, Graham Dellaire et David P. Bazett-Jones. « Transcriptional Regulation Is Affected by Subnuclear Targeting of Reporter Plasmids to PML Nuclear Bodies ». Molecular and Cellular Biology 26, n^o 23 (11 septembre 2006) : 8814–25. http://dx.doi.org/10.1128/mcb.00636-06.

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ABSTRACT Whereas the PML protein has been reported to have both transcriptional coactivator and corepressor potential, the contribution of the PML nuclear body (PML NB) itself to transcriptional regulation is not well understood. Here we demonstrate that plasmid DNA artificially tethered to PML or the PML NB-targeting domain of Sp100 is preferentially localized to PML NBs. Using the tethering technique, we targeted a simian virus 40 promoter-driven luciferase reporter plasmid to PML NBs, resulting in the repression of the transgene transcriptional activity. Conversely, the tethering of a cytomegalovirus promoter-containing reporter plasmid resulted in activation. Targeting a minimal eukaryotic promoter did not affect its activity. The expression of targeted promoters could be modulated by altering the cellular concentration of PML NB components, including Sp100 and isoforms of the PML protein. Finally, we demonstrate that ICP0, the promiscuous herpes simplex virus transactivator, increases the level of transcriptional activation of plasmid DNA tethered to the PML NB. We conclude that when PML NB components are artificially tethered to reporter plasmids, the PML NB contributes to the regulation of the tethered DNA in a promoter-dependent manner. Our findings demonstrate that transient transcription assays are sensitive to the subnuclear localization of the transgene plasmid.

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Pradella, Silke, Martin Allgaier, Christa Hoch, Orsola PÃ¯Â¿Â½uker, Erko Stackebrandt et Irene Wagner-DÃ¯Â¿Â½bler. « Genome Organization and Localization of the pufLM Genes of the Photosynthesis Reaction Center in Phylogenetically Diverse Marine Alphaproteobacteria ». Applied and Environmental Microbiology 70, n^o 6 (juin 2004) : 3360–69. http://dx.doi.org/10.1128/aem.70.6.3360-3369.2004.

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ABSTRACT Genome organization, plasmid content and localization of the pufLM genes of the photosynthesis reaction center were studied by pulsed-field gel electrophoresis (PFGE) in marine phototrophic Alphaproteobacteria. Both anaerobic phototrophs (Rhodobacter veldkampii and Rhodobacter sphaeroides) and strictly aerobic anoxygenic phototrophs from the Roseobacter-Sulfitobacter-Silicibacter clade (Roseivivax halodurans, Roseobacter litoralis, Staleya guttiformis, Roseovarius tolerans, and five new strains isolated from dinoflagellate cultures) were investigated. The complete genome size was estimated for R. litoralis DSM6996T to be 4,704 kb, including three linear plasmids. All strains contained extrachromosomal elements of various conformations (linear or circular) and lengths (between 4.35 and 368 kb). In strain DFL-12, a member of a putative new genus isolated from a culture of the toxic dinoflagellate Prorocentrum lima, seven linear plasmids were found, together comprising 860 kb of genetic information. Hybridization with probes against the pufLM genes of the photosynthesis gene cluster after Southern transfer of the genomic DNAs showed these genes to be located on a linear plasmid of 91 kb in R. litoralis and on a linear plasmid of 120 kb in S. guttiformis, theoretically allowing their horizontal transfer. In all other strains, the pufLM genes were detected on the bacterial chromosome. The large number and significant size of the linear plasmids found especially in isolates from dinoflagellates might account for the metabolic versatility and presumed symbiotic association with eukaryotic hosts in these bacteria.

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Huang, Zengwen, Juan Zhang, WuReliHazi Hazihan, Zhengyun Cai, Guosheng Xin, Xiaofang Feng et Yaling Gu. « Cloning expression and immunogenicity analysis of inhibin gene in Ye Mule Aries sheep ». PeerJ 7 (25 septembre 2019) : e7761. http://dx.doi.org/10.7717/peerj.7761.

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Background Ye Mule Aries sheep is one of the most important sheep breeds in Xinjiang, China. This breed is well adapted to harsh environmental conditions and displays strong disease resistance, fast growth, and high cold tolerance. To analyze the clonal expression and immunogenicity of the Ye Mule Aries sheep inhibin gene, total RNA was extracted from sheep ovarian tissue and used as a template to generate a eukaryotic expression vector and study inhibin immunogenicity. Methods Primers were designed to amplify the inhibin A gene via polymerase chain reaction and the amplified product was cloned between the ScalI and EcoRI restriction sites of the expression vector pEGFP-N1 to construct a recombinant plasmid, pEGFP-INHα. Following the validation of successful cloning, the pEGFP-INHα plasmid was transfected into BHK cells to verify expression in eukaryotes and subsequently utilized as an antigen in rabbits. Rabbits were tested for anti-inhibin antibodies and serum follicle-stimulating hormone (FSH) concentrations. Results The analysis of the INHα gene sequence revealed that INHα is 1109 bp long and is translated to an approximately 40 KDa protein. Bioinformatics approach indicated that the INHα gene is highly conserved between organisms. Immunization with the eukaryotic expression vector, pEGFP-INHα, which expresses the INHα gene elicited immune response and generatigeneration on of anti-INHα antibody. The antibody had a significant regulatory effect on the serum concentration of FSH in rabbits and led to higher levels of FSH, indicating increased ovary function. Conclusions The present work resulted in a successful construction of eukaryotic expression plasmid pEGFP-INHα and verified the immunogenicity of this highly conserved protein. Further, the expression of pEGFP-INHα was shown to have a significant impact on the secretion of FSH, indicating a potential regulatory role in ovarian function. In conclusion, our current findings can serve as a working model for studying the effect of INHα on the breeding performance of Ye Mule Aries sheep, providing a novel strategy to improve their reproduction rates.

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Shanmugam, Ananth, Meng-Jiao Shi, Lauren Yauch, Janet Stavnezer et Amy L. Kenter. « Evidence for Class-Specific Factors in Immunoglobulin Isotype Switching ». Journal of Experimental Medicine 191, n^o 8 (17 avril 2000) : 1365–80. http://dx.doi.org/10.1084/jem.191.8.1365.

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Immunoglobulin class switch recombination (SR) occurs by a B cell–specific, intrachromosomal deletional process between switch regions. We have developed a plasmid-based transient transfection assay for SR to test for the presence of transacting switch activities. The plasmids are novel in that they lack a eukaryotic origin of DNA replication. The recombination activity of these switch substrates is restricted to a subset of B cell lines that support isotype switching on their endogenous loci and to mitogen-activated normal splenic B cells. The factors required for extrachromosomal plasmid recombination are constitutively expressed in proliferating splenic B cells and in B cell lines capable of inducibly undergoing immunoglobulin SR on their chromosomal genes. These studies suggest that mitogens that induce switching on the chromosome induce accessibility rather than switch recombinase activity. Finally, we provide evidence for two distinct switching activities which independently mediate μ→α and μ→γ3 SR.

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Smith, Gregory A., et Lynn W. Enquist. « Construction and Transposon Mutagenesis inEscherichia coli of a Full-Length Infectious Clone of Pseudorabies Virus, an Alphaherpesvirus ». Journal of Virology 73, n^o 8 (1 août 1999) : 6405–14. http://dx.doi.org/10.1128/jvi.73.8.6405-6414.1999.

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ABSTRACT A full-length clone of the 142-kb pseudorabies virus (PRV) genome was constructed as a stable F plasmid in Escherichia coli. The clone, pBecker1, was colinear with PRV-Becker genomic DNA, lacking detectable rearrangements, deletions, or inversions. The transfection of pBecker1 into susceptible eukaryotic cells resulted in productive viral infection. Virus isolated following transfection was indistinguishable from wild-type virus in a rodent model of infection and spread to retinorecipient regions of the brain following inoculation in the vitreous body of the eye. Mutagenesis of pBecker1 inE. coli with a mini-Tn5-derived transposon enabled the rapid isolation of insertion mutants, identification of essential viral genes, and simplified construction of viral revertants. The serial passage of a viral insertion mutant demonstrated the transposon insertion to be stable. However, the F-plasmid insertion present in the viral gG locus was found to undergo a spontaneous deletion following transfection into eukaryotic cells. The implications of F-plasmid insertion into the viral genome with regard to phenotype and genomic stability are discussed.

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Monteiro, Gabriel A., et Sofia O. D. Duarte. « The Effect of Recombinant Protein Production in Lactococcus lactis Transcriptome and Proteome ». Microorganisms 10, n^o 2 (25 janvier 2022) : 267. http://dx.doi.org/10.3390/microorganisms10020267.

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Lactococcus lactis is a food-grade, and generally recognized as safe, bacterium, which making it ideal for producing plasmid DNA (pDNA) or recombinant proteins for industrial or pharmaceutical applications. The present paper reviews the major findings from L. lactis transcriptome and proteome studies, with an overexpression of native or recombinant proteins. These studies should provide important insights on how to engineer the plasmid vectors and/or the strains in order to achieve high pDNA or recombinant proteins yields, with high quality standards. L. lactis harboring high copy numbers of plasmids for DNA vaccines production showed altered proteome profiles, when compared with a smaller copy number plasmid. For live mucosal vaccination applications, the cell-wall anchored antigens had shown more promising results, when compared with intracellular or secreted antigens. However, previous transcriptome and proteome studies demonstrated that engineering L. lactis to express membrane proteins, mainly with a eukaryotic background, increases the overall cellular burden. Genome engineering strategies could be used to knockout or overexpress the pinpointed genes, so as to increase the profitability of the process. Studies about the effect of protein overexpression on Escherichia coli and Bacillus subtillis transcriptome and proteome are also included.

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Futcher, B., E. Reid et D. A. Hickey. « Maintenance of the 2 micron circle plasmid of Saccharomyces cerevisiae by sexual transmission : an example of a selfish DNA. » Genetics 118, n^o 3 (1 mars 1988) : 411–15. http://dx.doi.org/10.1093/genetics/118.3.411.

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Abstract Many eukaryotic mobile elements have been identified, but few have any obvious function. This has led to the proposal that many such elements may be parasitic DNA. We have used the 2 micron circle plasmid of Saccharomyces cerevisiae as a model system to investigate the maintenance of a cryptic genetic element. We find that under certain conditions this plasmid can spread through experimental populations despite demonstrable selection against it. This spread is dependent upon outbreeding, suggesting that cell to cell transmission of the plasmid during the yeast sexual cycle can counterbalance selection, and maintain the plasmid in populations. This result provides experimental support for the idea that some mobile elements may be parasitic DNA.

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Krupovic, Mart, et Eugene V. Koonin. « Polintons : a hotbed of eukaryotic virus, transposon and plasmid evolution ». Nature Reviews Microbiology 13, n^o 2 (22 décembre 2014) : 105–15. http://dx.doi.org/10.1038/nrmicro3389.

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Chen, Feng, Sheng-Song He, Rong-Yuan Qiu, Ran Pang, Juan-Juan Xu et Ji-Hua Dong. « Construction and expression of mouse TRAF6-shRNA eukaryotic expressing plasmid ». World Chinese Journal of Digestology 17, n^o 14 (2009) : 1406. http://dx.doi.org/10.11569/wcjd.v17.i14.1406.

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Weber, Martin, Michasia Welzeck, Kathrin Möller et Joechin Schorr. « Lipopolysaccharide contamination of plasmid DNA reduces eukaryotic cell transfection efficiency ». European Journal of Pharmaceutical Sciences 4 (septembre 1996) : S193. http://dx.doi.org/10.1016/s0928-0987(97)86590-6.

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Kumar, Priti, Arvindhan Nagarajan et Pradeep D. Uchil. « Calcium Phosphate-Mediated Transfection of Eukaryotic Cells with Plasmid DNAs ». Cold Spring Harbor Protocols 2019, n^o 10 (octobre 2019) : pdb.prot095430. http://dx.doi.org/10.1101/pdb.prot095430.

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Sambrook, Joseph, et David W. Russell. « Calcium-phosphate-mediated Transfection of Eukaryotic Cells with Plasmid DNAs ». Cold Spring Harbor Protocols 2006, n^o 1 (juin 2006) : pdb.prot3871. http://dx.doi.org/10.1101/pdb.prot3871.

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Morozova, Olga V., Tatyana G. Maksimova et Elena V. Kostenko. « EBV-Based Plasmid DNA Rearrangements after Transfection of Eukaryotic Cells ». Plasmid 43, n^o 3 (mai 2000) : 185–89. http://dx.doi.org/10.1006/plas.1999.1449.

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Mello, Francisco C. A., Nora Martel, Selma A. Gomes et Natalia M. Araujo. « Expression of Hepatitis B Virus Surface Antigen Containing Y100C Variant Frequently Detected in Occult HBV Infection ». Hepatitis Research and Treatment 2011 (6 février 2011) : 1–4. http://dx.doi.org/10.1155/2011/695859.

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Small hepatitis B virus surface protein (S-HBsAg) variant Y100C has been associated with HBsAg-negative phenotype. To determine whether Y100C substitution yields impaired HBsAg or small amounts of HBsAg that may reduce HBsAg detection by commercial anti-HBsAg antibodies, two eukaryotic expression plasmids, one containing a wild-type S and the other an S gene from a Y100C variant, were constructed and their levels of HBsAg compared by ELISA after transfection of HuH7 cells. Unexpectedly, the extracellular HBsAg levels detected with Y100C plasmid were higher than those observed with the wild-type plasmid, but without statistical significance. We concluded that the Y100C substitution alone did not play a role in reducing HBsAg amounts or HBsAg affinity by commercial ELISA assay. Further studies on in vitro replication fitness with the complete genome of HBV isolates displaying or not Y100C substitution may elucidate whether this mutation affects HBV replication and consequently HBsAg production.

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Soltysiak, Maximillian P. M., Rebecca S. Meaney, Samir Hamadache, Preetam Janakirama, David R. Edgell et Bogumil J. Karas. « Trans-Kingdom Conjugation within Solid Media from Escherichia coli to Saccharomyces cerevisiae ». International Journal of Molecular Sciences 20, n^o 20 (21 octobre 2019) : 5212. http://dx.doi.org/10.3390/ijms20205212.

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Conjugation is a bacterial mechanism for DNA transfer from a donor cell to a wide range of recipients, including both prokaryotic and eukaryotic cells. In contrast to conventional DNA delivery techniques, such as electroporation and chemical transformation, conjugation eliminates the need for DNA extraction, thereby preventing DNA damage during isolation. While most established conjugation protocols allow for DNA transfer in liquid media or on a solid surface, we developed a procedure for conjugation within solid media. Such a protocol may expand conjugation as a tool for DNA transfer to species that require semi-solid or solid media for growth. Conjugation within solid media could also provide a more stable microenvironment in which the conjugative pilus can establish and maintain contact with recipient cells for the successful delivery of plasmid DNA. Furthermore, transfer in solid media may enhance the ability to transfer plasmids and chromosomes greater than 100 kbp. Using our optimized method, plasmids of varying sizes were tested for transfer from Escherichia coli to Saccharomyces cerevisiae. We demonstrated that there was no significant change in conjugation frequency when plasmid size increased from 56.5 to 138.6 kbp in length. Finally, we established an efficient PCR-based synthesis protocol to generate custom conjugative plasmids.

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Carballido-López, Rut. « The Bacterial Actin-Like Cytoskeleton ». Microbiology and Molecular Biology Reviews 70, n^o 4 (décembre 2006) : 888–909. http://dx.doi.org/10.1128/mmbr.00014-06.

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SUMMARY Recent advances have shown conclusively that bacterial cells possess distant but true homologues of actin (MreB, ParM, and the recently uncovered MamK protein). Despite weak amino acid sequence similarity, MreB and ParM exhibit high structural homology to actin. Just like F-actin in eukaryotes, MreB and ParM assemble into highly dynamic filamentous structures in vivo and in vitro. MreB-like proteins are essential for cell viability and have been implicated in major cellular processes, including cell morphogenesis, chromosome segregation, and cell polarity. ParM (a plasmid-encoded actin homologue) is responsible for driving plasmid-DNA partitioning. The dynamic prokaryotic actin-like cytoskeleton is thought to serve as a central organizer for the targeting and accurate positioning of proteins and nucleoprotein complexes, thereby (and by analogy to the eukaryotic cytoskeleton) spatially and temporally controlling macromolecular trafficking in bacterial cells. In this paper, the general properties and known functions of the actin orthologues in bacteria are reviewed.

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Jamieson-Lane, Alastair D., et Bernd Blasius. « The gossip paradox : Why do bacteria share genes ? » Mathematical Biosciences and Engineering 19, n^o 6 (2022) : 5482–508. http://dx.doi.org/10.3934/mbe.2022257.

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<abstract><p>Bacteria, in contrast to eukaryotic cells, contain two types of genes: chromosomal genes that are fixed to the cell, and plasmids, smaller loops of DNA capable of being passed from one cell to another. The sharing of plasmid genes between individual bacteria and between bacterial lineages has contributed vastly to bacterial evolution, allowing specialized traits to 'jump ship' between one lineage or species and the next. The benefits of this generosity from the point of view of both recipient cell and plasmid are generally understood: plasmids receive new hosts and ride out selective sweeps across the population, recipient cells gain new traits (such as antibiotic resistance). Explaining this behavior from the point of view of donor cells is substantially more difficult. Donor cells pay a fitness cost in order to share plasmids, and run the risk of sharing advantageous genes with their competition and rendering their own lineage redundant, while seemingly receiving no benefit in return. Using both compartment based models and agent based simulations we demonstrate that 'secretive' genes which restrict horizontal gene transfer are favored over a wide range of models and parameter values, even when sharing carries no direct cost. 'Generous' chromosomal genes which are more permissive of plasmid transfer are found to have neutral fitness at best, and are generally disfavored by selection. Our findings lead to a peculiar paradox: given the obvious benefits of keeping secrets, why do bacteria share information so freely?</p></abstract>

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Jiang, Shimin, Akihiro Narita, David Popp, Umesh Ghoshdastider, Lin Jie Lee, Ramanujam Srinivasan, Mohan K. Balasubramanian et al. « Novel actin filaments fromBacillus thuringiensisform nanotubules for plasmid DNA segregation ». Proceedings of the National Academy of Sciences 113, n^o 9 (12 février 2016) : E1200—E1205. http://dx.doi.org/10.1073/pnas.1600129113.

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Here we report the discovery of a bacterial DNA-segregating actin-like protein (BtParM) fromBacillus thuringiensis, which forms novel antiparallel, two-stranded, supercoiled, nonpolar helical filaments, as determined by electron microscopy. TheBtParM filament features of supercoiling and forming antiparallel double-strands are unique within the actin fold superfamily, and entirely different to the straight, double-stranded, polar helical filaments of all other known ParMs and of eukaryotic F-actin. TheBtParM polymers show dynamic assembly and subsequent disassembly in the presence of ATP.BtParR, the DNA-BtParM linking protein, stimulated ATP hydrolysis/phosphate release byBtParM and paired two supercoiledBtParM filaments to form a cylinder, comprised of four strands with inner and outer diameters of 57 Å and 145 Å, respectively. Thus, in this prokaryote, the actin fold has evolved to produce a filament system with comparable features to the eukaryotic chromosome-segregating microtubule.

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Ayares, David, James Spencer, Faina Schwartz, Brian Morse et Raju Kucherlapati. « HOMOLOGOUS RECOMBINATION BETWEEN AUTONOMOUSLY REPLICATING PLASMIDS IN MAMMALIAN CELLS ». Genetics 111, n^o 2 (1 octobre 1985) : 375–88. http://dx.doi.org/10.1093/genetics/111.2.375.

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ABSTRACT The ability of autonomously replicating plasmids to recombine in mammalian cells was investigated. Two deletion plasmids of the eukaryotic-prokaryotic shuttle vector pSV2neo were cotransfected into transformed monkey COS cells. Examination of the low molecular weight DNA isolated after 48 hr of incubation revealed that recombination between the plasmids had occurred. The DNA was also used to transform recA- E. coli. Yield of neo R colonies signified homologous recombination. Examination of the plasmid DNA from these colonies confirmed this view. Double-strand breaks in one or both of the input plasmids at the sites of deletion resulted in an enhancement of recombination frequency. The recombination process yielded monomeric and dimeric molecules. Examination of these molecules revealed that reciprocal recombination as well as gene conversion events were involved in the generation of plasmids bearing an intact neo gene. The COS cell system we describe is analogous to study of bacteriophage recombination and yeast random-spore analysis.

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Davis, MG, et ES Huang. « Transfer and Expression of Plasmids Containing Human Cytomegalovirus Immediate‐Early Gene 1 Promoter‐Enhancer Sequences in Eukaryotic and Prokaryotic Cells ». Biotechnology and Applied Biochemistry 10, n^o 1 (février 1988) : 6–12. http://dx.doi.org/10.1111/j.1470-8744.1988.tb00001.x.

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The human cytomegalovirus immediate‐early gene region 1 promoter‐enhancer is active in bacteria and in many mammalian cells. Recombinant plasmids containing portions of this DNA can be used to promote the expression of foreign proteins in many cells. In this communication, we report the optimal conditions for transfer of plasmid DNA to cells by electroporation and the transient expression assays which document the activity of different promoter constructions. The observed activity of the human cytomegalovirus promoter is more than 100‐fold higher than the activity of the early promoter of SV40.

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Zhang, Xichen, Michael W. Epperly, Mark A. Kay, Zhi-Ying Chen, Tracy Smith, Darcy Franicola, Benjamin Greenberger, Paavani Komanduri et Joel S. Greenberger. « Minicircle Plasmid Containing the Human Manganese Superoxide Dismutase (MnSOD) Transgene Confers Radioprotection to Hematopoietic Progenitor Cell Line 32Dcl3. » Blood 110, n^o 11 (16 novembre 2007) : 5138. http://dx.doi.org/10.1182/blood.v110.11.5138.5138.

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Abstract Manganese superoxide dismutase plasmid/liposomes (MnSOD-PL) delivered by intratracheal, intraesophageal, or intraoral routes in rodent models has been demonstrated to confer organ specific ionizing irradiation protection. In addition intravenous injections of MnSOD-PL protect mice from whole body irradiation. Currently a seven week phase I/II clinical trial is in progress in lung cancer patients consisting of twice weekly swallowed MnSOD-PL for protection of the esophagus from chemoradiotherapy damage. To prepare for a potential clinical trial of systemic MnSOD-PL for radioprotection in humans, plasmid bacterial sequences were removed to diminish the immune response. The human MnSOD transgene attached to a CMV promoter and a poly A tail was inserted in the site between Spe I and Xho I into a eukaryotic expression cassette located in the p2ØC31 plasmid. The plasmid contains an endonuclease I-SceI gene which can be cleaved resulting in the formation of two minicircle plasmids. The smaller minicircle contains the eukaryotic expression cassette but no bacterial sequences while the larger minicircle plasmid contains the plasmid bacterial backbone. The minicircle MnSOD was purified and then co-transfected into 32Dcl3 murine hematopoietic progenitor cells with a plasmid containing the neo gene. Cells were selected in G418 (50μg/ml G418) and cloned by limiting dilution into 96 well plates. The clones were expanded and analyzed by PCR for the presence of the human MnSOD transgene using primers specific for the human MnSOD. One clone was chosen and the MnSOD biochemical activity was determined. The 32Dcl3 cells had a specific MnSOD activity of 2.7 ± 0.1 U/mg protein compared to 5.8 ± 0.5 U/mg protein for the 32D-mc-MnSOD clone (p=0.0039). To determine if the MnSOD transgene in the minicircle DNA retained radioprotective capacity 32D-mc-MnSOD, a clone transfected with a pRK5 plasmid containing the human MnSOD transgene (2C6), and parent 32Dcl3 cells were irradiated to doses of 0–8 Gy then grown at 37° C for 7 days at which time colonies of greater than 50 cells were counted. The data was analyzed by linear quadratic and single-hit, multi-target models. The 32D-mc-MnSOD cells were more radioresistant than 32Dcl3 cells as demonstrated by an increased shoulder on the irradiation survival curve (n = 4.8 ± 0.2 compared to 1.5 ± 0.5, respectively, p = 0.0078). In contrast, there was no significant reduction in the shoulder of the survival curve comparing 32D-mc-MnSOD and 2C6 (n = 4.8 ± 0.2 and 4.6 ± 0.2, respectively). In vivo C57BL/6NHsd mice received intraoral mc-MnSOD-PL, mc-DS-red-PL, MnSOD-PL or Blank-PL, swallowed the plasmid/liposome complexes and were then irradiated 24 hr later along with control mice to 31 Gy to the esophagus. Mice receiving mc-MnSOD-PL had increased survival compared to both the control mice or mice treated with mc-DS-red-PL (p = 0.0099 or 0.0391, respectively). There was significant and equivalent improved survival of mice injected with mc-MnSOD-PL compared to full length MnSOD-PL. Therefore minicircle DNA containing the human MnSOD transgene confers undiminished radioprotection to cells in vitro and the esophagus in vivo compared to a fully intact plasmid containing the MnSOD transgene.

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Rodolosse, Annie, Alain Barbat, Isabelle Chantret, Michel Lacasa, Edith Brot-Laroche, Alain Zweibaum et Monique Rousset. « Selecting agent hygromycin B alters expression of glucose-regulated genes in transfected Caco-2 cells ». American Journal of Physiology-Gastrointestinal and Liver Physiology 274, n^o 5 (1 mai 1998) : G931—G938. http://dx.doi.org/10.1152/ajpgi.1998.274.5.g931.

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Incorporation into plasmids of genes conferring resistance to aminoglycoside antibiotics such as hygromycin B is currently utilized for selection in experiments involving gene transfer in eukaryotic cells. Using a subclone of Caco-2 cells stably transfected with an episomal plasmid containing the hygromycin resistance gene, we observed that transformed cells subcultured in the presence of hygromycin B exhibit, compared with the same cells subcultured in antibiotic-free medium, a sixfold increase in the rates of glucose consumption and lactic acid production and dramatic changes, at mRNA and protein level, of the expressions of sucrase-isomaltase and hexose transporter GLUT-2, which are downregulated, contrasting with an upregulation of hexose transporter GLUT-1. This occurs without significant modifications of the differentiation status of the cells, as demonstrated by the normal expression of villin, ZO-1, dipeptidyl peptidase IV, or Na+-K+-ATPase. The plasmid copy number is, however, the same, whether or not the cells are cultured in the presence of hygromycin B. These results draw attention to the need to consider antibiotic-dependent alterations of metabolism and gene expression in transfection experiments.

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Lin, Yuh-ru, Mi-Young Hahn, Jung-Hye Roe, Tzu-Wen Huang, Hsiu-Hui Tsai, Yung-Feng Lin, Tsung-Sheng Su, Yu-Jiun Chan et Carton W. Chen. « Streptomyces Telomeres Contain a Promoter ». Journal of Bacteriology 191, n^o 3 (5 décembre 2008) : 773–81. http://dx.doi.org/10.1128/jb.01299-08.

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ABSTRACT Bidirectional replication of the linear chromosomes and plasmids of Streptomyces spp. results in single-strand overhangs at their 3′ ends, which contain extensive complex palindromic sequences. The overhangs are believed to be patched by DNA synthesis primed by a terminal protein that remains covalently bound to the 5′ ends of the telomeres. We discovered that in vitro a conserved 167-bp telomere DNA binds strongly to RNA polymerase holoenzyme and exhibits promoter activities stronger than those of an rRNA operon. In vivo, the telomere DNA exhibited promoter activity in both orientations on a circular plasmid in Streptomyces. The telomere promoter is also active on a linear plasmid during exponential growth. Such promoter activity in a telomere has not hitherto been observed in eukaryotic or prokaryotic replicons. Streptomyces telomere promoters may be involved in priming the terminal Okazaki fragment (during replication) replicative transfer (during conjugation), or expression of downstream genes (including a conserved ttrA helicase-like gene involved in conjugal transfer). Interestingly, the Streptomyces telomeres also function as a promoter in Escherichia coli and as a transcription enhancer in yeast.

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Yamamoto, K., N. Takahashi, H. Yoshikura et I. Kobayashi. « Homologous recombination involving a large heterology in Escherichia coli. » Genetics 119, n^o 4 (1 août 1988) : 759–69. http://dx.doi.org/10.1093/genetics/119.4.759.

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Abstract Recombination between two different deletion alleles of a gene (neo) for neomycin and kanamycin resistance was studied in an Escherichia coli sbcA- recB-C- strain. The two homologous regions were in an inverted orientation on the same plasmid molecule. Kanamycin-resistant plasmids were selected and analyzed. The rate of recombination to form kanamycin-resistant plasmids was decreased by mutations in the recE, recF and recJ genes, but was not decreased by a mutation in the recA gene. It was found that these plasmids often possessed one wild-type kanamycin-resistant allele (neo+) while the other neo allele was still in its original (deletion) form. Among kanamycin-resistant plasmids with one wild-type and one parental allele it was often found that the region between the inverted repeats had been flipped (turned around) with respect to sites outside the inverted repeats. These results were interpreted as follows. Gene conversion, analogous to gene conversion in eukaryotic meiosis, is responsible for a unidirectional transfer of information from one neo deletion allele to the other. The flipping of the region between the inverted repeats is interpreted as analogous to the crossing over associated with gene conversion in eukaryotic meiosis. In contrast with a rec+ strain, these products cannot be explained by two rounds of reciprocal crossing over involving a dimeric form as an intermediate. In the accompanying paper we present evidence that gene conversion by double-strand gap repair takes place in the same E. coli strain.

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Mo, Yongkai, Natalie M. Quanquin, William H. Vecino, Uma Devi Ranganathan, Lydia Tesfa, William Bourn, Keith M. Derbyshire, Norman L. Letvin, William R. Jacobs et Glenn J. Fennelly. « Genetic Alteration of Mycobacterium smegmatis To Improve Mycobacterium-Mediated Transfer of Plasmid DNA into Mammalian Cells and DNA Immunization ». Infection and Immunity 75, n^o 10 (30 juillet 2007) : 4804–16. http://dx.doi.org/10.1128/iai.01877-06.

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ABSTRACT Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines by Mycobacterium smegmatis or M. bovis BCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer using M. smegmatis or BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant—albeit only 1.7-fold—increase in the level of plasmid transfer to eukaryotic cells infected with M. smegmatis hyperconjugation mutants. M. smegmatis strains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation in oriM resulted in a threefold increase in gene transfer to J774 cells. Moreover, M. smegmatis strains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120h E) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P < 0.05) and threefold (P < 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude of these responses was approximately one-half of that observed after intramuscular immunization with pgp120h E. M. smegmatis and other nonpathogenic mycobacteria are promising candidate vectors for DNA vaccine delivery.

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Marie, Corinne, et Daniel Scherman. « Antibiotic-Free Gene Vectors : A 25-Year Journey to Clinical Trials ». Genes 15, n^o 3 (20 février 2024) : 261. http://dx.doi.org/10.3390/genes15030261.

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Until very recently, the major use, for gene therapy, specifically of linear or circular DNA, such as plasmids, was as ancillary products for viral vectors’ production or as a genetic template for mRNA production. Thanks to targeted and more efficient physical or chemical delivery techniques and to the refinement of their structure, non-viral plasmid DNA are now under intensive consideration as pharmaceutical drugs. Plasmids traditionally carry an antibiotic resistance gene for providing the selection pressure necessary for maintenance in a bacterial host. Nearly a dozen different antibiotic-free gene vectors have now been developed and are currently assessed in preclinical assays and phase I/II clinical trials. Their reduced size leads to increased transfection efficiency and prolonged transgene expression. In addition, associating non-viral gene vectors and DNA transposons, which mediate transgene integration into the host genome, circumvents plasmid dilution in dividing eukaryotic cells which generate a loss of the therapeutic gene. Combining these novel molecular tools allowed a significantly higher yield of genetically engineered T and Natural Killer cells for adoptive immunotherapies due to a reduced cytotoxicity and increased transposition rate. This review describes the main progresses accomplished for safer, more efficient and cost-effective gene and cell therapies using non-viral approaches and antibiotic-free gene vectors.

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Zheng, Fengrong, Xiuqin Sun, Xing'an Wu, Hongzhan Liu, Jiye Li, Suqi Wu et Jinxing Zhang. « Immune Efficacy of a Genetically Engineered Vaccine against Lymphocystis Disease Virus : Analysis of Different Immunization Strategies ». Evidence-Based Complementary and Alternative Medicine 2011 (2011) : 1–8. http://dx.doi.org/10.1155/2011/729216.

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Here, we report the construction of a vaccine against lymphocystis disease virus (LCDV) using nucleic acid vaccination technology. A fragment of the major capsid protein encoding gene from an LCDV isolated from China (LCDV-cn) was cloned into an eukaryotic expression vector pEGFP-N2, yielding a recombinant plasmid pEGFP-N2-LCDV-cn0.6 kb. This plasmid was immediately expressed after liposomal transfer into the Japanese flounder embryo cell line. The recombinant plasmid was inoculated into Japanese flounder via two routes (intramuscular injection and hypodermic injection) at three doses (0.1, 5, and 15 μg), and then T-lymphopoiesis in different tissues and antibodies raised against LCDV were evaluated. The results indicated that this recombinant plasmid induced unique humoral or cell-mediated immune responses depending on the inoculation route and conferred immune protection. Furthermore, the humoral immune responses and protective effects were significantly increased at higher vaccine doses via the two injection routes. Plasmid pEGFP-N2-LCDV0.6 kb is therefore a promising vaccine candidate against LCDV in Japanese flounder.

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Wang, Chen, Xiaokang Li, Chunjie Zhang, Tingcai Wu, Yinju Li et Xiangchao Cheng. « A Eukaryotic Expression Plasmid Carrying Chicken Interleukin-18 Enhances the Response to Newcastle Disease Virus Vaccine ». Clinical and Vaccine Immunology 22, n^o 1 (29 octobre 2014) : 56–64. http://dx.doi.org/10.1128/cvi.00636-14.

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ABSTRACTInterleukin-18 (IL-18) is an important cytokine involved in innate and acquired immunity. In this study, we cloned the full-length chicken IL-18 (ChIL-18) gene from specific-pathogen-free (SPF) chicken embryo spleen cells and provided evidence that the ChIL-18 gene in a recombinant plasmid was successfully expressed in chicken DT40 cells. ChIL-18 significantly enhanced gamma interferon (IFN-γ) mRNA expression in chicken splenocytes, which increased IFN-γ-induced nitric oxide (NO) synthesis by macrophages. The potential genetic adjuvant activity of the ChIL-18 plasmid was examined in chickens by coinjecting ChIL-18 plasmid and inactivated Newcastle disease virus (NDV) vaccine. ChIL-18 markedly elevated serum hemagglutination inhibition (HI) titers and anti-hemagglutinin-neuraminidase (anti-HN)-specific antibody levels, induced the secretion of both Th1- (IFN-γ) and Th2- (interleukin-4) type cytokines, promoted the proliferation of T and B lymphocytes, and increased the populations of CD3+T cells and their subsets, CD3+CD4+and CD3+CD8+T cells. Furthermore, a virus challenge revealed that ChIL-18 contributed to protection against Newcastle disease virus challenge. Taken together, our data indicate that the coadministration of ChIL-18 plasmid and NDV vaccine induces a strong immune response at both the humoral and cellular levels and that ChIL-18 is a novel immunoadjuvant suitable for NDV vaccination.

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Mikheev, A. A., E. V. Shmendel, G. V. Nazarov et M. A. Maslov. « Influence of Liposome Composition on Plasmid DNA Delivery to Eukaryotic Cells ». Russian Journal of Bioorganic Chemistry 47, n^o 5 (septembre 2021) : 1034–42. http://dx.doi.org/10.1134/s1068162021050319.

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Xu, Li-Hong, Yong Zheng, Ting Zhou, Rui Li et Ying Chen. « Construction and identification of rat pcDNA3.1(+)-antisense TβRI eukaryotic expressing plasmid ». World Chinese Journal of Digestology 14, n^o 1 (2006) : 39. http://dx.doi.org/10.11569/wcjd.v14.i1.39.

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Hense, Marc, Eugen Domann, Stefan Krusch, Petra Wachholz, Kurt E. J. Dittmar, Manfred Rohde, Jürgen Wehland, Trinad Chakraborty et Siegfried Weiss. « Eukaryotic expression plasmid transfer from the intracellular bacteriumListeria monocytogenesto host cells ». Cellular Microbiology 3, n^o 9 (septembre 2001) : 599–609. http://dx.doi.org/10.1046/j.1462-5822.2001.00138.x.

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Brandt, Curtis R., Franco M. Buonaguro, James K. McDougall et Denise A. Galloway. « Plasmid mediated mutagenesis of a cellular gene in transfected eukaryotic cells ». Nucleic Acids Research 15, n^o 2 (1987) : 561–73. http://dx.doi.org/10.1093/nar/15.2.561.

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Cab-Barrera, Eddy L., et Hugo A. Barrera-Saldaña. « Versatile plasmid vectors for use in studies of eukaryotic gene expression ». Gene 70, n^o 2 (octobre 1988) : 411–13. http://dx.doi.org/10.1016/0378-1119(88)90214-4.

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Kirchmaier, Ann L., et Bill Sugden. « Rep* : a Viral Element That Can Partially Replace the Origin of Plasmid DNA Synthesis of Epstein-Barr Virus ». Journal of Virology 72, n^o 6 (1 juin 1998) : 4657–66. http://dx.doi.org/10.1128/jvi.72.6.4657-4666.1998.

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ABSTRACT Replication of the Epstein-Barr viral (EBV) genome occurs once per cell cycle during latent infection. Similarly, plasmids containing EBV’s plasmid origin of replication, oriP, are replicated once per cell cycle. Replication from oriP requires EBV nuclear antigen 1 (EBNA-1) in trans; however, its contributions to this replication are unknown. oriPcontains 24 EBNA-1 binding sites; 20 are located within the family of repeats, and 4 are found within the dyad symmetry element. The site of initiation of DNA replication within oriP is at or near the dyad symmetry element. We have identified a plasmid that contains the family of repeats but lacks the dyad symmetry element whose replication can be detected for a limited number of cell cycles. The detection of short-term replication of this plasmid requires EBNA-1 and can be inhibited by a dominant-negative inhibitor of EBNA-1. We have identified two regions within this plasmid which can independently contribute to this replication in the absence of the dyad symmetry element of oriP. One region contains native EBV sequences within the BamHI C fragment of the B95-8 genome of EBV; the other contains sequences within the simian virus 40 genome. We have mapped the region contributing to replication within the EBV sequences to a 298-bp fragment, Rep*. Plasmids which contain three copies of Rep* plus the family of repeats support replication more efficiently than those with one copy, consistent with a stochastic model for the initiation of DNA synthesis. Plasmids with three copies of Rep* also support long-term replication in the presence of EBNA-1. These observations together indicate that the latent origin of replication of EBV is more complex than formerly appreciated; it is a multicomponent origin of which the dyad symmetry element is one efficient component. The experimental approach described here could be used to identify eukaryotic sequences which mediate DNA synthesis, albeit inefficiently.

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Sinegubova, Maria V., Nadezhda A. Orlova et Ivan I. Vorobiev. « Promoter from Chinese hamster elongation factor-1a gene and Epstein-Barr virus terminal repeats concatemer fragment maintain stable high-level expression of recombinant proteins ». PeerJ 11 (24 octobre 2023) : e16287. http://dx.doi.org/10.7717/peerj.16287.

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Background The Chinese hamster ovary (CHO) cell line is the main host for the high-titer production of therapeutic and diagnostic proteins in the biopharmaceutical industry. In most cases, plasmids for efficient protein expression in CHO cells are based on the cytomegalovirus (CMV) promoter. The autologous Chinese hamster eukaryotic translation elongation factor 1α (EEF1A1) promoter is a viable alternative to the CMV promoter in industrial applications. The EEF1A1 promoter and its surrounding DNA regions proved to be effective at maintaining high-level and stable expression of recombinant proteins in CHO cells. EEF1A1-based plasmids’ large size can lead to low transfection efficiency and hamper target gene amplification. We hypothesized that an efficient EEF1A1-based expression vector with a long terminal repeat fragment from the Epstein-Barr virus (EBVTR) could be truncated without affecting promoter strength or the long-term stability of target gene expression. Methods We made a series of deletions in the downstream flanking region of the EEF1A1 gene, and then in its upstream flanking region. The resulting plasmids, which coded for the enhanced green fluorescent protein (eGFP), were tested for the level of eGFP expression in the populations of stably transfected CHO DG44 cells and the stability of eGFP expression in the long-term culture in the absence of selection agents. Results It was shown that in the presence of the EBVTR fragment, the entire downstream flanking region of the EEF1A1 gene could be excluded from the plasmid vector. Shortening of the upstream flanking region of the EEF1A1 gene to a length of 2.5 kbp also had no significant effect on the level of eGFP expression or long-term stability. The EBVTR fragment significantly increased expression stability for both the CMV and EEF1A1 promoter-based plasmids, and the expression level drop during the two-month culture was more significant for both CMV promoter-based plasmids. Conclusion Target protein expression stability for the truncated plasmid, based on the EEF1A1 gene and EBVTR fragment, is sufficient for common biopharmaceutical applications, making these plasmid vectors a viable alternative to conventional CMV promoter-based vectors.

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Cornelis, Guy R., Anne Boland, Aoife P. Boyd, Cecile Geuijen, Maite Iriarte, Cécile Neyt, Marie-Paule Sory et Isabelle Stainier. « The Virulence Plasmid of Yersinia, an Antihost Genome ». Microbiology and Molecular Biology Reviews 62, n^o 4 (1 décembre 1998) : 1315–52. http://dx.doi.org/10.1128/mmbr.62.4.1315-1352.1998.

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SUMMARY The 70-kb virulence plasmid enables Yersinia spp. (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) to survive and multiply in the lymphoid tissues of their host. It encodes the Yop virulon, an integrated system allowing extracellular bacteria to disarm the cells involved in the immune response, to disrupt their communications, or even to induce their apoptosis by the injection of bacterial effector proteins. This system consists of the Yop proteins and their dedicated type III secretion apparatus, called Ysc. The Ysc apparatus is composed of some 25 proteins including a secretin. Most of the Yops fall into two groups. Some of them are the intracellular effectors (YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT), while the others (YopB, YopD, and LcrV) form the translocation apparatus that is deployed at the bacterial surface to deliver the effectors into the eukaryotic cells, across their plasma membrane. Yop secretion is triggered by contact with eukaryotic cells and controlled by proteins of the virulon including YopN, TyeA, and LcrG, which are thought to form a plug complex closing the bacterial secretion channel. The proper operation of the system also requires small individual chaperones, called the Syc proteins, in the bacterial cytosol. Transcription of the genes is controlled both by temperature and by the activity of the secretion apparatus. The virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis also encodes the adhesin YadA. The virulence plasmid contains some evolutionary remnants including, in Y. enterocolitica, an operon encoding resistance to arsenic compounds.

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Hou, Shuang, Chun Fang Wang, Yan Ru Zheng, Jia Ning Guan, Jia Ming Lin, Da Ming Gao, Yun Hang Gao, Hong Xia Ma et Xiu Yun Jiang. « Construction of Eukaryotic Expression Vector and Expression on Mycobacterium bovis ag85a and mpb70 Genes ». Applied Mechanics and Materials 421 (septembre 2013) : 308–12. http://dx.doi.org/10.4028/www.scientific.net/amm.421.308.

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Based on the polymerase chain reaction (PCR),ag85aandmpb70Fusion gene ofMycobacterium boviswere ligated and cloned into pMD18-T, then recombinant plasmid pMD-85a-70 was constructed. pMD-85a-70 and pVAX1-BMS were digested by double enzymesHindIII andEcoRI, the purified ag85a-mpb70 was subcloned into pVAX1-BMS, then recombinant plasmid pVAX1-BMS-85a-70 was constructed and transient expressed in Marc145 cell. These results laid solid foundations for further studies on ag85a-mpb70.

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Marquet, Magda, Sami Alouani et Stephen W. Brown. « Plasmid stability during continuous culture in aSaccharomyces cerevisiae double mutant transformed by a plasmid carrying a eukaryotic gene ». Biotechnology Letters 8, n^o 8 (août 1986) : 535–40. http://dx.doi.org/10.1007/bf01028078.

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Li, Xiaolin, Guozhong Jiang, Dan Wu, Xiuli Wang et Bingfang Zeng. « Construction of a Recombinant Eukaryotic Expression Plasmid Containing Human Calcitonin Gene and Its Expression in NIH3T3 Cells ». Journal of Biomedicine and Biotechnology 2009 (2009) : 1–7. http://dx.doi.org/10.1155/2009/241390.

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Aim. To construct a recombinant eukaryotic expression plasmid containing human calcitonin (hCT) gene and express the gene in murine fibroblast NIH3T3 cells.Materials and Methods. A murine Igκ-chain leader sequence and hCT gene were synthesized and cloned into pCDNA3.0 to form the pCDNA3.0-Igκ-hCT eukaryotic expression vector, which was transfected into NIH3T3 cells. The mRNA and protein expressions and secretion of hCT were detected. Primarily cultured osteoclasts were incubated with the supernatant of pCDNA3.0-Igk-hCT-transfected NIH3T3 cells, and their numbers were counted and morphology observed.Results. The expression and secretion of hCT were successfully detected in pCDNA3.0-Igk-hCT-transfected NIH3T3 cells. The number of osteoclasts was decreased and the cells became crumpled when they were incubated with the supernatant of pCDNA3.0-Igk-hCT-transfected NIH3T3 cells.Conclusion. A recombinant eukaryotic expression vector containing hCT gene was successfully constructed and expressed in NIH3T3 cells. The secreted recombinant hCT inhibited the growth and morphology of osteoclasts.

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