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Sample records for linear plasmid vector

  1. Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli.

    PubMed

    Godiska, Ronald; Mead, David; Dhodda, Vinay; Wu, Chengcang; Hochstein, Rebecca; Karsi, Attila; Usdin, Karen; Entezam, Ali; Ravin, Nikolai

    2010-04-01

    Despite recent advances in sequencing, complete finishing of large genomes and analysis of novel proteins they encode typically require cloning of specific regions. However, many of these fragments are extremely difficult to clone in current vectors. Superhelical stress in circular plasmids can generate secondary structures that are substrates for deletion, particularly in regions that contain numerous tandem or inverted repeats. Common vectors also induce transcription and translation of inserted fragments, which can select against recombinant clones containing open reading frames or repetitive DNA. Conversely, transcription from cloned promoters can interfere with plasmid stability. We have therefore developed a novel Escherichia coli cloning vector (termed 'pJAZZ' vector) that is maintained as a linear plasmid. Further, it contains transcriptional terminators on both sides of the cloning site to minimize transcriptional interference between vector and insert. We show that this vector stably maintains a variety of inserts that were unclonable in conventional plasmids. These targets include short nucleotide repeats, such as those of the expanded Fragile X locus, and large AT-rich inserts, such as 20-kb segments of genomic DNA from Pneumocystis, Plasmodium, Oxytricha or Tetrahymena. The pJAZZ vector shows decreased size bias in cloning, allowing more uniform representation of larger fragments in libraries. PMID:20040575

  2. Building mosaics of therapeutic plasmid gene vectors.

    PubMed

    Tolmachov, Oleg E

    2011-12-01

    Plasmids are circular or linear DNA molecules propagated extra-chromosomally in bacteria. Evolution shaped plasmids are inherently mosaic structures with individual functional units represented by distinct segments in the plasmid genome. The patchwork of plasmid genetic modules is a convenient template and a model for the generation of artificial plasmids used as vehicles for gene delivery into human cells. Plasmid gene vectors are an important tool in gene therapy and in basic biomedical research, where these vectors offer efficient transgene expression in many settings in vitro and in vivo. Plasmid vectors can be attached to nuclear directing ligands or transferred by electroporation as naked DNA to deliver the payload genes to the nuclei of the target cells. Transgene expression silencing by plasmid sequences of bacterial origin and immune stimulation by bacterial unmethylated CpG motifs can be avoided by the generation of plasmid-based minimized DNA vectors, such as minicircles. Systems of efficient site-specific integration into human chromosomes and stable episomal maintenance in human cells are being developed for further reduction of the chances for transgene silencing. The successful generation of plasmid vectors is governed by a number of vector design rules, some of which are common to all gene vectors, while others are specific to plasmid vectors. This review is focused both on the guiding principles and on the technical know-how of plasmid gene vector design. PMID:22023476

  3. The Development of a Novel Mycobacterium-Escherichia coli Shuttle Vector System Using pMyong2, a Linear Plasmid from Mycobacterium yongonense DSM 45126T

    PubMed Central

    Lee, Hyungki; Kim, Byoung-Jun; Kim, Bo-Ram; Kook, Yoon-Hoh; Kim, Bum-Joon

    2015-01-01

    The Mycobacterium-Escherichia coli shuttle vector system, equipped with the pAL5000 replicon, is widely used for heterologous gene expression and gene delivery in mycobacteria. Despite its extensive use, this system has certain limitations, which has led to the development of alternative mycobacterial vector systems. The present study describes the molecular structure and expression profiles of a novel 18-kb linear plasmid, pMyong2, from Mycobacterium yongonense. Sixteen open reading frames and a putative origin of replication were identified, and the compatibility of the pMyong2 and pAL5000 vector systems was demonstrated. In recombinant Mycobacterium smegmatis (rSmeg), the pMyong2 vector system showed a copy number that was approximately 37 times greater than that of pAL5000. Furthermore, pMyong2 increased the mRNA and protein expression of the human macrophage migration inhibitory factor (hMIF) over pAL5000 levels by approximately 10-fold and 50-fold, respectively, demonstrating the potential utility of the pMyong2 vector system in heterologous gene expression in mycobacteria. Successful delivery of the EGFP gene into mammalian cells via rSmeg carrying the pMyong2 vector system was also observed, demonstrating the feasibility of this system for DNA delivery. In conclusion, the pMyong2 vector system could be effectively used not only for the in vivo delivery of recombinant protein and DNA but also for mycobacterial genetic studies as an alternative or a complement to the pAL5000 vector system. PMID:25822634

  4. The development of a novel Mycobacterium-Escherichia coli shuttle vector system using pMyong2, a linear plasmid from Mycobacterium yongonense DSM 45126T.

    PubMed

    Lee, Hyungki; Kim, Byoung-Jun; Kim, Bo-Ram; Kook, Yoon-Hoh; Kim, Bum-Joon

    2015-01-01

    The Mycobacterium-Escherichia coli shuttle vector system, equipped with the pAL5000 replicon, is widely used for heterologous gene expression and gene delivery in mycobacteria. Despite its extensive use, this system has certain limitations, which has led to the development of alternative mycobacterial vector systems. The present study describes the molecular structure and expression profiles of a novel 18-kb linear plasmid, pMyong2, from Mycobacterium yongonense. Sixteen open reading frames and a putative origin of replication were identified, and the compatibility of the pMyong2 and pAL5000 vector systems was demonstrated. In recombinant Mycobacterium smegmatis (rSmeg), the pMyong2 vector system showed a copy number that was approximately 37 times greater than that of pAL5000. Furthermore, pMyong2 increased the mRNA and protein expression of the human macrophage migration inhibitory factor (hMIF) over pAL5000 levels by approximately 10-fold and 50-fold, respectively, demonstrating the potential utility of the pMyong2 vector system in heterologous gene expression in mycobacteria. Successful delivery of the EGFP gene into mammalian cells via rSmeg carrying the pMyong2 vector system was also observed, demonstrating the feasibility of this system for DNA delivery. In conclusion, the pMyong2 vector system could be effectively used not only for the in vivo delivery of recombinant protein and DNA but also for mycobacterial genetic studies as an alternative or a complement to the pAL5000 vector system.

  5. Electrotransfer of Plasmid Vector DNA into Muscle

    NASA Astrophysics Data System (ADS)

    Miyazaki, Satsuki; Miyazaki, Jun-Ichi

    Wolff et al. (1990) first reported that plasmid DNA injected into skeletal muscle is taken up by muscle cells and the genes in the plasmid are expressed for more than two months thereafter, although the transfected DNA does not usually undergo chromosomal integration (Wolff et al., 1991, 1992). However, the relatively low expression levels attained by this method have hampered its applications for uses other than as a DNA vaccine (Davis et al., 1995). There are a number of reports analyzing the conditions that affect the efficiency of gene transfer by intramuscular DNA injection and assessing the fine structures of expression plasmid vectors that may affect expression levels (Davis et al., 1993; Liang et al., 1996; Norman et al., 1997). Furthermore, various attempts were done to improve the efficiency of gene transfer by intramus cular DNA injection. Consequently, regenerating muscle was shown to produce 80-fold or more protein than did normal muscle, following injection of an expression plas-mid. Muscle regeneration was induced by treatment with cardiotoxin or bupivacaine (Wells, 1993; Vitadello et al., 1994). We previously demonstrated that by combining a strong promoter and bupivacaine pretreatment intramuscular injection of an IL-5 expression plasmid results in IL-5 production in muscle at a level sufficient to induce marked proliferation of eosinophils in the bone marrow and eosinophil infiltration of various organs (Tokui et al., 1997). It was also reported that a single intramuscular injection of an erythropoietin expression plasmid produced physiologically significant elevations in serum erythropoietin levels and increased hematocrits in adult mice (Tripathy et al., 1996). Hematocrits in these animals remained elevated at >60% for at least 90 days after a single injection. However, improvements to this method have not been sufficient to extend its applications including clinical use.

  6. N15: the linear phage-plasmid.

    PubMed

    Ravin, Nikolai V

    2011-03-01

    The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into chromosome but is a linear plasmid molecule with covalently closed ends. Upon infection the phage DNA circularises via cohesive ends, then phage-encoded enzyme, protelomerase, cuts at an inverted repeat site and forms hairpin ends (telomeres) of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally resulting in formation of duplicated telomeres. Then the N15 protelomerase cuts duplicated telomeres generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by partitioning operon similar to the F factor sop operon. Unlike F sop, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in N15 genome regions involved in phage replication and control of lysogeny, and binding of partition proteins at these sites regulates these processes. Two N15-related lambdoid Siphoviridae phages, φKO2 in Klebsiella oxytoca and pY54 in Yersinia enterocolitica, also lysogenize their hosts as linear plasmids, as well as Myoviridae marine phages VP882 and VP58.5 in Vibrio parahaemolyticus and ΦHAP-1 in Halomonas aquamarina. The genomes of all these phages contain similar protelomerase genes, lysogeny modules and replication genes, as well as plasmid-partitioning genes, suggesting that these phages may belong to a group diverged from a common ancestor.

  7. pLS101 plasmid vector

    DOEpatents

    Lacks, S.A.; Balganesh, T.S.

    1985-02-19

    Disclosed is recombinant plasmid pLS101, consisting essentially of a 2.0 Kb ma1M gene fragment ligated to a 4.4 Kb Tcr DNA fragment, which is particularly useful for transforming Gram-positive bacteria. This plasmid contains at least four restriction sites suitable for inserting exogeneous gene sequences. Also disclosed is a method for plasmid isolation by penicillin selection, as well as processes for enrichment of recombinant plasmids in Gram-positive bacterial systems. 5 figs., 2 tabs.

  8. pLS010 plasmid vector

    DOEpatents

    Lacks, Sanford A.; Balganesh, Tanjore S.

    1988-01-01

    Disclosed is recombinant plasmid pLS101, consisting essentially of a 2.0 Kb malM gene fragment ligated to a 4.4 Kb T.sub.c r DNA fragment, which is particularly useful for transforming Gram-positive bacteria. This plasmid contains at least four restriction sites suitable for inserting exogeneous gene sequences. Also disclosed is a method for plasmid isolation by penicillin selection, as well as processes for enrichment of recombinant plasmids in Gram-positive bacterial systems.

  9. Transformation of Saccharomyces cerevisiae and Schizosaccharomyces pombe with linear plasmids containing 2 micron sequences.

    PubMed Central

    Guerrini, A M; Ascenzioni, F; Tribioli, C; Donini, P

    1985-01-01

    Linear plasmids were constructed by adding telomeres prepared from Tetrahymena pyriformis rDNA to a circular hybrid Escherichia coli-yeast vector and transforming Saccharomyces cerevisiae. The parental vector contained the entire 2 mu yeast circle and the LEU gene from S. cerevisiae. Three transformed clones were shown to contain linear plasmids which were characterized by restriction analysis and shown to be rearranged versions of the desired linear plasmids. The plasmids obtained were imperfect palindromes: part of the parental vector was present in duplicated form, part as unique sequences and part was absent. The sequences that had been lost included a large portion of the 2 mu circle. The telomeres were approximately 450 bp longer than those of T. pyriformis. DNA prepared from transformed S. cerevisiae clones was used to transform Schizosaccharomyces pombe. The transformed S. pombe clones contained linear plasmids identical in structure to their linear parents in S. cerevisiae. No structural re-arrangements or integration into S. pombe was observed. Little or no telomere growth had occurred after transfer from S. cerevisiae to S. pombe. A model is proposed to explain the genesis of the plasmids. Images Fig. 1. Fig. 2. Fig. 4. PMID:3896773

  10. Linearized oncolytic adenoviral plasmid DNA delivered by bioreducible polymers

    PubMed Central

    Kim, Jaesung; Kim, Pyung-Hwan; Nam, Hye Yeong; Lee, Jung-Sun; Yun, Chae-Ok; Kim, Sung Wan

    2011-01-01

    As an effort to overcome limits of adenovirus (Ad) as a systemic delivery vector for cancer therapy, we developed a novel system using oncolytic Ad plasmid DNA with two bioreducible polymers: arginine-grafted bioreducible poly(disulfide amine)polymer (ABP) and PEG5k-conjugated ABP (ABP5k) in expectation of oncolytic effect caused by progeny viral production followed by replication. The linearized Ad DNAs for active viral replication polyplexed with each polymer were able to replicate only in humancancer cells and produce progeny viruses. The non-immunogenic polymers delivering the DNAs markedly elicited to evade the innate and adaptive immune response. The biodistribution ratio of the polyplexes administered systemically was approximately 99% decreased in liver when compared with naked Ad. Moreover, tumor-to-liver ratio of the Ad DNA delivered by ABP or ABP5k was significantly elevated at 229- or 419-fold greater than that of naked Ad, respectively. The ABP5k improved the chance of the DNA to localize within tumor versus liver with 1.8-fold increased ratio. In conclusion, the innovative and simple system for delivering oncolytic Ad plasmid DNA with the bioreducible polymers, skipping time-consuming steps such as generation and characterization of oncolytic Ad vectors, can be utilized as an alternative approach for cancer therapy. PMID:22207073

  11. Large linear plasmids of Borrelia species that cause relapsing fever.

    PubMed

    Miller, Shelley Campeau; Porcella, Stephen F; Raffel, Sandra J; Schwan, Tom G; Barbour, Alan G

    2013-08-01

    Borrelia species of relapsing fever (RF) and Lyme disease (LD) lineages have linear chromosomes and both linear and circular plasmids. Unique to RF species, and little characterized to date, are large linear plasmids of ∼160 kb, or ∼10% of the genome. By a combination of Sanger and next-generation methods, we determined the sequences of large linear plasmids of two New World species: Borrelia hermsii, to completion of its 174-kb length, and B. turicatae, partially to 114 kb of its 150 kb. These sequences were then compared to corresponding sequences of the Old World species B. duttonii and B. recurrentis and to plasmid sequences of LD Borrelia species. The large plasmids were largely colinear, except for their left ends, about 27 kb of which was inverted in New World species. Approximately 60% of the B. hermsii lp174 plasmid sequence was repetitive for 6 types of sequence, and half of its open reading frames encoded hypothetical proteins not discernibly similar to proteins in the database. The central ∼25 kb of all 4 linear plasmids was syntenic for orthologous genes for plasmid maintenance or partitioning in Borrelia species. Of all the sequenced linear and circular plasmids in Borrelia species, the large plasmid's putative partition/replication genes were most similar to those of the 54-kb linear plasmids of LD species. Further evidence for shared ancestry was the observation that two of the hypothetical proteins were predicted to be structurally similar to the LD species' CspA proteins, which are encoded on the 54-kb plasmids.

  12. Plasmid vector with temperature-controlled gene expression

    SciTech Connect

    Kravchenko, V.V.; Yamshchikov, V.F.; Pletnev, A.G.

    1986-02-01

    In plasmid pBR327, a fragment 169 b.p. long including promotor p/sub 3/ of the bla gene has been deleted. The deletional derivative so obtained (pSP2) has been used to construct a recombinant plasmid bearing a fragment of phage lambda DNA with the p/sub R/ promotor and the gene of the temperature-sensitive repressor cI. It has been shown that the plasmid vector so constructed (pCE119) with promotor cR performs repressor-cI-controlled transcription of the bla gene, as a result of which induction for an hour at 42/sup 0/C leads to an almost 100-fold increase in the amount of product of the bla gene as compared with that at 32/sup 0/C. The possibility of the use of plasmid cPE119 for the expression of other genes has been demonstrated for the case of the semisynthetic ..beta..-galactosidase gene of E. coli. In this case, on induction of the cells with recombinant plasmid pCEZ12 for 3 hours at 42/sup 0/C, a 300-fold increase in the amount of active ..beta..-galactosidase, as compared with that at 32/sup 0/C, was observed. It is important to point out that under these conditions (at 42/sup 0/C), at least 99% of the cells containing the plasmid retain the phenotype lacZ/sup +/, which indicates the stability of the proposed vector system

  13. The Standard European Vector Architecture (SEVA) plasmid toolkit.

    PubMed

    Durante-Rodríguez, Gonzalo; de Lorenzo, Víctor; Martínez-García, Esteban

    2014-01-01

    The Standard European Vector Architecture (SEVA) toolkit is a simple and powerful resource for constructing optimal plasmid vectors based on a backbone and three interchangeable modules flanked by uncommon restriction sites. Functional modules encode several origins of replication, diverse antibiotic selection markers, and a variety of cargoes with different applications. The backbone and DNA modules have been minimized and edited for flaws in their sequence and/or functionality. A protocol for the utilization of the SEVA platform to construct transcriptional and translational fusions between a promoter under study (the arsenic-responsive Pars of Pseudomonas putida KT2440) and the reporter lacZ gene is described. The resulting plasmid collection was instrumental to measure and compare the β-galactosidase activity that report gene expression (i.e., transcription and translation) in different genetic backgrounds.

  14. Involvement of Linear Plasmids in Aerobic Biodegradation of Vinyl Chloride

    SciTech Connect

    BRIGMON, ROBINL.

    2004-06-14

    Pseudomonas putida strain AJ and Ochrobactrum strain TD were isolated from hazardous waste sites based on their ability to use vinyl chloride (VC) as a sole source of carbon and energy under aerobic conditions. Strains AJ and TD also use ethene and ethylene oxide as growth substrates. Strain AJ contained a linear megaplasmid (approximately 260 kb) when grown on VC or ethene, but no circular plasmids. While growing on ethylene oxide, the size of the linear plasmid in strain AJ decreased to approximately 100 kb, although its ability to use VC as a substrate was retained. The linear plasmids in strain AJ were cured and its ability to consume VC, ethene, and ethylene oxide was lost following growth on a rich substrate (Luria-Bertani broth) through at least three transfers. Strain TD contained three linear plasmids, ranging in size from approximately 100 kb to 320 kb, when growing on VC or ethene. As with strain AJ, the linear plasmids in strain TD were cured following growth on Luria -Bertani broth and its ability to consume VC and ethene was lost. Further analysis of these linear plasmids may help reveal the pathway for VC biodegradation in strains AJ and TD and explain why this process occurs at many but not all sites where groundwater is contaminated with chloroethenes. Metabolism of VC and ethene by strains AJ and TD is initiated by an alkene monooxygenase. Their yields during growth on VC (0.15-0.20 mg total suspended solids per mg VC) are similar to the yields reported for other isolates i.e., Mycobacterium sp., Nocardioides sp., and Pseudomonas sp.

  15. Replication and Maintenance of Linear Phage-Plasmid N15.

    PubMed

    Ravin, Nikolai V

    2015-02-01

    The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into the chromosome but is a linear plasmid molecule with covalently closed ends (telomeres). Upon infection, the phage DNA circularizes via cohesive ends, and then a special phage enzyme of the tyrosine recombinase family, protelomerase, cuts at another site and joins the ends, forming hairpin telomeres of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally, resulting in the formation of duplicated telomeres. The N15 protelomerase cuts them, generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by a partitioning operon similar to the F factor sop operon. Unlike the F centromere, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in the N15 genome regions involved in phage replication and control of lytic development, and binding of partition proteins at these sites regulates these processes. The family of N15-like linear phage-plasmids includes lambdoid phages ɸKO2 and pY54, as well as Myoviridae phages ΦHAP-1, VHML, VP882, Vp58.5, and vB_VpaM_MAR of marine gamma-proteobacteria. The genomes of these phages contain similar protelomerase genes, lysogeny control modules, and replication genes, suggesting that these phages may belong to a group diverged from a common ancestor.

  16. Engineering and characterization of a symbiotic selection-marker-free vector-host system for therapeutic plasmid production.

    PubMed

    Shi, Xinchang; Wang, Junzhi

    2015-09-01

    The present study aimed to develop a symbiotic selection-marker-free plasmid and host system that would allow successful plasmid maintenance and amplification for use in gene therapy. Initially, the chromosomal aspartate‑semialdehyde dehydrogenase (asd) gene was disrupted in DH10B Escherichia coli using Red recombinase‑mediated homologous recombination. This method required the use of linear DNA fragments carrying kan‑kil genes, and/or homologous extensions to the targeted locus. The resultant auxotrophic cell wall‑deficient strain (DH10BΔasd) was evaluated as a symbiotic host for amplification of the marker‑free plasmid, allowing it to supply ASD activity. In order to construct the plasmid, an asd expression cassette was inserted, under the control of the nirB promoter, into a eukaryotic expression vector, and its kanamycin resistance gene was subsequently removed. The symbiotic plasmid and host system was assessed for numerous plasmid production and stability parameters, including structure, yield, plasmid‑retention rate, and bacterial storability, under various conditions. The presence of the plasmid was subsequently confirmed by growth test, restriction enzyme mapping, and sequencing. The plasmid yield and copy number produced in the symbiotic cells, in the absence of antibiotic selection, were shown to be similar to those produced under kanamycin selection, in the cells containing the precursor plasmid and kanamycin resistance gene. Furthermore, the results of the present study demonstrated that when inoculated with <1% inoculant volume, >98% of the cells in the culture retained the plasmid regardless of the number of passages. The strain was stable when stored at ‑70˚C, with negligible viability loss over 12 months. The constructed plasmid is stable and has potential in future gene therapy, while much work is still required.

  17. Plasmid-chromosome recombination of irradiated shuttle vector DNA in African Green Monkey kidney cells

    SciTech Connect

    Mudgett, J.S.

    1987-01-01

    An autonomously replicating shuttle vector was used to investigate the enhancement of plasmid-chromosome recombination in mammalian host cells by ultraviolet light and gamma radiation. Sequences homologous to the shuttle vector were stably inserted into the genome of African Green Monkey kidney cells to act as the target substrate for these recombination events. The SV40- and pBR322-derived plasmid DNA was irradiated with various doses of radiation before transfection into the transformed mammalian host cells. Ultraviolet light (UV) was found not to induce homologous plasmid-chromosome recombination, while gamma radiation increased the frequency of recombinant plasmids detected. The introduction of specific double-strand breaks in the plasmid or prolonging the time of plasmid residence in the mammalian host cells also enhanced plasmid-chromosome recombination. In contrast, plasmid mutagenesis was found to be increased by plasmid UV irradiation, but not to change with time. Plasmid survival, recombination, and mutagenesis were not affected by treating the mammalian host cells with UV light prior to plasmid transfection. The amp/sup r/ recombinant plasmid molecules analyzed were found to be mostly the result of nonconservative exchanges which appeared to involve both homologous and possibly nonhomologous interactions with the host chromosome.

  18. Plasmid-Chromosome Recombination of Irradiated Shuttle Vector DNA in African Green Monkey Kidney Cells.

    NASA Astrophysics Data System (ADS)

    Mudgett, John Stuart

    1987-09-01

    An autonomously replicating shuttle vector was used to investigate the enhancement of plasmid-chromosome recombination in mammalian host cells by ultraviolet light and gamma radiation. Sequences homologous to the shuttle vector were stably inserted into the genome of African Green Monkey kidney cells to act as the target substrate for these recombination events. The SV40- and pBR322-derived plasmid DNA was irradiated with various doses of radiation before transfection into the transformed mammalian host cells. The successful homologous transfer of the bacterial ampicillin resistance (amp^{rm r}) gene from the inserted sequences to replace a mutant amp^->=ne on the shuttle vector was identified by plasmid extraction and transformation into E. coli host cells. Ultraviolet light (UV) was found not to induce homologous plasmid-chromosome recombination, while gamma radiation increased the frequency of recombinant plasmids detected. The introduction of specific double -strand breaks in the plasmid or prolonging the time of plasmid residence in the mammalian host cells also enhanced plasmid-chromosome recombination. In contrast, plasmid mutagenesis was found to be increased by plasmid UV irradiation, but not to change with time. Plasmid survival, recombination, and mutagenesis were not affected by treating the mammalian host cells with UV light prior to plasmid transfection. The amp^{rm r} recombinant plasmid molecules analyzed were found to be mostly the result of nonconservative exchanges which appeared to involve both homologous and possibly nonhomologous interactions with the host chromosome. The observation that these recombinant structures were obtained from all of the plasmid alterations investigated suggests a common mechanistic origin for plasmid -chromosome recombination in these mammalian cells.

  19. Vector insert-targeted integrative antisense expression system for plasmid stabilization.

    PubMed

    Luke, Jeremy M; Carnes, Aaron E; Hodgson, Clague P; Williams, James A

    2011-01-01

    Some DNA vaccine and gene therapy vector-encoded transgenes are toxic to the E. coli plasmid production host resulting in poor production yields. For plasmid products undergoing clinical evaluation, sequence modification to eliminate toxicity is undesirable because an altered vector is a new chemical entity. We hypothesized that: (1) insert-encoded toxicity is mediated by unintended expression of a toxic insert-encoded protein from spurious bacterial promoters; and (2) that toxicity could be eliminated with antisense RNA-mediated translation inhibition. We developed the pINT PR PL vector, a chromosomally integrable RNA expression vector, and utilized it to express insert-complementary (anti-insert) RNA from a single defined site in the bacterial chromosome. Anti-insert RNA eliminated leaky fluorescent protein expression from a target plasmid. A toxic retroviral gag pol helper plasmid produced in a gag pol anti-insert strain had fourfold improved plasmid fermentation yields. Plasmid fermentation yields were also fourfold improved when a DNA vaccine plasmid containing a toxic Influenza serotype H1 hemagglutinin transgene was grown in an H1 sense strand anti-insert production strain, suggesting that in this case toxicity was mediated by an antisense alternative reading frame-encoded peptide. This anti-insert chromosomal RNA expression technology is a general approach to improve production yields with plasmid-based vectors that encode toxic transgenes, or toxic alternative frame peptides. PMID:20607625

  20. Plasmid vectors for Xylella fastidiosa utilizing a toxin-antitoxin system for plasmid stability in the absence of antibiotic selection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacte...

  1. A survey of drug resistance bla genes originating from synthetic plasmid vectors in six Chinese rivers.

    PubMed

    Chen, Jian; Jin, Min; Qiu, Zhi-Gang; Guo, Cong; Chen, Zhao-Li; Shen, Zhi-Qiang; Wang, Xin-Wei; Li, Jun-Wen

    2012-12-18

    Antibiotic resistance poses a significant challenge to human health and its rate continues to rise globally. While antibiotic-selectable synthetic plasmid vectors have proved invaluable tools of genetic engineering, this class of artificial recombinant DNA sequences with high expression of antibiotic resistance genes presents an unknown risk beyond the laboratory setting. Contamination of environmental microbes with synthetic plasmid vector-sourced antibiotic resistance genes may represent a yet unrecognized source of antibiotic resistance. In this study, PCR and real-time quantitative PCR were used to investigate the synthetic plasmid vector-originated ampicillin resistance gene, β-lactam antibiotic (blá), in microbes from six Chinese rivers with significant human interactions. Various levels of blá were detected in all six rivers, with the highest levels in the Pearl and Haihe rivers. To validate the blá pollution, environmental plasmids in the river samples were captured by the E. coli transformants from the community plasmid metagenome. The resultant plasmid library of 205 ampicillin-resistant E. coli (transformants) showed a blá-positive rate of 27.3% by PCR. Sequencing results confirmed the synthetic plasmid vector sources. In addition, results of the Kirby-Bauer disc-diffusion test reinforced the ampicillin-resistant functions of the environmental plasmids. The resistance spectrum of transformants from the Pearl and Haihe rivers, in particular, had expanded to the third- and fourth-generation of cephalosporin drugs, while that of other transformants mainly involved first- and second-generation cephalosporins. This study not only reveals environmental contamination of synthetic plasmid vector-sourced blá drug resistance genes in Chinese rivers, but also suggests that synthetic plasmid vectors may represent a source of antibiotic resistance in humans.

  2. The 2-micron plasmid as a nonselectable, stable, high copy number yeast vector

    NASA Technical Reports Server (NTRS)

    Ludwig, D. L.; Bruschi, C. V.

    1991-01-01

    The endogenous 2-microns plasmid of Saccharomyces cerevisiae has been used extensively for the construction of yeast cloning and expression plasmids because it is a native yeast plasmid that is able to be maintained stably in cells at high copy number. Almost invariably, these plasmid constructs, containing some or all 2-microns sequences, exhibit copy number levels lower than 2-microns and are maintained stably only under selective conditions. We were interested in determining if there was a means by which 2-microns could be utilized for vector construction, without forfeiting either copy number or nonselective stability. We identified sites in the 2-microns plasmid that could be used for the insertion of genetic sequences without disrupting 2-microns coding elements and then assessed subsequent plasmid constructs for stability and copy number in vivo. We demonstrate the utility of a previously described 2-microns recombination chimera, pBH-2L, for the manipulation and transformation of 2-microns as a pure yeast plasmid vector. We show that the HpaI site near the STB element in the 2-microns plasmid can be utilized to clone yeast DNA of at least 3.9 kb with no loss of plasmid stability. Additionally, the copy number of these constructs is as high as levels reported for the endogenous 2-microns.

  3. Isolation of a novel plasmid from Couchioplanes caeruleus and construction of two plasmid vectors for gene expression in Actinoplanes missouriensis.

    PubMed

    Jang, Moon-Sun; Fujita, Azusa; Ikawa, Satomi; Hanawa, Keitaro; Yamamura, Hideki; Tamura, Tomohiko; Hayakawa, Masayuki; Tezuka, Takeaki; Ohnishi, Yasuo

    2015-01-01

    To date, no plasmid vector has been developed for the rare actinomycete Actinoplanes missouriensis. Moreover, no small circular plasmid has been reported to exist in the genus Actinoplanes. Here, a novel plasmid, designated pCAZ1, was isolated from Couchioplanes caeruleus subsp. azureus via screening for small circular plasmids in Actinoplanes (57 strains) and Couchioplanes (2 strains). Nucleotide sequencing revealed that pCAZ1 is a 5845-bp circular molecule with a G + C content of 67.5%. The pCAZ1 copy number was estimated at 30 per chromosome. pCAZ1 contains seven putative open reading frames, one of which encodes a protein containing three motifs conserved among plasmid-encoded replication proteins that are involved in the rolling-circle mechanism of replication. Detection of single-stranded DNA intermediates in C. caeruleus confirmed that pCAZ1 replicates by this mechanism. The ColE1 origin from pBluescript SK(+) and the oriT sequence with the apramycin resistance gene aac(3)IV from pIJ773 were inserted together into pCAZ1, to construct the Escherichia coli-A. missouriensis shuttle vectors, pCAM1 and pCAM2, in which the foreign DNA fragment was inserted into pCAZ1 in opposite directions. pCAM1 and pCAM2 were successfully transferred to A. missouriensis through the E. coli-mediated conjugative transfer system. The copy numbers of pCAM1 and pCAM2 in A. missouriensis were estimated to be one and four per chromosome, respectively. Thus, these vectors can be used as effective genetic tools for homologous and heterologous gene expression studies in A. missouriensis.

  4. In vivo generation of linear plasmids with addition of telomeric sequences by Histoplasma capsulatum.

    PubMed

    Woods, J P; Goldman, W E

    1992-12-01

    Histoplasma capsulatum is a dimorphic pathogenic fungus that is a major cause of respiratory and systemic mycosis. We previously developed a transformation system for Histoplasma and demonstrated chromosomal integration of transforming plasmid sequences. In this study, we describe another Histoplasma mechanism for maintaining transforming DNA i.e. the generation of modified, multicopy linear plasmids carrying DNA from the transforming Escherichia coli plasmid. Under selective conditions, these linear plasmids were stable and capable of retransforming Histoplasma without further modification. In vivo modification of the transforming DNA included duplication of plasmid sequence and telomeric addition at the termini of linear DNA. Apparently Histoplasma telomerase, like that of other organisms such as humans and Tetrahymena, is able to act on non-telomeric substrates. The terminus of a Histoplasma linear plasmid was cloned and shown to contain multiple repeats of GGGTTA, the telomeric repeat unit also found in vertebrates, trypanosomes, and slime moulds. PMID:1474902

  5. Linear plasmids mobilize linear but not circular chromosomes in Streptomyces: support for the 'end first' model of conjugal transfer.

    PubMed

    Lee, Hsuan-Hsuan; Hsu, Chin-Chen; Lin, Yen-Ling; Chen, Carton W

    2011-09-01

    Gram-positive bacteria of the genus Streptomyces possess linear chromosomes and linear plasmids capped by terminal proteins covalently bound to the 5' ends of the DNA. The linearity of Streptomyces chromosomes raises the question of how they are transferred during conjugation, particularly when the mobilizing plasmids are also linear. The classical rolling circle replication model for transfer of circular plasmids and chromosomes from an internal origin cannot be applied to this situation. Instead it has been proposed that linear Streptomyces plasmids mobilize themselves and the linear chromosomes from their telomeres using terminal-protein-primed DNA synthesis. In support of this 'end first' model, we found that artificially circularized Streptomyces chromosomes could not be mobilized by linear plasmids (SLP2 and SCP1), while linear chromosomes could. In comparison, a circular plasmid (pIJ303) could mobilize both circular and linear chromosomes at the same efficiencies. Interestingly, artificially circularized SLP2 exhibited partial self-transfer capability, indicating that, being a composite replicon, it may have acquired the additional internal origin of transfer from an ancestral circular plasmid during evolution.

  6. Self-excising integrative yeast plasmid vectors containing an intronated recombinase gene.

    PubMed

    Agaphonov, Michael; Alexandrov, Alexander

    2014-11-01

    Site-specific recombinases are widely used for selectable marker recycling in molecular-genetic manipulations with eukaryotic cells. This usually involves the use of two genetic constructs, one of which possesses a selectable marker flanked by the recombinase recognition sequences, while the other one bears the recombinase gene. Combining the recombinase gene with its recognition sequences in one plasmid is usually avoided, as it may lead to undesirable recombination due to promoter leakage, while the plasmid is maintained in Escherichia coli cells. Here, we describe yeast vectors possessing Cre recombinase genes under control of regulatable yeast promoters and loxP sequences for the in vivo vector backbone excision. The plasmid stability in E. coli is ensured by the presence of an intron in the recombinase gene. Applicability of these vectors was validated by disruptions of the Hansenula polymorpha PMC1 and Saccharomyces cerevisiae HSP104 and PRB1 genes.

  7. Genetic transformation of Brassica nigra by agrobacterium based vector and direct plasmid uptake.

    PubMed

    Gupta, V; Lakshmi Sita, G; Shaila, M S; Jagannathan, V

    1993-05-01

    Genetic transformation systems have been established for Brassica nigra (cv. IC 257) by using an Agrobacterium binary vector as well as by direct DNA uptake of a plasmid vector. Both the type of vectors carried nptII gene and gus gene. For Agrobacterium mediated transformation, hypocotyl tissue explants were used, and up to 33% of the explants produced calli on selection medium. All of these expressed B-glucuronidase gene on histochemical staining. Protoplasts isolated from hypocotyl tissues of seedlings could be transformed with a plasmid vector by FEG mediated uptake of vector DNA. A number of fertile kanamycin resistant plants were obtained using both the methods, and their transformed nature was confirmed by Southern blot analysis and histochemical staining for GUS. Backcrossed and selfed progenies of these transformed plants showed the presence of npt and gus genes. PMID:24197344

  8. Isolation and characterization of linear deoxyribonucleic acid plasmids from Kluyveromyces lactis and the plasmid-associated killer character.

    PubMed Central

    Gunge, N; Tamaru, A; Ozawa, F; Sakaguchi, K

    1981-01-01

    Two linear deoxyribonucleic acid plasmids, designated pGK11 and pGK12, were isolated from the yeast Kluyveromyces lactis IFO 1267. pGK11 and pGK12 had molecular weights of 5.4 X 10(6) and 8.4 X 10(6), respectively. Both plasmids possessed the same density of 1.687 g/cm3, lighter than the densities of mitochondrial (1.692 g/cm3) and nuclear (1.699 g/cm3) deoxyribonucleic acids. A restriction map of pGK11 was constructed from digestions by EcoRI, HindIII, PstI, and BamHI. pGK12 was cleaved by EcoRI into seven fragments and by BamHI into two fragments K. lactis IFO 1267 killed Saccharomyces cerevisiae sensitive and killer strains and certain strains of Saccharomyces italicus, K. lactis, Kluyveromyces thermotolerans, and K. vanudenii. All K. lactis strains lacking the pGK1 plasmids were nonkillers. A hybrid was constructed between K. lactis IFO 1267 and a nonkiller K. lactis strain lacking the plasmids and subjected to tetrad analysis after sporulation. The killer character was extrachromosomally transmitted in all tetrads in association with the pGK1 plasmids. The double-stranded ribonucleic acid killer plasmid could not be detected in any K. lactis killer strains. It is thus highly probable that the killer character is mediated by the linear deoxyribonucleic acid plasmids. A single chromosomal gene was found which was responsible for the resistance to the K. lactis killer. Images PMID:6257636

  9. Limit cycles of linear vector fields on manifolds

    NASA Astrophysics Data System (ADS)

    Llibre, Jaume; Zhang, Xiang

    2016-10-01

    It is well known that linear vector fields on the manifold {{{R}}n} cannot have limit cycles, but this is not the case for linear vector fields on other manifolds. We study the periodic orbits of linear vector fields on different manifolds, and motivate and present an open problem on the number of limit cycles of linear vector fields on a class of {{C}1} connected manifold.

  10. Vectorization of linear discrete filtering algorithms

    NASA Technical Reports Server (NTRS)

    Schiess, J. R.

    1977-01-01

    Linear filters, including the conventional Kalman filter and versions of square root filters devised by Potter and Carlson, are studied for potential application on streaming computers. The square root filters are known to maintain a positive definite covariance matrix in cases in which the Kalman filter diverges due to ill-conditioning of the matrix. Vectorization of the filters is discussed, and comparisons are made of the number of operations and storage locations required by each filter. The Carlson filter is shown to be the most efficient of the filters on the Control Data STAR-100 computer.

  11. Streptomyces linear plasmids that contain a phage-like, centrally located, replication origin.

    PubMed

    Chang, P C; Kim, E S; Cohen, S N

    1996-12-01

    Unlike previously studied linear replicons containing 5' DNA termini covalently bound to protein, pSLA2, a 17 kb linear plasmid of Streptomyces rochei, initiates replication internally rather than at the telomeres (Chang and Cohen, 1994). Here we identify and characterize the replication origin of pSLA2, showing that it contains a series of direct repeats (iterons) within a centrally located gene encoding an essential DNA-binding protein (Rep1); a second essential protein (Rep2), which resembles prokaryotic DNA helicases and has ATPase activity stimulated by single-stranded DNA, is expressed from the same transcript. A 430 bp locus separated by almost 2 kb from the iterons of the origin specifies an as yet undefined additional function required in cis for plasmid replication. pSCL, a 12 kb linear plasmid of Streptomyces clavuligerus, contains, near the centre of the plasmid, a region configured like the pSLA2 origin. The replication regions of pSLA2 and pSCL, which are capable of propagating plasmid DNA in either a circular or linear form (Shiffman and Cohen, 1992; Chang and Cohen, 1994) resemble those of temperate bacteriophages of the Enterobacteriacae and Bacillus. Our observations suggest that Streptomyces linear plasmids may occupy an evolutionarily intermediate position between circular plasmids and linear phage replicons.

  12. Expression of cellulase genes in Rhodobacter capsulatus by use of plasmid expression vectors.

    PubMed Central

    Johnson, J A; Wong, W K; Beatty, J T

    1986-01-01

    Broad-host-range plasmid vectors were constructed for expression of heterologous genes in the photosynthetic bacterium Rhodobacter capsulatus. These plasmids utilize an RK2-derived replicon for maintenance and conjugative transfer and the R. capsulatus rxcA promoter to obtain transcription of genes within appropriately positioned DNA fragments. The expression vectors were used to obtain synthesis of endoglucanase and exoglucanase in R. capsulatus from cellulase genes present on exogenously derived DNA fragments. The cellulase genes were expressed either by use of their native translation initiation signals or by in-frame fusion with the rxcA B870 beta gene translation initiation signals to form a hybrid protein. The level of cellulase gene expression was found to be modulated in response to the extent of aeration of plasmid host cultures. Images PMID:3090019

  13. Optimizing a Method for the Quantification by Quantitative Real-Time Polymerase Chain Reaction of Host Cell DNA in Plasmid Vector Batches Used in Human Gene Therapy.

    PubMed

    Ferro, Serge; Fabre, Isabelle; Chenivesse, Xavier

    2016-08-01

    Gene therapy products are very complex advanced therapy medicinal products produced using different processes that require many chemical and biological reagents and production intermediates, such as producing cells. The quantification of residual impurities in gene therapy vectors is a major quality control step when these vectors are used for therapeutic purposes, whether or not they are derived from viruses. Indeed, in nonviral gene therapy products, particularly plasmid vectors used to transfer genetic material, the presence of host-cell DNA (HCDNA) from the bacterial cells used for the vector production is an important concern because of the risk of immunogenicity and insertional mutagenesis. Several methods have been developed to quantify residual HCDNA, but real-time quantitative polymerase chain reaction (qPCR) seems to be most suitable because it allows detecting traces of "contaminating" DNA. The French National Agency for Medicines and Health Products Safety (ANSM) ensures the quality and safety of gene transfer medicinal products and must be able to quantify, in its own laboratories, the amount of HCDNA present in plasmid vector batches. Therefore, we developed and validated a qPCR method to quantify at the femtogram level the presence of Escherichia coli residual DNA in plasmid vectors. This approach uses the capillary-based LightCycler 1.5 System (Roche) with SYBR Green I, a primer pair against the E. coli 23S ribosomal RNA gene and different concentrations of a linearized plasmid that contains the 23S target sequence, as standard. This qPCR method is linear on an 8-decade logarithmic scale, accurate, reproducible, and sensitive (quantification of up to 10 copies of 23S target sequence per reaction, or 1.4 E. coli genome, or 7 fg of bacterial DNA). This technique allows ensuring that batches of plasmid vectors to be used in clinical trials comply with the specifications on HCDNA content.

  14. Stable plasmid vectors for complementation of Xylella fastidiosa mutants in planta

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Current understanding of the mechanisms of Pierce’s Disease development has been significantly advanced by molecular genetic studies of the causal agent, Xylella fastidiosa (Xf). Plasmid vectors are an essential tool for studies of bacterial genetics and pathogenesis. However, most commonly used pla...

  15. Strategies and approaches in plasmidome studies-uncovering plasmid diversity disregarding of linear elements?

    PubMed

    Dib, Julián R; Wagenknecht, Martin; Farías, María E; Meinhardt, Friedhelm

    2015-01-01

    The term plasmid was originally coined for circular, extrachromosomal genetic elements. Today, plasmids are widely recognized not only as important factors facilitating genome restructuring but also as vehicles for the dissemination of beneficial characters within bacterial communities. Plasmid diversity has been uncovered by means of culture-dependent or -independent approaches, such as endogenous or exogenous plasmid isolation as well as PCR-based detection or transposon-aided capture, respectively. High-throughput-sequencing made possible to cover total plasmid populations in a given environment, i.e., the plasmidome, and allowed to address the quality and significance of self-replicating genetic elements. Since such efforts were and still are rather restricted to circular molecules, here we put equal emphasis on the linear plasmids which-despite their frequent occurrence in a large number of bacteria-are largely neglected in prevalent plasmidome conceptions. PMID:26074886

  16. Strategies and approaches in plasmidome studies—uncovering plasmid diversity disregarding of linear elements?

    PubMed Central

    Dib, Julián R.; Wagenknecht, Martin; Farías, María E.; Meinhardt, Friedhelm

    2015-01-01

    The term plasmid was originally coined for circular, extrachromosomal genetic elements. Today, plasmids are widely recognized not only as important factors facilitating genome restructuring but also as vehicles for the dissemination of beneficial characters within bacterial communities. Plasmid diversity has been uncovered by means of culture-dependent or -independent approaches, such as endogenous or exogenous plasmid isolation as well as PCR-based detection or transposon-aided capture, respectively. High-throughput-sequencing made possible to cover total plasmid populations in a given environment, i.e., the plasmidome, and allowed to address the quality and significance of self-replicating genetic elements. Since such efforts were and still are rather restricted to circular molecules, here we put equal emphasis on the linear plasmids which—despite their frequent occurrence in a large number of bacteria—are largely neglected in prevalent plasmidome conceptions. PMID:26074886

  17. Linear Plasmids and the Rate of Sequence Evolution in Plant Mitochondrial Genomes.

    PubMed

    Warren, Jessica M; Simmons, Mark P; Wu, Zhiqiang; Sloan, Daniel B

    2016-01-11

    The mitochondrial genomes of flowering plants experience frequent insertions of foreign sequences, including linear plasmids that also exist in standalone forms within mitochondria, but the history and phylogenetic distribution of plasmid insertions is not well known. Taking advantage of the increased availability of plant mitochondrial genome sequences, we performed phylogenetic analyses to reconstruct the evolutionary history of these plasmids and plasmid-derived insertions. Mitochondrial genomes from multiple land plant lineages (including liverworts, lycophytes, ferns, and gymnosperms) include fragmented remnants from ancient plasmid insertions. Such insertions are much more recent and widespread in angiosperms, in which approximately 75% of sequenced mitochondrial genomes contain identifiable plasmid insertions. Although conflicts between plasmid and angiosperm phylogenies provide clear evidence of repeated horizontal transfers, we were still able to detect significant phylogenetic concordance, indicating that mitochondrial plasmids have also experienced sustained periods of (effectively) vertical transmission in angiosperms. The observed levels of sequence divergence in plasmid-derived genes suggest that nucleotide substitution rates in these plasmids, which often encode their own viral-like DNA polymerases, are orders of magnitude higher than in mitochondrial chromosomes. Based on these results, we hypothesize that the periodic incorporation of mitochondrial genes into plasmids contributes to the remarkable heterogeneity in substitution rates among genes that has recently been discovered in some angiosperm mitochondrial genomes. In support of this hypothesis, we show that the recently acquired ψtrnP-trnW gene region in a maize linear plasmid is evolving significantly faster than homologous sequences that have been retained in the mitochondrial chromosome in closely related grasses.

  18. Linear Plasmids and the Rate of Sequence Evolution in Plant Mitochondrial Genomes.

    PubMed

    Warren, Jessica M; Simmons, Mark P; Wu, Zhiqiang; Sloan, Daniel B

    2016-02-01

    The mitochondrial genomes of flowering plants experience frequent insertions of foreign sequences, including linear plasmids that also exist in standalone forms within mitochondria, but the history and phylogenetic distribution of plasmid insertions is not well known. Taking advantage of the increased availability of plant mitochondrial genome sequences, we performed phylogenetic analyses to reconstruct the evolutionary history of these plasmids and plasmid-derived insertions. Mitochondrial genomes from multiple land plant lineages (including liverworts, lycophytes, ferns, and gymnosperms) include fragmented remnants from ancient plasmid insertions. Such insertions are much more recent and widespread in angiosperms, in which approximately 75% of sequenced mitochondrial genomes contain identifiable plasmid insertions. Although conflicts between plasmid and angiosperm phylogenies provide clear evidence of repeated horizontal transfers, we were still able to detect significant phylogenetic concordance, indicating that mitochondrial plasmids have also experienced sustained periods of (effectively) vertical transmission in angiosperms. The observed levels of sequence divergence in plasmid-derived genes suggest that nucleotide substitution rates in these plasmids, which often encode their own viral-like DNA polymerases, are orders of magnitude higher than in mitochondrial chromosomes. Based on these results, we hypothesize that the periodic incorporation of mitochondrial genes into plasmids contributes to the remarkable heterogeneity in substitution rates among genes that has recently been discovered in some angiosperm mitochondrial genomes. In support of this hypothesis, we show that the recently acquired ψtrnP-trnW gene region in a maize linear plasmid is evolving significantly faster than homologous sequences that have been retained in the mitochondrial chromosome in closely related grasses. PMID:26759362

  19. Linear Plasmids and the Rate of Sequence Evolution in Plant Mitochondrial Genomes

    PubMed Central

    Warren, Jessica M.; Simmons, Mark P.; Wu, Zhiqiang; Sloan, Daniel B.

    2016-01-01

    The mitochondrial genomes of flowering plants experience frequent insertions of foreign sequences, including linear plasmids that also exist in standalone forms within mitochondria, but the history and phylogenetic distribution of plasmid insertions is not well known. Taking advantage of the increased availability of plant mitochondrial genome sequences, we performed phylogenetic analyses to reconstruct the evolutionary history of these plasmids and plasmid-derived insertions. Mitochondrial genomes from multiple land plant lineages (including liverworts, lycophytes, ferns, and gymnosperms) include fragmented remnants from ancient plasmid insertions. Such insertions are much more recent and widespread in angiosperms, in which approximately 75% of sequenced mitochondrial genomes contain identifiable plasmid insertions. Although conflicts between plasmid and angiosperm phylogenies provide clear evidence of repeated horizontal transfers, we were still able to detect significant phylogenetic concordance, indicating that mitochondrial plasmids have also experienced sustained periods of (effectively) vertical transmission in angiosperms. The observed levels of sequence divergence in plasmid-derived genes suggest that nucleotide substitution rates in these plasmids, which often encode their own viral-like DNA polymerases, are orders of magnitude higher than in mitochondrial chromosomes. Based on these results, we hypothesize that the periodic incorporation of mitochondrial genes into plasmids contributes to the remarkable heterogeneity in substitution rates among genes that has recently been discovered in some angiosperm mitochondrial genomes. In support of this hypothesis, we show that the recently acquired ψtrnP-trnW gene region in a maize linear plasmid is evolving significantly faster than homologous sequences that have been retained in the mitochondrial chromosome in closely related grasses. PMID:26759362

  20. Construction and Characterization of an in-vivo Linear Covalently Closed DNA Vector Production System

    PubMed Central

    2012-01-01

    Background While safer than their viral counterparts, conventional non-viral gene delivery DNA vectors offer a limited safety profile. They often result in the delivery of unwanted prokaryotic sequences, antibiotic resistance genes, and the bacterial origins of replication to the target, which may lead to the stimulation of unwanted immunological responses due to their chimeric DNA composition. Such vectors may also impart the potential for chromosomal integration, thus potentiating oncogenesis. We sought to engineer an in vivo system for the quick and simple production of safer DNA vector alternatives that were devoid of non-transgene bacterial sequences and would lethally disrupt the host chromosome in the event of an unwanted vector integration event. Results We constructed a parent eukaryotic expression vector possessing a specialized manufactured multi-target site called “Super Sequence”, and engineered E. coli cells (R-cell) that conditionally produce phage-derived recombinase Tel (PY54), TelN (N15), or Cre (P1). Passage of the parent plasmid vector through R-cells under optimized conditions, resulted in rapid, efficient, and one step in vivo generation of mini lcc—linear covalently closed (Tel/TelN-cell), or mini ccc—circular covalently closed (Cre-cell), DNA constructs, separated from the backbone plasmid DNA. Site-specific integration of lcc plasmids into the host chromosome resulted in chromosomal disruption and 105 fold lower viability than that seen with the ccc counterpart. Conclusion We offer a high efficiency mini DNA vector production system that confers simple, rapid and scalable in vivo production of mini lcc DNA vectors that possess all the benefits of “minicircle” DNA vectors and virtually eliminate the potential for undesirable vector integration events. PMID:23216697

  1. Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection.

    PubMed

    Burbank, Lindsey P; Stenger, Drake C

    2016-08-01

    The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention.

  2. Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection.

    PubMed

    Burbank, Lindsey P; Stenger, Drake C

    2016-08-01

    The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention. PMID:27088393

  3. The covalent bioconjugate of multiwalled carbon nanotube and amino-modified linearized plasmid DNA for gene delivery.

    PubMed

    Geyik, Caner; Evran, Serap; Timur, Suna; Telefoncu, Azmi

    2014-01-01

    Carbon nanotubes (CNTs) are allotropes of carbon, which have unique physical, mechanical, and electronic properties. Among various biomedical applications, CNTs also attract interest as nonviral gene delivery systems. Functionalization of CNTs with cationic groups enables delivery of negatively charged DNA into cells. In contrast to this well-known strategy for DNA delivery, our approach included the covalent attachment of linearized plasmid DNA to carboxylated multiwalled CNTs (MWCNTs). Carboxyl groups were introduced onto MWCNTs by oxidative treatment, and then the carboxyl groups were activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The whole pQE-70 vector including the gene encoding green fluorescent protein (GFP) was subjected to polymerase chain reaction (PCR) using the modified nucleotide N6-(6-Amino)hexyl-2'-deoxyadenosine-5'-triphosphate. Hence, free amino groups were introduced onto the linearized plasmid. Covalent bonding between the amino-modified plasmid DNA and the carboxylated MWCNTs was achieved via EDC chemistry. The resulting bioconjugate was successfully transformed into chemically competent Escherichia coli cells, without necessity of a heat-shock step at 42°C. The presence of Ca(2+) in transformation medium was required to neutralize the electrostatic repulsion between DNA and negatively charged outer layer of E. coli. The transformants, which were able to express GFP were inspected manually on ampicillin agar plates. Our study represents a novelty with respect to other noncovalent CNT gene delivery systems. Considering the interest for delivery of linear DNA fragments, our study could give insights into further studies.

  4. Sequence analysis of a cryptic plasmid pKW2124 from Weissella cibaria KLC140 and construction of a surface display vector.

    PubMed

    Kim, Soo Young; Oh, Chang Geun; Lee, Young Joo; Choi, Kyu Ha; Shin, Doo Sik; Lee, Si Kyung; Park, Kab Joo; Shin, Hakdong; Park, Myeong Soo; Lee, Ju-Hoon

    2013-04-01

    Plasmid isolation of kimchi-derived Weissella cibaria KLC140 revealed six different plasmids. The smallest plasmid, pKW2124, was DNA sequenced and characterized, showing 2,124 bp with a GC content of 36.39% and five putative open reading frames (ORFs). In silico analysis of these ORFs showed ORF1 encodes a putative replication protein similar to rolling circular replication proteins from other lactic acid bacteria. However, a single-stranded intermediate was not detected when S1 nuclease was treated, suggesting it may follow theta replication. Interestingly, the replication initiation site of this plasmid is 100% identical to other plasmids from lactic acid bacteria, suggesting it may function for replication initiation. To construct a surface layer expression vector, pTSLGFP, slpA encoding the surface layer protein from Lactobacillus acidophilus was PCR amplified and fused with the gfp gene, forming a SLGFP fused gene. The plasmid pKW2124 was cloned into the XbaI site of pUC19, forming an Weissella-E. coli shuttle vector pKUW22. NheI-linearized pTSLGFP was ligated into pKUWCAT containing pKUW22 and the chloramphenicol acetyltransferase gene from pEK104, resulting in an 8.6 kb pKWCSLGFP surface layer expression vector. After transformation of this vector into W. cibaria KLC140, a GFP fluorescence signal was detected on the surface of the transformant, substantiating production of SLGFP fused protein and its secretion. This is the first report for construction of a Weissella surface layer expression vector, which may be useful for surface layer production of beneficial proteins in Weissella.

  5. A replicative plasmid vector allows efficient complementation of pathogenic Leptospira strains.

    PubMed

    Pappas, Christopher J; Benaroudj, Nadia; Picardeau, Mathieu

    2015-05-01

    Leptospirosis, an emerging zoonotic disease, remains poorly understood because of a lack of genetic manipulation tools available for pathogenic leptospires. Current genetic manipulation techniques include insertion of DNA by random transposon mutagenesis and homologous recombination via suicide vectors. This study describes the construction of a shuttle vector, pMaORI, that replicates within saprophytic, intermediate, and pathogenic leptospires. The shuttle vector was constructed by the insertion of a 2.9-kb DNA segment including the parA, parB, and rep genes into pMAT, a plasmid that cannot replicate in Leptospira spp. and contains a backbone consisting of an aadA cassette, ori R6K, and oriT RK2/RP4. The inserted DNA segment was isolated from a 52-kb region within Leptospira mayottensis strain 200901116 that is not found in the closely related strain L. mayottensis 200901122. Because of the size of this region and the presence of bacteriophage-like proteins, it is possible that this region is a result of a phage-related genomic island. The stability of the pMaORI plasmid within pathogenic strains was tested by passaging cultures 10 times without selection and confirming the presence of pMaORI. Concordantly, we report the use of trans complementation in the pathogen Leptospira interrogans. Transformation of a pMaORI vector carrying a functional copy of the perR gene in a null mutant background restores the expression of PerR and susceptibility to hydrogen peroxide comparable to that of wild-type cells. In conclusion, we demonstrate the replication of a stable plasmid vector in a large panel of Leptospira strains, including pathogens. The shuttle vector described will expand our ability to perform genetic manipulation of Leptospira spp.

  6. Genetic vaccination against murine cysticercosis by using a plasmid vector carrying Taenia solium paramyosin.

    PubMed

    Solís, Carlos F; Ostoa-Saloma, Pedro; Lugo-Martínez, Verónica H; Johnston, Stephen Albert; Laclette, Juan Pedro

    2005-03-01

    A plasmid vector carrying the immunoprotective amino-terminal fragment of Taenia solium paramyosin (VW2-1) was designed for genetic vaccination studies. Mice that were genetically immunized with VW2-1 and challenged by intraperitoneal inoculation of Taenia crassiceps cysticerci showed 43 to 48% reductions in the parasite burden, values which were similar to values obtained previously when the recombinant protein was used.

  7. Development of a plasmid vector for easy selection of strong promoters.

    PubMed

    Piñeiro, S A; Sordelli, D O; Centrón, D

    2000-05-01

    A promoter vector pACPR33 for Escherichia coli based on the promotorless ampicillin-resistance gene from pBR322 has been constructed. The promoter of the ampicillin-resistance gene was deleted and replaced by a suitable multiple cloning site. Molecular cloning of promoters into the polylinker resulted in activation of the ampicillin resistance in E. coli. The plasmid contains a functional origin of DNA replication and a tetracycline resistance gene for E. coli, and a chloramphenicol resistance gene for S. aureus. The vector permitted direct detection of promoter activity, especially strong promoters, by easy iodometric determination of beta-lactamase activity in liquid or solid media.

  8. Construction of small plasmid vectors for use in genetic improvement of the extremely acidophilic Acidithiobacillus caldus.

    PubMed

    Meng, Jianzhou; Wang, Huiyan; Liu, Xiangmei; Lin, Jianqun; Pang, Xin; Lin, Jianqiang

    2013-10-01

    The genetic improvement of biomining bacteria including Acidithiobacillus caldus could facilitate the bioleaching process of sulfur-containing minerals. However, the available vectors for use in A. caldus are very scanty and limited to relatively large broad-host-range IncQ plasmids. In this study, a set of small, mobilizable plasmid vectors (pBBR1MCS-6, pMSD1 and pMSD2) were constructed based on plasmid pBBR1MCS-2, which does not belong to the IncQ, IncW, or IncP groups. The function of the tac promoter on 5.8-kb pMSD2 was determined by inserting a kanamycin-resistant reporter gene. The resulting recombinant pMSD2-Km was successfully transferred by conjugation into A. caldus MTH-04 with transfer frequency of 1.38±0.64×10(-5). The stability and plasmid copy number of pMSD2-Km in A. caldus MTH-04 were 75±2.7% and 5-6 copies per cell, respectively. By inserting an arsABC operon into pMSD2, an arsenic-resistant recombinant pMSD2-As was constructed and transferred into A. caldus MTH-04 by conjugation. The arsenic tolerance of A. caldus MTH-04 containing pMSD2-As was obviously increased up to 45mM of NaAsO2. These vectors could be applied in genetic improvement of A. caldus as well as other bioleaching bacteria.

  9. Linear polyethylenimine-plasmid DNA nanoparticles are ototoxic to the cultured sensory epithelium of neonatal mice.

    PubMed

    Zhou, Han; Ma, Xiaofeng; Liu, Yongze; Dong, Lei; Luo, Yi; Zhu, Guangjie; Qian, Xiaoyun; Chen, Jie; Lu, Lin; Wang, Junguo; Gao, Xia

    2015-06-01

    Polyethylenimine (PEI) has been demonstrated to be an effective non‑viral synthetic polymer for efficient gene delivery amongst various cell types in vitro and in vivo. In the present study, 25 kDa linear PEI (L‑PEI) was used to transfer plasmid DNA (pDNA), encoding the enhanced green fluorescent protein reporter gene, into the cultured cochlear epithelium of neonatal mice. The 25 kDa L‑PEI/pDNA nanoparticles were generated in phosphate‑buffered saline prior to transfection. Sensory epithelial cells were transfected using an appropriate weight ratio of L‑PEI and pDNA. However, the use of higher L‑PEI/pDNA ratios, which result in the generation of a greater number of nanoparticles, induced significant damage to the sensory epithelial cells, as demonstrated by immunofluorescent and transmission electron microscopy analyses. These results indicated that precautionary measures are required with regard to the use of PEI nanoparticles in nanomedicine, and emphasized the requirement for thorough physicochemical characterization and toxicity testing for each polymer vector prior to the construction of nanotechnology systems for use in clinical applications. The development of effective management techniques for potential nano‑ototoxicity risks is of considerable significance to the clinical applications of nanoparticles. PMID:25652676

  10. Development of a novel plasmid vector pTIO-1 adapted for electrotransformation of Porphyromonas gingivalis.

    PubMed

    Tagawa, Junpei; Inoue, Tetsuyoshi; Naito, Mariko; Sato, Keiko; Kuwahara, Tomomi; Nakayama, Masaaki; Nakayama, Koji; Yamashiro, Takashi; Ohara, Naoya

    2014-10-01

    We report here the construction of a plasmid vector designed for the efficient electrotransformation of the periodontal pathogen Porphyromonas gingivalis. The novel Escherichia coli-Bacteroides/P. gingivalis shuttle vector, designated pTIO-1, is based on the 11.0-kb E. coli-Bacteroides conjugative shuttle vector, pVAL-1 (a pB8-51 derivative). To construct pTIO-1, the pB8-51 origin of replication and erythromycin resistance determinant of pVAL-1 were cloned into the E. coli cloning vector pBluescript II SK(-) and non-functional regions were deleted. pTIO-1 has an almost complete multiple cloning site from pBluescript II SK(-). The size of pTIO-1 is 4.5kb, which is convenient for routine gene manipulation. pTIO-1 was introduced into P. gingivalis via electroporation, and erythromycin-resistant transformants carrying pTIO-1 were obtained. We characterized the transformation efficiency, copy number, host range, stability, and insert size capacity of pTIO-1. An efficient plasmid electrotransformation of P. gingivalis will facilitate functional analysis and expression of P. gingivalis genes, including the virulence factors of this bacterium.

  11. Structure/load dependent vectors for linear structural dynamic analysis

    NASA Technical Reports Server (NTRS)

    Qin, Jiangning; Nguyen, Duc T.

    1992-01-01

    The dynamic solution vectors yielded by the present structure/load dependent-vectors method for large-scale linear structural dynamic analyses involving complex loadings can be used as starting vectors, so that both structure and load characteristics are encompassed by the basis vectors. The method is shown to entail fewer vectors than current alternatives for a given level of accuracy, especially in the cases of structures that have external concentrated masses. Numerical results are presented which illustrate the advantages of this dependent-vectors method relative to other reduction methods.

  12. Advances in host and vector development for the production of plasmid DNA vaccines.

    PubMed

    Mairhofer, Juergen; Lara, Alvaro R

    2014-01-01

    Recent developments in DNA vaccine research provide a new momentum for this rather young and potentially disruptive technology. Gene-based vaccines are capable of eliciting protective immunity in humans to persistent intracellular pathogens, such as HIV, malaria, and tuberculosis, for which the conventional vaccine technologies have failed so far. The recent identification and characterization of genes coding for tumor antigens has stimulated the development of DNA-based antigen-specific cancer vaccines. Although most academic researchers consider the production of reasonable amounts of plasmid DNA (pDNA) for immunological studies relatively easy to solve, problems often arise during this first phase of production. In this chapter we review the current state of the art of pDNA production at small (shake flasks) and mid-scales (lab-scale bioreactor fermentations) and address new trends in vector design and strain engineering. We will guide the reader through the different stages of process design starting from choosing the most appropriate plasmid backbone, choosing the right Escherichia coli (E. coli) strain for production, and cultivation media and scale-up issues. In addition, we will address some points concerning the safety and potency of the produced plasmids, with special focus on producing antibiotic resistance-free plasmids. The main goal of this chapter is to make immunologists aware of the fact that production of the pDNA vaccine has to be performed with as much as attention and care as the rest of their research.

  13. Delivery of an adenovirus vector plasmid by ultrapure oligochitosan based polyplexes.

    PubMed

    Agirre, Mireia; Zarate, Jon; Ojeda, Edilberto; Puras, Gustavo; Rojas, Luis A; Alemany, Ramón; Pedraz, José L

    2015-02-20

    Ultrapure oligochitosans have been recently reported as efficient non-viral vectors for the delivery of pCMS-EGFP plasmid (5.5kbp) to the cornea and retina. However, the delivery of oncolytic adenoviral plasmids (40kbp) represents a unique challenge. In this work, we elaborated self assembled O15 and O25 UOC/pAdTLRGD polyplexes, and we studied the influence of the N/P ratio, the pH of the transfection medium and the salt concentration on the particle size and zeta potential by an orthogonal experimental design. All polyplexes showed a particle size lower than 200nm and a positive zeta potential. These parameters were influenced by the N/P ratio, salt concentration, and pH of the transfection medium. The selected polyplexes were able to bind, release, and protect the plasmid from DNase degradation. Transfection experiments in HEK293 and A549 cell lines demonstrated that UOC/pAdTLRGD polyplexes were able to deliver the plasmid and transfect both cell lines. These results suggest that O15 and O25 UOC based polyplexes are suitable for future in vivo applications.

  14. A versatile low-copy-number cloning vector derived from plasmid F.

    PubMed

    Shi, J; Biek, D P

    1995-10-16

    We have constructed a cloning vector based on plasmid mini-F for use in Escherichia coli. Plasmid pZC320 consists of the ori-2 replication unit of F that confers very low copy number (lcn), and includes the sop partition functions to insure stable plasmid maintenance in the absence of selection. A multiple cloning site (MCS) containing 16 unique restriction sites is located within the 5' end of the lacZ alpha gene. Expression of lacZ alpha is under the control of the wild-type lactose operator/promoter (lacOP) region and is efficiently repressed by the lacI repressor. Clones containing inserts can be detected using the blue/white screen for beta-galactosidase (beta Gal). A T7 promoter allows transcription of cloned inserts in the presence of T7 RNA polymerase. We have demonstrated the use of this lcn vector for cloning the regulated tetracycline-resistance genes from Tn10, which confer only low-level resistance when present at high copy number.

  15. A Mini-intronic Plasmid (MIP): A Novel Robust Transgene Expression Vector In Vivo and In Vitro

    PubMed Central

    Lu, Jiamiao; Zhang, Feijie; Kay, Mark A

    2013-01-01

    The bacterial backbone (BB) sequences contained within a canonical plasmid DNA dampen exogenous transgene expression by tenfold to 1,000-fold over a period of a few weeks following transfection into quiescent tissues such as the liver. Minicircle DNA vectors devoid of bacterial plasmid backbone sequences overcome transgene silencing providing persistent transgene expression. Because, we recently established that the length rather than sequence of the DNA flanking the transgene expression cassette is the major parameter affecting transgene silencing, we developed an alternative plasmid propagation process in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within the eukaryotic expression cassette. As with the minicircle vector, the mini-intronic plasmid (MIP) vector system overcomes transgene silencing observed with plasmids but in addition provides between 2 and often 10 times or higher levels of transgene expression compared with minicircle vectors containing the same expression cassette in vivo and in vitro. These improved plasmids will benefit all studies involving gene transfer/therapy approaches. PMID:23459514

  16. A mini-intronic plasmid (MIP): a novel robust transgene expression vector in vivo and in vitro.

    PubMed

    Lu, Jiamiao; Zhang, Feijie; Kay, Mark A

    2013-05-01

    The bacterial backbone (BB) sequences contained within a canonical plasmid DNA dampen exogenous transgene expression by tenfold to 1,000-fold over a period of a few weeks following transfection into quiescent tissues such as the liver. Minicircle DNA vectors devoid of bacterial plasmid backbone sequences overcome transgene silencing providing persistent transgene expression. Because, we recently established that the length rather than sequence of the DNA flanking the transgene expression cassette is the major parameter affecting transgene silencing, we developed an alternative plasmid propagation process in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within the eukaryotic expression cassette. As with the minicircle vector, the mini-intronic plasmid (MIP) vector system overcomes transgene silencing observed with plasmids but in addition provides between 2 and often 10 times or higher levels of transgene expression compared with minicircle vectors containing the same expression cassette in vivo and in vitro. These improved plasmids will benefit all studies involving gene transfer/therapy approaches.

  17. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.

    PubMed

    Bierman, M; Logan, R; O'Brien, K; Seno, E T; Rao, R N; Schoner, B E

    1992-07-01

    We have constructed cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. All vectors contain the 760-bp oriT fragment from the IncP plasmid, RK2. Transfer functions need to be supplied in trans by the E. coli donor strain. We have incorporated into these vectors selectable antibiotic-resistance markers (AmR, ThR, SpR) that function in Streptomyces spp. and other features that should allow for: (i) integration via homologous recombination between cloned DNA and the Streptomyces spp. chromosome, (ii) autonomous replication, or (iii) site-specific integration at the bacteriophage phi C31 attachment site. Shuttle cosmids for constructing genomic libraries and bacteriophage P1 cloning vector capable of accepting approx. 100-kb fragments are also described. A simple mating procedure has been developed for the conjugal transfer of these vectors from E. coli to Streptomyces spp. that involves plating of the donor strain and either germinated spores or mycelial fragments of the recipient strain. We have shown that several of these vectors can be introduced into Streptomyces fradiae, a strain that is notoriously difficult to transform by PEG-mediated protoplast transformation.

  18. Multiple shRNA expressions in a single plasmid vector improve RNAi against the XPA gene

    SciTech Connect

    Nagao, Akihiro; Zhao, Xia; Takegami, Tsutomu; Nakagawa, Hideaki; Matsui, Shinobu; Matsunaga, Tsukasa; Ishigaki, Yasuhito

    2008-05-30

    To improve the efficiency of stable knockdown with short hairpin RNA (shRNA), we inserted multiple shRNA expression sequences into a single plasmid vector. In this study, the DNA repair factor XPA was selected as a target gene since it is not essential for cell viability and it is easy to check the functional knockdown of this gene. The efficiency of knockdown was compared among single and triple expression vectors. The single shRNA-expressing vector caused limited knockdown of the target protein in stable transfectants, however, the multiple expression vectors apparently increased the frequency of knockdown transfectants. There were correlations between the knockdown level and marker expression in multiple-expressing transfectants, whereas poorer correlations were observed in single vector transfectants. Multiple-transfectants exhibited reduced efficiency of repair of UV-induced DNA damage and an increased sensitivity to ultraviolet light-irradiation. We propose that multiple shRNA expression vectors might be a useful strategy for establishing knockdown cells.

  19. Linear plasmid DNAs of the plant pathogenic fungus Rhizoctonia solani with unique terminal structures.

    PubMed

    Miyashita, S; Hirochika, H; Ikeda, J E; Hashiba, T

    1990-01-01

    Three linear DNA plasmids were found in isolate RI-64 of anastomosis group 4 (AG-4) of Rhizoctonia solani. These plasmids, designated pRS64-1, -2, and -3, possessed the same size of 2.7 kb. Restriction mapping and Southern hybridization analysis of pRS64-1, -2, and -3 revealed the presence of homologous regions at both termini. The plasmid DNAs were resistant to both 3'-exonuclease and 5'-exonuclease even after treatment with proteinase K or alkali. The length of both terminal fragments that were generated by restriction endonuclease digestion was doubled under the denaturation condition, indicating that the linear plasmid DNAs have hairpin loops at both termini. Southern blotting analysis of total DNA showed the presence of two types of dimeric forms of pRS64 DNA. One is a head-to-head dimer and the other is a tail-to-tail dimer. The role of these unique DNA structures in replication of the plasmids is discussed.

  20. Successful transfer of plasmid DNA into in vitro cells transfected with an inorganic plasmid-Mg/Al-LDH nanobiocomposite material as a vector for gene expression

    NASA Astrophysics Data System (ADS)

    Jaffri Masarudin, Mas; Yusoff, Khatijah; Rahim, Raha Abdul; Zobir Hussein, Mohd

    2009-01-01

    The delivery of a full plasmid, encoding the green fluorescent protein gene into African monkey kidney (Vero3) cells, was successfully achieved using nanobiocomposites based on layered double hydroxides. This demonstrated the potential of using the system as an alternative DNA delivery vector. Intercalation of the circular plasmid DNA, pEGFP-N2, into Mg/Al-NO3- layered double hydroxides (LDH) was accomplished through anion exchange routes to form the nanobiocomposite material. The host was previously synthesized at the Mg2+ to Al3+ molar ratio Ri = 2 and subsequently intercalated with plasmid DNA. Size expansion of the interlamellae host from 8.8 Å in LDH to 42 Å was observed in the resulting nanobiocomposite, indicating stable hybridization of the plasmid DNA. The powder x-ray diffraction (PXRD) results, supplemented with Fourier-transform infrared (FTIR) spectroscopy, compositional and electrophoresis studies confirmed the encapsulation episode of the biomaterial. In order to elucidate the use of this resulting nanobiocomposite as a delivery vector, an MTT assay was performed to determine any cytotoxic effects of the host towards cells. The intercalated pEGFP-N2 anion was later successfully recovered through acidification with HNO3 after treatment with DNA-degrading enzymes, thus also showing the ability of the LDH host to protect the intercalated biomaterial from degradation. Cell transfection studies on Vero3 cells were then performed, where cells transfected with the nanobiocomposite exhibited fluorescence as early as 12 h post-treatment compared to naked delivery of the plasmid itself.

  1. Physical Characterization of Gemini Surfactant-Based Synthetic Vectors for the Delivery of Linear Covalently Closed (LCC) DNA Ministrings.

    PubMed

    Sum, Chi Hong; Nafissi, Nafiseh; Slavcev, Roderick A; Wettig, Shawn

    2015-01-01

    In combination with novel linear covalently closed (LCC) DNA minivectors, referred to as DNA ministrings, a gemini surfactant-based synthetic vector for gene delivery has been shown to exhibit enhanced delivery and bioavailability while offering a heightened safety profile. Due to topological differences from conventional circular covalently closed (CCC) plasmid DNA vectors, the linear topology of LCC DNA ministrings may present differences with regards to DNA interaction and the physicochemical properties influencing DNA-surfactant interactions in the formulation of lipoplexed particles. In this study, N,N-bis(dimethylhexadecyl)-α,ω-propanediammonium(16-3-16)gemini-based synthetic vectors, incorporating either CCC plasmid or LCC DNA ministrings, were characterized and compared with respect to particle size, zeta potential, DNA encapsulation, DNase sensitivity, and in vitro transgene delivery efficacy. Through comparative analysis, differences between CCC plasmid DNA and LCC DNA ministrings led to variations in the physical properties of the resulting lipoplexes after complexation with 16-3-16 gemini surfactants. Despite the size disparities between the plasmid DNA vectors (CCC) and DNA ministrings (LCC), differences in DNA topology resulted in the generation of lipoplexes of comparable particle sizes. The capacity for ministring (LCC) derived lipoplexes to undergo complete counterion release during lipoplex formation contributed to improved DNA encapsulation, protection from DNase degradation, and in vitro transgene delivery.

  2. Elucidation of insertion elements carried on plasmids and in vitro construction of shuttle vectors from the toxic cyanobacterium Planktothrix.

    PubMed

    Christiansen, Guntram; Goesmann, Alexander; Kurmayer, Rainer

    2014-08-01

    Several gene clusters that are responsible for toxin synthesis in bloom-forming cyanobacteria have been found to be associated with transposable elements (TEs). In particular, insertion sequence (IS) elements were shown to play a role in the inactivation or recombination of the genes responsible for cyanotoxin synthesis. Plasmids have been considered important vectors of IS element distribution to the host. In this study, we aimed to elucidate the IS elements propagated on the plasmids and the chromosome of the toxic cyanobacterium Planktothrix agardhii NIVA-CYA126/8 by means of high-throughput sequencing. In total, five plasmids (pPA5.5, pPA14, pPA50, pPA79, and pPA115, of 5, 6, 50, 79, and 120 kbp, respectively) were elucidated, and two plasmids (pPA5.5, pPA115) were found to propagate full IS element copies. Large stretches of shared DNA information between plasmids were constituted of TEs. Two plasmids (pPA5.5, pPA14) were used as candidates to engineer shuttle vectors (named pPA5.5SV and pPA14SV, respectively) in vitro by PCR amplification and the subsequent transposition of the Tn5 cat transposon containing the R6Kγ origin of replication of Escherichia coli. While pPA5.5SV was found to be fully segregated, pPA14SV consistently co-occurred with its wild-type plasmid even under the highest selective pressure. Interestingly, the Tn5 cat transposon became transferred by homologous recombination into another plasmid, pPA50. The availability of shuttle vectors is considered to be of relevance in investigating genome plasticity as a consequence of homologous recombination events. Combining the potential of high-throughput sequencing and in vitro production of shuttle vectors makes it simple to produce species-specific shuttle vectors for many cultivable prokaryotes.

  3. A replicating plasmid-based vector for GFP expression in Mycoplasma hyopneumoniae.

    PubMed

    Ishag, H Z A; Liu, M J; Yang, R S; Xiong, Q Y; Feng, Z X; Shao, G Q

    2016-01-01

    Mycoplasma hyopneumoniae (M. hyopneumoniae) causes porcine enzootic pneumonia (PEP) that significantly affects the pig industry worldwide. Despite the availability of the whole genome sequence, studies on the pathogenesis of this organism have been limited due to the lack of a genetic manipulation system. Therefore, the aim of the current study was to generate a general GFP reporter vector based on a replicating plasmid. Here, we describe the feasibility of GFP reporter expression in M. hyopneumoniae (strain 168L) controlled by the p97 gene promoter of this mycoplasma. An expression plasmid (pMD18-TOgfp) containing the p97 gene promoter, and origin of replication (oriC) of M. hyopneumoniae, tetracycline resistant marker (tetM), and GFP was constructed and used to transform competent M. hyopneumoniae cells. We observed green fluorescence in M. hyopneumoniae transformants under fluorescence microscopy, which indicates that there was expression of the GFP reporter that was driven by the p97 gene promoter. Additionally, an electroporation method for M. hyopneumoniae with an efficiency of approximately 1 x 10(-6) transformants/μg plasmid DNA was optimized and is described herein. In conclusion, our data demonstrate the susceptibility of M. hyopneumoniae to genetic manipulation whereby foreign genes are expressed. This work may encourage the development of genetic tools to manipulate the genome of M. hyopneumoniae for functional genomic analyses. PMID:27173288

  4. Determination of protein expression and plasmid copy number from cloned genes in Escherichia coli by flow injection analysis using an enzyme indicator vector.

    PubMed

    Schendel, F J; Baude, E J; Flickinger, M C

    1989-10-20

    On-line determination of expression rates from cloned genes in Escherichia coli and of plasmid copy number would be useful for monitoring accumulation of non-secreted proteins. As an initial model for monitoring gene expression in intact cells, a non-gene-fusion enzyme-based indicator plasmid has been constructed containing the phoA gene coding for alkaline phosphatase (AP) in pUCIS and pACYC184. The activity of AP can be rapidly determined in permeabilized cells. A flow injection analysis (FIA) assay has been developed which allows the direct real-time measurement of the AP activity during cell growth. A model target gene coding for E. coli cyanase (cynS) has been inserted in order to determine the ratio between the expression of the target and indicator, AP. A linear relationship has been found between plasmid copy number and AP activity for the high-copy pUC vector. To minimize indicator expression, transcription terminators have been inserted between the cynS and phoA genes, altering the target-to-indicator ratio by 10- to 40-fold. These vectors may be useful for the rapid continuous determination of plasmid copy number and target gene expression for nonsecreted proteins and would overcome the limitations of in situ probe biosensors for real-time determination of the accumulation of proteins from cloned genes in E. coli.

  5. Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines

    PubMed Central

    Börner, Kathleen; Niopek, Dominik; Cotugno, Gabriella; Kaldenbach, Michaela; Pankert, Teresa; Willemsen, Joschka; Zhang, Xian; Schürmann, Nina; Mockenhaupt, Stefan; Serva, Andrius; Hiet, Marie-Sophie; Wiedtke, Ellen; Castoldi, Mirco; Starkuviene, Vytaute; Erfle, Holger; Gilbert, Daniel F.; Bartenschlager, Ralf; Boutros, Michael; Binder, Marco; Streetz, Konrad; Kräusslich, Hans-Georg; Grimm, Dirk

    2013-01-01

    As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems. PMID:24049077

  6. Generation of mouse-induced pluripotent stem cells with plasmid vectors.

    PubMed

    Okita, Keisuke; Hong, Hyenjong; Takahashi, Kazutoshi; Yamanaka, Shinya

    2010-03-01

    Reprogramming of somatic cells into pluripotent stem cells has been reported by introducing a combination of several transcription factors (Oct3/4, Sox2, Klf4 and c-Myc). The induced pluripotent stem (iPS) cells from patient's somatic cells could be a useful source for drug discovery and cell transplantation therapies. However, to date, most iPS cells were made using viral vectors, such as retroviruses and lentiviruses. Here we describe an alternative method to generate iPS cells from mouse embryonic fibroblasts (MEFs) by continual transfection of plasmid vectors. This protocol takes around 2 months to complete, from MEF isolation to iPS cell establishment. Although the reprogramming efficiency of this protocol is still low, the established iPS cells are most likely free from plasmid integration. This virus-free technique reduces the safety concern for iPS cell generation and application, and provides a source of cells for the investigation of the mechanisms underlying reprogramming and pluripotency.

  7. Cloning vectors based on cryptic plasmids isolated from lactic acid bacteria: their characteristics and potential applications in biotechnology.

    PubMed

    Shareck, Julie; Choi, Young; Lee, Byong; Miguez, Carlos B

    2004-01-01

    Lactic acid bacteria (LAB) are Gram positive bacteria, widely distributed in nature, and industrially important as they are used in a variety of industrial food fermentations. The use of genetic engineering techniques is an effective means of enhancing the industrial applicability of LAB. However, when using genetic engineering technology, safety becomes an essential factor for the application of improved LAB to the food industry. Cloning and expression systems should be derived preferably from LAB cryptic plasmids that generally encode genes for which functions can be proposed, but no phenotypes can be observed. However, some plasmid-encoded functions have been discovered in cryptic plasmids originating from Lactobacillus, Streptococcus thermophilus, and Pediococcus spp. and can be used as selective marker systems in vector construction. This article presents information concerning LAB cryptic plasmids, and their structures, functions, and applications. A total of 134 cryptic plasmids collated are discussed.

  8. Extended Function of Plasmid Partition Genes: the Sop System of Linear Phage-Plasmid N15 Facilitates Late Gene Expression▿

    PubMed Central

    Ravin, Nikolai V.; Rech, Jérôme; Lane, David

    2008-01-01

    The mitotic stability of the linear plasmid-prophage N15 of Escherichia coli depends on a partition system closely related to that of the F plasmid SopABC. The two Sop systems are distinguished mainly by the arrangement of their centromeric SopB-binding sites, clustered in F (sopC) and dispersed in N15 (IR1 to IR4). Because two of the N15 inverted repeat (IR) sites are located close to elements presumed (by analogy with phage λ) to regulate late gene expression during the lytic growth of N15, we asked whether Sop partition functions play a role in this process. In N15, a putative Q antiterminator gene is located 6 kb upstream of the probable major late promoter and two intrinsic terminator-like sequences, in contrast to λ, where the Q gene is adjacent to the late promoter. Northern hybridization and lacZ reporter activity confirmed the identity of the N15 late promoter (p52), demonstrated antiterminator activity of the Q analogue, and located terminator sequences between p52 and the first open reading frame. Following prophage induction, N15 mutated in IR2 (downstream from gene Q) or IR3 (upstream of p52) showed a pronounced delay in lysis relative to that for wild-type N15. Expression of ir3−-p52::lacZ during N15 wild-type lytic growth was strongly reduced relative to the equivalent ir3+ fusion. The provision of Q protein and the IR2 and SopAB proteins in trans to ir3+-p52::lacZ increased expression beyond that seen in the absence of any one of these factors. These results indicate that the N15 Sop system has a dual role: partition and regulation of late gene transcription during lytic growth. PMID:18359814

  9. Extended function of plasmid partition genes: the Sop system of linear phage-plasmid N15 facilitates late gene expression.

    PubMed

    Ravin, Nikolai V; Rech, Jérôme; Lane, David

    2008-05-01

    The mitotic stability of the linear plasmid-prophage N15 of Escherichia coli depends on a partition system closely related to that of the F plasmid SopABC. The two Sop systems are distinguished mainly by the arrangement of their centromeric SopB-binding sites, clustered in F (sopC) and dispersed in N15 (IR1 to IR4). Because two of the N15 inverted repeat (IR) sites are located close to elements presumed (by analogy with phage lambda) to regulate late gene expression during the lytic growth of N15, we asked whether Sop partition functions play a role in this process. In N15, a putative Q antiterminator gene is located 6 kb upstream of the probable major late promoter and two intrinsic terminator-like sequences, in contrast to lambda, where the Q gene is adjacent to the late promoter. Northern hybridization and lacZ reporter activity confirmed the identity of the N15 late promoter (p52), demonstrated antiterminator activity of the Q analogue, and located terminator sequences between p52 and the first open reading frame. Following prophage induction, N15 mutated in IR2 (downstream from gene Q) or IR3 (upstream of p52) showed a pronounced delay in lysis relative to that for wild-type N15. Expression of ir3(-)-p52::lacZ during N15 wild-type lytic growth was strongly reduced relative to the equivalent ir3(+) fusion. The provision of Q protein and the IR2 and SopAB proteins in trans to ir3(+)-p52::lacZ increased expression beyond that seen in the absence of any one of these factors. These results indicate that the N15 Sop system has a dual role: partition and regulation of late gene transcription during lytic growth.

  10. Mutagenic specificity of a derivative of 3-nitrobenzanthrone in the supF shuttle vector plasmids.

    PubMed

    Kawanishi, M; Enya, T; Suzuki, H; Takebe, H; Matsui, S; Yagi, T

    1998-12-01

    3-Nitrobenzanthrone (NBA) is a powerful bacterial mutagen and a suspected human carcinogen present in diesel exhaust and airborne particulates [Enya, T., et al. (1997) Environ. Sci. Technol. 31, 2772-2776]. In the accompanying paper [Enya, T., et al. (1998) Chem. Res. Toxcol. 11, 1460-1467], N-acetoxy-N-acetyl-3-aminobenzanthrone (N-Aco-N-Ac-ABA) was synthesized to yield the DNA adducts of NBA. In this work, to investigate the mutagenic specificity of NBA in human cells, we analyzed mutations induced by N-Aco-N-Ac-ABA using the supF shuttle vector plasmids. Base sequence analysis of 110 and 100 plasmids with mutations in the supF gene propagated in normal cells [WI38-VA13] and nucleotide excision repair deficient cells [XP2OS(SV)], respectively, revealed that the majority of the mutations were base substitutions (85 and 90%) and the rest were deletions and insertions (10 and 15%) in both cell lines. About half of the mutant plasmids had a single base substitution. Of the base substitutions, the most frequent mutation was G.C to T.A transversion (41 and 51%), followed by G.C to A.T transitions (18 and 24%) in either cell. The mutations were distributed not randomly but located at several hot spots, and almost all (nine of ten) hot spots were at the sites of G.C base pairs. The polymerase stop assay in the supF gene revealed that N-Aco-N-Ac-ABA preferentially bound to guanine residues, and mutation sites were generally consistent with the sites where the guanine adducts were formed. PMID:9860489

  11. Kluyveromyces lactis killer system: analysis of cytoplasmic promoters of the linear plasmids.

    PubMed Central

    Schickel, J; Helmig, C; Meinhardt, F

    1996-01-01

    All of the 14 genes encoded by the cytoplasmic linear killer plasmids of Kluyveromyces lactis are preceded by upstream conserved sequences (UCSs), cis-acting elements involved in plasmid gene transcription. Using the bacterial glucose-dehydrogenase gene as a reporter, expression driven by seven cytoplasmic promoters was determined. The level of expression ranged from 0.5 to 6 nkat. The highest activity was displayed by UCS 6 of pGKL2 whereas the lowest level was obtained with UCS2 of pGKL2, all other values were in between. Sequences located 5' upstream the UCSs do not influence expression. As exemplified for UCS5 and UCS10, deletion led to an almost complete loss of expression. PMID:8657569

  12. Curing vector for IncI1 plasmids and its use to provide evidence for a metabolic burden of IncI1 CTX-M-1 plasmid pIFM3791 on Klebsiella pneumoniae.

    PubMed

    Freire Martín, Irene; Thomas, Christopher M; Laing, Emma; AbuOun, Manal; La Ragione, Roberto M; Woodward, Martin J

    2016-07-01

    Using a sequence-based approach we previously identified an IncI1 CTX-M-1 plasmid, pIFM3791, on a single pig farm in the UK that was harboured by Klebsiella pneumoniae, Escherichia coli and Salmonella enterica serotype 4,5,12:i:-. To test the hypothesis that the plasmid had spread rapidly into these differing host bacteria we wished to assess whether the plasmid conferred a fitness advantage. To do this an IncI1 curing vector was constructed and used to displace the IncI1 CTX-M-1 plasmids from K. pneumoniae strain B3791 and several other unrelated IncI1-harbouring strains indicating the potential wider application of the curing vector. The IncI1 CTX-M-1 plasmid was reintroduced by conjugation into the cured K. pneumoniae strain and also a naturally IncI1 plasmid free S. enterica serotype 4,5,12:i:-, S348/11. Original, cured and complemented strains were tested for metabolic competence using Biolog technology and in competitive growth, association to mammalian cells and biofilm formation experiments. The plasmid-cured K. pneumoniae strain grew more rapidly than either the original plasmid-carrying strain or plasmid-complemented strains in competition experiments. Additionally, the plasmid-cured strain was significantly better at respiring with l-sorbose as a carbon source and putrescine, γ-amino-n-butyric acid, l-alanine and l-proline as nitrogen sources. By contrast, no differences in phenotype were found when comparing plasmid-harbouring and plasmid-free S. enterica S348/11. In conclusion, the IncI1 curing vector successfully displaced multiple IncI plasmids. The IncI1 CTX-M1 plasmid conferred a growth disadvantage upon K. pneumoniae, possibly by imposing a metabolic burden, the mechanism of which remains to be determined. PMID:27166141

  13. Curing vector for IncI1 plasmids and its use to provide evidence for a metabolic burden of IncI1 CTX-M-1 plasmid pIFM3791 on Klebsiella pneumoniae.

    PubMed

    Freire Martín, Irene; Thomas, Christopher M; Laing, Emma; AbuOun, Manal; La Ragione, Roberto M; Woodward, Martin J

    2016-07-01

    Using a sequence-based approach we previously identified an IncI1 CTX-M-1 plasmid, pIFM3791, on a single pig farm in the UK that was harboured by Klebsiella pneumoniae, Escherichia coli and Salmonella enterica serotype 4,5,12:i:-. To test the hypothesis that the plasmid had spread rapidly into these differing host bacteria we wished to assess whether the plasmid conferred a fitness advantage. To do this an IncI1 curing vector was constructed and used to displace the IncI1 CTX-M-1 plasmids from K. pneumoniae strain B3791 and several other unrelated IncI1-harbouring strains indicating the potential wider application of the curing vector. The IncI1 CTX-M-1 plasmid was reintroduced by conjugation into the cured K. pneumoniae strain and also a naturally IncI1 plasmid free S. enterica serotype 4,5,12:i:-, S348/11. Original, cured and complemented strains were tested for metabolic competence using Biolog technology and in competitive growth, association to mammalian cells and biofilm formation experiments. The plasmid-cured K. pneumoniae strain grew more rapidly than either the original plasmid-carrying strain or plasmid-complemented strains in competition experiments. Additionally, the plasmid-cured strain was significantly better at respiring with l-sorbose as a carbon source and putrescine, γ-amino-n-butyric acid, l-alanine and l-proline as nitrogen sources. By contrast, no differences in phenotype were found when comparing plasmid-harbouring and plasmid-free S. enterica S348/11. In conclusion, the IncI1 curing vector successfully displaced multiple IncI plasmids. The IncI1 CTX-M1 plasmid conferred a growth disadvantage upon K. pneumoniae, possibly by imposing a metabolic burden, the mechanism of which remains to be determined.

  14. Elucidation of Insertion Elements Carried on Plasmids and In Vitro Construction of Shuttle Vectors from the Toxic Cyanobacterium Planktothrix

    PubMed Central

    Christiansen, Guntram; Goesmann, Alexander

    2014-01-01

    Several gene clusters that are responsible for toxin synthesis in bloom-forming cyanobacteria have been found to be associated with transposable elements (TEs). In particular, insertion sequence (IS) elements were shown to play a role in the inactivation or recombination of the genes responsible for cyanotoxin synthesis. Plasmids have been considered important vectors of IS element distribution to the host. In this study, we aimed to elucidate the IS elements propagated on the plasmids and the chromosome of the toxic cyanobacterium Planktothrix agardhii NIVA-CYA126/8 by means of high-throughput sequencing. In total, five plasmids (pPA5.5, pPA14, pPA50, pPA79, and pPA115, of 5, 6, 50, 79, and 120 kbp, respectively) were elucidated, and two plasmids (pPA5.5, pPA115) were found to propagate full IS element copies. Large stretches of shared DNA information between plasmids were constituted of TEs. Two plasmids (pPA5.5, pPA14) were used as candidates to engineer shuttle vectors (named pPA5.5SV and pPA14SV, respectively) in vitro by PCR amplification and the subsequent transposition of the Tn5 cat transposon containing the R6Kγ origin of replication of Escherichia coli. While pPA5.5SV was found to be fully segregated, pPA14SV consistently co-occurred with its wild-type plasmid even under the highest selective pressure. Interestingly, the Tn5 cat transposon became transferred by homologous recombination into another plasmid, pPA50. The availability of shuttle vectors is considered to be of relevance in investigating genome plasticity as a consequence of homologous recombination events. Combining the potential of high-throughput sequencing and in vitro production of shuttle vectors makes it simple to produce species-specific shuttle vectors for many cultivable prokaryotes. PMID:24907328

  15. Repeated capture of a cytoplasmic linear plasmid by the host nucleus in Debaryomyces hansenii.

    PubMed

    Satwika, Dhira; Klassen, Roland; Meinhardt, Friedhelm

    2012-03-01

    Debaryomyces hansenii is a halotolerant yeast species that has been shown to carry various nuclear genes of plasmid or viral origin (NUPAVs). However, a recent ancestor of such NUPAVs has not been identified. Here we determined for the first time the molecular structure of an entire cytoplasmic linear plasmid, pDH1A, indigenous to this species. The element is related to non-autonomous killer plasmids from Kluyveromyces lactis and Pichia acaciae and carries a B-type DNA polymerase as well as remnants of a killer toxin system, a secreted chitin-binding protein. Other essential toxin subunits or an immunity function, however, appear to be lost, while two additional small open reading frames are present. Transcripts for all four genes located on pDH1A could be verified by RT-PCR. Interestingly, all genes from pDH1A could be identified as ancestors of NUPAVs located at different chromosomes within the nucleus of D. hansenii, suggesting repeated nuclear capture of fragments originating from pDH1A. PMID:22434608

  16. A highly optimized protocol for reprogramming cancer cells to pluripotency using nonviral plasmid vectors.

    PubMed

    Zhao, Hongzhi; Davies, Timothy J; Ning, Jiaolin; Chang, Yanxu; Sachamitr, Patty; Sattler, Susanne; Fairchild, Paul J; Huang, Fang-Ping

    2015-02-01

    In spite of considerable interest in the field, reprogramming induced pluripotent stem cells (iPSCs) directly from cancer cells has encountered considerable challenges, including the extremely low reprogramming efficiency and instability of cancer-derived iPSCs (C-iPSCs). In this study, we aimed to identify the main obstacles that limit cancer cell reprogramming. Through a detailed multidimensional kinetic optimization, a highly optimized protocol is established for reprogramming C-iPSCs using nonviral plasmid vectors. We demonstrated how the initial cancer cell density seeded could be the most critical factor ultimately affecting C-iPSCs reprogramming. We have consistently achieved an unprecedented high C-iPSC reprogramming efficiency, establishing stable colonies with typical iPSC morphology, up to 50% of which express the iPSC phenotypic (Oct3/4, Sox2, Nanog) and enzymatic (alkaline phosphatase) markers. Furthermore, established C-iPSC lines were shown to be capable of forming teratomas in vivo, containing cell types and tissues from each of the embryonic germ layers, fully consistent with their acquisition of pluripotency. This protocol was tested and confirmed in two completely unrelated human lung adenocarcinoma (A549) and mouse melanoma (B16f10) cancer cell lines and thus offers a potentially valuable method for generating effectively virus-free C-iPSCs for future applications.

  17. Development of a Transformation System for Chlamydia trachomatis: Restoration of Glycogen Biosynthesis by Acquisition of a Plasmid Shuttle Vector

    PubMed Central

    Wang, Yibing; Kahane, Simona; Cutcliffe, Lesley T.; Skilton, Rachel J.; Lambden, Paul R.; Clarke, Ian N.

    2011-01-01

    Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB) is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB). Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of β-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active β-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP). As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome function by

  18. Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector.

    PubMed

    Wang, Yibing; Kahane, Simona; Cutcliffe, Lesley T; Skilton, Rachel J; Lambden, Paul R; Clarke, Ian N

    2011-09-01

    Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB) is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB). Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of β-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active β-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP). As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome function by

  19. Single inverted terminal repeats of the Junonia coenia Densovirus promotes somatic chromosomal integration of vector plasmids in insect cells and supports high efficiency expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plasmids that contain a disrupted genome of the Junonia coenia densovirus (JcDNV) integrate into the chromosomes of the somatic cells of insects. When subcloned individually, both the P9 inverted terminal repeat (P9-ITR) and the P93-ITR promote the chromosomal integration of vector plasmids in insec...

  20. A Simple and Inexpensive Method for Sending Binary Vector Plasmid DNA by Mail

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We describe a simple cost-effective technique for the transport of plasmid DNA by mail. Our results demonstrate that common multipurpose printing paper is a satisfactory substrate and superior to the more absorbent 3MM chromatography paper for the transport of plasmid DNA through the U.S. first clas...

  1. A stable shuttle vector for Xylella fastidiosa based on an endogenous incP-1 plasmid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xylella fastidiosa (Xf) strain RIV11 harbors a 25 kbp plasmid (pXFRIV11) belonging to the incP1 incompatibility group. Replication and stability factors of pXFRIV11 were identified and used to construct plasmids able to propagate in both Xf and Escherichia coli. Sequences required for replication i...

  2. Construction and application of an expression vector from the new plasmid pLAtc1 of Acidithiobacillus caldus.

    PubMed

    Zhang, Ming-Jiang; Jiang, Cheng-Ying; You, Xiao-Yan; Liu, Shuang-Jiang

    2014-05-01

    In this study, a recently sequenced 9.8-kb plasmid, pLAtc1, from Acidithiobacillus caldus strain SM-1 was characterized and developed into an expression vector. The pLAtc1 backbone carried an oriV, three rep genes, five mob genes, a Nic site, and an addiction system. Multilocus sequence analysis indicated that pLAtc1 was phylogenetically more related to the IncQ-like broad host range plasmids than to other IncQ plasmids. pLAtc1 was able to replicate and reside in Gram-negative Escherichia coli, Comamonas testosteroni, but not in Gram-positive Corynebacterium glutamicum. pLAtc1 was mobilized via conjugation into E. coli BL21 and A. caldus SM-1 from E. coli S17-1. Quantitative PCR revealed seven and four copies of plasmid in A. caldus and E. coli cells, respectively. The expression vector pLAtcE was constructed from pLAtc1 by introducing a regulatable promoter (P tetH ), a transcriptional terminator, a multiple cloning site, a kanamycin resistance gene, and a streptomycin resistance gene. The functionality of pLAtcE was demonstrated by expressing a gene encoding enhanced green fluorescence protein in E. coli and in A. caldus. pLAtcE was used to express α-ketoglutarate dehydrogenase (sucAB) and succinate dehydrogenase (sdhA) genes in A. caldus. The newly engineered strain that harbored sucAB and sdhA on a plasmid pLAtcE-sucA-sucB-sdhA grew better than the parent strain SM-1/pLAtcE in tetrathionate and glucose-supplemented medium and produced more acidity and resulted in a more oxidative environment. This study created a useful molecular tool for genetic manipulation of the thermoacidophilic and autotrophic A. caldus.

  3. Highly linear high-density vector quantiser and vector-matrix multiplier

    NASA Astrophysics Data System (ADS)

    Pedroni, V. A.

    1994-06-01

    Simplicity is a key factor in the development of high-density systems. The authors discuss a balanced, four-quadrant, fully-analogue vector-matrix multiplier (VMM) and a vector quantiser (VQ) which require very small silicon area for their implementations, while presenting high linearity, a totally flexible input dynamic range, a symmetric power consumption behaviour, and are inherently suitable for parallel operation. The circuits require only four transistors per synapse in the VMM and two in the VQ, plus two (small) refresh transistors.

  4. Systematic Comparisons of Formulations of Linear Oligolysine Peptides with siRNA and Plasmid DNA.

    PubMed

    Kwok, Albert; McCarthy, David; Hart, Stephen L; Tagalakis, Aristides D

    2016-05-01

    The effects of lysine peptide lengths on DNA and siRNA packaging and delivery were studied using four linear oligolysine peptides with 8 (K8), 16 (K16), 24 (K24) and 32 (K32) lysines. Oligolysine peptides with 16 lysines or longer were effective for stable monodisperse particle formation and optimal transfection efficiency with plasmid DNA (pDNA), but K8 formulations were less stable under anionic heparin challenge and consequently displayed poor transfection efficiency. However, here we show that the oligolysines were not able to package siRNA to form stable complexes, and consequently, siRNA transfection was unsuccessful. These results indicate that the physical structure and length of cationic peptides and their charge ratios are critical parameters for stable particle formation with pDNA and siRNA and that without packaging, delivery and transfection cannot be achieved. PMID:26684657

  5. Fasciation induction by the phytopathogen Rhodococcus fascians depends upon a linear plasmid encoding a cytokinin synthase gene.

    PubMed Central

    Crespi, M; Messens, E; Caplan, A B; van Montagu, M; Desomer, J

    1992-01-01

    Rhodococcus fascians is a nocardiform bacteria that induces leafy galls (fasciation) on dicotyledonous and several monocotyledonous plants. The wild-type strain D188 contained a conjugative, 200 kb linear extrachromosomal element, pFiD188. Linear plasmid-cured strains were avirulent and reintroduction of this linear element restored virulence. Pulsed field electrophoresis indicated that the chromosome might also be a linear molecule of 4 megabases. Three loci involved in phytopathogenicity have been identified by insertion mutagenesis of this Fi plasmid. Inactivation of the fas locus resulted in avirulent strains, whereas insertions in the two other loci affected the degree of virulence, yielding attenuated (att) and hypervirulent (hyp) bacteria. One of the genes within the fas locus encoded an isopentenyltranferase (IPT) with low homology to analogous proteins from Gram-negative phytopathogenic bacteria. IPT activity was detected after expression of this protein in Escherichia coli cells. In R.fascians, ipt expression could only be detected in bacteria induced with extracts from fasciated tissue. R.fascians strains without the linear plasmid but containing this fas locus alone could not provoke any phenotype on plants, indicating additional genes from the linear plasmid were also essential for virulence. These studies, the first genetic analysis of the interaction of a Gram-positive bacterium with plants, suggest that a novel mechanism for plant tumour induction has evolved in R.fascians independently from the other branches of the eubacteria. Images PMID:1547783

  6. Development of a Ti plasmid vector for plant genetic engineering. Progress report, April 15, 1981-April 14, 1984

    SciTech Connect

    Chilton, M.D.

    1984-02-01

    The Agrobacterium Ti plasmids have the natural ability to insert a specific portion of their DNA, called T-DNA, into the nuclear genome of host plant cells, where it is maintained, replicated and transcribed into mRNA that is translated into foreign protein products. These pathogenic Ti plasmids in nature are acting as gene vectors, for they insert genes that benefit Agrobacterium in several ways. They encode novel enzymes that divert host plant metabolites into storage forms (compounds called opines) that are inaccessible to the plant and to other organisms, but that are specifically catabolized by Agrobacterium. Other genes in the transferred DNA (T-DNA) bring about elevated auxin and cytokinin biosynthesis, causing the tumorous cells to grow autonomously (increasing the size of the opine factory). The present project, begun three years ago, had as its objective the domestication of the Ti plasmid pTi T37 as a gene vector for introduction of desirable genes into higher plants. Three obstacles had to be overcome to this end: (1) insertion of foreign DNA precisely into T-DNA (technically challenging because of the enormous size of the Ti plasmid); (2) developing a disarmed form of the T-DNA that would not cause cells to grow as tumors (a problem because tumor cells could not regenerate into complete plants); and (3) developing new selectable or screenable markers to allow facile isolation of the transformed cells containing genetically engineered T-DNA necessary because the tumorous trait is not usable in the disarmed vector. All of these objectives have been accomplished in the course of the last three years.

  7. Improvement of pBBR1MCS plasmids, a very useful series of broad-host-range cloning vectors.

    PubMed

    Obranić, Sonja; Babić, Fedora; Maravić-Vlahoviček, Gordana

    2013-09-01

    pBBR1MCS vectors are small in size, contain unique cloning sites within the lacZα gene, and are mobilizable and compatible with various plasmid incompatibility groups. We cloned four genes for aminoglycoside resistance methyltransferases from the Arm and Kam families into pBBR1MCS-3 and expressed them in Escherichia coli. The activity of two of these enzymes was impaired because of the fusion with the first 20 amino acids of the β-galactosidase α-peptide derived from the pBBR1MCS-3 vector. In order to overcome this problem, we introduced by site-directed mutagenesis a new NdeI restriction site into pBBR1MCS-3 to generate a start codon directly at the beginning of lacZα gene. We modified the pBBR1MCS-2, 4 and 5 plasmids in the same manner and obtained the enhanced pBBR1MCS_START vector series that retains all the useful features of the previous vectors, but eliminates the unknown effect of the fusion with the β-galactosidase α-peptide.

  8. Characterization of plasmid pXL100 from Amycolatopsis orientalis HCCB10007 and construction of a shuttle vector.

    PubMed

    Xu, Li; Li, Yanmei; Zhu, Li; Zhao, Wei; Chen, Daijie; Huang, Weiyi; Yang, Sheng

    2015-02-01

    Many strains of Amycolatopsis, such as Amycolatopsis orientalis, A. balhimycina, and A. mediterranei, are important antibiotic producers. Three indigenous plasmids, pMEA100, pMEA300, and pA387, found in this genus have been sequenced. However, only some vectors based on pA387 have been widely applied in Amycolatopsis research. An indigenous plasmid, pXL100, was isolated from the vancomycin producer A. orientalis HCCB10007. Sequence analysis of pXL100 revealed its total length to be 33,499 bp and GC content to be 68.9%. A 2830-bp fragment containing three ORFs has been identified as essential for replication in A. orientalis, but it has no significant similarity to any known replicons. A vector, pLYZW7-3, was constructed based on the pXL100 replicon and could be transferred into A. mediterranei and A. orientalis by electroporation or conjugation with high frequency. A mutant with a disrupted gene was successfully complemented with the pLYZW7-3 vector, indicating that the vector is potentially useful in Amycolatopsis research.

  9. An Invertron-Like Linear Plasmid Mediates Intracellular Survival and Virulence in Bovine Isolates of Rhodococcus equi

    PubMed Central

    Valero-Rello, Ana; Hapeshi, Alexia; Anastasi, Elisa; Alvarez, Sonsiray; Scortti, Mariela; Meijer, Wim G.; MacArthur, Iain

    2015-01-01

    We report a novel host-associated virulence plasmid in Rhodococcus equi, pVAPN, carried by bovine isolates of this facultative intracellular pathogenic actinomycete. Surprisingly, pVAPN is a 120-kb invertron-like linear replicon unrelated to the circular virulence plasmids associated with equine (pVAPA) and porcine (pVAPB variant) R. equi isolates. pVAPN is similar to the linear plasmid pNSL1 from Rhodococcus sp. NS1 and harbors six new vap multigene family members (vapN to vapS) in a vap pathogenicity locus presumably acquired via en bloc mobilization from a direct predecessor of equine pVAPA. Loss of pVAPN rendered R. equi avirulent in macrophages and mice. Mating experiments using an in vivo transconjugant selection strategy demonstrated that pVAPN transfer is sufficient to confer virulence to a plasmid-cured R. equi recipient. Phylogenetic analyses assigned the vap multigene family complement from pVAPN, pVAPA, and pVAPB to seven monophyletic clades, each containing plasmid type-specific allelic variants of a precursor vap gene carried by the nearest vap island ancestor. Deletion of vapN, the predicted “bovine-type” allelic counterpart of vapA, essential for virulence in pVAPA, abrogated pVAPN-mediated intramacrophage proliferation and virulence in mice. Our findings support a model in which R. equi virulence is conferred by host-adapted plasmids. Their central role is mediating intracellular proliferation in macrophages, promoted by a key vap determinant present in the common ancestor of the plasmid-specific vap islands, with host tropism as a secondary trait selected during coevolution with specific animal species. PMID:25895973

  10. Thermodynamic study of the interaction between linear plasmid DNA and an anion exchange support under linear and overloaded conditions.

    PubMed

    Aguilar, P A; Twarda, A; Sousa, F; Dias-Cabral, A C

    2014-11-01

    Anion-exchange chromatography has been successfully used in plasmid DNA (pDNA) purification. However, pDNA adsorption mechanism using this method is still not completely understood, and the prediction of the separation behavior is generally unreliable. Flow microcalorimetry (FMC) has proven its ability to provide an improved understanding of the driving forces and mechanisms involved in the adsorption process of biomolecules onto several chromatographic systems. Thus, using FMC, this study aims to understand the adsorption mechanism of linear pDNA (pVAX1-LacZ) onto the anion-exchange support Fast Flow (FF) Q-Sepharose. Static binding capacity studies have shown that the mechanism of pDNA adsorption onto Q-Sepharose follows a Langmuir isotherm. FMC experiments resulted in thermograms that comprised endothermic and exothermic heats. Endothermic heat major contributor was suggested to be the desolvation process. Exothermic heats were related to the interaction between pDNA and Q-Sepharose primary and secondary adsorption. Furthermore, FMC revealed that the overall adsorption process is exothermic, as expected for an anion-exchange interaction. Nevertheless, there are evidences of the presence of nonspecific effects, such as reorientation and electrostatic repulsive forces. PMID:25465014

  11. Characterization of a small cryptic plasmid from endophytic Pantoea agglomerans and its use in the construction of an expression vector

    PubMed Central

    de Lima Procópio, Rudi Emerson; Araújo, Welington Luiz; Andreote, Fernando Dini; Azevedo, João Lúcio

    2011-01-01

    A circular cryptic plasmid named pPAGA (2,734 bp) was isolated from Pantoea agglomerans strain EGE6 (an endophytic bacterial isolate from eucalyptus). Sequence analysis revealed that the plasmid has a G+C content of 51% and contains four potential ORFs, 238(A), 250(B), 131(C), and 129(D) amino acids in length without homology to known proteins. The shuttle vector pLGM1 was constructed by combining the pPAGA plasmid with pGFPmut3.0 (which harbors a gene encoding green fluorescent protein, GFP), and the resulting construct was used to over-express GFP in E. coli and P. agglomerans cells. GFP production was used to monitor the colonization of strain EGE6gfp in various plant tissues by fluorescence microscopy. Analysis of EGE6gfp colonization showed that 14 days after inoculation, the strain occupied the inner tissue of Eucalyptus grandis roots, preferentially colonizing the xylem vessels of the host plants. PMID:21637551

  12. Transcriptional Profiling the 150 kb Linear Megaplasmid of Borrelia turicatae Suggests a Role in Vector Colonization and Initiating Mammalian Infection

    PubMed Central

    Wilder, Hannah K.; Raffel, Sandra J.; Barbour, Alan G.; Porcella, Stephen F.; Sturdevant, Daniel E.; Vaisvil, Benjamin; Kapatral, Vinayak; Schmitt, Daniel P.; Schwan, Tom G.; Lopez, Job E.

    2016-01-01

    Adaptation is key for survival as vector-borne pathogens transmit between the arthropod and vertebrate, and temperature change is an environmental signal inducing alterations in gene expression of tick-borne spirochetes. While plasmids are often associated with adaptation, complex genomes of relapsing fever spirochetes have hindered progress in understanding the mechanisms of vector colonization and transmission. We utilized recent advances in genome sequencing to generate the most complete version of the Borrelia turicatae 150 kb linear megaplasmid (lp150). Additionally, a transcriptional analysis of open reading frames (ORFs) in lp150 was conducted and identified regions that were up-regulated during in vitro cultivation at tick-like growth temperatures (22°C), relative to bacteria grown at 35°C and infected murine blood. Evaluation of the 3’ end of lp150 identified a cluster of ORFs that code for putative surface lipoproteins. With a microbe’s surface proteome serving important roles in pathogenesis, we confirmed the ORFs expression in vitro and in the tick compared to spirochetes infecting murine blood. Transcriptional evaluation of lp150 indicates the plasmid likely has essential roles in vector colonization and/or initiating mammalian infection. These results also provide a much needed transcriptional framework to delineate the molecular mechanisms utilized by relapsing fever spirochetes during their enzootic cycle. PMID:26845332

  13. Investigation of the origin and transmission of linear mitochondrial plasmid based on phylogenetic analysis in Japanese rapeseed varieties.

    PubMed

    Handa, Hirokazu

    2007-02-01

    A linear mitochondrial plasmid is present in some varieties of rapeseed. To elucidate its origin and transmission the author investigated types of mitochondrial genome and the presence of plasmid in 78 rapeseed varieties and landraces in Japan and carried out a comparative analysis using the breeding history of Japanese rapeseed varieties. The mitochondrial genome of rapeseed was classified roughly into 2 types, type I (nap) and type II (cam). Type II rapeseed mitochondria closely resembles that of Brassica rapa, which is a related species of rapeseed. In this study, the author found that all varieties with type II mitochondria originated from interspecific crosses between rapeseed (B. napus) and B. rapa. This indicates that type II cytoplasm was introduced to rapeseed through a breeding program. The presence of plasmid was limited to B. rapa landraces and rapeseed varieties that arose by interspecific crosses between B. napus and B. rapa. The results suggest that mitochondrial plasmid is of B. rapa origin and that it has been introduced into rapeseed by interspecific crosses in a modern breeding program, as in the case of the mitochondrial genome. Phylogenetic study of Japanese rapeseed varieties suggests the participation not of the mitochondrial genome but, rather, the nuclear genome for the perpetuation of plasmid in progeny varieties.

  14. A single-plasmid vector for transgene amplification using short hairpin RNA targeting the 3'-UTR of amplifiable dhfr.

    PubMed

    Kang, Shin-Young; Kim, Yeon-Gu; Lee, Hong Weon; Lee, Eun Gyo

    2015-12-01

    Gene amplification using dihydrofolate reductase gene (dhfr) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRNA) that targets the 3'-untranslated region (UTR) of endogenous dhfr. Thus, shRNAs were designed to target the 3'-UTR of endogenous dhfr, and shRNA-2 efficiently down-regulated dhfr expression in CHO-K1 cells. A single gene copy of shRNA-2 also decreased the translational level of DHFR by 80% in Flp-In CHO cells. shRNA-2 was then incorporated into a plasmid vector expressing human erythropoietin (EPO) and an exogenous DHFR to develop EPO-producing cells in the Flp-In system. The specific EPO productivity (q EPO) was enhanced by stepwise increments of MTX concentration, and differences in the amplification rate were observed in Flp-In CHO cells that expressed shRNA-2. In addition, the q EPO increased by more than 2.5-fold in the presence of 500 nM MTX. The mRNA expression level and gene copy numbers of dhfr were correlated with increased productivity in the cells, which is influenced by inhibition of endogenous dhfr. This study reveals that an expression vector including shRNA that targets the 3'-UTR of endogenous dhfr can enhance the transgene amplification rate and productivity by generating DHFR-deficient cells. This approach may be applied for amplifying the foreign gene in wild-type cell lines as a versatile single-plasmid vector.

  15. A single-plasmid vector for transgene amplification using short hairpin RNA targeting the 3'-UTR of amplifiable dhfr.

    PubMed

    Kang, Shin-Young; Kim, Yeon-Gu; Lee, Hong Weon; Lee, Eun Gyo

    2015-12-01

    Gene amplification using dihydrofolate reductase gene (dhfr) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRNA) that targets the 3'-untranslated region (UTR) of endogenous dhfr. Thus, shRNAs were designed to target the 3'-UTR of endogenous dhfr, and shRNA-2 efficiently down-regulated dhfr expression in CHO-K1 cells. A single gene copy of shRNA-2 also decreased the translational level of DHFR by 80% in Flp-In CHO cells. shRNA-2 was then incorporated into a plasmid vector expressing human erythropoietin (EPO) and an exogenous DHFR to develop EPO-producing cells in the Flp-In system. The specific EPO productivity (q EPO) was enhanced by stepwise increments of MTX concentration, and differences in the amplification rate were observed in Flp-In CHO cells that expressed shRNA-2. In addition, the q EPO increased by more than 2.5-fold in the presence of 500 nM MTX. The mRNA expression level and gene copy numbers of dhfr were correlated with increased productivity in the cells, which is influenced by inhibition of endogenous dhfr. This study reveals that an expression vector including shRNA that targets the 3'-UTR of endogenous dhfr can enhance the transgene amplification rate and productivity by generating DHFR-deficient cells. This approach may be applied for amplifying the foreign gene in wild-type cell lines as a versatile single-plasmid vector. PMID:26245680

  16. A method to facilitate and monitor expression of exogenous genes in the rat kidney using plasmid and viral vectors

    PubMed Central

    Corridon, Peter R.; Rhodes, George J.; Leonard, Ellen C.; Basile, David P.; Gattone, Vincent H.; Bacallao, Robert L.

    2013-01-01

    Gene therapy has been proposed as a novel alternative to treat kidney disease. This goal has been hindered by the inability to reliably deliver transgenes to target cells throughout the kidney, while minimizing injury. Since hydrodynamic forces have previously shown promising results, we optimized this approach and designed a method that utilizes retrograde renal vein injections to facilitate transgene expression in rat kidneys. We show, using intravital fluorescence two-photon microscopy, that fluorescent albumin and dextrans injected into the renal vein under defined conditions of hydrodynamic pressure distribute broadly throughout the kidney in live animals. We found injection parameters that result in no kidney injury as determined by intravital microscopy, histology, and serum creatinine measurements. Plasmids, baculovirus, and adenovirus vectors, designed to express EGFP, EGFP-actin, EGFP-occludin, EGFP-tubulin, tdTomato-H2B, or RFP-actin fusion proteins, were introduced into live kidneys in a similar fashion. Gene expression was then observed in live and ex vivo kidneys using two-photon imaging and confocal laser scanning microscopy. We recorded widespread fluorescent protein expression lasting more than 1 mo after introduction of transgenes. Plasmid and adenovirus vectors provided gene transfer efficiencies ranging from 50 to 90%, compared with 10–50% using baculovirus. Using plasmids and adenovirus, fluorescent protein expression was observed 1) in proximal and distal tubule epithelial cells; 2) within glomeruli; and 3) within the peritubular interstitium. In isolated kidneys, fluorescent protein expression was observed from the cortex to the papilla. These results provide a robust approach for gene delivery and the study of protein function in live mammal kidneys. PMID:23467422

  17. pEVL: A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly(A) Sequences

    PubMed Central

    Grier, Alexandra E; Burleigh, Stephen; Sahni, Jaya; Clough, Courtnee A; Cardot, Victoire; Choe, Dongwook C; Krutein, Michelle C; Rawlings, David J; Jensen, Michael C; Scharenberg, Andrew M; Jacoby, Kyle

    2016-01-01

    Increasing demand for large-scale synthesis of in vitro transcribed (IVT) mRNA is being driven by the increasing use of mRNA for transient gene expression in cell engineering and therapeutic applications. An important determinant of IVT mRNA potency is the 3′ polyadenosine (poly(A)) tail, the length of which correlates with translational efficiency. However, present methods for generation of IVT mRNA rely on templates derived from circular plasmids or PCR products, in which homopolymeric tracts are unstable, thus limiting encoded poly(A) tail lengths to ~120 base pairs (bp). Here, we have developed a novel method for generation of extended poly(A) tracts using a previously described linear plasmid system, pJazz. We find that linear plasmids can successfully propagate poly(A) tracts up to ~500 bp in length for IVT mRNA production. We then modified pJazz by removing extraneous restriction sites, adding a T7 promoter sequence upstream from an extended multiple cloning site, and adding a unique type-IIS restriction site downstream from the encoded poly(A) tract to facilitate generation of IVT mRNA with precisely defined encoded poly(A) tracts and 3′ termini. The resulting plasmid, designated pEVL, can be used to generate IVT mRNA with consistent defined lengths and terminal residue(s). PMID:27093168

  18. Genetic transformation of a clinical (genital tract), plasmid-free isolate of Chlamydia trachomatis: engineering the plasmid as a cloning vector.

    PubMed

    Wang, Yibing; Kahane, Simona; Cutcliffe, Lesley T; Skilton, Rachel J; Lambden, Paul R; Persson, Kenneth; Bjartling, Carina; Clarke, Ian N

    2013-01-01

    Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide "proof of principle" that it is possible to "knock out" selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb β-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed β-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active β-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need for

  19. pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation.

    PubMed

    Kugeratski, Fernanda G; Batista, Michel; Inoue, Alexandre Haruo; Ramos, Bruno Dias; Krieger, Marco Aurelio; Marchini, Fabricio K

    2015-08-01

    The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzigenes and is freely available for scientific community.

  20. Generation of Footprint-Free Induced Pluripotent Stem Cells from Human Fibroblasts Using Episomal Plasmid Vectors.

    PubMed

    Ovchinnikov, Dmitry A; Sun, Jane; Wolvetang, Ernst J

    2015-01-01

    Human induced pluripotent stem cells (hiPSCs) have provided novel insights into the etiology of disease and are set to transform regenerative medicine and drug screening over the next decade. The generation of human iPSCs free of a genetic footprint of the reprogramming process is crucial for the realization of these potential uses. Here we describe in detail the generation of human iPSC from control and disease-carrying individuals' fibroblasts using episomal plasmids.

  1. Transformation of a plasmid-free, genital tract isolate of Chlamydia trachomatis with a plasmid vector carrying a deletion in CDS6 revealed that this gene regulates inclusion phenotype.

    PubMed

    Wang, Yibing; Cutcliffe, Lesley T; Skilton, Rachel J; Persson, Kenneth; Bjartling, Carina; Clarke, Ian N

    2013-03-01

    The development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis provides the basis for the detailed investigation of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In this study we constructed a plasmid vector with CDS6 deleted (pCDS6KO) from the original Escherichia coli/C. trachomatis shuttle vector pGFP::SW2. pCDS6KO was transformed into a clinical isolate of C. trachomatis from Sweden that is plasmid-free (C. trachomatis SWFP-). Penicillin-resistant transformants expressing the green fluorescent protein were selected. These transformants did not stain with iodine, indicating that this property is regulated by CDS6 or its gene product. In addition, mature inclusions of C. trachomatis SWFP- transformed by pCDS6KO displayed an identical morphological phenotype to the untransformed plasmid-free recipient host. In this phenotype the morphology of inclusions was altered with the chlamydiae lining the periphery of the inclusion leaving a 'hole' in the centre. These green fluorescent inclusions appear 'doughnut-shaped' with an empty centre when examined under blue light, giving rise to a characteristic 'black hole' phenotype. Our study demonstrates the power of the new genetic system for investigating chlamydial gene function using gene deletion technology. PMID:23620154

  2. Transformation of a plasmid-free, genital tract isolate of Chlamydia trachomatis with a plasmid vector carrying a deletion in CDS6 revealed that this gene regulates inclusion phenotype

    PubMed Central

    Wang, Yibing; Cutcliffe, Lesley T; Skilton, Rachel J; Persson, Kenneth; Bjartling, Carina; Clarke, Ian N

    2013-01-01

    The development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis provides the basis for the detailed investigation of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In this study we constructed a plasmid vector with CDS6 deleted (pCDS6KO) from the original Escherichia coli/C. trachomatis shuttle vector pGFP::SW2. pCDS6KO was transformed into a clinical isolate of C. trachomatis from Sweden that is plasmid-free (C. trachomatis SWFP–). Penicillin-resistant transformants expressing the green fluorescent protein were selected. These transformants did not stain with iodine, indicating that this property is regulated by CDS6 or its gene product. In addition, mature inclusions of C. trachomatis SWFP– transformed by pCDS6KO displayed an identical morphological phenotype to the untransformed plasmid-free recipient host. In this phenotype the morphology of inclusions was altered with the chlamydiae lining the periphery of the inclusion leaving a ‘hole’ in the centre. These green fluorescent inclusions appear ‘doughnut-shaped’ with an empty centre when examined under blue light, giving rise to a characteristic ‘black hole’ phenotype. Our study demonstrates the power of the new genetic system for investigating chlamydial gene function using gene deletion technology. PMID:23620154

  3. Construction of a shuttle vector based on the small cryptic plasmid pJY33 from Weissella cibaria 33.

    PubMed

    Park, Ji Yeong; Jeong, Seon-Ju; Sa, Hyun Deok; Lee, Jae Yong; Liu, Xiaoming; Cho, Min Jeong; Lee, Kang Wook; Kim, Jeong Hwan

    2015-05-01

    A cryptic plasmid, pJY33, from Weissella cibaria 33 was characterized. pJY33 was 2365 bp in size with a GC content of 41.27% and contained two putative open reading frames (ORFs). orf1 encoded a putative hypothetical protein of 134 amino acids. orf2 was 849 bp in size, and its putative translation product exhibited 87% identity with a replication initiation factor from a plasmid from W. cibaria KLC140. A Weissella-Escherichia coli shuttle vector, pJY33E (6.5 kb, Em(r)), was constructed by ligation of pJY33 with pBluescript II SK(-) and an erythromycin resistance gene (Em(r)). pJY33E replicated in Lactococcus lactis, Leuconostoc citreum, Lactobacillus brevis, Lactobacillus plantarum, and Weissella confusa. A single-stranded DNA intermediate was detected from Lb. brevis 2.14 harbouring pJY33E, providing evidence for rolling-circle replication of pJY33. Most Lb. brevis 2.14 cells (85.9%) retained pJY33E after one week of daily culturing in MRS broth without Em. An aga gene encoding α-galactosidase (α-Gal) from Leuconostoc mesenteroides was successfully expressed in Lb. brevis 2.14 using pJY33E, and the highest level of α-Gal activity (36.13 U/mg protein) was observed when cells were grown on melibiose.

  4. Plasmid Biopharmaceuticals.

    PubMed

    Prazeres, Duarte Miguel F; Monteiro, Gabriel A

    2014-12-01

    Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its mission to produce pharmaceutical proteins, antibodies, vaccines, industrial enzymes, and molecular diagnostics, to name a few key products. Furthermore, plasmids have gradually stepped up in the past 20 years as useful biopharmaceuticals in the context of gene therapy and DNA vaccination interventions. This review provides a concise coverage of the scientific progress that has been made since the emergence of what are called today plasmid biopharmaceuticals. The most relevant topics are discussed to provide researchers with an updated overview of the field. A brief outline of the initial breakthroughs and innovations is followed by a discussion of the motivation behind the medical uses of plasmids in the context of therapeutic and prophylactic interventions. The molecular characteristics and rationale underlying the design of plasmid vectors as gene transfer agents are described and a description of the most important methods used to deliver plasmid biopharmaceuticals in vivo (gene gun, electroporation, cationic lipids and polymers, and micro- and nanoparticles) is provided. The major safety issues (integration and autoimmunity) surrounding the use of plasmid biopharmaceuticals is discussed next. Aspects related to the large-scale manufacturing are also covered, and reference is made to the plasmid products that have received marketing authorization as of today.

  5. Genetic Transformation of a Clinical (Genital Tract), Plasmid-Free Isolate of Chlamydia trachomatis: Engineering the Plasmid as a Cloning Vector

    PubMed Central

    Wang, Yibing; Kahane, Simona; Cutcliffe, Lesley T.; Skilton, Rachel J.; Lambden, Paul R.; Persson, Kenneth; Bjartling, Carina; Clarke, Ian N.

    2013-01-01

    Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide “proof of principle” that it is possible to “knock out” selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb β-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed β-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active β-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need

  6. Linear and nonlinear structural identifications using the support vector regression

    NASA Astrophysics Data System (ADS)

    Zhang, Jian; Sato, Tadanobu

    2006-03-01

    Robust and efficient identification methods are necessary to study in the structural health monitoring field, especially when the I/O data are accompanied by high-level noise and the structure studied is a large-scale one. The Support vector Regression (SVR) is a promising nonlinear modeling method that has been found working very well in many fields, and has a powerful potential to be applied in system identifications. The SVR-based methods are provided in this article to make linear large-scale structural identification and nonlinear hysteretic structural identifications. The LS estimator is a cornerstone of statistics but less robust to outliers. Instead of the classical Gaussian loss function without regularization used in the LS method, a novel e-insensitive loss function is employed in the SVR. Meanwhile, the SVR adopts the 'max-margined' idea to search for an optimum hyper-plane separating the training data into two subsets by maximizing the margin between them. Therefore, the SVR-based structural identification approach is robust and accuracy even though the observation data involve different kinds and high-level noise. By means of the local strategy, the linear large-scale structural identification approach based on the SVR is first investigated. The novel SVR can identify structural parameters directly by writing structural observation equations in linear equations with respect to unknown structural parameters. Furthermore, the substrutural idea employed reduces the number of unknown parameters seriously to guarantee the SVR work in a low dimension and to focus the identification on a local arbitrary subsystem. It is crucial to make nonlinear structural identification also, because structures exhibit highly nonlinear characters under severe loads such as strong seismic excitations. The Bouc-Wen model is often utilized to describe structural nonlinear properties, the power parameter of the model however is often assumed as known even though it is unknown

  7. Circular and linear plasmids of Lyme disease spirochetes have extensive homology: characterization of a repeated DNA element.

    PubMed Central

    Zückert, W R; Meyer, J

    1996-01-01

    We have cloned three copies of a repeated DNA segment from Borrelia burgdorferi sensu stricto strain B31, present on both circular and linear plasmids of this and other B. burgdorferi sensu lato strains. The DNA sequences are characterized by a highly homologous segment containing two open reading frames (ORFs), ORF-A and ORF-B. Five additional ORFs can be found on the slightly less homologous flanking sequences: ORF-G on the opposite strand upstream of ORF-A, and ORF-C, ORF-D, ORF-E, and ORF-F downstream of ORF-B. The 4.6-kb-long element containing ORF-A through ORF-E is flanked by approximately 180-bp-long imperfect inverted repeats (IRs). The putative gene product of ORF-C displays homology to proteins involved in plasmid maintenance in a number of gram-positive and gram-negative bacteria. ORF-E features several short, highly homologous direct repeats. ORF-A, ORF-B, and ORF-D are homologous to three ORFs on a recently described 8.3-kb circular plasmid of Borrelia afzelii Ip21 that are flanked by similar IRs (J. J. Dunn, S. R. Buchstein, L.-L. Butler, S. Fisenne, D. S. Polin, B. N. Lade, and B. J. Luft, J. Bacteriol. 176:2706-2717,1994). ORF-C and ORF-E, however, are missing from this region on the Ip21 plasmid. Furthermore, the repeated DNA element as defined by the IRs is present in opposite orientations relative to the flanking sequences on the B31 and Ip21 plasmids. PMID:8636030

  8. The mean-square error optimal linear discriminant function and its application to incomplete data vectors

    NASA Technical Reports Server (NTRS)

    Walker, H. F.

    1979-01-01

    In many pattern recognition problems, data vectors are classified although one or more of the data vector elements are missing. This problem occurs in remote sensing when the ground is obscured by clouds. Optimal linear discrimination procedures for classifying imcomplete data vectors are discussed.

  9. Plasmids for increased efficiency of vector construction and genetic engineering in filamentous fungi.

    PubMed

    Schoberle, Taylor J; Nguyen-Coleman, C Kim; May, Gregory S

    2013-01-01

    Fungal species are continuously being studied to not only understand disease in humans and plants but also to identify novel antibiotics and other metabolites of industrial importance. Genetic manipulations, such as gene deletion, gene complementation, and gene over-expression, are common techniques to investigate fungal gene functions. Although advances in transformation efficiency and promoter usage have improved genetic studies, some basic steps in vector construction are still laborious and time-consuming. Gateway cloning technology solves this problem by increasing the efficiency of vector construction through the use of λ phage integrase proteins and att recombination sites. We developed a series of Gateway-compatible vectors for use in genetic studies in a range of fungal species. They contain nutritional and drug-resistance markers and can be utilized to manipulate different filamentous fungal genomes.

  10. Hawking radiation of massive vector particles from the linear dilaton black holes

    NASA Astrophysics Data System (ADS)

    Li, Ran; Zhao, Junkun

    2016-07-01

    By using the tunneling formalism, we calculated the massive vector particles' Hawking radiation from the non-rotating and rotating linear dilaton black holes. By applying the WKB approximation to the Proca field equation that govern the dynamics of massive vector bosons, we derive the tunneling probabilities and radiation spectrums of the emitted vector particles from the linear dilaton black holes. The Hawking temperatures of the linear dilaton black holes have been recovered, which are consistent with the previous results in the literature. This means that the vector particles' tunneling method can also be used in studying the Hawking radiation of asymptotically non-flat and non-AdS black holes.

  11. HygR and PurR plasmid vectors for episomal transfection of Trypanosoma cruzi.

    PubMed

    Nóbrega, Otávio T; Santana, Jaime M; Sturm, Nancy R; Teixeira, Antônio R L; Campbell, David A

    2004-08-01

    This work describes the development and functional testing of two episomes for stable transfection of Trypanosoma cruzi. pHygD contained the 5'- and 3'- flanking regions of the gene encoding the cathepsin B-like protease of T. cruzi as functional trans-splicing and polyadenylation signals for the hygR ORF. Evidence is presented to support extrachromosomal maintenance and organization as tandem repeats in transfected parasites. pPac was derived from pHygD by replacement of the entire hygR ORF with a purR coding region. The ability to modify pHygD and the availability of the complete DNA sequence make these plasmids useful tools for the genetic manipulation of T. cruzi.

  12. Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome

    PubMed Central

    Stadelmann, Britta; Birkestedt, Sandra; Hellman, Ulf; Svärd, Staffan G.

    2012-01-01

    In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide–glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG–tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes. PMID:22611020

  13. Identification of facultatively heterotrophic, N/sub 2/-fixing cyanobacteria able to receive plasmid vectors from Escherichia coli by conjugation. [Anabaena spp; Nostoc

    SciTech Connect

    Flores, E.; Wolk, C.P.

    1985-06-01

    Plasmid vectors transferable by conjugation from Escherichia coli to obligately photoautotrophic strains of Anabaena spp. are also transferred to and maintained in heterotrophic, filamentous cyanobacteria of the genus Nostoc. These organisms can be used for the genetic analysis of oxygenic photosynthesis, chromatic adaptation, nitrogen fixation, and heterocyst development.

  14. Eukaryotic gene invasion by a bacterial mobile insertion sequence element IS2 during cloning into a plasmid vector.

    PubMed

    Senejani, Alireza G; Sweasy, Joann B

    2010-01-01

    Escherichia coli (E. coli) are commonly used as hosts for DNA cloning and sequencing. Upon transformation of E. coli with recombined vector carrying a gene of interest, the bacteria multiply the gene of interest while maintaining the integrity of its content. During the subcloning of a mouse genomic fragment into a plasmid vector, we noticed that the size of the insert increased significantly upon replication in E. coli. The sequence of the insert was determined and found to contain a novel DNA sequence within the mouse genomic insert. A BLAST search of GenBank revealed the novel sequence to be that of the Insertion Sequence 2 (IS2) element from E. coli that was likely inserted during replication in that organism. Importantly, a detailed search of GenBank shows that the IS2 is present within many eukaryotic nucleotide sequences, and in many cases, has been annotated as being part of the protein. The results of this study suggest that one must perform additional careful analysis of the sequence results using BLAST comparisons, and further verification of gene annotation before submission into the GenBank. PMID:20678256

  15. Galactomannan-PEI based non-viral vectors for targeted delivery of plasmid to macrophages and hepatocytes.

    PubMed

    Bansal, Ruby; Singh, A K; Gandhi, R P; Pant, A B; Kumar, P; Gupta, K C

    2014-08-01

    Intracellular nature and diversified locations of infectious and parasitic diseases such as leishmaniasis, trypanosomiasis, tuberculosis and hepatitis B and C pose a significant global burden and challenge to the scientists working in the area of drug discovery and drug delivery. The macrophages and hepatocytes are considered as potential target sites as they together play an important role in various infectious diseases. The present study scrutinizes the applicability of a natural biopolymer-based chemical vectors, capable of targeting both macrophages and hepatocytes, that can form a complex with plasmid and administer it into cells to produce a desired protein. The investigations were made to develop a novel series of gene carriers by conjugating depolymerized galactomannan (guar gum), a biocompatible polysaccharide with low molecular weight branched PEI (LMWP). A series of conjugates were developed and characterized using physicochemical techniques. All the GP/pDNA complexes showed significantly higher transfection efficiency with GP-3/pDNA, one of the best formulations, showed ~2.0-7.7-folds higher transfection efficacy when compared with the standard transfection reagents. Further, GP-3/pDNA displayed significantly higher target specific transfection efficiency under both in vitro and in vivo conditions. The data demonstrate the potential of GP vectors to deliver nucleic acids simultaneously to macrophages and hepatocytes in gene delivery applications.

  16. Delivery of plasmid DNA expression vector for keratinocyte growth factor-1 using electroporation to improve cutaneous wound healing in a septic rat model.

    PubMed

    Lin, Michael P; Marti, Guy P; Dieb, Rami; Wang, Jiaai; Ferguson, Mark; Qaiser, Rabia; Bonde, Pramod; Duncan, Mark D; Harmon, John W

    2006-01-01

    We have previously shown that wound healing was improved in a diabetic mouse model of impaired wound healing following transfection with keratinocyte growth factor-1 (KGF-1) cDNA. We now extend these findings to the characterization of the effects of DNA plasmid vectors delivered to rats using electroporation (EP) in vivo in a sepsis-based model of impaired wound healing. To assess plasmid transfection and wound healing, gWIZ luciferase and PCDNA3.1/KGF-1 expression vectors were used, respectively. Cutaneous wounds were produced using an 8 mm-punch biopsy in Sprague-Dawley rats in which healing was impaired by cecal ligation-induced sepsis. We used National Institutes of Health image analysis software and histologic assessment to analyze wound closure and found that EP increased expression of gWIZ luciferase vector up to 53-fold compared with transfection without EP (p < 0.001). EP-assisted plasmid transfection was found to be localized to skin. Septic rats had a 4.7 times larger average wound area on day 9 compared with control (p < 0.001). Rats that underwent PCDNA3.1/KGF-1 transfection with EP had 60% smaller wounds on day 12 compared with vector without EP (p < 0.009). Quality of healing with KGF-1 vector plus EP scored 3.0 +/- 0.3 and was significantly better than that of 1.8 +/- 0.3 for treatment with vector alone (p < 0.05). We conclude that both the rate and quality of healing were improved with DNA plasmid expression vector for growth factor delivered with EP to septic rats.

  17. [Sop proteins can cause transcriptional silencing of genes located close to the centromere sites of linear plasmid N15].

    PubMed

    Mardanov, A V; Lane, D; Ravin, N V

    2010-01-01

    Stable inheritance of bacterial chromosomes and low copy number plasmids is ensured by accurate partitioning of replicated molecules between the daughter cells at division. Partitioning of the prophage of the temperate bacteriophage N15, which exists as a linear plasmid molecule with covalently closed ends, depends on the sop locus, comprising genes sopA and sopB, as well as four centromere sites located in different regions of the N15 genome essential for replication and the control of lysogeny. We found that binding of SopB to the centromere can silence centromere-proximal promoters, presumably due to subsequent polymerizing of SopB along the DNA. Close to the IR4 centromere site we identified a promoter, P59, able to drive expression of phage late genes encoding the structural proteins of virion. We found that following binding to IR4 the N15 Sop proteins can cause repression of this promoter. The repression depends on SopB and became stronger in the presence of SopA. Sop-dependent silencing of centromere-proximal promoters control gene expression in phage N15, particularly preventing undesired expression of late genes in the N15 prophage. Thus, the phage N15 sop system not only ensures plasmid partitioning but is also involved in the genetic network controlling prophage replication and the maintenance of lysogeny.

  18. Characterization of the minimal replicon of a cryptic Deinococcus radiodurans SARK plasmid and development of versatile Escherichia coli-D. radiodurans shuttle vectors.

    PubMed

    Meima, R; Lidstrom, M E

    2000-09-01

    The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus. Annotation of the sequence revealed 12 possible open reading frames. Among these are the repU and resU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in Escherichia coli. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D. radiodurans strain R1. The minimal region required for autonomous replication in D. radiodurans was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E. coli and D. radiodurans. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus promoter was shown to efficiently express LacZ.

  19. Characterization of the Minimal Replicon of a Cryptic Deinococcus radiodurans SARK Plasmid and Development of Versatile Escherichia coli-D. radiodurans Shuttle Vectors

    PubMed Central

    Meima, Rob; Lidstrom, Mary E.

    2000-01-01

    The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus. Annotation of the sequence revealed 12 possible open reading frames. Among these are the repU and resU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in Escherichia coli. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D. radiodurans strain R1. The minimal region required for autonomous replication in D. radiodurans was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E. coli and D. radiodurans. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus promoter was shown to efficiently express LacZ. PMID:10966401

  20. Transformation of microorganisms with the plasmid vector with the replicon from pAC1 from Acetobacter pasteurianus.

    PubMed

    Grones, J; Turna, J

    1995-01-26

    A number of gram-negative and gram-positive bacteria species was screened for the expression of the gram-negative plasmid pACK5 and pACT72 with replicon of pAC1 plasmid from Acetobacter pasteurianus. As was described previously, both plasmids were expressed in Escherichia coli, Acetobacter pasteurianus, Acetobacter aceti, Shigella spp. and Citrobacter spp. Expressions of plasmids were successful in twelve species tested, Comamonas terrigena, Salmonella typhimurium, Serratia marcescens, Bacillus cereus, Bacillus megatericum, Bacillus subtilis, Lactobacillus helveticus, Micrococcus luteus, Sarcina lutea, Staphylococcus aureus, Staphylococcus epidermidis, Streptoccocus feacalis, and the stability of plasmid DNA was tested after cultivation in non-selective conditions.

  1. Structural analysis of the 6 kb cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 and construction of Escherichia coli-Rhodococcus shuttle vectors.

    PubMed

    De Mot, R; Nagy, I; De Schrijver, A; Pattanapipitpaisal, P; Schoofs, G; Vanderleyden, J

    1997-10-01

    The complete nucleotide sequence of the 5936 bp cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 was determined. Based on the characteristics of its putative replication genes, repA and repB, pFAJ2600 was assigned to the family of pAL5000-related small replicons identified in Mycobacterium (pAL5000), Corynebacterium (pXZ10142), Brevibacterium (pRBL1), Bifidobacterium (pMB1) and Neisseria (pJD1). The replication systems of these plasmids show striking similarities to the ones used by the ColE2 family of plasmids from Enterobacteria with respect to both trans-acting factors and ori sequences. Two possible plasmid stabilization systems are encoded on pFAJ2600: a site-specific recombinase (PmrA) related to the Escherichia coli Xer proteins for plasmid multimer resolution and an ATPase (ParA) related to the A-type of proteins in sop/par partitioning systems. The proposed replication termination region of pFAJ2600 has features in common with the Ter loci of Bacillus subtilis. Chimeras composed of a pUC18-Cmr derivative inserted in the parA-repA intergenic region of vector pFAJ2600 produced vectors that could be shuttled between Escherichia coli and several Rhodococcus species (R. erythropolis, R. fascians, R. rhodochrous, R. ruber). The pFAJ2600-based shuttle vector pFAJ2574 was stably maintained in R. erythropolis and R. fascians growing under non-selective conditions.

  2. 3D Linear Transformations in the Form of Matrix and Vector

    NASA Astrophysics Data System (ADS)

    Zhang, Hua

    2008-11-01

    In this article, results of four 3D linear transformations (translation, rotation, scale and shear) in the form of matrix and vector are simplified into a same 3D physical coordinates system. Comparing the simplified results of those four linear transformations, the results obtained from matrix form are exactly the same as what obtained from vector algebra in final expressions, although they are different from original expressions.

  3. Construction of a stable plasmid vector for industrial production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by a recombinant Cupriavidus necator H16 strain.

    PubMed

    Sato, Shunsuke; Fujiki, Tetsuya; Matsumoto, Keiji

    2013-12-01

    A new stable plasmid vector (pCUP3) was developed for high and stable production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) using Cupriavidus necator H16 as the host strain. In pCUP3, it was found that the plasmid partition and replication region of the megaplasmid pMOL28 in the Cupriavidus metallidurans CH34 strain plays an important role in plasmid stability in C. necator H16. Moreover, the partition locus (comprising parA28 and parB28 and the parS28 region) is essential for plasmid maintenance under high-PHBH-accumulation. PHBH productivity by the C. necator H16/ds strain (phaC1 deactivated mutant strain) harboring a phaCAc NSDG within pCUP3 was identical to the productivity of poly(3-hydroxybutyrate) by the C. necator H16 strain when palm kernel oil was used as the sole carbon source without any antibiotics. This new vector is important for industrial mass production of polyhydroxyalkanoates using the C. necator H16 strain as the host, dispensing the necessity of the application of selective pressure such as antibiotics.

  4. A novel plasmid vector designed for chromosomal gene integration and expression: use for developing a genetically stable Escherichia coli melanin production strain.

    PubMed

    Sabido, Andrea; Martínez, Luz María; de Anda, Ramón; Martínez, Alfredo; Bolívar, Francisco; Gosset, Guillermo

    2013-01-01

    Recombinant Escherichia coli strains for the production of valuable products are usually generated by transformation with plasmid expression vectors. However, in spite of their usefulness, common problems associated with plasmid use include segregrational and structural instability as well as undesired copy-number effects. A viable alternative to plasmid use is chromosomal gene integration. With the purpose of facilitating the process of stable strain generation, a novel chromosomal integration vector was developed and tested. We describe the construction and use of novel expression vector pLoxGentrc that contains the strong trc promoter (P(trc)), a multiple cloning site, the T1 and T2 rrnB terminator sequences, the lacI(q) gene and the aacC1 gene conferring gentamicin resistance flanked by two loxP sites. As a demonstration of utility, melanin-producing strains of E. coli were generated employing this vector. Melanin is a polymer synthesized by the enzyme tyrosinase using l-tyrosine as substrate. The melA gene encoding a tyrosinase from Rhizobium etli was ligated to pLoxGentrc to generate pLoxGentrcmelA. This plasmid was transformed into E. coli W3110 to generate a melanin-producing strain. A region from this plasmid including P(trc)melA, T1 and T2 rrnB and the aacC1 gene was amplified by PCR employing primers with 45 b regions of homology to the lacZ gene. The PCR product was electroporated into strain W3110 that expressed the λ-Red enzymes. From this experiment, strain W3110P(tr)(c)melA, was obtained having the melA gene inserted in the lacZ locus. Fermentor cultures with strain W3110/pLoxGentrcmelA grown in the presence and absence of gentamicin as well as W3110P(tr)(c)melA without antibiotic revealed that the latter displays high genetic stability as well as the highest melanin titer. Vector pLoxGentrc should be useful during strain generation processes, enabling direct comparison of plasmid and chromosome-based production systems. PMID:22884755

  5. A novel plasmid vector designed for chromosomal gene integration and expression: use for developing a genetically stable Escherichia coli melanin production strain.

    PubMed

    Sabido, Andrea; Martínez, Luz María; de Anda, Ramón; Martínez, Alfredo; Bolívar, Francisco; Gosset, Guillermo

    2013-01-01

    Recombinant Escherichia coli strains for the production of valuable products are usually generated by transformation with plasmid expression vectors. However, in spite of their usefulness, common problems associated with plasmid use include segregrational and structural instability as well as undesired copy-number effects. A viable alternative to plasmid use is chromosomal gene integration. With the purpose of facilitating the process of stable strain generation, a novel chromosomal integration vector was developed and tested. We describe the construction and use of novel expression vector pLoxGentrc that contains the strong trc promoter (P(trc)), a multiple cloning site, the T1 and T2 rrnB terminator sequences, the lacI(q) gene and the aacC1 gene conferring gentamicin resistance flanked by two loxP sites. As a demonstration of utility, melanin-producing strains of E. coli were generated employing this vector. Melanin is a polymer synthesized by the enzyme tyrosinase using l-tyrosine as substrate. The melA gene encoding a tyrosinase from Rhizobium etli was ligated to pLoxGentrc to generate pLoxGentrcmelA. This plasmid was transformed into E. coli W3110 to generate a melanin-producing strain. A region from this plasmid including P(trc)melA, T1 and T2 rrnB and the aacC1 gene was amplified by PCR employing primers with 45 b regions of homology to the lacZ gene. The PCR product was electroporated into strain W3110 that expressed the λ-Red enzymes. From this experiment, strain W3110P(tr)(c)melA, was obtained having the melA gene inserted in the lacZ locus. Fermentor cultures with strain W3110/pLoxGentrcmelA grown in the presence and absence of gentamicin as well as W3110P(tr)(c)melA without antibiotic revealed that the latter displays high genetic stability as well as the highest melanin titer. Vector pLoxGentrc should be useful during strain generation processes, enabling direct comparison of plasmid and chromosome-based production systems.

  6. Linear matrix inequalities for analysis and control of linear vector second-order systems

    SciTech Connect

    Adegas, Fabiano D.; Stoustrup, Jakob

    2014-10-06

    Many dynamical systems are modeled as vector second-order differential equations. This paper presents analysis and synthesis conditions in terms of LMI with explicit dependence in the coefficient matrices of vector second-order systems. These conditions benefit from the separation between the Lyapunov matrix and the system matrices by introducing matrix multipliers, which potentially reduce conservativeness in hard control problems. Multipliers facilitate the usage of parameter-dependent Lyapunov functions as certificates of stability of uncertain and time-varying vector second-order systems. The conditions introduced in this work have the potential to increase the practice of analyzing and controlling systems directly in vector second-order form.

  7. Implementation of iterative methods for large sparse nonsymmetric linear systems on a parallel vector machine

    SciTech Connect

    Ma, S.; Chronopoulos, A.T. )

    1990-01-01

    This paper reports on the restructure of three outstanding iterative methods for large space nonsymmetric linear systems. These methods are CGS (conjugate gradient squared), CRS (conjugate residual squared), and Orthomin(k). The restructured methods are more suitable for vector and parallel processing. The authors implemented these methods on a parallel vector system. The linear systems for the numerical tests are obtained from discretizing four two- dimensional elliptic partial differential equations by finite difference and finite element methods. A vectorizable and parallelizable version of incomplete LU preconditioning is used. The authors restructured the subroutines to enhance the data locality in vector machines with storage hierarchy. Speedup was measured for multitasking by four processors.

  8. Generation of plasmid vectors expressing FLAG-tagged proteins under the regulation of human elongation factor-1α promoter using Gibson assembly.

    PubMed

    Grozdanov, Petar N; MacDonald, Clinton C

    2015-02-09

    Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1α) promoter to drive gene expression. Plasmid vectors containing hEF1α are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1α promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1α promoter part 1, hEF1α promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

  9. Generation of plasmid vectors expressing FLAG-tagged proteins under the regulation of human elongation factor-1α promoter using Gibson assembly.

    PubMed

    Grozdanov, Petar N; MacDonald, Clinton C

    2015-01-01

    Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1α) promoter to drive gene expression. Plasmid vectors containing hEF1α are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1α promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1α promoter part 1, hEF1α promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification. PMID:25742071

  10. Recombinational cloning of the antibiotic biosynthetic gene clusters in linear plasmid SCP1 of Streptomyces coelicolor A3(2).

    PubMed

    Zhang, Ran; Xia, Haiyang; Xu, Qingyu; Dang, Fujun; Qin, Zhongjun

    2013-08-01

    The model organism Streptomyces coelicolor A3(2) harbors a 356-kb linear plasmid, SCP1. We report here development of a recombinational cloning method for deleting large segment from one telomere of SCP1 followed by replacing with the telomere of pSLA2 and sequentially inserting with the overlapping cosmids in vivo. The procedure depends on homologous recombination coupled with cleavage at telomere termini by telomere terminal protein. Using this procedure, we cloned the 81-kb avermectin and the 76-kb spinosad biosynthetic gene clusters into SCP1. Heterologous expression of avermectin production in S. coelicolor was detected. These results demonstrate the utility of SCP1 for cloning large DNA segments such as antibiotic biosynthetic gene clusters.

  11. arsRBOCT Arsenic Resistance System Encoded by Linear Plasmid pHZ227 in Streptomyces sp. Strain FR-008

    PubMed Central

    Wang, Lianrong; Chen, Shi; Xiao, Xiang; Huang, Xi; You, Delin; Zhou, Xiufen; Deng, Zixin

    2006-01-01

    In the arsenic resistance gene cluster from the large linear plasmid pHZ227, two novel genes, arsO (for a putative flavin-binding monooxygenase) and arsT (for a putative thioredoxin reductase), were coactivated and cotranscribed with arsR1-arsB and arsC, respectively. Deletion of the ars gene cluster on pHZ227 in Streptomyces sp. strain FR-008 resulted in sensitivity to arsenic, and heterologous expression of the ars gene cluster in the arsenic-sensitive Streptomyces strains conferred resistance on the new hosts. The pHZ227 ArsB protein showed homology to the yeast arsenite transporter Acr3p. The pHZ227 ArsC appears to be a bacterial thioredoxin-dependent ArsC-type arsenate reductase with four conserved cysteine thioredoxin-requiring motifs. PMID:16672525

  12. Characterization of pMC11, a plasmid with dual origins of replication isolated from Lactobacillus casei MCJ and construction of shuttle vectors with each replicon.

    PubMed

    Chen, Zhengjun; Lin, Jinzhong; Ma, Chengjie; Zhao, Shumiao; She, Qunxin; Liang, Yunxiang

    2014-07-01

    Many lactic acid bacteria carry different plasmids, particularly those that replicate via a theta mechanism. Here we describe Lactobacillus casei MCJ(CCTCC AB20130356), a new isolate that contains pMC11, carrying two distinct theta-type replicons. Each replicon contained an iteron in the origin of replication (oriV1 or oriV2) and a gene coding for the replicase (RepA_1 or RepB_1), both of which are essential for plasmid replication. Escherichia coli/Lactobacillus shuttle vectors were constructed with each replicon, yielding pEL5.7 and pEL5.6 that are based on oriV2 and oriV1 replicons, respectively. These plasmids showed distinct properties: pEL5.7 was capable of replicating in L. casei MCJΔ1 and Lactobacillus delbrueckii subsp. lactic LBCH-1 but failed to do so in two other tested lactobacilli strains whereas pEL5.6 replicated in three different strains, including L. casei MCJΔ1, L. casei NJ, Lactobacillus paracasei LPC-37 and L. delbrueckii subsp. lactic LBCH-1. Plasmid stability was studied: pEL5.6 and pEL5.7 were very stably maintained in L. casei, as the loss rate was lower than 1 % per generation. pEL5.7 was also stable in L. delbrueckii subsp. lactic LBCH-1 with the loss rate estimated to be 3 %. These vectors were employed to express a green fluorescent protein (GFP) using the promoter of S-layer protein SlpA from Lactobacillus acidophilus. And a growth-phase regulated expression of GFP was observed in different strains. In conclusion, these shuttle vectors provide efficient genetic tools for DNA cloning and heterologous gene expression in lactobacilli.

  13. A cloning vector for creation of Escherichia coli lacZ translational fusions and generation of linear template for chromosomal integration.

    PubMed

    Uhlich, Gaylen A; Chen, Chin-Yi

    2012-05-01

    A novel cloning vector to aid in the construction of single copy β-galactosidase reporter systems for gene expression studies in lactose metabolizing Escherichia coli strains, including STEC, is described. The plasmid allows construction of translational fusions of cloned gene promoters to a short segment of E. coli lacZ. A selectable spectinomycin resistance marker flanked by a short lacI segment is positioned 5' to the cloning site. PCR amplification using opposing primers complementary to the upstream lacI fragment and the downstream lacZ fragment generates a linear template suitable for integration using pRedET recombination. Integration of linear template derived from the recombinant plasmid into host strains replaces the entire native lacZ promoter and fuses the promoter of interest in-frame with the lacZ gene, thus simultaneously producing a single-copy, chromosomal reporter system and eliminating background lacZ expression. Studies comparing ahpC expression from a chromosomal fusion in the lac open with that on a plasmid in E. coli strain EDL933 are shown. PMID:22197962

  14. Food-grade host/vector expression system for Lactobacillus casei based on complementation of plasmid-associated phospho-beta-galactosidase gene lacG.

    PubMed

    Takala, T M; Saris, P E J; Tynkkynen, S S H

    2003-01-01

    A new food-grade host/vector system for Lactobacillus casei based on lactose selection was constructed. The wild-type non-starter host Lb. casei strain E utilizes lactose via a plasmid-encoded phosphotransferase system. For food-grade cloning, a stable lactose-deficient mutant was constructed by deleting a 141-bp fragment from the phospho-beta-galactosidase gene lacG via gene replacement. The deletion resulted in an inactive phospho-beta-galactosidase enzyme with an internal in-frame deletion of 47 amino acids. A complementation plasmid was constructed containing a replicon from Lactococcus lactis, the lacG gene from Lb. casei, and the constitutive promoter of pepR for lacG expression from Lb. rhamnosus. The expression of the lacG gene from the resulting food-grade plasmid pLEB600 restored the ability of the lactose-negative mutant strain to grow on lactose to the wild-type level. The vector pLEB600 was used for expression of the proline iminopeptidase gene pepI from Lb. helveticus in Lb. casei. The results show that the food-grade expression system reported in this paper can be used for expression of foreign genes in Lb. casei. PMID:12536257

  15. The use of the replication region of plasmid pRS7 from Oenococcus oeni as a putative tool to generate cloning vectors for lactic acid bacteria.

    PubMed

    Rodríguez, M Carmen; Alegre, M Teresa; Martín, M Cruz; Mesas, Juan M

    2015-01-01

    A chimeric plasmid, pRS7Rep (6.1 kb), was constructed using the replication region of pRS7, a large plasmid from Oenococcus oeni, and pEM64, a plasmid derived from pIJ2925 and containing a gene for resistance to chloramphenicol. pRS7Rep is a shuttle vector that replicates in Escherichia coli using its pIJ2925 component and in lactic acid bacteria (LAB) using the replication region of pRS7. High levels of transformants per µg of DNA were obtained by electroporation of pRS7Rep into Pediococcus acidilactici (1.5 × 10(7)), Lactobacillus plantarum (5.7 × 10(5)), Lactobacillus casei (2.3 × 10(5)), Leuconostoc citreum (2.7 × 10(5)), and Enterococcus faecalis (2.4 × 10(5)). A preliminary optimisation of the technical conditions of electrotransformation showed that P. acidilactici and L. plantarum are better transformed at a later exponential phase of growth, whereas L. casei requires the early exponential phase for better electrotransformation efficiency. pRS7Rep contains single restriction sites useful for cloning purposes, BamHI, XbaI, SalI, HincII, SphI and PstI, and was maintained at an acceptable rate (>50%) over 100 generations without selective pressure in L. plantarum, but was less stable in L. casei and P. acidilactici. The ability of pRS7Rep to accept and express other genes was assessed. To the best of our knowledge, this is the first time that the replication region of a plasmid from O. oeni has been used to generate a cloning vector.

  16. Natural plasmids of filamentous fungi.

    PubMed Central

    Griffiths, A J

    1995-01-01

    Among eukaryotes, plasmids have been found in fungi and plants but not in animals. Most plasmids are mitochondrial. In filamentous fungi, plasmids are commonly encountered in isolates from natural populations. Individual populations may show a predominance of one type, but some plasmids have a global distribution, often crossing species boundaries. Surveys have shown that strains can contain more than one type of plasmid and that different types appear to be distributed independently. In crosses, plasmids are generally inherited maternally. Horizontal transmission is by cell contact. Circular plasmids are common only in Neurospora spp., but linear plasmids have been found in many fungi. Circular plasmids have one open reading frame (ORF) coding for a DNA polymerase or a reverse transcriptase. Linear plasmids generally have two ORFs, coding for presumptive DNA and RNA polymerases with amino acid motifs showing homology to viral polymerases. Plasmids often attain a high copy number, in excess of that of mitochondrial DNA. Linear plasmids have a protein attached to their 5' end, and this is presumed to act as a replication primer. Most plasmids are neutral passengers, but several linear plasmids integrate into mitochondrial DNA, causing death of the host culture. Inferred amino acid sequences of linear plasmid ORFs have been used to plot phylogenetic trees, which show a fair concordance with conventional trees. The circular Neurospora plasmids have replication systems that seem to be evolutionary intermediates between the RNA and the DNA worlds. PMID:8531891

  17. High and prolonged sulfamidase secretion by the liver of MPS-IIIA mice following hydrodynamic tail vein delivery of antibiotic-free pFAR4 plasmid vector.

    PubMed

    Quiviger, M; Arfi, A; Mansard, D; Delacotte, L; Pastor, M; Scherman, D; Marie, C

    2014-12-01

    Mucopolysaccharidosis type IIIA (MPS-IIIA) or Sanfilippo A syndrome is a lysosomal storage genetic disease that results from the deficiency of the N-sulfoglucosamine sulfohydrolase (SGSH) protein, a sulfamidase required for the degradation of heparan sulfate glycosaminoglycans (GAGs). The accumulation of these macromolecules leads to somatic organ pathologies, severe neurodegeneration and death. To assess a novel gene therapy approach based on prolonged secretion of the missing enzyme by the liver, mediated by hydrodynamic gene delivery, we first compared a kanamycin and an antibiotic-free expression plasmid vector, called pFAR4. Thanks to the reduced vector size, pFAR4 derivatives containing either a ubiquitous or a liver-specific promoter mediated a higher reporter gene expression level than the control plasmid. Hydrodynamic delivery of SGSH-encoding pFAR4 into MPS-IIIA diseased mice led to high serum levels of sulfamidase protein that was efficiently taken up by neighboring organs, as shown by the correction of GAG accumulation. A similar reduction in GAG content was also observed in the brain, at early stages of the disease. Thus, this study contributes to the effort towards the development of novel biosafe non-viral gene vectors for therapeutic protein expression in the liver, and represents a first step towards an alternative gene therapy approach for the MPS-IIIA disease.

  18. Design and in vitro characterization of a single regulatory module for efficient control of gene expression in both plasmid DNA and a self-inactivating lentiviral vector.

    PubMed Central

    Ogueta, S. B.; Yao, F.; Marasco, W. A.

    2001-01-01

    BACKGROUND: Regulation of transgene expression in target cells represents a critical and challenging aspect of gene therapy. Recently, a two-plasmid tetracycline-inducible system was developed in which the tetracycline repressor (tetR) alone, rather than the tetR-VP16 fusion derivative, was shown to function as a potent trans-modulator of a second plasmid that contains two tandem repeats of the tetracycline operator (tetO) inserted between the TATA box and the transcription start site of the hCMV major immediate-early promoter. A technological advance in this area would be the development of a single autoregulatory cassette that incorporates both of these components into nonviral and viral gene transfer vectors. For the latter, an inducible lentiviral vector that is capable of temporal and quantitative control of gene expression in either dividing or nondividing cells is highly desirable. MATERIALS AND METHODS: A one-piece inducible (1Pi) autoregulatory cassette was constructed to provide IRES-mediated translation of the tetR as well as tight control over the tetO unit preventing transcription initiation of the first cistron in the absence of the tetracycline. To increase efficiency of tetR-mediated repression, a nuclear localization signal was incorporated at the 3' end of the tetR gene. Regulation of gene expression at the transcriptional and protein level was analyzed in transient transfection experiments using plasmid DNA. Construction of a self-inactivating lentiviral vector containing this 1Pi cassette allowed the study of its long-term effectiveness in primary human cells. RESULTS: The 1Pi autoregulatory cassette when incorporated into plasmid DNA allows efficient control of the secretable hEGF as well as eGFP expression in a variety of cell types. Transient transfection studies demonstrated that the time course of repression is different for the 1Pi and two-plasmid system (2Pi). In the 2Pi system, greater repression is seen with the first 24-48 hr; however

  19. Linear Test Bed. Volume 2: Test Bed No. 2. [linear aerospike test bed for thrust vector control

    NASA Technical Reports Server (NTRS)

    1974-01-01

    Test bed No. 2 consists of 10 combustors welded in banks of 5 to 2 symmetrical tubular nozzle assemblies, an upper stationary thrust frame, a lower thrust frame which can be hinged, a power package, a triaxial combustion wave ignition system, a pneumatic control system, pneumatically actuated propellant valves, a purge and drain system, and an electrical control system. The power package consists of the Mark 29-F fuel turbopump, the Mark 29-0 oxidizer turbopump, a gas generator assembly, and propellant ducting. The system, designated as a linear aerospike system, was designed to demonstrate the feasibility of the concept and to explore technology related to thrust vector control, thrust vector optimization, improved sequencing and control, and advanced ignition systems. The propellants are liquid oxygen/liquid hydrogen. The system was designed to operate at 1200-psia chamber pressure at an engine mixture ratio of 5.5. With 10 combustors, the sea level thrust is 95,000 pounds.

  20. Gene Amplification by PCR and Subcloning into a GFP-Fusion Plasmid Expression Vector as a Molecular Biology Laboratory Course

    ERIC Educational Resources Information Center

    Bornhorst, Joshua A.; Deibel, Michael A.; Mulnix, Amy B.

    2004-01-01

    A novel experimental sequence for the advanced undergraduate laboratory course has been developed at Earlham College. Utilizing recent improvements in molecular techniques for a time-sensitive environment, undergraduates were able to create a chimera of a selected gene and green fluorescent protein (GFP) in a bacterial expression plasmid over the…

  1. Removal of Inserted BAC after linearizatiON (RIBON)—a novel strategy to excise the mini-F sequences from viral BAC vectors

    PubMed Central

    ISHIHARA, Yukari; ESAKI, Motoyuki; YASUDA, Atsushi

    2016-01-01

    The bacterial artificial chromosome (BAC) technology has been a mainstay approach for generating recombinant viruses, and several methods for excision of the mini-F sequences from the viral BAC vectors have been developed. However, these strategies either require complicated procedures or leave scars of inserted sequences. To overcome these problems, a new method to excise the mini-F sequences from viral BAC vectors based on the Removal of Inserted BAC after linearizatiON (RIBON) strategy was developed in this study for herpesvirus of turkeys (HVT). Enhanced green fluorescent protein (eGFP) DNA and the mini-F sequences were inserted into the gene encoding HVT thymidine kinase (TK) by homologous recombination in chicken embryo fibroblasts (CEFs), and the constructed HVT-BAC vector was used to transform Escherichia coli (pHVT-BAC). To remove the inserted eGFP and mini-F sequences, pHVT-BAC was linearized using a homing endonuclease I-SceI and used to cotransfect CEFs together with a plasmid containing the TK gene of HVT. The obtained viruses (44%) did not express eGFP, and DNA sequencing of isolated clones revealed that they were completely free of the inserted BAC sequences. Moreover, growth kinetics and plaque morphology of reconstituted viruses were comparable with those of the parental HVT. The results of this study demonstrate that the novel RIBON approach to remove mini-F sequences from the viral genome is simple and effective. PMID:27041357

  2. Scalar mesons in a linear sigma model with (axial-)vector mesons

    SciTech Connect

    Parganlija, D.; Kovacs, P.; Wolf, Gy.; Giacosa, F.; Rischke, D. H.

    2013-03-25

    The structure of the scalar mesons has been a subject of debate for many decades. In this work we look for qq states among the physical resonances using an extended Linear Sigma Model that contains scalar, pseudoscalar, vector, and axial-vector mesons both in the non-strange and strange sectors. We perform global fits of meson masses, decay widths and amplitudes in order to ascertain whether the scalar qq states are below or above 1 GeV. We find the scalar states above 1 GeV to be preferred as qq states.

  3. Algorithms for solving large sparse systems of simultaneous linear equations on vector processors

    NASA Technical Reports Server (NTRS)

    David, R. E.

    1984-01-01

    Very efficient algorithms for solving large sparse systems of simultaneous linear equations have been developed for serial processing computers. These involve a reordering of matrix rows and columns in order to obtain a near triangular pattern of nonzero elements. Then an LU factorization is developed to represent the matrix inverse in terms of a sequence of elementary Gaussian eliminations, or pivots. In this paper it is shown how these algorithms are adapted for efficient implementation on vector processors. Results obtained on the CYBER 200 Model 205 are presented for a series of large test problems which show the comparative advantages of the triangularization and vector processing algorithms.

  4. Compound gravity receptor polarization vectors evidenced by linear vestibular evoked potentials

    NASA Technical Reports Server (NTRS)

    Jones, S. M.; Jones, T. A.; Bell, P. L.; Taylor, M. J.

    2001-01-01

    The utricle and saccule are gravity receptor organs of the vestibular system. These receptors rely on a high-density otoconial membrane to detect linear acceleration and the position of the cranium relative to Earth's gravitational vector. The linear vestibular evoked potential (VsEP) has been shown to be an effective non-invasive functional test specifically for otoconial gravity receptors (Jones et al., 1999). Moreover, there is some evidence that the VsEP can be used to independently test utricular and saccular function (Taylor et al., 1997; Jones et al., 1998). Here we characterize compound macular polarization vectors for the utricle and saccule in hatchling chickens. Pulsed linear acceleration stimuli were presented in two axes, the dorsoventral (DV, +/- Z axis) to isolate the saccule, and the interaural (IA, +/- Y axis) to isolate the utricle. Traditional signal averaging was used to resolve responses recorded from the surface of the skull. Latency and amplitude of eighth nerve components of the linear VsEP were measured. Gravity receptor responses exhibited clear preferences for one stimulus direction in each axis. With respect to each utricular macula, lateral translation in the IA axis produced maximum ipsilateral response amplitudes with substantially greater amplitude intensity (AI) slopes than medially directed movement. Downward caudal motions in the DV axis produced substantially larger response amplitudes and AI slopes. The results show that the macula lagena does not contribute to the VsEP compound polarization vectors of the sacculus and utricle. The findings suggest further that preferred compound vectors for the utricle depend on the pars externa (i.e. lateral hair cell field) whereas for the saccule they depend on pars interna (i.e. superior hair cell fields). These data provide evidence that maculae saccule and utricle can be selectively evaluated using the linear VsEP.

  5. Compound gravity receptor polarization vectors evidenced by linear vestibular evoked potentials.

    PubMed

    Jones, S M; Jones, T A; Bell, P L; Taylor, M J

    2001-04-01

    The utricle and saccule are gravity receptor organs of the vestibular system. These receptors rely on a high-density otoconial membrane to detect linear acceleration and the position of the cranium relative to Earth's gravitational vector. The linear vestibular evoked potential (VsEP) has been shown to be an effective non-invasive functional test specifically for otoconial gravity receptors (Jones et al., 1999). Moreover, there is some evidence that the VsEP can be used to independently test utricular and saccular function (Taylor et al., 1997; Jones et al., 1998). Here we characterize compound macular polarization vectors for the utricle and saccule in hatchling chickens. Pulsed linear acceleration stimuli were presented in two axes, the dorsoventral (DV, +/- Z axis) to isolate the saccule, and the interaural (IA, +/- Y axis) to isolate the utricle. Traditional signal averaging was used to resolve responses recorded from the surface of the skull. Latency and amplitude of eighth nerve components of the linear VsEP were measured. Gravity receptor responses exhibited clear preferences for one stimulus direction in each axis. With respect to each utricular macula, lateral translation in the IA axis produced maximum ipsilateral response amplitudes with substantially greater amplitude intensity (AI) slopes than medially directed movement. Downward caudal motions in the DV axis produced substantially larger response amplitudes and AI slopes. The results show that the macula lagena does not contribute to the VsEP compound polarization vectors of the sacculus and utricle. The findings suggest further that preferred compound vectors for the utricle depend on the pars externa (i.e. lateral hair cell field) whereas for the saccule they depend on pars interna (i.e. superior hair cell fields). These data provide evidence that maculae saccule and utricle can be selectively evaluated using the linear VsEP. PMID:11423215

  6. Compound gravity receptor polarization vectors evidenced by linear vestibular evoked potentials.

    PubMed

    Jones, S M; Jones, T A; Bell, P L; Taylor, M J

    2001-04-01

    The utricle and saccule are gravity receptor organs of the vestibular system. These receptors rely on a high-density otoconial membrane to detect linear acceleration and the position of the cranium relative to Earth's gravitational vector. The linear vestibular evoked potential (VsEP) has been shown to be an effective non-invasive functional test specifically for otoconial gravity receptors (Jones et al., 1999). Moreover, there is some evidence that the VsEP can be used to independently test utricular and saccular function (Taylor et al., 1997; Jones et al., 1998). Here we characterize compound macular polarization vectors for the utricle and saccule in hatchling chickens. Pulsed linear acceleration stimuli were presented in two axes, the dorsoventral (DV, +/- Z axis) to isolate the saccule, and the interaural (IA, +/- Y axis) to isolate the utricle. Traditional signal averaging was used to resolve responses recorded from the surface of the skull. Latency and amplitude of eighth nerve components of the linear VsEP were measured. Gravity receptor responses exhibited clear preferences for one stimulus direction in each axis. With respect to each utricular macula, lateral translation in the IA axis produced maximum ipsilateral response amplitudes with substantially greater amplitude intensity (AI) slopes than medially directed movement. Downward caudal motions in the DV axis produced substantially larger response amplitudes and AI slopes. The results show that the macula lagena does not contribute to the VsEP compound polarization vectors of the sacculus and utricle. The findings suggest further that preferred compound vectors for the utricle depend on the pars externa (i.e. lateral hair cell field) whereas for the saccule they depend on pars interna (i.e. superior hair cell fields). These data provide evidence that maculae saccule and utricle can be selectively evaluated using the linear VsEP.

  7. SIMULTANEOUSLY SPARSE SOLUTIONS TO LINEAR INVERSE PROBLEMS WITH MULTIPLE SYSTEM MATRICES AND A SINGLE OBSERVATION VECTOR*

    PubMed Central

    ZELINSKI, ADAM C.; GOYAL, VIVEK K.; ADALSTEINSSON, ELFAR

    2010-01-01

    A problem that arises in slice-selective magnetic resonance imaging (MRI) radio-frequency (RF) excitation pulse design is abstracted as a novel linear inverse problem with a simultaneous sparsity constraint. Multiple unknown signal vectors are to be determined, where each passes through a different system matrix and the results are added to yield a single observation vector. Given the matrices and lone observation, the objective is to find a simultaneously sparse set of unknown vectors that approximately solves the system. We refer to this as the multiple-system single-output (MSSO) simultaneous sparse approximation problem. This manuscript contrasts the MSSO problem with other simultaneous sparsity problems and conducts an initial exploration of algorithms with which to solve it. Greedy algorithms and techniques based on convex relaxation are derived and compared empirically. Experiments involve sparsity pattern recovery in noiseless and noisy settings and MRI RF pulse design. PMID:20445814

  8. pSLA2-M of Streptomyces rochei is a composite linear plasmid characterized by self-defense genes and homology with pSLA2-L.

    PubMed

    Yang, Yingjie; Kurokawa, Toru; Takahama, Yoshifumi; Nindita, Yosi; Mochizuki, Susumu; Arakawa, Kenji; Endo, Satoru; Kinashi, Haruyasu

    2011-01-01

    The 113,463-bp nucleotide sequence of the linear plasmid pSLA2-M of Streptomyces rochei 7434AN4 was determined. pSLA2-M had a 69.7% overall GC content, 352-bp terminal inverted repeats with 91% (321/352) identity at both ends, and 121 open reading frames. The rightmost 14.6-kb sequence was almost (14,550/14,555) identical to that of the coexisting 211-kb linear plasmid pSLA2-L. Adjacent to this homologous region an 11.8-kb CRISPR cluster was identified, which is known to function against phage infection in prokaryotes. This cluster region as well as another one containing two large membrane protein genes (orf78 and orf79) were flanked by direct repeats of 194 and 566 bp respectively. Hence the insertion of circular DNAs containing each cluster by homologous recombination was suggested. In addition, the orf71 encoded a Ku70/Ku80-like protein, known to function in the repair of double-strand DNA breaks in eukaryotes, but disruption of it did not affect the radiation sensitivity of the mutant. A pair of replication initiation genes (orf1-orf2) were identified at the extreme left end. Thus, pSLA2-M proved to be a composite linear plasmid characterized by self-defense genes and homology with pSLA2-L that might have been generated by multiple recombination events.

  9. A general theory of linear cosmological perturbations: scalar-tensor and vector-tensor theories

    NASA Astrophysics Data System (ADS)

    Lagos, Macarena; Baker, Tessa; Ferreira, Pedro G.; Noller, Johannes

    2016-08-01

    We present a method for parametrizing linear cosmological perturbations of theories of gravity, around homogeneous and isotropic backgrounds. The method is sufficiently general and systematic that it can be applied to theories with any degrees of freedom (DoFs) and arbitrary gauge symmetries. In this paper, we focus on scalar-tensor and vector-tensor theories, invariant under linear coordinate transformations. In the case of scalar-tensor theories, we use our framework to recover the simple parametrizations of linearized Horndeski and ``Beyond Horndeski'' theories, and also find higher-derivative corrections. In the case of vector-tensor theories, we first construct the most general quadratic action for perturbations that leads to second-order equations of motion, which propagates two scalar DoFs. Then we specialize to the case in which the vector field is time-like (à la Einstein-Aether gravity), where the theory only propagates one scalar DoF. As a result, we identify the complete forms of the quadratic actions for perturbations, and the number of free parameters that need to be defined, to cosmologically characterize these two broad classes of theories.

  10. A bivalent typhoid live vector vaccine expressing both chromosome- and plasmid-encoded Yersinia pestis antigens fully protects against murine lethal pulmonary plague infection.

    PubMed

    Galen, James E; Wang, Jin Yuan; Carrasco, Jose A; Lloyd, Scott A; Mellado-Sanchez, Gabriela; Diaz-McNair, Jovita; Franco, Olga; Buskirk, Amanda D; Nataro, James P; Pasetti, Marcela F

    2015-01-01

    Live attenuated bacteria hold great promise as multivalent mucosal vaccines against a variety of pathogens. A major challenge of this approach has been the successful delivery of sufficient amounts of vaccine antigens to adequately prime the immune system without overattenuating the live vaccine. Here we used a live attenuated Salmonella enterica serovar Typhi strain to create a bivalent mucosal plague vaccine that produces both the protective F1 capsular antigen of Yersinia pestis and the LcrV protein required for secretion of virulence effector proteins. To reduce the metabolic burden associated with the coexpression of F1 and LcrV within the live vector, we balanced expression of both antigens by combining plasmid-based expression of F1 with chromosomal expression of LcrV from three independent loci. The immunogenicity and protective efficacy of this novel vaccine were assessed in mice by using a heterologous prime-boost immunization strategy and compared to those of a conventional strain in which F1 and LcrV were expressed from a single low-copy-number plasmid. The serum antibody responses to lipopolysaccharide (LPS) induced by the optimized bivalent vaccine were indistinguishable from those elicited by the parent strain, suggesting an adequate immunogenic capacity maintained through preservation of bacterial fitness; in contrast, LPS titers were 10-fold lower in mice immunized with the conventional vaccine strain. Importantly, mice receiving the optimized bivalent vaccine were fully protected against lethal pulmonary challenge. These results demonstrate the feasibility of distributing foreign antigen expression across both chromosomal and plasmid locations within a single vaccine organism for induction of protective immunity.

  11. Mutagenic specificity of N-acetoxy-3-aminobenzanthrone, a major metabolically activated form of 3-nitrobenzanthrone, in shuttle vector plasmids propagated in human cells.

    PubMed

    Nishida, Hiroshi; Kawanishi, Masanobu; Takamura-Enya, Takeji; Yagi, Takashi

    2008-06-30

    3-Nitrobenzanthrone (3-NBA) is a potent environmental mutagen and a potential human carcinogen present in diesel exhaust and airborne particulates. N-acetoxy-3-aminobenzanthrone (N-Aco-ABA) has been shown to be a major reactive metabolite of 3-NBA, which mainly produces adducts with guanine and adenine in cellular DNA. Here we analyzed mutations induced by N-Aco-ABA using supF shuttle vector plasmids to elucidate the mutagenic specificity of 3-NBA in human cells. Base sequence analysis of more than 100 plasmids with supF mutations induced in wildtype and DNA repair-deficient XP cells revealed that the major mutation was base substitutions of which the majority (42 and 38%, respectively) were G:C to T:A transversions. The next major mutation was G:C to A:T and A:T to G:C base substitutions in wildtype and XP cells, respectively. The DNA polymerase stop assay using N-Aco-ABA-treated plasmids as a template showed that most stop signals, i.e., adducted sites, appeared at G:C sites. These results suggest that N-Aco-ABA binds preferably to guanine rather than adenine, and adducted adenine is repaired more efficiently by the nucleotide excision repair. Error-prone DNA polymerases could insert adenine at sites opposite to N-Aco-ABA-adducted guanine, which leads to G:C to T:A transversion. These findings could be very important to evaluate the human lung cancer risk of environmental 3-NBA. PMID:18583182

  12. Characterization of a cryptic plasmid from a Greenland ice core Arthrobacter isolate and construction of a shuttle vector that replicates in psychrophilic high G+C Gram-positive recipients.

    PubMed

    Miteva, Vanya; Lantz, Sarah; Brenchley, Jean

    2008-05-01

    Over 60 Greenland glacial isolates were screened for plasmids and antibiotic resistance/sensitivity as the first step in establishing a genetic system. Sequence analysis of a small, cryptic, 1,950 bp plasmid, p54, from isolate GIC54, related to Arthrobacter agilis, showed a region similar to that found in theta replicating Rhodococcus plasmids. A 6,002 bp shuttle vector, pSVJ21, was constructed by ligating p54 and pUC18 and inserting a chloramphenicol acetyl transferase (CAT) cassette conferring chloramphenicol resistance. Candidate Gram-positive recipients were chosen among glacial isolates based on phylogenetic relatedness, relatively short doubling times at low temperatures, sensitivity to antibiotics, and absence of indigenous plasmids. We developed an electroporation protocol and transformed seven isolates related to members of the Arthrobacter, Microbacterium, Curtobacterium, and Rhodoglobus genera with pSVJ21. Plasmid stability was demonstrated by successive transformation into Escherichia coli and four Gram-positive isolates, growth without antibiotic, and plasmid re-isolation. This shuttle vector and our transformation protocol provide the basis for genetic experiments with different high G+C Gram-positive hosts to study cold adaptation and expression of cold-active enzymes at low temperatures.

  13. The primer vector in linear, relative-motion equations. [spacecraft trajectory optimization

    NASA Technical Reports Server (NTRS)

    1980-01-01

    Primer vector theory is used in analyzing a set of linear, relative-motion equations - the Clohessy-Wiltshire equations - to determine the criteria and necessary conditions for an optimal, N-impulse trajectory. Since the state vector for these equations is defined in terms of a linear system of ordinary differential equations, all fundamental relations defining the solution of the state and costate equations, and the necessary conditions for optimality, can be expressed in terms of elementary functions. The analysis develops the analytical criteria for improving a solution by (1) moving any dependent or independent variable in the initial and/or final orbit, and (2) adding intermediate impulses. If these criteria are violated, the theory establishes a sufficient number of analytical equations. The subsequent satisfaction of these equations will result in the optimal position vectors and times of an N-impulse trajectory. The solution is examined for the specific boundary conditions of (1) fixed-end conditions, two-impulse, and time-open transfer; (2) an orbit-to-orbit transfer; and (3) a generalized rendezvous problem. A sequence of rendezvous problems is solved to illustrate the analysis and the computational procedure.

  14. Vector magnetometry based on electromagnetically induced transparency in linearly polarized light

    SciTech Connect

    Yudin, V. I.; Taichenachev, A. V.; Dudin, Y. O.; Velichansky, V. L.; Zibrov, A. S.; Zibrov, S. A.

    2010-09-15

    We develop a generalized principle of electromagnetically induced transparency (EIT) vector magnetometry based on high-contrast EIT resonances and the symmetry of atom-light interaction in the linearly polarized bichromatic fields. Operation of such vector magnetometer on the D{sub 1} line of {sup 87}Rb has been demonstrated. The proposed compass-magnetometer has an increased immunity to shifts produced by quadratic Zeeman and ac-Stark effects, as well as by atom-buffer gas and atom-atom collisions. In our proof-of-principle experiment the detected angular sensitivity to magnetic field orientation is 10{sup -3} deg/Hz{sup 1/2}, which is limited by laser intensity fluctuations, light polarization quality, and magnitude of the magnetic field.

  15. Analysis of chromosomal integration and deletions of yeast plasmids.

    PubMed Central

    Cameron, J R; Philippsen, P; Davis, R W

    1977-01-01

    Plasmid DNAs from six strains of Saccharomyces cerevisiae were compared. Three different plasmids were found, designated Scp 1, Scp 2 and Scp 3, with monomer lengths of 6.19, 6.06 and 5.97 kilobases as referenced to sequenced phiX174 DNA. DNA from each of the plasmids was inserted into a lambda vector DNA. Hybrid phage containing inserted DNA of the desired size were enriched by genetic selection and their DNAs analysed by rapid techniques. All three plasmids share the same organization, two unique sequences separated by two inverted repeats, and share basically the same DNA sequences. Scp 2 and Scp 3 differ from Scp 1 by missing a unique HpaI site and by having small overlapping deletions in the same region. The HpaI site in Scp 1 is, therefore, in a nonessential region and suitable for insertion of foreign DNA in the potential use of the yeast plasmid as a vector. Hybridization of labelled cloned plasmid DNA to restriction fragments of linear yeast DNA separated on agarose gels showed that the plasmid DNA was not stably integrated into the yeast chromosomal DNA. Images PMID:331256

  16. Determination of Meta-Parameters for Support Vector Machine Linear Combinations.

    PubMed

    Jasial, Swarit; Balfer, Jenny; Vogt, Martin; Bajorath, Jürgen

    2015-02-01

    Support vector machines (SVMs) are among the most popular machine learning methods for compound classification and other chemoinformatics tasks such as, for example, the prediction of ligand-target pairs or compound activity profiles. Depending on the specific applications, different SVM strategies can be used. For example, in the context of potency-directed virtual screening, linear combinations of multiple SVM models have been shown to enrich database selection sets with potent compounds compared to individual models. An open question concerning the use of SVM linear combinations (SVM-LCs) is how to best weight the models on a relative scale. Typically, linear weights are subjectively set. Herein, preferred weighting factors for SVM-LC were systematically determined. Therefore, weights were treated as meta-parameters and optimized by machine learning to enrich data set rankings with highly active compounds. The meta-parameter approach has been applied to 10 screening data sets and found to further improve SVM performance over other SVM-LCs and support vector regression (SVR) models. The results show that optimal weights depend on data set characteristics and chosen molecular representations. In addition, individual models often do not contribute to the performance of SVM-LCs. Taken together, these findings emphasize the need for systematic meta-parameter estimation.

  17. Determination of Meta-Parameters for Support Vector Machine Linear Combinations.

    PubMed

    Jasial, Swarit; Balfer, Jenny; Vogt, Martin; Bajorath, Jürgen

    2015-02-01

    Support vector machines (SVMs) are among the most popular machine learning methods for compound classification and other chemoinformatics tasks such as, for example, the prediction of ligand-target pairs or compound activity profiles. Depending on the specific applications, different SVM strategies can be used. For example, in the context of potency-directed virtual screening, linear combinations of multiple SVM models have been shown to enrich database selection sets with potent compounds compared to individual models. An open question concerning the use of SVM linear combinations (SVM-LCs) is how to best weight the models on a relative scale. Typically, linear weights are subjectively set. Herein, preferred weighting factors for SVM-LC were systematically determined. Therefore, weights were treated as meta-parameters and optimized by machine learning to enrich data set rankings with highly active compounds. The meta-parameter approach has been applied to 10 screening data sets and found to further improve SVM performance over other SVM-LCs and support vector regression (SVR) models. The results show that optimal weights depend on data set characteristics and chosen molecular representations. In addition, individual models often do not contribute to the performance of SVM-LCs. Taken together, these findings emphasize the need for systematic meta-parameter estimation. PMID:27490035

  18. Characterization of a linear DNA plasmid from the filamentous fungal plant pathogen Glomerella musae [Anamorph: Colletotrichum musae (Berk. and Curt.) arx.

    USGS Publications Warehouse

    Freeman, S.; Redman, R.S.; Grantham, G.; Rodriguez, R.J.

    1997-01-01

    A 7.4-kilobase (kb) DNA plasmid was isolated from Glomerella musae isolate 927 and designated pGML1. Exonuclease treatments indicated that pGML1 was a linear plasmid with blocked 5' termini. Cell-fractionation experiments combined with sequence-specific PCR amplification revealed that pGML1 resided in mitochondria. The pGML1 plasmid hybridized to cesium chloride-fractionated nuclear DNA but not to A + T-rich mitochondrial DNA. An internal 7.0-kb section of pGML1 was cloned and did not hybridize with either nuclear or mitochondrial DNA from G. musae. Sequence analysis revealed identical terminal inverted repeats (TIR) of 520 bp at the ends of the cloned 7.0-kb section of pGML1. The occurrence of pGML1 did not correspond with the pathogenicity of G. musae on banana fruit. Four additional isolates of G. musae possessed extrachromosomal DNA fragments similar in size and sequence to pGML1.

  19. High-frequency transformation of Brevibacterium lactofermentum protoplasts by plasmid DNA.

    PubMed Central

    Santamaria, R I; Gil, J A; Martin, J F

    1985-01-01

    An efficient polyethylene glycol-assisted method for transformation of Brevibacterium lactofermentum protoplasts that uses plasmid vectors has been developed. Two small plasmids, pUL330 (5.2 kilobases) and pUL340 (5.8 kilobases), both containing the kanamycin resistance gene from transposon Tn5 and the replication origin of the natural plasmid pBL1 of B. lactofermentum, were selected as vectors. Supercoiled forms of the plasmids yielded a 100-fold higher transformation frequency than did linear forms. The optimal transformation frequency was achieved with 10 ng of DNA in 1 ml of transformation buffer. Higher concentrations of plasmid DNA resulted in a decrease in transformation frequency per microgram of DNA. Optimal transformation was obtained with 25 to 35% polyethylene glycol 6000. Under optimal conditions, 10(6) transformants per microgram of DNA were obtained. PMID:3980445

  20. Multi-cavity complex controller with vector simulator for TESLA technology linear accelerator

    NASA Astrophysics Data System (ADS)

    Czarski, Tomasz; Pozniak, Krzysztof T.; Romaniuk, Ryszard S.; Szewinski, Jaroslaw

    2008-01-01

    A digital control, as the main part of the Low Level RF system, for superconducting cavities of a linear accelerator is presented. The FPGA based controller, supported by MATLAB system, was developed to investigate a novel firmware implementation. The complex control algorithm based on the non-linear system identification is the proposal verified by the preliminary experimental results. The general idea is implemented as the Multi-Cavity Complex Controller (MCC) and is still under development. The FPGA based controller executes procedure according to the prearranged control tables: Feed-Forward, Set-Point and Corrector unit, to fulfill the required cavity performance: driving in the resonance during filling and field stabilization for the flattop range. Adaptive control algorithm is applied for the feed-forward and feedback modes. The vector Simulator table has been introduced for an efficient verification of the FPGA controller structure. Experimental results of the internal simulation, are presented for a cavity representative condition.

  1. Vector Sum Excited Linear Prediction (VSELP) speech coding at 4.8 kbps

    NASA Technical Reports Server (NTRS)

    Gerson, Ira A.; Jasiuk, Mark A.

    1990-01-01

    Code Excited Linear Prediction (CELP) speech coders exhibit good performance at data rates as low as 4800 bps. The major drawback to CELP type coders is their larger computational requirements. The Vector Sum Excited Linear Prediction (VSELP) speech coder utilizes a codebook with a structure which allows for a very efficient search procedure. Other advantages of the VSELP codebook structure is discussed and a detailed description of a 4.8 kbps VSELP coder is given. This coder is an improved version of the VSELP algorithm, which finished first in the NSA's evaluation of the 4.8 kbps speech coders. The coder uses a subsample resolution single tap long term predictor, a single VSELP excitation codebook, a novel gain quantizer which is robust to channel errors, and a new adaptive pre/postfilter arrangement.

  2. Peaceful coexistence amongst Borrelia plasmids: getting by with a little help from their friends?

    PubMed

    Chaconas, George; Norris, Steven J

    2013-09-01

    Borrelia species comprise a unique genus of bacterial pathogens. These organisms contain a segmented genome with up to two dozen plasmids ranging in size from 5 kb up to about 200 kb. The plasmids have also been referred to as mini-chromosomes or essential genetic elements, as some of them carry information important for infection of vertebrates or for survival in the tick vector. Most of the plasmids are linear with covalently closed hairpin telomeres and these linear plasmids are in a constant state of genetic rearrangement. The mechanisms of plasmid replication, maintenance and partitioning remain largely obscure and are complicated by a long doubling time, the requirement for expensive media and inefficient genetic manipulation. A set of five parologous protein families (PFs) are believed to confer the ability for autonomous replication and plasmid maintenance. The number of plasmids also complicates analyses because of the possibility that PFs from one plasmid may sometimes function in trans on other plasmids. Two papers in the last year have moved the field forward and their combined data suggest that trans complementation amongst Borrelia plasmids may sometimes occur.

  3. Highly Effective Non-Viral Antitumor Gene Therapy System Comprised of Biocompatible Small Plasmid Complex Particles Consisting of pDNA, Anionic Polysaccharide, and Fully Deprotected Linear Polyethylenimine.

    PubMed

    Koyama, Yoshiyuki; Sugiura, Kikuya; Yoshihara, Chieko; Inaba, Toshio; Ito, Tomoko

    2015-07-23

    We have reported that ternary complexes of plasmid DNA with conventional linear polyethylenimine (l-PEI) and certain polyanions were very stably dispersed, and, with no cryoprotectant, they could be freeze-dried and re-hydrated without the loss of transfection ability. These properties enabled the preparation of a concentrated suspension of very small pDNA complex, by preparing the complexes at highly diluted conditions, followed by condensation via lyophilization-and-rehydration procedure. Recently, a high potency linear polyethylenimine having no residual protective groups, i.e., Polyethylenimine "Max" (PEI "Max"), is available, which has been reported to induce much higher gene expression than conventional l-PEI. We tried to prepare the small DNA/PEI "Max"/polyanion complexes by a similar freeze-drying method. Small complex particles could be obtained without apparent aggregation, but transfection activity of the rehydrated complexes was severely reduced. Complex-preparation conditions were investigated in details to achieve the freeze-dried DNA/PEI "Max"/polyanion small ternary complexes with high transfection efficiency. DNA/PEI "Max"/polyanion complexes containing cytokine-coding plasmids were then prepared, and their anti-tumor therapeutic efficacy was examined in tumor-bearing mice.

  4. Highly Effective Non-Viral Antitumor Gene Therapy System Comprised of Biocompatible Small Plasmid Complex Particles Consisting of pDNA, Anionic Polysaccharide, and Fully Deprotected Linear Polyethylenimine

    PubMed Central

    Koyama, Yoshiyuki; Sugiura, Kikuya; Yoshihara, Chieko; Inaba, Toshio; Ito, Tomoko

    2015-01-01

    We have reported that ternary complexes of plasmid DNA with conventional linear polyethylenimine (l-PEI) and certain polyanions were very stably dispersed, and, with no cryoprotectant, they could be freeze-dried and re-hydrated without the loss of transfection ability. These properties enabled the preparation of a concentrated suspension of very small pDNA complex, by preparing the complexes at highly diluted conditions, followed by condensation via lyophilization-and-rehydration procedure. Recently, a high potency linear polyethylenimine having no residual protective groups, i.e., Polyethylenimine “Max” (PEI “Max”), is available, which has been reported to induce much higher gene expression than conventional l-PEI. We tried to prepare the small DNA/PEI “Max”/polyanion complexes by a similar freeze-drying method. Small complex particles could be obtained without apparent aggregation, but transfection activity of the rehydrated complexes was severely reduced. Complex-preparation conditions were investigated in details to achieve the freeze-dried DNA/PEI “Max”/polyanion small ternary complexes with high transfection efficiency. DNA/PEI “Max”/polyanion complexes containing cytokine-coding plasmids were then prepared, and their anti-tumor therapeutic efficacy was examined in tumor-bearing mice. PMID:26213961

  5. Expression of the major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis in Escherichia coli using an arabinose-inducible plasmid vector.

    PubMed

    Hoelzle, L E; Hoelzle, K; Wittenbrink, M M

    2003-10-01

    The ompA genes encoding the 40 kDa major outer membrane protein (MOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were cloned into the arabinose-inducible plasmid vector pBADMycHis, and recombinant MOMPs (rMOMP) from the three chlamydial species were expressed at high levels in Escherichia (E.) coli. The proteins lacking the 22 aa N-terminal signal peptide were expressed as insoluble cytoplasmic inclusion bodies which were readily purified using immobilized metal-affinity chromatography. The rMOMPs including the N-terminal signal peptide were expressed and translocated as a surface-exposed immunoaccessible protein into the outer membrane of E. coli. Transformants expressing this full-length rMOMP were significantly reduced in viability. Purified native elementary bodies (EB) and rMOMPs of the three chlamydial species purified from the E. coli cytoplasm were used for immunization of rabbits. The resulting sera were analysed for their ability to recognize homologous and heterologous rMOMP and native EB. When testing rMOMP antisera against rMOMP and EB antigens, marked cross-reactivities were detected between the three species. Using EB antisera and rMOMPs as antigens, a significant species-specific reactivity was measured.

  6. [Construction of genomic library of L. interrogans serovar lai using lambda gt11 as the vector and a study of recombiant plasmid pDL121].

    PubMed

    Liu, H; Dai, B; Jing, B; Wu, W; Li, S; Fang, Z; Zhao, H; Ye, D; Yan, R; Liu, J; Song, S; Yang, Y; Zhang, Y; Liu, F; Tu, Y; Yang, H; Huang, Z; Liang, L; Hu, L; Zhao, M

    1997-03-01

    A genomic library of L. interrogans serovar lai strain 017 has been constructed using lambda gt11 as the vector. DNA was partially digested by two blunt-end restriction enzymes, then methylated with EcoR I methylase; after EcoR I linker was added to the DNA, the linker-ended DNA was ligated to the dephosphorylated EcoR I digested lambda gt11 arms. The recombined DNA was packaged in vitro, and used to transduct E. coli Y1090 for amplification. There were 2.1 x 10(6) recombinant bacteriophages as recognized by their ability to form white plaques plated on Lac host in the presence of both IPTG and X-Ga1. A positive clone, designated lambda DL12, was screened with a rabbit anti-serum against L. interrogans serovar lai from the genomic library. The DNA from lambda DL12 was subcloned into plasmid pUC18. A recombinant (designated as pDL121) was obtained. SDS-PAGE analysis indicated that a 23 kd was expressed in E. coli JM 103 harboring pDL121. Western blotting analysis showed that a specific protein band molecular weight of 23 kd could be recognized by the rabbit antiserum against L. interrogans serovar lai strain 017. PMID:10684055

  7. A review of vector convergence acceleration methods, with applications to linear algebra problems

    NASA Astrophysics Data System (ADS)

    Brezinski, C.; Redivo-Zaglia, M.

    In this article, in a few pages, we will try to give an idea of convergence acceleration methods and extrapolation procedures for vector sequences, and to present some applications to linear algebra problems and to the treatment of the Gibbs phenomenon for Fourier series in order to show their effectiveness. The interested reader is referred to the literature for more details. In the bibliography, due to space limitation, we will only give the more recent items, and, for older ones, we refer to Brezinski and Redivo-Zaglia, Extrapolation methods. (Extrapolation Methods. Theory and Practice, North-Holland, 1991). This book also contains, on a magnetic support, a library (in Fortran 77 language) for convergence acceleration algorithms and extrapolation methods.

  8. A Vector Study of Linearized Supersonic Flow Applications to Nonplanar Problems

    NASA Technical Reports Server (NTRS)

    Martin, John C

    1953-01-01

    A vector study of the partial-differential equation of steady linearized supersonic flow is presented. General expressions which relate the velocity potential in the stream to the conditions on the disturbing surfaces, are derived. In connection with these general expressions the concept of the finite part of an integral is discussed. A discussion of problems dealing with planar bodies is given and the conditions for the solution to be unique are investigated. Problems concerning nonplanar systems are investigated, and methods are derived for the solution of some simple nonplanar bodies. The surface pressure distribution and the damping in roll are found for rolling tails consisting of four, six, and eight rectangular fins for the Mach number range where the region of interference between adjacent fins does not affect the fin tips.

  9. Bounded solutions of fermions in the background of mixed vector-scalar inversely linear potentials

    SciTech Connect

    Castro, Antonio S. de . E-mail: castro@feg.unesp.br

    2005-04-01

    The problem of a fermion subject to a general mixing of vector and scalar potentials in a two-dimensional world is mapped into a Sturm-Liouville problem. Isolated bounded solutions are also searched. For the specific case of an inversely linear potential, which gives rise to an effective Kratzer potential in the Sturm-Liouville problem, exact bounded solutions are found in closed form. The case of a pure scalar potential with their isolated zero-energy solutions, already analyzed in a previous work, is obtained as a particular case. The behavior of the upper and lower components of the Dirac spinor is discussed in detail and some unusual results are revealed. The nonrelativistic limit of our results adds a new support to the conclusion that even-parity solutions to the nonrelativistic one-dimensional hydrogen atom do not exist.

  10. Vector meson and associated strangeness production using a linearly polarized photon beam at Jefferson Lab

    SciTech Connect

    Philip L. Cole

    2004-09-01

    The set of experiments forming the g8a run took place in the summer of 2001 in Hall B of Jefferson Lab. The g8a run was the commissioning experiment for the linearly-polarized photon beam at CLAS. The aim of these experiments is to improve the understanding of the underlying symmetry of the quark degrees of freedom in the nucleon, the nature of the parity exchange between the incident photon and the target nucleon, and the mechanism of associated strangeness production in electromagnetic reactions. A beam of tagged and collimated linearly polarized photons (energy range 1.8-2.2 GeV) in conjunction with the large solid angle coverage of CLAS make possible the extraction of the differential cross-sections and polarization observables for the photoproduction of vector mesons and kaons. The reaction channels are under investigation to search for possibly missing nucleon resonances. An overview of the experiment and preliminary results on the measurement of the photon asymmetries of the aforementioned reactions will be presented in this paper.

  11. A cloning vector for creation of Escherichia coli lacZ translational fusions and generation of linear template for chromosomal integrations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel cloning vector to aid in the construction of ß-galactosidase reporter systems for gene expression studies in lactose metabolizing strains of Shiga toxin producing Escherichia coli is described. The plasmid allows construction of translational fusions of cloned gene promoters with a short seg...

  12. A novel method to convert a DNA fragment inserted into a plasmid to an inverted repeat structure.

    PubMed

    Tomimoto, Kazuya; Fujita, Kosuke; Ishibashi, Jun; Imanishi, Shigeo; Yamakawa, Minoru; Tanaka, Hiromitsu

    2012-01-01

    Transfection of an expression plasmid possessing inverted repeat (IR) DNA into cultured cells leads to the overexpression of hairpin RNA and efficient suppression of target gene expression. Such DNA vector-based RNA interference (RNAi) is widely used for characterizing genes of interest in cultured cell lines. In this study, we developed a new method to convert an inserted DNA fragment (IDF) in specially designed plasmid vectors into an IR structure by using nicking endonucleases and BcaBEST DNA polymerase. This method consists of the following steps: (1) linearization of the plasmid with a nick by using a restriction enzyme and a nicking endonuclease, (2) formation of the hairpin-loop DNA at the end near the IDF of the linearized plasmid, (3) insertion of a nick at the other end of the IDF by a nicking endonuclease, (4) execution of the strand displacement reaction from the nick to synthesize IR DNA, and (5) self-ligation of the linear double-stranded DNA. The IR DNA containing expression plasmids constructed by this method effectively induced target-specific RNAi in a silkworm cell line. We further established a method to purify expression plasmids containing IR DNA. Our new methods provide techniques for the construction of long hairpin RNA (lhRNA) expression plasmids for silencing specific genes in silkworms and other organisms, and offer a fundamental methodology for constructing an lhRNA expression library from a cDNA plasmid library. PMID:21516519

  13. A novel method to convert a DNA fragment inserted into a plasmid to an inverted repeat structure.

    PubMed

    Tomimoto, Kazuya; Fujita, Kosuke; Ishibashi, Jun; Imanishi, Shigeo; Yamakawa, Minoru; Tanaka, Hiromitsu

    2012-01-01

    Transfection of an expression plasmid possessing inverted repeat (IR) DNA into cultured cells leads to the overexpression of hairpin RNA and efficient suppression of target gene expression. Such DNA vector-based RNA interference (RNAi) is widely used for characterizing genes of interest in cultured cell lines. In this study, we developed a new method to convert an inserted DNA fragment (IDF) in specially designed plasmid vectors into an IR structure by using nicking endonucleases and BcaBEST DNA polymerase. This method consists of the following steps: (1) linearization of the plasmid with a nick by using a restriction enzyme and a nicking endonuclease, (2) formation of the hairpin-loop DNA at the end near the IDF of the linearized plasmid, (3) insertion of a nick at the other end of the IDF by a nicking endonuclease, (4) execution of the strand displacement reaction from the nick to synthesize IR DNA, and (5) self-ligation of the linear double-stranded DNA. The IR DNA containing expression plasmids constructed by this method effectively induced target-specific RNAi in a silkworm cell line. We further established a method to purify expression plasmids containing IR DNA. Our new methods provide techniques for the construction of long hairpin RNA (lhRNA) expression plasmids for silencing specific genes in silkworms and other organisms, and offer a fundamental methodology for constructing an lhRNA expression library from a cDNA plasmid library.

  14. Linear FMCW Laser Radar for Precision Range and Vector Velocity Measurements

    NASA Technical Reports Server (NTRS)

    Pierrottet, Diego; Amzajerdian, Farzin; Petway, Larry; Barnes, Bruce; Lockhard, George; Rubio, Manuel

    2008-01-01

    An all fiber linear frequency modulated continuous wave (FMCW) coherent laser radar system is under development with a goal to aide NASA s new Space Exploration initiative for manned and robotic missions to the Moon and Mars. By employing a combination of optical heterodyne and linear frequency modulation techniques and utilizing state-of-the-art fiber optic technologies, highly efficient, compact and reliable laser radar suitable for operation in a space environment is being developed. Linear FMCW lidar has the capability of high-resolution range measurements, and when configured into a multi-channel receiver system it has the capability of obtaining high precision horizontal and vertical velocity measurements. Precision range and vector velocity data are beneficial to navigating planetary landing pods to the preselected site and achieving autonomous, safe soft-landing. The all-fiber coherent laser radar has several important advantages over more conventional pulsed laser altimeters or range finders. One of the advantages of the coherent laser radar is its ability to measure directly the platform velocity by extracting the Doppler shift generated from the motion, as opposed to time of flight range finders where terrain features such as hills, cliffs, or slopes add error to the velocity measurement. Doppler measurements are about two orders of magnitude more accurate than the velocity estimates obtained by pulsed laser altimeters. In addition, most of the components of the device are efficient and reliable commercial off-the-shelf fiber optic telecommunication components. This paper discusses the design and performance of a second-generation brassboard system under development at NASA Langley Research Center as part of the Autonomous Landing and Hazard Avoidance (ALHAT) project.

  15. vSmartMOM: A vector matrix operator method-based radiative transfer model linearized with respect to aerosol properties

    NASA Astrophysics Data System (ADS)

    Sanghavi, Suniti; Davis, Anthony B.; Eldering, Annmarie

    2014-01-01

    In this paper, we build up on the scalar model smartMOM to arrive at a formalism for linearized vector radiative transfer based on the matrix operator method (vSmartMOM). Improvements have been made with respect to smartMOM in that a novel method of computing intensities for the exact viewing geometry (direct raytracing) without interpolation between quadrature points has been implemented. Also, the truncation method employed for dealing with highly peaked phase functions has been changed to a vector adaptation of Wiscombe's delta-m method. These changes enable speedier and more accurate radiative transfer computations by eliminating the need for a large number of quadrature points and coefficients for generalized spherical functions. We verify our forward model against the benchmarking results of Kokhanovsky et al. (2010) [22]. All non-zero Stokes vector elements are found to show agreement up to mostly the seventh significant digit for the Rayleigh atmosphere. Intensity computations for aerosol and cloud show an agreement of well below 0.03% and 0.05% at all viewing angles except around the solar zenith angle (60°), where most radiative models demonstrate larger variances due to the strongly forward-peaked phase function. We have for the first time linearized vector radiative transfer based on the matrix operator method with respect to aerosol optical and microphysical parameters. We demonstrate this linearization by computing Jacobian matrices for all Stokes vector elements for a multi-angular and multispectral measurement setup. We use these Jacobians to compare the aerosol information content of measurements using only the total intensity component against those using the idealized measurements of full Stokes vector [I,Q,U,V] as well as the more practical use of only [I,Q,U]. As expected, we find for the considered example that the accuracy of the retrieved parameters improves when the full Stokes vector is used. The information content for the full Stokes

  16. Construction of Plasmid Insulin Gene Vector Containing Metallothionein IIA (pcDNAMTChIns) and Carbohydrate Response Element (ChoRE), and Its Expression in NIH3T3 Cell Line

    PubMed Central

    Piri, Hossein; Kazemi, Bahram; Rezaei, Mohsen; Bandehpour, Mojgan; Khodadadi, Iraj; Hassanzadeh, Taghi; Karimi, Jamshid; Yarian, Fatemeh; Peirovi, Habibollah; Tavakoli, Amir Hossein; Goodarzi, Mohammad Taghi

    2012-01-01

    Background Type 1 diabetes mellitus is one of the metabolic diseases that cause insulin-producing pancreatic ß cells be destroyed by immune system self-reactive T cells. Recent­ly, new treatment methods have been developed including use of the stem cells, ß islet cells transplantation and gene therapy by viral and non-viral gene constructs. Objectives The aim of this project was preparing the non-viral vector containing the glucose inducible insulin gene and using it in the NIH3T3 cell line. Materials and Methods Cloning was carried out by standard methods. Total RNA was extracted from pancreatic tissue, RNA was converted to cDNA using RT-PCR reaction and preproinsulin gene was amplified using specific primers. PNMTCH plasmid was extract­ed and digested by NotI, HindIII, and MTIIA and ChoRE genes were purified and cloned into pcDNA3.1 (-) plasmid and named pcDNAMTCh. Finally, the preproinsulin genes were cloned into pcDNA3.1 (-) plasmid and pcDNAMTChIns was built. Results The cloned gene constructs were evaluated by restriction enzyme digestion and RT-PCR. The NIH3T3 cells were transfected by plasmid naked DNA containing preproinsu­lin gene and expression was confirmed by Reverse Transcriptase PCR and Western Blot­ting Techniques. Conclusions Gel electrophoresis of PCR products confirmed that cloning was per­formed correctly. The expression of preproinsulin gene in recombinant plasmid in NI­H3T3 cell line was observed for the first time. The findings in this study can be the basis of further research on diabetes mellitus type 1 gene therapy on animals. PMID:23843817

  17. Complete nucleotide sequence of the linear DNA plasmid pRS224 with hairpin loops from Rhizoctonia solani and its unique transcriptional form.

    PubMed

    Katsura, K; Sasaki, A; Nagasaka, A; Fuji, M; Miyake, Y; Hashiba, T

    2001-10-01

    The complete nucleotide sequence of the linear DNA plasmid (pRS224-1) from the plant-pathogenic fungus Rhizoctonia solani isolate H-16 was determined; and its unique RNA transcripts were characterized. The pRS224-1 DNA consists of 4,986 nucleotides. A computer-based study of the folding of pRS224-1 at both termini predicted hairpin-loop structures. The hairpin loops consisted of the left and right termini of 236 and 264 nucleotides, respectively, and share no sequence homology. Unique poly(A) RNAs, 4.7 kb and 7.4 kb in length and hybridizing with the pRS224 DNA, were found in mycelial cells of R. solani H-16. Transcript product-mapping allowed the prediction of the locations of different expression signals. The 7.4-kb transcript is generated from the left terminal region of the complementary strand, via the full-length sense-strand, to the right terminal region of the complementary strand. The 4.7-kb transcript is generated from the center region of the sense strand to the right terminal region of the complementary strand. One open reading frame (ORF) found in pRS224-1 is 887 amino acids long and has a potential coding capacity of 102 kDa. The ORF contains the highly conserved domains characteristic of reverse transcriptase sequences, including the highly conserved YXDD sequence.

  18. Dynamic plasmonic beam shaping by vector beams with arbitrary locally linear polarization states

    SciTech Connect

    Man, Zhongsheng; Zhang, Yuquan; Zhang, Chonglei; Du, Luping; Min, Changjun E-mail: xcyuan@szu.edu.cn; Yuan, X.-C. E-mail: xcyuan@szu.edu.cn; Zhu, Siwei; Paul Urbach, H.

    2014-07-07

    Vector beams, which have space-variant state of polarization (SOP) comparing with scalar beams with spatially homogeneous SOP, are used to manipulate surface plasmon polarizations (SPPs). We find that the excitation, orientation, and distribution of the focused SPPs excited in a high numerical aperture microscopic configuration highly depend on the space-variant polarization of the incident vector beam. When it comes to vector beam with axial symmetry, multi-foci of SPPs with the same size and uniform intensity can be obtained, and the number of foci is depending on the polarization order n. Those properties can be of great value in biological sensor and plasmonic tweezers applications.

  19. f(R) gravity on non-linear scales: the post-Friedmann expansion and the vector potential

    SciTech Connect

    Thomas, D.B.; Bruni, M.; Koyama, K.; Li, B.; Zhao, G.-B. E-mail: marco.bruni@port.ac.uk E-mail: baojiu.li@durham.ac.uk

    2015-07-01

    Many modified gravity theories are under consideration in cosmology as the source of the accelerated expansion of the universe and linear perturbation theory, valid on the largest scales, has been examined in many of these models. However, smaller non-linear scales offer a richer phenomenology with which to constrain modified gravity theories. Here, we consider the Hu-Sawicki form of f(R) gravity and apply the post-Friedmann approach to derive the leading order equations for non-linear scales, i.e. the equations valid in the Newtonian-like regime. We reproduce the standard equations for the scalar field, gravitational slip and the modified Poisson equation in a coherent framework. In addition, we derive the equation for the leading order correction to the Newtonian regime, the vector potential. We measure this vector potential from f(R) N-body simulations at redshift zero and one, for two values of the f{sub R{sub 0}} parameter. We find that the vector potential at redshift zero in f(R) gravity can be close to 50% larger than in GR on small scales for |f{sub R{sub 0}}|=1.289 × 10{sup −5}, although this is less for larger scales, earlier times and smaller values of the f{sub R{sub 0}} parameter. Similarly to in GR, the small amplitude of this vector potential suggests that the Newtonian approximation is highly accurate for f(R) gravity, and also that the non-linear cosmological behaviour of f(R) gravity can be completely described by just the scalar potentials and the f(R) field.

  20. Transformation of Azotobacter vinelandii OP with a broad host range plasmid containing a cloned chromosomal nif-DNA marker.

    PubMed

    Bingle, W H

    1988-05-01

    The non-nitrogen-fixing (Nif-) strain UW10 of Azotobacter vinelandii OP (UW) was naturally induced to competence and transformed with broad host range plasmid pKT210 containing the cloned wild-type nif-10 locus from A. vinelandii UW (Nif+); this marker was unable to complement the nif-10 mutation in trans, but could through recombination with the chromosome. The most frequent type of transformation event observed was recombination between the homologous regions of the plasmid and chromosome (producing Nif+ transformants) with loss of the plasmid vector. At a substantially lower frequency, transformants expressing the plasmid-encoded antibiotic resistance determinants were isolated which were phenotypically Nif-. Agarose gel electrophoresis showed that these transformants contained a plasmid migrating with the same mobility as the original donor plasmid. During culture these transformants acquired a Nif+ phenotype without the loss of the plasmid, as judged by the use of a hybridization probe specific for the cloned nif-DNA fragment. These data indicate that plasmids carrying sequences homologous to chromosomal sequences could be maintained in recombination-proficient A. vinelandii UW. The introduction of plasmids containing sequences homologous to chromosomal sequences was facilitated by prelinearization of the plasmid using a restriction endonuclease generating cohesive ends. Because the site of linearization could be chosen outside the region of shared homology, it was unlikely that the route of plasmid establishment occurred via a homology-facilitated transformation mechanism. The data also indicated that A. vinelandii UW could harbor broad host range cloning vectors based on plasmid RSF1010 without significant impairment of its nitrogen-fixation ability.

  1. Significance analysis of qualitative mammographic features, using linear classifiers, neural networks and support vector machines.

    PubMed

    Mavroforakis, Michael; Georgiou, Harris; Dimitropoulos, Nikos; Cavouras, Dionisis; Theodoridis, Sergios

    2005-04-01

    models, namely linear classifiers, neural networks and support vector machines, were employed to investigate the true efficiency of each one of them, as well as the overall complexity of the diagnostic task of mammographic tumor characterization. Both the statistical and the classification results have proven the explicit correlation of all the selected features with the final diagnosis, qualifying them as an adequate input base for any type of similar automated diagnosis system. The underlying complexity of the diagnostic task has justified the high value of sophisticated pattern recognition architectures. PMID:15797296

  2. Primer vector theory applied to the linear relative-motion equations. [for N-impulse space trajectory optimization

    NASA Technical Reports Server (NTRS)

    Jezewski, D.

    1980-01-01

    Prime vector theory is used in analyzing a set of linear relative-motion equations - the Clohessy-Wiltshire (C/W) equations - to determine the criteria and necessary conditions for an optimal N-impulse trajectory. The analysis develops the analytical criteria for improving a solution by: (1) moving any dependent or independent variable in the initial and/or final orbit, and (2) adding intermediate impulses. If these criteria are violated, the theory establishes a sufficient number of analytical equations. The subsequent satisfaction of these equations will result in the optimal position vectors and times of an N-impulse trajectory. The solution is examined for the specific boundary conditions of: (1) fixed-end conditions, two impulse, and time-open transfer; (2) an orbit-to-orbit transfer; and (3) a generalized renezvous problem.

  3. Why Close a Bacterial Genome? The Plasmid of Alteromonas Macleodii HOT1A3 is a Vector for Inter-Specific Transfer of a Flexible Genomic Island

    PubMed Central

    Fadeev, Eduard; De Pascale, Fabio; Vezzi, Alessandro; Hübner, Sariel; Aharonovich, Dikla; Sher, Daniel

    2016-01-01

    Genome sequencing is rapidly becoming a staple technique in environmental and clinical microbiology, yet computational challenges still remain, leading to many draft genomes which are typically fragmented into many contigs. We sequenced and completely assembled the genome of a marine heterotrophic bacterium, Alteromonas macleodii HOT1A3, and compared its full genome to several draft genomes obtained using different reference-based and de novo methods. In general, the de novo assemblies clearly outperformed the reference-based or hybrid ones, covering >99% of the genes and representing essentially all of the gene functions. However, only the fully closed genome (∼4.5 Mbp) allowed us to identify the presence of a large, 148 kbp plasmid, pAM1A3. While HOT1A3 belongs to A. macleodii, typically found in surface waters (“surface ecotype”), this plasmid consists of an almost complete flexible genomic island (fGI), containing many genes involved in metal resistance previously identified in the genomes of Alteromonas mediterranea (“deep ecotype”). Indeed, similar to A. mediterranea, A. macleodii HOT1A3 grows at concentrations of zinc, mercury, and copper that are inhibitory for other A. macleodii strains. The presence of a plasmid encoding almost an entire fGI suggests that wholesale genomic exchange between heterotrophic marine bacteria belonging to related but ecologically different populations is not uncommon. PMID:27014193

  4. Hard-sphere dispersions: Small-wave-vector structure-factor measurements in a linear shear flow

    NASA Astrophysics Data System (ADS)

    Ackerson, Bruce J.; van der Werff, Jos; de Kruif, C. G.

    1988-06-01

    Small-scattering-wave-vector structure-factor measurements have been made for model hard-sphere suspensions undergoing a steady linear shear flow. The samples are comprised of sterically stabilized silica particles in cyclohexane and have been well characterized previously by rheological, light scattering, and neutron scattering measurements. These combined measurements provide a strict test of recent theories of microscopic order in suspensions undergoing shear and suggest a picture which unifies several intuitive notions about suspensions undergoing shear flow: distortion of the pair correlation function, clustering, layering, and nonequilibrium phase transitions.

  5. Walking Drosophila align with the e-vector of linearly polarized light through directed modulation of angular acceleration

    PubMed Central

    Velez, Mariel M.; Wernet, Mathias F.; Clark, Damon A.

    2014-01-01

    Understanding the mechanisms that link sensory stimuli to animal behavior is a central challenge in neuroscience. The quantitative description of behavioral responses to defined stimuli has led to a rich understanding of different behavioral strategies in many species. One important navigational cue perceived by many vertebrates and insects is the e-vector orientation of linearly polarized light. Drosophila manifests an innate orientation response to this cue (‘polarotaxis’), aligning its body axis with the e-vector field. We have established a population-based behavioral paradigm for the genetic dissection of neural circuits guiding polarotaxis to both celestial as well as reflected polarized stimuli. However, the behavioral mechanisms by which flies align with a linearly polarized stimulus remain unknown. Here, we present a detailed quantitative description of Drosophila polarotaxis, systematically measuring behavioral parameters that are modulated by the stimulus. We show that angular acceleration is modulated during alignment, and this single parameter may be sufficient for alignment. Furthermore, using monocular deprivation, we show that each eye is necessary for modulating turns in the ipsilateral direction. This analysis lays the foundation for understanding how neural circuits guide these important visual behaviors. PMID:24810784

  6. Existence of the critical endpoint in the vector meson extended linear sigma model

    NASA Astrophysics Data System (ADS)

    Kovács, P.; Szép, Zs.; Wolf, Gy.

    2016-06-01

    The chiral phase transition of the strongly interacting matter is investigated at nonzero temperature and baryon chemical potential (μB) within an extended (2 +1 ) flavor Polyakov constituent quark-meson model that incorporates the effect of the vector and axial vector mesons. The effect of the fermionic vacuum and thermal fluctuations computed from the grand potential of the model is taken into account in the curvature masses of the scalar and pseudoscalar mesons. The parameters of the model are determined by comparing masses and tree-level decay widths with experimental values in a χ2-minimization procedure that selects between various possible assignments of scalar nonet states to physical particles. We examine the restoration of the chiral symmetry by monitoring the temperature evolution of condensates and the chiral partners' masses and of the mixing angles for the pseudoscalar η -η' and the corresponding scalar complex. We calculate the pressure and various thermodynamical observables derived from it and compare them to the continuum extrapolated lattice results of the Wuppertal-Budapest collaboration. We study the T -μB phase diagram of the model and find that a critical endpoint exists for parameters of the model, which give acceptable values of χ2.

  7. Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes.

    PubMed Central

    Ward, J E; Dale, E M; Christie, P J; Nester, E W; Binns, A N

    1990-01-01

    The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the approximately 9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virB11, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHo1 insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirB11-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A. tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virB11 genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virB11, the virB9 and virB10 genes are required for tumorigenicity. Images PMID:2203743

  8. Characterization of four plasmids harboured in a Lactobacillus brevis strain encoding a novel bacteriocin, brevicin 925A, and construction of a shuttle vector for lactic acid bacteria and Escherichia coli.

    PubMed

    Wada, Takaomi; Noda, Masafumi; Kashiwabara, Fumi; Jeon, Hyung Joon; Shirakawa, Ayano; Yabu, Hironori; Matoba, Yasuyuki; Kumagai, Takanori; Sugiyama, Masanori

    2009-05-01

    In this study we isolated over 250 lactic acid bacteria (LAB) candidates from fruit, flowers, vegetables and a fermented food to generate an LAB library. One strain, designated 925A, isolated from kimchi (a traditional Korean fermented dish made from Chinese cabbage) produced a novel type of bacteriocin, brevicin 925A, which is effective against certain LAB, including strains of Lactobacillus, Enterococcus, Streptococcus, Bacillus and Listeria. Strain 925A, identified as Lactobacillus brevis, harboured at least four plasmids and we determined the entire nucleotide sequence of each one. The four plasmids were designated pLB925A01-04, and have molecular sizes of 1815, 3524, 8881 and 65 037 bp, respectively. We obtained bacteriocin non-producing derivatives by treatment of strain 925A with novobiocin. All of these derivatives, which were susceptible to their own antibacterial product, lost the largest plasmid, pLB925A04, suggesting that the genes for bacteriocin biosynthesis (breB and breC) and immunity (breE) are located on pLB925A04. The partial amino acid sequence of purified brevicin 925A and sequence analysis of pLB925A04 showed that breB is the structural gene for brevicin 925A. We constructed a shuttle vector (pLES003, 6134 bp) that can replicate in both Escherichia coli and LAB such as Lactobacillus plantarum, Lb. brevis, Lactobacillus helveticus, Lactobacillus hilgardii and Enterococcus hirae. To determine the function of gene breE, which displays no significant similarity to any other sequences in the blast search database, the gene was inserted into pLES003. A pLB925A04-cured derivative transformed with pLES003 carrying breE acquired immunity to brevicin 925A, suggesting that breE encodes an immunity protein.

  9. Historical Events That Spawned the Field of Plasmid Biology.

    PubMed

    Kado, Clarence I

    2014-10-01

    This chapter revisits the historical development and outcome of studies focused on the transmissible, extrachromosomal genetic elements called plasmids. Early work on plasmids involved structural and genetic mapping of these molecules, followed by the development of an understanding of how plasmids replicate and segregate during cell division. The intriguing property of plasmid transmission between bacteria and between bacteria and higher cells has received considerable attention. The utilitarian aspects of plasmids are described, including examples of various plasmid vector systems. This chapter also discusses the functional attributes of plasmids needed for their persistence and survival in nature and in man-made environments. The term plasmid biology was first conceived at the Fallen Leaf Lake Conference on Promiscuous Plasmids, 1990, Lake Tahoe, California. The International Society for Plasmid Biology was established in 2004 (www.ISPB.org).

  10. Historical Events That Spawned the Field of Plasmid Biology.

    PubMed

    Kado, Clarence I

    2014-10-01

    This chapter revisits the historical development and outcome of studies focused on the transmissible, extrachromosomal genetic elements called plasmids. Early work on plasmids involved structural and genetic mapping of these molecules, followed by the development of an understanding of how plasmids replicate and segregate during cell division. The intriguing property of plasmid transmission between bacteria and between bacteria and higher cells has received considerable attention. The utilitarian aspects of plasmids are described, including examples of various plasmid vector systems. This chapter also discusses the functional attributes of plasmids needed for their persistence and survival in nature and in man-made environments. The term plasmid biology was first conceived at the Fallen Leaf Lake Conference on Promiscuous Plasmids, 1990, Lake Tahoe, California. The International Society for Plasmid Biology was established in 2004 (www.ISPB.org). PMID:26104369

  11. Acoustic Biometric System Based on Preprocessing Techniques and Linear Support Vector Machines.

    PubMed

    del Val, Lara; Izquierdo-Fuente, Alberto; Villacorta, Juan J; Raboso, Mariano

    2015-06-17

    Drawing on the results of an acoustic biometric system based on a MSE classifier, a new biometric system has been implemented. This new system preprocesses acoustic images, extracts several parameters and finally classifies them, based on Support Vector Machine (SVM). The preprocessing techniques used are spatial filtering, segmentation-based on a Gaussian Mixture Model (GMM) to separate the person from the background, masking-to reduce the dimensions of images-and binarization-to reduce the size of each image. An analysis of classification error and a study of the sensitivity of the error versus the computational burden of each implemented algorithm are presented. This allows the selection of the most relevant algorithms, according to the benefits required by the system. A significant improvement of the biometric system has been achieved by reducing the classification error, the computational burden and the storage requirements.

  12. Acoustic Biometric System Based on Preprocessing Techniques and Linear Support Vector Machines

    PubMed Central

    del Val, Lara; Izquierdo-Fuente, Alberto; Villacorta, Juan J.; Raboso, Mariano

    2015-01-01

    Drawing on the results of an acoustic biometric system based on a MSE classifier, a new biometric system has been implemented. This new system preprocesses acoustic images, extracts several parameters and finally classifies them, based on Support Vector Machine (SVM). The preprocessing techniques used are spatial filtering, segmentation—based on a Gaussian Mixture Model (GMM) to separate the person from the background, masking—to reduce the dimensions of images—and binarization—to reduce the size of each image. An analysis of classification error and a study of the sensitivity of the error versus the computational burden of each implemented algorithm are presented. This allows the selection of the most relevant algorithms, according to the benefits required by the system. A significant improvement of the biometric system has been achieved by reducing the classification error, the computational burden and the storage requirements. PMID:26091392

  13. Acoustics vector sensor linear array passive ranging based on waveguide invariant

    NASA Astrophysics Data System (ADS)

    Li, Jian; Sun, Guiqing; Han, Qingbang; Zhang, Chunhua

    2012-11-01

    A passive ranging method is proposed based on waveguide invariant analysis. The received Low Frequency Analysis Record (LOFAR) spectrum contains parabolic striations when a wideband target passes by the Closest Point of Approach (CPA). We can extract the striations through a suitable image processing technique such as the HOUGH transform, and we can then derive the waveguide invariant. Finally we can estimate the range of the target. A vector LOFARgram containing particle velocity information has higher SNR than a scalar LOFARgram, and this information can improve the precision of range estimate. This method can estimate the range of the CPA with high precision for both simulation and experimental data. In estimating the CPA range, both the experimental value and the measured value of the waveguide invariant are used. The results show that the measured value is more credible.

  14. Combination of the somatic cell nuclear transfer method and RNAi technology for the production of a prion gene-knockdown calf using plasmid vectors harboring the U6 or tRNA promoter.

    PubMed

    Wongsrikeao, Pimprapar; Sutou, Shizuyo; Kunishi, Miho; Dong, Ya Juan; Bai, Xuejin; Otoi, Takeshige

    2011-01-01

    By combining RNAi technology with SCNT method, we attempted to produce transgenic calves with knocked down bPRNP for technological assessments. The respective utilities of type II (tRNA) and type III (hU6) Pol III promoters in mediating plasmid vector-based RNAi for the production of a bPRNP-knockdown calf were compared. Plasmid harboring DNA for siRNA expression was introduced stably into the genome of primary cultured bovine cells. By inserting the transgenic cell into an enucleated bovine egg, SCNT embryos were produced. The ability for SCNT embryos to develop to blastocysts was higher in hU6 based vector groups (44-53%) than in a tRNA group (32%). In all, 30 hU6-embryos and 12 tRNA-embryos were transferred to 11 recipients. Only tRNA-embryos were able to impregnate recipients (6 out of 11 transfers), resulting in four aborted fetuses, one stillbirth, and one live-born calf. The expression of EGFP, a marker, was detected in all six. The bPRNP transcript levels in the nervous tissues (brain, cerebellum, spinal bulb, and spinal cord) from the calf, which was killed 20 days after birth, were reduced to 35% of those of the control calf on average, as determined by qRT-PCR. The PrPC levels, as estimated by western blot were reduced to 86% on average in the nervous tissues. These findings suggest that SCNT technology remains immature, that the tRNA promoter is useful, and that RNAi can significantly reduce PRNP mRNA levels, but insufficient reduction of PrPC levels exists in cattle under these conditions.

  15. Linear and Nonlinear Support Vector Machine for the Classification of Human 5-HT1A Ligand Functionality.

    PubMed

    Wang, Lirong; Ma, Chao; Wipf, Peter; Xie, Xiang-Qun

    2012-01-01

    Upon binding to a receptor, agonists and antagonists can induce distinct biological functions and thus lead to significantly different pharmacological responses. Thus, in silico prediction or in vitro characterization of ligand agonistic or antagonistic functionalities is an important step toward identifying specific pharmacological therapeutics. In this study, we investigated the molecular properties of agonists and antagonists of human 5-hydroxytryptamine receptor subtype 1A (5-HT1A ). Subsequently, intrinsic functions of these ligands (agonists/antagonists) were modeled by support vector machine (SVM), using five 2D molecular fingerprints and the 3D Topomer distance. Five kernel functions, including linear, polynomial, RBF, Tanimoto and a novel Topomer kernel based on Topomer 3D similarity were used to develop linear and nonlinear classifiers. These classifiers were validated through cross-validation, yielding a classification accuracy ranging from 80.4 % to 92.3 %. The performance of different kernels and fingerprints was analyzed and discussed. Linear and nonlinear models were further interpreted through the illustration of underlying classification mechanism. The computation protocol has been automated and demonstrated through our online service. This study expands the scope and applicability of similarity-based methods in cheminformatics, which are typically used for the identification of active molecules against a target protein. Our findings provide a good starting point for further systematic classifications of other GPCR ligands and for the data mining of large chemical libraries.

  16. Complete genome sequencing of Agrobacterium sp. H13-3, the former Rhizobium lupini H13-3, reveals a tripartite genome consisting of a circular and a linear chromosome and an accessory plasmid but lacking a tumor-inducing Ti-plasmid.

    PubMed

    Wibberg, Daniel; Blom, Jochen; Jaenicke, Sebastian; Kollin, Florian; Rupp, Oliver; Scharf, Birgit; Schneiker-Bekel, Susanne; Sczcepanowski, Rafael; Goesmann, Alexander; Setubal, Joao Carlos; Schmitt, Rüdiger; Pühler, Alfred; Schlüter, Andreas

    2011-08-20

    Agrobacterium sp. H13-3, formerly known as Rhizobium lupini H13-3, is a soil bacterium that was isolated from the rhizosphere of Lupinus luteus. The isolate has been established as a model system for studying novel features of flagellum structure, motility and chemotaxis within the family Rhizobiaceae. The complete genome sequence of Agrobacterium sp. H13-3 has been established and the genome structure and phylogenetic assignment of the organism was analysed. For de novo sequencing of the Agrobacterium sp. H13-3 genome, a combined strategy comprising 454-pyrosequencing on the Genome Sequencer FLX platform and PCR-based amplicon sequencing for gap closure was applied. The finished genome consists of three replicons and comprises 5,573,770 bases. Based on phylogenetic analyses, the isolate could be assigned to the genus Agrobacterium biovar I and represents a genomic species G1 strain within this biovariety. The highly conserved circular chromosome (2.82 Mb) of Agrobacterium sp. H13-3 mainly encodes housekeeping functions characteristic for an aerobic, heterotrophic bacterium. Agrobacterium sp. H13-3 is a motile bacterium driven by the rotation of several complex flagella. Its behaviour towards external stimuli is regulated by a large chemotaxis regulon and a total of 17 chemoreceptors. Comparable to the genome of Agrobacterium tumefaciens C58, Agrobacterium sp. H13-3 possesses a linear chromosome (2.15 Mb) that is related to its reference replicon and features chromosomal and plasmid-like properties. The accessory plasmid pAspH13-3a (0.6 Mb) is only distantly related to the plasmid pAtC58 of A. tumefaciens C58 and shows a mosaic structure. A tumor-inducing Ti-plasmid is missing in the sequenced strain H13-3 indicating that it is a non-virulent isolate. PMID:21329740

  17. Combining support vector machines with linear quadratic regulator adaptation for the online design of an automotive active suspension system

    NASA Astrophysics Data System (ADS)

    Chiou, J.-S.; Liu, M.-T.

    2008-02-01

    As a powerful machine-learning approach to pattern recognition problems, the support vector machine (SVM) is known to easily allow generalization. More importantly, it works very well in a high-dimensional feature space. This paper presents a nonlinear active suspension controller which achieves a high level performance by compensating for actuator dynamics. We use a linear quadratic regulator (LQR) to ensure optimal control of nonlinear systems. An LQR is used to solve the problem of state feedback and an SVM is used to address the question of the estimation and examination of the state. These two are then combined and designed in a way that outputs feedback control. The real-time simulation demonstrates that an active suspension using the combined SVM-LQR controller provides passengers with a much more comfortable ride and better road handling.

  18. A polymerase chain reaction-based method for constructing a linear vector with site-specific DNA methylation.

    PubMed

    Arakawa, Toshiya; Ohta, Tohru; Abiko, Yoshihiro; Okayama, Miki; Mizoguchi, Itaru; Takuma, Taishin

    2011-09-15

    DNA methylation is an important epigenetic modification that leads to a wide variety of biological functions, including transcription, growth and development, and diseases associated with altered gene expression such as cancers. However, tools to insert site-specific methylation into DNA for analyzing epigenetic functions are limited. Here we describe a novel polymerase chain reaction (PCR)-based approach to provide site-specific DNA methylation at any site, including CpG or CpNpG islands. This method is simple and versatile, and it consists of four steps to construct the DNA methylation vector: (I) design and synthesis of methylated primers, (II) PCR amplification, (III) isolation of single-stranded DNA, and (IV) annealing and ligation of isolated single-stranded DNAs. First we produced and validated a linear green fluorescence protein (GFP) vector by this method. Next we applied this method to introduce methyl groups into the promoter of the cyclooxygenase-2 (COX-2) gene and found that site-specific DNA methylation at the CRE element significantly altered COX-2 gene expression. These results demonstrate that this PCR-based approach is useful for the analysis of biological functions that depend on DNA methylation. PMID:21669180

  19. Moving Kriging shape function modeling of vector TARMA models for modal identification of linear time-varying structural systems

    NASA Astrophysics Data System (ADS)

    Yang, Wu; Liu, Li; Zhou, Si-Da; Ma, Zhi-Sai

    2015-10-01

    This work proposes a Moving Kriging (MK) shape function modeling method for modal identification of linear time-varying (LTV) structural systems based on vector time-dependent autoregressive moving average (VTARMA) models. It aims to avoid the functional subspaces selection of the conventional functional series VTARMA (FS-VTARMA) models. Instead of the common basis functions, it constructs the time-varying coefficients on the time nodes with the MK shape functions in a compact support domain. The merit of the MK shape function is to determine its shape parameters upon vector random vibration signals adaptively. Model identification is effectively dealt with through an optimization scheme that decomposes the identification problem into two subproblems: estimating model parameters via two-stage least squares (2SLS) method and estimating shape function parameters via a discrete-continuous-variable hybrid optimization. In addition, the model order selection is achieved by the optimization scheme. This method has been validated by a Monte Carlo study of simulation case and further by an experimental test case, and the performance and potential advantages are illustrated.

  20. Targeted therapy via oral administration of attenuated Salmonella expression plasmid-vectored Stat3-shRNA cures orthotopically transplanted mouse HCC.

    PubMed

    Tian, Y; Guo, B; Jia, H; Ji, K; Sun, Y; Li, Y; Zhao, T; Gao, L; Meng, Y; Kalvakolanu, D V; Kopecko, D J; Zhao, X; Zhang, L; Xu, D

    2012-06-01

    The development of RNA interference-based cancer gene therapies has been delayed due to the lack of effective tumor-targeting delivery systems. Attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) has a natural tropism for solid tumors. We report here the use of attenuated S. Typhimurium as a vector to deliver shRNA directly into tumor cells. Constitutively activated signal transducer and activator of transcription 3 (Stat3) is a key transcription factor involved in both hepatocellular carcinoma (HCC) growth and metastasis. In this study, attenuated S. Typhimurium was capable of delivering shRNA-expressing vectors to the targeted cancer cells and inducing RNA interference in vivo. More importantly, a single oral dose of attenuated S. Typhimurium carrying shRNA-expressing vectors targeting Stat3 induced remarkably delayed and reduced HCC (in 70% of mice). Cancer in these cured mice did not recur over 2 years following treatment. These data demonstrated that RNA interference combined with Salmonella as a delivery system may offer a novel clinical approach for cancer gene therapy. PMID:22555509

  1. The fully non-linear post-Friedmann frame-dragging vector potential: magnitude and time evolution from N-body simulations

    NASA Astrophysics Data System (ADS)

    Thomas, Daniel B.; Bruni, Marco; Wands, David

    2015-09-01

    Newtonian simulations are routinely used to examine the matter dynamics on non-linear scales. However, even on these scales, Newtonian gravity is not a complete description of gravitational effects. A post-Friedmann approach shows that the leading-order correction to Newtonian theory is a vector potential in the metric. This vector potential can be calculated from N-body simulations, requiring a method for extracting the velocity field. Here, we present the full details of our calculation of the post-Friedmann vector potential, using the Delaunay Tessellation Field Estimator code. We include a detailed examination of the robustness of our numerical result, including the effects of box size and mass resolution on the extracted fields. We present the power spectrum of the vector potential and find that the power spectrum of the vector potential is ˜105 times smaller than the power spectrum of the fully non-linear scalar gravitational potential at redshift zero. Comparing our numerical results to perturbative estimates, we find that the fully non-linear result can be more than an order of magnitude larger than the perturbative estimate on small scales. We extend the analysis of the vector potential to multiple redshifts, showing that this ratio persists over a range of scales and redshifts. We also comment on the implications of our results for the validity and interpretation of Newtonian simulations.

  2. Enhancing immune responses of EV71 VP1 DNA vaccine by co-inoculating plasmid IL-12 or GM-CSF expressing vector in mice.

    PubMed

    Peng, X; Fang, X; Li, J; Kong, L; Li, B; Ding, X

    2016-01-01

    Enterovirus 71 (EV71) is a major causative viral agent for large outbreaks of hand, foot, and mouth disease in children and infants, yet there is no vaccine or effective antiviral treatment for severe EV71 infection. The immunogenicity of EV71 VP1 DNA vaccine and the immunoregulatory activity of interleukin-12 (IL-12) or granulocyte-monocyte colony stimulating factor (GM-CSF) were investigated. DNA vaccine plasmids, pcDNA-VP1, pcDNA-IL-12 and pcDNA-GM-CSF were constructed and inoculated into BALB/c mice with or without pcDNA-IL-12 or pcDNA-GM-CSF by intramuscular injection. Cellular and humoral immune responses were assessed by indirect ELISA, lymphocyte proliferation assays, cytokine release assay and FACS. The VP1 DNA vaccine had good immunogenicity and can induce specific humoral and cellular immunity in BALB/c mice, while IL-2 or GM-CSF plays an immunoadjuvant role and enhances specific immune responses. This study provides a frame of reference for the design of DNA vaccines against EV71. PMID:27188732

  3. Enhancing immune responses of EV71 VP1 DNA vaccine by co-inoculating plasmid IL-12 or GM-CSF expressing vector in mice.

    PubMed

    Peng, X; Fang, X; Li, J; Kong, L; Li, B; Ding, X

    2016-01-01

    Enterovirus 71 (EV71) is a major causative viral agent for large outbreaks of hand, foot, and mouth disease in children and infants, yet there is no vaccine or effective antiviral treatment for severe EV71 infection. The immunogenicity of EV71 VP1 DNA vaccine and the immunoregulatory activity of interleukin-12 (IL-12) or granulocyte-monocyte colony stimulating factor (GM-CSF) were investigated. DNA vaccine plasmids, pcDNA-VP1, pcDNA-IL-12 and pcDNA-GM-CSF were constructed and inoculated into BALB/c mice with or without pcDNA-IL-12 or pcDNA-GM-CSF by intramuscular injection. Cellular and humoral immune responses were assessed by indirect ELISA, lymphocyte proliferation assays, cytokine release assay and FACS. The VP1 DNA vaccine had good immunogenicity and can induce specific humoral and cellular immunity in BALB/c mice, while IL-2 or GM-CSF plays an immunoadjuvant role and enhances specific immune responses. This study provides a frame of reference for the design of DNA vaccines against EV71.

  4. The replication origin of a repABC plasmid

    PubMed Central

    2011-01-01

    Background repABC operons are present on large, low copy-number plasmids and on some secondary chromosomes in at least 19 α-proteobacterial genera, and are responsible for the replication and segregation properties of these replicons. These operons consist, with some variations, of three genes: repA, repB, and repC. RepA and RepB are involved in plasmid partitioning and in the negative regulation of their own transcription, and RepC is the limiting factor for replication. An antisense RNA encoded between the repB-repC genes modulates repC expression. Results To identify the minimal region of the Rhizobium etli p42d plasmid that is capable of autonomous replication, we amplified different regions of the repABC operon using PCR and cloned the regions into a suicide vector. The resulting vectors were then introduced into R. etli strains that did or did not contain p42d. The minimal replicon consisted of a repC open reading frame under the control of a constitutive promoter with a Shine-Dalgarno sequence that we designed. A sequence analysis of repC revealed the presence of a large A+T-rich region but no iterons or DnaA boxes. Silent mutations that modified the A+T content of this region eliminated the replication capability of the plasmid. The minimal replicon could not be introduced into R. etli strain containing p42d, but similar constructs that carried repC from Sinorhizobium meliloti pSymA or the linear chromosome of Agrobacterium tumefaciens replicated in the presence or absence of p42d, indicating that RepC is an incompatibility factor. A hybrid gene construct expressing a RepC protein with the first 362 amino acid residues from p42d RepC and the last 39 amino acid residues of RepC from SymA was able to replicate in the presence of p42d. Conclusions RepC is the only element encoded in the repABC operon of the R. etli p42d plasmid that is necessary and sufficient for plasmid replication and is probably the initiator protein. The oriV of this plasmid resides

  5. Characteristic element of matrix attachment region mediates vector attachment and enhances nerve growth factor expression in Chinese hamster ovary cells.

    PubMed

    Wang, X Y; Zhang, J H; Sun, Q L; Yao, Z Y; Deng, B G; Guo, W Y; Wang, L; Dong, W H; Wang, F; Zhao, C P; Wang, T Y

    2015-08-07

    Preliminary studies have suggested that a characteristic element of the matrix attachment region (MAR) in human interferon-β mediates the adhesion of vectors to Chinese hamster ovary (CHO) cells. In this study, we investigated if vector adhesion increased nerve growth factor (NGF) expression in CHO cells. The MAR characteristic element sequence of human interferon-β was inserted into the multiple-cloning site of the pEGFP-C1 vector. The target NGF gene was inserted upstream of the MAR characteristic element sequence to construct the MAR/NGF expression vector. The recombinant plasmid was transfected into CHO cells and stable monoclonal cells were selected using G418. NGF mRNA and protein expression was detected by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Plasmid reduction experiments were used to determine the state of transfected plasmid in mammalian cells. The insertion of MAR into the vector increased NGF expression levels in CHO cells (1.93- fold) compared to the control. The recombinant plasmid expressing the MAR sequence was digested into a linear space vector. The inserted MAR and NGF sequences were consistent with those inserted into the plasmid before recombination. Therefore, we concluded that the MAR characteristic element mediates vector adhesion to CHO cells and enhances the stability and efficiency of the target gene expression.

  6. Upper bounds for convergence rates of vector extrapolation methods on linear systems with initial iterations. Ph.D. Thesis

    NASA Technical Reports Server (NTRS)

    Sidi, Avram; Shapira, Yair

    1992-01-01

    The application of the minimal polynomial extrapolation (MPE) and the reduced rank extrapolation (RRE) to a vector sequence obtained by the linear iterative technique x(sub j) + 1 = Ax(sub j) = b,j = 1,2,..., is considered. Both methods produce a two dimensional array of approximations s(sub n,k) to the solution of the system (I - A)x = b. Here, s(sub n,k) is obtained from the vectors x(sub j), n is less than or equal to j is less than or equal to n + k + 1. It was observed in an earlier publication by the first author that the sequence s(sub n,k), k = 1,2,..., for n greater than 0, but fixed, possesses better convergence properties than the sequence s(sub 0,k), k = 1,2,.... A detailed theoretical explanation for this phenomenon is provided in the present work. This explanation is heavily based on approximations by incomplete polynomials. It is demonstrated by numerical examples when the matrix A is sparse that cycling with s(sub n,k) for n greater than 0, but fixed, produces better convergence rates and costs less computationally than cycling with s(sub 0,k). It is also illustrated numerically with a convection-diffusion problem that the former may produce excellent results where the latter may fail completely. As has been shown in an earlier publication, the results produced by s(sub 0,k) are identical to the corresponding results obtained by applying the Arnoldi method or generalized minimal residual scheme (GMRES) to the system (I - A)x = b.

  7. Preparation of Pichia pastoris expression plasmids.

    PubMed

    Logez, Christel; Alkhalfioui, Fatima; Byrne, Bernadette; Wagner, Renaud

    2012-01-01

    When planning any heterologous expression experiment, the very first critical step is related to the design of the overall strategy, hence to the selection of the most adapted expression vector. The very flexible Pichia pastoris system offers a broad range of possibilities for the production of secreted, endogenous or membrane proteins thanks to a combination of various plasmid backbones, selection markers, promoters and fusion sequences introduced into dedicated host strains. The present chapter provides some guidelines on the choice of expression vectors and expression strategies. It also brings the reader a complete toolbox from which plasmids and fusion sequences can be picked and assembled to set up appropriate expression vectors. Finally, it provides standard starting protocols for the preparation of the selected plasmids and their use for host strain transformation.

  8. Molecular mechanisms of intramolecular recombination-dependent recircularization of linearized plasmid DNA in Escherichia coli: requirements for the ruvA, ruvB, recG, recF and recR gene products.

    PubMed

    McFarlane, R J; Saunders, J R

    1996-10-24

    Intramolecular recombinogenic recircularization (IRR) of linearized plasmid DNA was used to study mechanistic relationships between recombination functions in Escherichia coli in vivo. Homology requirement for IRR ranges from 1 to 11 bp, and does not exhibit any notable strain to strain variability, with recombination occurring at a large number of possible sites within the plasmid molecule. We show that recF- and recR-deficient strains exhibit greatly reduced IRR efficiency, although neither gene product is totally essential. Mutation of recF and recR does not alter the distribution of recombination sites nor the range of molecules produced during IRR. A recO-deficient strain did not exhibit dramatic reduction in efficiency of IRR, implying that RecF and RecR proteins maintain function during this mechanism in the absence of functional RecO. The main IRR mechanism is ruvA-, ruvB- and recG-dependent and there is a lower efficiency second IRR mechanism operating in ruvA, ruvB and recG mutants. Some evidence suggests that this second mechanism involves functions associated with the replisome. PMID:8921869

  9. Plasmids from Euryarchaeota.

    PubMed

    Forterre, Patrick; Krupovic, Mart; Raymann, Kasie; Soler, Nicolas

    2014-12-01

    Many plasmids have been described in Euryarchaeota, one of the three major archaeal phyla, most of them in salt-loving haloarchaea and hyperthermophilic Thermococcales. These plasmids resemble bacterial plasmids in terms of size (from small plasmids encoding only one gene up to large megaplasmids) and replication mechanisms (rolling circle or theta). Some of them are related to viral genomes and form a more or less continuous sequence space including many integrated elements. Plasmids from Euryarchaeota have been useful for designing efficient genetic tools for these microorganisms. In addition, they have also been used to probe the topological state of plasmids in species with or without DNA gyrase and/or reverse gyrase. Plasmids from Euryarchaeota encode both DNA replication proteins recruited from their hosts and novel families of DNA replication proteins. Euryarchaeota form an interesting playground to test evolutionary hypotheses on the origin and evolution of viruses and plasmids, since a robust phylogeny is available for this phylum. Preliminary studies have shown that for different plasmid families, plasmids share a common gene pool and coevolve with their hosts. They are involved in gene transfer, mostly between plasmids and viruses present in closely related species, but rarely between cells from distantly related archaeal lineages. With few exceptions (e.g., plasmids carrying gas vesicle genes), most archaeal plasmids seem to be cryptic. Interestingly, plasmids and viral genomes have been detected in extracellular membrane vesicles produced by Thermococcales, suggesting that these vesicles could be involved in the transfer of viruses and plasmids between cells.

  10. Use of an endogenous plasmid locus for stable in trans complementation in Borrelia burgdorferi.

    PubMed

    Kasumba, Irene N; Bestor, Aaron; Tilly, Kit; Rosa, Patricia A

    2015-02-01

    Targeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirochete Borrelia burgdorferi. A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers than B. burgdorferi plasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle. B. burgdorferi has over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochete in vivo but relatively unstable during in vitro cultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number and in vivo stability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into the bbe02 locus, a site on lp25 that was previously shown to be nonessential during both in vitro and in vivo growth. We demonstrate the functional utility of this strategy by restoring infectivity to an ospC mutant through complementation at this site on lp25 and stable maintenance of the ospC gene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation in B. burgdorferi.

  11. COMSAT: Residue contact prediction of transmembrane proteins based on support vector machines and mixed integer linear programming.

    PubMed

    Zhang, Huiling; Huang, Qingsheng; Bei, Zhendong; Wei, Yanjie; Floudas, Christodoulos A

    2016-03-01

    In this article, we present COMSAT, a hybrid framework for residue contact prediction of transmembrane (TM) proteins, integrating a support vector machine (SVM) method and a mixed integer linear programming (MILP) method. COMSAT consists of two modules: COMSAT_SVM which is trained mainly on position-specific scoring matrix features, and COMSAT_MILP which is an ab initio method based on optimization models. Contacts predicted by the SVM model are ranked by SVM confidence scores, and a threshold is trained to improve the reliability of the predicted contacts. For TM proteins with no contacts above the threshold, COMSAT_MILP is used. The proposed hybrid contact prediction scheme was tested on two independent TM protein sets based on the contact definition of 14 Å between Cα-Cα atoms. First, using a rigorous leave-one-protein-out cross validation on the training set of 90 TM proteins, an accuracy of 66.8%, a coverage of 12.3%, a specificity of 99.3% and a Matthews' correlation coefficient (MCC) of 0.184 were obtained for residue pairs that are at least six amino acids apart. Second, when tested on a test set of 87 TM proteins, the proposed method showed a prediction accuracy of 64.5%, a coverage of 5.3%, a specificity of 99.4% and a MCC of 0.106. COMSAT shows satisfactory results when compared with 12 other state-of-the-art predictors, and is more robust in terms of prediction accuracy as the length and complexity of TM protein increase. COMSAT is freely accessible at http://hpcc.siat.ac.cn/COMSAT/. PMID:26756402

  12. Validation of non-REM sleep stage decoding from resting state fMRI using linear support vector machines.

    PubMed

    Altmann, A; Schröter, M S; Spoormaker, V I; Kiem, S A; Jordan, D; Ilg, R; Bullmore, E T; Greicius, M D; Czisch, M; Sämann, P G

    2016-01-15

    A growing body of literature suggests that changes in consciousness are reflected in specific connectivity patterns of the brain as obtained from resting state fMRI (rs-fMRI). As simultaneous electroencephalography (EEG) is often unavailable, decoding of potentially confounding sleep patterns from rs-fMRI itself might be useful and improve data interpretation. Linear support vector machine classifiers were trained on combined rs-fMRI/EEG recordings from 25 subjects to separate wakefulness (S0) from non-rapid eye movement (NREM) sleep stages 1 (S1), 2 (S2), slow wave sleep (SW) and all three sleep stages combined (SX). Classifier performance was quantified by a leave-one-subject-out cross-validation (LOSO-CV) and on an independent validation dataset comprising 19 subjects. Results demonstrated excellent performance with areas under the receiver operating characteristics curve (AUCs) close to 1.0 for the discrimination of sleep from wakefulness (S0|SX), S0|S1, S0|S2 and S0|SW, and good to excellent performance for the classification between sleep stages (S1|S2:~0.9; S1|SW:~1.0; S2|SW:~0.8). Application windows of fMRI data from about 70 s were found as minimum to provide reliable classifications. Discrimination patterns pointed to subcortical-cortical connectivity and within-occipital lobe reorganization of connectivity as strongest carriers of discriminative information. In conclusion, we report that functional connectivity analysis allows valid classification of NREM sleep stages. PMID:26596551

  13. Use of multivariate linear regression and support vector regression to predict functional outcome after surgery for cervical spondylotic myelopathy.

    PubMed

    Hoffman, Haydn; Lee, Sunghoon I; Garst, Jordan H; Lu, Derek S; Li, Charles H; Nagasawa, Daniel T; Ghalehsari, Nima; Jahanforouz, Nima; Razaghy, Mehrdad; Espinal, Marie; Ghavamrezaii, Amir; Paak, Brian H; Wu, Irene; Sarrafzadeh, Majid; Lu, Daniel C

    2015-09-01

    This study introduces the use of multivariate linear regression (MLR) and support vector regression (SVR) models to predict postoperative outcomes in a cohort of patients who underwent surgery for cervical spondylotic myelopathy (CSM). Currently, predicting outcomes after surgery for CSM remains a challenge. We recruited patients who had a diagnosis of CSM and required decompressive surgery with or without fusion. Fine motor function was tested preoperatively and postoperatively with a handgrip-based tracking device that has been previously validated, yielding mean absolute accuracy (MAA) results for two tracking tasks (sinusoidal and step). All patients completed Oswestry disability index (ODI) and modified Japanese Orthopaedic Association questionnaires preoperatively and postoperatively. Preoperative data was utilized in MLR and SVR models to predict postoperative ODI. Predictions were compared to the actual ODI scores with the coefficient of determination (R(2)) and mean absolute difference (MAD). From this, 20 patients met the inclusion criteria and completed follow-up at least 3 months after surgery. With the MLR model, a combination of the preoperative ODI score, preoperative MAA (step function), and symptom duration yielded the best prediction of postoperative ODI (R(2)=0.452; MAD=0.0887; p=1.17 × 10(-3)). With the SVR model, a combination of preoperative ODI score, preoperative MAA (sinusoidal function), and symptom duration yielded the best prediction of postoperative ODI (R(2)=0.932; MAD=0.0283; p=5.73 × 10(-12)). The SVR model was more accurate than the MLR model. The SVR can be used preoperatively in risk/benefit analysis and the decision to operate. PMID:26115898

  14. Use of multivariate linear regression and support vector regression to predict functional outcome after surgery for cervical spondylotic myelopathy

    PubMed Central

    Hoffman, Haydn; Lee, Sunghoon Ivan; Garst, Jordan H.; Lu, Derek S.; Li, Charles H.; Nagasawa, Daniel T.; Ghalehsari, Nima; Jahanforouz, Nima; Razaghy, Mehrdad; Espinal, Marie; Ghavamrezaii, Amir; Paak, Brian H.; Wu, Irene; Sarrafzadeh, Majid; Lu, Daniel C.

    2016-01-01

    This study introduces the use of multivariate linear regression (MLR) and support vector regression (SVR) models to predict postoperative outcomes in a cohort of patients who underwent surgery for cervical spondylotic myelopathy (CSM). Currently, predicting outcomes after surgery for CSM remains a challenge. We recruited patients who had a diagnosis of CSM and required decompressive surgery with or without fusion. Fine motor function was tested preoperatively and postoperatively with a handgrip-based tracking device that has been previously validated, yielding mean absolute accuracy (MAA) results for two tracking tasks (sinusoidal and step). All patients completed Oswestry disability index (ODI) and modified Japanese Orthopaedic Association questionnaires preoperatively and postoperatively. Preoperative data was utilized in MLR and SVR models to predict postoperative ODI. Predictions were compared to the actual ODI scores with the coefficient of determination (R2) and mean absolute difference (MAD). From this, 20 patients met the inclusion criteria and completed follow-up at least 3 months after surgery. With the MLR model, a combination of the preoperative ODI score, preoperative MAA (step function), and symptom duration yielded the best prediction of postoperative ODI (R2 = 0.452; MAD = 0.0887; p = 1.17 × 10−3). With the SVR model, a combination of preoperative ODI score, preoperative MAA (sinusoidal function), and symptom duration yielded the best prediction of postoperative ODI (R2 = 0.932; MAD = 0.0283; p = 5.73 × 10−12). The SVR model was more accurate than the MLR model. The SVR can be used preoperatively in risk/benefit analysis and the decision to operate. PMID:26115898

  15. COMSAT: Residue contact prediction of transmembrane proteins based on support vector machines and mixed integer linear programming.

    PubMed

    Zhang, Huiling; Huang, Qingsheng; Bei, Zhendong; Wei, Yanjie; Floudas, Christodoulos A

    2016-03-01

    In this article, we present COMSAT, a hybrid framework for residue contact prediction of transmembrane (TM) proteins, integrating a support vector machine (SVM) method and a mixed integer linear programming (MILP) method. COMSAT consists of two modules: COMSAT_SVM which is trained mainly on position-specific scoring matrix features, and COMSAT_MILP which is an ab initio method based on optimization models. Contacts predicted by the SVM model are ranked by SVM confidence scores, and a threshold is trained to improve the reliability of the predicted contacts. For TM proteins with no contacts above the threshold, COMSAT_MILP is used. The proposed hybrid contact prediction scheme was tested on two independent TM protein sets based on the contact definition of 14 Å between Cα-Cα atoms. First, using a rigorous leave-one-protein-out cross validation on the training set of 90 TM proteins, an accuracy of 66.8%, a coverage of 12.3%, a specificity of 99.3% and a Matthews' correlation coefficient (MCC) of 0.184 were obtained for residue pairs that are at least six amino acids apart. Second, when tested on a test set of 87 TM proteins, the proposed method showed a prediction accuracy of 64.5%, a coverage of 5.3%, a specificity of 99.4% and a MCC of 0.106. COMSAT shows satisfactory results when compared with 12 other state-of-the-art predictors, and is more robust in terms of prediction accuracy as the length and complexity of TM protein increase. COMSAT is freely accessible at http://hpcc.siat.ac.cn/COMSAT/.

  16. [Methods for construction of transgenic plant expression vector: a review].

    PubMed

    Zhang, Yangpu; Yang, Shushen

    2015-03-01

    Construction of recombinant plasmid vector for gene expression is a key step in making transgenic plants and important to study gene function and plant genetic engineering. A right choice of gene construction method can be cost-effective and achieve more diverse recombinant plasmids. In addition to the traditional methods in construction of plant gene expression vectors, such as Gateway technology, three DNA method and one step cloning, a few novel methods have been developed in recent years. These methods include oligonucleotide synthesis-based construction of small fragment gene expression vectors via competitive connection; construction of small RNA expression vector using pre-microRNA; recombination-fusion PCR method which inserts DNA fragments of multiple restriction sites into the target vector; and insertion of a DNA fragment into any region of a linear vector via In-Fusion Kit. Construction of complex vectors with many fragments uses sequence and ligation-independent cloning method, Gibson isothermal assembly or Golden Gate assembly. This paper summarizes our working experience in the area of recombinant vector construction and reports from others with an intention to disseminate ideas about currently widely used DNA recombination methods for plant transformation.

  17. [Construction of PPENK-MIDGE-NLS gene vector and the expression in rat].

    PubMed

    Chen, Xi; Xu, Xuemin; Peng, Xijuan; Jiang, Wei; Yao, Linong

    2015-02-01

    Increasing the production and secretion of endogenous opioid peptide by immune cell can significantly induce myocardial protective effects against ischemia-reperfusion injury. Gene therapy is promising to increase endogenous enkephalin (ENK). However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, gene recombination, oncogene activation, the production of antibacterial antibody and changes in physiological gene expression. Minimalistic immunologically defined gene expression (MIDGE) can overcome all the deficients of viral and plasmid vectors. The exon of rat's preproenkephalin (PPENK) gene was amplified by PCR and the fragments were cloned into pEGFP-N1 plasmids. The recombined plasmids were digested with enzymes to obtain a linear vector contained promoter, preproenkephalin gene, RNA stable sequences and oligodesoxy nucleotides (ODNs) added to both ends of the gene vector to protect gene vector from exonuclease degradation. A nuclear localization sequence (NLS) was attached to an ODN to ensure the effective transport to the nucleus and transgene expression. Flow cytometry, laser confocal microscopy and Western blotting demonstrated that PPENK-MIDGE-NLS can transfect leukocyte of rat in vivo, increase the expression of proenkephalin (PENK) in tissue, and the transfection efficiency depends on gene vector's dosage. These results indicate that PPENK-MIDGE-NLS could be an innovative method to protect and treatment of myocardial ischemia-reperfusion injury.

  18. Chromosome and Plasmids of the Tick-Borne Relapsing Fever Agent Borrelia hermsii

    PubMed Central

    2016-01-01

    The zoonotic pathogen Borrelia hermsii bears its multiple paralogous genes for variable antigens on several linear plasmids. Application of combined long-read and short-read next-generation sequencing provided complete sequences for antigen-encoding plasmids as well as other linear and circular plasmids and the linear chromosome of the genome. PMID:27284141

  19. Plasmid mapping computer program.

    PubMed

    Nolan, G P; Maina, C V; Szalay, A A

    1984-01-11

    Three new computer algorithms are described which rapidly order the restriction fragments of a plasmid DNA which has been cleaved with two restriction endonucleases in single and double digestions. Two of the algorithms are contained within a single computer program (called MPCIRC). The Rule-Oriented algorithm, constructs all logical circular map solutions within sixty seconds (14 double-digestion fragments) when used in conjunction with the Permutation method. The program is written in Apple Pascal and runs on an Apple II Plus Microcomputer with 64K of memory. A third algorithm is described which rapidly maps double digests and uses the above two algorithms as adducts. Modifications of the algorithms for linear mapping are also presented. PMID:6320105

  20. Design and implementation of cost-effective algorithms for direct solution of banded linear systems on the vector processor system 32 supercomputer

    SciTech Connect

    Samba, A.S.

    1985-01-01

    The problem of solving banded linear systems by direct (non-iterative) techniques on the Vector Processor System (VPS) 32 supercomputer is considered. Two efficient direct methods for solving banded linear systems on the VPS 32 are described. The vector cyclic reduction (VCR) algorithm is discussed in detail. The performance of the VCR on a three parameter model problem is also illustrated. The VCR is an adaptation of the conventional point cyclic reduction algorithm. The second direct method is the Customized Reduction of Augmented Triangles' (CRAT). CRAT has the dominant characteristics of an efficient VPS 32 algorithm. CRAT is tailored to the pipeline architecture of the VPS 32 and as a consequence the algorithm is implicitly vectorizable.

  1. Safety and Tolerability of Conserved Region Vaccines Vectored by Plasmid DNA, Simian Adenovirus and Modified Vaccinia Virus Ankara Administered to Human Immunodeficiency Virus Type 1-Uninfected Adults in a Randomized, Single-Blind Phase I Trial

    PubMed Central

    Hayton, Emma-Jo; Rose, Annie; Ibrahimsa, Umar; Del Sorbo, Mariarosaria; Capone, Stefania; Crook, Alison; Black, Antony P.; Dorrell, Lucy; Hanke, Tomáš

    2014-01-01

    Trial Design HIV-1 vaccine development has advanced slowly due to viral antigenic diversity, poor immunogenicity and recently, safety concerns associated with human adenovirus serotype-5 vectors. To tackle HIV-1 variation, we designed a unique T-cell immunogen HIVconsv from functionally conserved regions of the HIV-1 proteome, which were presented to the immune system using a heterologous prime-boost combination of plasmid DNA, a non-replicating simian (chimpanzee) adenovirus ChAdV-63 and a non-replicating poxvirus, modified vaccinia virus Ankara. A block-randomized, single-blind, placebo-controlled phase I trial HIV-CORE 002 administered for the first time candidate HIV-1- vaccines or placebo to 32 healthy HIV-1/2-uninfected adults in Oxford, UK and elicited high frequencies of HIV-1-specific T cells capable of inhibiting HIV-1 replication in vitro. Here, detail safety and tolerability of these vaccines are reported. Methods Local and systemic reactogenicity data were collected using structured interviews and study-specific diary cards. Data on all other adverse events were collected using open questions. Serum neutralizing antibody titres to ChAdV-63 were determined before and after vaccination. Results Two volunteers withdrew for vaccine-unrelated reasons. No vaccine-related serious adverse events or reactions occurred during 190 person-months of follow-up. Local and systemic events after vaccination occurred in 27/32 individuals and most were mild (severity grade 1) and predominantly transient (<48 hours). Myalgia and flu-like symptoms were more strongly associated with MVA than ChAdV63 or DNA vectors and more common in vaccine recipients than in placebo. There were no intercurrent HIV-1 infections during follow-up. 2/24 volunteers had low ChAdV-63-neutralizing titres at baseline and 7 increased their titres to over 200 with a median (range) of 633 (231-1533) post-vaccination, which is of no safety concern. Conclusions These data demonstrate safety and good

  2. Retroviral vector production.

    PubMed

    Miller, A Dusty

    2014-01-01

    In this unit, the basic protocol generates stable cell lines that produce retroviral vectors that carry selectable markers. Also included are an alternate protocol that applies when the retroviral vector does not carry a selectable marker, and another alternate protocol for rapidly generating retroviral vector preparations by transient transfection. A support protocol describes construction of the retroviral vectors. The methods for generating virus from retroviral vector plasmids rely on the use of packaging cells that synthesize all of the retroviral proteins but do not produce replication-competent virus. Additional protocols detail plasmid transfection, virus titration, assay for replication-competent virus, and histochemical staining to detect transfer of a vector encoding alkaline phosphatase.

  3. Expression Plasmids for Use in Candida glabrata

    PubMed Central

    Zordan, Rebecca E.; Ren, Yuxia; Pan, Shih-Jung; Rotondo, Giuseppe; Peñas, Alejandro De Las; Iluore, Joseph; Cormack, Brendan P.

    2013-01-01

    We describe a series of CEN/ARS episomal plasmids containing different Candida glabrata promoters, allowing for a range of constitutive or regulated expression of proteins in C. glabrata. The set of promoters includes three constitutive promoters (EGD2pr, HHT2pr, PDC1pr), two macrophage/phagocytosis-induced promoters (ACO2pr, LYS21pr), and one nutritionally regulated promoter (MET3pr). Each promoter was cloned into two plasmid backbones that differ in their selectable marker, URA3, or the dominant-selectable NAT1 gene, which confers resistance to the drug nourseothricin. Expression from the 12 resulting plasmids was assessed using GFP as a reporter and flow cytometry or quantitative reverse-transcription polymerase chain reaction to assess expression levels. Together this set of plasmids expands the toolkit of expression vectors available for use with C. glabrata. PMID:23934995

  4. Responses to rotating linear acceleration vectors considered in relation to a model of the otolith organs. [human oculomotor response to transverse acceleration stress

    NASA Technical Reports Server (NTRS)

    Benson, A. J.; Barnes, G. R.

    1973-01-01

    Human subjects were exposed to a linear acceleration vector that rotated in the transverse plane of the skull without angular counterrotation. Lateral eye movements showed a sinusoidal change in slow phase velocity and an asymmetry or bias in the same direction as vector rotation. A model is developed that attributes the oculomotor response to otolithic mechanisms. It is suggested that the bias component is the manifestation of torsion of the statoconial plaque relative to the base of the utricular macula and that the sinusoidal component represents the translational oscillation of the statoconia. The model subsumes a hypothetical neural mechanism which allows x- and y-axis accelerations to be resolved. Derivation of equations of motion for the statoconial plaque in torsion and translation, which take into account forces acting in shear and normal to the macula, yield estimates of bias and sinusoidal components that are in qualitative agreement with the diverse experimental findings.

  5. Degradative plasmids from sphingomonads.

    PubMed

    Stolz, Andreas

    2014-01-01

    Large plasmids ('megaplasmids') are commonly found in members of the Alphaproteobacterial family Sphingomonadaceae ('sphingomonads'). These plasmids contribute to the extraordinary catabolic flexibility of this group of organisms, which degrade a broad range of recalcitrant xenobiotic compounds. The genomes of several sphingomonads have been sequenced during the last years. In the course of these studies, also the sequences of several plasmids have been determined. The analysis of the published information and the sequences deposited in the public databases allowed a first classification of these plasmids into a restricted number of groups according to the proteins involved in the initiation of replication, plasmid partition and conjugation. The sequence comparisons demonstrated that the plasmids from sphingomonads encode for four main groups of replication initiation (Rep) proteins. These Rep proteins belong to the protein superfamilies RepA_C (Pfam 04796), Rep_3 (Pfam 01051), RPA (Pfam 10134) and HTH-36 (Pfam 13730). The 'degradative megaplasmids' pNL2, pCAR3, pSWIT02, pCHQ1, pISP0, and pISP1, which code for genes involved in the degradation of aromatic hydrocarbons, carbazole, dibenzo-p-dioxin and γ-hexachlorocyclohexane, carry Rep proteins which either belong to the RepA_C- (plasmids pNL2, pCAR3, pSWIT02), Rep-3- (plasmids pCHQ1, pISP0) or RPA-superfamily (pISP1). The classification of these 'degradative megaplasmids' into three groups is also supported by sequence comparisons of the proteins involved in plasmid partition (ParAB) and the organization of the three genes on the respective plasmids. All analysed 'degradative megaplasmids' carry genes, which might allow a conjugative transfer of the plasmids. Sequence comparisons of these genes suggest the presence of at least two types of transfer functions, which either are closer related to the tra- or vir-genes previously described for plasmids from other sources.

  6. Singular vectors of a linear imaging system as efficient channels for the ideal observer in detection tasks involving non-Gaussian distributed lumpy images

    NASA Astrophysics Data System (ADS)

    Witten, Joel M.; Park, Subok; Myers, Kyle J.

    2008-03-01

    The Bayesian ideal observer sets an upper bound for diagnostic performance of an imaging system in binary detection tasks. Thus, this observer should be used for image quality assessment whenever possible. However, it is difficult to compute ideal-observer performance because the probability density functions of the data, required for the observer, are often unknown in tasks involving complex backgrounds. Furthermore, the dimension of the integrals that need to be calculated for the observer is huge. To attempt to reduce the dimensionality of the problem, and yet still approximate ideal-observer performance, a channelized-ideal observer (CIO) with Laguerre-Gauss channels was previously investigated for detecting a Gaussian signal at a known location in non-Gaussian lumpy images. While the CIO with Laguerre-Gauss channels had, in some cases, approximated ideal-observer performance, there was still a gap between the mean performance of the ideal observer and the CIO. Moreover, it is not clear how to choose efficient channels for the ideal observer. In the current work, we investigate the use of singular vectors of a linear imaging system as efficient channels for the ideal observer in the same tasks. Singular value decomposition of the imaging system is performed to obtain its singular vectors. Singular vectors most relevant to the signal and background images are chosen as candidate channels. Results indicate that the singular vectors are not only more efficient than Laguerre-Gauss channels, but are also highly efficient for the ideal observer. The results further demonstrate that singular vectors strongly associated with the signal-only image are the most efficient channels.

  7. Establishment of an entirely plasmid-based reverse genetics system for Bluetongue virus.

    PubMed

    Pretorius, Jakobus M; Huismans, Henk; Theron, Jacques

    2015-12-01

    Bluetongue virus (BTV), the type species of the genus Orbivirus within the family Reoviridae, has a genome consisting of 10 linear double-stranded RNA genome segments. Current reverse genetics approaches for engineering the BTV genome rely upon in vitro synthesis of capped RNA transcripts from cloned cDNA corresponding to viral genome segments. In an effort to expand the utility of BTV reverse genetics, we constructed a reverse genetics vector containing a T7 RNA polymerase promoter, hepatitis delta ribozyme sequence and T7 RNA polymerase terminator sequence. Viable virus was recovered following transfection of mammalian cells, expressing T7 RNA polymerase, with 10 plasmid constructs representing the cloned BTV-1 genome. Furthermore, the plasmid-based reverse genetics system was used successfully to isolate viable cross-serotype reassortant viruses and a mutant virus containing a defined mutation in the replicating viral genome. The new reverse genetics platform established here for BTV is likely applicable to other orbiviruses.

  8. Identification and Characterization of a Gene stp17 Located on the Linear Plasmid pBSSB1 as an Enhanced Gene of Growth and Motility in Salmonella enterica Serovar Typhi

    PubMed Central

    Zhang, Haifang; Zhu, Yunxia; Xie, Xiaofang; Wang, Min; Du, Hong; Xu, Shungao; Zhang, Ying; Gong, Mingyu; Ni, Bin; Xu, Huaxi; Huang, Xinxiang

    2016-01-01

    The linear plasmid pBSSB1 mediates the flagellar phase variation in H:z66 positive Salmonella enterica serovar Typhi (S. Typhi). The gene named stp17 (S. Typhi plasmid number 17 gene) is located on pBSSB1 and encodes the protein STP17. The expression pattern at the protein-level and function of STP17 remains unknown. In this study, the recombinant protein STP17His6 was expressed, purified and used to prepare the polyclonal anti-STP17 antibody. We detected protein-level expression of stp17 in S. Typhi and further investigated the protein expression characteristics of stp17 in different growth phases by western blot analysis. The effects of STP17 on bacterial growth and motility were analyzed. In addition, the structure of STP17 was predicted and the active site of STP17 was identified by site-directed mutagenesis. The results showed that STP17 was expressed stably in the wild type strain of S. Typhi. STP17 expression at the protein level peaks when cultures reach an OD600 value of 1.2. The growth rate and motility of the Δstp17 strain were significantly decreased compared with the wild type strain (P < 0.05) and this phenotype was restored in the stp17 complementary strain. Moreover, the growth rate and motility of the stp17 over-expression strain was greater than the wild type strain. STP17 contains nine Helix segments, six Stand segments and some Coil segments in the secondary structural level. The top-ranked 3-D structure of STP17 predicted by I-TASSER contains a putative ATPase domain and the amino acid residues of GLY16, GLY19, LYS20, ASN133, LYS157, and LYS158 may be the active site residues of STP17. Finally, STP17 was able to catalyze the ATP to ADP reaction, suggesting that STP17 may be an ATPase. To our knowledge, this is the first report describing the protein expression characteristics of STP17 in S. Typhi, showing that STP17 promotes bacterial growth and motility, which may be associated with its potential ATPase activity. PMID:27761429

  9. On Duffin-Kemmer-Petiau particles with a mixed minimal-nonminimal vector coupling and the nondegenerate bound-states for the one-dimensional inversely linear background

    SciTech Connect

    Castro, A. S. de

    2010-10-15

    The problem of spin-0 and spin-1 bosons in the background of a general mixing of minimal and nonminimal vector inversely linear potentials is explored in a unified way in the context of the Duffin-Kemmer-Petiau theory. It is shown that spin-0 and spin-1 bosons behave effectively in the same way. An orthogonality criterion is set up and it is used to determine uniquely the set of solutions as well as to show that even-parity solutions do not exist.

  10. Plasmids with E2 epitope tags: tagging modules for N- and C-terminal PCR-based gene targeting in both budding and fission yeast, and inducible expression vectors for fission yeast.

    PubMed

    Tamm, Tiina

    2009-01-01

    A single-step PCR-based epitope tagging enables fast and efficient gene targeting with various epitope tags. This report presents a series of plasmids for the E2 epitope tagging of proteins in Saccharomyces cerevisiae and Schizosaccharomyces pombe. E2Tags are 10-amino acids (epitope E2a: SSTSSDFRDR)- and 12 amino acids (epitope E2b: GVSSTSSDFRDR)-long peptides derived from the E2 protein of bovine papillomavirus type 1. The modules for C-terminal tagging with E2a and E2b epitopes were constructed by the modification of the pYM-series plasmid. The N-terminal E2a and E2b tagging modules were based on pOM-series plasmid. The pOM-series plasmids were selected for this study because of their use of the Cre-loxP recombination system. The latter enables a marker cassette to be removed after integration into the loci of interest and, thereafter, the tagged protein is expressed under its endogenous promoter. Specifically for fission yeast, high copy pREP plasmids containing the E2a epitope tag as an N-terminal or C-terminal tag were constructed. The properties of E2a and E2b epitopes and the sensitivity of two anti-E2 monoclonal antibodies (5E11 and 3F12) were tested using several S. cerevisiae and Sz. pombe E2-tagged strains. PMID:19180640

  11. SPP1-mediated plasmid transduction.

    PubMed Central

    Canosi, U; Lüder, G; Trautner, T A

    1982-01-01

    The virulent Bacillus subtilis phage SPP1 transduces plasmid DNA. Plasmid-transducing phages contain only plasmid DNA. Such DNA represents a concatemer of monomeric plasmid molecules with the molecular weight of mature SPP1 DNA. Biological parameters of plasmid transduction are described. Images PMID:6292508

  12. Plasmidal maintenance of composite DNA derived from polyoma related plasmid, L factor.

    PubMed Central

    Saito, H; Uehara, H; Kusano, T; Oishi, M

    1987-01-01

    Recently, we reported a multicopy mammalian plasmid with a structure related to polyoma. The plasmid, named L factor, was found at a high copy number (5,000 or more per cell) in a subclone derived from mouse L cells. We attempted to utilize L factor as a plasmid vector for mammalian cells. A series of composite DNA consisting of L factor and a foreign (herpes simplex virus tk) were constructed. These DNA could be established as plasmids after transfection to several mouse cell lines, although the copy number of the re-established plasmids was considerably less than that observed for the original subclone. The composite DNA maintained the structure of the original DNA after prolonged culture and the copy number remained constant even with no selective pressure. A composite DNA, with no DNA sequence corresponding to polyoma T antigen, could also be established as a plasmid in a mouse L cell line in which polyoma T antigen is expressed. The potential use of the plasmid is discussed. Images PMID:2825120

  13. Multiplex protein pattern unmixing using a non-linear variable-weighted support vector machine as optimized by a particle swarm optimization algorithm.

    PubMed

    Yang, Qin; Zou, Hong-Yan; Zhang, Yan; Tang, Li-Juan; Shen, Guo-Li; Jiang, Jian-Hui; Yu, Ru-Qin

    2016-01-15

    Most of the proteins locate more than one organelle in a cell. Unmixing the localization patterns of proteins is critical for understanding the protein functions and other vital cellular processes. Herein, non-linear machine learning technique is proposed for the first time upon protein pattern unmixing. Variable-weighted support vector machine (VW-SVM) is a demonstrated robust modeling technique with flexible and rational variable selection. As optimized by a global stochastic optimization technique, particle swarm optimization (PSO) algorithm, it makes VW-SVM to be an adaptive parameter-free method for automated unmixing of protein subcellular patterns. Results obtained by pattern unmixing of a set of fluorescence microscope images of cells indicate VW-SVM as optimized by PSO is able to extract useful pattern features by optimally rescaling each variable for non-linear SVM modeling, consequently leading to improved performances in multiplex protein pattern unmixing compared with conventional SVM and other exiting pattern unmixing methods.

  14. Helper plasmid cloning in Streptococcus sanguis: cloning of a tetracycline resistance determinant from the Streptococcus mutans chromosome.

    PubMed

    Tobian, J A; Macrina, F L

    1982-10-01

    A model system for testing the helper plasmid cloning system of Gryczan et al. (Mol. Gen. Genet. 177:459-467, 1980) was devised for the Streptococcus sanguis (Challis) host-vector system. In this system, linearized pVA736 plasmid efficiently transformed an S. sanguis (Challis) host containing a homologous plasmid, pVA380-1, but did not transform a plasmidless host or a host containing a nonhomologous plasmid, pVA380. In addition, whereas monomeric circular pVA736 transformed a plasmidless host with two-hit kinetics, it transformed a pVA380-1-containing host with one-hit kinetics. This helper plasmid cloning system was used to isolate two HindIII fragments (5.0 megadaltons [Mdal] and 1.9 Mdal in size) from the chromosome of Streptococcus mutans V825 which conferred high-level tetracycline resistance. One tetracycline-resistant clone was examined and found to contain three plasmids which were sized and designated pVA868 (9.0 Mdal), pVA869 (9.5 Mdal), and pVA870 (9.8 Mdal). Results of Southern blot hybridization and restriction endonuclease digestion confirmed that all three chimeras were composed of two HindIII fragments of the S. mutans V825 chromosome, as well as a large portion, varying in size for each chimera, of the 2.8 Mdal cloning vector, pVA380-1. Incompatibility observed between pVA380-1 and each of the chimeras indicated that replication of the chimeras was governed by the pVA380-1 replicative origin. Southern blotting experiments revealed that the chimeras hybridized to Tn916, providing the first evidence that transposon-related genes of enteric streptococcal origin are disseminated among oral streptococci.

  15. Ovary-drip transformation: a simple method for directly generating vector- and marker-free transgenic maize (Zea mays L.) with a linear GFP cassette transformation.

    PubMed

    Yang, Aifu; Su, Qiao; An, Lijia

    2009-03-01

    The presence of selectable marker genes and vector backbone sequences has affected the safe assessment of transgenic plants. In this study, the ovary-drip method for directly generating vector- and selectable marker-free transgenic plants was described, by which maize was transformed with a linear GFP cassette (Ubi-GFP-nos). The key features of this method center on the complete removal of the styles and the subsequent application of a DNA solution directly to the ovaries. The movement of the exogenous DNA was monitored using fluorescein isothiocyanate-labeled DNA, which showed that the time taken by the exogenous DNA to enter the ovaries was shortened compared to that of the pollen-tube pathway. This led to an improved transformation frequency of 3.38% compared to 0.86% for the pollen-tube pathway as determined by PCR analysis. The use of 0.05% surfactant Silwet L-77 + 5% sucrose as a transformation solution further increased the transformation frequency to 6.47%. Southern blot analysis showed that the transgenic plants had low transgene copy number and simple integration pattern. Green fluorescence was observed in roots and immature embryos of transgenic plants by fluorescence microscopy. Progeny analysis showed that GFP insertions were inherited in T(1) generation. The ovary-drip method would become a favorable choice for directly generating vector- and marker-free transgenic maize expressing functional genes of agronomic interest.

  16. Gene transcription from the linear plasmid pBClin15 leads to cell lysis and extracellular DNA-dependent aggregation of Bacillus cereus ATCC 14579 in response to quinolone-induced stress.

    PubMed

    Vörös, Aniko; Simm, Roger; Kroeger, Jasmin K; Kolstø, Anne-Brit

    2013-11-01

    The Bacillus cereus type strain ATCC 14579 harbours pBClin15, a linear plasmid with similar genome organization to tectiviruses. Since phage morphogenesis is not known to occur it has been suggested that pBClin15 may be a defect relic of a tectiviral prophage without relevance for the bacterial physiology. However, in this paper, we demonstrate that a pBClin15-cured strain is more tolerant to antibiotics interfering with DNA integrity than the WT strain. Growth in the presence of crystal violet or the quinolones nalidixic acid, norfloxacin or ciprofloxacin resulted in aggregation and lysis of the WT strain, whereas the pBClin15-cured strain was unaffected. Microarray analysis comparing the gene expression in the WT and pBClin15-cured strains showed that pBClin15 gene expression was strongly upregulated in response to norfloxacin stress, and coincided with lysis and aggregation of the WT strain. The aggregating bacteria experienced a significant survival benefit compared with the planktonic counterparts in the presence of norfloxacin. There was no difference between the WT and pBClin15-cured strains during growth in the absence of norfloxacin, the pBClin15 genes were moderately expressed, and no effect was observed on chromosomal gene expression. These data demonstrate for the first time that although pBClin15 may be a remnant of a temperate phage, it negatively affects the DNA stress tolerance of B. cereus ATCC 14579. Furthermore, our results warrant a recommendation to always verify the presence of pBClin15 following genetic manipulation of B. cereus ATCC 14579.

  17. PCG: A software package for the iterative solution of linear systems on scalar, vector and parallel computers

    SciTech Connect

    Joubert, W.; Carey, G.F.

    1994-12-31

    A great need exists for high performance numerical software libraries transportable across parallel machines. This talk concerns the PCG package, which solves systems of linear equations by iterative methods on parallel computers. The features of the package are discussed, as well as techniques used to obtain high performance as well as transportability across architectures. Representative numerical results are presented for several machines including the Connection Machine CM-5, Intel Paragon and Cray T3D parallel computers.

  18. Pathway of plasmid transformation in pneumococcus

    SciTech Connect

    Guild, W.R.; Saunders, C.W.

    1981-01-01

    Plasmids transform Streptococcus pneumoniae by a process involving low efficiency assembly of replicons from fragments of single strands that have entered the cell separately. Transformation of preexisting replicons is much more efficient. We have cloned the erm gene of pIP501 into pMV158, which so far as we know is the first example of cloning in a pneumococcus host-vector system.

  19. Chlamydial plasmids and bacteriophages.

    PubMed

    Pawlikowska-Warych, Małgorzata; Śliwa-Dominiak, Joanna; Deptuła, Wiesław

    2015-01-01

    Chlamydia are absolute pathogens of humans and animals; despite being rather well recognised, they are still open for discovery. One such discovery is the occurrence of extrachromosomal carriers of genetic information. In prokaryotes, such carriers include plasmids and bacteriophages, which are present only among some Chlamydia species. Plasmids were found exclusively in Chlamydia (C.) trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum and C. caviae. In prokaryotic organisms, plasmids usually code for genes that facilitate survival of the bacteria in the environment (although they are not essential). In chlamydia, their role has not been definitely recognised, apart from the fact that they participate in the synthesis of glycogen and encode proteins responsible for their virulence. Furthermore, in C. suis it was evidenced that the plasmid is integrated in a genomic island and contains the tetracycline-resistance gene. Bacteriophages specific for chlamydia (chlamydiaphages) were detected only in six species: C. psittaci, C. abortus, C. felis, C. caviae C. pecorum and C. pneumoniae. These chlamydiaphages cause inhibition of the developmental cycle, and delay transformation of reticulate bodies (RBs) into elementary bodies (EBs), thus reducing the possibility of infecting other cells in time. Plasmids and bacteriophages can be used in the diagnostics of chlamydioses; although especially in the case of plasmids, they are already used for detection of chlamydial infections. In addition, bacteriophages could be used as therapeutic agents to replace antibiotics, potentially addressing the problem of increasing antibiotic-resistance among chlamydia.

  20. Plasmid partitioning systems of conjugative plasmids from Clostridium perfringens.

    PubMed

    Adams, Vicki; Watts, Thomas D; Bulach, Dieter M; Lyras, Dena; Rood, Julian I

    2015-07-01

    Many pathogenic strains of Clostridium perfringens carry several highly similar toxin or antibiotic resistance plasmids that have 35 to 40 kb of very closely related syntenous sequences, including regions that carry the genes encoding conjugative transfer, plasmid replication and plasmid maintenance functions. Key questions are how are these closely related plasmids stably maintained in the same cell and what is the basis for plasmid incompatibility in C. perfringens. Comparative analysis of the Rep proteins encoded by these plasmids suggested that this protein was not the basis for plasmid incompatibility since plasmids carried in a single strain often encoded an almost identical Rep protein. These plasmids all carried a similar, but not identical, parMRC plasmid partitioning locus. Phylogenetic analysis of the deduced ParM proteins revealed that these proteins could be divided into ten separate groups. Importantly, in every strain that carried more than one of these plasmids, the respective ParM proteins were from different phylogenetic groups. Similar observations were made from the analysis of phylogenetic trees of the ParR proteins and the parC loci. These findings provide evidence that the basis for plasmid incompatibility in the conjugative toxin and resistance plasmid family from C. perfringens resides in subtle differences in the parMRC plasmid partitioning loci carried by these plasmids.

  1. Nucleotide sequence of a small cryptic plasmid from Acidithiobacillus ferrooxidans strain A-6

    SciTech Connect

    F. Roberto

    2003-10-01

    A 2.1 kb cryptic plasmid from Acidithiobacillus ferrooxidans strain A-6 was isolated and cloned into the E. coli vector plasmid, pUC128. The cloned plasmid was mapped by restriction enzyme fragment analysis and subsequently sequenced. At this time over half the plasmid sequence has been determined and compared to sequences in the GenBank nucleotide and protein sequence databases. Much of the plasmid remains cryptic, but substantial nucleotide and protein sequence similarities have been observed to the putative replication protein, RepA, of the small cryptic plasmids pAYS and pAYL found in the ammonia-oxidizing Nitrosomonas sp. Strain ENI-11. These results suggest an entirely new class of plasmid is maintained in at least one strain of Acidithiobacillus ferrooxidans and other acidophilic bacteria, and raises interesting questions about the origin of this plasmid in acidic environments.

  2. Pharmaceutical grade large-scale plasmid DNA manufacturing process.

    PubMed

    Schmeer, Marco; Schleef, Martin

    2014-01-01

    For pharmaceutical applications of plasmid DNA, either direct or indirect, certain quality standards are required. Whereas for direct gene transfer into human "Good Manufacturing Practice" (GMP) grade is mandatory, for GMP production of, e.g., viral vectors (AAV, etc.) the plasmid DNA used needs not necessarily be produced under GMP. Besides such regulatory aspects up-scaling of the plasmid DNA production process from research laboratory scale (up to a few milligrams) to industrial scales (milligram to gram scales) is an issue that is addressed here.

  3. Plasmid-Mediated Antimicrobial Resistance in Staphylococci and Other Firmicutes.

    PubMed

    Schwarz, Stefan; Shen, Jianzhong; Wendlandt, Sarah; Fessler, Andrea T; Wang, Yang; Kadlec, Kristina; Wu, Cong-Ming

    2014-12-01

    In staphylococci and other Firmicutes, resistance to numerous classes of antimicrobial agents, which are commonly used in human and veterinary medicine, is mediated by genes that are associated with mobile genetic elements. The gene products of some of these antimicrobial resistance genes confer resistance to only specific members of a certain class of antimicrobial agents, whereas others confer resistance to the entire class or even to members of different classes of antimicrobial agents. The resistance mechanisms specified by the resistance genes fall into any of three major categories: active efflux, enzymatic inactivation, and modification/replacement/protection of the target sites of the antimicrobial agents. Among the mobile genetic elements that carry such resistance genes, plasmids play an important role as carriers of primarily plasmid-borne resistance genes, but also as vectors for nonconjugative and conjugative transposons that harbor resistance genes. Plasmids can be exchanged by horizontal gene transfer between members of the same species but also between bacteria belonging to different species and genera. Plasmids are highly flexible elements, and various mechanisms exist by which plasmids can recombine, form cointegrates, or become integrated in part or in toto into the chromosomal DNA or into other plasmids. As such, plasmids play a key role in the dissemination of antimicrobial resistance genes within the gene pool to which staphylococci and other Firmicutes have access. This chapter is intended to provide an overview of the current knowledge of plasmid-mediated antimicrobial resistance in staphylococci and other Firmicutes.

  4. Plasmids encoding therapeutic agents

    DOEpatents

    Keener, William K.

    2007-08-07

    Plasmids encoding anti-HIV and anti-anthrax therapeutic agents are disclosed. Plasmid pWKK-500 encodes a fusion protein containing DP178 as a targeting moiety, the ricin A chain, an HIV protease cleavable linker, and a truncated ricin B chain. N-terminal extensions of the fusion protein include the maltose binding protein and a Factor Xa protease site. C-terminal extensions include a hydrophobic linker, an L domain motif peptide, a KDEL ER retention signal, another Factor Xa protease site, an out-of-frame buforin II coding sequence, the lacZ.alpha. peptide, and a polyhistidine tag. More than twenty derivatives of plasmid pWKK-500 are described. Plasmids pWKK-700 and pWKK-800 are similar to pWKK-500 wherein the DP178-encoding sequence is substituted by RANTES- and SDF-1-encoding sequences, respectively. Plasmid pWKK-900 is similar to pWKK-500 wherein the HIV protease cleavable linker is substituted by a lethal factor (LF) peptide-cleavable linker.

  5. A 1.83 μJ/Classification, 8-Channel, Patient-Specific Epileptic Seizure Classification SoC Using a Non-Linear Support Vector Machine.

    PubMed

    Bin Altaf, Muhammad Awais; Yoo, Jerald

    2016-02-01

    A non-linear support vector machine (NLSVM) seizure classification SoC with 8-channel EEG data acquisition and storage for epileptic patients is presented. The proposed SoC is the first work in literature that integrates a feature extraction (FE) engine, patient specific hardware-efficient NLSVM classification engine, 96 KB SRAM for EEG data storage and low-noise, high dynamic range readout circuits. To achieve on-chip integration of the NLSVM classification engine with minimum area and energy consumption, the FE engine utilizes time division multiplexing (TDM)-BPF architecture. The implemented log-linear Gaussian basis function (LL-GBF) NLSVM classifier exploits the linearization to achieve energy consumption of 0.39 μ J/operation and reduces the area by 28.2% compared to conventional GBF implementation. The readout circuits incorporate a chopper-stabilized DC servo loop to minimize the noise level elevation and achieve noise RTI of 0.81 μ Vrms for 0.5-100 Hz bandwidth with an NEF of 4.0. The 5 × 5 mm (2) SoC is implemented in a 0.18 μm 1P6M CMOS process consuming 1.83 μ J/classification for 8-channel operation. SoC verification has been done with the Children's Hospital Boston-MIT EEG database, as well as with a specific rapid eye-blink pattern detection test, which results in an average detection rate, average false alarm rate and latency of 95.1%, 0.94% (0.27 false alarms/hour) and 2 s, respectively. PMID:25700471

  6. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR.

    PubMed

    D'Costa, Susan; Blouin, Veronique; Broucque, Frederic; Penaud-Budloo, Magalie; François, Achille; Perez, Irene C; Le Bec, Christine; Moullier, Philippe; Snyder, Richard O; Ayuso, Eduard

    2016-01-01

    Clinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new "Free-ITR" qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field.

  7. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

    PubMed Central

    D’Costa, Susan; Blouin, Veronique; Broucque, Frederic; Penaud-Budloo, Magalie; François, Achille; Perez, Irene C; Le Bec, Christine; Moullier, Philippe; Snyder, Richard O; Ayuso, Eduard

    2016-01-01

    Clinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new “Free-ITR” qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field. PMID:27069952

  8. Mechanisms of plasmid segregation: have multicopy plasmids been overlooked?

    PubMed

    Million-Weaver, Samuel; Camps, Manel

    2014-09-01

    Plasmids are self-replicating pieces of DNA typically bearing non-essential genes. Given that plasmids represent a metabolic burden to the host, mechanisms ensuring plasmid transmission to daughter cells are critical for their stable maintenance in the population. Here we review these mechanisms, focusing on two active partition strategies common to low-copy plasmids: par systems type I and type II. Both involve three components: an adaptor protein, a motor protein, and a centromere, which is a sequence area in the plasmid that is recognized by the adaptor protein. The centromere-bound adaptor nucleates polymerization of the motor, leading to filament formation, which can pull plasmids apart (par I) or push them towards opposite poles of the cell (par II). No such active partition mechanisms are known to occur in high copy number plasmids. In this case, vertical transmission is generally considered stochastic, due to the random distribution of plasmids in the cytoplasm. We discuss conceptual and experimental lines of evidence questioning the random distribution model and posit the existence of a mechanism for segregation in high copy number plasmids that moves plasmids to cell poles to facilitate transmission to daughter cells. This mechanism would involve chromosomally-encoded proteins and the plasmid origin of replication. Modulation of this proposed mechanism of segregation could provide new ways to enhance plasmid stability in the context of recombinant gene expression, which is limiting for large-scale protein production and for bioremediation.

  9. Cloning in Streptococcus lactis of plasmid-mediated UV resistance and effect on prophage stability

    SciTech Connect

    Chopin, M.C.; Chopin, A.; Rouault, A.; Simon, D.

    1986-02-01

    Plasmid pIL7 (33 kilobases) from Streptococcus lactis enhances UV resistance and prophage stability. A 5.4-kilobase pIL7 fragment carrying genes coding for both characters was cloned into S. lactis, using plasmid pHV1301 as the cloning vector. The recombinant plasmid was subsequently transferred to three other S. lactis strains by transformation or protoplast fusion. Cloned genes were expressed in all tested strains.

  10. Plasmid Stability in Pseudomonas fluorescens in the Rhizosphere

    PubMed Central

    van der Bij, A. J.; de Weger, L. A.; Tucker, W. T.; Lugtenberg, B.

    1996-01-01

    Plasmids belonging to various incompatibility (Inc) groups were introduced into the efficiently root-colonizing strain Pseudomonas fluorescens WCS365, and their stabilities in complex and minimal media and in the rhizospheres of tomato, wheat, and potato plants grown under gnotobiotic conditions without selection pressure were tested. The IncP plasmid was found to be highly unstable under all conditions tested, whereas the IncQ and IncW plasmids showed intermediate stabilities and the plasmids pVSP41 and pWTT2081, for which the Inc group is unknown, both containing the origin of replication (rep) and stability (sta) regions of the Pseudomonas aeruginosa pVS1 replicon, were stably maintained under all conditions tested. Growth experiments in which cells of strain WCS365 carrying the plasmid pWTT2081 were grown in the presence of WCS365 without the plasmid showed that the presence of pWTT2081 acts as a burden. We conclude that pVSP41 and pWTT2081 are valuable as stable vectors for the functional analysis of genes involved in root colonization, provided that control cells carry the empty vector. PMID:16535259

  11. Partial Least-Squares and Linear Support Vector Regression Chemometric Methods for Simultaneous Determination of Amoxicillin Trihydrate and Dicloxacillin Sodium in the Presence of Their Common Impurity.

    PubMed

    Naguib, Ibrahim A; Abdelaleem, Eglal A; Zaazaa, Hala E; Hussein, Essraa A

    2016-07-01

    Two multivariate chemometric models, namely, partial least-squares regression (PLSR) and linear support vector regression (SVR), are presented for the analysis of amoxicillin trihydrate and dicloxacillin sodium in the presence of their common impurity (6-aminopenicillanic acid) in raw materials and in pharmaceutical dosage form via handling UV spectral data and making a modest comparison between the two models, highlighting the advantages and limitations of each. For optimum analysis, a three-factor, four-level experimental design was established, resulting in a training set of 16 mixtures containing different ratios of interfering species. To validate the prediction ability of the suggested models, an independent test set consisting of eight mixtures was used. The presented results show the ability of the two proposed models to determine the two drugs simultaneously in the presence of small levels of the common impurity with high accuracy and selectivity. The analysis results of the dosage form were statistically compared to a reported HPLC method, with no significant difference regarding accuracy and precision, indicating the ability of the suggested multivariate calibration models to be reliable and suitable for routine analysis of the drug product. Compared to the PLSR model, the SVR model gives more accurate results with a lower prediction error, as well as high generalization ability; however, the PLSR model is easy to handle and fast to optimize. PMID:27305461

  12. Toxin plasmids of Clostridium perfringens.

    PubMed

    Li, Jihong; Adams, Vicki; Bannam, Trudi L; Miyamoto, Kazuaki; Garcia, Jorge P; Uzal, Francisco A; Rood, Julian I; McClane, Bruce A

    2013-06-01

    In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract.

  13. A series of template plasmids for Escherichia coli genome engineering.

    PubMed

    Deb, Shalini S; Reshamwala, Shamlan M S; Lali, Arvind M

    2016-06-01

    Metabolic engineering strategies often employ multi-copy episomal vectors to overexpress genes. However, chromosome-based overexpression is preferred as it avoids the use of selective pressure and reduces metabolic burden on the cell. We have constructed a series of template plasmids for λ Red-mediated Escherichia coli genome engineering. The template plasmids allow construction of genome integrating cassettes that can be used to integrate single copies of DNA sequences at predetermined sites or replace promoter regions. The constructed cassettes provide flexibility in terms of expression levels achieved and antibiotics used for selection, as well as allowing construction of marker-free strains. The modular design of the template plasmids allows replacement of genetic parts to construct new templates. Gene integration and promoter replacement using the template plasmids are illustrated. PMID:27071533

  14. Plasmid maintenance systems suitable for GMO-based bacterial vaccines.

    PubMed

    Spreng, Simone; Viret, Jean-François

    2005-03-18

    Live carrier-based bacterial vaccines represent a vaccine strategy that offers exceptional flexibility. Commensal or attenuated strains of pathogenic bacteria can be used as live carriers to present foreign antigens from unrelated pathogens to the immune system, with the aim of eliciting protective immune responses. As for oral immunisation, such an approach obviates the usual loss of antigen integrity observed during gastrointestinal passage and allows the delivery of a sufficient antigen dose to the mucosal immune system. Antibiotic and antibiotic-resistance genes have traditionally been used for the maintenance of recombinant plasmid vectors in bacteria used for biotechnological purposes. However, their continued use may appear undesirable in the field of live carrier-based vaccine development. This review focuses on strategies to omit antibiotic resistance determinants in live bacterial vaccines and discusses several balanced lethal-plasmid stabilisation systems with respect to maintenance of plasmid inheritance and antigenicity of plasmid-encoded antigen in vivo.

  15. Plasmid CDS5 influences infectivity and virulence in a mouse model of Chlamydia trachomatis urogenital infection.

    PubMed

    Ramsey, K H; Schripsema, J H; Smith, B J; Wang, Y; Jham, B C; O'Hagan, K P; Thomson, N R; Murthy, A K; Skilton, R J; Chu, P; Clarke, I N

    2014-08-01

    The native plasmid of both Chlamydia muridarum and Chlamydia trachomatis has been shown to control virulence and infectivity in mice and in lower primates. We recently described the development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis that for the first time provides a platform for the molecular dissection of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In the present study, we transformed a plasmid-free lymphogranuloma venereum isolate of C. trachomatis, serovar L2, with either the original shuttle vector (pGFP::SW2) or a derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene (pCDS5KO). Female mice were inoculated with these strains either intravaginally or transcervically. We found that transformation of the plasmid-free isolate with the intact pGFP::SW2 vector significantly enhanced infectivity and induction of host inflammatory responses compared to the plasmid-free parental isolate. Transformation with pCDS5KO resulted in infection courses and inflammatory responses not significantly different from those observed in mice infected with the plasmid-free isolate. These results indicate a critical role of plasmid CDS5 in in vivo fitness and in induction of inflammatory responses. To our knowledge, these are the first in vivo observations ascribing infectivity and virulence to a specific plasmid gene. PMID:24866804

  16. Plasmid CDS5 Influences Infectivity and Virulence in a Mouse Model of Chlamydia trachomatis Urogenital Infection

    PubMed Central

    Schripsema, J. H.; Smith, B. J.; Wang, Y.; Jham, B. C.; O'Hagan, K. P.; Thomson, N. R.; Murthy, A. K.; Skilton, R. J.; Chu, P.; Clarke, I. N.

    2014-01-01

    The native plasmid of both Chlamydia muridarum and Chlamydia trachomatis has been shown to control virulence and infectivity in mice and in lower primates. We recently described the development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis that for the first time provides a platform for the molecular dissection of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In the present study, we transformed a plasmid-free lymphogranuloma venereum isolate of C. trachomatis, serovar L2, with either the original shuttle vector (pGFP::SW2) or a derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene (pCDS5KO). Female mice were inoculated with these strains either intravaginally or transcervically. We found that transformation of the plasmid-free isolate with the intact pGFP::SW2 vector significantly enhanced infectivity and induction of host inflammatory responses compared to the plasmid-free parental isolate. Transformation with pCDS5KO resulted in infection courses and inflammatory responses not significantly different from those observed in mice infected with the plasmid-free isolate. These results indicate a critical role of plasmid CDS5 in in vivo fitness and in induction of inflammatory responses. To our knowledge, these are the first in vivo observations ascribing infectivity and virulence to a specific plasmid gene. PMID:24866804

  17. Plasmid Copy Number Determination by Quantitative Polymerase Chain Reaction.

    PubMed

    Anindyajati; Artarini, A Anita; Riani, Catur; Retnoningrum, Debbie S

    2016-01-01

    Recombinant therapeutic proteins are biopharmaceutical products that develop rapidly for years. Recombinant protein production in certain hosts requires vector expression harboring the gene encoding the corresponding protein. Escherichia coli is the prokaryote organism mostly used in recombinant protein production, commonly using a plasmid as the expression vector. Recombinant protein production is affected by plasmid copy number harboring the encoded gene, hence the determination of plasmid copy number also plays an important role in establishing a recombinant protein production system. On the industrial scale, a low copy number of plasmids are more suitable due to their better stability. In the previous study we constructed pCAD, a plasmid derived from the low copy number pBR322 plasmid. This study was aimed to confirm pCAD's copy number by quantitative polymerase chain reaction (qPCR). Plasmid copy number was determined by comparing the quantification signal from the plasmid to those from the chromosome. Copy number was then calculated by using a known copy number plasmid as a standard. Two pairs of primers, called tdk and ori, were designed for targeting a single gene tdk in the chromosome and a conserved domain in the plasmid's ori, respectively. Primer quality was analyzed in silico using PrimerSelect DNASTAR and PraTo software prior to in vitro evaluation on primer specificity and efficiency as well as optimization of qPCR conditions. Plasmid copy number determination was conducted on E. coli lysates harboring each plasmid, with the number of cells ranging from 10(2)-10(5) cells/μL. Cells were lysed by incubation at 95ºC for 10 minutes, followed by immediate freezing at -4°C. pBR322 plasmid with the copy number of ~19 copies/cell was used as the standard, while pJExpress414-sod plasmid possessing the high copy number pUC ori was also determined to test the method being used. In silico analysis based on primer-primer and primer-template interactions showed

  18. Characterization of the Lactobacillus plantarum plasmid pCD033 and generation of the plasmid free strain L. plantarum 3NSH.

    PubMed

    Heiss, Silvia; Grabherr, Reingard; Heinl, Stefan

    2015-09-01

    Lactobacillus plantarum CD033, a strain isolated from grass silage in Austria, harbors a 7.9 kb plasmid designated pCD033. Sequence analysis identified 14 open reading frames and 8 of these were supposed to be putative coding sequences. Gene annotation revealed no putative essential genes being plasmid encoded, but a plasmid addiction system based on a PemI/PemK-like toxin-antitoxin system, able to stabilize plasmid maintenance. Absence of a replication initiation protein, a double strand origin as well as a single strand origin on plasmid pCD033 suggests replication via a new type of theta mechanism, whereby plasmid replication is potentially initiated and regulated by non-coding RNA. Detailed examination of segregational stability of plasmid vectors consisting of pCD033-fragments, combined with a selection marker, resulted in definition of a stably maintained minimal replicon. A gene encoding a RepB/OrfX-like protein was found to be not essential for plasmid replication. Alignment of the amino acid sequence of this protein with related proteins unveiled a highly conserved amino acid motif (LLDQQQ). L. plantarum CD033 was cured of pCD033 resulting in the novel plasmid free strain L. plantarum 3NSH. Plasmid curing demonstrated that no essential features are provided by pCD033 under laboratory conditions.

  19. Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli

    PubMed Central

    Ali, Syed A.; Chew, Yik Wei; Omar, Tasyriq Che; Azman, Nizuwan

    2015-01-01

    Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing β-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system. PMID:26642325

  20. Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli.

    PubMed

    Ali, Syed A; Chew, Yik Wei; Omar, Tasyriq Che; Azman, Nizuwan

    2015-01-01

    Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing β-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system.

  1. Characterization of two novel plasmids from Geobacillus sp. 610 and 1121 strains.

    PubMed

    Kananavičiūtė, Rūta; Butaitė, Elena; Citavičius, Donaldas

    2014-01-01

    We describe two cryptic low molecular weight plasmids, pGTD7 (3279bp) and pGTG5 (1540bp), isolated from Geobacillus sp. 610 and 1121 strains, respectively. Homology analysis of the replication protein (Rep) sequences and detection of ssDNA indicate that both of them replicate via rolling circle mechanism. As revealed by sequence similarities of dso region and Rep protein, plasmid pGTD7 belongs to pC194/pUB110 plasmid family. The replicon of pGTD7 was proved to be functional in another Geobacillus host. For this purpose, a construct pUCK7, containing a replicon of the analyzed plasmid, was created and transferred to G. stearothermophilus NUB3621R strain by electroporation. Plasmid pGTG5, based on Rep protein sequence similarity, was found to be related mostly to some poorly characterized bacterial plasmids. Rep proteins encoded by these plasmids contain conservative motifs that are most similar to those of Microviridae phages. This feature suggests that pGTG5, together with other plasmids containing the same motifs, could constitute a new family of bacterial plasmids. To date, pGTG5 is the smallest plasmid identified in bacteria belonging to the genus Geobacillus. The two plasmids described in this study can be used for the construction of new vectors suitable for biotechnologically important bacteria of the genus Geobacillus.

  2. Exogenous Isolation of Mobilizing Plasmids from Polluted Soils and Sludges

    PubMed Central

    Top, Eva; De Smet, Ingrid; Verstraete, Willy; Dijkmans, Roger; Mergeay, Max

    1994-01-01

    Exogenous plasmid isolation was used to assess the presence of mobilizing plasmids in several soils and activated sludges. Triparental matings were performed with Escherichia coli (a member of the γ subgroup of the Proteobacteria) as the donor of an IncQ plasmid (pMOL155, containing the heavy metal resistance genes czc: Cor, Znr, and Cdr), Alcaligenes eutrophus (a member of the β subgroup of the Proteobacteria) as the recipient, and indigenous microorganisms from soil and sludge samples as helper strains. We developed an assay to assess the plasmid mobilization potential of a soil ecosystem on the basis of the number of transconjugants obtained after exogenous isolations. After inoculation into soil of several concentrations of a helper strain (E. coli CM120 harboring IncP [IncP1] mobilizing plasmid RP4), the log numbers of transconjugants obtained from exogenous isolations with different soil samples were a linear function of the log numbers of helper strain CM120(RP4) present in the soils. Four soils were analyzed for the presence of mobilizing elements, and mobilizing plasmids were isolated from two of these soils. Several sludge samples from different wastewater treatment plants yielded much higher numbers of transconjugants than the soil samples, indicating that higher numbers of mobilizing strains were present. The mobilizing plasmids isolated from Gent-O sludge and one plasmid isolated from Eislingen soil hybridized to the repP probe, whereas the plasmids isolated from Essen soil did not hybridize to a large number of rep probes (repFIC, repHI1, repH12, repL/M, repN, repP, repT, repU, repW, repX). This indicates that in Essen soil, broad-host-range mobilizing plasmids belonging to other incompatibility groups may be present. Images PMID:16349216

  3. Plasmid DNA production for therapeutic applications.

    PubMed

    Lara, Alvaro R; Ramírez, Octavio T; Wunderlich, Martin

    2012-01-01

    Plasmid DNA (pDNA) is the base for promising DNA vaccines and gene therapies against many infectious, acquired, and genetic diseases, including HIV-AIDS, Ebola, Malaria, and different types of cancer, enteric pathogens, and influenza. Compared to conventional vaccines, DNA vaccines have many advantages such as high stability, not being infectious, focusing the immune response to only those antigens desired for immunization and long-term persistence of the vaccine protection. Especially in developing countries, where conventional effective vaccines are often unavailable or too expensive, there is a need for both new and improved vaccines. Therefore the demand of pDNA is expected to rise significantly in the near future. Since the injection of pDNA usually only leads to a weak immune response, several milligrams of DNA vaccine are necessary for immunization protection. Hence, there is a special interest to raise the product yield in order to reduce manufacturing costs. In this chapter, the different stages of plasmid DNA production are reviewed, from the vector design to downstream operation options. In particular, recent advances on cell engineering for improving plasmid DNA production are discussed. PMID:22160904

  4. Bacterial plasmid transfer under space flight conditions: The Mobilisatsia experience

    NASA Astrophysics Data System (ADS)

    de Boever, P.; Ilyin, V.; Mahillon, J.; Mergeay, M.

    Background Microorganisms are subject to a genetic evolution which may lead to the capacity to colonize new environments and to cause infections Central players in this evolutionary process are mobile genetic elements phages plasmids and transposons The latter help to mobilize and reorganize genes be it within a given genome intragenomic mobility or between bacterial cells intercellular mobility Confined environment and space flight related factors such as microgravity and cosmic radiation may influence the frequency with which mobile genetic elements are exchanged between microorganisms Aim Within the frame of the Mobilisatsia experiment a triparental microbial plasmid transfer was promoted aboard the International Space Station ISS The efficiency of the plasmid exchange process was compared with a synchronously performed ground control experiment An experiment was carried out with well-characterized Gram-negative test strains and one experiment was done with Gram-positive test strains Results The experiment took place during the Soyouz Mission 8 to the ISS from April 19th until April 30th 2004 Liquid cultures of the bacterial strains Cupriavidus metallidurans AE815 final recipient Escherichia coli CM1962 carrying a mobilisable vector with a nickel-resistance marker and E coli CM140 carrying the Broad Host Range plasmid RP4 for the Gram-negative experiment and Bacillus thuringiensis Bti AND931 carrying the conjugative plasmid pXO16 Bti 4Q7 with mobilisable vector pC194 carrying a resistance to chloramphenicol and Bti GBJ002

  5. Synthesis of hybrid bacterial plasmids containing highly repeated satellite DNA.

    PubMed

    Brutlag, D; Fry, K; Nelson, T; Hung, P

    1977-03-01

    Hybrid plasmid molecules containing tandemly repeated Drosophila satellite DNA were constructed using a modification of the (dA)-(dT) homopolymer procedure of Lobban and Kaiser (1973). Recombinant plasmids recovered after transformation of recA bacteria contained 10% of the amount of satellite DNA present in the transforming molecules. The cloned plasmids were not homogenous in size. Recombinant plasmids isolated from a single colony contained populations of circular molecules which varied both in the length of the satellite region and in the poly(dA)-(dt) regions linking satellite and vector. While subcloning reduced the heterogeneity of these plasmid populations, continued cell growth caused further variations in the size of the repeated regions. Two different simple sequence satellites of Drosophila melanogaster (1.672 and 1.705 g/cm3) were unstable in both recA and recBC hosts and in both pSC101 and pCR1 vectors. We propose that this recA-independent instability of tandemly repeated sequences is due to unequal intramolecular recombination events in replicating DNA molecules, a mechanism analogous to sister chromatid exchange in eucaryotes. PMID:403010

  6. Genetic transformation of Rhodopseudomonas sphaeroides by plasmid DNA.

    PubMed Central

    Fornari, C S; Kaplan, S

    1982-01-01

    A broad-host-range cloning vector, pUI81, was constructed in vitro from plasmids RSF1010 and pSL25 (a pBR322 derivative) and used to assay for transformation in Rhodopseudomonas sphaeroides. Washing cells with 500 mM Tris was an effective means of inducing competence for DNA uptake. Transformation frequencies as high as 10(-5) (transformants per viable cell) have been achieved by incubating Tris-treated cells with plasmid DNA, 100 mM CaCl2, and 20% polyethylene glycol 6000. Maximum frequencies were obtained when recipient cells were spread onto selective media after a 6.5-h outgrowth period in antibiotic-free medium. The structure (open circular versus closed, covalent circular), size, and concentration of plasmid DNA all significantly affected the transformation frequency. Four different plasmids, all small and suitable as cloning vectors, have been introduced by transformation into several different R. sphaeroides strains. Recombinant DNA carried on small, nonconjugative plasmids with broad host ranges can now be directly transferred to R. sphaeroides by this method. Images PMID:6981642

  7. The Addgene repository: an international nonprofit plasmid and data resource

    PubMed Central

    Kamens, Joanne

    2015-01-01

    The Addgene Repository (http://www.addgene.org) was founded to accelerate research and discovery by improving access to useful, high-quality research materials and information. The repository archives plasmids generated by scientists, conducts quality control, annotates the associated data and makes the plasmids and their data available to the scientific community. Plasmid associated data undergoes ongoing curation by members of the scientific community and by Addgene scientists. The growing database contains information on >31 000 unique plasmids spanning most experimental biological systems and organisms. The library includes a large number of plasmid tools for use in a wide variety of research areas, such as empty backbones, lentiviral resources, fluorescent protein vectors and genome engineering tools. The Addgene Repository database is always evolving with new plasmid deposits so it contains currently pertinent resources while ensuring the information on earlier deposits is still available. Custom search and browse features are available to access information on the diverse collection. Extensive educational materials and information are provided by the database curators to support the scientists that are accessing the repository's materials and data. PMID:25392412

  8. Transformation of Rhizobium meliloti 41 with plasmid DNA.

    PubMed Central

    Kiss, G B; Kálmán, Z

    1982-01-01

    Plasmid pGV1106, a derivative of the wide-host-range plasmid S-a of the W incompatibility group, was introduced into Rhizobium meliloti 41 by plasmid-mediated mobilization to overcome the restriction of foreign DNA. The mobilized plasmid pKK2 differed from the original pGV1106 by an extra piece of DNA of 1.3 kilobase pairs which supposedly originated from pJB3JI used for mobilization. If pKK2 was isolated from R. meliloti 41, it could be successfully reintroduced by transformation. The transformation frequency was low (10 to 54 colonies per micrograms of plasmid DNA) but reproducible, and several lines of evidence showed that it was the consequence of plasmid DNA uptake. The small size (10.3 kilobases) and elevated copy number (10 to 15 copies per cell) of pKK2 make it a potentially useful cloning vector for the study of symbiotic nitrogen fixation genes of R. meliloti 41. Images PMID:6279558

  9. Toxin Plasmids of Clostridium perfringens

    PubMed Central

    Li, Jihong; Adams, Vicki; Bannam, Trudi L.; Miyamoto, Kazuaki; Garcia, Jorge P.; Uzal, Francisco A.; Rood, Julian I.

    2013-01-01

    SUMMARY In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  10. Introduction of temperature-sensitive helper and donor plasmids into Bac-to-Bac baculovirus expression systems.

    PubMed

    Huang, Zhihong; Li, Ao; Pan, Mengjia; Wu, Wenbi; Yuan, Meijin; Yang, Kai

    2015-10-01

    In the baculovirus shuttle vector (bacmid) system, a helper plasmid and a donor plasmid are employed to insert heterologous genes into a cloned baculovirus genome via Tn7 transposition in Escherichia coli. The helper and donor plasmids are usually cotransfected with constructed bacmids into insect cells, which will lead to integration of these plasmids into the viral genome, and hence to the production of defective virions. In this study, to facilitate the preparation of plasmid-free recombinant bacmids, we modified a set of helper and donor plasmids by replacing their replication origins with that of a temperature-sensitive (ts) plasmid, pSIM6. Using the resulting ts helper plasmid pMON7124(ts) and the ts donor plasmid pFB1(ts)-PH-GFP, a recombinant bacmid, bAcWT-PG(-), was constructed, and the transposition efficiency was found to be 33.1%. The plasmids were then removed by culturing at 37 °C. For bAcWT-PG(-), the infectious progeny virus titer and the protein expression level under the control of the polyhedrin promoter were similar to those of a bacmid constructed with unmodified helper and donor plasmids. These ts plasmids will be useful for obtaining plasmid-free bacmids for both heterologous protein production and fundamental studies of baculovirus biology.

  11. Stable transformation of a mosquito cell line results in extraordinarily high copy numbers of the plasmid.

    PubMed Central

    Monroe, T J; Muhlmann-Diaz, M C; Kovach, M J; Carlson, J O; Bedford, J S; Beaty, B J

    1992-01-01

    Stable incorporation of high copy numbers (greater than 10,000 per cell) of a plasmid vector containing a gene conferring resistance to the antibiotic hygromycin was achieved in a cell line derived from the Aedes albopictus mosquito. Plasmid sequences were readily observed by ethidium bromide staining of cellular DNA after restriction endonuclease digestion and agarose gel electrophoresis. The plasmid was demonstrated by in situ hybridization to be present in large arrays integrated in metaphase chromosomes and in minute and double-minute replicating elements. In one subclone, approximately 60,000 copies of the plasmid were organized in a large array that resembles a chromosome, morphologically and in the segregation of its chromatids during anaphase. The original as well as modified versions of the plasmid were rescued by transformation of Escherichia coli using total cellular DNA. Southern blot analyses of recovered plasmids indicate the presence of mosquito-derived sequences. Images PMID:1631052

  12. Construction of a bioluminescence reporter plasmid for Francisella tularensis.

    PubMed

    Bina, Xiaowen R; Miller, Mark A; Bina, James E

    2010-11-01

    A Francisella tularensis shuttle vector that constitutively expresses the Photorhabdus luminescens lux operon in type A and type B strains of F. tularensis was constructed. The bioluminescence reporter plasmid was introduced into the live vaccine strain of F. tularensis and used to follow F. tularensis growth in a murine intranasal challenge model in real-time by bioluminescence imaging. The results show that the new bioluminescence reporter plasmid represents a useful tool for tularemia research that is suitable for following F. tularensis growth in both in vitro and in vivo model systems. PMID:20620161

  13. A linear programming manual

    NASA Technical Reports Server (NTRS)

    Tuey, R. C.

    1972-01-01

    Computer solutions of linear programming problems are outlined. Information covers vector spaces, convex sets, and matrix algebra elements for solving simultaneous linear equations. Dual problems, reduced cost analysis, ranges, and error analysis are illustrated.

  14. GeneGuard: A modular plasmid system designed for biosafety.

    PubMed

    Wright, Oliver; Delmans, Mihails; Stan, Guy-Bart; Ellis, Tom

    2015-03-20

    Synthetic biology applications in biosensing, bioremediation, and biomining envision the use of engineered microbes beyond a contained laboratory. Deployment of such microbes in the environment raises concerns of unchecked cellular proliferation or unwanted spread of synthetic genes. While antibiotic-resistant plasmids are the most utilized vectors for introducing synthetic genes into bacteria, they are also inherently insecure, acting naturally to propagate DNA from one cell to another. To introduce security into bacterial synthetic biology, we here took on the task of completely reformatting plasmids to be dependent on their intended host strain and inherently disadvantageous for others. Using conditional origins of replication, rich-media compatible auxotrophies, and toxin-antitoxin pairs we constructed a mutually dependent host-plasmid platform, called GeneGuard. In this, replication initiators for the R6K or ColE2-P9 origins are provided in trans by a specified host, whose essential thyA or dapA gene is translocated from a genomic to a plasmid location. This reciprocal arrangement is stable for at least 100 generations without antibiotic selection and is compatible for use in LB medium and soil. Toxin genes ζ or Kid are also employed in an auxiliary manner to make the vector disadvantageous for strains not expressing their antitoxins. These devices, in isolation and in concert, severely reduce unintentional plasmid propagation in E. coli and B. subtilis and do not disrupt the intended E. coli host's growth dynamics. Our GeneGuard system comprises several versions of modular cargo-ready vectors, along with their requisite genomic integration cassettes, and is demonstrated here as an efficient vector for heavy-metal biosensors.

  15. The genetic basis of plasmid tropism between Chlamydia trachomatis and Chlamydia muridarum.

    PubMed

    Wang, Yibing; Cutcliffe, Lesley T; Skilton, Rachel J; Ramsey, Kyle H; Thomson, Nicholas R; Clarke, Ian N

    2014-10-01

    The development of genetic transformation technology for Chlamydia trachomatis using its endogenous plasmid has recently been described. Chlamydia muridarum cannot be transformed by the C. trachomatis plasmid, indicating a barrier between chlamydial species. To determine which regions of the plasmid conferred the species specificity, we used the novel approach of transforming wild-type C. muridarum carrying the endogenous plasmid pNigg and forced recombination with the C. trachomatis vector pGFP::SW2 which carries the complete C. trachomatis plasmid (pSW2). Penicillin and chloramphenicol-resistant transformants expressing the green fluorescent protein were selected. Recovery of plasmids from these transformants showed they were recombinants. The differences between the pSW2 and pNigg allowed identification of the recombination breakpoints and showed that pGFP::SW2 had exchanged a ~ 1 kbp region with pNigg covering CDS 2. The recombinant plasmid (pSW2NiggCDS2) is maintained under antibiotic selection when transformed into plasmid-cured C. muridarum. The ability to select for recombinants in C. muridarum shows that the barrier is not at transformation, but at the level of plasmid replication or maintenance. Our studies show that CDS 2, together with adjoining sequences, is the main determinant of plasmid tropism. PMID:24700815

  16. The genetic basis of plasmid tropism between Chlamydia trachomatis and Chlamydia muridarum

    PubMed Central

    Wang, Yibing; Cutcliffe, Lesley T; Skilton, Rachel J; Ramsey, Kyle H; Thomson, Nicholas R; Clarke, Ian N

    2014-01-01

    The development of genetic transformation technology for Chlamydia trachomatis using its endogenous plasmid has recently been described. Chlamydia muridarum cannot be transformed by the C. trachomatis plasmid, indicating a barrier between chlamydial species. To determine which regions of the plasmid conferred the species specificity, we used the novel approach of transforming wild-type C. muridarum carrying the endogenous plasmid pNigg and forced recombination with the C. trachomatis vector pGFP::SW2 which carries the complete C. trachomatis plasmid (pSW2). Penicillin and chloramphenicol-resistant transformants expressing the green fluorescent protein were selected. Recovery of plasmids from these transformants showed they were recombinants. The differences between the pSW2 and pNigg allowed identification of the recombination breakpoints and showed that pGFP::SW2 had exchanged a ∼ 1 kbp region with pNigg covering CDS 2. The recombinant plasmid (pSW2NiggCDS2) is maintained under antibiotic selection when transformed into plasmid-cured C. muridarum. The ability to select for recombinants in C. muridarum shows that the barrier is not at transformation, but at the level of plasmid replication or maintenance. Our studies show that CDS 2, together with adjoining sequences, is the main determinant of plasmid tropism. PMID:24700815

  17. Introducing Vectors.

    ERIC Educational Resources Information Center

    Roche, John

    1997-01-01

    Suggests an approach to teaching vectors that promotes active learning through challenging questions addressed to the class, as opposed to subtle explanations. Promotes introducing vector graphics with concrete examples, beginning with an explanation of the displacement vector. Also discusses artificial vectors, vector algebra, and unit vectors.…

  18. Phenotypic plasticity in bacterial plasmids.

    PubMed Central

    Turner, Paul E

    2004-01-01

    Plasmid pB15 was previously shown to evolve increased horizontal (infectious) transfer at the expense of reduced vertical (intergenerational) transfer and vice versa, a key trade-off assumed in theories of parasite virulence. Whereas the models predict that susceptible host abundance should determine which mode of transfer is selectively favored, host density failed to mediate the trade-off in pB15. One possibility is that the plasmid's transfer deviates from the assumption that horizontal spread (conjugation) occurs in direct proportion to cell density. I tested this hypothesis using Escherichia coli/pB15 associations in laboratory serial culture. Contrary to most models of plasmid transfer kinetics, my data show that pB15 invades static (nonshaking) bacterial cultures only at intermediate densities. The results can be explained by phenotypic plasticity in traits governing plasmid transfer. As cells become more numerous, the plasmid's conjugative transfer unexpectedly declines, while the trade-off between transmission routes causes vertical transfer to increase. Thus, at intermediate densities the plasmid's horizontal transfer can offset selection against plasmid-bearing cells, but at high densities pB15 conjugates so poorly that it cannot invade. I discuss adaptive vs. nonadaptive causes for the phenotypic plasticity, as well as potential mechanisms that may lead to complex transfer dynamics of plasmids in liquid environments. PMID:15166133

  19. Plasmid acquisition in microgravity

    NASA Technical Reports Server (NTRS)

    Juergensmeyer, Margaret A.; Juergensmeyer, Elizabeth A.; Guikema, James A.

    1995-01-01

    In microgravity, bacteria often show an increased resistance to antibiotics. Bacteria can develop resistance to an antibiotic after transformation, the acquisition of DNA, usually in the form of a plasmid containing a gene for resistance to one or more antibiotics. In order to study the capacity of bacteria to become resistant to antibiotics in microgravity, we have modified the standard protocol for transformation of Escherichia coli for use in the NASA-flight-certified hardware package, The Fluid Processing Apparatus (FPA). Here we report on the ability of E. coli to remain competent for long periods of time at temperatures that are readily available on the Space Shuttle, and present some preliminary flight results.

  20. A plasmid in Streptococcus pneumoniae.

    PubMed Central

    Smith, M D; Guild, W R

    1979-01-01

    Plasmid deoxyribonucleic acid has been detected in three related laboratory strains of Streptococcus pneumoniae. Strains D39S, R36, and R36NC each contain a minimum of two copies per cell of a 2.0-megadalton plasmid (pDP1). A plasmid twice as large as this smaller one is also present in much lower quantity in these strains, but neither plasmid is present in four strains related to these or in a drug-resistant clinical isolate from Paris. The plasmid yield was not amplified in the presence of chloramphenicol. No phenotype has been correlated with the presence of pDP1, which has existed in strains carried for many years in laboratory collections. Images PMID:33961

  1. Characterization of ESBL disseminating plasmids.

    PubMed

    Brolund, Alma; Sandegren, Linus

    2016-01-01

    Bacteria producing extended-spectrum β-lactamases (ESBLs) constitute a globally increasing problem that contributes to treatment complications and elevated death rates. The extremely successful dissemination by ESBL-producing Enterobacteriaceae during the latest decades is a result of the combination of mobilization, evolution and horizontal spread of β-lactamase genes on plasmids. In parallel, spread of these plasmids to particularly well-adapted bacterial clones (outbreak clones) has expanded. In this review we describe ESBL-producing bacteria and the genetic mechanisms for dissemination of ESBL resistance. We describe available methodology for studying plasmids and the importance of including plasmids in epidemiological typing as natural parts of the organisms. Plasmids play a fundamental role in how resistance arises and disseminates.

  2. Construction of the recombinant broad-host-range plasmids providing their bacterial hosts arsenic resistance and arsenite oxidation ability.

    PubMed

    Drewniak, Lukasz; Ciezkowska, Martyna; Radlinska, Monika; Sklodowska, Aleksandra

    2015-02-20

    The plasmid pSinA of Sinorhizobium sp. M14 was used as a source of functional phenotypic modules, encoding proteins involved in arsenite oxidation and arsenic resistance, to obtain recombinant broad-host-range plasmids providing their bacterial hosts arsenic resistance and arsenite oxidative ability. An arsenite oxidation module was cloned into pBBR1MCS-2 vector yielding plasmid vector pAIO1, while an arsenic resistance module was cloned into pCM62 vector yielding plasmid pARS1. Both plasmid constructs were introduced (separately and together) into the cells of phylogenetically distant (representing Alpha-, Beta-, and Gammaproteobacteria) and physiologically diversified (unable to oxidize arsenite and susceptible/resistant to arsenite and arsenate) bacteria. Functional analysis of the modified strains showed that: (i) the plasmid pARS1 can be used for the construction of strains with an increased resistance to arsenite [up to 20mM of As(III), (ii) the presence of the plasmid pAIO1 in bacteria previously unable to oxidize As(III) to As(V), contributes to the acquisition of arsenite oxidation abilities by these cells, (iii) the highest arsenite utilization rate are observed in the culture of strains harbouring both the plasmids pAIO1 and pARS1, (iv) the strains harbouring the plasmid pAIO1 were able to grow on arsenic-contaminated mine waters (∼ 3.0 mg As L(-1)) without any supplementation.

  3. Genome Sequence of Listeria monocytogenes Plasmid pLM-C-273 Carrying Genes Related to Stress Resistance

    PubMed Central

    Liang, Lindsay; Gnaneshan, Saravanamuttu; Garduño, Rafael A.

    2016-01-01

    Mobile genetic elements in bacteria, such as plasmids, act as important vectors for the transfer of antibiotic resistance, virulence, and metal resistance genes. Here, we report the genome sequence of a new plasmid pLM-C-273, identified in a Listeria monocytogenes strain isolated from a clinical sample in Ontario, Canada. PMID:27738039

  4. Optimization of a one-step heat-inducible in vivo mini DNA vector production system.

    PubMed

    Nafissi, Nafiseh; Sum, Chi Hong; Wettig, Shawn; Slavcev, Roderick A

    2014-01-01

    While safer than their viral counterparts, conventional circular covalently closed (CCC) plasmid DNA vectors offer a limited safety profile. They often result in the transfer of unwanted prokaryotic sequences, antibiotic resistance genes, and bacterial origins of replication that may lead to unwanted immunostimulatory responses. Furthermore, such vectors may impart the potential for chromosomal integration, thus potentiating oncogenesis. Linear covalently closed (LCC), bacterial sequence free DNA vectors have shown promising clinical improvements in vitro and in vivo. However, the generation of such minivectors has been limited by in vitro enzymatic reactions hindering their downstream application in clinical trials. We previously characterized an in vivo temperature-inducible expression system, governed by the phage λ pL promoter and regulated by the thermolabile λ CI[Ts]857 repressor to produce recombinant protelomerase enzymes in E. coli. In this expression system, induction of recombinant protelomerase was achieved by increasing culture temperature above the 37°C threshold temperature. Overexpression of protelomerase led to enzymatic reactions, acting on genetically engineered multi-target sites called "Super Sequences" that serve to convert conventional CCC plasmid DNA into LCC DNA minivectors. Temperature up-shift, however, can result in intracellular stress responses and may alter plasmid replication rates; both of which may be detrimental to LCC minivector production. We sought to optimize our one-step in vivo DNA minivector production system under various induction schedules in combination with genetic modifications influencing plasmid replication, processing rates, and cellular heat stress responses. We assessed different culture growth techniques, growth media compositions, heat induction scheduling and temperature, induction duration, post-induction temperature, and E. coli genetic background to improve the productivity and scalability of our system

  5. Extrachromosomal plasmids in the plant pathogenic fungus Rhizoctonia solani.

    PubMed

    Jabaji-Hare, S H; Burger, G; Forget, L; Lang, B F

    1994-05-01

    Extrachromosomal DNA elements were found in field isolates of Rhizoctonia solani belonging to anastomosis groups (AG) 1-5. An isolate of AG-5 (Rh41) contains a 3.6-kbp plasmid (pRS188) which has a similar A+T content to mitochondrial DNA. pRS188 is linear and has knob structures at its ends, as revealed by electron microscopy. Exonuclease digestions show that the linear ends of pRS188 are protected, and remain protected even after proteinase K digestion. pRS188 does not hybridise to nuclear or mitochondrial DNAs of its host isolate (Rh41), to total DNAs of other plasmid-less AG-5 isolates, or to total DNA of plasmid-harbouring isolates belonging to different AGs. Cellular-fractionation experiments suggest that pRS188 is associated with mitochondria, but it remains undecided whether this occurs inside or outside of the organelles. The nucleotide sequence of about 60% of the plasmid has been determined, revealing no open reading frame longer than 91 amino acids, and no known gene or genetic element is detected in the sequence contigs of 300-1572 bp length. Similar studies were performed with the plasmid pRS104 present in an isolate of AG-4 (Rh36), the sequence of which exhibits essentially the same features as pRS188 except that its A+T content resembles that of nuclear DNA. Pathogenicity tests reveal that the isolates Rh41 and R36 are as virulent as the plasmid-less isolates of AG-4 and -5, indicating that the plasmids do not play any role in pathogenicity.

  6. Change in the plasmid copy number in acetic acid bacteria in response to growth phase and acetic acid concentration.

    PubMed

    Akasaka, Naoki; Astuti, Wiwik; Ishii, Yuri; Hidese, Ryota; Sakoda, Hisao; Fujiwara, Shinsuke

    2015-06-01

    Plasmids pGE1 (2.5 kb), pGE2 (7.2 kb), and pGE3 (5.5 kb) were isolated from Gluconacetobacter europaeus KGMA0119, and sequence analyses revealed they harbored 3, 8, and 4 genes, respectively. Plasmid copy numbers (PCNs) were determined by real-time quantitative PCR at different stages of bacterial growth. When KGMA0119 was cultured in medium containing 0.4% ethanol and 0.5% acetic acid, PCN of pGE1 increased from 7 copies/genome in the logarithmic phase to a maximum of 12 copies/genome at the beginning of the stationary phase, before decreasing to 4 copies/genome in the late stationary phase. PCNs for pGE2 and pGE3 were maintained at 1-3 copies/genome during all phases of growth. Under a higher concentration of ethanol (3.2%) the PCN for pGE1 was slightly lower in all the growth stages, and those of pGE2 and pGE3 were unchanged. In the presence of 1.0% acetic acid, PCNs were higher for pGE1 (10 copies/genome) and pGE3 (6 copies/genome) during the logarithmic phase. Numbers for pGE2 did not change, indicating that pGE1 and pGE3 increase their PCNs in response to acetic acid. Plasmids pBE2 and pBE3 were constructed by ligating linearized pGE2 and pGE3 into pBR322. Both plasmids were replicable in Escherichia coli, Acetobacter pasteurianus and G. europaeus, highlighting their suitability as vectors for acetic acid bacteria.

  7. Change in the plasmid copy number in acetic acid bacteria in response to growth phase and acetic acid concentration.

    PubMed

    Akasaka, Naoki; Astuti, Wiwik; Ishii, Yuri; Hidese, Ryota; Sakoda, Hisao; Fujiwara, Shinsuke

    2015-06-01

    Plasmids pGE1 (2.5 kb), pGE2 (7.2 kb), and pGE3 (5.5 kb) were isolated from Gluconacetobacter europaeus KGMA0119, and sequence analyses revealed they harbored 3, 8, and 4 genes, respectively. Plasmid copy numbers (PCNs) were determined by real-time quantitative PCR at different stages of bacterial growth. When KGMA0119 was cultured in medium containing 0.4% ethanol and 0.5% acetic acid, PCN of pGE1 increased from 7 copies/genome in the logarithmic phase to a maximum of 12 copies/genome at the beginning of the stationary phase, before decreasing to 4 copies/genome in the late stationary phase. PCNs for pGE2 and pGE3 were maintained at 1-3 copies/genome during all phases of growth. Under a higher concentration of ethanol (3.2%) the PCN for pGE1 was slightly lower in all the growth stages, and those of pGE2 and pGE3 were unchanged. In the presence of 1.0% acetic acid, PCNs were higher for pGE1 (10 copies/genome) and pGE3 (6 copies/genome) during the logarithmic phase. Numbers for pGE2 did not change, indicating that pGE1 and pGE3 increase their PCNs in response to acetic acid. Plasmids pBE2 and pBE3 were constructed by ligating linearized pGE2 and pGE3 into pBR322. Both plasmids were replicable in Escherichia coli, Acetobacter pasteurianus and G. europaeus, highlighting their suitability as vectors for acetic acid bacteria. PMID:25575969

  8. Genome Stability of Lyme Disease Spirochetes: Comparative Genomics of Borrelia burgdorferi Plasmids

    SciTech Connect

    Casjens S. R.; Dunn J.; Mongodin, E. F.; Qiu, W.-G.; Luft, B. J.; Schutzer, S. E.; Gilcrease, E. B.; Huang, W. M.; Vujadinovic, M.; Aron, J. K.; Vargas, L. C.; Freeman, S.; Radune, D.; Weidman, J. F.; Dimitrov, G. I.; Khouri, H. M.; Sosa, J. E.; Halpin, R. A.; Fraser, C. M.

    2012-03-14

    Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33-40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi {approx}900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short {le}20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant.

  9. Transformation of Streptococcus sanguis Challis by plasmid deoxyribonucleic acid from Streptococcus faecalis.

    PubMed Central

    LeBlanc, D J; Hassell, F P

    1976-01-01

    Plasmid deoxyribonucleic acid (DNA) from Streptococcus faecalis, strain DS5, was transferred to the Challis strain of Streptococcus sanguis by transformation. Two antibiotic resistance markers carried by the beta plasmid from strain DS5, erythromycin and lincomycin, were transferred to S. sanguis at a maximum frequency of 1.8 x 10-5/colony-forming unit. Approximately 70% of the covalently closed circular DNA isolated from transformant cultures by dye buoyant density gradients was shown to be hybridizable to beta plasmid DNA. Two major differences were observed between the beta plasmid from S. faecalis and the plasmid isolated from transformed S. sanguis: (i) the beta plasmid from strain DS5 sedimented in velocity gradients at 43S, whereas the covalently closed circular DNA from transformed Challis sedimented at 41S, suggesting a 1.5-Mdal deletion from the beta plasmid occurred; (ii) although the 43S beta plasmid remained in the supercoiled configuration for several weeks after isolation, the 41S plasmid was rapidly converted to a linear double-stranded molecule. Attempts to transform S. sanguis with the alpha plasmid from S. faecalis, strain DS5, were unsuccessful. PMID:824275

  10. Analysis of replication region of the cryptic plasmid pAG20 from Acetobacter aceti 3620.

    PubMed

    Kretová, Miroslava; Szemes, Tomás; Laco, Juraj; Gronesová, Paulína; Grones, Jozef

    2005-03-01

    The DNA sequence of small cryptic plasmid pAG20 in Acetobacter aceti was determined at 3064 bp with 51.6% GC pairs. The plasmid encoded a 186 amino acid protein which is important for plasmid replication in Gram-negative bacteria except Escherichia coli. Two 21 bp large direct repeat sequence 1 and two 13 bp direct repeat sequence 2 were determined in the regulation region upstream from gene encoded Rep protein. Vector pAG24 with kanamycin gene and two deletion derivatives pAG25 and pAG26 without rep gene from plasmid pAG20 were constructed. Plasmid pAG24 was replicated in a broad host range like E. coli, Acetobacter pasteurianus, A. aceti, Comanomonas spp., Serratia marcescens, and Shigella spp.

  11. Plasmid-mediated quinolone resistance

    PubMed Central

    Jacoby, George A.; Strahilevitz, Jacob; Hooper, David C.

    2014-01-01

    Summary Three mechanisms for plasmid-mediated quinolone resistance (PMQR) have been discovered since 1998. Plasmid genes qnrA, qnrB, qnrC, qnrD, qnrS, and qnrVC code for proteins of the pentapeptide repeat family that protects DNA gyrase and topoisomerase IV from quinolone inhibition. The qnr genes appear to have been acquired from chromosomal genes in aquatic bacteria, are usually associated with mobilizing or transposable elements on plasmids, and are often incorporated into sul1-type integrons. The second plasmid-mediated mechanism involves acetylation of quinolones with an appropriate amino nitrogen target by a variant of the common aminoglycoside acetyltransferase AAC(6′)-Ib. The third mechanism is enhanced efflux produced by plasmid genes for pumps QepAB and OqxAB. PMQR has been found in clinical and environmental isolates around the world and appears to be spreading. The plasmid-mediated mechanisms provide only low-level resistance that by itself does not exceed the clinical breakpoint for susceptibility but nonetheless facilitates selection of higher-level resistance and makes infection by pathogens containing PMQR harder to treat. PMID:25584197

  12. A rational proposal for plasmid nomenclature.

    PubMed

    Campos, P; Martín Luengo, F

    1989-09-01

    We propose a more rational system for nomenclature of wild plasmids of bacteria. With this proposal for nomenclature of bacterial plasmids, it is established in an unambiguous way: 1) if a plasmid is wild or derivative, and 2) in which species and bacterial strain it was found (in the case of wild plasmids).

  13. Transformation of Shewanella baltica with ColE1-like and P1 plasmids and their maintenance during bacterial growth in cultures.

    PubMed

    Milewska, Klaudia; Węgrzyn, Grzegorz; Szalewska-Pałasz, Agnieszka

    2015-09-01

    The presence of natural plasmids has been reported for many Shewanella isolates. However, knowledge about plasmid replication origin and segregation mechanisms is not extensive for this genus. Shewanella baltica is an important species in the marine environment due to its denitrification ability in oxygen-deficient zones and the potential role in bioremediation processes. However, no information about possible use of plasmid vectors in this species has been reported to date. Here we report that plasmids with ColE1-type and plasmid P1 origin can transform S. baltica and replicate in this bacterium. Without the antibiotic selection pressure plasmid maintenance is less efficient than in Escherichia coli. Nevertheless, cultivation of S. baltica in the presence of appropriate antibiotics caused relatively stable maintenance of ColE1-like and P1-derived plasmids. This indicates that plasmid-based genetic manipulations and gene transfer in S. baltica are possible.

  14. Transformation of Shewanella baltica with ColE1-like and P1 plasmids and their maintenance during bacterial growth in cultures.

    PubMed

    Milewska, Klaudia; Węgrzyn, Grzegorz; Szalewska-Pałasz, Agnieszka

    2015-09-01

    The presence of natural plasmids has been reported for many Shewanella isolates. However, knowledge about plasmid replication origin and segregation mechanisms is not extensive for this genus. Shewanella baltica is an important species in the marine environment due to its denitrification ability in oxygen-deficient zones and the potential role in bioremediation processes. However, no information about possible use of plasmid vectors in this species has been reported to date. Here we report that plasmids with ColE1-type and plasmid P1 origin can transform S. baltica and replicate in this bacterium. Without the antibiotic selection pressure plasmid maintenance is less efficient than in Escherichia coli. Nevertheless, cultivation of S. baltica in the presence of appropriate antibiotics caused relatively stable maintenance of ColE1-like and P1-derived plasmids. This indicates that plasmid-based genetic manipulations and gene transfer in S. baltica are possible. PMID:26170108

  15. Radiosensitivity of plasmid DNA: role of topology and concentration

    NASA Astrophysics Data System (ADS)

    Giustranti, C.; Pérez, C.; Rousset, S.; Balanzat, E.; Sage, E.

    1999-01-01

    Using the plasmid relaxation assay, the induction of single strand breaks (SSB) by ionizing radiation was investigated in two plasmids of different length, pBS and pSP189. The dose-response was linear for both plasmids but pSP189 exhibited a three times higher sensitivity than pBS. This disparity may be explained by a reduced accessibility to hydroxyl radicals due to a different topology of each plasmid, i.e. degree of compaction, as observed with electron microscopy. pBS plasmid was also exposed at various DNA concentrations to rays. The yield of SSB decreased with increasing concentration, suggesting a diminution in the amount of hydroxyl radicals efficient for radiolytic attack. This effect of concentration was also observed with densely ionizing radiation. In conclusion, the accessibility of DNA is a key-parameter in the formation of damage in vitro and in vivo as well. En utilisant la technique de relaxation de plasmide, l'induction de cassures simple brin (SSB) par les radiations ? a été comparée dans deux plasmides de taille différente, pSP189 et pBS. La relation dose-effet est linéaire pour les deux plasmides, mais il se forme trois fois plus de SSB dans pSP189 que dans pBS. Cette disparité semble pouvoir être reliée au degré de compaction différent des plasmides, observé en microscopie électronique. Elle s'expliquerait en terme d'accessibilité aux espèces radicalaires formées lors de la radiolyse de l'eau. Le plasmide pBS, à différentes concentrations, a été ensuite exposé aux radiations γ. Le taux de cassures décroit lorsque la concentration en ADN croit, suggérant une diminution du nombre de radicaux pouvant efficacement réagir avec l'ADN. Cet effet a également été mis en évidence lors d'une irradiation avec des particules de TEL élevé. En conclusion, l'accessibilité de l'ADN est un paramètre- clé dans la formation des dommages, tant in vitro que in vivo.

  16. Bacteriophage lambda-based expression vectors.

    PubMed

    Christensen, A C

    2001-03-01

    Bacteriophage lambda has been in use as a cloning vector for over 25 years, and has been used extensively as an expression vector. The efficiency of packaging and infection, and the simplicity of plaque screening are advantages of lambda as a cloning vector. A number of ingenious modifications help overcome the disadvantages associated with its mode of growth and its size. Some lambda vectors have been designed to be readily converted into plasmids or phagemids, and there are a variety of promoters and fusions that can be used to drive expression of foreign genes. Screening lambda libraries with antibodies or ligands is a powerful way of identifying novel genes. PMID:11434310

  17. Anion exchange purification of plasmid DNA using expanded bed adsorption.

    PubMed

    Ferreira, G N; Cabral, J M; Prazeres, D M

    2000-01-01

    Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity of process streams is of major concern in the purification of plasmids, since it can cause high back pressures in column operations, thus limiting the throughput. In order to avoid these high back pressures, expanded bed anion exchange chromatography was evaluated as an alternative to fixed bed chromatography. A Streamline 25 column filled with 100 ml of Streamline QXL media, was equilibrated with 0.5 M NaCl in TE (10 mM Tris, 1 mM EDTA, pH = 8.0) buffer at an upward flow of 300 cmh-1, E. coli lysates (obtained from up to 3 liters of fermentation broth) were injected in the column. After washing out the unbound material, the media was allowed to sediment and the plasmid was eluted with 1 M NaCl in TE buffer at a downward flow of 120 cmh-1. Purification factors of 36 +/- 1 fold, 26 +/- 0.4 plasmid purity, and close to 100% yields were obtained when less than one settled column volume of plasmid feed was injected. However, both recovery yield and purity abruptly decreased when larger amounts were processed-values of 35 +/- 2 and 5 +/- 0.7 were obtained for the recovery yield and purity, respectively, when 250 ml of feedstock were processed. In these cases, gel clogging and expansion collapse were observed. The processing of larger volumes, thus larger plasmid quantities, was only possible by performing an isopropanol precipitation step prior to the chromatographic step. This step led to an enhancement of the purification step.

  18. Plasmids of Carotenoid-Producing Paracoccus spp. (Alphaproteobacteria) - Structure, Diversity and Evolution

    PubMed Central

    Maj, Anna; Dziewit, Lukasz; Czarnecki, Jakub; Wlodarczyk, Miroslawa; Baj, Jadwiga; Skrzypczyk, Grazyna; Giersz, Dorota; Bartosik, Dariusz

    2013-01-01

    Plasmids are components of many bacterial genomes. They enable the spread of a large pool of genetic information via lateral gene transfer. Many bacterial strains contain mega-sized replicons and these are particularly common in Alphaproteobacteria. Considerably less is known about smaller alphaproteobacterial plasmids. We analyzed the genomes of 14 such plasmids residing in 4 multireplicon carotenoid-producing strains of the genus Paracoccus (Alphaproteobacteria): P. aestuarii DSM 19484, P. haeundaensis LG P-21903, P. marcusii DSM 11574 and P. marcusii OS22. Comparative analyses revealed mosaic structures of the plasmids and recombinational shuffling of diverse genetic modules involved in (i) plasmid replication, (ii) stabilization (including toxin-antitoxin systems of the relBE/parDE, tad-ata, higBA, mazEF and toxBA families) and (iii) mobilization for conjugal transfer (encoding relaxases of the MobQ, MobP or MobV families). A common feature of the majority of the plasmids is the presence of AT-rich sequence islets (located downstream of exc1-like genes) containing genes, whose homologs are conserved in the chromosomes of many bacteria (encoding e.g. RelA/SpoT, SMC-like proteins and a retron-type reverse transcriptase). The results of this study have provided insight into the diversity and plasticity of plasmids of Paracoccus spp., and of the entire Alphaproteobacteria. Some of the identified plasmids contain replication systems not described previously in this class of bacteria. The composition of the plasmid genomes revealed frequent transfer of chromosomal genes into plasmids, which significantly enriches the pool of mobile DNA that can participate in lateral transfer. Many strains of Paracoccus spp. have great biotechnological potential, and the plasmid vectors constructed in this study will facilitate genetic studies of these bacteria. PMID:24260361

  19. Plasmids of carotenoid-producing Paracoccus spp. (Alphaproteobacteria) - structure, diversity and evolution.

    PubMed

    Maj, Anna; Dziewit, Lukasz; Czarnecki, Jakub; Wlodarczyk, Miroslawa; Baj, Jadwiga; Skrzypczyk, Grazyna; Giersz, Dorota; Bartosik, Dariusz

    2013-01-01

    Plasmids are components of many bacterial genomes. They enable the spread of a large pool of genetic information via lateral gene transfer. Many bacterial strains contain mega-sized replicons and these are particularly common in Alphaproteobacteria. Considerably less is known about smaller alphaproteobacterial plasmids. We analyzed the genomes of 14 such plasmids residing in 4 multireplicon carotenoid-producing strains of the genus Paracoccus (Alphaproteobacteria): P. aestuarii DSM 19484, P. haeundaensis LG P-21903, P. marcusii DSM 11574 and P. marcusii OS22. Comparative analyses revealed mosaic structures of the plasmids and recombinational shuffling of diverse genetic modules involved in (i) plasmid replication, (ii) stabilization (including toxin-antitoxin systems of the relBE/parDE, tad-ata, higBA, mazEF and toxBA families) and (iii) mobilization for conjugal transfer (encoding relaxases of the MobQ, MobP or MobV families). A common feature of the majority of the plasmids is the presence of AT-rich sequence islets (located downstream of exc1-like genes) containing genes, whose homologs are conserved in the chromosomes of many bacteria (encoding e.g. RelA/SpoT, SMC-like proteins and a retron-type reverse transcriptase). The results of this study have provided insight into the diversity and plasticity of plasmids of Paracoccus spp., and of the entire Alphaproteobacteria. Some of the identified plasmids contain replication systems not described previously in this class of bacteria. The composition of the plasmid genomes revealed frequent transfer of chromosomal genes into plasmids, which significantly enriches the pool of mobile DNA that can participate in lateral transfer. Many strains of Paracoccus spp. have great biotechnological potential, and the plasmid vectors constructed in this study will facilitate genetic studies of these bacteria.

  20. Production of lentiviral vectors

    PubMed Central

    Merten, Otto-Wilhelm; Hebben, Matthias; Bovolenta, Chiara

    2016-01-01

    Lentiviral vectors (LV) have seen considerably increase in use as gene therapy vectors for the treatment of acquired and inherited diseases. This review presents the state of the art of the production of these vectors with particular emphasis on their large-scale production for clinical purposes. In contrast to oncoretroviral vectors, which are produced using stable producer cell lines, clinical-grade LV are in most of the cases produced by transient transfection of 293 or 293T cells grown in cell factories. However, more recent developments, also, tend to use hollow fiber reactor, suspension culture processes, and the implementation of stable producer cell lines. As is customary for the biotech industry, rather sophisticated downstream processing protocols have been established to remove any undesirable process-derived contaminant, such as plasmid or host cell DNA or host cell proteins. This review compares published large-scale production and purification processes of LV and presents their process performances. Furthermore, developments in the domain of stable cell lines and their way to the use of production vehicles of clinical material will be presented. PMID:27110581

  1. [Chromatographic separation of plasmid DNA by anion-exchange cryogel].

    PubMed

    Guo, Yantao; Shen, Shaochuan; Yun, Junxian; Yao, Kejian

    2012-08-01

    Plasmid DNA (pDNA) is used as an important vector for gene therapy, and its wide application is restricted by the purity and yield. To obtain high-purity pDNA, a chromatographic method based on anion-exchange supermacroporous cryogel was explored. The anion-exchange cryogel was prepared by grafting diethylaminoethyl-dextran to the epoxide groups of polyacrylamide-based matrix and pUC19 plasmid was used as a target to test the method. The plasmid was transferred into Escherichia coli DH5alpha, cultivated, harvested and lysed. The obtained culture was centrifuged and the supernatant was used as the plasmid feedstock, which was loaded into the anion-exchange cryogel bed for chromatographic separation. By optimizing the pH of running buffer and the elution conditions, high-purity pDNA was obtained by elution with 0.5 mol/L sodium chloride solution at pH 6.6. Compared to the traditional methods for purification of pDNA, animal source enzymes and toxic reagents were not involved in the present separation process, ensuring the safety of both the purification operations and the obtained pDNA.

  2. [Chromatographic separation of plasmid DNA by anion-exchange cryogel].

    PubMed

    Guo, Yantao; Shen, Shaochuan; Yun, Junxian; Yao, Kejian

    2012-08-01

    Plasmid DNA (pDNA) is used as an important vector for gene therapy, and its wide application is restricted by the purity and yield. To obtain high-purity pDNA, a chromatographic method based on anion-exchange supermacroporous cryogel was explored. The anion-exchange cryogel was prepared by grafting diethylaminoethyl-dextran to the epoxide groups of polyacrylamide-based matrix and pUC19 plasmid was used as a target to test the method. The plasmid was transferred into Escherichia coli DH5alpha, cultivated, harvested and lysed. The obtained culture was centrifuged and the supernatant was used as the plasmid feedstock, which was loaded into the anion-exchange cryogel bed for chromatographic separation. By optimizing the pH of running buffer and the elution conditions, high-purity pDNA was obtained by elution with 0.5 mol/L sodium chloride solution at pH 6.6. Compared to the traditional methods for purification of pDNA, animal source enzymes and toxic reagents were not involved in the present separation process, ensuring the safety of both the purification operations and the obtained pDNA. PMID:23185899

  3. An improved method for including upper size range plasmids in metamobilomes.

    PubMed

    Norman, Anders; Riber, Leise; Luo, Wenting; Li, Li Li; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    2014-01-01

    Two recently developed isolation methods have shown promise when recovering pure community plasmid DNA (metamobilomes/plasmidomes), which is useful in conducting culture-independent investigations into plasmid ecology. However, both methods employ multiple displacement amplification (MDA) to ensure suitable quantities of plasmid DNA for high-throughput sequencing. This study demonstrates that MDA greatly favors smaller circular DNA elements (<10 Kbp), which, in turn, leads to stark underrepresentation of upper size range plasmids (>10 Kbp). Throughout the study, we used two model plasmids, a 4.4 Kbp cloning vector (pBR322), and a 56 Kbp conjugative plasmid (pKJK10), to represent lower- and upper plasmid size ranges, respectively. Subjecting a mixture of these plasmids to the overall isolation protocol revealed a 34-fold over-amplification of pBR322 after MDA. To address this bias, we propose the addition of an electroelution step that separates different plasmid size ranges prior to MDA in order to reduce size-dependent competition during incubation. Subsequent analyses of metamobilome data from wastewater spiked with the model plasmids showed in silica recovery of pKJK10 to be very poor with the established method and a 1,300-fold overrepresentation of pBR322. Conversely, complete recovery of pKJK10 was enabled with the new modified protocol although considerable care must be taken during electroelution to minimize cross-contamination between samples. For further validation, non-spiked wastewater metamobilomes were mapped to more than 2,500 known plasmid genomes. This displayed an overall recovery of plasmids well into the upper size range (median size: 30 kilobases) with the modified protocol. Analysis of de novo assembled metamobilome data also suggested distinctly better recovery of larger plasmids, as gene functions associated with these plasmids, such as conjugation, was exclusively encoded in the data output generated through the modified protocol. Thus

  4. Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a differ...

  5. Data mining methods in the prediction of Dementia: A real-data comparison of the accuracy, sensitivity and specificity of linear discriminant analysis, logistic regression, neural networks, support vector machines, classification trees and random forests

    PubMed Central

    2011-01-01

    Background Dementia and cognitive impairment associated with aging are a major medical and social concern. Neuropsychological testing is a key element in the diagnostic procedures of Mild Cognitive Impairment (MCI), but has presently a limited value in the prediction of progression to dementia. We advance the hypothesis that newer statistical classification methods derived from data mining and machine learning methods like Neural Networks, Support Vector Machines and Random Forests can improve accuracy, sensitivity and specificity of predictions obtained from neuropsychological testing. Seven non parametric classifiers derived from data mining methods (Multilayer Perceptrons Neural Networks, Radial Basis Function Neural Networks, Support Vector Machines, CART, CHAID and QUEST Classification Trees and Random Forests) were compared to three traditional classifiers (Linear Discriminant Analysis, Quadratic Discriminant Analysis and Logistic Regression) in terms of overall classification accuracy, specificity, sensitivity, Area under the ROC curve and Press'Q. Model predictors were 10 neuropsychological tests currently used in the diagnosis of dementia. Statistical distributions of classification parameters obtained from a 5-fold cross-validation were compared using the Friedman's nonparametric test. Results Press' Q test showed that all classifiers performed better than chance alone (p < 0.05). Support Vector Machines showed the larger overall classification accuracy (Median (Me) = 0.76) an area under the ROC (Me = 0.90). However this method showed high specificity (Me = 1.0) but low sensitivity (Me = 0.3). Random Forest ranked second in overall accuracy (Me = 0.73) with high area under the ROC (Me = 0.73) specificity (Me = 0.73) and sensitivity (Me = 0.64). Linear Discriminant Analysis also showed acceptable overall accuracy (Me = 0.66), with acceptable area under the ROC (Me = 0.72) specificity (Me = 0.66) and sensitivity (Me = 0.64). The remaining classifiers showed

  6. Computer Program For Linear Algebra

    NASA Technical Reports Server (NTRS)

    Krogh, F. T.; Hanson, R. J.

    1987-01-01

    Collection of routines provided for basic vector operations. Basic Linear Algebra Subprogram (BLAS) library is collection from FORTRAN-callable routines for employing standard techniques to perform basic operations of numerical linear algebra.

  7. Effect of the atmospheric pressure nonequilibrium plasmas on the conformational changes of plasmid DNA

    SciTech Connect

    Yan Xu; He Guangyuan; Shi Mengjun; Gao Xuan; Li Yin; Ma Fengyun; Yu Men; Wang Changdong; Wang Yuesheng; Yang Guangxiao; Zou Fei; Lu Xinpei; Xiong Qing; Xiong Zilan

    2009-08-24

    The cold atmospheric pressure plasma, which has been widely used for biomedical applications, may potentially affect the conformation of DNA. In this letter, an atmospheric pressure plasma plume is used to investigate its effects on the conformational changes of DNA of plasmid pAHC25. It is found that the plasma plume could cause plasmid DNA topology alteration, resulting in the percentage of the supercoiled plasmid DNA form decreased while that of the open circular and linearized form of plasmid DNA increased as detected by agrose gel electrophoresis. On the other hand, further investigation by using polymerase chain reaction method shows that the atmospheric pressure plasma jet treatments under proper conditions does not affect the genes of the plasmid DNA, which may have potential application in increasing the transformation frequency by genetic engineering.

  8. Prevalence and significance of plasmid maintenance functions in the virulence plasmids of pathogenic bacteria.

    PubMed

    Sengupta, Manjistha; Austin, Stuart

    2011-07-01

    Virulence functions of pathogenic bacteria are often encoded on large extrachromosomal plasmids. These plasmids are maintained at low copy number to reduce the metabolic burden on their host. Low-copy-number plasmids risk loss during cell division. This is countered by plasmid-encoded systems that ensure that each cell receives at least one plasmid copy. Plasmid replication and recombination can produce plasmid multimers that hinder plasmid segregation. These are removed by multimer resolution systems. Equitable distribution of the resulting monomers to daughter cells is ensured by plasmid partition systems that actively segregate plasmid copies to daughter cells in a process akin to mitosis in higher organisms. Any plasmid-free cells that still arise due to occasional failures of replication, multimer resolution, or partition are eliminated by plasmid-encoded postsegregational killing systems. Here we argue that all of these three systems are essential for the stable maintenance of large low-copy-number plasmids. Thus, they should be found on all large virulence plasmids. Where available, well-annotated sequences of virulence plasmids confirm this. Indeed, virulence plasmids often appear to contain more than one example conforming to each of the three system classes. Since these systems are essential for virulence, they can be regarded as ubiquitous virulence factors. As such, they should be informative in the search for new antibacterial agents and drug targets.

  9. Supercoiling, knotting and replication fork reversal in partially replicated plasmids

    PubMed Central

    Olavarrieta, L.; Martínez-Robles, M. L.; Sogo, J. M.; Stasiak, A.; Hernández, P.; Krimer, D. B.; Schvartzman, J. B.

    2002-01-01

    To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18. The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy. Replication forks stop at the Ter–TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.26 times the mass of non-replicating plasmids for pBR18-TerE@StyI and 1.57 times for pBR18-TerE@EcoRI. The number of knotted bubbles detected after digestion with ScaI and the number and electrophoretic mobility of undigested partially replicated topoisomers reflect the changes in plasmid topology that occur in DNA molecules replicated to different extents. Exposure to increasing concentrations of chloroquine or ethidium bromide revealed that partially replicated topoisomers (CCCRIs) do not sustain positive supercoiling as efficiently as their non-replicating counterparts. It was suggested that this occurs because in partially replicated plasmids a positive ΔLk is absorbed by regression of the replication fork. Indeed, we showed by electron microscopy that, at least in the presence of chloroquine, some of the CCCRIs of pBR18-Ter@StyI formed Holliday-like junction structures characteristic of reversed forks. However, not all the positive supercoiling was absorbed by fork reversal in the presence of high concentrations of ethidium bromide. PMID:11809877

  10. Construction of three new Gateway® expression plasmids for Trypanosoma cruzi.

    PubMed

    Alonso, Victoria L; Ritagliati, Carla; Cribb, Pamela; Serra, Esteban C

    2014-12-01

    We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX). PMID:25424446

  11. Construction of three new Gateway® expression plasmids for Trypanosoma cruzi

    PubMed Central

    Alonso, Victoria L; Ritagliati, Carla; Cribb, Pamela; Serra, Esteban C

    2014-01-01

    We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX). PMID:25424446

  12. CpG-free plasmid expression cassettes for cystic fibrosis gene therapy.

    PubMed

    Pringle, Ian A; Hyde, Stephen C; Connolly, Mary M; Lawton, Anna E; Xu, Bihui; Nunez-Alonso, Graciela; Davies, Lee A; Sumner-Jones, Stephanie G; Gill, Deborah R

    2012-10-01

    Clinical studies are underway for the aerosol delivery of plasmid DNA complexed with Genzyme Lipid GL67A to the lungs of patients with cystic fibrosis (CF). Plasmid vectors contain several functional elements all of which play a role in determining the efficacy of the final clinical product. To optimise the final plasmid, variations of CpG-free 5' enhancer elements and 3'UTR regions were inserted into a common CpG-free, plasmid backbone containing Luciferase or CFTR transgenes. Plasmids were compared in immortalised cell culture, human airway liquid interface primary cell cultures, and mouse lung models to determine which design directed optimal transgene expression. Following aerosol delivery to mouse lung, plasmids containing the murine CMV enhancer showed higher peak Luciferase activity than the human CMV enhancer, but the human version resulted in persistent expression. In cell culture, the SV40 3'UTR and a novel BGH2 3'UTR exhibited up to 20-fold higher Luciferase activity than the commonly used BGH 3'UTR, but in mouse lung aerosol studies the activity and duration was greater for BGH 3'UTR. Systematic evaluation of each functional component of the plasmid has resulted in an improved design, exhibiting superior levels and duration of lung gene expression. PMID:22727465

  13. Identification and characterization of plasmids from the western aster yellows mycoplasmalike organism.

    PubMed Central

    Kuske, C R; Kirkpatrick, B C

    1990-01-01

    Supercoiled double-stranded DNA molecules (plasmids) were isolated from plants infected with three laboratory strains of western aster yellows mycoplasma-like organism (AY-MLO) by using cesium chloride-ethidium bromide density gradients. Southern blot analysis, using plasmids from the severe strain of AY-MLO (SAY-MLO) as the probe, identified at least four plasmids in celery, aster, and periwinkle plants and in Macrosteles severini leafhopper vectors infected with either the dwarf AY-MLO, Tulelake AY-MLO, or SAY-MLO strain. Plasmids were also detected in two California field isolates of AY-MLO but not in plants infected with the beet leafhopper-transmitted virescence agent, western X, or elm yellows MLOs. SAY-MLO plasmids were 5.2, 4.9, 3.4, and 1.7 kilobase pairs in size. Plasmids isolated from dwarf AY- and Tulelake AY-MLOs were 7.4, 5.1, 3.5, and 1.7 kilobase pairs in size. No evidence was obtained for integration of SAY-MLO plasmids into the MLO chromosome. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5 PMID:2307660

  14. Animal-free production of ccc-supercoiled plasmids for research and clinical applications.

    PubMed

    Schleef, Martin; Schmidt, Torsten

    2004-02-01

    The topological structure of plasmid DNA can be characterized by capillary gel electrophoresis (CGE analysis)-an important tool for quality control and stability assessments in DNA storage or application. Hence, a large-scale manufacturing process was developed that allows the removal of undesired open circular (oc) or linear plasmid topologies, bacterial genomic DNA, RNA, proteins as well as lipopolysaccharides (endotoxins) and results in obtaining supercoiled (covalently closed circular, ccc) plasmid DNA in a pure form without using any animal-derived substances. Using CGE, the development and in-line monitoring for pharmaceutical plasmid production starting from fermentation control throughout the whole manufacturing process including the formulated and filled product can be performed the first time in a way conforming to good manufacturing practices (GMP). Plasmid stability data were obtained from analysis of shear effects influencing the plasmid quality in DNA drug delivery formulation and application (e.g. gene gun or jet injection). The physical stability of plasmid DNA is for the first time evaluated in DNA storage experiments on the level of different plasmid forms.

  15. Origin and Evolution of Rickettsial Plasmids

    PubMed Central

    El Karkouri, Khalid; Pontarotti, Pierre; Raoult, Didier; Fournier, Pierre-Edouard

    2016-01-01

    Background Rickettsia species are strictly intracellular bacteria that have undergone a reductive genomic evolution. Despite their allopatric lifestyle, almost half of the 26 currently validated Rickettsia species have plasmids. In order to study the origin, evolutionary history and putative roles of rickettsial plasmids, we investigated the evolutionary processes that have shaped 20 plasmids belonging to 11 species, using comparative genomics and phylogenetic analysis between rickettsial, microbial and non-microbial genomes. Results Plasmids were differentially present among Rickettsia species. The 11 species had 1 to 4 plasmid (s) with a size ranging from 12 kb to 83 kb. We reconstructed pRICO, the last common ancestor of the current rickettsial plasmids. pRICO was vertically inherited mainly from Rickettsia/Orientia chromosomes and diverged vertically into a single or multiple plasmid(s) in each species. These plasmids also underwent a reductive evolution by progressive gene loss, similar to that observed in rickettsial chromosomes, possibly leading to cryptic plasmids or complete plasmid loss. Moreover, rickettsial plasmids exhibited ORFans, recent gene duplications and evidence of horizontal gene transfer events with rickettsial and non-rickettsial genomes mainly from the α/γ-proteobacteria lineages. Genes related to maintenance and plasticity of plasmids, and to adaptation and resistance to stress mostly evolved under vertical and/or horizontal processes. Those involved in nucleotide/carbohydrate transport and metabolism were under the influence of vertical evolution only, whereas genes involved in cell wall/membrane/envelope biogenesis, cycle control, amino acid/lipid/coenzyme and secondary metabolites biosynthesis, transport and metabolism underwent mainly horizontal transfer events. Conclusion Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via

  16. Phytoplasma PMU1 exists as linear chromosomal and circular extrachromosomal elements and has enhanced expression in insect vectors compared with plant hosts.

    PubMed

    Toruño, Tania Y; Musić, Martina Seruga; Simi, Silvia; Nicolaisen, Mogens; Hogenhout, Saskia A

    2010-09-01

    Phytoplasmas replicate intracellularly in plants and insects and are dependent on both hosts for dissemination in nature. Phytoplasmas have small genomes lacking genes for major metabolic pathways. Nevertheless, their genomes harbour multicopy gene clusters that were named potential mobile units (PMUs). PMU1 is the largest most complete repeat among the PMUs in the genome of Aster Yellows phytoplasma strain Witches' Broom (AY-WB). PMU1 is c. 20 kb in size and contains 21 genes encoding DNA replication and predicted membrane-targeted proteins. Here we show that AY-WB has a chromosomal linear PMU1 (L-PMU1) and an extrachromosomal circular PMU1 (C-PMU1). The C-PMU1 copy number was consistently higher by in average approximately fivefold in insects compared with plants and PMU1 gene expression levels were also considerably higher in insects indicating that C-PMU1 synthesis and expression are regulated. We found that the majority of AY-WB virulence genes lie on chromosomal PMU regions that have similar gene content and organization as PMU1 providing evidence that PMUs contribute to phytoplasma host adaptation and have integrated into the AY-WB chromosome.

  17. Proteomic profiling of salivary gland after nonviral gene transfer mediated by conventional plasmids and minicircles

    PubMed Central

    Geguchadze, Ramaz; Wang, Zhimin; Zourelias, Lee; Perez-Riveros, Paola; Edwards, Paul C; Machen, Laurie; Passineau, Michael J

    2014-01-01

    In this study, we compared gene transfer efficiency and host response to ultrasound-assisted, nonviral gene transfer with a conventional plasmid and a minicircle vector in the submandibular salivary glands of mice. Initially, we looked at gene transfer efficiency with equimolar amounts of the plasmid and minicircle vectors, corroborating an earlier report showing that minicircle is more efficient in the context of a physical method of gene transfer. We then sought to characterize the physiological response of the salivary gland to exogenous gene transfer using global proteomic profiling. Somewhat surprisingly, we found that sonoporation alone, without a gene transfer vector present, had virtually no effect on the salivary gland proteome. However, when a plasmid vector was used, we observed profound perturbations of the salivary gland proteome that compared in magnitude to that seen in a previous report after high doses of adeno-associated virus. Finally, we found that gene transfer with a minicircle induces only minor proteomic alterations that were similar to sonoporation alone. Using mass spectrometry, we assigned protein IDs to 218 gel spots that differed between plasmid and minicircle. Bioinformatic analysis of these proteins demonstrated convergence on 68 known protein interaction pathways, most notably those associated with innate immunity, cellular stress, and morphogenesis. PMID:25414909

  18. Plasmids containing the gene for DNA polymerase I from Streptococcus pneumoniae

    DOEpatents

    Lacks, S.A.; Martinez, S.; Lopez, P.; Espinosa, M.

    1987-08-28

    A method is disclosed for cloning the gene which encodes a DNA polymerase-exonuclease of /und Streptococcus/ /und pneumoniae/. Plasmid pSM22, the vector containing the pneumococcal polA gene, facilitates the expression of 50-fold greater amounts of the PolI enzyme. 1 fig., 1 tab.

  19. Plasmids containing the gene for DNA polymerase I from Streptococcus pneumoniae

    DOEpatents

    Lacks, S.A.; Martinez, S.; Lopez, P.; Espinosa, M.

    1991-03-26

    A method is disclosed for cloning the gene which encodes a DNA polymerase-exonuclease of Streptococcus pneumoniae. Plasmid pSM22, the vector containing the pneumocccal polA gene, facilitates the expression of 50-fold greater amounts of the PolI enzyme. 1 figure.

  20. Facile Recovery of Individual High-Molecular-Weight, Low-Copy-Number Natural Plasmids for Genomic Sequencing

    SciTech Connect

    Williams, L.E.; Detter, C,; Barrie, K.; Lapidus, A.; Summers, A.O.

    2006-06-01

    Sequencing of the large (>50 kb), low-copy-number (<5 per cell) plasmids that mediate horizontal gene transfer has been hindered by the difficulty and expense of isolating DNA from individual plasmids of this class. We report here that a kit method previously devised for purification of bacterial artificial chromosomes (BACs) can be adapted for effective preparation of individual plasmids up to 220 kb from wild gram-negative and gram-positive bacteria. Individual plasmid DNA recovered from less than 10 ml of Escherichia coli, Staphylococcus, and Corynebacterium cultures was of sufficient quantity and quality for construction of highcoverage libraries, as shown by sequencing five native plasmids ranging in size from 30 kb to 94 kb. We also report recommendations for vector screening to optimize plasmid sequence assembly, preliminary annotation of novel plasmid genomes, and insights on mobile genetic element biology derived from these sequences. Adaptation of this BAC method for large plasmid isolation removes one major technical hurdle to expanding our knowledge of the natural plasmid gene pool.

  1. Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community

    PubMed Central

    Cormier, Catherine Y.; Mohr, Stephanie E.; Zuo, Dongmei; Hu, Yanhui; Rolfs, Andreas; Kramer, Jason; Taycher, Elena; Kelley, Fontina; Fiacco, Michael; Turnbull, Greggory; LaBaer, Joshua

    2010-01-01

    The Protein Structure Initiative Material Repository (PSI-MR; http://psimr.asu.edu) provides centralized storage and distribution for the protein expression plasmids created by PSI researchers. These plasmids are a resource that allows the research community to dissect the biological function of proteins whose structures have been identified by the PSI. The plasmid annotation, which includes the full length sequence, vector information and associated publications, is stored in a freely available, searchable database called DNASU (http://dnasu.asu.edu). Each PSI plasmid is also linked to a variety of additional resources, which facilitates cross-referencing of a particular plasmid to protein annotations and experimental data. Plasmid samples can be requested directly through the website. We have also developed a novel strategy to avoid the most common concern encountered when distributing plasmids namely, the complexity of material transfer agreement (MTA) processing and the resulting delays this causes. The Expedited Process MTA, in which we created a network of institutions that agree to the terms of transfer in advance of a material request, eliminates these delays. Our hope is that by creating a repository of expression-ready plasmids and expediting the process for receiving these plasmids, we will help accelerate the accessibility and pace of scientific discovery. PMID:19906724

  2. Conditional gene vectors regulated in cis

    PubMed Central

    Pich, Dagmar; Humme, Sibille; Spindler, Mark-Peter; Schepers, Aloys; Hammerschmidt, Wolfgang

    2008-01-01

    Non-integrating gene vectors, which are stably and extrachromosomally maintained in transduced cells would be perfect tools to support long-term expression of therapeutic genes but preserve the genomic integrity of the cellular host. Small extrachromosomal plasmids share some of these ideal characteristics but are primarily based on virus blueprints. These plasmids are dependent on viral trans-acting factors but they can replicate their DNA molecules in synchrony with the chromosome of the cellular host and segregate to daughter cells in an autonomous fashion. On the basis of the concept of the latent origin of DNA replication of Epstein-Barr virus, oriP, we devised novel derivatives, which exclusively rely on an artificial replication factor for both nuclear retention and replication of plasmid DNA. In addition, an allosteric switch regulates the fate of the plasmid molecules, which are rapidly lost upon addition of doxycycline. Conditional maintenance of these novel plasmid vectors allows the reversible transfer of genetic information into target cells for the first time. PMID:18566006

  3. Improving DNA vaccine performance through vector design.

    PubMed

    Williams, James A

    2014-01-01

    DNA vaccines are a rapidly deployed next generation vaccination platform for treatment of human and animal disease. DNA delivery devices, such as electroporation and needle free jet injectors, are used to increase gene transfer. This results in higher antigen expression which correlates with improved humoral and cellular immunity in humans and animals. This review highlights recent vector and transgene design innovations that improve DNA vaccine performance. These new vectors improve antigen expression, increase plasmid manufacturing yield and quality in bioreactors, and eliminate antibiotic selection and other potential safety issues. A flowchart for designing synthetic antigen transgenes, combining antigen targeting, codon-optimization and bioinformatics, is presented. Application of improved vectors, of antibiotic free plasmid production, and cost effective manufacturing technologies will be critical to ensure safety, efficacy, and economically viable manufacturing of DNA vaccines currently under development for infectious disease, cancer, autoimmunity, immunotolerance and allergy indications.

  4. Plasmids as stochastic model systems

    NASA Astrophysics Data System (ADS)

    Paulsson, Johan

    2003-05-01

    Plasmids are self-replicating gene clusters present in on average 2-100 copies per bacterial cell. To reduce random fluctuations and thereby avoid extinction, they ubiquitously autoregulate their own synthesis using negative feedback loops. Here I use van Kampen's Ω-expansion for a two-dimensional model of negative feedback including plasmids and ther replication inhibitors. This analytically summarizes the standard perspective on replication control -- including the effects of sensitivity amplification, exponential time-delays and noisy signaling. I further review the two most common molecular sensitivity mechanisms: multistep control and cooperativity. Finally, I discuss more controversial sensitivity schemes, such as noise-enhanced sensitivity, the exploitation of small-number combinatorics and double-layered feedback loops to suppress noise in disordered environments.

  5. Transformation of haploid Datura innoxia protoplasts and analysis of the plasmid integration pattern in regenerated transgenic plants.

    PubMed

    Schmidt-Rogge, T; Meixner, M; Srivastava, V; Guha-Mukherjee, S; Schieder, O

    1993-05-01

    We developed a highly efficient transformation protocol for the PEG-mediated direct transfer of plasmid DNA into protoplasts of haploid Datura innoxia. Vectors harbouring a neomycin phosphotransferase II gene or a hygromycin B phosphotransferase gene under the control of different promoters were used in the transformation experiments. Various amounts of plasmid DNA were applied without any carrier DNA to show the direct influence of the plasmid DNA concentration on the transformation efficiency. Approximately 95% of the selected calli were regenerated to plants; 20% of them remained haploid. Total DNA of different transgenic plants was analysed with regard to the integration pattern of the plasmid DNA. Plants carrying only one or two copies of the vector DNA were observed as well as individuals with multi-copy integration (up to ten or more copies).

  6. Gyromagnetic gs factors of the spin-1/2 particles in the (1/2+-1/2--3/2-) triad of the four-vector spinor, ψμ, irreducibility and linearity

    NASA Astrophysics Data System (ADS)

    Delgado Acosta, E. G.; Banda Guzmán, V. M.; Kirchbach, M.

    2015-07-01

    The gauged Klein-Gordon equation, extended by a gsσμνFμν/4 interaction, the contraction of the electromagnetic field strength tensor, Fμν, with the generators, σμν/2, of the Lorentz group in (1/2, 0) ⊕ (0, 1/2), and gs being the gyromagnetic factor, is examined with the aim to find out as to what extent it qualifies as a wave equation for general relativistic spin-1/2 particles transforming as (1/2, 0) ⊕ (0, 1/2) and possibly distinct from the Dirac fermions. This equation can be viewed as the generalization of the gs = 2 case, known under the name of the Feynman-Gell-Mann equation, the only one which allows for a bilinearization into the gauged Dirac equation and its conjugate. At the same time, it is well-known a fact that a gs = 2 value can also be obtained upon the bilinearization of the nonrelativistic Schrödinger into nonrelativistic Pauli equations. The inevitable conclusion is that it must not be necessarily relativity which fixes the gyromagnetic factor of the electron to g(1/2) = 2, but rather the specific form of the primordial quadratic wave equation obeyed by it, that is amenable to a linearization. The fact is that space-time symmetries alone define solely the kinematic properties of the particles and neither fix the values of their interacting constants, nor do they necessarily prescribe linear Lagrangians. Information on such properties has to be obtained from additional physical inputs involving the dynamics. We here provide an example in support of the latter statement. Our case is that the spin-1/2- fermion residing within the four-vector spinor triad, ψμ (1/2+-1/2--3/2-), whose sectors at the free particle level are interconnected by spin-up and spin-down ladder operators, does not allow for a description within a linear framework at the interacting level. Upon gauging, despite transforming according to the irreducible (1/2, 1) ⊕ (1, 1/2) building block of ψμ, and being described by 16-dimensional four-vector spinors, though

  7. Factors affecting plasmid production in Escherichia coli from a resource allocation standpoint

    PubMed Central

    Cunningham, Drew S; Koepsel, Richard R; Ataai, Mohammad M; Domach, Michael M

    2009-01-01

    Background Plasmids are being reconsidered as viable vector alternatives to viruses for gene therapies and vaccines because they are safer, non-toxic, and simpler to produce. Accordingly, there has been renewed interest in the production of plasmid DNA itself as the therapeutic end-product of a bioprocess. Improvement to the best current yields and productivities of such emerging processes would help ensure economic feasibility on the industrial scale. Our goal, therefore, was to develop a stoichiometric model of Escherichia coli metabolism in order to (1) determine its maximum theoretical plasmid-producing capacity, and to (2) identify factors that significantly impact plasmid production. Results Such a model was developed for the production of a high copy plasmid under conditions of batch aerobic growth on glucose minimal medium. The objective of the model was to maximize plasmid production. By employing certain constraints and examining the resulting flux distributions, several factors were determined that significantly impact plasmid yield. Acetate production and constitutive expression of the plasmid's antibiotic resistance marker exert negative effects, while low pyruvate kinase (Pyk) flux and the generation of NADPH by transhydrogenase activity offer positive effects. The highest theoretical yield (592 mg/g) resulted under conditions of no marker or acetate production, nil Pyk flux, and the maximum allowable transhydrogenase activity. For comparison, when these four fluxes were constrained to wild-type values, yields on the order of tens of mg/g resulted, which are on par with the best experimental yields reported to date. Conclusion These results suggest that specific plasmid yields can theoretically reach 12 times their current experimental maximum (51 mg/g). Moreover, they imply that abolishing Pyk activity and/or transhydrogenase up-regulation would be useful strategies to implement when designing host strains for plasmid production; mutations that

  8. Targeting relaxase genes for classification of the predominant plasmids in Enterobacteriaceae.

    PubMed

    Compain, Fabrice; Poisson, Agathe; Le Hello, Simon; Branger, Catherine; Weill, François-Xavier; Arlet, Guillaume; Decré, Dominique

    2014-05-01

    Plasmids are the main vectors of antimicrobial drug resistance and virulence genes, especially in Enterobacteriaceae. Identification and classification of plasmids is essential for analysis of their distribution. The most widely used typing method is PCR-based replicon typing (PBRT). A new classification scheme based on relaxase gene typing has been described recently. We propose a practical application of this method, with the development of a multiplex PCR set targeting relaxase genes found on plasmids most frequently encountered in Enterobacteriaceae. This method, here called "plasmid relaxase gene typing" (PRaseT), was validated with 60 transconjugants and transformants harboring various replicon types. The method was tested with 39 multidrug-resistant clinical isolates including Escherichia coli, Klebsiella pneumoniae and Salmonella enterica subsp. enterica carrying 1-7 replicons as well as with 17 plasmids non-typeable using PBRT; all replicons were tested in parallel with PBRT for comparison. Six multiplex PCRs and one simplex PCR, including 24 pairs of primers, recognized plasmids of groups A/C, B/O, colE, FIA, FIB, FIC, FV, FIIk, HI1, HI2, I1, K, L/M, N, P1α, Q1, U, W, X1, X2, X3 and X4. There was perfect correlation between PRaseT and PBRT results in 31/39 (79.5%) clinical isolates. Moreover, 11/17 (64.7%) plasmids non-typeable by PBRT could be typed by PRaseT. Our set of multiplex PCRs showed high sensitivity and specificity for the classification of resistance plasmids. It has proved complementary to the widely used PBRT and will improve the monitoring of plasmid distribution in every-day practice.

  9. [The relationship of plasmids from environmental Yersinia isolates and the virulence plasmid of enteropathogenic Yersinia strains].

    PubMed

    Hoffmann, B; Strauch, E; Appel, B; Nattermann, H

    2002-01-01

    The human pathogenic strains of Yersinia harbour a conserved plasmid carrying the Yop virulon. The virulence plasmid of Yersinia enterocolitica strains belonging to the serogroups O:3 and O:9 were used as probes to detect homologous sequences in plasmids of "avirulent" Yersinia strains. "Avirulent" Yersinia strains (Y. enterocolitica biogroup 1A, Y. intermedia, Y. kristensenii and Y. frederiksenii) lack the virulence plasmid. They are widely distributed in the environment and can frequently be isolated from clinical samples. Hybridisation experiments revealed a number of common genetic elements of the virulence plasmid and the plasmids of "avirulent" Yersinia strains. These elements were identified as genes involved in plasmid replication, as an endonuclease gene and as mobile genetic elements. However, none of the plasmid encoded virulence genes was present in the plasmids of "avirulent" Yersinia strains. The frequent occurrence and the possible etiological relevance of "avirulent" isolates will be discussed.

  10. Plasmid Diversity and Adaptation Analyzed by Massive Sequencing of Escherichia coli Plasmids.

    PubMed

    de Toro, María; Garcilláon-Barcia, M Pilar; De La Cruz, Fernando

    2014-12-01

    Whole-genome sequencing is revolutionizing the analysis of bacterial genomes. It leads to a massive increase in the amount of available data to be analyzed. Bacterial genomes are usually composed of one main chromosome and a number of accessory chromosomes, called plasmids. A recently developed methodology called PLACNET (for plasmid constellation networks) allows the reconstruction of the plasmids of a given genome. Thus, it opens an avenue for plasmidome analysis on a global scale. This work reviews our knowledge of the genetic determinants for plasmid propagation (conjugation and related functions), their diversity, and their prevalence in the variety of plasmids found by whole-genome sequencing. It focuses on the results obtained from a collection of 255 Escherichia coli plasmids reconstructed by PLACNET. The plasmids found in E. coli represent a nonaleatory subset of the plasmids found in proteobacteria. Potential reasons for the prevalence of some specific plasmid groups will be discussed and, more importantly, additional questions will be posed.

  11. Virulence Plasmids of Spore-Forming Bacteria.

    PubMed

    Adams, Vicki; Li, Jihong; Wisniewski, Jessica A; Uzal, Francisco A; Moore, Robert J; McClane, Bruce A; Rood, Julian I

    2014-12-01

    Plasmid-encoded virulence factors are important in the pathogenesis of diseases caused by spore-forming bacteria. Unlike many other bacteria, the most common virulence factors encoded by plasmids in Clostridium and Bacillus species are protein toxins. Clostridium perfringens causes several histotoxic and enterotoxin diseases in both humans and animals and produces a broad range of toxins, including many pore-forming toxins such as C. perfringens enterotoxin, epsilon-toxin, beta-toxin, and NetB. Genetic studies have led to the determination of the role of these toxins in disease pathogenesis. The genes for these toxins are generally carried on large conjugative plasmids that have common core replication, maintenance, and conjugation regions. There is considerable functional information available about the unique tcp conjugation locus carried by these plasmids, but less is known about plasmid maintenance. The latter is intriguing because many C. perfringens isolates stably maintain up to four different, but closely related, toxin plasmids. Toxin genes may also be plasmid-encoded in the neurotoxic clostridia. The tetanus toxin gene is located on a plasmid in Clostridium tetani, but the botulinum toxin genes may be chromosomal, plasmid-determined, or located on bacteriophages in Clostridium botulinum. In Bacillus anthracis it is well established that virulence is plasmid determined, with anthrax toxin genes located on pXO1 and capsule genes on a separate plasmid, pXO2. Orthologs of these plasmids are also found in other members of the Bacillus cereus group such as B. cereus and Bacillus thuringiensis. In B. thuringiensis these plasmids may carry genes encoding one or more insecticidal toxins.

  12. Enhanced gene disruption by programmable nucleases delivered by a minicircle vector.

    PubMed

    Dad, A-B K; Ramakrishna, S; Song, M; Kim, H

    2014-11-01

    Targeted genetic modification using programmable nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) is of great value in biomedical research, medicine and biotechnology. Minicircle vectors, which lack extraneous bacterial sequences, have several advantages over conventional plasmids for transgene delivery. Here, for the first time, we delivered programmable nucleases into human cells using transient transfection of a minicircle vector and compared the results with those obtained using a conventional plasmid. Surrogate reporter assays and T7 endonuclease analyses revealed that cells in the minicircle vector group displayed significantly higher mutation frequencies at the target sites than those in the conventional plasmid group. Quantitative PCR and reverse transcription-PCR showed higher vector copy number and programmable nuclease transcript levels, respectively, in 293T cells after minicircle versus conventional plasmid vector transfection. In addition, tryphan blue staining and flow cytometry after annexin V and propidium iodide staining showed that cell viability was also significantly higher in the minicircle group than in the conventional plasmid group. Taken together, our results show that gene disruption using minicircle vector-mediated delivery of ZFNs and TALENs is a more efficient, safer and less toxic method than using a conventional plasmid, and indicate that the minicircle vector could serve as an advanced delivery method for programmable nucleases.

  13. The sex pheromone system of Enterococcus faecalis. More than just a plasmid-collection mechanism?

    PubMed

    Wirth, R

    1994-06-01

    The sex pheromone system of Enterococcus faecalis was discovered by observing a clumping reaction of E. faecalis strains during conjugative transfer of plasmids. It was found that only a special type of E. faecalis plasmids, the so-called sex pheromone plasmids, are transferred via this mechanism. Various experiments, especially by the group of D. B. Clewell, led to the formulation of a model describing how the sex pheromone system works. Small linear peptides, the so-called sex pheromones, are excreted by strains not possessing the corresponding sex pheromone plasmid. Donor strains harboring the plasmid do not produce the corresponding sex pheromone; they react to the presence of the peptide by production of a plasmid-encoded adhesin, the so-called aggregation substance. This adhesin allows contact between the non-motile mating partners; after conjugative transfer of the plasmid, the former recipient possesses and replicates the new plasmid. Thereby the population of E. faecalis strains is shifted to a high percentage of donor strains. This is especially true because a donor strain will still excrete sex pheromones corresponding to plasmids it does not harbor; therefore, such a strain can also function as recipient for other sex pheromone plasmids it does not possess. Various aspects of this unique plasmid collection mechanism have been studied during the last few years. The data indicate that, with the exception of pAM373, all sex pheromone plasmids possess one DNA region which is highly similar to and codes for the adhesin. It is also becoming more and more clear that regulatory functions/proteins are not conserved between different sex pheromone plasmids. Induction of adhesin synthesis needs the action of a regulatory cascade composed of unique features; at the moment we are just beginning to understand this cascade. By sequencing the first structural gene for one of those adhesins, we realized that the aggregation substance might act also as an adhesin for

  14. MEGAWHOP cloning: a method of creating random mutagenesis libraries via megaprimer PCR of whole plasmids.

    PubMed

    Miyazaki, Kentaro

    2011-01-01

    MEGAWHOP allows for the cloning of DNA fragments into a vector and is used for conventional restriction digestion/ligation-based procedures. In MEGAWHOP, the DNA fragment to be cloned is used as a set of complementary primers that replace a homologous region in a template vector through whole-plasmid PCR. After synthesis of a nicked circular plasmid, the mixture is treated with DpnI, a dam-methylated DNA-specific restriction enzyme, to digest the template plasmid. The DpnI-treated mixture is then introduced into competent Escherichia coli cells to yield plasmids carrying replaced insert fragments. Plasmids produced by the MEGAWHOP method are virtually free of contamination by species without any inserts or with multiple inserts, and also the parent. Because the fragment is usually long enough to not interfere with hybridization to the template, various types of fragments can be used with mutations at any site (either known or unknown, random, or specific). By using fragments having homologous sequences at the ends (e.g., adaptor sequence), MEGAWHOP can also be used to recombine nonhomologous sequences mediated by the adaptors, allowing rapid creation of novel constructs and chimeric genes. PMID:21601687

  15. Plasmid Conjugation from Proteobacteria as Evidence for the Origin of Xenologous Genes in Cyanobacteria

    PubMed Central

    Encinas, David; Garcillán-Barcia, M. Pilar; Santos-Merino, María; Delaye, Luis; Moya, Andrés

    2014-01-01

    Comparative genomics have shown that 5% of Synechococcus elongatus PCC 7942 genes are of probable proteobacterial origin. To investigate the role of interphylum conjugation in cyanobacterial gene acquisition, we tested the ability of a set of prototype proteobacterial conjugative plasmids (RP4, pKM101, R388, R64, and F) to transfer DNA from Escherichia coli to S. elongatus. A series of BioBrick-compatible, mobilizable shuttle vectors was developed. These vectors were based on the putative origin of replication of the Synechococcus resident plasmid pANL. Not only broad-host-range plasmids, such as RP4 and R388, but also narrower-host-range plasmids, such as pKM101, all encoding MPFT-type IV secretion systems, were able to transfer plasmid DNA from E. coli to S. elongatus by conjugation. Neither MPFF nor MPFI could be used as interphylum DNA delivery agents. Reciprocally, pANL-derived cointegrates could be introduced in E. coli by electroporation, where they conferred a functional phenotype. These results suggest the existence of potentially ample channels of gene flow between proteobacteria and cyanobacteria and point to MPFT-based interphylum conjugation as a potential mechanism to explain the proteobacterial origin of a majority of S. elongatus xenologous genes. PMID:24509315

  16. Reduction-responsive cross-linked stearyl peptide for effective delivery of plasmid DNA.

    PubMed

    Yao, Chong; Tai, Zongguang; Wang, Xiaoyu; Liu, Jiyong; Zhu, Quangang; Wu, Xin; Zhang, Lijuan; Zhang, Wei; Tian, Jing; Gao, Yuan; Gao, Shen

    2015-01-01

    Low efficiency and significant toxicity are the main obstacles to successful gene delivery. We have developed a cationic reduction-responsive vector based on a disulfide cross-linked stearylated polyarginine peptide modified with histidine (C-SHR) for DNA delivery. The structure of the C-SHR was characterized, and the in vitro and in vivo transfection efficiency and cytotoxicity of C-SHR/plasmid DNA complexes were examined. Compared with non-cross-linked stearylated polyarginine peptide (SHR), C-SHR increased the intracellular uptake and dissociation behavior of the complexes. In addition, the gene transfection efficiency of C-SHR/plasmid DNA complexes in HEK293 and HeLa cells was improved and was comparable with that of bPEI-25K/plasmid DNA complexes, and the cytotoxicity of C-SHR was significantly less than that of bPEI-25K. Importantly, the in vivo gene transfection efficiency of C-SHR/plasmid DNA complexes was five fold higher than that of SHR/plasmid DNA complexes, suggesting that C-SHR is an efficient non-viral vector for DNA delivery.

  17. Positive epistasis between co-infecting plasmids promotes plasmid survival in bacterial populations.

    PubMed

    San Millan, Alvaro; Heilbron, Karl; MacLean, R Craig

    2014-03-01

    Plasmids have a key role in the horizontal transfer of genes among bacteria. Although plasmids are catalysts for bacterial evolution, it is challenging to understand how they can persist in bacterial populations over the long term because of the burden they impose on their hosts (the 'plasmid paradox'). This paradox is especially perplexing in the case of 'small' plasmids, which are unable to self-transfer by conjugation. Here, for the first time, we investigate how interactions between co-infecting plasmids influence plasmid persistence. Using an experimental model system based on interactions between a diverse assemblage of 'large' plasmids and a single small plasmid, pNI105, in the pathogenic bacterium Pseudomonas aeruginosa, we demonstrate that positive epistasis minimizes the cost associated with carrying multiple plasmids over the short term and increases the stability of the small plasmid over a longer time scale. In support of these experimental data, bioinformatic analysis showed that associations between small and large plasmids are more common than would be expected owing to chance alone across a range of families of bacteria; more generally, we find that co-infection with multiple plasmids is more common than would be expected owing to chance across a wide range of bacterial phyla. Collectively, these results suggest that positive epistasis promotes plasmid stability in bacterial populations. These findings pave the way for future mechanistic studies aimed at elucidating the molecular mechanisms of plasmid-plasmid interaction, and evolutionary studies aimed at understanding how the coevolution of plasmids drives the spread of plasmid-encoded traits.

  18. Effects of genes exerting growth inhibition and plasmid stability on plasmid maintenance.

    PubMed

    Boe, L; Gerdes, K; Molin, S

    1987-10-01

    Plasmid stabilization mediated by the parA+ and parB+ genes of the R1 plasmid and the ccd+ and sop+ genes of the F plasmid was tested on a mini-R1 plasmid and a pBR322 plasmid derivative. The mini-R1 plasmid is thought to be unstably inherited owing to a low copy number and to random segregation of the plasmid at cell division, whereas cells harboring the pBR322 derivative used in this work are lost through competition with plasmid-free cells, mainly as a result of the shorter generation time of cells without plasmids. The pBR322 derivative carries a fusion between part of the atp operon of Escherichia coli and the bacteriophage lambda pR promoter, and the cI857 repressor gene. The insertion of sop+ from the F plasmid or parB+ from the R1 plasmid reduced the loss frequency by a factor of 10(3) for the pBR322 derivative and by at least a factor of 10(2) for the mini-R1 plasmid. Insertion of parA+ from the R1 plasmid decreased the loss frequency of the pBR322 derivative by a factor of 10 and that of the mini-R1 plasmid by a factor of 50. When ccd+ from the F plasmid was inserted, the loss frequency of the pBR322 derivative was decreased by a factor of 10, but it had only a marginal effect on the stability of the mini-R1 plasmid. In no case was any significant structural instability of the plasmids observed.

  19. Preparation and quantification of pseudotyped retroviral vector.

    PubMed

    Yu, Hong; Kwon, Young Jik

    2008-01-01

    Retroviral vectors have been widely used for research and clinical trials in gene therapy because of their high transduction efficiency. Retroviruses interact with target cells through their surface molecules (i.e., envelope proteins) and cellular receptors, which limit the susceptibility of target cells to retroviral vectors. Murine leukemia retrovirus (MuLV) pseudotyped with vesicular stomatitis virus G glycoprotein (VSV-G) overcomes the species barrier and is more resistant to mechanical and biochemical inactivation. A cell line producing VSV-G pseudotyped MuLV vector can be established by transfecting 293T cells expressing Gag, Pol, and VSV-G (293 GPG cell line) with a retroviral vector plasmid. Transduction potency of the resulting VSV-G pseudotyped MuLV retroviral supernatant can be quantified by titration, electron microscopy (EM), and the reverse transcriptase (RT) assay. These protocols provide methods to prepare and quantify a pseudotyped retroviral vector with high transduction rates for most types of target cells.

  20. The complete nucleotide sequence of a plant root-inducing (Ri) plasmid indicates its chimeric structure and evolutionary relationship between tumor-inducing (Ti) and symbiotic (Sym) plasmids in Rhizobiaceae.

    PubMed

    Moriguchi, K; Maeda, Y; Satou, M; Hardayani, N S; Kataoka, M; Tanaka, N; Yoshida, K

    2001-03-30

    The Ri (root-inducing) plasmid in Agrobacterium rhizogenes and Ti (tumor-inducing) plasmid in Agrobacterium tumefaciens have provided the fundamental basis for the construction of plant vectors and transgenic plants. Recently, the determination of the first complete nucleotide sequence of the Ti plasmid (pTi-SAKURA) has been successful. To understand the general structure of these oncogenic T-DNA transfer plasmids, the whole nucleotide sequence of a mikimopine-type Ri plasmid, pRi1724, was analyzed. The plasmid is 217,594 bp in size, and has 173 open reading frames (ORFs) in total, which are asymmetrically distributed. Except for 27 ORFs, which are unknown, 173 ORFs were classified into 12 groups as follows: three for DNA replication, nine for plasmid modification, 22 for conjugation, 26 for virulence, 11 for T-DNA gene, 19 for mikimopine/mikimopine-lactam transport, ten for an unknown opine metabolism, seven for transcriptional regulator, five for sugar transport, five for glycerol metabolism, four for chemoreceptor and 32 for others. The elucidated chimeric structure of pRi1724 interestingly indicates that the evolution of Rhizobiaceae plasmids seems to have kept interactions among the plasmids; especially, the genes and elements for a conjugal transfer of pRi1724 had clearly closer kinship to those of a Sym (symbiotic) plasmid, pNGR234a in Rhizobium sp. than those of Ti plasmids. By using sequencing and Northern analysis, we examined the metabolic pathway and gene expression of mikimopine, which is probably an Ri-specific opine. PMID:11273700

  1. Cyanobacterium sp. host cell and vector for production of chemical compounds in Cyanobacterial cultures

    DOEpatents

    Piven, Irina; Friedrich, Alexandra; Duhring, Ulf; Uliczka, Frank; Baier, Kerstin; Inaba, Masami; Shi, Tuo; Wang, Kui; Enke, Heike; Kramer, Dan

    2016-04-19

    A cyanobacterial host cell, Cyanobacterium sp., that harbors at least one recombinant gene for the production of a chemical compounds is provided, as well as vectors derived from an endogenous plasmid isolated from the cell.

  2. Cyanobacterium sp. host cell and vector for production of chemical compounds in cyanobacterial cultures

    SciTech Connect

    Piven, Irina; Friedrich, Alexandra; Duhring, Ulf; Uliczka, Frank; Baier, Kerstin; Inaba, Masami; Shi, Tuo; Wang, Kui; Enke, Heike; Kramer, Dan

    2014-09-30

    A cyanobacterial host cell, Cyanobacterium sp., that harbors at least one recombinant gene for the production of a chemical compounds is provided, as well as vectors derived from an endogenous plasmid isolated from the cell.

  3. Microwave effects on plasmid DNA

    SciTech Connect

    Sagripanti, J.L.; Swicord, M.L.; Davis, C.C.

    1987-05-01

    The exposure of purified plasmid DNA to microwave radiation at nonthermal levels in the frequency range from 2.00 to 8.75 GHz produces single- and double-strand breaks that are detected by agarose gel electrophoresis. Microwave-induced damage to DNA depends on the presence of small amounts of copper. This effect is dependent upon both the microwave power and the duration of the exposure. Cuprous, but not cupric, ions were able to mimic the effects produced by microwaves on DNA.

  4. Characterization of plasmids in bacterial fish pathogen.

    PubMed Central

    Toranzo, A E; Barja, J L; Colwell, R R; Hetrick, F M

    1983-01-01

    Plasmid profiles of representative fish pathogens, Aeromonas salmonicida, Aeromonas hydrophila, Vibrio anguillarum, Pasteurella piscicida, Yersinia ruckeri, Edwardsiella tarda, and Renibacterium salmoninarum, were determined by agarose gel electrophoresis with four different plasmid detection methods. A combination of two methods was required to detect the plasmids present in these strains and to calculate precisely the molecular weights of the plasmids. Of 38 strains, 28 harbored one or more plasmids, with the majority of strains demonstrating multiplasmid banding. Similarity in plasmid banding between strains was noted and related to geographic source. Five strains of A. salmonicida possessed six plasmid bands having molecular weights of 8.6 X 10(6), 8.4 X 10(6), 8.1 X 10(6), 3.6 X 10(6), 3.5 X 10(6), and 3.4 X 10(6). Four P. piscicida isolates shared three plasmid bands having molecular weights of 37 X 10(6), 15 X 10(6), and 5 X 10(6), and five A. hydrophila strains harbored a common plasmid having a molecular weight of ca. 20 X 10(6) to 30 X 10(6). The highest-molecular-weight plasmids (145 X 10(6) and 130 X 10(6) were detected in V. anguillarum. From curing experiments, it was found that in A. hydrophila strain 79-62, a loss of resistance to tetracycline was associated with loss of plasmid content in all susceptible derivatives, suggesting plasmid-mediated tetracycline resistance. Cell surface characteristics and metabolic properties were also modified in cured derivatives of A. hydrophila strain 79-62. Images PMID:6822413

  5. Superporous agarose anion exchangers for plasmid isolation.

    PubMed

    Tiainen, Peter; Gustavsson, Per-Erik; Ljunglöf, Anders; Larsson, Per-Olof

    2007-01-01

    Superporous agarose beads have wide, connecting flow pores allowing large molecules such as plasmids to be transported into the interior of the beads by convective flow. The pore walls provide additional surface for plasmid binding thus increasing the binding capacity of the adsorbent. Novel superporous agarose anion exchangers have been prepared, differing with respect to bead diameter, superpore diameter and type of anion-exchange functional group (poly(ethyleneimine) and quaternary amine). The plasmid binding capacities were obtained from breakthrough curves and compared with the binding capacity of homogeneous agarose beads of the same particle size. Significantly, the smaller diameter superporous agarose beads were found to have four to five times higher plasmid binding capacity than the corresponding homogeneous agarose beads. The experimentally determined plasmid binding capacity was compared with the theoretically calculated surface area for each adsorbent and fair agreement was found. Confocal microscopy studies of beads with adsorbed, fluorescently labelled plasmids aided in the interpretation of the results. Superporous poly(ethyleneimine)-substituted beads with a high ion capacity (230 micromol/ml) showed a plasmid binding of 3-4 mg/ml adsorbent. Superporous quaternary amine-substituted beads had a lower ion capacity (81 micromol/ml) and showed a correspondingly lower plasmid binding capacity (1-2 mg/ml adsorbent). In spite of the lower capacity, the beads with quaternary amine ligand were preferred, due to their much better plasmid recovery (70-100% recovery). Interestingly, both capacity and recovery was improved when the plasmid adsorption step was carried out in the presence of a moderate salt concentration. The most suitable superporous bead type (45-75 microm diameter beads; 4 microm superpores; quaternary amine ligand) was chosen for the capture of plasmid DNA from a clarified alkaline lysate. Two strategies were evaluated, one with and one

  6. FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli

    PubMed Central

    Ali, Syed A.; Chew, Yik Wei

    2015-01-01

    Antibiotic resistance genes and antibiotics are frequently used to maintain plasmid vectors in bacterial hosts such as Escherichia coli. Due to the risk of spread of antibiotic resistance, the regulatory authorities discourage the use of antibiotic resistance genes/antibiotics for the maintenance of plasmid vectors in certain biotechnology applications. Overexpression of E. coli endogenous fabI gene and subsequent selection on Triclosan has been proposed as a practical alternative to traditional antibiotic selection systems. Unfortunately, overexpression of fabI cannot be used to select medium –copy number plasmids, typically used for the expression of heterologous proteins in E. coli. Here we report that Vibrio cholera FabV, a functional homologue of E. coli FabI, can be used as a suitable marker for the selection and maintenance of both high and medium -copy number plasmid vectors in E. coli. PMID:26057251

  7. Generation of helper-dependent adenoviral vectors by homologous recombination.

    PubMed

    Toietta, Gabriele; Pastore, Lucio; Cerullo, Vincenzo; Finegold, Milton; Beaudet, Arthur L; Lee, Brendan

    2002-02-01

    Helper-dependent adenoviral vectors (HD-Ad) represent a potentially valuable tool for safe and prolonged gene expression in vivo. The current approach for generating these vectors is based on ligation of the expression cassette into large plasmids containing the viral inverted terminal repeats flanking "stuffer" DNA to maintain a final size above the lower limit for efficient packaging into the adenovirus capsid (approximately 28 kb). The ligation to produce the viral plasmid is generally very inefficient. Similar problems in producing first-generation adenoviral (FG-Ad) vectors were circumvented with the development of a system taking advantage of efficient homologous recombination between a shuttle plasmid containing the expression cassette and a FG-Ad vector backbone in the Escherichia coli strain BJ5183. Here we describe a method for fast and efficient generation of HD-Ad vector plasmids that can accommodate expression cassettes of any size up to 35 kb. To validate the system, we generated a HD-Ad vector expressing the fusion protein between beta-galactosidase and neomycin resistance genes under the control of the SR alpha promoter, and one expressing the enhanced green fluorescent protein under the control of the cytomegalovirus promoter. The viruses were rescued and tested in vitro and for in vivo expression in mice. The data collected indicate the possibility for achieving a high level of hepatocyte transduction using HD-Ad vectors derived from plasmids obtained by homologous recombination in E. coli, with no significant alteration of liver enzymes and a less severe, transient thrombocytopenia in comparison with previous reports with similar doses of a FG-Ad vector. PMID:11829528

  8. Hyperbolic-symmetry vector fields.

    PubMed

    Gao, Xu-Zhen; Pan, Yue; Cai, Meng-Qiang; Li, Yongnan; Tu, Chenghou; Wang, Hui-Tian

    2015-12-14

    We present and construct a new kind of orthogonal coordinate system, hyperbolic coordinate system. We present and design a new kind of local linearly polarized vector fields, which is defined as the hyperbolic-symmetry vector fields because the points with the same polarization form a series of hyperbolae. We experimentally demonstrate the generation of such a kind of hyperbolic-symmetry vector optical fields. In particular, we also study the modified hyperbolic-symmetry vector optical fields with the twofold and fourfold symmetric states of polarization when introducing the mirror symmetry. The tight focusing behaviors of these vector fields are also investigated. In addition, we also fabricate micro-structures on the K9 glass surfaces by several tightly focused (modified) hyperbolic-symmetry vector fields patterns, which demonstrate that the simulated tightly focused fields are in good agreement with the fabricated micro-structures.

  9. Construction and identification of multiple genes Co silence of plasmid shRNA.

    PubMed

    Sun, Jin; Wang, Liang; Dong, Ming-Min; Cao, Hua; Tian, Xiu-Fen

    2015-01-01

    Objective To construct and identify the eukaryotic vector expressing shRNA (Plasmid-1), which expressed the VEGF, C-myc, Survivin and hTERT gene at the same time. To detect its interference effects on the nasopharyngeal carcinoma cell line (CNE-2Z) compared with single gene plasmid VEFG (Plasmid-2). Methods According to the sequence of VEGF, C-myc, Survivin and hTERT gene, we designed 2 oligonucleotide sequences and synthesized a complementary DNA chain, then inserted it into the eukaryotic vector expressing pGenesil 1. The cell proliferation activity was detected by MTT method. The interference efficacy on human nasopharyngeal carcinoma cell line (CNE-2Z) in the level of mRNA and protein were detected by RT-PCR and Western-bolt. The inhibitory effect of plasmid on tumor in nude mice was also observed in vivo. Results The restriction enzyme digestion and sequencing technologies confirmed the construction of recombinant eukaryotic vector expressing was correct. The plasmid was transfected into CNE-2Z cells, green fluorescence can be seen clearly in the single gene and multi gene transfected cells under fluorescent microscope. MTT showed that the proliferation of cell was inhibited, the invasive ability was decreased in vitro, and the inhibition effects of single gene plasmid on the growth and proliferation of cells were lower than multi gene. Real-time-PCR and Western-bolt confirmed that the expression of target gene was decreased in the level of mRNA and protein, and the interference effect of multi gene was better than the single gene. The nude mice experiment showed that the interference effect of shRNA plasmid on the growth of tumor cell was better than single gene plasmid Conclusion We constructed a shRNA plasmid encoded four different genes successfully. After transfected with nasopharyngeal carcinoma cells, it can interfere the expression of VEGF, C-myc, Survivin and hTERT gene at the same time. And the interference effect was better than silence VEGF alone

  10. Cloning vector

    DOEpatents

    Guilfoyle, R.A.; Smith, L.M.

    1994-12-27

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site. 2 figures.

  11. Cloning vector

    DOEpatents

    Guilfoyle, Richard A.; Smith, Lloyd M.

    1994-01-01

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site.

  12. Plasmids from Food Lactic Acid Bacteria: Diversity, Similarity, and New Developments

    PubMed Central

    Cui, Yanhua; Hu, Tong; Qu, Xiaojun; Zhang, Lanwei; Ding, Zhongqing; Dong, Aijun

    2015-01-01

    Plasmids are widely distributed in different sources of lactic acid bacteria (LAB) as self-replicating extrachromosomal genetic materials, and have received considerable attention due to their close relationship with many important functions as well as some industrially relevant characteristics of the LAB species. They are interesting with regard to the development of food-grade cloning vectors. This review summarizes new developments in the area of lactic acid bacteria plasmids and aims to provide up to date information that can be used in related future research. PMID:26068451

  13. Plasmids from Food Lactic Acid Bacteria: Diversity, Similarity, and New Developments.

    PubMed

    Cui, Yanhua; Hu, Tong; Qu, Xiaojun; Zhang, Lanwei; Ding, Zhongqing; Dong, Aijun

    2015-06-10

    Plasmids are widely distributed in different sources of lactic acid bacteria (LAB) as self-replicating extrachromosomal genetic materials, and have received considerable attention due to their close relationship with many important functions as well as some industrially relevant characteristics of the LAB species. They are interesting with regard to the development of food-grade cloning vectors. This review summarizes new developments in the area of lactic acid bacteria plasmids and aims to provide up to date information that can be used in related future research.

  14. Application of least squares support vector regression and linear multiple regression for modeling removal of methyl orange onto tin oxide nanoparticles loaded on activated carbon and activated carbon prepared from Pistacia atlantica wood.

    PubMed

    Ghaedi, M; Rahimi, Mahmoud Reza; Ghaedi, A M; Tyagi, Inderjeet; Agarwal, Shilpi; Gupta, Vinod Kumar

    2016-01-01

    Two novel and eco friendly adsorbents namely tin oxide nanoparticles loaded on activated carbon (SnO2-NP-AC) and activated carbon prepared from wood tree Pistacia atlantica (AC-PAW) were used for the rapid removal and fast adsorption of methyl orange (MO) from the aqueous phase. The dependency of MO removal with various adsorption influential parameters was well modeled and optimized using multiple linear regressions (MLR) and least squares support vector regression (LSSVR). The optimal parameters for the LSSVR model were found based on γ value of 0.76 and σ(2) of 0.15. For testing the data set, the mean square error (MSE) values of 0.0010 and the coefficient of determination (R(2)) values of 0.976 were obtained for LSSVR model, and the MSE value of 0.0037 and the R(2) value of 0.897 were obtained for the MLR model. The adsorption equilibrium and kinetic data was found to be well fitted and in good agreement with Langmuir isotherm model and second-order equation and intra-particle diffusion models respectively. The small amount of the proposed SnO2-NP-AC and AC-PAW (0.015 g and 0.08 g) is applicable for successful rapid removal of methyl orange (>95%). The maximum adsorption capacity for SnO2-NP-AC and AC-PAW was 250 mg g(-1) and 125 mg g(-1) respectively. PMID:26414425

  15. Efficient non-enzymatic cleavage of Staphylococcus aureus plasmid DNAs mediated by neodymium ions.

    PubMed

    Zovčáková, Monika; Španová, Alena; Pantůček, Roman; Doškař, Jiří; Rittich, Bohuslav

    2016-08-15

    Staphylococcus aureus plasmids are the main factor in the spreading of antibacterial resistance among bacterial strains that has emerged on a worldwide scale. Plasmids recovered from 12 clinical and food isolates of S. aureus were treated with 10 mM free lanthanide Nd(3+) ions (non-enzymatic cleavage agent) in Hepes buffer (pH 7.5) at 70 °C. Topological forms of plasmids-closed circular (ccc), open circular (oc), and linear (lin)-produced by cleavage at different times were separated using pulsed-field agarose gel electrophoresis. The method is proposed to detect and differentiate several plasmids in the same bacterial strain according to their size. PMID:27237372

  16. Efficient non-enzymatic cleavage of Staphylococcus aureus plasmid DNAs mediated by neodymium ions.

    PubMed

    Zovčáková, Monika; Španová, Alena; Pantůček, Roman; Doškař, Jiří; Rittich, Bohuslav

    2016-08-15

    Staphylococcus aureus plasmids are the main factor in the spreading of antibacterial resistance among bacterial strains that has emerged on a worldwide scale. Plasmids recovered from 12 clinical and food isolates of S. aureus were treated with 10 mM free lanthanide Nd(3+) ions (non-enzymatic cleavage agent) in Hepes buffer (pH 7.5) at 70 °C. Topological forms of plasmids-closed circular (ccc), open circular (oc), and linear (lin)-produced by cleavage at different times were separated using pulsed-field agarose gel electrophoresis. The method is proposed to detect and differentiate several plasmids in the same bacterial strain according to their size.

  17. DNA Minicircle Technology Improves Purity of Adeno-associated Viral Vector Preparations

    PubMed Central

    Schnödt, Maria; Schmeer, Marco; Kracher, Barbara; Krüsemann, Christa; Espinosa, Laura Escalona; Grünert, Anja; Fuchsluger, Thomas; Rischmüller, Anja; Schleef, Martin; Büning, Hildegard

    2016-01-01

    Adeno-associated viral (AAV) vectors are considered as one of the most promising delivery systems in human gene therapy. In addition, AAV vectors are frequently applied tools in preclinical and basic research. Despite this success, manufacturing pure AAV vector preparations remains a difficult task. While empty capsids can be removed from vector preparations owing to their lower density, state-of-the-art purification strategies as of yet failed to remove antibiotic resistance genes or other plasmid backbone sequences. Here, we report the development of minicircle (MC) constructs to replace AAV vector and helper plasmids for production of both, single-stranded (ss) and self-complementary (sc) AAV vectors. As bacterial backbone sequences are removed during MC production, encapsidation of prokaryotic plasmid backbone sequences is avoided. This is of particular importance for scAAV vector preparations, which contained an unproportionally high amount of plasmid backbone sequences (up to 26.1% versus up to 2.9% (ssAAV)). Replacing standard packaging plasmids by MC constructs not only allowed to reduce these contaminations below quantification limit, but in addition improved transduction efficiencies of scAAV preparations up to 30-fold. Thus, MC technology offers an easy to implement modification of standard AAV packaging protocols that significantly improves the quality of AAV vector preparations.

  18. Characterization of the theta replication plasmid pGR7 from Acetobacter aceti CCM 3610.

    PubMed

    Grones, Peter; Grones, Jozef

    2012-07-01

    A cryptic plasmid of Acetobacter aceti CCM 3610, designated pGR7, was sequenced and characterized. It is a 2446-bp circular molecule with a G + C content of 30%, which is unusual when compared to the already known plasmids isolated from Acetobacter genera. Sequence analysis of pGR7 revealed three putative open reading frames (ORFs). ORF1 displays low similarity with other Acetobacter plasmid replication proteins. The other two ORFs show similarities only to hypothetical proteins and do not encode any important protein. The replication module comprises a DnaA box-like sequence, indirect repeats, a potential prokaryotic promoter and the rep gene. The rep module organization is similar to that found in other theta-replicating plasmids from acetic acid bacteria that stably maintain in both Acetobacter and Escherichia coli, with two repeated sequences containing modules. Nevertheless, the pGR7 plasmid could replicate and be stably maintained only in Acetobacter strains and not in E. coli, another uncommon feature of this plasmid. The Rep protein was cloned into the pET30a + expression vector and purified by high-performance liquid chromatography. The helicase activity was determined and the ability of the protein to bind to the plasmid regulation region was confirmed by an electrophoretic mobility shift assay. The plasmid was stable in the Acetobacter cells after cultivation under nonselective conditions. By real-time polymerase chain reaction, the relative copy number of pGR7 was estimated to be seven copies per host chromosome equivalent.

  19. Vector quantization

    NASA Technical Reports Server (NTRS)

    Gray, Robert M.

    1989-01-01

    During the past ten years Vector Quantization (VQ) has developed from a theoretical possibility promised by Shannon's source coding theorems into a powerful and competitive technique for speech and image coding and compression at medium to low bit rates. In this survey, the basic ideas behind the design of vector quantizers are sketched and some comments made on the state-of-the-art and current research efforts.

  20. Sequence comparisons of plasmids pBJS-O of Spiroplasma citri and pSKU146 of S. kunkelii: implications for plasmid evolution

    PubMed Central

    Joshi, Bharat D; Berg, Michael; Rogers, Janet; Fletcher, Jacqueline; Melcher, Ulrich

    2005-01-01

    Background Spiroplasma citri BR3-3X and S. kunkelii CR2-3X cause serious diseases worldwide on citrus and maize species, respectively. S. citri BR3-3X harbors a plasmid, pBJS-Original (pBJS-O), that encodes the spiroplasma adhesion related protein 1 (SARP1), a protein implicated in binding of the pathogen to cells of its leafhopper vector, Circulifer tenellus. The S. kunkelii CR2-3X plasmid, pSKU146, encodes a homolog of SARP1, Sk-ARP1. Due to the close phylogenetic relationship of the two pathogens, we hypothesized that the two plasmids are closely related as well. Results The nucleotide sequence of pBJS-O was determined and compared to the sequences of a plasmid from BR3-T (pBJS-T), which is a multiply passaged leafhopper transmissible derivative of BR3-3X, and to known plasmid sequences including that of pSKU146. In addition to arp1, the 13,374 bp pBJS-O sequence putatively contains nine genes, recognized as open reading frames (ORFs). Several pBJS-O ORFs have homologs on pSKU146. However, the sequences flanking soj-like genes on both plasmids were found to be more distant from one another than sequences in any other region. Further, unlike pSKU146, pBJS-O lacks the conserved oriT region characteristic of the IncP group of bacterial plasmids. We were unable to identify a region in pBJS-O resembling a known plasmid origin of transfer. In regions where sequence was available for the plasmid from both BR3-3X and BR3-T, the pBJS-T sequence had a 0.4 kb deletion relative to its progenitor, pBJS-O. Southern blot hybridization of extrachromosomal DNA from various S. citri strains and spiroplasma species to an arp-specific probe and a probe made from the entire plasmid DNA of BR3-3X revealed limited conservation of both sequences in the genus Spiroplasma. Finally, we also report the presence on the BR3-3X chromosome of arp2, an S. citri homolog of arp1 that encodes the predicted protein SARP2. The C-terminal domain of SARP2 is homologous to that of SARP1, but its N

  1. A bacteriocin gene cluster able to enhance plasmid maintenance in Lactococcus lactis

    PubMed Central

    2014-01-01

    Background Lactococcus lactis is widely used as a dairy starter and has been extensively studied. Based on the acquired knowledge on its physiology and metabolism, new applications have been envisaged and there is an increasing interest of using L. lactis as a cell factory. Plasmids constitute the main toolbox for L. lactis genetic engineering and most rely on antibiotic resistant markers for plasmid selection and maintenance. In this work, we have assessed the ability of the bacteriocin Lactococcin 972 (Lcn972) gene cluster to behave as a food-grade post-segregational killing system to stabilize recombinant plasmids in L. lactis in the absence of antibiotics. Lcn972 is a non-lantibiotic bacteriocin encoded by the 11-kbp plasmid pBL1 with a potent antimicrobial activity against Lactococcus. Results Attempts to clone the full lcn972 operon with its own promoter (P972), the structural gene lcn972 and the immunity genes orf2-orf3 in the unstable plasmid pIL252 failed and only plasmids with a mutated promoter were recovered. Alternatively, cloning under other constitutive promoters was approached and achieved, but bacteriocin production levels were lower than those provided by pBL1. Segregational stability studies revealed that the recombinant plasmids that yielded high bacteriocin titers were maintained for at least 200 generations without antibiotic selection. In the case of expression vectors such as pTRL1, the Lcn972 gene cluster also contributed to plasmid maintenance without compromising the production of the fluorescent mCherry protein. Furthermore, unstable Lcn972 recombinant plasmids became integrated into the chromosome through the activity of insertion sequences, supporting the notion that Lcn972 does apply a strong selective pressure against susceptible cells. Despite of it, the Lcn972 gene cluster was not enough to avoid the use of antibiotics to select plasmid-bearing cells right after transformation. Conclusions Inserting the Lcn972 cluster into

  2. Hygromycin-resistance vectors for gene expression in Pichia pastoris.

    PubMed

    Yang, Junjie; Nie, Lei; Chen, Biao; Liu, Yingmiao; Kong, Yimeng; Wang, Haibin; Diao, Liuyang

    2014-04-01

    Pichia pastoris is a common host organism for heterologous protein expression and metabolic engineering. Zeocin-, G418-, nourseothricin- and blasticidin-resistance genes are the only dominant selectable markers currently available for selecting P. pastoris transformants. We describe here new P. pastoris expression vectors that confer a hygromycin resistance base on the Klebsiella pneumoniae hph gene. To demonstrate the application of the vectors for intracellular and secreted protein expression, green fluorescent protein (GFP) and human serum albumin (HSA) were cloned into the vectors and transformed into P. pastoris cells. The resulting strains expressed GFP and HSA constitutively or inducibly. The hygromycin resistance marker was also suitable for post-transformational vector amplication (PTVA) for obtaining strains with high plasmid copy numbers. A strain with multiple copies of the HSA expression cassette after PTVA had increased HSA expression compared with a strain with a single copy of the plasmid. To demonstrate compatibility of the new vectors with other vectors bearing antibiotic-resistance genes, P. pastoris was transformed with the Saccharomyces cerevisiae genes GSH1, GSH2 or SAM2 on plasmids containing genes for resistance to Zeocin, G418 or hygromycin. The resulting strain produced glutathione and S-adenosyl-L-methionine at levels approximately twice those of the parent strain. The new hygromycin-resistance vectors allow greater flexibility and potential applications in recombinant protein production and other research using P. pastoris. PMID:24822243

  3. Thermosensitive antibiotic resistance plasmids in enterobacteria.

    PubMed

    Smith, H W; Parsell, Z; Green, P

    1978-11-01

    Of 775 conjugative plasmids found in enterobacteria mediating antibiotic resistance, 24 (3.1%) were thermosensitive (ts); they were most common in Klebsiella pneumoniae. Ts plasmids were also found in all the samples of sewage and river water examined. Over half of 73 ts plasmids from unrelated sources mediated resistance to chloramphenicol in addition to several other antibiotics. Many of them mediated resistance to mercury (53.4%), arsenite (38.4%) and tellurite (79.5%) but not to copper, cobalt and silver. Fifty-eight belonged to incompatibility group H2 and 12 belonged to the H1 group. Resistance to mercury, arsenite and tellurite was common in strains containing H2 plasmids but not in H1 plasmids. The 73 plasmids transferred at high rates at 22 and 28 degrees C and at lower rates at 15 degrees C; they transferred at very low rates or not at all at 37 degrees C. They could be divided into two sets according to whether they transferred at a high or at a low rate at 33 degrees C. Unlike the prototype plasmid, Rts 1, they were solely or mainly ts for transfer and not for replication and only one of them brought about a marked reduction in growth rate of its host organism at 42 degrees C. None of the 73 plasmids mediated colicin or haemolysin production. Three plasmids, all from K. pneumoniae, mediated utilization of lactose, two of sucrose and raffinose and three, all belonging to group H1, of citrate. None of the plasmids increased the pathogenicity of Salmonella typhimurium for chicks or Escherichia coli K12 for mice.

  4. Stable transformation of tobacco by electroporation: evidence for plasmid concatenation.

    PubMed Central

    Riggs, C D; Bates, G W

    1986-01-01

    Electroporation (electric field-mediated DNA transfer) of tobacco protoplasts in the presence of the linearized plasmid pMON200 has led to the formation of transgenic plants. Defined electric shocks were delivered by capacitive discharges with readily available, low-cost electrical components. This transformation procedure is simple and efficient and may suggest a quick method for determining the appropriate electric fields for new cell systems. An optimal transformation frequency of 2.2 X 10(-4) (based on the number of cells subjected to the shock) was obtained with a single 2000-V/cm, 250-microseconds-duration capacitive discharge. Calli transformed to kanamycin resistance have been regenerated into whole plants. Southern blots of DNA from the transgenic plants demonstrate the integration of the selectable marker gene (neomycin phosphotransferase) at single or multiple genomic sites. In some cases, the plasmid appears to be integrated intact; in others, it is rearranged. The blots also provide evidence of plasmid recircularization and/or the formation of head-to-head and head-to-tail concatemers in most of the plants analyzed. Although some plants apparently have multiple integration sites, analysis of progeny obtained by self-fertilization of the transgenic plants indicates that the kanamycin-resistance marker is inherited as a single dominant gene. Images PMID:3016708

  5. Factors influencing the titer and infectivity of lentiviral vectors.

    PubMed

    Logan, Aaron C; Nightingale, Sarah J; Haas, Dennis L; Cho, Gerald J; Pepper, Karen A; Kohn, Donald B

    2004-10-01

    Lentiviral vectors have undergone several generations of design improvement to enhance their biosafety and expression characteristics, and have been approved for use in human clinical studies. Most preclinical studies with these vectors have employed easily assayed marker genes for the purpose of determining vector titers and transduction efficiencies. Naturally, the adaptation of these vector systems to clinical use will increasingly involve the transfer of genes whose products may not be easily measured, meaning that the determination of vector titer will be more complicated. One method for determining vector titer that can be universally employed on all human immunodeficiency virus type 1-based lentiviral vector supernatants involves the measurement of Gag (p24) protein concentration in vector supernatants by immunoassay. We have studied the effects that manipulation of several variables involved in vector design and production by transient transfection have on vector titer and infectivity. We have determined that manipulation of the amount of transfer vector, packaging, and envelope plasmids used to transfect the packaging cells does not alter vector infectivity, but does influence vector titer. We also found that modifications to the transfer vector construct, such as replacing the internal promoter or transgene, do not generally alter vector infectivity, whereas inclusion of the central polypurine tract in the transfer vector increases vector infectivity on HEK293 cells and human umbilical cord blood CD34+ hematopoietic progenitor cells (HPCs). The infectivities of vector supernatants can also be increased by harvesting at early time points after the initiation of vector production, collection in serum-free medium, and concentration by ultracentrifugation. For the transduction of CD34+ HPCs, we found that the simplest method of increasing vector infectivity is to pseudotype vector particles with the RD114 envelope instead of vesicular stomatitis virus G

  6. High-Level Production of Plasmid DNA by Escherichia coli DH5α ΩsacB by Introducing inc Mutations

    PubMed Central

    Trivedi, Ram Narayan; Akhtar, Parvez; Meade, Jonathan; Bartlow, Patrick; Ataai, Mohammad M.; Khan, Saleem A.

    2014-01-01

    For small-copy-number pUC-type plasmids, the inc1 and inc2 mutations, which deregulate replication, were previously found to increase the plasmid copy number 6- to 7-fold. Because plasmids can exert a growth burden, it was not clear if further amplification of copy number would occur due to inc mutations when the starting point for plasmid copy number was orders of magnitude higher. To investigate further the effects of the inc mutations and the possible limits of plasmid synthesis, the parent plasmid pNTC8485 was used as a starting point. It lacks an antibiotic resistance gene and has a copy number of ∼1,200 per chromosome. During early stationary-phase growth in LB broth at 37°C, inc2 mutants of pNTC8485 exhibited a copy number of ∼7,000 per chromosome. In minimal medium at late log growth, the copy number was found to be significantly increased, to approximately 15,000. In an attempt to further increase the plasmid titer (plasmid mass/culture volume), enzymatic hydrolysis of the selection agent, sucrose, at late log growth extended growth and tripled the total plasmid amount such that an approximately 80-fold gain in total plasmid was obtained compared to the value for typical pUC-type vectors. Finally, when grown in minimal medium, no detectable impact on the exponential growth rate or the fidelity of genomic or plasmid DNA replication was found in cells with deregulated plasmid replication. The use of inc mutations and the sucrose degradation method presents a simplified way for attaining high titers of plasmid DNA for various applications. PMID:25217014

  7. Lentiviral vectors.

    PubMed

    Giry-Laterrière, Marc; Verhoeyen, Els; Salmon, Patrick

    2011-01-01

    Lentiviral vectors have evolved over the last decade as powerful, reliable, and safe tools for stable gene transfer in a wide variety of mammalian cells. Contrary to other vectors derived from oncoretroviruses, they allow for stable gene delivery into most nondividing primary cells. In particular, lentivectors (LVs) derived from HIV-1 have gradually evolved to display many desirable features aimed at increasing both their safety and their versatility. This is why lentiviral vectors are becoming the most useful and promising tools for genetic engineering, to generate cells that can be used for research, diagnosis, and therapy. This chapter describes protocols and guidelines, for production and titration of LVs, which can be implemented in a research laboratory setting, with an emphasis on standardization in order to improve transposability of results between laboratories. We also discuss latest designs in LV technology.

  8. Replication and Control of Circular Bacterial Plasmids

    PubMed Central

    del Solar, Gloria; Giraldo, Rafael; Ruiz-Echevarría, María Jesús; Espinosa, Manuel; Díaz-Orejas, Ramón

    1998-01-01

    An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. In this review, we focus on replication and its control in circular plasmids. Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer. This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming (theta and strand displacement replication) or (ii) cleavage of one of the DNA strands to generate a 3′-OH end (rolling-circle replication). Initiation is catalyzed most frequently by one or a few plasmid-encoded initiation proteins that recognize plasmid-specific DNA sequences and determine the point from which replication starts (the origin of replication). In some cases, these proteins also participate directly in the generation of the primer. These initiators can also play the role of pilot proteins that guide the assembly of the host replisome at the plasmid origin. Elongation of plasmid replication is carried out basically by DNA polymerase III holoenzyme (and, in some cases, by DNA polymerase I at an early stage), with the participation of other host proteins that form the replisome. Termination of replication has specific requirements and implications for reinitiation, studies of which have started. The initiation stage plays an additional role: it is the stage at which mechanisms controlling replication operate. The objective of this control is to maintain a fixed concentration of plasmid molecules in a growing bacterial population (duplication of the plasmid pool paced with duplication of the bacterial population

  9. High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe.

    PubMed Central

    Okazaki, K; Okazaki, N; Kume, K; Jinno, S; Tanaka, K; Okayama, H

    1990-01-01

    We describe a highly efficient alkali cation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of fission yeast Schizosaccharomyces pombe mutants. cDNA libraries constructed with the pcD or pcD2 vector are transduced into yeast by cotransfection with a linearized vector, which allows an enhanced homologous recombination between the yeast vector and the library plasmid leading to the efficient formation of concatemers containing pcD molecules. The transformation frequencies obtained by the method are 10(6) colonies per 10(8) cells transfected with 2 micrograms of library and 1 microgram of vector, 50-60% of which contain pcD molecules. The high-efficiency alkali cation method circumvents many of the shortcomings of the spheroplast method generally used for Schiz. pombe transfection. The vectors are maximized for the efficiency of library transduction and minimized for the rearrangements of pcD molecules during propagation in yeast. This system allows rapid screening of multi-million cDNA clone libraries for rare cDNAs in a routine scale of experiments. Using this system, various mammalian cDNAs that are extremely difficult, time-consuming, or unclonable to clone by other methods have been cloned. Images PMID:2251111

  10. Ada Linear-Algebra Program

    NASA Technical Reports Server (NTRS)

    Klumpp, A. R.; Lawson, C. L.

    1988-01-01

    Routines provided for common scalar, vector, matrix, and quaternion operations. Computer program extends Ada programming language to include linear-algebra capabilities similar to HAS/S programming language. Designed for such avionics applications as software for Space Station.

  11. Colliders and brane vector phenomenology

    SciTech Connect

    Clark, T. E.; Love, S. T.; Xiong, C.; Nitta, Muneto; Veldhuis, T. ter

    2008-12-01

    Brane world oscillations manifest themselves as massive vector gauge fields. Their coupling to the standard model is deduced using the method of nonlinear realizations of the spontaneously broken higher dimensional space-time symmetries. Brane vectors are stable and weakly interacting and therefore escape particle detectors unnoticed. LEP and Tevatron data on the production of a single photon in conjunction with missing energy are used to delineate experimentally excluded regions of brane vector parameter space. The additional region of parameter space accessible to the LHC as well as a future lepton linear collider is also determined by means of this process.

  12. Thermosensitive H1 plasmids determining citrate utilization.

    PubMed

    Smith, H W; Parsell, Z; Green, P

    1978-12-01

    Twelve thermosensitive H1 plasmids from strains of Salmonella typhi that had caused outbreaks of chloramphenicol-resistant typhoid fever in Vietnam, Thailand and India mediated citrate utilization (Cit+) in a prototrophic Escherichia coli K12 strain but not in the S. typhi strains from which they were derived. Four H1 plasmids from a similar outbreak in Mexico differed from the Far Eastern plasmids in not mediating citrate utlization but in mediating mercury resistance. H1 plasmids resembling the Far Eastern and the Mexican plasmids in regard to citrate utilization and mercury resistance were found in sewage in Britain. Citrate utilization was transferred to eight pathogenic strains of E. coli and to one strain each of Shigella flexneri and Shigella sonnei. Cultures of Cit+ bacteria grew more rapidly in citrate media at 28 degrees C than at 37 degrees C. Plasmid mutants that were more efficient at utilizing citrate were present in all such cultures--they grew equally well or better at 37 degrees C than at 28 degrees C. None of 222 strains of E. coli or Shigella that contained a variety of different plasmids were able to utilize citrate. This property was not transferred to the prototrophic E. coli K12 strain from Citrobacter (3 strains), Salmonella (39 strains), Proteus (44 strains), Klebsiella pneumoniae (33 strains) or Pseudomonas aeruginosa (44 strains).

  13. Rolling-circle replication of bacterial plasmids.

    PubMed Central

    Khan, S A

    1997-01-01

    Many bacterial plasmids replicate by a rolling-circle (RC) mechanism. Their replication properties have many similarities to as well as significant differences from those of single-stranded DNA (ssDNA) coliphages, which also replicate by an RC mechanism. Studies on a large number of RC plasmids have revealed that they fall into several families based on homology in their initiator proteins and leading-strand origins. The leading-strand origins contain distinct sequences that are required for binding and nicking by the Rep proteins. Leading-strand origins also contain domains that are required for the initiation and termination of replication. RC plasmids generate ssDNA intermediates during replication, since their lagging-strand synthesis does not usually initiate until the leading strand has been almost fully synthesized. The leading- and lagging-strand origins are distinct, and the displaced leading-strand DNA is converted to the double-stranded form by using solely the host proteins. The Rep proteins encoded by RC plasmids contain specific domains that are involved in their origin binding and nicking activities. The replication and copy number of RC plasmids, in general, are regulated at the level of synthesis of their Rep proteins, which are usually rate limiting for replication. Some RC Rep proteins are known to be inactivated after supporting one round of replication. A number of in vitro replication systems have been developed for RC plasmids and have provided insight into the mechanism of plasmid RC replication. PMID:9409148

  14. Internal ribosome entry site (IRES) from Encephalomyocarditis virus (EMCV) as a tool for shuttle expression plasmids.

    PubMed

    Telpalo-Carpio, Sandra Aurora; Diaz-Mitoma, Francisco; Moreno-Cuevas, Jorge Eugenio; Aguilar-Yáñez, José Manuel

    2015-12-25

    In eukaryotes, IRES sequences aid the recruitment of factors needed for translation to occur, enabling protein production independent of 5' capped mRNA. Many patents and commercially available plasmids exploit their properties for polycistronic expression of recombinant proteins. However, these applications have been restricted to eukaryotic organisms, since it was thought that elements of this origin were essential for their activity. Here, using two tricistronic vectors designed for expression in mammalian hosts, we present evidence of EMCV IRES activity in prokaryotes. This finding enables the development of new and more versatile plasmid vectors for the production of recombinant proteins in multiple hosts from a single construct. Additionally, it provides new hints for the elaboration of alternative models describing the molecular mechanism of EMCV IRES mediated translation, in the absence of eukaryotic elements that were considered indispensable for its function.

  15. Effect of innate glutathione levels on activity of redox-responsive gene delivery vectors

    PubMed Central

    Manickam, Devika S.; Li, Jing; Putt, David A.; Zhou, Qing-Hui; Wu, Chao; Lash, Lawrence H.; Oupický, David

    2009-01-01

    Redox-responsive polyplexes represent a promising class of non-viral gene delivery vectors. The reducible disulfide bonds in the polyplexes undergo intracellular reduction owing to the presence of high concentrations of reduced glutathione (GSH). Available evidence suggests improved transfection activity of redox-sensitive polyplexes upon artificial modulation of intracellular GSH. This study investigates the effect of innate differences in GSH concentration in a panel of human pancreatic cancer cell lines on activity of reducible polyplexes of the four major classes of nucleic acid therapeutics: plasmid DNA (pDNA), messenger RNA (mRNA), antisense oligodeoxynucleotides (AON) and siRNA. In general, reducible polyplexes of linear poly(amido amines) (PAA) show improved activity compared to non-reducible polyplexes of PAA. Results demonstrate that increased GSH levels are associated with improved transfection of mRNA polyplexes but no clear trend is observed for pDNA, AON and siRNA polyplexes. PMID:19720098

  16. RNA1 is sufficient to mediate plasmid ColE1 incompatibility in vivo.

    PubMed

    Fitzwater, T; Tamm, J; Polisky, B

    1984-05-25

    The multicopy plasmid ColE1 specifies a small RNA designated RNA1 that has been implicated in copy number control and incompatibility. We have inserted a 148 base-pair ColE1 DNA fragment containing a promoter-less RNA1 gene into a plasmid vector downstream from the tryptophan promoter of Serratia marcesens . The ColE1 RNA1 produced by this plasmid is not functional in vivo due to the presence of 49 nucleotides appended to the 5'-terminus of the wild-type RNA1 sequence. Deletions of these sequences by Bal3l nuclease in vitro and genetic selection for ColE1 incompatibility function in vivo permitted isolation of a plasmid expressing wild-type ColE1 RNA1 initiated properly from the S. marcesens trp promoter. These experiments demonstrate that RNA1 is sufficient to mediate ColE1 incompatibility in vivo. In addition, several plasmids were isolated that contain altered RNA1 genes. These alterations consist of additions or deletions of sequences at the 5'-terminus of RNA1. Analysis of the ability of these altered RNA1 molecules to express incompatibility in vivo suggests that the 5'-terminal region of RNA1 is crucial for its function.

  17. Contribution of Epigenetic Modifications to the Decline in Transgene Expression from Plasmid DNA in Mouse Liver

    PubMed Central

    Zang, Lei; Nishikawa, Makiya; Ando, Mitsuru; Takahashi, Yuki; Takakura, Yoshinobu

    2015-01-01

    Short-term expression of transgenes is one of the problems frequently associated with non-viral in vivo gene transfer. To obtain experimental evidence for the design of sustainable transgene expression systems, the contribution of epigenetic modifications to the decline in transgene expression needs to be investigated. Bisulfite sequencing and reactivation by hydrodynamic injection of isotonic solution were employed to investigate methylation statues of CpG in transiently expressing plasmid, pCMV-Luc, in mouse liver after hydrodynamic delivery. The cytosines of CpGs in the promoter region of pCMV-Luc were methylated in mouse liver, but the methylation was much later than the decline in the expression. The expression from pre-methylated pCMV-Luc was insensitive to reactivation. Neither an inhibitor of DNA methylation nor an inhibitor of histone deacetylation had significant effects on transgene expression after hydrodynamic injection of pCMV-Luc. Partial hepatectomy, which reduces the transgene expression from the non-integrated vector into the genome, significantly reduced the transgene expression of human interferon γ from a long-term expressing plasmid pCpG-Huγ, suggesting that the CpG-reduced plasmid was not significantly integrated into the genomic DNA. These results indicate that the CpG-reduced plasmids achieve prolonged transgene expression without integration into the host genome, although the methylation status of CpG sequences in plasmids will not be associated with the prolonged expression. PMID:26262639

  18. Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.

    PubMed

    McDaniel, Jonathan R; Mackay, J Andrew; Quiroz, Felipe García; Chilkoti, Ashutosh

    2010-04-12

    This paper reports a new strategy, recursive directional ligation by plasmid reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats. PRe-RDL has several unique features that stem from the use of type IIs restriction endonucleases: first, PRe-RDL is a seamless cloning method that leaves no extraneous nucleotides at the ligation junction. Because it uses type IIs endonucleases to ligate the two halves of the plasmid, PRe-RDL also addresses the major limitation of RDL in that it abolishes any restriction on the gene sequence that can be oligomerized. The reconstitution of a functional plasmid only upon successful ligation in PRe-RDL also addresses two other limitations of RDL: the significant background from self-ligation of the vector observed in RDL, and the decreased efficiency of ligation due to nonproductive circularization of the insert. PRe-RDL can also be used to assemble genes that encode different sequences in a predetermined order to encode block copolymers or append leader and trailer peptide sequences to the oligomerized gene. PMID:20184309

  19. Fibrin-embedded administration of VEGF plasmid enhances skin flap survival.

    PubMed

    Michlits, Wolfgang; Mittermayr, Rainer; Schäfer, Romana; Redl, Heinz; Aharinejad, Seyedhossein

    2007-01-01

    The aim of the present study was to experimentally evaluate whether topical fibrin-mediated administration of a vascular endothelial growth factor (VEGF)-A plasmid to the wound bed can protect skin flaps from necrosis. A plasmid expression vector containing the VEGF-A cDNA was constructed. The plasmid was then administered to the wound bed of rat abdominal skin flaps in a fibrin sealant. The percentage of viable, ischemic and necrotic tissue was assessed postoperatively as a baseline and after 3 and 7 days using digital surface area morphometry. Laser Doppler imaging of the flaps and VEGF-A Western blot analysis of flap tissue were performed to assess angiogenesis and VEGF-A tissue levels. Flaps treated with VEGF plasmids in the presence of uptake enhancing Lipofectamine transfection reagent increased flap survival 7 days postoperatively significantly associated with markedly elevated tissue perfusion and enhanced tissue VEGF-A protein expression. Our results indicate that topical fibrin-mediated administration of a VEGF-A plasmid may serve as an alternative to previous strategies in treating ischemic skin flaps. The suggested therapeutic approach is easily applicable and inexpensive in preparation. Thus, this protocol may also enhance wound healing in posttrauma skin lacerations or in skin grafts.

  20. Generalized transduction of small Yersinia enterocolitica plasmids.

    PubMed

    Hertwig, S; Popp, A; Freytag, B; Lurz, R; Appel, B

    1999-09-01

    To study phage-mediated gene transfer in Yersinia, the ability of Yersinia phages to transduce naturally occurring plasmids was investigated. The transduction experiments were performed with a temperate phage isolated from a pathogenic Yersinia enterocolitica strain and phage mixtures isolated from sewage. Small plasmids (4.3 and 5.8 kb) were transduced at a frequency of 10(-5) to 10(-7)/PFU. However, we could not detect the transduction of any indigenous virulence plasmid (ca. 72 kb) in pathogenic Yersinia strains. Transductants obtained by infection with the temperate phage were lysogenic and harbored the phage genome in their chromosomes.

  1. ColE1-Plasmid Production in Escherichia coli: Mathematical Simulation and Experimental Validation

    PubMed Central

    Freudenau, Inga; Lutter, Petra; Baier, Ruth; Schleef, Martin; Bednarz, Hanna; Lara, Alvaro R.; Niehaus, Karsten

    2015-01-01

    Plasmids have become very important as pharmaceutical gene vectors in the fields of gene therapy and genetic vaccination in the past years. In this study, we present a dynamic model to simulate the ColE1-like plasmid replication control, once for a DH5α-strain carrying a low copy plasmid (DH5α-pSUP 201-3) and once for a DH5α-strain carrying a high copy plasmid (DH5α-pCMV-lacZ) by using ordinary differential equations and the MATLAB software. The model includes the plasmid replication control by two regulatory RNA molecules (RNAI and RNAII) as well as the replication control by uncharged tRNA molecules. To validate the model, experimental data like RNAI- and RNAII concentration, plasmid copy number (PCN), and growth rate for three different time points in the exponential phase were determined. Depending on the sampled time point, the measured RNAI- and RNAII concentrations for DH5α-pSUP 201-3 reside between 6 ± 0.7 and 34 ± 7 RNAI molecules per cell and 0.44 ± 0.1 and 3 ± 0.9 RNAII molecules per cell. The determined PCNs averaged between 46 ± 26 and 48 ± 30 plasmids per cell. The experimentally determined data for DH5α-pCMV-lacZ reside between 345 ± 203 and 1086 ± 298 RNAI molecules per cell and 22 ± 2 and 75 ± 10 RNAII molecules per cell with an averaged PCN of 1514 ± 1301 and 5806 ± 4828 depending on the measured time point. As the model was shown to be consistent with the experimentally determined data, measured at three different time points within the growth of the same strain, we performed predictive simulations concerning the effect of uncharged tRNA molecules on the ColE1-like plasmid replication control. The hypothesis is that these tRNA molecules would have an enhancing effect on the plasmid production. The in silico analysis predicts that uncharged tRNA molecules would indeed increase the plasmid DNA production. PMID:26389114

  2. Recombinant plasmids for encoding restriction enzymes DpnI and DpnII of streptococcus pneumontae

    DOEpatents

    Lacks, Sanford A.

    1990-01-01

    Chromosomal DNA cassettes containing genes encoding either the DpnI or DpnII restriction endonucleases from Streptococcus pneumoniae are cloned into a streptococcal vector, pLS101. Large amounts of the restriction enzymes are produced by cells containing the multicopy plasmids, pLS202 and pLS207, and their derivatives pLS201, pLS211, pLS217, pLS251 and pLS252.

  3. Recombinant plasmids for encoding restriction enzymes DpnI and DpnII of Streptococcus pneumontae

    DOEpatents

    Lacks, S.A.

    1990-10-02

    Chromosomal DNA cassettes containing genes encoding either the DpnI or DpnII restriction endonucleases from Streptococcus pneumoniae are cloned into a streptococcal vector, pLS101. Large amounts of the restriction enzymes are produced by cells containing the multicopy plasmids, pLS202 and pLS207, and their derivatives pLS201, pLS211, pLS217, pLS251 and pLS252. 9 figs.

  4. Poly(lactic acid)-poly(ethylene glycol) nanoparticles as new carriers for the delivery of plasmid DNA.

    PubMed

    Perez, C; Sanchez, A; Putnam, D; Ting, D; Langer, R; Alonso, M J

    2001-07-10

    The purpose of the present work was to produce and characterize poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) nanoparticles (size lower than 300 nm) containing a high loading of plasmid DNA in a free form or co-encapsulated with either poly(vinyl alcohol) (PVA) or poly(vinylpyrrolidone) (PVP). The plasmid alone or with PVA or PVP was encapsulated by two different techniques: an optimized w/o/w emulsion-solvent evaporation technique as well as by a new w/o emulsion-solvent diffusion technique. Particle size, zeta potential, plasmid DNA loading and in vitro release were determined for the three plasmid-loaded formulations. The influence of the initial plasmid loadings (5, 10, 20 microg plasmid DNA/mg PLA-PEG) on those parameters was also investigated. The plasmid loaded into the nanoparticles and released in vitro was quantified by fluorimetry and the different molecular forms were identified by gel electrophoresis. PLA-PEG nanoparticles containing plasmid DNA in a free form or co-encapsulated with PVA or PVP were obtained in the range size of 150-300 nm and with a negative zeta potential, both parameters being affected by the preparation technique. Encapsulation efficiencies were high irrespective of the presence of PVA or PVP (60-90%) and were slightly affected by the preparation technique and by the initial loading. The final plasmid DNA loading in the nanoparticles was up to 10-12 microg plasmid DNA/mg polymer. Plasmid DNA release kinetics varied depending on the plasmid incorporation technique: nanoparticles prepared by the w/o diffusion technique released their content rapidly whereas those obtained by the w/o/w showed an initial burst followed by a slow release for at least 28 days. No significant influence of the plasmid DNA loading and of the co-encapsulation of PVP or PVA on the in vitro release rate was observed. In all cases the conversion of the supercoiled form to the open circular and linear forms was detected. In conclusion, plasmid DNA can be

  5. Adsorption of plasmid DNA to mineral surfaces and protection against DNase I.

    PubMed Central

    Romanowski, G; Lorenz, M G; Wackernagel, W

    1991-01-01

    The adsorption of [3H]thymidine-labeled plasmid DNA (pHC314; 2.4 kb) of different conformations to chemically pure sand was studied in a flowthrough microenvironment. The extent of adsorption was affected by the concentration and valency of cations, indicating a charge-dependent process. Bivalent cations (Mg2+, Ca2+) were 100-fold more effective than monovalent cations (Na+, K+, NH4+). Quantitative adsorption of up to 1 microgram of negatively supercoiled or linearized plasmid DNA to 0.7 g of sand was observed in the presence of 5 mM MgCl2 at pH 7. Under these conditions, more than 85% of DNA adsorbed within 60 s. Maximum adsorption was 4 micrograms of DNA to 0.7 g of sand. Supercoil molecules adsorbed slightly less than linearized or open circular plasmids. An increase of the pH from 5 to 9 decreased adsorption at 0.5 mM MgCl2 about eightfold. It is concluded that adsorption of plasmid DNA to sand depends on the neutralization of negative charges on the DNA molecules and the mineral surfaces by cations. The results are discussed on the grounds of the polyelectrolyte adsorption model. Sand-adsorbed DNA was 100 times more resistant against DNase I than was DNA free in solution. The data support the idea that plasmid DNA can enter the extracellular bacterial gene pool which is located at mineral surfaces in natural bacterial habitats. PMID:1647748

  6. Plasmids as Tools for Containment.

    PubMed

    García, José L; Díaz, Eduardo

    2014-10-01

    Active containment systems are a major tool for reducing the uncertainty associated with the introduction of monocultures, genetically engineered or not, into target habitats for a large number of biotechnological applications (e.g., bioremediation, bioleaching, biopesticides, biofuels, biotransformations, live vaccines, etc.). While biological containment reduces the survival of the introduced organism outside the target habitat and/or upon completion of the projected task, gene containment strategies reduce the lateral spread of the key genetic determinants to indigenous microorganisms. In fundamental research, suicide circuits become relevant tools to address the role of gene transfer, mainly plasmid transfer, in evolution and how this transfer contributes to genome plasticity and to the rapid adaptation of microbial communities to environmental changes. Many lethal functions and regulatory circuits have been used and combined to design efficient containment systems. As many new genomes are being sequenced, novel lethal genes and regulatory elements are available, e.g., new toxin-antitoxin modules, and they could be used to increase further the current containment efficiencies and to expand containment to other organisms. Although the current containment systems can increase the predictability of genetically modified organisms in the environment, containment will never be absolute, due to the existence of mutations that lead to the appearance of surviving subpopulations. In this sense, orthogonal systems (xenobiology) appear to be the solution for setting a functional genetic firewall that will allow absolute containment of recombinant organisms. PMID:26104372

  7. Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with various plasmids.

    PubMed

    Serror, Pascale; Sasaki, Takashi; Ehrlich, S Dusko; Maguin, Emmanuelle

    2002-01-01

    We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 10(4) transformants per microg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a good basis for developing molecular tools for L. delbrueckii and open the field of genetic studies in L. delbrueckii.

  8. Using Plasmids as DNA Vaccines for Infectious Diseases.

    PubMed

    Tregoning, John S; Kinnear, Ekaterina

    2014-12-01

    DNA plasmids can be used to induce a protective (or therapeutic) immune response by delivering genes encoding vaccine antigens. That naked DNA (without the refinement of coat proteins or host evasion systems) can cross from outside the cell into the nucleus and be expressed is particularly remarkable given the sophistication of the immune system in preventing infection by pathogens. As a result of the ease, low cost, and speed of custom gene synthesis, DNA vaccines dangle a tantalizing prospect of the next wave of vaccine technology, promising individual designer vaccines for cancer or mass vaccines with a rapid response time to emerging pandemics. There is considerable enthusiasm for the use of DNA vaccination as an approach, but this enthusiasm should be tempered by the successive failures in clinical trials to induce a potent immune response. The technology is evolving with the development of improved delivery systems that increase expression levels, particularly electroporation and the incorporation of genetically encoded adjuvants. This review will introduce some key concepts in the use of DNA plasmids as vaccines, including how the DNA enters the cell and is expressed, how it induces an immune response, and a summary of clinical trials with DNA vaccines. The review also explores the advances being made in vector design, delivery, formulation, and adjuvants to try to realize the promise of this technology for new vaccines. If the immunogenicity and expression barriers can be cracked, then DNA vaccines may offer a step change in mass vaccination.

  9. Mining Environmental Plasmids for Synthetic Biology Parts and Devices.

    PubMed

    Martínez-García, Esteban; Benedetti, Ilaria; Hueso, Angeles; De Lorenzo, Víctor

    2015-02-01

    The scientific and technical ambition of contemporary synthetic biology is the engineering of biological objects with a degree of predictability comparable to those made through electric and industrial manufacturing. To this end, biological parts with given specifications are sequence-edited, standardized, and combined into devices, which are assembled into complete systems. This goal, however, faces the customary context dependency of biological ingredients and their amenability to mutation. Biological orthogonality (i.e., the ability to run a function in a fashion minimally influenced by the host) is thus a desirable trait in any deeply engineered construct. Promiscuous conjugative plasmids found in environmental bacteria have evolved precisely to autonomously deploy their encoded activities in a variety of hosts, and thus they become excellent sources of basic building blocks for genetic and metabolic circuits. In this article we review a number of such reusable functions that originated in environmental plasmids and keep their properties and functional parameters in a variety of hosts. The properties encoded in the corresponding sequences include inter alia origins of replication, DNA transfer machineries, toxin-antitoxin systems, antibiotic selection markers, site-specific recombinases, effector-dependent transcriptional regulators (with their cognate promoters), and metabolic genes and operons. Several of these sequences have been standardized as BioBricks and/or as components of the SEVA (Standard European Vector Architecture) collection. Such formatting facilitates their physical composability, which is aimed at designing and deploying complex genetic constructs with new-to-nature properties. PMID:26104565

  10. Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae

    PubMed Central

    Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

    2011-01-01

    Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3+ clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway. PMID:21853137

  11. SIMPLAS: A Simulation of Bacterial Plasmid Maintenance.

    ERIC Educational Resources Information Center

    Dunn, A.; And Others

    1988-01-01

    This article describes a computer simulation of bacterial physiology during growth in a chemostat. The program was designed to help students to appreciate and understand the related effects of parameters which influence plasmid persistence in bacterial populations. (CW)

  12. Protein diversity confers specificity in plasmid segregation.

    PubMed

    Fothergill, Timothy J G; Barillà, Daniela; Hayes, Finbarr

    2005-04-01

    The ParG segregation protein (8.6 kDa) of multidrug resistance plasmid TP228 is a homodimeric DNA-binding factor. The ParG dimer consists of intertwined C-terminal domains that adopt a ribbon-helix-helix architecture and a pair of flexible, unstructured N-terminal tails. A variety of plasmids possess partition loci with similar organizations to that of TP228, but instead of ParG homologs, these plasmids specify a diversity of unrelated, but similarly sized, partition proteins. These include the proteobacterial pTAR, pVT745, and pB171 plasmids. The ParG analogs of these plasmids were characterized in parallel with the ParG homolog encoded by the pseudomonal plasmid pVS1. Like ParG, the four proteins are dimeric. No heterodimerization was detectable in vivo among the proteins nor with the prototypical ParG protein, suggesting that monomer-monomer interactions are specific among the five proteins. Nevertheless, as with ParG, the ParG analogs all possess significant amounts of unordered amino acid residues, potentially highlighting a common structural link among the proteins. Furthermore, the ParG analogs bind specifically to the DNA regions located upstream of their homologous parF-like genes. These nucleoprotein interactions are largely restricted to cognate protein-DNA pairs. The results reveal that the partition complexes of these and related plasmids have recruited disparate DNA-binding factors that provide a layer of specificity to the macromolecular interactions that mediate plasmid segregation. PMID:15805511

  13. Comparison of 10 IncP plasmids: homology in the regions involved in plasmid replication.

    PubMed Central

    Chikami, G K; Guiney, D G; Schmidhauser, T J; Helinski, D R

    1985-01-01

    We have examined the DNA homology in the replication regions of 10 IncP plasmids independently isolated from several different countries. Two regions of RK2, the best-studied plasmid of this group, are required for vegetative DNA replication: the origin of replication (oriV) and the trfA region, which codes for a gene product necessary for replication. Six of nine IncP plasmids studied were identical to RK2 in the oriV and trfA regions as shown by Southern hybridization. Three P plasmids, R751, R772, and R906, showed weaker homology with the RK2 trfA, region and hybridized to different-sized HaeII fragments than the other six plasmids. R751, R772, and R906 hybridized to the region of the RK2 replication origin which expresses P incompatibility but differed markedly from RK2 and the other six plasmids in the GC-rich region of the origin required for replication. These data indicate that the P-group plasmids can be divided into two subgroups: IncP alpha, which includes the RK2-like plasmids, and IncP beta which includes the R751-like plasmids. Images PMID:2985542

  14. Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

  15. Plasmid-mediated mineralization of 4-chlorobiphenyl.

    PubMed Central

    Shields, M S; Hooper, S W; Sayler, G S

    1985-01-01

    Strains of Alcaligenes and Acinetobacter spp. were isolated from a mixed culture already proven to be proficient at complete mineralization of monohalogenated biphenyls. These strains were shown to harbor a 35 X 10(6)-dalton plasmid mediating a complete pathway for 4-chlorobiphenyl (4CB) oxidation. Subsequent plasmid curing of these bacteria resulted in the abolishment of the 4CB mineralization phenotype and loss of even early 4CB metabolism by Acinetobacter spp. Reestablishment of the Alcaligenes plasmid, denoted pSS50, in the cured Acinetobacter spp. via filter surface mating resulted in the restoration of 4CB mineralization abilities. 4CB mineralization, however, proved to be an unstable characteristic in some subcultured strains. Such loss was not found to coincide with any detectable alteration in plasmid size. Cultures capable of complete mineralization, as well as those limited to partial metabolism of 4CB, produced 4-chlorobenzoate as a metabolite. Demonstration of mineralization of a purified 14C-labeled chlorobenzoate showed it to be a true intermediate in 4CB mineralization. Unlike the mineralization capability, the ability to produce a metabolite has proven to be stable on subculture. These results indicate the occurrence of a novel plasmid, or evolved catabolic plasmid, that mediates the complete mineralization of 4CB. Images PMID:2993249

  16. Clostridium perfringens type A-E toxin plasmids.

    PubMed

    Freedman, John C; Theoret, James R; Wisniewski, Jessica A; Uzal, Francisco A; Rood, Julian I; McClane, Bruce A

    2015-05-01

    Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell.

  17. Kinetic modeling of plasmid bioproduction in Escherichia coli DH5α cultures over different carbon-source compositions.

    PubMed

    Lopes, Marta B; Martins, Gabriel; Calado, Cecília R C

    2014-09-30

    The need for the development of economic high plasmid production in Escherichia coli cultures is emerging, as a result of the latest advances in DNA vaccination and gene therapy. In order to contribute to achieve that, a model describing the kinetics involved in the bioproduction of plasmid by recombinant E. coli DH5α is presented, as an attempt to understand the complex and non-linear metabolic relationships and the plasmid production occurring in dynamic batch culture environments, run under different media compositions of glucose and glycerol, that result in distinct maximum biomass growths (between 8.2 and 12.8 g DCW/L) and specific plasmid productions (between 1.1 and 7.4 mg/g DCW). The model based on mass balance equations for biomass, glucose, glycerol, acetate and plasmid accurately described different culture behaviors, using either glucose or glycerol as carbon source, or mixtures of both. From the 17 parameters obtained after model simplification, the following 10 parameters were found to be independent of the carbon source composition: the substrate affinity constants, the inhibitory constants of biomass growth on glycerol by glucose, of biomass growth on acetate by glycerol and the global biomass growth by acetate, and the yields of biomass on acetate, acetate on glucose and glycerol, and plasmid on glucose. The parameters that depend on the culture composition, and that might explain the differences found between cultures, were: maximum specific growth rates on glucose, glycerol and acetate; biomass yield on glucose and glycerol; and plasmid yield on glycerol and acetate. Moreover, a crucial role of acetate in the plasmid production was revealed by the model, with most of plasmid production being associated to the acetate consumption. The model provides meaningful insight on the E. coli dynamic cell behavior concerning the plasmid bioproduction, which might lead to important guidelines for culture optimization and process scale-up and control.

  18. Monitoring plasmid replication in live mammalian cells over multiple generations by fluorescence microscopy.

    PubMed

    Norby, Kathryn; Chiu, Ya-Fang; Sugden, Bill

    2012-01-01

    Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies (1-3). These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres (4-8). Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell. Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest (9). The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed. This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to

  19. A highly effective and adjustable dual plasmid system for O-GlcNAcylated recombinant protein production in E. coli.

    PubMed

    Han, Cuifang; Shan, Hui; Bi, Chuanlin; Zhang, Xinling; Qi, Jieqiong; Zhang, Boyuan; Gu, Yuchao; Yu, Wengong

    2015-06-01

    O-GlcNAcylation is a ubiquitous, dynamic and reversible post-translational protein modification in metazoans, and it is catalysed and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase, respectively. Prokaryotes lack endogenous OGT activity. It has been reported that coexpression of mammalian OGT with its target substrates in Escherichia coli produce O-GlcNAcylated recombinant proteins, but the plasmids used were not compatible, and the expression of both OGT and its target protein were induced by the same inducer. Here, we describe a compatible dual plasmid system for coexpression of OGT and its target substrate for O-GlcNAcylated protein production in E. coli. The approach was validated using the CKII and p53 protein as control. This compatible dual plasmid system contains an arabinose-inducible OGT expression vector with a pUC origin and an isopropyl β-d-thiogalactopyranoside-inducible OGT target substrate expression vector bearing a p15A origin. The dual plasmid system produces recombinant proteins with varying O-GlcNAcylation levels by altering the inducer concentration. More importantly, the O-GlcNAcylation efficiency was much higher than the previously reported system. Altogether, we established an adjustable compatible dual plasmid system that can effectively yield O-GlcNAcylated proteins in E. coli.

  20. Artificial plasmid labeled with 5-bromo-2'-deoxyuridine: a universal molecular system for strand break detection.

    PubMed

    Zylicz-Stachula, Agnieszka; Polska, Katarzyna; Skowron, Piotr; Rak, Janusz

    2014-07-01

    DNA strand breaks (SBs) are among the most cytotoxic forms of DNA damage, and their residual levels correlate directly with cell death. Hence, the type and amount of SBs is directly related to the efficacy of a given anticancer therapy. In this study, we describe a molecular tool that can differentiate between single (SSBs) and double (DSBs) strand breaks and also assess them quantitatively. Our method involves PCR amplification of a linear DNA fragment labeled with a sensitizing nucleotide, circularization of that fragment, and enzymatic introduction of supercoils to transform the circular relaxed form of the synthesized plasmid into a supercoiled one. After exposure of the molecule to a damaging factor, SSB and DSB levels can be easily assayed with gel electrophoresis. We applied this method to prepare an artificial plasmid labeled with 5-bromo-2'-deoxyuridine and to assay SBs photoinduced in the synthesized plasmid.

  1. Transduction of plasmid DNA in Streptomyces spp. and related genera by bacteriophage FP43.

    PubMed

    McHenney, M A; Baltz, R H

    1988-05-01

    A segment (hft) of bacteriophage FP43 DNA cloned into plasmid pIJ702 mediated high-frequency transduction of the resulting plasmid (pRHB101) by FP43 in Streptomyces griseofuscus. The transducing particles contained linear concatemers of plasmid DNA. Lysates of FP43 prepared on S. griseofuscus containing pRHB101 also transduced many other Streptomyces species, including several that restrict plaque formation by FP43 and at least two that produce restriction endonucleases that cut pRHB101 DNA. Transduction efficiencies in different species were influenced by the addition of anti-FP43 antiserum to the transduction plates, the temperature for cell growth before transduction, the multiplicity of infection, and the host on which the transducing lysate was prepared. FP43 lysates prepared on S. griseofuscus(pRHB101) also transduced species of Streptoverticillium, Chainia, and Saccharopolyspora.

  2. Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204.

    PubMed Central

    Eaton, R W; Timmis, K N

    1986-01-01

    A Pseudomonas putida strain designated RE204, able to utilize isopropylbenzene as the sole carbon and energy source, was isolated. Tn5 transposon mutagenesis by means of the suicide transposon donor plasmid pLG221 yielded mutant derivatives defective in isopropylbenzene metabolism. These were characterized by the identification of the products which they accumulated when grown in the presence of isopropylbenzene and by the assay of enzyme activities in cell extracts. Based on the results obtained, the following metabolic pathway is proposed: isopropylbenzene----2,3-dihydro -2,3-dihydroxyisopropylbenzene----3-isopropylcatechol----2 -hydroxy-6-oxo-7-methylocta-2,4-dienoate----isobutyrate + 2-oxopent-4-enoate----amphibolic intermediates. Plasmid DNA was isolated from strain RE204 and mutant derivatives and characterized by restriction enzyme cleavage analysis. Isopropylbenzene-negative isolates carried a Tn5 insert within a 15-kilobase region of a 105-kilobase plasmid designated pRE4. DNA fragments of pRE4 carrying genes encoding isopropylbenzene catabolic enzymes were cloned in Escherichia coli with various plasmid vectors; clones were identified by (i) selection for Tn5-encoded kanamycin resistance in the case of Tn5 mutant plasmids, (ii) screening for isopropylbenzene dioxygenase-catalyzed oxidation of indole to indigo, and (iii) use of a Tn5-carrying restriction fragment, derived from a pRE4::Tn5 mutant plasmid, as a probe for clones carrying wild-type restriction fragments. These clones were subsequently used to generate a transposon insertion and restriction enzyme cleavage map of the isopropylbenzene metabolic region of pRE4. Images PMID:3019995

  3. Protection from ischemic heart injury by a vigilant heme oxygenase-1 plasmid system.

    PubMed

    Tang, Yao Liang; Tang, Yi; Zhang, Y Clare; Qian, Keping; Shen, Leping; Phillips, M Ian

    2004-04-01

    Although human heme oxygenase-1 (hHO-1) could provide a useful approach for cellular protection in the ischemic heart, constitutive overexpression of hHO-1 may lead to unwanted side effects. To avoid this, we designed a hypoxia-regulated hHO-1 gene therapy system that can be switched on and off. This vigilant plasmid system is composed of myosin light chain-2v promoter and a gene switch that is based on an oxygen-dependent degradation domain from the hypoxia inducible factor-1-alpha. The vector can sense ischemia and switch on the hHO-1 gene system, specifically in the heart. In an in vivo experiment, the vigilant hHO-1 plasmid or saline was injected intramyocardially into myocardial infarction mice or sham operation mice. After gene transfer, expression of hHO-1 was only detected in the ischemic heart treated with vigilant hHO-1 plasmids. Masson trichrome staining showed significantly fewer fibrotic areas in vigilant hHO-1 plasmids-treated mice compared with saline control (43.0%+/-4.8% versus 62.5%+/-3.3%, P<0.01). The reduction of interstitial fibrosis is accompanied by an increase in myocardial hHO-1 expression in peri-infarct border areas, concomitant with higher Bcl-2 levels and lower Bax, Bak, and caspase 3 levels in the ischemic myocardium compared with saline control. By use of a cardiac catheter, heart from vigilant hHO-1 plasmids-treated mice showed improved recovery of contractile and diastolic performance after myocardial infarction compared with saline control. This study documents the beneficial regulation and therapeutic potential of vigilant plasmid-mediated hHO-1 gene transfer. This novel gene transfer strategy can provide cardiac-specific protection from future repeated bouts of ischemic injury.

  4. Cloning, nucleotide sequence, and expression of the chromate resistance determinant of Pseudomonas aeruginosa plasmid pUM505.

    PubMed Central

    Cervantes, C; Ohtake, H; Chu, L; Misra, T K; Silver, S

    1990-01-01

    The chromate resistance determinant of Pseudomonas aeruginosa plasmid pUM505 was cloned into broad-host-range vector pSUP104. The hybrid plasmid containing an 11.1-kilobase insert conferred chromate resistance and reduced uptake of chromate in P. aeruginosa PAO1. Resistance to chromate was not expressed in Escherichia coli. Contiguous 1.6- and 6.3-kilobase HindIII fragments from this plasmid hybridized to pUM505 but not to P. aeruginosa chromosomal DNA and only weakly to chromate resistance plasmids pLHB1 and pMG6. Further subcloning produced a plasmid with an insert of 2,145 base pairs, which was sequenced. Analysis of deletions revealed that a single open reading frame was sufficient to determine chromate resistance. This open reading frame encodes a highly hydrophobic polypeptide, ChrA, of 416 amino acid residues that appeared to be expressed in E. coli under control of the T7 promoter. No significant homology was found between ChrA and proteins in the amino acid sequence libraries, but 29% amino acid identity was found with the ChrA amino acid sequence for another chromate resistance determinant sequenced in this laboratory from an Alcaligenes eutrophus plasmid (A. Nies, D. Nies, and S. Silver, submitted for publication). Images FIG. 3 FIG. 5 PMID:2152903

  5. Hyperbranched polysiloxysilane nanoparticles for nonviral gene delivery vectors and nanoprobes

    NASA Astrophysics Data System (ADS)

    Kim, Won Jin; Bonoiu, Adela C.; Lee, Kwang-Sup; Hayakawa, Teruaki; Xia, Cheng; Kakimoto, Masa-aki; Pudavar, Haridas E.; Prasad, Paras N.

    2009-08-01

    We report an approach to produce predefined surface charge tunable gene delivery vectors using siloxysilsane-based polymer for gene delivery studies. To obtain nonviral vectors, new series of hyperbranched polysiloxysilane (HBPS) were synthesized, and the end groups in polymer structures have modified with hydrophilic molecules; in other words, carboxylic acid and quaternary ammonium groups were employed into terminal structures to give the amphiphilicity. The novelty of these amphiphilic HBPS polymers lies in the fact that nanoparticles with different zeta potential (surface charge density) can be easily tailored and functionalized. These polymeric nanoparticles which containing various chemical groups on the surface indicated altered surface charge distributions (from -40 to +64mV). Finally, the use of these nanoparticles as efficient gene delivery vectors was demonstrated by means of in vitro transfection study using β- galactosidase plasmid and pEGFP-N1 plasmid, and the most efficient combination was obtained using HBPS-CN30:70.

  6. FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium-Copy Number Plasmids in Escherichia coli.

    PubMed

    Ali, Syed A; Chew, Yik Wei

    2015-01-01

    Antibiotic resistance genes and antibiotics are frequently used to maintain plasmid vectors in bacterial hosts such as Escherichia coli. Due to the risk of spread of antibiotic resistance, the regulatory authorities discourage the use of antibiotic resistance genes/antibiotics for the maintenance of plasmid vectors in certain biotechnology applications. Overexpression of E. coli endogenous fabI gene and subsequent selection on Triclosan has been proposed as a practical alternative to traditional antibiotic selection systems. Unfortunately, overexpression of fabI cannot be used to select medium-copy number plasmids, typically used for the expression of heterologous proteins in E. coli. Here we report that Vibrio cholera FabV, a functional homologue of E. coli FabI, can be used as a suitable marker for the selection and maintenance of both high and medium-copy number plasmid vectors in E. coli.

  7. TraG from RP4 and TraG and VirD4 from Ti Plasmids Confer Relaxosome Specificity to the Conjugal Transfer System of pTiC58

    PubMed Central

    Hamilton, Claire M.; Lee, Hyewon; Li, Pei-Li; Cook, David M.; Piper, Kevin R.; von Bodman, Susanne Beck; Lanka, Erich; Ream, Walt; Farrand, Stephen K.

    2000-01-01

    Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraGRP4) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraGRP4. A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraGRP4 was expressed in the donors. Mutations in traGRP4 with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraGRP4 nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traGRP4 mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraGRP4-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraGRP4 and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the

  8. Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA

    PubMed Central

    Case, Mary E.; Schweizer, Michael; Kushner, Sidney R.; Giles, Norman H.

    1979-01-01

    An efficient transformation system has been developed for Neurospora crassa that uses spheroplasts and pVK88 plasmid DNA. pVK88 is a recombinant Escherichia coli plasmid carrying the N. crassa qa-2+ gene which encodes catabolic dehydroquinase (3-dehydroquinate hydro-lyase, EC 4.2.1.10) and is part of the qa gene cluster. The recipient strain carries a stable qa-2- mutation and an arom-9- mutation, thus lacking both catabolic and biosynthetic dehydroquinase activities. Transformants were selected as colonies able to grow in the absence of an aromatic amino acid supplement. These colonies were qa-2+ and had normal levels of catabolic dehydroquinase. DNA·DNA hybridization evidence with appropriate labeled probes indicates clearly that in some instances transformation involves the integration of bacterial plasmid sequences together with the qa-2+ gene into the N. crassa genome. On the basis of genetic, enzyme assay, and DNA hybridization data, at least three types of transformation events can be distinguished: (i) replacement of the qa-2- gene by the qa-2+ gene without any effect on the expression of the other genes in the qa cluster, (ii) linked insertion of a normal qa-2+ gene accompanied by inactivation of the adjacent qa-4+ gene, and (iii) insertion of a normal qa-2+ gene at an unlinked site in the N. crassa genome. This newly integrated qa-2+ genetic material is inherited in a typical Mendelian fashion. A low level of transformation has also been obtained by using linear total N. crassa DNA. Two such qa-2+ transformants are unlinked to the qa-2- gene of the recipient. Images PMID:159454

  9. Deletions at short direct repeats and base substitutions are characteristic mutations for bleomycin-induced double- and single-strand breaks, respectively, in a human shuttle vector system

    NASA Technical Reports Server (NTRS)

    Dar, M. E.; Jorgensen, T. J.

    1995-01-01

    Using the radiomimetic drug, bleomycin, we have determined the mutagenic potential of DNA strand breaks in the shuttle vector pZ189 in human fibroblasts. The bleomycin treatment conditions used produce strand breaks with 3'-phosphoglycolate termini as > 95% of the detectable dose-dependent lesions. Breaks with this end group represent 50% of the strand break damage produced by ionizing radiation. We report that such strand breaks are mutagenic lesions. The type of mutation produced is largely determined by the type of strand break on the plasmid (i.e. single versus double). Mutagenesis studies with purified DNA forms showed that nicked plasmids (i.e. those containing single-strand breaks) predominantly produce base substitutions, the majority of which are multiples, which presumably originate from error-prone polymerase activity at strand break sites. In contrast, repair of linear plasmids (i.e. those containing double-strand breaks) mainly results in deletions at short direct repeat sequences, indicating the involvement of illegitimate recombination. The data characterize the nature of mutations produced by single- and double-strand breaks in human cells, and suggests that deletions at direct repeats may be a 'signature' mutation for the processing of DNA double-strand breaks.

  10. CRISPR/Cas9-based genome editing in mice by single plasmid injection.

    PubMed

    Fujihara, Yoshitaka; Ikawa, Masahito

    2014-01-01

    CRISPR/Cas-mediated genome modification has opened a new era for elucidating gene function. Gene knockout mice can be generated by injecting humanized Cas9 (hCas9) mRNA and guide RNA (sgRNA) into fertilized eggs. However, delivery of RNA instead of DNA to the fertilized oocyte requires extra preparation and extra care with storage. To simplify the method of delivery, we injected the circular pX330 plasmids expressing both hCas9 and sgRNA and found that mutant mice were generated as efficiently as with RNA injection. Different from the linearized plasmid, the circular plasmid decreased the chance of integration into the host genome. We also developed the pCAG-EGxxFP reporter plasmid for evaluating the sgRNA activity by observing EGFP fluorescence in HEK293T cells. The combination of these techniques allowed us to develop a rapid, easy, and reproducible strategy for targeted mutagenesis in living mice. This chapter provides an experimental protocol for the design of sgRNAs, the construction of pX330-sgRNA and pCAG-EGxxFP-target plasmids, the validation of cleavage efficiency in vitro, and the generation of targeted gene mutant mice. These mice can be generated within a month.

  11. Human clinical trials of plasmid DNA vaccines.

    PubMed

    Liu, Margaret A; Ulmer, Jeffrey B

    2005-01-01

    This article gives an overview of DNA vaccines with specific emphasis on the development of DNA vaccines for clinical trials and an overview of those trials. It describes the preclinical research that demonstrated the efficacy of DNA vaccines as well as an explication of the immunologic mechanisms of action. These include the induction of cognate immune responses, such as the generation of cytolytic T lymphocytes (CTL) as well as the effect of the plasmid DNA upon the innate immune system. Specific issues related to the development of DNA as a product candidate are then discussed, including the manufacture of plasmid, the qualification of the plasmid DNA product, and the safety testing necessary for initiating clinical trials. Various human clinical trials for infectious diseases and cancer have been initiated or completed, and an overview of these trials is given. Finally, because the early clinical trials have shown less than optimal immunogenicity, methods to increase the potency of the vaccines are described. PMID:16291211

  12. Distribution of small native plasmids in Streptococcus pyogenes in India.

    PubMed

    Bergmann, René; Nerlich, Andreas; Chhatwal, Gursharan S; Nitsche-Schmitz, D Patric

    2014-05-01

    Complete characterization of a Streptococcus pyogenes population from a defined geographic region comprises information on the plasmids that circulate in these bacteria. Therefore, we determined the distribution of small plasmids (<5kb) in a collection of 279 S. pyogenes isolates from India, where diversity of strains and incidence rates of S. pyogenes infections are high. The collection comprised 77 emm-types. For plasmid detection and discrimination, we developed PCRs for different plasmid replication initiation protein genes, the putative repressor gene copG and bacteriocin genes dysA and scnM57. Plasmid distribution was limited to 13 emm-types. Co-detection analysis using aforementioned PCRs revealed four distinct plasmid sub-types, two of which were previously unknown. Representative plasmids pA852 and pA996 of the two uncharacterized plasmid sub-types were sequenced. These two plasmids could be assigned to the pMV158 and the pC194/pUB110 family of rolling-circle plasmids, respectively. The majority of small plasmids found in India belonged to the two newly characterized sub-types, with pA852- and pA996-like plasmids amounting to 42% and 22% of all detected plasmids, respectively. None of the detected plasmids coded for a known antibiotic resistance gene. Instead, all of the four plasmid sub-types carried known or potential bacteriocin genes. These genes may have influence on the evolutionary success of certain S. pyogenes genotypes. Notably, pA852-like plasmids were found in all isolates of the most prevalent emm-type 11.0. Together, a priori fitness of this genotype and increased fitness due to the acquired plasmids may have rendered type emm11.0 successful and caused the prevalence of pA852-like plasmids in India.

  13. Construction of a new GFP vector and its use for Fusaruim oxysporum transformation

    PubMed Central

    Zhang, Shuo; Zhao, Baixia; Liu, Xian; Gao, Zenggui; Huang, Xinyang

    2012-01-01

    In this study, the gfp fragment as a reporter gene had integrated into the form plasmid vector pBC-hygro which contains an expressive promoter of the fungus to facilitate the transformation of Fusarium oxysporum. The resultant plasmid pBC-hygro-GFP was identified by digestion with enzymes. Binary plasmids pBC-hygro-GFP were transformed into F. oxysporum by using the PEG–CaCl2 mediated transformation technique. Results show that the recombinant plasmid pBC-hygro-GFP was constructed correctly. The gfp gene was stably maintained and did not convey any significant loss of phenotype which would affect the survival and behaviour of the tagged strains. Introduction of the gfp gene into F. oxysporum provides a simple, specific and cost-effective method of strain identification for ecological studies. Transcriptional reporter vectors were constructed for using the green fluorescent protein (GFP) reporter. PMID:23961215

  14. Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204

    SciTech Connect

    Eaton, R.W.; Timmis, K.N.

    1986-10-01

    A Pseudomonas putida strain designated RE204, able to utilize isopropylbenzene as the sole carbon and energy source, was isolated. Tn5 transposon mutagenesis by means of the suicide transposon donor plasmid pLG221 yielded mutant derivatives defective in isopropylbenzene metabolism. These were characterized by the identification of the products which they accumulated when grown in the presence of isopropylbenzene and by the assay of enzyme activities in cell extracts. Based on the results obtained, the following metabolic pathway is proposed: isopropylbenzene ..-->.. 2,3-dihydro-2,3-dihydroxyisopropylbenzene ..-->.. 3-isopropylcatechol ..-->.. 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate ..-->.. isobutyrate + 2-oxopent-4-enoate ..-->.. amphibolic intermediates. Plasmid DNA was isolated from strain RE204 and mutant derivatives and characterized by restriction enzyme cleavage analysis. Isopropylbenzene-negative isolates carried a Tn5 insert within a 15-kilobase region of a 105-kilobase plasmid designated pRE4. DNA fragments of pRE4 carrying genes encoding isopropylbenzene catabolic enzymes were cloned in Escherichia coli with various plasmid vectors. These clones were subsequently used to generate a transposon insertion and restriction enzyme cleavage map of the isopropylbenzene metabolic region of pRE4.

  15. Plasmid-borne prokaryotic gene expression: Sources of variability and quantitative system characterization

    NASA Astrophysics Data System (ADS)

    Bagh, Sangram; Mazumder, Mostafizur; Velauthapillai, Tharsan; Sardana, Vandit; Dong, Guang Qiang; Movva, Ashok B.; Lim, Len H.; McMillen, David R.

    2008-02-01

    One aim of synthetic biology is to exert systematic control over cellular behavior, either for medical purposes or to “program” microorganisms. An engineering approach to the design of biological controllers demands a quantitative understanding of the dynamics of both the system to be controlled and the controllers themselves. Here we focus on a widely used method of exerting control in bacterial cells: plasmid vectors bearing gene-promoter pairs. We study two variants of the simplest such element, an unregulated promoter constitutively expressing its gene, against the varying genomic background of four Escherichia coli cell strains. Absolute protein numbers and rates of expression vary with both cell strain and plasmid type, as does the variability of expression across the population. Total variability is most strongly coupled to the cell division process, and after cell size is scaled away, plasmid copy number regulation emerges as a significant effect. We present simple models that capture the main features of the system behavior. Our results confirm that complex interactions between plasmids and their hosts can have significant effects on both expression and variability, even in deliberately simplified systems.

  16. Characterization of the transgenic rice event TT51-1 and construction of a reference plasmid.

    PubMed

    Cao, Yinglong; Wu, Gang; Wu, Yuhua; Nie, Shujing; Zhang, Li; Lu, Changming

    2011-08-24

    Transgenic rice TT51-1 (BT63) is an insect resistant strain that was granted for safety certificate in China in 2009. This study characterizes the transgenic event TT51-1 using a GenomeWalker strategy. The organization of the transgenes indicated that the transgenes on two plasmids, pFHBT1 and pGL2RC7, had been integrated at the same locus. The sequence of the event TT51-1 spanned 8725 bp, including a truncated Cry1Ab/Ac cassette, an intact Cry1Ab/Ac cassette, two Amp gene segments, and an Hph gene segment. The 5' and 3' plant flanking sequences were isolated and used to locate the transgenes to chromosome 10 in TT51-1. The isolated TT51-1 fragment and a fragment of the rice PLD gene were integrated into a plasmid vector, to create plasmid pK-TT51 as a calibrator for detecting rice containing TT51-1. Analysis of unknown samples indicated that the reference plasmid was a reliable alternative to TT51-1 genomic DNA.

  17. Using Pulmozyme DNase treatment in lentiviral vector production.

    PubMed

    Shaw, Aaron; Bischof, Daniela; Jasti, Aparna; Ernstberger, Aaron; Hawkins, Troy; Cornetta, Kenneth

    2012-02-01

    In the production of lentiviral vector for clinical studies the purity of the final product is of vital importance. To remove plasmid and producer cell line DNA, investigators have incubated the vector product with Benzonase, a bacterially derived DNase. As an alternative we investigated the use of Pulmozyme, a U.S. Food and Drug Administration-approved human DNase for the treatment of cystic fibrosis, by comparing the efficiency of DNA removal from lentiviral vector preparations. A green fluorescent protein-expressing lentiviral vector was prepared by transient calcium phosphate transfection of HEK 293T cells and DNA removal was compared when treating vector after harvest or immediately after transfection. The effectiveness of DNase treatment was measured by quantitative PCR using primers for vesicular stomatitis virus glycoprotein G viral envelope plasmid. When treating the final product, 1-hr incubations (37°C) with Pulmozyme at 20 U/ml reduced plasmid DNA to undetectable levels. Longer incubations (up to 4 hr) did not improve DNA removal at lower concentrations and the effectiveness was equivalent to or better than Benzonase at 50 U/ml. Attempting to use Pulmozyme immediately after transfection, but before final medium change, as a means to decrease Pulmozyme concentration in the final product provided a 2-log reduction in DNA but was inferior to treatment at the end of production. Pulmozyme, at concentrations up to 100 U/ml, had no measurable effect on infectious titer of the final vector product. The use of Pulmozyme is likely to increase the cost of DNase treatment when preparing vector product and should be considered when generating clinical-grade vector products. PMID:22428981

  18. Spread of Plasmids Carrying Multiple GES Variants.

    PubMed

    Cuzon, Gaelle; Bogaerts, Pierre; Bauraing, Caroline; Huang, Te-Din; Bonnin, Rémy A; Glupczynski, Youri; Naas, Thierry

    2016-08-01

    Five GES-producing Enterobacteriaceae isolates that displayed an extended-spectrum β-lactamase (ESBL) phenotype harbored two GES variants: GES-7 ESBL and GES-6 carbapenemase. In all isolates, the two GES alleles were located on the same integron that was inserted into an 80-kb IncM1 self-conjugative plasmid. Whole-genome sequencing suggested in vivo horizontal gene transfer of the plasmid along with clonal diffusion of Enterobacter cloacae To our knowledge, this is the first description in Europe of clustered Enterobacteriaceae isolates carrying two GES β-lactamases, of which one has extended activity toward carbapenems. PMID:27216071

  19. Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10.

    PubMed Central

    Hill, K E; Weightman, A J; Fry, J C

    1992-01-01

    This study examined the potential of bacteria from river epilithon to mobilize a recombinant catabolic plasmid, pD10, encoding 3-chlorobenzoate degradation and kanamycin resistance. Fifty-four mobilizing plasmids were exogenously isolated by triparental matings between strains of Pseudomonas putida and epilithic bacteria from the River Taff (South Wales, United Kingdom). Frequencies for mobilization ranged from 1.7 x 10(-8) to 4.5 x 10(-3) per recipient at 20 degrees C. The sizes of the mobilizing plasmids isolated ranged from 40 kb to over 200 kb, and 19 of 54 were found to encode mercury resistance. Plasmid-encoded resistance to tetracycline and streptomycin was also found but not resistance to UV light or various heavy metals. Eight plasmids of epilithic bacteria, analyzed by comparing restriction fragmentation patterns, showed significant differences between those isolated from different independent matings. Optimal temperatures for mobilization of pD10 were between 15 and 25 degrees C. Four mercury resistance plasmids were found to be broad host range, transferring mercury resistance and mobilizing pD10 readily to representative species of beta- and gamma-purple bacteria. In general, frequencies of pD10 mobilization by plasmids of epilithic bacteria were 2 to 3 orders of magnitude lower than conjugal transfer frequencies. Thus, there is a high potential for exchange of recombinant genes introduced into the epilithon by mobilization between a variety of bacterial species. Images PMID:1599248

  20. DNA Targeting Sequence Improves Magnetic Nanoparticle-Based Plasmid DNA Transfection Efficiency in Model Neurons

    PubMed Central

    Vernon, Matthew M.; Dean, David A.; Dobson, Jon

    2015-01-01

    Efficient non-viral plasmid DNA transfection of most stem cells, progenitor cells and primary cell lines currently presents an obstacle for many applications within gene therapy research. From a standpoint of efficiency and cell viability, magnetic nanoparticle-based DNA transfection is a promising gene vectoring technique because it has demonstrated rapid and improved transfection outcomes when compared to alternative non-viral methods. Recently, our research group introduced oscillating magnet arrays that resulted in further improvements to this novel plasmid DNA (pDNA) vectoring technology. Continued improvements to nanomagnetic transfection techniques have focused primarily on magnetic nanoparticle (MNP) functionalization and transfection parameter optimization: cell confluence, growth media, serum starvation, magnet oscillation parameters, etc. Noting that none of these parameters can assist in the nuclear translocation of delivered pDNA following MNP-pDNA complex dissociation in the cell’s cytoplasm, inclusion of a cassette feature for pDNA nuclear translocation is theoretically justified. In this study incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid improved transfection efficiency in model neurons, presumably from increased nuclear translocation. This observation became most apparent when comparing the response of the dividing SH-SY5Y precursor cell to the non-dividing and differentiated SH-SY5Y neuroblastoma cells. PMID:26287182

  1. DNA Targeting Sequence Improves Magnetic Nanoparticle-Based Plasmid DNA Transfection Efficiency in Model Neurons.

    PubMed

    Vernon, Matthew M; Dean, David A; Dobson, Jon

    2015-01-01

    Efficient non-viral plasmid DNA transfection of most stem cells, progenitor cells and primary cell lines currently presents an obstacle for many applications within gene therapy research. From a standpoint of efficiency and cell viability, magnetic nanoparticle-based DNA transfection is a promising gene vectoring technique because it has demonstrated rapid and improved transfection outcomes when compared to alternative non-viral methods. Recently, our research group introduced oscillating magnet arrays that resulted in further improvements to this novel plasmid DNA (pDNA) vectoring technology. Continued improvements to nanomagnetic transfection techniques have focused primarily on magnetic nanoparticle (MNP) functionalization and transfection parameter optimization: cell confluence, growth media, serum starvation, magnet oscillation parameters, etc. Noting that none of these parameters can assist in the nuclear translocation of delivered pDNA following MNP-pDNA complex dissociation in the cell's cytoplasm, inclusion of a cassette feature for pDNA nuclear translocation is theoretically justified. In this study incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid improved transfection efficiency in model neurons, presumably from increased nuclear translocation. This observation became most apparent when comparing the response of the dividing SH-SY5Y precursor cell to the non-dividing and differentiated SH-SY5Y neuroblastoma cells. PMID:26287182

  2. Development of a doxycycline-inducible lentiviral plasmid with an instant regulatory feature.

    PubMed

    Yang, Tian; Burrows, Christopher; Park, Jeong Hyeon

    2014-03-01

    Lentiviruses provide highly efficient gene delivery vehicles in both dividing and non-dividing cells. Inducible gene expression systems often employ a specific cell line that constitutively expresses a regulatory protein for transgene expression. As one of such inducible expression systems the Tet-On system uses a cell line expressing reverse tetracycline-responsive transcriptional activator (rtTA). The rtTA protein binds to the tetracycline-responsive element (TRE) in the promoter and activates transcription of a transgene in a doxycycline-dependent manner. To establish a universal and instant regulatory system without generating Tet-On cell lines, the cDNAs of rtTA and a testing target gene (PPM1B) were cloned in the bi-directional TRE-containing promoters. Here, we examined whether a basal leaky expression of rtTA allows instantly inducible expression of both rtTA itself and the target gene, PPM1B in a single plasmid using the two mini-CMV promoters. Transient transfection of the lentiviral plasmids into human embryonic kidney HEK293T cells showed a significant induction of PPM1B expression in response to doxycycline, suggesting that these lentiviral plasmids can be used as an instantly inducible mammalian expression vector. However, the expression of rtTA by lentiviral transduction shows a minimal expression without a consistent response to doxycycline, suggesting that the utility of these lentiviral vectors is limited. A potential solution to overcome lentiviral transgene inactivation is proposed. PMID:24727543

  3. Plasmid introduction in metal-stressed, subsurface-derived microcosms: plasmid fate and community response.

    PubMed

    Smets, Barth F; Morrow, Jayne B; Arango Pinedo, Catalina

    2003-07-01

    The nonconjugal IncQ plasmids pMOL187 and pMOL222, which contain the metal resistance-encoding genes czc and ncc, were introduced by using Escherichia coli as a transitory delivery strain into microcosms containing subsurface-derived parent materials. The microcosms were semicontinuously dosed with an artificial groundwater to set a low-carbon flux and a target metal stress (0, 10, 100, and 1,000 micro M CdCl(2)), permitting long-term community monitoring. The broad-host-range IncPalpha plasmid RP4 was also transitorily introduced into a subset of microcosms. No novel community phenotype was detected after plasmid delivery, due to the high background resistances to Cd and Ni. At fixed Cd doses, however, small but consistent increases in Cd(r) or Ni(r) density were measured due to the introduction of a single pMOL plasmid, and this effect was enhanced by the joint introduction of RP4; the effects were most significant at the highest Cd doses. The pMOL plasmids introduced could, however, be monitored via czc- and ncc-targeted infinite-dilution PCR (ID-PCR) methods, because these genes were absent from the indigenous community: long-term presence of czc (after 14 or 27 weeks) was contingent on the joint introduction of RP4, although RP4 cointroduction was not yet required to ensure retention of ncc after 8 weeks. Plasmids isolated from Ni(r) transconjugants further confirmed the presence and retention of a pMOL222-sized plasmid. ID-PCR targeting the RP4-specific trafA gene revealed retention of RP4 for at least 8 weeks. Our findings confirm plasmid transfer and long-term retention in low-carbon-flux, metal-stressed subsurface communities but indicate that the subsurface community examined has limited mobilization potential for the IncQ plasmids employed.

  4. Novel reticular cyclen-based polymer as gene vector in DNA transfection.

    PubMed

    Zhou, Li-Hong; Yang, Mu; Zhou, Hong; Zhang, Ji; Li, Kun; Xiang, Yong-Zhe; Wang, Na; Tian, Yun-Fei; Yu, Xiao-Qi

    2009-02-01

    This study provided an experimental evidence for the use of cyclen (1, 4, 7, 10-tetraazacyclododecane)-based polymer for gene delivery. The interesting interaction of the polymer with plasmid DNA was studied by using fluorescence titration, circular dichroism spectra, agarose gel electrophoresis and atomic force microscopy. It was found that polyplex was formed between the polycation and plasmid DNA. The results demonstrated that the cyclen-based polymer could act as non-viral gene vector with relatively low cytotoxicity.

  5. Improved vector for promoter screening in lactococci.

    PubMed Central

    Bojovic, B; Djordjevic, G; Topisirovic, L

    1991-01-01

    Fragments of Lactococcus lactis subsp. lactis NP45 chromosomal DNA provided promoter activity in Escherichia coli when cloned into the promoter probe vector pGKV210. Only 13% of these recombinant plasmids promoted detectable cat-86 activity when transferred to L. lactis, i.e., expressed chloramphenicol resistance. In these promoter-containing versions of pGKV210, the cat-86 gene specifies chloramphenicol-inducible chloramphenicol acetyltransferase expression. This could be a limiting factor for cloning of promoters with lower activity in L. lactis. Therefore, we have constructed a new promoter probe vector, pBV5030, with the mutated version of the cat-86 gene, which is constitutively expressed when transcriptionally activated by the insertion of a promoter. We found that in L. lactis IL1403 the constitutively expressed cat-86 gene (on a pBV5030 derivative) has four times higher activity than the inducible version of the same gene (on a pGKV210 derivative) when both have the same promoter inserted upstream of the cat-86 gene. These results suggest that plasmid pBV5030 could be a more efficient vector for the cloning of promoters from lactococci. PMID:1901705

  6. Increased tumor distribution and expression of histidine-rich plasmid polyplexes

    PubMed Central

    Leng, Qixin; Chou, Szu-Ting; Scaria, Puthupparampil V; Woodle, Martin C; Mixson, A James

    2014-01-01

    Background Selecting nonviral carriers for in vivo gene delivery is often dependent on determining the optimal carriers from transfection assays in vitro. The rationale behind this in vitro strategy is to cast a net sufficiently wide to identify the few effective carriers of plasmids for in vivo studies. Nevertheless, many effective in vivo carriers may be overlooked by this strategy because of the marked differences between in vitro and in vivo assays. Methods After solid-phase synthesis of linear and branched histidine/lysine (HK) peptides, the two peptide carriers were compared for their ability to transfect MDA-MB-435 tumor cells in vitro and then in vivo. Results By contrast to their transfection activity in vitro, the linear H2K carrier of plasmids was far more effective in vivo compared to the branch H2K4b. Surprisingly, negatively-charged polyplexes formed by the linear H2K peptide gave higher transfection in vivo than did those with a positive surface charge. To examine the distribution of plasmid expression within the tumor from H2K polyplexes, we found widespread expression by immunohistochemical staining. With a fluorescent tdTomato expressing-plasmid, we confirmed a pervasive distribution and gene expression within the tumor mediated by the H2K polyplex. Conclusions Although mechanisms underlying the efficiency of gene expression are probably multifactorial, unpacking of the H2K polyplex within the tumor appears to have a significant role. Further development of these H2K polyplexes represents an attractive approach for plasmid-based therapies of cancer. © 2014 The Authors. The Journal of Gene Medicine published by John Wiley & Sons, Ltd. PMID:25303767

  7. Single primer-mediated circular polymerase chain reaction for hairpin DNA cloning and plasmid editing.

    PubMed

    Huang, Jiansheng; Khan, Inamullah; Liu, Rui; Yang, Yan; Zhu, Naishuo

    2016-05-01

    We developed and validated a universal polymerase chain reaction (PCR) method, single primer circular (SPC)-PCR, using single primer to simultaneously insert and amplify a short hairpin sequence into a vector with a high success rate. In this method, the hairpin structure is divided into two parts and fused into a vector by PCR. Then, a single primer is used to cyclize the chimera into a mature short hairpin RNA (shRNA) expression vector. It is not biased by loop length or palindromic structures. Six hairpin DNAs with short 4-nucleotide loops were successfully cloned. Moreover, SPC-PCR was also applied to plasmid editing within 3 h with a success rate higher than 95%.

  8. Single primer-mediated circular polymerase chain reaction for hairpin DNA cloning and plasmid editing.

    PubMed

    Huang, Jiansheng; Khan, Inamullah; Liu, Rui; Yang, Yan; Zhu, Naishuo

    2016-05-01

    We developed and validated a universal polymerase chain reaction (PCR) method, single primer circular (SPC)-PCR, using single primer to simultaneously insert and amplify a short hairpin sequence into a vector with a high success rate. In this method, the hairpin structure is divided into two parts and fused into a vector by PCR. Then, a single primer is used to cyclize the chimera into a mature short hairpin RNA (shRNA) expression vector. It is not biased by loop length or palindromic structures. Six hairpin DNAs with short 4-nucleotide loops were successfully cloned. Moreover, SPC-PCR was also applied to plasmid editing within 3 h with a success rate higher than 95%. PMID:26792375

  9. Novel linear megaplasmid from Brevibacterium sp. isolated from extreme environment.

    PubMed

    Dib, Julián Rafael; Wagenknecht, Martin; Hill, Russell T; Farías, María Eugenia; Meinhardt, Friedhelm

    2010-06-01

    Brevibacterium sp. Ap13, isolated from flamingo's feces in Laguna Aparejos, a high-altitude lake located at approximately 4,200 m in the northwest of Argentina was previously found to be resistant to multiple antibiotics, and was therefore screened for plasmids that may be implicated in antibiotic resistance. Brevibacterium sp. Ap13 was found to contain two plasmids of approximately 87 and 436 kb, designated pAP13 and pAP13c, respectively. Only pAP13 was stably maintained and was extensively characterized by pulsed-field gel electrophoresis to reveal that this plasmid is linear and likely has covalently linked terminal proteins associated with its 5' ends. This is the first report of a linear plasmid in the genus Brevibacterium and may provide a new tool for genetic manipulation of this commercially important genus. PMID:20473959

  10. PC Basic Linear Algebra Subroutines

    1992-03-09

    PC-BLAS is a highly optimized version of the Basic Linear Algebra Subprograms (BLAS), a standardized set of thirty-eight routines that perform low-level operations on vectors of numbers in single and double-precision real and complex arithmetic. Routines are included to find the index of the largest component of a vector, apply a Givens or modified Givens rotation, multiply a vector by a constant, determine the Euclidean length, perform a dot product, swap and copy vectors, andmore » find the norm of a vector. The BLAS have been carefully written to minimize numerical problems such as loss of precision and underflow and are designed so that the computation is independent of the interface with the calling program. This independence is achieved through judicious use of Assembly language macros. Interfaces are provided for Lahey Fortran 77, Microsoft Fortran 77, and Ryan-McFarland IBM Professional Fortran.« less

  11. Belos Block Linear Solvers Package

    2004-03-01

    Belos is an extensible and interoperable framework for large-scale, iterative methods for solving systems of linear equations with multiple right-hand sides. The motivation for this framework is to provide a generic interface to a collection of algorithms for solving large-scale linear systems. Belos is interoperable because both the matrix and vectors are considered to be opaque objects--only knowledge of the matrix and vectors via elementary operations is necessary. An implementation of Balos is accomplished viamore » the use of interfaces. One of the goals of Belos is to allow the user flexibility in specifying the data representation for the matrix and vectors and so leverage any existing software investment. The algorithms that will be included in package are Krylov-based linear solvers, like Block GMRES (Generalized Minimal RESidual) and Block CG (Conjugate-Gradient).« less

  12. P1 plasmid replication requires methylated DNA.

    PubMed Central

    Abeles, A L; Austin, S J

    1987-01-01

    Plasmids driven by the plasmid replication origin of bacteriophage P1 cannot be established in Escherichia coli strains that are defective for the DNA adenine methylase (dam). Using a composite plasmid that has two origins, we show that the P1 origin cannot function even in a plasmid that is already established in a dam strain. An in vitro replication system for the P1 origin was developed that uses as a substrate M13 replicative-form DNA containing the minimal P1 origin. The reaction mixture contains a crude extract of E. coli and purified P1 RepA protein. In addition to being RepA dependent, synthesis was shown to be dependent on methylation of the dam methylase-sensitive sites of the substrate DNA. As the P1 origin contains five such sites in a small region known to be critical for origin function, it can be concluded that methylation of these sites is a requirement for initiation. This suggests that the postreplicational methylation of the origin may control reinitiation and contribute to the accuracy of the highly stringent copy-number control of the origin in vivo. PMID:2826133

  13. Transformation of Thermoanaerobacterium sp. strain JW/SL-YS485 with plasmid pIKM1 conferring kanamycin resistance

    SciTech Connect

    Mai, V.; Lorenz, W.W.; Wiegel, J.

    1997-12-31

    The industrial application of thermophilic (eu)bacteria is hampered by the lack of genetic systems for these bacteria. We report here the first unequivocal transformation of a Gram-positive, thermophilic, anaerobic microorganism, Thermoanaerobacterium, with the kanamycin resistance-mediating plasmid pIKM1. The construct pIKM1 is based on the Escherichia coli-Clostridium acetobutylicum shuttle vector pIMP1 and contains the thermostable kanamycin cassette from S. faecalis plasmid pKD102. Using electrotransformation, plasmid pIKM1 mediated kanamycin resistance in Thermoanaerobacterium sp. strain JW/SL-YS485 up to 400 {mu}g ml{sup -1} at 48{degrees}C and 200 {mu}g ml{sup -1} at 60{degrees}C.

  14. Cloning and expression in Escherichia coli cells of a plasmid pBS195 gene that determines the activity of oxygenase

    SciTech Connect

    Kozlova, E.V.; Suvorova, E.S.; Romanov, V.P.; Boronin, A.M.

    1995-02-01

    Plasmid pBS195, detected in a strain of Lactobacillus sp. isolated from long-living persons, has a broad host range, including Gram-positive and Gram-negative microorganisms. Plasmid-harboring colonies of the strain Escherichia coli HB101 give a color reaction with catechol. This indicates that genes mediating the activity of oxygenase are present in this plasmid. The high activity level of this enzyme, mediated by pBS195, and substrate specificity, which has not been detected in any known metapyrocatechases, were found in cells of E. coli. Hybridization with a {sup 32}P-labeled fragment containing the NahC gene revealed a region of homology with a 1.6-kb EcoR I-BamH I fragment of plasmid pBS195. Deletion variants of this plasmid that lost oxygenase activity confirmed the location of the oxygenase gene in this region. The gene responsible for oxygenase activity in the plasmid was cloned on the pUC19 vector in E. coli cells. The expression of the cloned gene is controlled by the lac promoter of this vector. Physical, hybridization, and deletion analyses as well as analysis of polypeptides, which are synthesized in E. coli minicells, showed that this activity requires the participation of a polypeptide with molecular mass of 34 kDa. 9 refs., 3 figs., 1 tab.

  15. Simple method for identification of plasmid-coded proteins.

    PubMed

    Sancar, A; Hack, A M; Rupp, W D

    1979-01-01

    Proteins encoded by plasmid DNA are specifically labeled in UV-irradiated cells of Escherichia coli carrying recA and uvrA mutations because extensive degradation of the chromosome DNA occurs concurrently with amplification of plasmid DNA.

  16. Plasmids spread very fast in heterogeneous bacterial communities.

    PubMed Central

    Dionisio, Francisco; Matic, Ivan; Radman, Miroslav; Rodrigues, Olivia R; Taddei, François

    2002-01-01

    Conjugative plasmids can mediate gene transfer between bacterial taxa in diverse environments. The ability to donate the F-type conjugative plasmid R1 greatly varies among enteric bacteria due to the interaction of the system that represses sex-pili formations (products of finOP) of plasmids already harbored by a bacterial strain with those of the R1 plasmid. The presence of efficient donors in heterogeneous bacterial populations can accelerate plasmid transfer and can spread by several orders of magnitude. Such donors allow millions of other bacteria to acquire the plasmid in a matter of days whereas, in the absence of such strains, plasmid dissemination would take years. This "amplification effect" could have an impact on the evolution of bacterial pathogens that exist in heterogeneous bacterial communities because conjugative plasmids can carry virulence or antibiotic-resistance genes. PMID:12524329

  17. A novel method of plasmid isolation using laundry detergent.

    PubMed

    Yadav, P; Yadav, A; Garg, V; Datta, T K; Goswami, S L; De, S

    2011-07-01

    Since the discovery of plasmid, various methods have been developed to isolate plasmid DNA. All the methods have one common and important target of isolating plasmid DNA of high quality and quantity in less time. These methods are not completely safe because of use of toxic chemicals compounds. The developed protocol for plasmid extraction is based on the alkaline lysis method of plasmid preparation (extraction atpH 8.0) with slight modifications. Cell lysis reagent sodium dodecyl sulfate is replaced by lipase enzyme present in laundry detergent. A good plasmid preparation can be made, which is well suited for subsequent molecular biology applications. By taking safety measures on count, contaminants like, RNA and protein can be completely avoided with maximized plasmid yield. The resultant plasmid quality and quantity can be well comparable to other prevalent methods.

  18. Complex in vivo Ligation Using Homologous Recombination and High-efficiency Plasmid Rescue from Saccharomyces cerevisiae

    PubMed Central

    Finnigan, Gregory C.; Thorner, Jeremy

    2015-01-01

    The protocols presented here allow for the facile generation of a wide variety of complex multipart DNA constructs (tagged gene products, gene fusions, chimeric proteins, and other variants) using homologous recombination and in vivo ligation in budding yeast (Saccharomyces cerevisiae). This method is straightforward, efficient and cost-effective, and can be used both for vector creation and for subsequent one-step, high frequency integration into a chromosomal locus in yeast. The procedure utilizes PCR with extended oligonucleotide “tails” of homology between multiple fragments to allow for reassembly in yeast in a single transformation followed by a method for highly efficient plasmid extraction from yeast (for transformation into bacteria). The latter is an improvement on existing methods of yeast plasmid extraction, which, historically, has been a limiting step in recovery of desired constructs. We describe the utility and convenience of our techniques, and provide several examples. PMID:26523287

  19. Characterization of the chromosomal integration of Saccharopolyspora plasmid pCM32 and its application to improve production of spinosyn in Saccharopolyspora spinosa.

    PubMed

    Chen, Jian; Xia, Haiyang; Dang, Fujun; Xu, Qingyu; Li, Wenjun; Qin, Zhongjun

    2015-12-01

    Saccharopolyspora spinosa produces tetra-cyclic macrolide spinosyns, a group of highly efficient pesticidal agents. However, this species lacks efficient vectors for genetic manipulation. In this study, the circular plasmid pCM32 was newly isolated from Saccharopolyspora endophytica YIM 61095. The complete nucleotide sequence of pCM32 consists of 14,611 bp and is predicted to encode 17 open reading frames (ORFs). Interestingly, a putative int gene in pCM32 was predicted by homologous alignment to encode an integrase belonging to the tyrosine family of integrases/recombinases. Plasmid pCM238 containing this int locus derived from pCM32 could be transferred by conjugation from Escherichia coli into Sa. spinosa at a high frequency. Integration of pCM238 in the host chromosome was demonstrated as site-specific recombination (at the tRNA (Ser) gene) via a 56-bp core sequence within the attP/attB sites. Plasmid pCM265, a shuttle vector containing the int and attP sequences of pCM32, was constructed to introduce foreign genes into Sa. spinosa. The production of spinosad approximately doubled in Sa. spinosa NRRL18395 after introducing pCM265-derived plasmids carrying the genes for phosphofructokinase (PFK) or anthranilate synthase. These results indicate that plasmid pCM32 is an actinomycete integrative and conjugative element (AICE) and that its derived integrative vectors are useful for efficiently introducing foreign DNA into Sa. spinosa.

  20. Transient gene expression mediated by integrase-defective retroviral vectors.

    PubMed

    Yu, Seung Shin; Dan, Kazuyuki; Chono, Hideto; Chatani, Emi; Mineno, Junichi; Kato, Ikunoshin

    2008-04-18

    Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.

  1. Presence of a virulence-associated plasmid in Yersinia pseudotuberculosis.

    PubMed Central

    Gemski, P; Lazere, J R; Casey, T; Wohlhieter, J A

    1980-01-01

    We have shown that Yersinia pseudotuberculosis can possess plasmids which are similar in size and function to the previously described Vwa plasmids of Y. enterocolitica. These plasmids are associated with the production of V and W antigens (calcium dependency) and pathogenicity of the organism. Further investigation of these plasmids from Y. pseudotuberculosis and Y. enterocolitica with restriction endonucleases revealed significant differences in their fragmentation pattern. Images Fig. 1 Fig. 2 Fig. 3 PMID:6249747

  2. Demonstration of plasmid-mediated drug resistance in Mycobacterium abscessus.

    PubMed

    Matsumoto, Cristianne Kayoko; Bispo, Paulo José Martins; Santin, Katiane; Nogueira, Christiane Lourenço; Leão, Sylvia Cardoso

    2014-05-01

    Plasmid-mediated kanamycin resistance was detected in a strain of Mycobacterium abscessus subsp. bolletii responsible for a nationwide epidemic of surgical infections in Brazil. The plasmid did not influence susceptibility to tobramycin, streptomycin, trimethoprim-sulfamethoxazole, clarithromycin, or ciprofloxacin. Plasmid-mediated drug resistance has not been described so far in mycobacteria. PMID:24574286

  3. Plasmid mediated enhancement of uv resistance in Streptococcus faecalis

    SciTech Connect

    Miehl, R.; Miller, M.; Yasbin, R.E.

    1980-01-01

    A 38.5-Mdal plasmid of Streptococcus faecalis subdp. zymogenes has been shown to enhance survival following uv irradiation. In addition, the presence of this plasmid increases the mutation frequencies following uv irradiation and enhanced W-reactivation. The data presented indicate that S. faecalis has an inducible error-prone repair system and that the plasmid enhances these repair functions.

  4. Gauge Theories of Vector Particles

    DOE R&D Accomplishments Database

    Glashow, S. L.; Gell-Mann, M.

    1961-04-24

    The possibility of generalizing the Yang-Mills trick is examined. Thus we seek theories of vector bosons invariant under continuous groups of coordinate-dependent linear transformations. All such theories may be expressed as superpositions of certain "simple" theories; we show that each "simple theory is associated with a simple Lie algebra. We may introduce mass terms for the vector bosons at the price of destroying the gauge-invariance for coordinate-dependent gauge functions. The theories corresponding to three particular simple Lie algebras - those which admit precisely two commuting quantum numbers - are examined in some detail as examples. One of them might play a role in the physics of the strong interactions if there is an underlying super-symmetry, transcending charge independence, that is badly broken. The intermediate vector boson theory of weak interactions is discussed also. The so-called "schizon" model cannot be made to conform to the requirements of partial gauge-invariance.

  5. Lack of dependence on p53 for DNA double strand break repair of episomal vectors in human lymphoblasts

    NASA Technical Reports Server (NTRS)

    Kohli, M.; Jorgensen, T. J.

    1999-01-01

    The p53 tumor suppressor gene has been shown to be involved in a variety of repair processes, and recent findings have suggested that p53 may be involved in DNA double strand break repair in irradiated cells. The role of p53 in DNA double strand break repair, however, has not been fully investigated. In this study, we have constructed a novel Epstein-Barr virus (EBV)-based shuttle vector, designated as pZEBNA, to explore the influence of p53 on DNA strand break repair in human lymphoblasts, since EBV-based vectors do not inactivate the p53 pathway. We have compared plasmid survival of irradiated, restriction enzyme linearized, and calf intestinal alkaline phosphatase (CIP)-treated pZEBNA with a Simian virus 40 (SV40)-based shuttle vector, pZ189, in TK6 (wild-type p53) and WTK1 (mutant p53) lymphoblasts and determined that p53 does not modulate DNA double strand break repair in these cell lines. Copyright 1999 Academic Press.

  6. Efficient production of dual recombinant adeno-associated viral vectors for factor VIII delivery.

    PubMed

    Wang, Qizhao; Dong, Biao; Firrman, Jenni; Roberts, Sean; Moore, Andrea Rossi; Cao, Wenjing; Diao, Yong; Kapranov, Philipp; Xu, Ruian; Xiao, Weidong

    2014-08-01

    Recombinant adeno-associated viral (rAAV) vectors have gained attention for human gene therapy because of their high safety and clinical efficacy profile. For factor VIII gene delivery, splitting the coding region between two AAV vectors remains a viable strategy to avoid the packaging capacity limitation (∼5.0 kb). However, it is time-consuming and labor-intensive to produce two rAAV vectors in separate batches. Here we demonstrated successful production of dual rAAV vectors for hemophilia A gene therapy in a single preparation. When the AAV vector plasmids carrying the human factor VIII heavy chain (hHC) and the light chain (hLC) expression cassettes were cotransfected into 293 cells along with the AAV rep&cap and mini-adenovirus helper plasmids, both rAAV-hHC and rAAV-hLC were produced at the desired ratio and in high titer. Interestingly, the rAAV-hHC vectors always yielded higher titers than rAAV-hLC vectors as a result of more efficient replication of rAAV-hHC genomes. The resulting vectors were effective in transducing the tissue culture cells in vitro. When these vectors were administered to hemophilia A mice, factor VIII was detected in the mouse plasma by both the activated partial thromboplastin time assay and enzyme-linked immunosorbent assay. The functional activity as well as the antigen levels of secreted factor VIII were similar to those of vectors produced by the traditional method. The dual-vector production method has been successfully extended to both AAV2 and AAV8 serotypes. In conclusion, cotransfection of vector plasmids presents an efficient method for producing dual or multiple AAV vectors at significantly reduced cost and labor.

  7. Revealing the latent mobilization capability of the staphylococcal bacteriocinogenic plasmid pRJ9.

    PubMed

    Coutinho, Bruna Gonçalves; Coelho, Marcus Lívio Varella; Ceotto, Hilana; Bastos, Maria do Carmo de Freire

    2011-01-01

    Plasmid pRJ9 is a non-self-mobilizable bacteriocinogenic plasmid from Staphylococcus aureus. Despite this feature, DNA sequencing and RT-PCR experiments showed that it presents a Mob region with three genes (mobCAB), transcribed as an operon. In silico analysis of the Mob proteins encoded by pRJ9 showed that they present all the conserved functional features reported until present as being essential for plasmid mobilization. Moreover, they showed a high identity to Mob proteins encoded by mobilizable plasmids from Staphylococcus spp., especially to those encoded by plasmid pRJ6, which presents four mob genes (mobCDAB). A putative oriT region was also found upstream of the pRJ9 mob operon. pRJ9 could only be successfully mobilized by pGO1 when pRJ6 was present in the same strain. Further experiments showed that the pRJ9 oriT can be recognized by the pRJ6 Mob proteins, confirming its functionality. As pRJ9 does not possess a mobD gene while pRJ6 does, the absence of this gene was believed to be responsible for its lack of mobilization. However, conjugation experiments with a donor strain carrying also mobD cloned into an S. aureus vector showed that pRJ9 does not become mobilized even in the presence of the protein MobD encoded by pRJ6. Therefore, the reasons for pRJ9 failure to be mobilized are presently unknown. PMID:22286044

  8. Linear Accelerators

    SciTech Connect

    Sidorin, Anatoly

    2010-01-05

    In linear accelerators the particles are accelerated by either electrostatic fields or oscillating Radio Frequency (RF) fields. Accordingly the linear accelerators are divided in three large groups: electrostatic, induction and RF accelerators. Overview of the different types of accelerators is given. Stability of longitudinal and transverse motion in the RF linear accelerators is briefly discussed. The methods of beam focusing in linacs are described.

  9. High Prevalence of Plasmid-Mediated Quinolone Resistance and IncQ Plasmids Carrying qnrS2 Gene in Bacteria from Rivers near Hospitals and Aquaculture in China.

    PubMed

    Wen, Yanping; Pu, Xiaoying; Zheng, Wei; Hu, Guang

    2016-01-01

    Effluents from hospital and aquaculture are considered important sources of quinolone resistance. However, little information is available on the impact of this effluent on nearby rivers. In this study, 188 ciprofloxacin-resistant bacterial isolates obtained from rivers near hospitals and aquaculture were screened for plasmid-mediated quinolone resistance (PMQR) genes. Species identification, antibiotic susceptibility testing, and PMQR gene transferability assessment were conducted for PMQR-positive bacteria. Representative qnrS2-encoding plasmids were subsequently sequenced using a primer-walking approach. In total, 44 isolates (23.4%) were positive for qnr genes (16 qnrB2, 3 qnrS1, and 25 qnrS2) and 32 isolates (17.0%) were positive for aac(6')-Ib-cr. Other PMQR genes were not detected. The qnrB2 and aac(6')-Ib-cr genes had a higher prevalence in aquaculture samples than in hospital samples, and were significantly associated with Enterobacteriaceae (p < 0.05). In contrast, the prevalence of qnrS2 was not site-related, but was significantly associated with Aeromonas spp. (p < 0.05). All PMQR isolates were resistant to three or more classes of antibiotics. Eleven qnrS2-harboring plasmids from Aeromonas spp., including a novel conjugative plasmid pHP18, were selected for sequencing. These plasmids were small in size (6,388-16,197 bp) and belonged to the IncQ or IncU plasmid family, with qnrS2 being part of a mobile insertion cassette. Taken together, our findings suggest that aquaculture is a possible source for aac(6')-Ib-cr and qnrB2 dissemination, and demonstrate the ubiquity of qnrS2 in aquatic environments. Finally, Aeromonas spp. served as vectors for qnrS2 with the help of IncQ-type plasmids.

  10. High Prevalence of Plasmid-Mediated Quinolone Resistance and IncQ Plasmids Carrying qnrS2 Gene in Bacteria from Rivers near Hospitals and Aquaculture in China

    PubMed Central

    Wen, Yanping; Pu, Xiaoying; Zheng, Wei

    2016-01-01

    Effluents from hospital and aquaculture are considered important sources of quinolone resistance. However, little information is available on the impact of this effluent on nearby rivers. In this study, 188 ciprofloxacin-resistant bacterial isolates obtained from rivers near hospitals and aquaculture were screened for plasmid-mediated quinolone resistance (PMQR) genes. Species identification, antibiotic susceptibility testing, and PMQR gene transferability assessment were conducted for PMQR-positive bacteria. Representative qnrS2-encoding plasmids were subsequently sequenced using a primer-walking approach. In total, 44 isolates (23.4%) were positive for qnr genes (16 qnrB2, 3 qnrS1, and 25 qnrS2) and 32 isolates (17.0%) were positive for aac(6′)-Ib-cr. Other PMQR genes were not detected. The qnrB2 and aac(6′)-Ib-cr genes had a higher prevalence in aquaculture samples than in hospital samples, and were significantly associated with Enterobacteriaceae (p < 0.05). In contrast, the prevalence of qnrS2 was not site-related, but was significantly associated with Aeromonas spp. (p < 0.05). All PMQR isolates were resistant to three or more classes of antibiotics. Eleven qnrS2-harboring plasmids from Aeromonas spp., including a novel conjugative plasmid pHP18, were selected for sequencing. These plasmids were small in size (6,388–16,197 bp) and belonged to the IncQ or IncU plasmid family, with qnrS2 being part of a mobile insertion cassette. Taken together, our findings suggest that aquaculture is a possible source for aac(6′)-Ib-cr and qnrB2 dissemination, and demonstrate the ubiquity of qnrS2 in aquatic environments. Finally, Aeromonas spp. served as vectors for qnrS2 with the help of IncQ-type plasmids. PMID:27427763

  11. High Prevalence of Plasmid-Mediated Quinolone Resistance and IncQ Plasmids Carrying qnrS2 Gene in Bacteria from Rivers near Hospitals and Aquaculture in China.

    PubMed

    Wen, Yanping; Pu, Xiaoying; Zheng, Wei; Hu, Guang

    2016-01-01

    Effluents from hospital and aquaculture are considered important sources of quinolone resistance. However, little information is available on the impact of this effluent on nearby rivers. In this study, 188 ciprofloxacin-resistant bacterial isolates obtained from rivers near hospitals and aquaculture were screened for plasmid-mediated quinolone resistance (PMQR) genes. Species identification, antibiotic susceptibility testing, and PMQR gene transferability assessment were conducted for PMQR-positive bacteria. Representative qnrS2-encoding plasmids were subsequently sequenced using a primer-walking approach. In total, 44 isolates (23.4%) were positive for qnr genes (16 qnrB2, 3 qnrS1, and 25 qnrS2) and 32 isolates (17.0%) were positive for aac(6')-Ib-cr. Other PMQR genes were not detected. The qnrB2 and aac(6')-Ib-cr genes had a higher prevalence in aquaculture samples than in hospital samples, and were significantly associated with Enterobacteriaceae (p < 0.05). In contrast, the prevalence of qnrS2 was not site-related, but was significantly associated with Aeromonas spp. (p < 0.05). All PMQR isolates were resistant to three or more classes of antibiotics. Eleven qnrS2-harboring plasmids from Aeromonas spp., including a novel conjugative plasmid pHP18, were selected for sequencing. These plasmids were small in size (6,388-16,197 bp) and belonged to the IncQ or IncU plasmid family, with qnrS2 being part of a mobile insertion cassette. Taken together, our findings suggest that aquaculture is a possible source for aac(6')-Ib-cr and qnrB2 dissemination, and demonstrate the ubiquity of qnrS2 in aquatic environments. Finally, Aeromonas spp. served as vectors for qnrS2 with the help of IncQ-type plasmids. PMID:27427763

  12. A unified development of several techniques for the representation of random vectors and data sets

    NASA Technical Reports Server (NTRS)

    Bundick, W. T.

    1973-01-01

    Linear vector space theory is used to develop a general representation of a set of data vectors or random vectors by linear combinations of orthonormal vectors such that the mean squared error of the representation is minimized. The orthonormal vectors are shown to be the eigenvectors of an operator. The general representation is applied to several specific problems involving the use of the Karhunen-Loeve expansion, principal component analysis, and empirical orthogonal functions; and the common properties of these representations are developed.

  13. An E3-14.7K peptide that promotes microtubules-mediated transport of plasmid DNA increases polyplexes transfection efficiency.

    PubMed

    Pigeon, Lucie; Gonçalves, Cristine; Gosset, David; Pichon, Chantal; Midoux, Patrick

    2013-11-25

    Chemical vectors as cationic polymers and cationic lipids are promising alternatives to viral vectors for gene therapy. Beside endosome escape and nuclear import, plasmid DNA (pDNA) migration in the cytosol toward the nuclear envelope is also regarded as a limiting step for efficient DNA transfection with non-viral vectors. Here, the interaction between E3-14.7K and FIP-1 to favor migration of pDNA along microtubules is exploited. E3-14.7K is an early protein of human adenoviruses that interacts via FIP-1 (Fourteen.7K Interacting Protein 1) protein with the light-chain components of the human microtubule motor protein dynein (TCTEL1). This peptide is conjugated with pDNA and mediates interaction of pDNA in vitro with isolated microtubules as well as with microtubules in cellulo. Videomicroscopy and tracking treatment of images clearly demonstrate that P79-98/pDNA conjugate exhibits a linear transport with large amplitude along microtubules upon 2 h transfection with polyplexes whereas control pDNA conjugate exhibits small non-directional movements in the cytoplasm. Remarkably, P79-98/peGFP polyplexes enhance by a factor 2.5 (up to 76%) the number of transfected cells. The results demonstrate, for the first time, that the transfection efficiency of polyplexes can be drastically increased when the microtubules migration of pDNA is facilitated by a peptide allowing pDNA docking to TCTEL1. This is a real breakthrough in the non viral gene delivery field that opens hope to build artificial viruses.

  14. Thermoresponsive polymers as gene delivery vectors: cell viability, DNA transport and transfection studies.

    PubMed

    Twaites, Beverley R; de Las Heras Alarcón, Carolina; Lavigne, Matthieu; Saulnier, Annabelle; Pennadam, Sivanand S; Cunliffe, David; Górecki, Dariusz C; Alexander, Cameron

    2005-11-28

    A range of gene delivery vectors containing the thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAm) was evaluated for effects on cell viability, intracellular trafficking and transgene expression in C2C12 mouse muscle cells. Polymers were complexed with plasmid DNA at pH 7.4 and the ability of the resulting particles to transfect cells was assessed via confocal microscopy and protein expression studies in tissue culture. Cell viability assays indicated that these polymers were toxic at high concentrations when not complexed to DNA or at certain polymer:DNA ratios. Poly(ethyleneimine) co-polymers with side-chain grafted PNIPAm were shown to be less toxic than poly(ethyleneimine) alone or PNIPAm-co-(N,N'-dimethylaminoethylmethacrylate) linear co-polymers and the effects were concentration dependent. Confocal micrographs of labeled polymers and DNA indicated rapid cellular entry for all the complexes but expression of Green Fluorescent Protein was achieved only when the branched PEI-PNIPAm co-polymers were used as vectors. The results indicate that design of appropriate co-polymer components and overall polymer architecture can be used to mediate, and perhaps ultimately control, DNA transport and transgene expression. PMID:16214254

  15. Characterization of the replication, transfer, and plasmid/lytic phage cycle of the Streptomyces plasmid-phage pZL12.

    PubMed

    Zhong, Li; Cheng, Qiuxiang; Tian, Xinli; Zhao, Liqian; Qin, Zhongjun

    2010-07-01

    We report here the isolation and recombinational cloning of a large plasmid, pZL12, from endophytic Streptomyces sp. 9R-2. pZL12 comprises 90,435 bp, encoding 112 genes, 30 of which are organized in a large operon resembling bacteriophage genes. A replication locus (repA) and a conjugal transfer locus (traA-traC) were identified in pZL12. Surprisingly, the supernatant of a 9R-2 liquid culture containing partially purified phage particles infected 9R-2 cured of pZL12 (9R-2X) to form plaques, and a phage particle (phiZL12) was observed by transmission electron microscopy. Major structural proteins (capsid, portal, and tail) of phiZL12 virions were encoded by pZL12 genes. Like bacteriophage P1, linear phiZL12 DNA contained ends from a largely random pZL12 sequence. There was also a hot end sequence in linear phiZL12. phiZL12 virions efficiently infected only one host, 9R-2X, but failed to infect and form plaques in 18 other Streptomyces strains. Some 9R-2X spores rescued from lysis by infection of phiZL12 virions contained a circular pZL12 plasmid, completing a cycle comprising autonomous plasmid pZL12 and lytic phage phiZL12. These results confirm pZL12 as the first example of a plasmid-phage in Streptomyces.

  16. Cloning of a Thiobacillus ferrooxidans plasmid in Escherichia coli

    SciTech Connect

    Holmes, D.S.; Lobos, J.H.; Bopp, L.H.; Welch, G.C.

    1984-01-01

    Three separate plasmids of 6, 7, 16, and >23 kilobases were purified from a single clone of Thiobacillus ferrooxidans ATCC 33020 grown in the presence of uranium. The 6.7-kilobase plasmid (pTfl) was cloned separately into the HindIII or BamHI site of Escherichia coli plasmid pBR322. Restriction maps of the recombinant plasmids, termed pTf100 and pTf110, respectively, were constructed, creating potential cloning vehicles for exchanging genetic information between E. coli and T. ferrooxidans. Evidence from restriction enzyme analysis and Southern blot DNA-DNA hybridization indicates that the three native plasmids share little sequence homology.

  17. Genetically engineering adenoviral vectors for gene therapy.

    PubMed

    Coughlan, Lynda

    2014-01-01

    Adenoviral (Ad) vectors are commonly used for various gene therapy applications. Significant advances in the genetic engineering of Ad vectors in recent years has highlighted their potential for the treatment of metastatic disease. There are several methods to genetically modify the Ad genome to incorporate retargeting peptides which will redirect the natural tropism of the viruses, including homologous recombination in bacteria or yeast. However, homologous recombination in yeast is highly efficient and can be achieved without the need for extensive cloning strategies. In addition, the method does not rely on the presence of unique restriction sites within the Ad genome and the reagents required for this method are widely available and inexpensive. Large plasmids containing the entire adenoviral genome (~36 kbp) can be modified within Saccharomyces cerevisiae yeast and genomes easily rescued in Escherichia coli hosts for analysis or amplification. A method for two-step homologous recombination in yeast is described in this chapter.

  18. Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16.

    PubMed

    Gruber, Steffen; Hagen, Jeremias; Schwab, Helmut; Koefinger, Petra

    2014-09-30

    The Gram-negative β-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. The majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmid loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16, a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. In particular, the use of promoters derived from the bacteriophage T5 was described for the first time in this work, characterizing the j5 promoter as the strongest promoter yet to be applied in R. eutropha H16. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96 h. The utility of the new vector family in R. eutropha H16 is demonstrated by providing expression data with different model proteins and consequently further raises the value of this organism as cell factory for biotechnological applications including protein and metabolite production.

  19. Reprint of "versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16".

    PubMed

    Gruber, Steffen; Hagen, Jeremias; Schwab, Helmut; Koefinger, Petra

    2014-12-20

    The Gram-negative β-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. The majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmid loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16, a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. In particular, the use of promoters derived from the bacteriophage T5 was described for the first time in this work, characterizing the j5 promoter as the strongest promoter yet to be applied in R. eutropha H16. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96h. The utility of the new vector family in R. eutropha H16 is demonstrated by providing expression data with different model proteins and consequently further raises the value of this organism as cell factory for biotechnological applications including protein and metabolite production.

  20. Sequence analysis of plasmid pKJ50 from Bifidobacterium longum.

    PubMed

    Park, M S; Shin, D W; Lee, K H; Ji, G E

    1999-03-01

    The complete nucleotide sequence of a plasmid, pKJ50, isolated from an intestinal bacterium, Bifidobacterium longum KJ, has been determined. The plasmid was analysed and found to be 4960 bp in size with a G+C content of 61.7 mol%. Computer analysis of sequence data revealed three major ORFs encoding putative proteins of 31.5 (ORFI), 24.5 (ORFII) and 38.6 kDa (ORFIII). ORFI encodes a protein with a pI of 10.18 and shows relatively high amino acid sequence similarity (more than 60%) with several plasmid replication proteins from Gram-positive and -negative bacteria. Southern blot analysis showed that pKJ50 accumulates an ssDNA intermediate, suggesting that it replicates by a rolling-circle mechanism. Upstream of ORFI, three sets of repeated sequences resembling iteron structures of related plasmids were identified. ORFIII encodes a protein with a pI of 10.97. It also shows a high level of amino acid sequence similarity with some plasmid mobilization proteins. Upstream of ORFIII, a 12 bp stretch resembles an oriT DNA sequence with inverted repeats identical to those found in conjugative plasmids. Hydropathy plot analysis of ORFII, encoding an acidic protein (pI = 4.95), suggests it is a transmembrane protein. Several interesting palindromic sequences, repeat sequences and hairpin-loop structures around ORFI, which might confer regulatory effects on the replication of the plasmid, were also noted. Reverse transcriptase PCR (RT-PCR) and in vitro translation confirmed the expression of ORFI and ORFII. RT-PCR produced amplified DNA fragments of the expected sizes, corresponding to ORFI and ORFII. However, no RT-PCR product corresponding to ORFIII was obtained. In vitro translation showed protein bands of the expected sizes, corresponding to each ORF. A shuttle vector capable of transforming Bifidobacterium animalis MB209 was constructed by cloning pKJ50 and a chloramphenicol resistance gene into pBR322.

  1. Characterization of the stable maintenance properties of the par region of broad-host-range plasmid RK2.

    PubMed Central

    Sobecky, P A; Easter, C L; Bear, P D; Helinski, D R

    1996-01-01

    A 3.2-kb fragment encoding five genes, parCBA/DE, in two divergently transcribed operons promotes stable maintenance of the replicon of the broad-host-range plasmid RK2 in a vector-independent manner in Escherichia coli. The parDE operon has been shown to contribute to stabilization through the postsegregational killing of plasmid-free daughter cells, while the parCBA operon encodes a resolvase, ParA, that mediates the resolution of plasmid multimers through site-specific recombination. To date, evidence indicates that multimer resolution alone does not play a significant role in RK2 stable maintenance by the parCBA operon in E. coli. It has been proposed, instead, that the parCBA region encodes an additional stability mechanism, a partition system, that ensures that each daughter cell receives a plasmid copy at cell division. However, studies carried out to date have not directly determined the plasmid stabilization activity of the parCBA operon alone. An assessment was made of the relative contributions of postsegregational killing (parDE) and the putative partitioning system (parCBA) to the stabilization of mini-RK2 replicons in E. coli. Mini-RK2 replicons carrying either the entire 3.2-kb (parCBA/DE) fragment or the 2.3-kb parCBA region alone were found to be stably maintained in two E. coli strains tested. The stabilization found is not due to resolution of multimers. The stabilizing effectiveness of parCBA was substantially reduced when the plasmid copy number was lowered, as in the case of E. coli cells carrying a temperature-sensitive mini-RK2 replicon grown at a nonpermissive temperature. The presence of the entire 3.2-kb region effectively stabilized the replicon, however, under both low- and high-copy-number-conditions. In those instances of decreased plasmid copy number, the postsegregational killing activity, encoded by parDE, either as part of the 3.2-kb fragment or alone played the major role in the stabilization of mini-RK2 replicons within the

  2. Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET), a new method for plasmid reconstruction from whole genome sequences.

    PubMed

    Lanza, Val F; de Toro, María; Garcillán-Barcia, M Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M; de la Cruz, Fernando

    2014-12-01

    Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages. PMID:25522143

  3. Intrauterine Infection with Plasmid-Free Chlamydia muridarum Reveals a Critical Role of the Plasmid in Chlamydial Ascension and Establishes a Model for Evaluating Plasmid-Independent Pathogenicity

    PubMed Central

    Chen, Jianlin; Yang, Zhangsheng; Sun, Xin; Tang, Lingli; Ding, Yiling; Xue, Min; Zhou, Zhiguang; Baseman, Joel

    2015-01-01

    Intravaginal infection with plasmid-competent but not plasmid-free Chlamydia muridarum induces hydrosalpinx in mouse upper genital tract, indicating a critical role of the plasmid in chlamydial pathogenicity. To evaluate the contribution of the plasmid to chlamydial ascension and activation of tubal inflammation, we delivered plasmid-free C. muridarum directly into the endometrium by intrauterine inoculation. We found that three of the six mouse strains tested, including CBA/J, C3H/HeJ, and C57BL/6J, developed significant hydrosalpinges when 1 × 107 inclusion-forming units (IFU) of plasmid-free C. muridarum were intrauterinally inoculated. Even when the inoculum was reduced to 1 × 104 IFU, the CBA/J mice still developed robust hydrosalpinx. The hydrosalpinx development in CBA/J mice correlated with increased organism ascension to the oviduct following the intrauterine inoculation. The CBA/J mice intravaginally infected with the same plasmid-free C. muridarum strain displayed reduced ascending infection and failed to develop hydrosalpinx. These observations have demonstrated a critical role of the plasmid in chlamydial ascending infection. The intrauterine inoculation of the CBA/J mice with plasmid-free C. muridarum not only resulted in more infection in the oviduct but also stimulated more inflammatory infiltration and cytokine production in the oviduct than the intravaginal inoculation, suggesting that the oviduct inflammation can be induced by plasmid-independent factors, which makes the hydrosalpinx induction in CBA/J mice by intrauterine infection with plasmid-free C. muridarum a suitable model for investigating plasmid-independent pathogenic mechanisms. PMID:25870225

  4. Plasmid Flux in Escherichia coli ST131 Sublineages, Analyzed by Plasmid Constellation Network (PLACNET), a New Method for Plasmid Reconstruction from Whole Genome Sequences

    PubMed Central

    Garcillán-Barcia, M. Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M.; de la Cruz, Fernando

    2014-01-01

    Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ–proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages. PMID:25522143

  5. Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET), a new method for plasmid reconstruction from whole genome sequences.

    PubMed

    Lanza, Val F; de Toro, María; Garcillán-Barcia, M Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M; de la Cruz, Fernando

    2014-12-01

    Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

  6. Chloramphenicol Resistance Plasmids in Escherichia coli Isolated from Diseased Piglets

    PubMed Central

    Jørgensen, Sigrid Tue

    1978-01-01

    The plasmids in 19 chloramphenicol-resistant Escherichia coli strains of three pig pathogenic antigen types were studied in conjugation and transduction experiments. The plasmids had identical resistance patterns: streptomycin, spectinomycin, sulfonamides, and chloramphenicol (Sm, Sp, Su, Cm) and belonged to IncFII. One plasmid carried ampicillin resistance in addition. Restriction enzyme analysis of the deoxyribonucleic acid from five of the plasmids originating from the same herd showed that their digestion patterns with EcoRI were indistinguishable. EcoRI cleaved the deoxyribonucleic acid of a sixth plasmid from the same herd and displayed nine of the ten bands of the other five plasmids plus an additional six. It appears that the five plasmids with identical restriction patterns have a common origin and may be copies of the same plasmid from which the sixth may have developed. Four strains carried two plasmids each. In two of these strains, a plasmid with a tetracycline marker (Tc), or possibly the tetracycline marker alone, recombined frequently with the Sm Sp Su Cm plasmid without destroying any known function of the latter. The possibility that Tc is carried on a translocation sequence is discussed. Images PMID:352263

  7. Functionalized organic nanotubes as tubular nonviral gene transfer vector.

    PubMed

    Ding, Wuxiao; Wada, Momoyo; Kameta, Naohiro; Minamikawa, Hiroyuki; Shimizu, Toshimi; Masuda, Mitsutoshi

    2011-11-30

    Tubular nanomaterials are expected to represent a novel nonviral gene transfer vectors due to the unique morphology and potential biological functionalities. Here we rationally constructed functionalized organic nanotubes (ONTs) for gene delivery through the co-assembly of bipolar glycolipid, arginine-lipid and PEG-lipid. The arginine- and PEG-functionalized ONTs efficiently formed complexes with plasmid DNA without aggregation, and protect DNA from enzymatic degradation; while the arginine-functionalized ONTs aggregated with DNA as large bundles. Long ONTs exceeding 1μm in length was rarely taken up into the cells, while those with a length of 400-800nm could effectively deliver plasmid DNA into cells and induce high transgene expression of green fluorescense protein. This study demonstrated the usefulness of functionalized ONT in gene delivery, and the functionalized ONT represents a novel type of tubular nonviral gene transfer vector.

  8. Plasmid-associated aggregation in Thermus thermophilus HB8

    SciTech Connect

    Mather, M.W.; Fee, J.A. )

    1990-01-01

    Thermus thermophilus HB8, a moderate thermophile, exhibits visible aggregation when growing on a rich broth. Strain HB8 also contains two cryptic plasmids. The authors isolated cured strains from HB8 and observed that loss of the 47-MDa plasmid was correlated with loss of aggregation. An enrichment procedure was developed for aggregating cells and used to demonstrate that aggregation was restored upon transformation of a cured strain with plasmid DNA. The aggregation phenotype of transformed cells was variably stable; most did not retain either the plasmid or the phenotype for prolonged periods of growth. Hybridization experiments using a partial sequence from the 47-MDa plasmid suggested the presence of a repeated DNA sequence on this plasmid and on the chromosome. This is the first report of a phenotype associated with a plasmid from a Thermus strain.

  9. The Influence of Biofilms in the Biology of Plasmids.

    PubMed

    Cook, Laura C C; Dunny, Gary M

    2014-10-01

    The field of plasmid biology has historically focused on bacteria growing in liquid culture. Surface-attached communities of bacterial biofilms have recently been understood to be the normal environment of bacteria in the natural world. Thus, studies examining plasmid replication, maintenance, and transfer in biofilms are essential for a true understanding of bacterial plasmid biology. This article reviews the current knowledge of the interplay between bacterial biofilms and plasmids, focusing on the role of plasmids in biofilm development and the role of biofilms in plasmid maintenance, copy-number control, and transfer. The studies examined herein highlight the importance of biofilms as an important ecological niche in which bacterial plasmids play an essential role.

  10. Construction of a stable expression vector carrying sop genes [ZJ1].

    PubMed

    Du, X; Ding, J; Wu, X; Yu, Z; Men, D

    1996-01-01

    Mini-F, the fifth fragment of F plasmid from EcoRI digestion, is known to carry an efficient partitioning function. Two pBR322 plasmid derivatives, pDMC32 and pDMC311, have been constructed from this fragment. The plasmid pDMC32 carries all the relative genes for plasmid stability, ccd, repD, and sop genes (sopA, sopB, and sopC), along with oriS and oriV, while pDMC311 carries only sop genes (sopA, sopB, and sopC). The plasmid maintenance proportions for pDMC32 and pDMC311 in E. coli were 93% and 100%, respectively, after 100 generations continuous cultivation of cells harboring the derivatives, MI32 (pDMC32) and MI311 (pDMC311), in a phosphate-limited basal medium. As a control, the maintenance proportion of plasmid pBR322 dropped down to a low of 10% at generation 55 of continuous cultivation of E. coli MIR322 (pBR322) in the same medium. In order to make a stable expression vector that carries only sop genes, plasmid pDMC40 was constructed by adding a trp promoter from pDR720 to pBR322. The stable expression vector pDMC48 was then derived from pDMC40 by inserting sop genes into it from pDMC311. The maintenance proportion of plasmid pDMC48 in E. coli was still 100% after 100 generations of continuous cultivation of cells harboring the plasmid in phosphate-limited basal medium.

  11. A calibrator plasmid for quantitative analysis of insect resistant maize (Yieldgard MON 810).

    PubMed

    Ballari, Rajashekhar V; Martin, Asha; Gowda, Lalitha R

    2013-09-01

    Real-time PCR (RT-PCR) is the preferred method for the quantification of genetically modified organisms (GMOs) and implementation of labeling regulations. The precision, sensitivity, and reproducibility of RT-PCR data depend on the use of external calibrators. In this investigation, a dual target plasmid designated pRSETMON-02 comprising of MON 810 maize event specific and endogenous zein gene sequences in 1:1 ratio in tandem was constructed and validated. Commutability of plasmid DNA (pDNA) and genomic DNA (gDNA) calibrators for the quantification of MON 810 maize was assessed by employing a TaqMan RT-PCR targeting the P-35S and zein gene. Higher PCR efficiencies, good linearity and lower relative standard deviation (RSD) values were associated with pRSETMON-02 as opposed to gDNA calibrants. pDNA calibrants exhibited better performance characteristic in terms of closeness to the expected value of unknown samples than their genomic counterparts. Short term stability study of the pRSETMON-02 plasmid stored at different temperatures showed that pDNA is stable for 45 days at -20, and 4 °C. The results demonstrated that the developed dual target plasmid pRSETMON-02 is fit for the intended use of quantifying MON 810 maize and is a better alternative to conventional seed powder calibrants. PMID:23578657

  12. Recombinant plasmids containing Xenopus laevis globin structural genes derived from complementary DNA.

    PubMed Central

    Humphries, P; Old, R; Coggins, L W; McShane, T; Watson, C; Paul, J

    1978-01-01

    Details are presented of the in vitro synthesis of double-stranded DNA complementary to purified Xenopus globin messenger RNA, using a combination of reverse transcriptase, fragment 'A' of E. coli DNA polymerase 1 and S1 endonuclease. After selection of duplex DNA molecules approaching the length of Xenopus globin messenger RNA by sedimentation of the DNA through neutral sucrose gradients, the 3'-OH termini of the synthetic globin gene sequences were extended with short tracts of oligo dGMP using terminal transferase. This material was integrated into oligo dCMP-extended linear pCR1 plasmid DNA and amplified by transfection of E. coli. Plasmids carrying globin sequences were identified by hybridization of 32P-labelled globin mRNA to total cellular DNA in situ, by hybridization of purified plasmids to globin cDNA in solution, by analysis of recombinant DNA on polyacrylamide and agarose gels, and by heteroduplex mapping. The results show that extensive DNA copies of Xenopus globin mRNA have been integrated into recombinant plasmids. Images PMID:347404

  13. Toxin-antitoxin-stabilized reporter plasmids for biophotonic imaging of Group A streptococcus.

    PubMed

    Loh, Jacelyn M S; Proft, Thomas

    2013-11-01

    Bioluminescence is a rapid and cost-efficient optical imaging technology that allows the detection of bacteria in real-time during disease development. Here, we report a novel strategy to generate a wide range of bioluminescent group A streptococcus (GAS) strains by using a toxin-antitoxin-stabilized plasmid. The bacterial luciferin-luciferase operon (lux) or the firefly luciferase gene (ffluc) was introduced into GAS via a stabilized plasmid. The FFluc reporter gave significantly stronger bioluminescent signals than the Lux reporter, and was generally more stable. Plasmid-based luciferase reporters could easily be introduced into a variety of GAS strains and the signals correlated linearly with viable cell counts. Co-expression of the streptococcal ω-ε-ζ toxin-antitoxin operon provided segregational stability in the absence of antibiotics for at least 17 passages in vitro and up to 7 days in a mouse infection model. In addition, genome-integrated reporter constructs were also generated by site-specific recombination, but were found to be technically more challenging. The quick and efficient generation of various M-type GAS strains expressing plasmid-based luciferase reporters with comparable and quantifiable bioluminescence signals allows for comparative analysis of different GAS strains in vitro and in vivo.

  14. A calibrator plasmid for quantitative analysis of insect resistant maize (Yieldgard MON 810).

    PubMed

    Ballari, Rajashekhar V; Martin, Asha; Gowda, Lalitha R

    2013-09-01

    Real-time PCR (RT-PCR) is the preferred method for the quantification of genetically modified organisms (GMOs) and implementation of labeling regulations. The precision, sensitivity, and reproducibility of RT-PCR data depend on the use of external calibrators. In this investigation, a dual target plasmid designated pRSETMON-02 comprising of MON 810 maize event specific and endogenous zein gene sequences in 1:1 ratio in tandem was constructed and validated. Commutability of plasmid DNA (pDNA) and genomic DNA (gDNA) calibrators for the quantification of MON 810 maize was assessed by employing a TaqMan RT-PCR targeting the P-35S and zein gene. Higher PCR efficiencies, good linearity and lower relative standard deviation (RSD) values were associated with pRSETMON-02 as opposed to gDNA calibrants. pDNA calibrants exhibited better performance characteristic in terms of closeness to the expected value of unknown samples than their genomic counterparts. Short term stability study of the pRSETMON-02 plasmid stored at different temperatures showed that pDNA is stable for 45 days at -20, and 4 °C. The results demonstrated that the developed dual target plasmid pRSETMON-02 is fit for the intended use of quantifying MON 810 maize and is a better alternative to conventional seed powder calibrants.

  15. Rotations with Rodrigues' Vector

    ERIC Educational Resources Information Center

    Pina, E.

    2011-01-01

    The rotational dynamics was studied from the point of view of Rodrigues' vector. This vector is defined here by its connection with other forms of parametrization of the rotation matrix. The rotation matrix was expressed in terms of this vector. The angular velocity was computed using the components of Rodrigues' vector as coordinates. It appears…

  16. Invasion of E. coli biofilms by antibiotic resistance plasmids.

    PubMed

    Król, Jaroslaw E; Wojtowicz, Andrzej J; Rogers, Linda M; Heuer, Holger; Smalla, Kornelia; Krone, Stephen M; Top, Eva M

    2013-07-01

    In spite of the contribution of plasmids to the spread of antibiotic resistance in human pathogens, little is known about the transferability of various drug resistance plasmids in bacterial biofilms. The goal of this study was to compare the efficiency of transfer of 19 multidrug resistance plasmids into Escherichia coli recipient biofilms and determine the effects of biofilm age, biofilm-donor exposure time, and donor-to-biofilm attachment on this process. An E. coli recipient biofilm was exposed separately to 19 E. coli donors, each with a different plasmid, and transconjugants were determined by plate counting. With few exceptions, plasmids that transferred well in a liquid environment also showed the highest transferability in biofilms. The difference in transfer frequency between the most and least transferable plasmid was almost a million-fold. The 'invasibility' of the biofilm by plasmids, or the proportion of biofilm cells that acquired plasmids within a few hours, depended not only on the type of plasmid, but also on the time of biofilm exposure to the donor and on the ability of the plasmid donor to attach to the biofilm, yet not on biofilm age. The efficiency of donor strain attachment to the biofilm was not affected by the presence of plasmids. The most invasive plasmid was pHH2-227, which based on genome sequence analysis is a hybrid between IncU-like and IncW plasmids. The wide range in transferability in an E. coli biofilm among plasmids needs to be taken into account in our fight against the spread of drug resistance.

  17. Linear Colliders

    NASA Astrophysics Data System (ADS)

    Yamamoto, Akira; Yokoya, Kaoru

    2015-02-01

    An overview of linear collider programs is given. The history and technical challenges are described and the pioneering electron-positron linear collider, the SLC, is first introduced. For future energy frontier linear collider projects, the International Linear Collider (ILC) and the Compact Linear Collider (CLIC) are introduced and their technical features are discussed. The ILC is based on superconducting RF technology and the CLIC is based on two-beam acceleration technology. The ILC collaboration completed the Technical Design Report in 2013, and has come to the stage of "Design to Reality." The CLIC collaboration published the Conceptual Design Report in 2012, and the key technology demonstration is in progress. The prospects for further advanced acceleration technology are briefly discussed for possible long-term future linear colliders.

  18. Linear Colliders

    NASA Astrophysics Data System (ADS)

    Yamamoto, Akira; Yokoya, Kaoru

    An overview of linear collider programs is given. The history and technical challenges are described and the pioneering electron-positron linear collider, the SLC, is first introduced. For future energy frontier linear collider projects, the International Linear Collider (ILC) and the Compact Linear Collider (CLIC) are introduced and their technical features are discussed. The ILC is based on superconducting RF technology and the CLIC is based on two-beam acceleration technology. The ILC collaboration completed the Technical Design Report in 2013, and has come to the stage of "Design to Reality." The CLIC collaboration published the Conceptual Design Report in 2012, and the key technology demonstration is in progress. The prospects for further advanced acceleration technology are briefly discussed for possible long-term future linear colliders.

  19. Model suicide vector for containment of genetically engineered microorganisms.

    PubMed

    Bej, A K; Perlin, M H; Atlas, R M

    1988-10-01

    A model suicide vector (pBAP19h), designed for the potential containment of genetically engineered microorganisms, was made by constructing a plasmid with the hok gene, which codes for a lethal polypeptide, under the control of the lac promoter. The vector plasmid also codes for carbenicillin resistance. In the absence of carbenicillin, induction of the hok gene in vitro caused elimination of all detectable cells containing the suicide vector; pBAP19h-free cells of the culture survived and grew exponentially. In the presence of carbenicillin, however, the number of cells containing pBAP19h initially declined after induction of hok but then multiplied exponentially. The surviving cells still had a fully functional hok gene and had apparently developed resistance to the action of the Hok polypeptide. Thus, high selective pressure against the loss of the suicide vector led to a failure of the system. Soil microcosm experiments confirmed the ability of a suicide vector to restrict the growth of a genetically engineered microorganism in the absence of selective pressure against the loss of the plasmid, with 90 to 99% elimination of hok-bearing cells within 24 h of hok induction. However, some pBAP19h-bearing cells survived in the soil microcosms after hok induction. The surviving cells contained an active hok gene but were not capable of normal growth even after elimination of the hok gene; it appears that a mutation that made them Hok resistant also reduced their capacity for membrane functions needed for energy generation and exponential cell growth. Thus, the model suicide vector was shown to be functional in soil as well as in vitro.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3060017

  20. Transposon-containing DNA cloning vector and uses thereof

    DOEpatents

    Berg, Claire M.; Berg, Douglas E.; Wang, Gan

    1997-01-01

    The present invention discloses a rapid method of restriction mapping, sequencing or localizing genetic features in a segment of deoxyribonucleic acid (DNA) that is up to 42 kb in size. The method in part comprises cloning of the DNA segment in a specialized cloning vector and then isolating nested deletions in either direction in vivo by intramolecular transposition into the cloned DNA. A plasmid has been prepared and disclosed.

  1. Transposon-containing DNA cloning vector and uses thereof

    DOEpatents

    Berg, C.M.; Berg, D.E.; Wang, G.

    1997-07-08

    The present invention discloses a rapid method of restriction mapping, sequencing or localizing genetic features in a segment of deoxyribonucleic acid (DNA) that is up to 42 kb in size. The method in part comprises cloning of the DNA segment in a specialized cloning vector and then isolating nested deletions in either direction in vivo by intramolecular transposition into the cloned DNA. A plasmid has been prepared and disclosed. 4 figs.

  2. First Report of Complete Sequence of a blaNDM-13-Harboring Plasmid from an Escherichia coli ST5138 Clinical Isolate

    PubMed Central

    Lv, Jingnan; Qi, Xiuqin; Zhang, Dan; Zheng, Zhou; Chen, Yuehui; Guo, Yinjuan; Wang, Shanshan; Chen, Liang; Kreiswirth, Barry N.; Tang, Yi-Wei; Chen, Zengqiang; Hu, Longhua; Wang, Liangxing; Yu, Fangyou

    2016-01-01

    Since the first report of blaNDM-1, 16 blaNDM variants have been identified among Gram-negative bacteria worldwide. Recently, a novel blaNDM variant, blaNDM-13, was identified in the chromosome of an ST101 Escherichia coli isolate from Nepal. Here we first reported plasmid-mediated blaNDM-13 in a carbapenem-resistant E. coli ST5138 clinical isolate associated with hospital-acquired urinary tract infection from China. blaNDM-13 and blaSHV-12 coexisted on the a ~54 Kb self-transferable plasmid. Compared with NDM-1, NDM-13, NDM-3, and NDM-4 had two amino acid substitutions (D95N and M154L), one amino acid substitution (D95N) and one amino acid substitutions (M154L), respectively. Complete plasmid sequencing showed that blaNDM-13-harboring plasmid (pNDM13-DC33) was highly similar to the blaNDM-1-harboring IncX3 plasmid pNDM-HN380, a common blaNDM-harboring vector circulating in China. In accordance with the structure of pNDM-HN380, pNDM13-DC33 consists of a 33-kb backbone encoding plasmid replication (repB), stability partitioning, and transfer (tra, trb, and pil) functions, and a 21-kb antimicrobial resistance region with high GC content between umuD and mpr genes. In conclusion, the present study is the first report of a plasmid-encoded blaNDM-13 and the complete sequence of a blaNDM-13-harboring plasmid (pNDM13-DC33). blaNDM-13 maybe originate from blaNDM-1 located on a pNDM-HN380-like plasmid by sequential mutations.

  3. Improved Primate Foamy Virus Vectors and Packaging Constructs

    PubMed Central

    Heinkelein, Martin; Dressler, Marco; Jármy, Gergely; Rammling, Matthias; Imrich, Horst; Thurow, Jana; Lindemann, Dirk; Rethwilm, Axel

    2002-01-01

    Foamy virus (FV) vectors that have minimal cis-acting sequences and are devoid of residual viral gene expression were constructed and analyzed by using a packaging system based on transient cotransfection of vector and different packaging plasmids. Previous studies indicated (i) that FV gag gene expression requires the presence of the R region of the long terminal repeat and (ii) that RNA from packaging constructs is efficiently incorporated into vector particles. Mutants with changes in major 5′ splice donor (SD) site located in the R region identified this sequence element as responsible for regulating gag gene expression by an unidentified mechanism. Replacement of the FV 5′ SD with heterologous splice sites enabled expression of the gag and pol genes. The incorporation of nonvector RNA into vector particles could be reduced to barely detectable levels with constructs in which the human immunodeficiency virus 5′ SD or an unrelated intron sequence was substituted for the FV 5′ untranslated region and in which gag expression and pol expression were separated on two different plasmids. By this strategy, efficient vector transfer was achieved with constructs that have minimal genetic overlap. PMID:11907217

  4. Improved primate foamy virus vectors and packaging constructs.

    PubMed

    Heinkelein, Martin; Dressler, Marco; Jármy, Gergely; Rammling, Matthias; Imrich, Horst; Thurow, Jana; Lindemann, Dirk; Rethwilm, Axel

    2002-04-01

    Foamy virus (FV) vectors that have minimal cis-acting sequences and are devoid of residual viral gene expression were constructed and analyzed by using a packaging system based on transient cotransfection of vector and different packaging plasmids. Previous studies indicated (i) that FV gag gene expression requires the presence of the R region of the long terminal repeat and (ii) that RNA from packaging constructs is efficiently incorporated into vector particles. Mutants with changes in major 5' splice donor (SD) site located in the R region identified this sequence element as responsible for regulating gag gene expression by an unidentified mechanism. Replacement of the FV 5' SD with heterologous splice sites enabled expression of the gag and pol genes. The incorporation of nonvector RNA into vector particles could be reduced to barely detectable levels with constructs in which the human immunodeficiency virus 5' SD or an unrelated intron sequence was substituted for the FV 5' untranslated region and in which gag expression and pol expression were separated on two different plasmids. By this strategy, efficient vector transfer was achieved with constructs that have minimal genetic overlap.

  5. Linear Collisions

    ERIC Educational Resources Information Center

    Walkiewicz, T. A.; Newby, N. D., Jr.

    1972-01-01

    A discussion of linear collisions between two or three objects is related to a junior-level course in analytical mechanics. The theoretical discussion uses a geometrical approach that treats elastic and inelastic collisions from a unified point of view. Experiments with a linear air track are described. (Author/TS)

  6. Antimicrobial resistance and the ecology of Escherichia coli plasmids.

    PubMed Central

    Platt, D. J.; Sommerville, J. S.; Kraft, C. A.; Timbury, M. C.

    1984-01-01

    Four hundred and seven clinical isolates of Escherichia coli were examined for the presence of plasmids. These isolates comprised 189 which were collected irrespective of antimicrobial resistance (VP) and 218 which were collected on the basis of high-level trimethoprim resistance (TPR). The VP isolates were divided into drug sensitive (VPS) and drug-resistant (VPR) subpopulations. Plasmids were detected in 88% of VP isolates (81% of VPS and 94% of VPR) and 98% of TPR isolates. The distribution of plasmids in both groups and subpopulations was very similar. However, there were small but statistically significant differences between the plasmid distributions. These showed that more isolates in the resistant groups harboured plasmids than in the sensitive subpopulation (VPS) and that the number of plasmids carried by resistant isolates was greater. Multiple drug resistance was significantly more common among TPR isolates than the VPR subpopulation and this was paralleled by increased numbers of plasmids. Fifty-eight per cent of VPR and 57% of TPR isolates transferred antimicrobial resistance and plasmids to E. coli K12. Of the R+ isolates, 60% carried small plasmids (MW less than 20Md) and 52% of these co-transferred with R-plasmids. These results are discussed. PMID:6389695

  7. A vector platform for the rapid and efficient engineering of stable complex transgenes

    PubMed Central

    Jäckel, Carsten; Nogueira, Melanie Schmitt; Ehni, Nadja; Kraus, Christiane; Ranke, Julius; Dohmann, Maike; Noessner, Elfriede; Nelson, Peter J.

    2016-01-01

    We describe the generation of a set of plasmid vector tools that allow the rapid generation of complex-interacting stable transgenes in immortalized and primary cells. Of particular importance is inclusion of a mechanism to monitor the activation status of regulatory pathways via a reporter cassette (using Gaussia Luciferase), with control of additional transgene expression through doxycycline de-repression. The resulting vectors can be used to assess regulatory pathway activation and are well suited for regulatory pathway crosstalk studies. The system incorporates MultiSite-Gateway cloning for the rapid generation of vectors allowing flexible choice of promoters and transgenes, and Sleeping Beauty transposase technology for efficient incorporation of multiple transgenes in into host cell DNA. The vectors and a library of compatible Gateway Entry clones are available from the non-profit plasmid repository Addgene. PMID:27694838

  8. Sustaining protein synthesis in the absence of rapid cell division: an investigation of plasmid-encoded protein expression in Escherichia coli during very slow growth.

    PubMed

    Flickinger, M C; Rouse, M P

    1993-01-01

    The minimum growth rate capable of supporting plasmid-encoded gene expression is determined using continuous cultures of Escherichia coli MZ9387 at dilution rates (D) as low as 5% of the maximum specific growth rate. Expression from a low copy number plasmid, pMPR166, encoding cyanase under the control of P(lac) is investigated in order to study plasmid-encoded gene expression under conditions approaching starvation. Plasmid copy number was stabilized by selection in the presence of 500 micrograms/mL chloramphenicol by constitutive expression of chloramphenicol acetyl transferase (CAT). Plasmid retention was determined by dot-blot hybridization and chloramphenicol resistance. The contribution of plasmid maintenance and cyanase expression to the maximum cell yield (Y'x/s) and the maintenance coefficient (ms) was determined for MZ9387 and MZ9387:pMPR166 under uninduced and IPTG-induced conditions. The values of Y'x/s and ms for non-plasmid-bearing cultures were 0.56 g of cell dry mass (DCM)/g of glucose and 0.26 g of glucose/g of DCM.h, respectively. The cell yield for plasmid-bearing cultures under uninduced conditions (Y 0'x/s) was 0.28 g of DCM/g of glucose, with m0s = 0.08 g of glucose/g of DCM.h. These values decreased following induction of cyanase expression. Glucose consumption in the presence of IPTG was linearly related to the growth rate at D < 0.28 h-1 but nonlinear at dilution rates greater than 50% of the maximum specific growth rate, indicating that cyanase expression alters metabolism and glucose consumption. The fraction of plasmid-free cells decreased with decreasing Damköhler number (Da). These data confirm the usefulness of Da for predicting the relationship between plasmid-free and plasmid-bearing cells where plasmids are stabilized by concentrations of antibiotic greater than the minimum plasmid-free host cell growth inhibitory concentration. Specific cyanase expression increased as the dilution rate decreased to D = 0.15 h-1. Between D = 0

  9. Promoters of the Broad Host Range Plasmid Rk2: Analysis of Transcription (Initiation) in Five Species of Gram-Negative Bacteria

    PubMed Central

    Greener, A.; Lehman, S. M.; Helinski, D. R.

    1992-01-01

    A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined. PMID:1732166

  10. Plasmid transfection in mammalian cells spatiotemporally tracked by a gold nanoparticle.

    PubMed

    Muroski, Megan E; Carnevale, Kate J F; Riskowski, Ryan A; Strouse, Geoffrey F

    2015-01-27

    Recent advances in cell transfection have suggested that delivery of a gene on a gold nanoparticle (AuNP) can enhance transfection efficiency. The mechanism of transfection is poorly understood, particularly when the gene is appended to a AuNP, as expression of the desired exogenous protein is dependent not only on the efficiency of the gene being taken into the cell but also on efficient endosomal escape and cellular processing of the nucleic acid. Design of a multicolor surface energy transfer (McSET) molecular beacon by independently dye labeling a linearized plasmid and short duplex DNA (sdDNA) appended to a AuNP allows spatiotemporal profiling of the transfection events, providing insight into package uptake, disassembly, and final plasmid expression. Delivery of the AuNP construct encapsulated in Lipofectamine2000 is monitored in Chinese hamster ovary cells using live-cell confocal microscopy. The McSET beacon signals the location and timing of the AuNP release and endosomal escape events for the plasmid and the sdDNA discretely, which are correlated with plasmid transcription by fluorescent protein expression within the cell. It is observed that delivery of the construct leads to endosomal release of the plasmid and sdDNA from the AuNP surface at different rates, prior to endosomal escape. Slow cytosolic diffusion of the nucleic acids is believed to be the limiting step for transfection, impacting the time-dependent expression of protein. The overall protein expression yield is enhanced when delivered on a AuNP, possibly due to better endosomal escape or lower degradation prior to endosomal escape.

  11. Nonviral Vectors for Gene Delivery

    NASA Astrophysics Data System (ADS)

    Baoum, Abdulgader Ahmed

    2011-12-01

    The development of nonviral vectors for safe and efficient gene delivery has been gaining considerable attention recently. An ideal nonviral vector must protect the gene against degradation by nuclease in the extracellular matrix, internalize the plasma membrane, escape from the endosomal compartment, unpackage the gene at some point and have no detrimental effects. In comparison to viruses, nonviral vectors are relatively easy to synthesize, less immunogenic, low in cost, and have no limitation in the size of a gene that can be delivered. Significant progress has been made in the basic science and applications of various nonviral gene delivery vectors; however, the majority of nonviral approaches are still inefficient and often toxic. To this end, two nonviral gene delivery systems using either biodegradable poly(D,L-lactide- co-glycolide) (PLG) nanoparticles or cell penetrating peptide (CPP) complexes have been designed