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Sample records for rpa mediates recombination

  1. RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells

    Sleeth, Kate M; Srensen, Claus Storgaard; Issaeva, Natalia; Dziegielewski, Jaroslaw; Bartek, Jiri; Helleday, Thomas

    2007-01-01

    The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited...... the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation...... and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a...

  2. RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells.

    Sleeth, Kate M; Srensen, Claus Storgaard; Issaeva, Natalia; Dziegielewski, Jaroslaw; Bartek, Jiri; Helleday, Thomas

    2007-10-12

    The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role of RPA in homologous recombination in assembly of the RAD51 and RAD52 proteins. Furthermore, our data suggest that replacement of RPA with the RAD51 and RAD52 proteins is affected by checkpoint signalling. PMID:17765923

  3. RPA homologs and ssDNA processing during meiotic recombination.

    Ribeiro, Jonathan; Abby, Emilie; Livera, Gabriel; Martini, Emmanuelle

    2016-06-01

    Meiotic homologous recombination is a specialized process that involves homologous chromosome pairing and strand exchange to guarantee proper chromosome segregation and genetic diversity. The formation and repair of DNA double-strand breaks (DSBs) during meiotic recombination differs from those during mitotic recombination in that the homologous chromosome rather than the sister chromatid is the preferred repair template. The processing of single-stranded DNA (ssDNA) formed on intermediate recombination structures is central to driving the specific outcomes of DSB repair during meiosis. Replication protein A (RPA) is the main ssDNA-binding protein complex involved in DNA metabolism. However, the existence of RPA orthologs in plants and the recent discovery of meiosis specific with OB domains (MEIOB), a widely conserved meiosis-specific RPA1 paralog, strongly suggest that multiple RPA complexes evolved and specialized to subdivide their roles during DNA metabolism. Here we review ssDNA formation and maturation during mitotic and meiotic recombination underlying the meiotic specific features. We describe and discuss the existence and properties of MEIOB and multiple RPA subunits in plants and highlight how they can provide meiosis-specific fates to ssDNA processing during homologous recombination. Understanding the functions of these RPA homologs and how they interact with the canonical RPA subunits is of major interest in the fields of meiosis and DNA repair. PMID:26520106

  4. In Vitro Analysis of the Role of Replication Protein A (RPA) and RPA Phosphorylation in ATR-mediated Checkpoint Signaling*

    Lindsey-Boltz, Laura A.; Reardon, Joyce T.; Wold, Marc S.; Sancar, Aziz

    2012-01-01

    Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair. PMID:22948311

  5. Molecular anatomy of the recombination mediator function of Saccharomyces cerevisiae Rad52

    Seong, C.; Sehorn, M.G.; Plate, Iben; Shi, I.; Song, B.; Chi, P.; Mortensen, Uffe Hasbro; Sung, P.; Krejci, L.

    2008-01-01

    A helical filament of Rad51 on single-strand DNA (ssDNA), called the presynaptic filament, catalyzes DNA joint formation during homologous recombination. Rad52 facilitates presynaptic filament assembly, and this recombination mediator activity is thought to rely on the interactions of Rad52 with...... Rad51, the ssDNA-binding protein RPA, and ssDNA. The N-terminal region of Rad52, which has DNA binding activity and an oligomeric structure, is thought to be crucial for mediator activity and recombination. Unexpectedly, we find that the C-terminal region of Rad52 also harbors a DNA binding function....... Importantly, the Rad52 C-terminal portion alone can promote Rad51 presynaptic filament assembly. The middle portion of Rad52 associates with DNA-bound RPA and contributes to the recombination mediator activity. Accordingly, expression of a protein species that harbors the middle and C-terminal regions of Rad...

  6. The sensitivity of R_pA to color recombination effects

    Zapp, Korinna Christina; Wiedemann, Urs Achim

    2015-01-01

    In hadronization models with color recombination, partons are allowed to regroup into color singlet structures that are different from those determined by the perturbative parton shower. This aims at modeling the possibility that soft interactions of partons with the underlying event can change color connections. If such an effect is at play in proton-proton collisions, it may be expected to be enhanced in proton-nucleus collisions due to the higher color charge density in the underlying event. Here, we provide a qualitative argument that color recombination effects could lead to a multiplicity dependent hardening of single inclusive hadron spectra that dies out very weakly with increasing transverse momentum. We present results of a (conservative) model implementation in the cluster hadronization model of the SHERPA event generator. In this model, we find that color recombination effects harden indeed the single inclusive hadron spectra without affecting the jet spectra, but that this effect does not depend ...

  7. The self consistent RPA

    Following the equation of motion method and the variation principle we derive the self consistent RPA equations including a generalized mean field equation coupled to the RPA fluctuations. We show some of its properties in three exactly solvable models of particle-hole particle-particle and superfluid correlations. (Author)

  8. Anharmonicity in extended RPA

    An analytical formula for the calculation of the anharmonicity of the two-phonon state is presented which is based on a variational derivation of RPA-like equations for a many fermion system. Analytical estimations of the anharmonicity have been made using the Lipkin model and a degenerated schematic model. The formula obtained, together with the extended RPA predictions, have been checked by comparison with the exact solutions of the models. A rough estimation of the anharmonicity of the two phonon state built on the Giant Dipole Resonance and on the Giant Quadrupole Resonance has been deduced. (authors) 32 refs., 6 figs

  9. BRCA2 regulates DMC1-mediated recombination through the BRC repeats.

    Martinez, Juan S; von Nicolai, Catharina; Kim, Taeho; Ehlén, Åsa; Mazin, Alexander V; Kowalczykowski, Stephen C; Carreira, Aura

    2016-03-29

    In somatic cells, BRCA2 is needed for RAD51-mediated homologous recombination. The meiosis-specific DNA strand exchange protein, DMC1, promotes the formation of DNA strand invasion products (joint molecules) between homologous molecules in a fashion similar to RAD51. BRCA2 interacts directly with both human RAD51 and DMC1; in the case of RAD51, this interaction results in stimulation of RAD51-promoted DNA strand exchange. However, for DMC1, little is known regarding the basis and functional consequences of its interaction with BRCA2. Here we report that human DMC1 interacts directly with each of the BRC repeats of BRCA2, albeit most tightly with repeats 1-3 and 6-8. However, BRC1-3 bind with higher affinity to RAD51 than to DMC1, whereas BRC6-8 bind with higher affinity to DMC1, providing potential spatial organization to nascent filament formation. With the exception of BRC4, each BRC repeat stimulates joint molecule formation by DMC1. The basis for this stimulation is an enhancement of DMC1-ssDNA complex formation by the stimulatory BRC repeats. Lastly, we demonstrate that full-length BRCA2 protein stimulates DMC1-mediated DNA strand exchange between RPA-ssDNA complexes and duplex DNA, thus identifying BRCA2 as a mediator of DMC1 recombination function. Collectively, our results suggest unique and specialized functions for the BRC motifs of BRCA2 in promoting homologous recombination in meiotic and mitotic cells. PMID:26976601

  10. Stn1-Ten1 is an Rpa2-Rpa3-like complex at telomeres

    Sun, Jia; Yu, Eun Young; Yang, Yuting; Confer, Laura A; Sun, Steven H; Wan, Ke; Lue, Neal F; Lei, Ming; (Weill); (Michigan-Med)

    2010-09-02

    In budding yeast, Cdc13, Stn1, and Ten1 form a heterotrimeric complex (CST) that is essential for telomere protection and maintenance. Previous bioinformatics analysis revealed a putative oligonucleotide/oligosaccharide-binding (OB) fold at the N terminus of Stn1 (Stn1N) that shows limited sequence similarity to the OB fold of Rpa2, a subunit of the eukaryotic ssDNA-binding protein complex replication protein A (RPA). Here we present functional and structural analyses of Stn1 and Ten1 from multiple budding and fission yeast. The crystal structure of the Candida tropicalis Stn1N complexed with Ten1 demonstrates an Rpa2N-Rpa3-like complex. In both structures, the OB folds of the two components pack against each other through interactions between two C-terminal helices. The structure of the C-terminal domain of Saccharomyces cerevisiae Stn1 (Stn1C) was found to comprise two related winged helix-turn-helix (WH) motifs, one of which is most similar to the WH motif at the C terminus of Rpa2, again supporting the notion that Stn1 resembles Rpa2. The crystal structure of the fission yeast Schizosaccharomyces pombe Stn1N-Ten1 complex exhibits a virtually identical architecture as the C. tropicalis Stn1N-Ten1. Functional analyses of the Candida albicans Stn1 and Ten1 proteins revealed critical roles for these proteins in suppressing aberrant telomerase and recombination activities at telomeres. Mutations that disrupt the Stn1-Ten1 interaction induce telomere uncapping and abolish the telomere localization of Ten1. Collectively, our structural and functional studies illustrate that, instead of being confined to budding yeast telomeres, the CST complex may represent an evolutionarily conserved RPA-like telomeric complex at the 3' overhangs that works in parallel with or instead of the well-characterized POT1-TPP1/TEBP{alpha}-{beta} complex.

  11. Cross-talk and regulatory interactions between the essential response regulator RpaB and cyanobacterial circadian clock output

    Espinosa, Javier; Boyd, Joseph S.; Cantos, Raquel; Salinas, Paloma; Golden, Susan S.; Contreras, Asuncion

    2015-01-01

    The response regulator RpaB (regulator of phycobilisome associated B), part of an essential two-component system conserved in cyanobacteria that responds to multiple environmental signals, has recently been implicated in the control of cell dimensions and of circadian rhythms of gene expression in the model cyanobacterium Synechococcus elongatus PCC 7942. However, little is known of the molecular mechanisms that underlie RpaB functions. In this study we show that the regulation of phenotypes by RpaB is intimately connected with the activity of RpaA (regulator of phycobilisome associated A), the master regulator of circadian transcription patterns. RpaB affects RpaA activity both through control of gene expression, a function requiring an intact effector domain, and via altering RpaA phosphorylation, a function mediated through the N-terminal receiver domain of RpaB. Thus, both phosphorylation cross-talk and coregulation of target genes play a role in the genetic interactions between the RpaA and RpaB pathways. In addition, RpaB?P levels appear critical for survival under light:dark cycles, conditions in which RpaB phosphorylation is environmentally driven independent of the circadian clock. We propose that the complex regulatory interactions between the essential and environmentally sensitive NblS-RpaB system and the SasA-RpaA clock output system integrate relevant extra- and intracellular signals to the circadian clock. PMID:25653337

  12. ATR Prohibits Replication Catastrophe by Preventing Global Exhaustion of RPA

    Toledo Lazaro, Luis Ignacio; Altmeyer, Matthias; Rask, Maj-Britt; Lukas, Claudia; Larsen, Dorthe Helena; Povlsen, Lou Klitgaard; Bekker-Jensen, Simon; Mailand, Niels; Bartek, Jiri; Lukas, Jiri

    2013-01-01

    ATR, activated by replication stress, protects replication forks locally and suppresses origin firing globally. Here, we show that these functions of ATR are mechanistically coupled. Although initially stable, stalled forks in ATR-deficient cells undergo nucleus-wide breakage after unscheduled...... origin firing generates an excess of single-stranded DNA that exhausts the nuclear pool of RPA. Partial reduction of RPA accelerated fork breakage, and forced elevation of RPA was sufficient to delay such "replication catastrophe" even in the absence of ATR activity. Conversely, unscheduled origin firing...... induced breakage of stalled forks even in cells with active ATR. Thus, ATR-mediated suppression of dormant origins shields active forks against irreversible breakage via preventing exhaustion of nuclear RPA. This study elucidates how replicating genomes avoid destabilizing DNA damage. Because cancer cells...

  13. Mechanochemical regulations of RPA's binding to ssDNA

    Chen, Jin; Le, Shimin; Basu, Anindita; Chazin, Walter J.; Yan, Jie

    2015-03-01

    Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein that serves to protect ssDNA from degradation and annealing, and as a template for recruitment of many downstream factors in virtually all DNA transactions in cell. During many of these transactions, DNA is tethered and is likely subject to force. Previous studies of RPA's binding behavior on ssDNA were conducted in the absence of force; therefore the RPA-ssDNA conformations regulated by force remain unclear. Here, using a combination of atomic force microscopy imaging and mechanical manipulation of single ssDNA tethers, we show that force mediates a switch of the RPA bound ssDNA from amorphous aggregation to a much more regular extended conformation. Further, we found an interesting non-monotonic dependence of the binding affinity on monovalent salt concentration in the presence of force. In addition, we discovered that zinc in micromolar concentrations drives ssDNA to a unique, highly stiff and more compact state. These results provide new mechanochemical insights into the influences and the mechanisms of action of RPA on large single ssDNA.

  14. GENERATION OF RECOMBINANT BACULOVIRUS VIA LIPOSOME MEDIATED TRANSFECTION

    Baculovirus expression vectors have become a popular method of producing recombinant proteins. Production of recombinant virus requires the transfection of both the native viral DNA and a transfer plasmid into insect cells where recombination takes place. While several methods of...

  15. Bistability-mediated carrier recombination at light-induced boron-oxygen complexes in silicon.

    Du, Mao-Hua; Branz, Howard M; Crandall, Richard S; Zhang, S B

    2006-12-22

    A first-principles study of the BO2 complex in B-doped Czochralski Si reveals a defect-bistability-mediated carrier recombination mechanism, which contrasts with the standard fixed-level Shockley-Read-Hall model of recombination. An O2 dimer distant from B causes only weak carrier recombination, which nevertheless drives O2 diffusion under light to form the BO2 complex. Although BO2 and O2 produce nearly identical defect levels in the band gap, the recombination at BO2 is substantially faster than at O2 because the charge state of the latter inhibits the hole capture step of recombination. PMID:17280375

  16. RPA calculations with Gaussian expansion method

    Nakada, H.; Mizuyama, K.; Yamagami, M; Matsuo, M.

    2009-01-01

    The Gaussian expansion method (GEM) is extensively applied to the calculations in the random-phase approximation (RPA). We adopt the mass-independent basis-set that has been tested in the mean-field calculations. By comparing the RPA results with those obtained by several other available methods for Ca isotopes, using a density-dependent contact interaction and the Woods-Saxon single-particle states, we confirm that energies, transition strengths and widths of their distribution are described...

  17. Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats.

    Warmerdam, Daniël O; van den Berg, Jeroen; Medema, René H

    2016-03-22

    rDNA repeats constitute the most heavily transcribed region in the human genome. Tumors frequently display elevated levels of recombination in rDNA, indicating that the repeats are a liability to the genomic integrity of a cell. However, little is known about how cells deal with DNA double-stranded breaks in rDNA. Using selective endonucleases, we show that human cells are highly sensitive to breaks in 45S but not the 5S rDNA repeats. We find that homologous recombination inhibits repair of breaks in 45S rDNA, and this results in repeat loss. We identify the structural maintenance of chromosomes protein 5 (SMC5) as contributing to recombination-mediated repair of rDNA breaks. Together, our data demonstrate that SMC5-mediated recombination can lead to error-prone repair of 45S rDNA repeats, resulting in their loss and thereby reducing cellular viability. PMID:26972008

  18. Homologous recombination-mediated cloning and manipulation of genomic DNA regions using Gateway and recombineering systems

    Kagale Sateesh

    2008-11-01

    Full Text Available Abstract Background Employing genomic DNA clones to characterise gene attributes has several advantages over the use of cDNA clones, including the presence of native transcription and translation regulatory sequences as well as a representation of the complete repertoire of potential splice variants encoded by the gene. However, working with genomic DNA clones has traditionally been tedious due to their large size relative to cDNA clones and the presence, absence or position of particular restriction enzyme sites that may complicate conventional in vitro cloning procedures. Results To enable efficient cloning and manipulation of genomic DNA fragments for the purposes of gene expression and reporter-gene studies we have combined aspects of the Gateway system and a bacteriophage-based homologous recombination (i.e. recombineering system. To apply the method for characterising plant genes we developed novel Gateway and plant transformation vectors that are of small size and incorporate selectable markers which enable efficient identification of recombinant clones. We demonstrate that the genomic coding region of a gene can be directly cloned into a Gateway Entry vector by recombineering enabling its subsequent transfer to Gateway Expression vectors. We also demonstrate how the coding and regulatory regions of a gene can be directly cloned into a plant transformation vector by recombineering. This construct was then rapidly converted into a novel Gateway Expression vector incorporating cognate 5' and 3' regulatory regions by using recombineering to replace the intervening coding region with the Gateway Destination cassette. Such expression vectors can be applied to characterise gene regulatory regions through development of reporter-gene fusions, using the Gateway Entry clones of GUS and GFP described here, or for ectopic expression of a coding region cloned into a Gateway Entry vector. We exemplify the utility of this approach with the Arabidopsis PAP85 gene and demonstrate that the expression profile of a PAP85::GUS transgene highly corresponds with native PAP85 expression. Conclusion We describe a novel combination of the favourable attributes of the Gateway and recombineering systems to enable efficient cloning and manipulation of genomic DNA clones for more effective characterisation of gene function. Although the system and plasmid vectors described here were developed for applications in plants, the general approach is broadly applicable to gene characterisation studies in many biological systems.

  19. Retrovirus-Associated Heparan Sulfate Mediates Immobilization and Gene Transfer on Recombinant Fibronectin

    Lei, Pedro; Bajaj, Bharat; Andreadis, Stelios T.

    2002-01-01

    Recombinant retroviruses have been shown to bind to fibronectin (FN) and increase the efficiency of gene transfer to a variety of cell types. Despite recent work to optimize gene transfer on recombinant FN, the mechanism of retrovirus binding to FN and the interactions of target cells with the bound virus remain elusive. We investigated the roles of virus surface glycoprotein (gp70), cell-conditioned medium, and proteoglycans in mediating retrovirus binding to FN. We also examined the role of...

  20. Microbial antigenic variation mediated by homologous DNA recombination.

    Vink, Cornelis; Rudenko, Gloria; Seifert, H Steven

    2012-09-01

    Pathogenic microorganisms employ numerous molecular strategies in order to delay or circumvent recognition by the immune system of their host. One of the most widely used strategies of immune evasion is antigenic variation, in which immunogenic molecules expressed on the surface of a microorganism are continuously modified. As a consequence, the host is forced to constantly adapt its humoral immune response against this pathogen. An antigenic change thus provides the microorganism with an opportunity to persist and/or replicate within the host (population) for an extended period of time or to effectively infect a previously infected host. In most cases, antigenic variation is caused by genetic processes that lead to the modification of the amino acid sequence of a particular antigen or to alterations in the expression of biosynthesis genes that induce changes in the expression of a variant antigen. Here, we will review antigenic variation systems that rely on homologous DNA recombination and that are found in a wide range of cellular, human pathogens, including bacteria (such as Neisseria spp., Borrelia spp., Treponema pallidum, and Mycoplasma spp.), fungi (such as Pneumocystis carinii) and parasites (such as the African trypanosome Trypanosoma brucei). Specifically, the various DNA recombination-based antigenic variation systems will be discussed with a focus on the employed mechanisms of recombination, the DNA substrates, and the enzymatic machinery involved. PMID:22212019

  1. Genetic analysis of yeast RPA1 reveals its multiple functions in DNA metabolism

    Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 104 to 105 times increased sensitivity to these agents. Some of the UV- and MMSsensitive mutants were killed by an HO-induced double-strand break atMAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages. (author)

  2. Expression of an ortholog of replication protein A1 (RPA1) is induced by gibberellin in deepwater rice

    van der Knaap, Esther; Jagoueix, Sandrine; Kende, Hans

    1997-01-01

    Internodes of deepwater rice are induced to grow rapidly when plants become submerged. This adaptation enables deepwater rice to keep part of its foliage above the rising flood waters during the monsoon season and to avoid drowning. This growth response is, ultimately, elicited by the plant hormone gibberellin (GA). The primary target tissue for GA action is the intercalary meristem of the internode. Using differential display of mRNA, we have isolated a number of genes whose expression in the intercalary meristem is regulated by GA. The product of one of these genes was identified as an ortholog of replication protein A1 (RPA1). RPA is a heterotrimeric protein involved in DNA replication, recombination, and repair and also in regulation of transcription. A chimeric construct, in which the single-stranded DNA-binding domain of rice RPA1 was spliced into the corresponding region of yeast RPA1, was able to complement a yeast rpa1 mutant. The transcript level of rice RPA1 is high in tissues containing dividing cells. RPA1 mRNA levels increase rapidly in the intercalary meristem during submergence and treatment with GA before the increase in the level of histone H3 mRNA, a marker for DNA replication. PMID:9275237

  3. TALEN-mediated homologous recombination in Daphnia magna

    Takashi Nakanishi; Yasuhiko Kato; Tomoaki Matsuura; Hajime Watanabe

    2015-01-01

    Transcription Activator-Like Effector Nucleases (TALENs) offer versatile tools to engineer endogenous genomic loci in various organisms. We established a homologous recombination (HR)-based knock-in using TALEN in the crustacean Daphnia magna, a model for ecological and toxicological genomics. We constructed TALENs and designed the 67 bp donor insert targeting a point deletion in the eyeless mutant that shows eye deformities. Co-injection of the TALEN mRNA with donor DNA into eggs led to the ...

  4. Recombinant adenovirus-mediated gene transfer suppresses experimental arthritis

    E. Quattrocchi; Feldmann, M

    2011-01-01

    Collagen Induced Arthritis (CIA) is a widely studied animal model to develop and test novel therapeutic approaches for treating Rheumatoid Arthritis (RA) in humans. Soluble Cytotoxic T-Lymphocyte Antigen 4 (CTLA4-Ig), which binds B7 molecule on antigen presenting cells and blocks CD28 mediated T-lymphocyte activation, has been shown to ameliorate experimental autoimmune diseases such as lupus, diabetes and CIA. Objective of our research was to investigate in vivo the effectiveness of blocking...

  5. Physical and unphysical solutions of RPA equation

    Nakada, H

    2016-01-01

    Properties of solutions of the RPA equation is reanalyzed mathematically, which is defined as a generalized eigenvalue problem of the stability matrix $\\mathsf{S}$ with the norm matrix $\\mathsf{N}=\\mathrm{diag.}(1,-1)$. As well as physical solutions, unphysical solutions are examined in detail, with taking the possibility of Jordan blocks of the matrix $\\mathsf{N\\,S}$ into consideration. Two types of duality of eigenvectors and basis vectors of the Jordan blocks are pointed out and explored, which disclose many basic properties of the RPA solutions.

  6. Overexpressed of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability

    Schild, David; Wiese, Claudia

    2009-10-15

    RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of single-stranded DNA (ss-DNA) coated by RPA (a ss-DNA binding protein). Displacement of RPA by RAD51 is highly regulated and facilitated by a number of different proteins known as the 'recombination mediators'. To assist these recombination mediators, a second group of proteins also is required and we are defining these proteins here as 'recombination co-mediators'. Defects in either recombination mediators or comediators, including BRCA1 and BRCA2, lead to impaired HRR that can genetically be complemented for (i.e. suppressed) by overexpression of RAD51. Defects in HRR have long been known to contribute to genomic instability leading to tumor development. Since genomic instability also slows cell growth, precancerous cells presumably require genomic restabilization to gain a growth advantage. RAD51 is overexpressed in many tumors, and therefore, we hypothesize that the complementing ability of elevated levels of RAD51 in tumors with initial HRR defects limits genomic instability during carcinogenic progression. Of particular interest, this model may also help explain the high frequency of TP53 mutations in human cancers, since wild-type p53 represses RAD51.

  7. TALEN-mediated homologous recombination in Daphnia magna.

    Nakanishi, Takashi; Kato, Yasuhiko; Matsuura, Tomoaki; Watanabe, Hajime

    2015-01-01

    Transcription Activator-Like Effector Nucleases (TALENs) offer versatile tools to engineer endogenous genomic loci in various organisms. We established a homologous recombination (HR)-based knock-in using TALEN in the crustacean Daphnia magna, a model for ecological and toxicological genomics. We constructed TALENs and designed the 67 bp donor insert targeting a point deletion in the eyeless mutant that shows eye deformities. Co-injection of the TALEN mRNA with donor DNA into eggs led to the precise integration of the donor insert in the germ line, which recovered eye deformities in offspring. The frequency of HR events in the germ line was 2% by using both plasmid and single strand oligo DNA with 1.5 kb and 80 nt homology to the target. Deficiency of ligase 4 involved in non-homologous end joining repair did not increase the HR efficiency. Our data represent efficient HR-based knock-in by TALENs in D. magna, which is a promising tool to understand Daphnia gene functions. PMID:26674741

  8. Recombinant adenovirus-mediated gene transfer suppresses experimental arthritis

    E. Quattrocchi

    2011-09-01

    Full Text Available Collagen Induced Arthritis (CIA is a widely studied animal model to develop and test novel therapeutic approaches for treating Rheumatoid Arthritis (RA in humans. Soluble Cytotoxic T-Lymphocyte Antigen 4 (CTLA4-Ig, which binds B7 molecule on antigen presenting cells and blocks CD28 mediated T-lymphocyte activation, has been shown to ameliorate experimental autoimmune diseases such as lupus, diabetes and CIA. Objective of our research was to investigate in vivo the effectiveness of blocking the B7/CD28 T-lymphocyte co-stimulatory pathway, utilizing a gene transfer technology, as a therapeutic strategy against CIA. Replication-deficient adenoviruses encoding a chimeric CTLA4-Ig fusion protein, or β-galactosidase as control, have been injected intravenously once at arthritis onset. Disease activity has been monitored by the assessment of clinical score, paw thickness and type II collagen (CII specific cellular and humoral immune responses for 21 days. The adenovirally delivered CTLA4-Ig fusion protein at a dose of 2×108 pfu suppressed established CIA, whereas the control β-galactosidase did not significantly affect the disease course. CII-specific lymphocyte proliferation, IFNg production and anti-CII antibodies were significantly reduced by CTLA4-Ig treatment. Our results demonstrate that blockade of the B7/CD28 co-stimulatory pathway by adenovirus-mediated CTLA4-Ig gene transfer is effective in treating established CIA suggesting its potential in treating RA.

  9. Extended GIPQ description of the RPA excitations

    The work presents an application of the GIPQ quantization method, extended to include the wave function quantization. By explicit calculation on a particular proton-neutron system it is shown that the extended quantization gives the same excitation operator and energy as the RPA. (authors)

  10. A recurrent translocation is mediated by homologous recombination between HERV-H elements

    Hermetz Karen E

    2012-01-01

    Full Text Available Abstract Background Chromosome rearrangements are caused by many mutational mechanisms; of these, recurrent rearrangements can be particularly informative for teasing apart DNA sequence-specific factors. Some recurrent translocations are mediated by homologous recombination between large blocks of segmental duplications on different chromosomes. Here we describe a recurrent unbalanced translocation casued by recombination between shorter homologous regions on chromosomes 4 and 18 in two unrelated children with intellectual disability. Results Array CGH resolved the breakpoints of the 6.97-Megabase (Mb loss of 18q and the 7.30-Mb gain of 4q. Sequencing across the translocation breakpoints revealed that both translocations occurred between 92%-identical human endogenous retrovirus (HERV elements in the same orientation on chromosomes 4 and 18. In addition, we find sequence variation in the chromosome 4 HERV that makes one allele more like the chromosome 18 HERV. Conclusions Homologous recombination between HERVs on the same chromosome is known to cause chromosome deletions, but this is the first report of interchromosomal HERV-HERV recombination leading to a translocation. It is possible that normal sequence variation in substrates of non-allelic homologous recombination (NAHR affects the alignment of recombining segments and influences the propensity to chromosome rearrangement.

  11. Human replication protein A: Global fold of the N-terminal RPA-70 domain reveals a basic cleft and flexible C-terminal linker+

    Human Replication Protein A (hsRPA) is required for multiple cellular processes in DNA metabolism including DNA repair, replication and recombination. It binds single-stranded DNA with high affinity and interacts specifically with multiple proteins. hsRPA forms a heterotrimeric complex composed of 70-, 32- and 14-kDa subunits (henceforth RPA70, RPA32, and RPA14). The N-terminal 168 residues of RPA70 form a structurally distinct domain that stimulates DNA polymerase α activity, interacts with several transcriptional activators including tumor suppressor p53, and during the cell cycle it signals escape from the DNA damage induced G2/M checkpoint. We have solved the global fold of the fragment corresponding to this domain (RPA70Δ169) and we find residues 8-108 of the N-terminal domain are structured. The remaining C-terminal residues are unstructured and may form a flexible linker to the DNA-binding domain of RPA70. The globular region forms a five-stranded anti-parallel β-barrel. The ends of the barrel are capped by short helices. Two loops on one side of the barrel form a large basic cleft which is a likely site for binding the acidic motifs of transcriptional activators. Many lethal or conditional lethal yeast point mutants map to this cleft, whereas no mutations with severe phenotype have been found in the linker region

  12. Partial Depletion of Histone H4 Increases Homologous Recombination-Mediated Genetic Instability

    Prado, Félix; Aguilera, Andrés

    2005-01-01

    DNA replication can be a source of genetic instability. Given the tight connection between DNA replication and nucleosome assembly, we analyzed the effect of a partial depletion of histone H4 on genetic instability mediated by homologous recombination. A Saccharomyces cerevisiae strain was constructed in which the expression of histone H4 was driven by the regulated tet promoter. In agreement with defective nucleosome assembly, partial depletion of histone H4 led to subtle changes in plasmid ...

  13. Hin-mediated DNA knotting and recombining promote replicon dysfunction and mutation

    Mann Jennifer K

    2007-05-01

    Full Text Available Abstract Background The genetic code imposes a dilemma for cells. The DNA must be long enough to encode for the complexity of an organism, yet thin and flexible enough to fit within the cell. The combination of these properties greatly favors DNA collisions, which can knot and drive recombination of the DNA. Despite the well-accepted propensity of cellular DNA to collide and react with itself, it has not been established what the physiological consequences are. Results Here we analyze the effects of recombined and knotted plasmids in E. coli using the Hin site-specific recombination system. We show that Hin-mediated DNA knotting and recombination (i promote replicon loss by blocking DNA replication; (ii block gene transcription; and (iii cause genetic rearrangements at a rate three to four orders of magnitude higher than the rate for an unknotted, unrecombined plasmid. Conclusion These results show that DNA reactivity leading to recombined and knotted DNA is potentially toxic and may help drive genetic evolution.

  14. RPA calculations with Gaussian expansion method

    The Gaussian expansion method (GEM) is applied to calculations of the nuclear excitations in the random-phase approximation (RPA). We adopt the mass-independent basis-set that is successful in the mean-field calculations. The RPA results obtained by the GEM are compared with those obtained by several other available methods in Ca isotopes, by using a density-dependent contact interaction along with the Woods-Saxon single-particle states. It is confirmed that energies, transition strengths and widths of their distribution are described by the GEM with good precision, for the 1-, 2+ and 3- collective states. The GEM is then applied to the self-consistent RPA calculations with the finite-range Gogny D1S interaction. The spurious center-of-mass motion is well separated from the physical states in the E1 response, and the energy-weighted sum rules for the isoscalar transitions are fulfilled reasonably well. Properties of low-energy transitions in 60Ca are investigated in some detail.

  15. RPA calculations with Gaussian expansion method

    Nakada, H; Yamagami, M; Matsuo, M

    2009-01-01

    The Gaussian expansion method (GEM) is extensively applied to the calculations in the random-phase approximation (RPA). We adopt the mass-independent basis-set that has been tested in the mean-field calculations. By comparing the RPA results with those obtained by several other available methods for Ca isotopes, using a density-dependent contact interaction and the Woods-Saxon single-particle states, we confirm that energies, transition strengths and widths of their distribution are described by the GEM bases to good precision, for the $1^-$, $2^+$ and $3^-$ collective states. The GEM is then applied to the self-consistent RPA calculations with the finite-range Gogny D1S interaction. The spurious center-of-mass motion is well separated from the physical states in the $E1$ response, and the energy-weighted sum rules for the isoscalar transitions are fulfilled reasonably well. Properties of low-energy transitions in $^{60}$Ca are argued in some detail.

  16. RPA regulates telomerase action by providing Est1p access to chromosome ends.

    Schramke, Vera; Luciano, Pierre; Brevet, Vanessa; Guillot, Sylvine; Corda, Yves; Longhese, Maria Pia; Gilson, Eric; Gli, Vincent

    2004-01-01

    Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein involved in DNA replication, recombination and repair. We show here that RPA is present at the telomeres of the budding yeast Saccharomyces cerevisiae, with a maximal association in S phase. A truncation of the N-terminal region of Rfa2p (associated with the rfa2Delta40 mutated allele) results in severe telomere shortening caused by a defect in the in vivo regulation of telomerase activity. Cells carrying rfa2Delta40 show impaired binding of the protein Est1p, which is required for telomerase action. In addition, normal telomere length can be restored by expressing a Cdc13-Est1p hybrid protein. These findings indicate that RPA activates telomerase by loading Est1p onto telomeres during S phase. We propose a model of in vivo telomerase action that involves synergistic action of RPA and Cdc13p at the G-rich 3' overhang of telomeric DNA. PMID:14702040

  17. Regulating infidelity: RNA-mediated recruitment of AID to DNA during class switch recombination.

    DiMenna, Lauren J; Chaudhuri, Jayanta

    2016-03-01

    The mechanism by which the DNA deaminase activation-induced cytidine deaminase (AID) is specifically recruited to repetitive switch region DNA during class switch recombination is still poorly understood. Work over the past decade has revealed a strong link between transcription and RNA polymerase-associated factors in AID recruitment, yet none of these processes satisfactorily explain how AID specificity is affected. Here, we review a recent finding wherein AID is guided to switch regions not by a protein factor but by an RNA moiety, and especially one associated with a noncoding RNA that has been long thought of as being inert. This work explains the long-standing requirement of splicing of noncoding transcripts during class switching, and has implications in both B cell-mediated immunity as well as the underlying pathological syndromes associated with the recombination reaction. PMID:26799454

  18. Recombinant equine interleukin-1? induces putative mediators of articular cartilage degradation in equine chondrocytes

    Tung, J. T.; Fenton, J. I.; Arnold, C.; Alexander, L.; Yuzbasiyan-Gurkan, V.; Venta, P. J.; Peters, T. L.; Orth, M. W.; Richardson, D. W.; Caron, J. P.

    2002-01-01

    Interleukin-1 is considered a central mediator of cartilage loss in osteoarthritis in several species, however an equine recombinant form of this cytokine is not readily available for in vitro use in equine osteoarthritis research. Equine recombinant interleukin-1? was cloned and expressed and its effects on the expression and activity of selected chondrocytic proteins implicated in cartilage matrix degradation were characterized. Reverse transcriptase polymerase chain reaction methods were used to amplify the entire coding region of the equine IL-1? mRNA, which was cloned into an expression vector, expressed in E. coli, and purified using a Ni2+ chromatographic method. The effects of the recombinant peptide on chondrocyte gene expression were determined by Northern blotting using RNA from equine chondrocyte cultures hybridized to probes for matrix metalloproteinases (MMP 1, MMP 3, MMP 13), tissue inhibitor of matrix metalloproteinases 1 (TIMP 1) and cyclooxygenase 2 (COX 2). Effects on selected mediators of cartilage degradation (nitrite concentrations and MMP activity) were determined using conditioned medium from reIL-1?-treated equine cartilage explant cultures. A recombinant peptide of approximately 21 kd was obtained. Northern blotting analyses revealed a marked up-regulation of expression of all MMPs, TIMP 1, and COX 2 in mRNA from treated chondrocytes. Furthermore, cartilage explants exposed to reIL-1? had augmented collagenase/gelatinase and stromelysin activities as well as increased concentration of nitrite in conditioned media. The development of a biologically active, species-specific IL-1? provides a valuable tool in the study of osteoarthritis pathophysiology and its treatment in horses. PMID:11858644

  19. Recombinant equine interleukin-1beta induces putative mediators of articular cartilage degradation in equine chondrocytes.

    Tung, J T; Fenton, J I; Arnold, C; Alexander, L; Yuzbasiyan-Gurkan, V; Venta, P J; Peters, T L; Orth, M W; Richardson, D W; Caron, J P

    2002-01-01

    Interleukin-1 is considered a central mediator of cartilage loss in osteoarthritis in several species, however an equine recombinant form of this cytokine is not readily available for in vitro use in equine osteoarthritis research. Equine recombinant interleukin-1beta was cloned and expressed and its effects on the expression and activity of selected chondrocytic proteins implicated in cartilage matrix degradation were characterized. Reverse transcriptase polymerase chain reaction methods were used to amplify the entire coding region of the equine IL-1beta mRNA, which was cloned into an expression vector, expressed in E. coli, and purified using a Ni2+ chromatographic method. The effects of the recombinant peptide on chondrocyte gene expression were determined by Northern blotting using RNA from equine chondrocyte cultures hybridized to probes for matrix metalloproteinases (MMP 1, MMP 3, MMP 13), tissue inhibitor of matrix metalloproteinases 1 (TIMP 1) and cyclooxygenase 2 (COX 2). Effects on selected mediators of cartilage degradation (nitrite concentrations and MMP activity) were determined using conditioned medium from reIL-1beta-treated equine cartilage explant cultures. A recombinant peptide of approximately 21 kd was obtained. Northern blotting analyses revealed a marked up-regulation of expression of all MMPs, TIMP 1, and COX 2 in mRNA from treated chondrocytes. Furthermore, cartilage explants exposed to reIL-1beta had augmented collagenase/gelatinase and stromelysin activities as well as increased concentration of nitrite in conditioned media. The development of a biologically active, species-specific IL-1beta provides a valuable tool in the study of osteoarthritis pathophysiology and its treatment in horses. PMID:11858644

  20. Self-consistent RPA calculations with Skyrme-type interactions: The skyrme_rpa program

    Colò, Gianluca; Cao, Ligang; Van Giai, Nguyen; Capelli, Luigi

    2013-01-01

    Random Phase Approximation (RPA) calculations are nowadays an indispensable tool in nuclear physics studies. We present here a complete version implemented with Skyrme-type interactions, with the spherical symmetry assumption, that can be used in cases where the effects of pairing correlations and of deformation can be ignored. The full self-consistency between the Hartree-Fock mean field and the RPA excitations is enforced, and it is numerically controlled by comparison with energy-weighted sum rules. The main limitations are that charge-exchange excitations and transitions involving spin operators are not included in this version. Program summaryProgram title: skyrme_rpa (v 1.00) Catalogue identifier: AENF_v1_0 Program summary URL: http://cpc.cs.qub.ac.uk/summaries/AENF_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 5531 No. of bytes in distributed program, including test data, etc.: 39435 Distribution format: tar.gz Programming language: FORTRAN-90/95; easily downgradable to FORTRAN-77. Computer: PC with Intel Celeron, Intel Pentium, AMD Athlon and Intel Core Duo processors. Operating system: Linux, Windows. RAM: From 4 MBytes to 150 MBytes, depending on the size of the nucleus and of the model space for RPA. Word size: The code is written with a prevalent use of double precision or REAL(8) variables; this assures 15 significant digits. Classification: 17.24. Nature of problem: Systematic observations of excitation properties in finite nuclear systems can lead to improved knowledge of the nuclear matter equation of state as well as a better understanding of the effective interaction in the medium. This is the case of the nuclear giant resonances and low-lying collective excitations, which can be described as small amplitude collective motions in the framework of the Random Phase Approximation (RPA). This work provides a tool where one starts from an assumed form of nuclear effective interaction (the Skyrme forces) and builds the self-consistent Hartree-Fock mean field of a given nucleus, and then the RPA multipole excitations of that nucleus. Solution method: The Hartree-Fock (HF) equations are solved in a radial mesh, using a Numerov algorithm. The solutions are iterated until self-consistency is achieved (in practice, when the energy eigenvalues are stable within a desired accuracy). In the obtained mean field, unoccupied states necessary for the RPA calculations are found. For all single-particle states, box boundary conditions are assumed. To solve the RPA problem for a given value of total angular momentum and parity Jπ a coupled basis is constructed and the RPA matrix is diagonalized (protons and neutrons are treated explicitly, and no approximation related to the use of isospin formalism is introduced). The transition amplitudes and transition strengths associated to given external operators are calculated. The HF densities and RPA transition densities are also evaluated. Restrictions: The main restrictions are related to the assumed spherical symmetry and absence of pairing correlations. Running time: The typical running time depends strongly on the nucleus, on the multipolarity, on the choice of the model space and of course on the computer. It can vary from a few minutes to several hours.

  1. Multiple Rad5 activities mediate sister chromatid recombination to bypass DNA damage at stalled replication forks.

    Minca, Eugen C; Kowalski, David

    2010-06-11

    DNA damage that blocks replication is bypassed in order to complete chromosome duplication and preserve cell viability and genome stability. Rad5, a PCNA polyubiquitin ligase and DNA-dependent ATPase in yeast, is orthologous to putative tumor suppressors and controls error-free damage bypass by an unknown mechanism. To identify the mechanism in vivo, we investigated the roles of Rad5 and analyzed the DNA structures that form during damage bypass at site-specific stalled forks present at replication origins. Rad5 mediated the formation of recombination-dependent, X-shaped DNA structures containing Holliday junctions between sister chromatids. Mutants lacking these damage-induced chromatid junctions were defective in resolving stalled forks, restarting replication, and completing chromosome duplication. Rad5 polyubiquitin ligase and ATPase domains both contributed to replication fork recombination. Our results indicate that multiple activities of Rad5 function coordinately with homologous recombination factors to enable replication template switch events that join sister chromatids at stalled forks and bypass DNA damage. PMID:20541998

  2. RPA prevents G-rich structure formation at lagging-strand telomeres to allow maintenance of chromosome ends.

    Audry, Julien; Maestroni, Laetitia; Delagoutte, Emmanuelle; Gauthier, Tiphaine; Nakamura, Toru M; Gachet, Yannick; Saintom, Carole; Gli, Vincent; Coulon, Stphane

    2015-07-14

    Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination, and repair. In fission yeast, the Rpa1-D223Y mutation provokes telomere shortening. Here, we show that this mutation impairs lagging-strand telomere replication and leads to the accumulation of secondary structures and recruitment of the homologous recombination factor Rad52. The presence of these secondary DNA structures correlates with reduced association of shelterin subunits Pot1 and Ccq1 at telomeres. Strikingly, heterologous expression of the budding yeast Pif1 known to efficiently unwind G-quadruplex rescues all the telomeric defects of the D223Y cells. Furthermore, in vitro data show that the identical D to Y mutation in human RPA specifically affects its ability to bind G-quadruplex. We propose that RPA prevents the formation of G-quadruplex structures at lagging-strand telomeres to promote shelterin association and facilitate telomerase action at telomeres. PMID:26041456

  3. Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA)

    Cottingham, Matthew G; Andersen, Rikke F; Spencer, Alexandra J; Saurya, Saroj; Furze, Julie; Hill, Adrian V S; Gilbert, Sarah C

    2008-01-01

    -length, rescuable clones were obtained, which had indistinguishable immunogenicity in mice. One clone was shotgun sequenced and found to be identical to the parent. We employed GalK recombination-mediated genetic engineering (recombineering) of MVA-BAC to delete five selected viral genes. Deletion of C12L, A44L, A...... using GalK counterselection to insert an antigen expression cassette lacking a tandem marker gene into the traditional thymidine kinase locus of MVA-BAC. MVA continues to feature prominently in clinical trials of recombinant vaccines against diseases such as HIV-AIDS, malaria and tuberculosis. Here we...... demonstrate in proof-of-concept experiments that MVA-BAC recombineering is a viable route to more rapid and efficient generation of new candidate mutant and recombinant vaccines based on a clinically deployable viral vector....

  4. Extended RPA study of nuclear collective phenomena

    A fully microscopic study of nuclear collective phenomena is presented within the framework of an extended RPA which includes 1p-1h and 2p-2h excitations in a consistent way. This theory allows us to obtain a very realistic description of various excitation spectra. As a result, a strong evidence of correlation effects beyond mean-field theory emerges. The effective interaction used is a G-matrix derived from the meson-exchange potential. The extended theory introduces also additional correlations which screen the long-large part of the effective interaction. This effect significantly enhances the stability of the ground state against density fluctuations. In this connection a possible importance of relativistic effects is also discussed. 99 refs., 19 figs., 5 tabs. (author)

  5. RPA correction to the optical potential

    Bauge E.

    2010-03-01

    Full Text Available In studies of nucleon elastic scattering, a correction to the microscopic optical potential built from Melbourne g-matrix was found to be necessary at low nucleon incident energy [1,2]. Indeed, at energies lower than 60 MeV, the absorption generated from Melbourne g-matrix is too weak within 25%. Coupling to collective excited states of the target nucleus are not included in the g-matrix and could explain the missing absorption. We propose to calculate this correction to the optical potential using the Gogny D1S effective nucleon-nucleon interaction in the coupling to excited states of the target. We use the Random Phase Approximation (RPA description of the excited states of the target with the same interaction.

  6. Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions

    Vasquez, Karen M.; Christensen, Jesper; Li, Lei; Finch, Rick A.; Glazer, Peter M.

    2002-04-01

    Nucleotide excision repair (NER) plays a central role in maintaining genomic integrity by detecting and repairing a wide variety of DNA lesions. Xeroderma pigmentosum complementation group A protein (XPA) is an essential component of the repair machinery, and it is thought to be involved in the initial step as a DNA damage recognition and/or confirmation factor. Human replication protein A (RPA) and XPA have been reported to interact to form a DNA damage recognition complex with greater specificity for damaged DNA than XPA alone. The mechanism by which these two proteins recognize such a wide array of structures resulting from different types of DNA damage is not known. One possibility is that they recognize a common feature of the lesions, such as distortions of the helical backbone. We have tested this idea by determining whether human XPA and RPA proteins can recognize the helical distortions induced by a DNA triple helix, a noncanonical DNA structure that has been shown to induce DNA repair, mutagenesis, and recombination. We measured binding of XPA and RPA, together or separately, to substrates containing triplexes with three, two, or no strands covalently linked by psoralen conjugation and photoaddition. We found that RPA alone recognizes all covalent triplex structures, but also forms multivalent nonspecific DNA aggregates at higher concentrations. XPA by itself does not recognize the substrates, but it binds them in the presence of RPA. Addition of XPA decreases the nonspecific DNA aggregate formation. These results support the hypothesis that the NER machinery is targeted to helical distortions and demonstrate that RPA can recognize damaged DNA even without XPA.

  7. A pink mouse reports the switch from red to green fluorescence upon Cre-mediated recombination

    Hartwich Heiner

    2012-06-01

    Full Text Available Abstract Background Targeted genetic modification in the mouse becomes increasingly important in biomedical and basic science. This goal is most often achieved by use of the Cre/loxP system and numerous Cre-driver mouse lines are currently generated. Their initial characterization requires reporter mouse lines to study the in vivo spatiotemporal activity of Cre. Findings Here, we report a dual fluorescence reporter mouse line, which switches expression from the red fluorescent protein mCherry to eGFP after Cre-mediated recombination. Both fluorescent proteins are expressed from the ubiquitously active and strong CAGGS promoter. Among the founders, we noticed a pink mouse line, expressing high levels of the red fluorescent protein mCherry throughout the entire body. Presence of mCherry in the living animal as well as in almost all organs was clearly visible without optical equipment. Upon Cre-activity, mCherry expression was switched to eGFP, demonstrating functionality of this reporter mouse line. Conclusions The pink mouse presented here is an attractive novel reporter line for fluorescence-based monitoring of Cre-activity. The high expression of mCherry, which is visible to the naked eye, facilitates breeding and crossing, as no genotyping is required to identify mice carrying the reporter allele. The presence of two fluorescent proteins allows in vivo monitoring of recombined and non-recombined cells. Finally, the pink mouse is an eye-catching animal model to demonstrate the power of transgenic techniques in teaching courses.

  8. Radiosensitivity enhancement of recombinant adenoviral-mediated retinoblastoma 94 gene in human esophageal carcinoma cells

    Objective: To study the radiosensitivity enhancement effects of recombinant adenovirus mediated retinoblastoma 94 gene on the growth of esophageal carcinoma cells EC109. Methods: EC109 cells was transfected with recombinant Rb94 gene adenovirus and irradiated by 137Cs γ-rays. The cohorts were divided into groups as blank control, Ad-LacZ, Ad-Rb94, radiation and Ad-Rb94 combined with radiation. Cell inhibition ratio, cell cycle and expression of retinoblastoma protein of EC109 cells were analyzed. Results: The growth of EC109 cells transfected with Ad-Rb94, radiation and Ad-Rb94 combined with radiation group was all inhibited. The group of Ad-Rb94 combined with radiation resulted in greater inhibition of cells growth compared with Ad-Rb94 group and radiation group (F=23.31, P<0.05). Cells of G2 phases of EC109 cancinoma cells for Ad-Rb94 combined with radiation group were the highest, which was 50%. The combination of Ad-Rb94 and radiation group resulted in the greatest expression of retinoblastoma protein, which reached 71%, significantly higher than Ad-Rb94 infection and radiation groups (χ2=8.31, 6.73, P<0.05). Conclusions: Retinoblastoma 94 gene combined with ionizing radiation can enhance the radiation sensitivity of EC109 cells. (authors)

  9. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression

    Fujiuchi, Naomichi; Matoba, Nobuyuki; Matsuda, Ryo

    2016-01-01

    Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: (1) recombinant protein content per unit biomass and (2) recombinant protein productivity per unit area–time at the end of the upstream production. Because environmental factors in the upstream production have impacts on these parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and postinoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Preinoculation environmental factors associated with planting density, light quality, and nutrient supply affect plant characteristics, such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, postinoculation environmental factors, such as temperature, light intensity, and humidity, have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the postinoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain high recombinant protein content and productivity, thus enhancing the utility of plant-based transient expression systems as recombinant protein factories.

  10. Radiosensitization effect of recombinant adenoviral-mediated PUMA gene on pancreatic carcinoma cells

    Objective: To study the effect of PUMA gene mediated by recombinant adenovirus vector combined with radiation on the pancreatic carcinoma. Methods: The PANC-1 cells were infected with Ad- PUMA (MOI=10, 50 and 100, respectively) for 48 h. The expression of PUMA mRNA and protein was detected by RT-PCR and Western blot, respectively. PANC-1 cells were divided into 4 groups: control group, transfection group, irradiation group and combined treatment group. The cell growth inhibition rate and apoptotic rate of PANC-1 cells were assessed by MTT assay and flow cytometry. Human pancreatic carcinomas were transplanted subcutaneously in nude mice, which were randomized into 4 groups: control group, transfection group, irradiation group and combined treatment group. Tumor growth rate and apoptotic index at different time points were recorded in 35 days. Results: The expression of PUMA mRNA and protein was increased with the increase of MOI of Ad-PUMA, which was does-dependant (MOI=10, mRNA=0.46± 0.02, protein=0.75± 0.09; MOI=50, mRNA=1.12±0.09, protein=1.01±0.18; MOI=100, mRNA=1.50±0.08, protein= 1.80±0.15; P3, (39.5±9.23)mm3, (33.6±10.3)mm3 and (52.0±11.43)mm3, respectively, P<0.05]. And the apoptotic index was increased in the same manner (AI=0.43±0.05, 0.29±0.10, 0.24±0.05 and 0.00±0.00, respectively, P<0.05). Conclusions: Recombinant adenoviral-mediated PUMA gene combined with irradiation could increase the cell-killing effect on pancreatic carcinoma. It is better than that of either one kind of therapy. (authors)

  11. 76 FR 54195 - 2010 Resources Planning Act (RPA) Assessment Draft

    2011-08-31

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF AGRICULTURE Forest Service 2010 Resources Planning Act (RPA) Assessment Draft AGENCY: Forest Service, USDA. ACTION... analyses of forests, rangelands, wildlife and fish, biodiversity, water, outdoor recreation,...

  12. RPA correlations and nuclear densities in relativistic mean field approach

    Van Giai, N. [Institut de Physique Nucleaire, CNRS, UMR 8608, F-91406 Orsay (France)]|[Universite Paris-Sud, F-91406 Orsay (France); Liang, H.Z. [Institut de Physique Nucleaire, CNRS, UMR 8608, F-91406 Orsay (France)]|[Universite Paris-Sud, F-91406 Orsay (France)]|[School of Physics, Peking University, 100871 Beijing (China); Meng, J. [School of Physics, Peking University, 100871 Beijing (China)

    2007-02-15

    The relativistic mean field approach (RMF) is well known for describing accurately binding energies and nucleon distributions in atomic nuclei throughout the nuclear chart. The random phase approximation (RPA) built on top of the RMF is also a good framework for the study of nuclear excitations. Here, we examine the consequences of long range correlations brought about by the RPA on the neutron and proton densities as given by the RMF approach. (authors)

  13. RPA equations and the instantaneous Bethe-Salpeter equation

    Resag, J

    1993-01-01

    We give a derivation of the particle-hole RPA equations for an interacting multi-fermion system by applying the instantaneous approximation to the amputated two-fermion propagator of the system. In relativistic field theory the same approximation leads from the fermion-antifermion Bethe-Salpeter equation to the Salpeter equation. We show that RPA equations and Salpeter equation are indeed equivalent.

  14. SITE-SPECIFIC RECOMBINATION FOR PLANT GENETIC ENGINEERING: STRATEGY FOR AGRO-MEDIATED GENE STACKING

    The precise rearrangement of DNA in planta can be achieved through site-specific recombination. For the past decade and a half, laboratory experiments have shown that site-specific recombination can delete genomic DNA, regulate gene expression, recombine chromosomes, and target new DNA into designat...

  15. Matrix metalloproteinase-mediation of tumor targeting human recombinant tumor necrosis factor-α fusion protein.

    Ren, Hui; Shao, Xin; Zeng, Liang; Wang, Fa; Huang, Di-Nan; Hou, Gan

    2015-08-01

    The aim of the present study was to use genetic engineering in order to establish an efficient tumor necrosis factor (TNF)-α fusion protein with low toxicity, which may be used to target tumors. Four types of matrix metalloproteinase (MMP)-mediated tumor targeting human recombinant TNF-α (rhTNF-α) fusion protein vectors were constructed. These were subsequently introduced into Escherichia coli. rhTNF-α fusion protein with a glutathione S-transferase (GST)-tag was purified using GST resin affinity chromatography, and GST-tags were digested using factor Xa. The cytotoxic effects of the fusion protein on L929 cells were determined using MTT assays. At a concentration of 1 pM, the GST-tagged fusion protein exerted no cytotoxic effects on the cells, compared with the negative control cells (P=0.975>0.05). However, at a concentration of 1000 pM, the deblocking fusion protein exerted greater cytotoxic effects on L929 cells, compared with positive control cells (Peffects on healthy cells. PMID:25891416

  16. Characterization of Fabry mice treated with recombinant adeno-associated virus 2/8-mediated gene transfer

    Choi Jin-Ok; Lee Mi; Park Hae-Young; Jung Sung-Chul

    2010-01-01

    Abstract Background Enzyme replacement therapy (ERT) with ?-galactosidase A (?-Gal A) is currently the most effective therapeutic strategy for patients with Fabry disease, a lysosomal storage disease. However, ERT has limitations of a short half-life, requirement for frequent administration, and limited efficacy for patients with renal failure. Therefore, we investigated the efficacy of recombinant adeno-associated virus (rAAV) vector-mediated gene therapy for a Fabry disease mouse model and ...

  17. Recombinant antibody mediated delivery of organelle-specific DNA pH sensors along endocytic pathways

    Modi, Souvik; Halder, Saheli; Nizak, Clment; Krishnan, Yamuna

    2013-12-01

    DNA has been used to build nanomachines with potential in cellulo and in vivo applications. However their different in cellulo applications are limited by the lack of generalizable strategies to deliver them to precise intracellular locations. Here we describe a new molecular design of DNA pH sensors with response times that are nearly 20 fold faster. Further, by changing the sequence of the pH sensitive domain of the DNA sensor, we have been able to tune their pH sensitive regimes and create a family of DNA sensors spanning ranges from pH 4 to 7.6. To enable a generalizable targeting methodology, this new sensor design also incorporates a `handle' domain. We have identified, using a phage display screen, a set of three recombinant antibodies (scFv) that bind sequence specifically to the handle domain. Sequence analysis of these antibodies revealed several conserved residues that mediate specific interactions with the cognate DNA duplex. We also found that all three scFvs clustered into different branches indicating that their specificity arises from mutations in key residues. When one of these scFvs is fused to a membrane protein (furin) that traffics via the cell surface, the scFv-furin chimera binds the `handle' and ferries a family of DNA pH sensors along the furin endocytic pathway. Post endocytosis, all DNA nanodevices retain their functionality in cellulo and provide spatiotemporal pH maps of retrogradely trafficking furin inside living cells. This new molecular technology of DNA-scFv-protein chimeras can be used to site-specifically complex DNA nanostructures for bioanalytical applications.DNA has been used to build nanomachines with potential in cellulo and in vivo applications. However their different in cellulo applications are limited by the lack of generalizable strategies to deliver them to precise intracellular locations. Here we describe a new molecular design of DNA pH sensors with response times that are nearly 20 fold faster. Further, by changing the sequence of the pH sensitive domain of the DNA sensor, we have been able to tune their pH sensitive regimes and create a family of DNA sensors spanning ranges from pH 4 to 7.6. To enable a generalizable targeting methodology, this new sensor design also incorporates a `handle' domain. We have identified, using a phage display screen, a set of three recombinant antibodies (scFv) that bind sequence specifically to the handle domain. Sequence analysis of these antibodies revealed several conserved residues that mediate specific interactions with the cognate DNA duplex. We also found that all three scFvs clustered into different branches indicating that their specificity arises from mutations in key residues. When one of these scFvs is fused to a membrane protein (furin) that traffics via the cell surface, the scFv-furin chimera binds the `handle' and ferries a family of DNA pH sensors along the furin endocytic pathway. Post endocytosis, all DNA nanodevices retain their functionality in cellulo and provide spatiotemporal pH maps of retrogradely trafficking furin inside living cells. This new molecular technology of DNA-scFv-protein chimeras can be used to site-specifically complex DNA nanostructures for bioanalytical applications. Electronic supplementary information (ESI) available: Detailed description of all oligonucleotide sequences used in this study; list of figures that support claims from the main text. Mainly these show sensor sequences, phage display results, scFv purification and binding data, cell images clamped at different pH and co-localization studies with endocytic tracers. See DOI: 10.1039/c3nr03769j

  18. Heterosubtypic immunity to influenza mediated by liposome adjuvanted H5N1 recombinant protein vaccines.

    Thueng-in, Kanyarat; Maneewatch, Santi; Srimanote, Potjanee; Songserm, Thaweesak; Tapchaisri, Pramuan; Sookrung, Nitat; Tongtawe, Pongsri; Channarong, Sunee; Chaicumpa, Wanpen

    2010-09-24

    A non-egg, non-culture based influenza vaccine that intervenes large influenza outbreaks and protects against heterosubtypic infections is needed. Candidates of such vaccine are likely to be conserved influenza virus proteins or their coding DNA. The vaccine must be conveniently produced at reasonable cost, safe, highly immunogenic and should be able to recall rapidly the immunological memory upon the antigenic re-exposure. In this study vaccines made of full length recombinant NP and M2 of the H5N1 influenza A virus were entrapped either alone or together into liposome (L) made of phosphatidylcholine and cholesterol. The vaccines (L-NP, L-M2 or L-NP+M2) and mocks (L or PBS) were safe without causing any adverse reaction in the intramuscularly injected mice. They were readily immunogenic at a single dose and a recalled response could be detected within one day post booster. Cytokine and antibody data indicated that the vaccines induced a Th1 bias immune response. NP containing vaccines stimulated a marked increase of cytotoxic lymphocytes, i.e., CD8(+), intracellular IFN?(+) cells, while M2 containing vaccines elicited good antibody response which neutralized infectivity of heterologous influenza viruses. Although the three vaccines elicited different immunological defense factors; nevertheless, they similarly and readily abrogated lung histopathology mediated by viruses belonging to different H5N1 clade/subclade and heterosubtypes including swine H1N1 and human H1N1/2009 viruses. They protected the vaccinated mice against lethal challenges with mouse adapted avian H5N1 virus. The liposome adjuvanted vaccines which demonstrated high protective efficacy in mice warrant testing further in a non-rodent model as well as in humans. PMID:20688037

  19. RPA instabilities in finite nuclei at low density

    Early development of the instabilities in a dilute nuclear source is investigated using a finite temperature quantal Random Phase Approximation (RPA) approach for different systems. The growth rates of the unstable collective modes are determined by solving a dispersion relation. Calculations indicate that, for an expanding source, unstable modes show a transition from surface to volume character. (author)

  20. RPA for Light-Front Hamiltonian Field Theory

    Harada, Koji

    1999-01-01

    A self-consistent random phase approximation (RPA) is proposed as an effective Hamiltonian method in Light-Front Field Theory (LFFT). We apply the general idea to the light-front massive Schwinger model to obtain a new bound state equation and solve it numerically.

  1. Binding polarity of RPA to telomeric sequences and influence of G-quadruplex stability.

    Safa, Layal; Delagoutte, Emmanuelle; Petruseva, Irina; Alberti, Patrizia; Lavrik, Olga; Riou, Jean-Franois; Saintom, Carole

    2014-08-01

    Replication protein A (RPA) is a single-stranded DNA binding protein that plays an essential role in telomere maintenance. RPA binds to and unfolds G-quadruplex (G4) structures formed in telomeric DNA, thus facilitating lagging strand DNA replication and telomerase activity. To investigate the effect of G4 stability on the interactions with human RPA (hRPA), we used a combination of biochemical and biophysical approaches. Our data revealed an inverse relationship between G4 stability and ability of hRPA to bind to telomeric DNA; notably small G4 ligands that enhance G4 stability strongly impaired G4 unfolding by hRPA. To gain more insight into the mechanism of binding and unfolding of telomeric G4 structures by RPA, we carried out photo-crosslinking experiments to elucidate the spatial arrangement of the RPA subunits along the DNA strands. Our results showed that RPA1 and RPA2 are arranged from 5' to 3' along the unfolded telomeric G4, as already described for unstructured single-stranded DNA, while no contact is possible with RPA3 on this short oligonucleotide. In addition, these data are compatible with a 5' to 3' directionality in G4 unfolding by hRPA. PMID:24747047

  2. A set of vectors for introduction of antibiotic resistance genes by in vitro Cre-mediated recombination

    Vassetzky Yegor S

    2008-12-01

    Full Text Available Abstract Background Introduction of new antibiotic resistance genes in the plasmids of interest is a frequent task in molecular cloning practice. Classical approaches involving digestion with restriction endonucleases and ligation are time-consuming. Findings We have created a set of insertion vectors (pINS carrying genes that provide resistance to various antibiotics (puromycin, blasticidin and G418 and containing a loxP site. Each vector (pINS-Puro, pINS-Blast or pINS-Neo contains either a chloramphenicol or a kanamycin resistance gene and is unable to replicate in most E. coli strains as it contains a conditional R6Kγ replication origin. Introduction of the antibiotic resistance genes into the vector of interest is achieved by Cre-mediated recombination between the replication-incompetent pINS and a replication-competent target vector. The recombination mix is then transformed into E. coli and selected by the resistance marker (kanamycin or chloramphenicol present in pINS, which allows to recover the recombinant plasmids with 100% efficiency. Conclusion Here we propose a simple strategy that allows to introduce various antibiotic-resistance genes into any plasmid containing a replication origin, an ampicillin resistance gene and a loxP site.

  3. Construction of targeted single copy lac fusions using lambda Red and FLP-mediated site-specific recombination in bacteria.

    Ellermeier, Craig D; Janakiraman, Anuradha; Slauch, James M

    2002-05-15

    A simple method for the construction of targeted transcriptional and translational fusions to the lac operon using FLP mediated site-specific recombination is described. Conditional plasmids containing promoterless lacZY genes and the FLP recognition target (FRT) site in both orientations were constructed for generating transcriptional fusions. Similarly, a plasmid used to create translational fusions was constructed in which the endogenous translational start of lacZ has been removed. These plasmids can be transformed into strains containing a single FRT site, which was previously integrated downstream of the promoter of interest using the lambda Red recombination method. The FLP protein produced from a helper plasmid that contains a conditional origin of replication promotes site-specific recombination between the FRT sites, resulting in an integrated lac fusion to the gene of interest. Transcriptional fusions to the Salmonella typhimurium genes sodCII and sitA were constructed using this method and shown to respond appropriately to mutations in the respective regulatory genes, rpoS and fur. Translational fusions were also constructed using this method. In this case, expression of beta-galactosidase was dependent on translation of the target protein. Given that the FLP recombinase does not require host factors for function and that this method requires no molecular cloning, this method should be applicable for the analysis of gene expression in a variety of organisms. PMID:12062810

  4. BLM and RMI1 Alleviate RPA Inhibition of TopoIIIa Decatenase Activity

    Yang, Jay; Bachrati, Csanad Z; Hickson, Ian D; Brown, Grant W

    2012-01-01

    of topoisomerase IIIa. We found that RPA and other ssDNA binding proteins inhibit decatenation by topoisomerase IIIa. Complex formation between BLM, TopoIIIa, and RMI1 ablates inhibition of decatenation by ssDNA binding proteins. Together, these data indicate that inhibition by RPA does not involve...... species-specific interactions between RPA and BLM-TopoIIIa-RMI1, which contrasts with RPA modulation of double Holliday junction dissolution. We propose that topoisomerase IIIa and RPA compete to bind to single-stranded regions of catenanes. Interactions with BLM and RMI1 enhance toposiomerase IIIa...

  5. FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori

    A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they h...

  6. Various applications of TALEN- and CRISPR/Cas9-mediated homologous recombination to modify the Drosophila genome

    Zhongsheng Yu

    2014-03-01

    Full Text Available Modifying the genomes of many organisms is becoming as easy as manipulating DNA in test tubes, which is made possible by two recently developed techniques based on either the customizable DNA binding protein, TALEN, or the CRISPR/Cas9 system. Here, we describe a series of efficient applications derived from these two technologies, in combination with various homologous donor DNA plasmids, to manipulate the Drosophila genome: (1 to precisely generate genomic deletions; (2 to make genomic replacement of a DNA fragment at single nucleotide resolution; and (3 to generate precise insertions to tag target proteins for tracing their endogenous expressions. For more convenient genomic manipulations, we established an easy-to-screen platform by knocking in a white marker through homologous recombination. Further, we provided a strategy to remove the unwanted duplications generated during the “ends-in” recombination process. Our results also indicate that TALEN and CRISPR/Cas9 had comparable efficiency in mediating genomic modifications through HDR (homology-directed repair; either TALEN or the CRISPR/Cas9 system could efficiently mediate in vivo replacement of DNA fragments of up to 5 kb in Drosophila, providing an ideal genetic tool for functional annotations of the Drosophila genome.

  7. Skyrme RPA for spherical and axially symmetric nuclei

    Repko, Anton; Nesterenko, V O; Reinhard, P -G

    2015-01-01

    Random Phase Approximation (RPA) is the basic method for calculation of excited states of nuclei over the Hartree-Fock ground state, suitable also for energy density functionals (EDF or DFT). We developed a convenient formalism for expressing densities and currents in a form of reduced matrix elements, which allows fast calculation of spectra for spherical nuclei. All terms of Skyrme functional were taken into account, so it is possible to calculate electric, magnetic and vortical/toroidal/compression transitions and strength functions of any multipolarity. Time-odd (spin) terms in Skyrme functional become important for magnetic M1 and isovector toroidal E1 transitions. It was also found that transition currents in pygmy region (low-lying part of E1 resonance) exhibit isoscalar toroidal flow, so the previously assumed picture of neutron-skin vibration is not the only mechanism present in pygmy transitions. RPA calculations with heavy axially-symmetric nuclei now become feasible on ordinary PC. Detailed formul...

  8. Properties of magnetized neutral mesons within a full RPA evaluation

    Avancini, Sidney S; Pinto, Marcus B

    2016-01-01

    We consider the two flavor Nambu--Jona-Lasinio model within the RPA framework to evaluate the masses of the $\\sigma$ and $\\pi^0$ mesons and the $\\pi^0$ decay constant in the presence of a magnetic field at vanishing temperatures and baryonic densities. The present work extends other RPA applications by fully considering the external momenta, which enter the integrals representing the magnetized polarization tensor, so that more accurate results can be obtained in the evaluation of physical quantities containing pionic contributions. As we show, this technical improvement generates results which agree well with those produced by lattice simulations and chiral perturbation theory. Our method may also prove to be useful in future evaluations of quantities such as the shear viscosity and the equation of state of magnetized quark matter with mesonic contributions.

  9. Properties of magnetized neutral mesons within a full RPA evaluation

    Avancini, Sidney S.; Tavares, William R.; Pinto, Marcus B.

    2016-01-01

    We consider the two-flavor Nambu-Jona-Lasinio model within the random phase approximation (RPA) framework to evaluate the masses of the σ and π0 mesons and the π0 decay constant in the presence of a magnetic field at vanishing temperatures and baryonic densities. The present work extends other RPA applications by fully considering the external momenta which enter the integrals representing the magnetized polarization tensor. We employ a a field-independent regularization scheme so that more accurate results can be obtained in the evaluation of physical quantities containing pionic contributions. As we show, this technical improvement generates results which agree well with those produced by lattice simulations and chiral perturbation theory. Our method may also prove to be useful in future evaluations of quantities, such as the shear viscosity and the equation of state of magnetized quark matter with mesonic contributions.

  10. Random-Matrix Approach to RPA equations. I

    Barillier-Pertuisel, X; Weidenmller, H A

    2008-01-01

    We study the RPA equations in their most general form by taking the matrix elements appearing in the RPA equations as random. This yields either a unitarily or an orthogonally invariant random-matrix model which is not of the Cartan type. The average spectrum of the model is studied with the help of a generalized Pastur equation. Two independent parameters govern the behaviour of the system: The strength $\\alpha^2$ of the coupling between positive- and negative-energy states and the distance between the origin and the centers of the two semicircles that describe the average spectrum for $\\alpha^2 = 0$, the latter measured in units of the equal radii of the two semicircles. With increasing $\\alpha^2$, positive- and negative-energy states become mixed and ever more of the spectral strength of the positive-energy states is transferred to those at negative energy, and vice versa. The two semicircles are deformed and pulled toward each other. As they begin to overlap, the RPA equations yield non--real eigenvalues:...

  11. Recombinase polymerase amplification (RPA) of CaMV-35S promoter and nos terminator for rapid detection of genetically modified crops.

    Xu, Chao; Li, Liang; Jin, Wujun; Wan, Yusong

    2014-01-01

    Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that enables the amplification of DNA within 30 min at a constant temperature of 37-42 °C by simulating in vivo DNA recombination. In this study, based on the regulatory sequence of the cauliflower mosaic virus 35S (CaMV-35S) promoter and the Agrobacterium tumefaciens nopaline synthase gene (nos) terminator, which are widely incorporated in genetically modified (GM) crops, we designed two sets of RPA primers and established a real-time RPA detection method for GM crop screening and detection. This method could reliably detect as few as 100 copies of the target molecule in a sample within 15-25 min. Furthermore, the real-time RPA detection method was successfully used to amplify and detect DNA from samples of four major GM crops (maize, rice, cotton, and soybean). With this novel amplification method, the test time was significantly shortened and the reaction process was simplified; thus, this method represents an effective approach to the rapid detection of GM crops. PMID:25310647

  12. Recombinase Polymerase Amplification (RPA of CaMV-35S Promoter and nos Terminator for Rapid Detection of Genetically Modified Crops

    Chao Xu

    2014-10-01

    Full Text Available Recombinase polymerase amplification (RPA is a novel isothermal DNA amplification and detection technology that enables the amplification of DNA within 30 min at a constant temperature of 37–42 °C by simulating in vivo DNA recombination. In this study, based on the regulatory sequence of the cauliflower mosaic virus 35S (CaMV-35S promoter and the Agrobacterium tumefaciens nopaline synthase gene (nos terminator, which are widely incorporated in genetically modified (GM crops, we designed two sets of RPA primers and established a real-time RPA detection method for GM crop screening and detection. This method could reliably detect as few as 100 copies of the target molecule in a sample within 15–25 min. Furthermore, the real-time RPA detection method was successfully used to amplify and detect DNA from samples of four major GM crops (maize, rice, cotton, and soybean. With this novel amplification method, the test time was significantly shortened and the reaction process was simplified; thus, this method represents an effective approach to the rapid detection of GM crops.

  13. Generation of TALEN-mediated GRdim knock-in rats by homologous recombination.

    Ponce de León V.; Mérillat A.M.; Tesson L.; Anegón I.; Hummler E.

    2014-01-01

    Transcription Activator-Like Effector Nucleases (TALEN) are potential tools for precise genome engineering of laboratory animals. We report the first targeted genomic integration in the rat using TALENs (Transcription Activator-Like Effector Nucleases) by homology-derived recombination (HDR). We assembled TALENs and designed a linear donor insert targeting a pA476T mutation in the rat Glucocorticoid Receptor (Nr3c1) namely GR(dim), that prevents receptor homodimerization in the mouse. TALEN m...

  14. Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination.

    Thomas-Claudepierre, Anne-Sophie; Robert, Isabelle; Rocha, Pedro P; Raviram, Ramya; Schiavo, Ebe; Heyer, Vincent; Bonneau, Richard; Luo, Vincent M; Reddy, Janardan K; Borggrefe, Tilman; Skok, Jane A; Reina-San-Martin, Bernardo

    2016-03-01

    Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation. PMID:26903242

  15. Long-term Cre-mediated Retrograde Tagging of Neurons Using a Novel Recombinant Pseudorabies Virus

    Hassana Oyibo

    2014-09-01

    Full Text Available Brain regions contain diverse populations of neurons that project to different long-range targets. The study of these subpopulations in circuit function and behavior requires a toolkit to characterize and manipulate their activity in vivo. We have developed a novel set of reagents based on Pseudorabies Virus (PRV for efficient and long-term genetic tagging of neurons based on their projection targets. By deleting IE180, the master transcriptional regulator in the PRV genome, we have produced a mutant virus capable of infection and transgene expression in neurons but unable to replicate in or spread from those neurons. IE180-null mutants showed no cytotoxicity, and infected neurons exhibited normal physiological function more than 45 days after infection, indicating the utility of these engineered viruses for chronic experiments. To enable rapid and convenient construction of novel IE180-null recombinants, we engineered a bacterial artificial chromosome (BAC shuttle-vector system for moving new constructs into the PRV IE180-null genome. Using this system we generated an IE180-null recombinant virus expressing the site-specific recombinase Cre. This Cre-expressing virus (PRV-hSyn-Cre efficiently and robustly infects neurons in vivo and activates transgene expression from Cre-dependent vectors in local and retrograde projecting populations of neurons in the mouse. We also generated an assortment of recombinant viruses expressing fluorescent proteins (mCherry, EGFP, ECFP. These viruses exhibit long-term labeling of neurons in vitro but transient labeling in vivo. Together these novel IE180-null PRV reagents expand the toolkit for targeted gene expression in the brain, facilitating functional dissection of neuronal circuits in vivo.

  16. Development of recombinant antibody mediated resistance against Tomato yellow leaf curl virus

    Safarnejad, Mohammad Reza

    2008-01-01

    In this study, we exploited the expression of specific scFv fragment in plant cells for suppression of disease symptoms caused by TYLCV infection. The C1, V1 and V2 genes encoding Rep, CP and MP, respectively, were cloned into the TOPO vector and subsequently into pGEX-5x3 and pMALc2x expression vectors. Recombinant proteins were expressed in E. coli as C-terminal fusion with GST or MBP and purified proteins obtained by affinity chromatography method. In addition, the amino terminal part of C...

  17. Recombinant dimeric IgA antibodies against the epidermal growth factor receptor mediate effective tumor cell killing.

    Lohse, Stefan; Derer, Stefanie; Beyer, Thomas; Klausz, Katja; Peipp, Matthias; Leusen, Jeanette H W; van de Winkel, Jan G J; Dechant, Michael; Valerius, Thomas

    2011-03-15

    Dimeric IgA Abs contribute significantly to the humoral part of the mucosal immune system. However, their potential as immunotherapeutic agent has hardly been explored. In this article, we describe the production, purification, and functional evaluation of recombinant dimeric IgA against the epidermal growth factor receptor. Human joining chain-containing IgA was produced by nonadherent Chinese hamster ovarian (CHO)-K1 cells under serum-free conditions. Purification by anti-human ? and anti-His-tag affinity, as well as size exclusion chromatography, resulted in a homogenous preparation of highly pure IgA dimers. Functional studies demonstrated dimeric IgA to be at least as effective as monomeric IgA in triggering Ab-dependent cellular cytotoxicity by isolated monocytes or polymorphonuclear cell and in human whole-blood assays. Importantly, dimeric IgA was more effective in F(ab)-mediated killing mechanisms, such as inhibition of ligand binding, receptor downmodulation, and growth inhibition. Furthermore, only dimeric but not monomeric IgA or IgG was directionally transported by the polymeric Ig receptor through an epithelial cell monolayer. Together, these studies demonstrate that recombinant dimeric IgA Abs recruit a distinct repertoire of effector functions compared with monomeric IgA or IgG1 Abs. PMID:21317397

  18. P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish structure-function relationships

    Di Pietro A.

    1999-01-01

    Full Text Available Resistance to chemotherapy in cancer cells is mainly mediated by overexpression of P-glycoprotein (Pgp, a plasma membrane ATP-binding cassette (ABC transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. Pgp consists of two homologous halves each containing a transmembrane domain and a cytosolic nucleotide-binding domain (NBD which contains two consensus Walker motifs, A and B, involved in ATP binding and hydrolysis. The protein also contains an S signature characteristic of ABC transporters. The molecular mechanism of Pgp-mediated drug transport is not known. Since the transporter has an extraordinarily broad substrate specificity, its cellular function has been described as a "hydrophobic vacuum cleaner". The limited knowledge about the mechanism of Pgp, partly due to the lack of a high-resolution structure, is well reflected in the failure to efficiently inhibit its activity in cancer cells and thus to reverse multidrug resistance (MDR. In contrast to the difficulties encountered when studying the full-length Pgp, the recombinant NBDs can be obtained in large amounts as soluble proteins. The biochemical and biophysical characterization of recombinant NBDs is shown here to provide a suitable alternative route to establish structure-function relationships. NBDs were shown to bind ATP and analogues as well as potent modulators of MDR, such as hydrophobic steroids, at a region close to the ATP site. Interestingly, flavonoids also bind to NBDs with high affinity. Their binding site partly overlaps both the ATP-binding site and the steroid-interacting region. Therefore flavonoids constitute a new promising class of bifunctional modulators of Pgp.

  19. Microhomology-mediated end joining in fission yeast is repressed by pku70 and relies on genes involved in homologous recombination.

    Decottignies, Anabelle

    2007-01-01

    Two DNA repair pathways are known to mediate DNA double-strand-break (DSB) repair: homologous recombination (HR) and nonhomologous end joining (NHEJ). In addition, a nonconservative backup pathway showing extensive nucleotide loss and relying on microhomologies at repair junctions was identified in NHEJ-deficient cells from a variety of organisms and found to be involved in chromosomal translocations. Here, an extrachromosomal assay was used to characterize this microhomology-mediated end-joi...

  20. Microhomology-Mediated End Joining in Fission Yeast Is Repressed by Pku70 and Relies on Genes Involved in Homologous Recombination

    Decottignies, Anabelle

    2007-01-01

    Two DNA repair pathways are known to mediate DNA double-strand-break (DSB) repair: homologous recombination (HR) and nonhomologous end joining (NHEJ). In addition, a nonconservative backup pathway showing extensive nucleotide loss and relying on microhomologies at repair junctions was identified in NHEJ-deficient cells from a variety of organisms and found to be involved in chromosomal translocations. Here, an extrachromosomal assay was used to characterize this microhomology-mediated end-joi...

  1. Finite temperature RPA in symmetric nuclear matter with Skyrme interactions

    We investigate the RPA response for thermally excited nuclear matter interacting through Skyrme interactions. Closed analytical expressions are obtained for the dynamic susceptibility in each spin-isopspin channel. We compute the strength as a function of energy, transferred momentum and temperature, and examine the evolution of collective states, when present. The energy weighted sum rules Mk, for k = -1, 1 and 3 are also shown to possess explicit expressions as functions of both momentum and temperature. It is seen that thermal effects on the susceptibility are as important as dynamical ones associated to momentum transfer, at least for temperatures as high as 20% of the Fermi energy. (orig.)

  2. Microscopic description of hot nuclei: the SPA+RPA approach

    We discuss the static path plus random phase approximation (SPA+RPA) to the partition function of warm finite nuclei. The method, derived from the auxiliary field path integral representation of the partition function, takes into account large amplitude statistical fluctuations around the mean field, and is able to provide an accurate evaluation of thermodynamics properties within finite configuration spaces. We present some recent improvements which include the treatment of repulsive terms in the interaction, the exact implementation in canonical and in restricted grand canonical ensembles with fixed number parity, and the evaluation of response and strength functions

  3. Recombinant Adeno-Associated Virus-Mediated microRNA Delivery into the Postnatal Mouse Brain Reveals a Role for miR-134 in Dendritogenesis in Vivo

    Christensen, Mette; Larsen, Lars A; Kauppinen, Sakari; Schratt, Gerhard

    2010-01-01

    delivery of microRNAs in vivo by use of recombinant adeno-associated virus (rAAV). rAAV-mediated overexpression of miR-134 in neurons of the postnatal mouse brain provided evidence for a negative role of miR-134 in dendritic arborization of cortical layer V pyramidal neurons in vivo, thereby confirming...

  4. Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling

    Choi, Jun-Hyuk; Lindsey-Boltz, Laura A.; Kemp, Michael; Mason, Aaron C.; Wold, Marc S.; Sancar, Aziz

    2010-01-01

    ATR kinase is a critical upstream regulator of the checkpoint response to various forms of DNA damage. Previous studies have shown that ATR is recruited via its binding partner ATR-interacting protein (ATRIP) to replication protein A (RPA)-covered single-stranded DNA (RPA-ssDNA) generated at sites of DNA damage where ATR is then activated by TopBP1 to phosphorylate downstream targets including the Chk1 signal transducing kinase. However, this critical feature of the human ATR-initiated DNA damage checkpoint signaling has not been demonstrated in a defined system. Here we describe an in vitro checkpoint system in which RPA-ssDNA and TopBP1 are essential for phosphorylation of Chk1 by the purified ATR-ATRIP complex. Checkpoint defective RPA mutants fail to activate ATR kinase in this system, supporting the conclusion that this system is a faithful representation of the in vivo reaction. Interestingly, we find that an alternative form of RPA (aRPA), which does not support DNA replication, can substitute for the checkpoint function of RPA in vitro, thus revealing a potential role for aRPA in the activation of ATR kinase. We also find that TopBP1 is recruited to RPA-ssDNA in a manner dependent on ATRIP and that the N terminus of TopBP1 is required for efficient recruitment and activation of ATR kinase. PMID:20616048

  5. Inhibition of corneal neovascularization by recombinant adenovirus-mediated sFlk-1 expression

    The interaction of vascular endothelial growth factor (VEGF) and its receptors (Flt-1, Flk-1/KDR) is correlated with neovascularization in the eyes. Therefore, blocking the binding of VEGF and the corresponding receptor has become critical for inhibiting corneal neovascularization. In this study, we have expressed the cDNA for sFlk-1 under the control of cytomegalovirus immediate-early promoter (CMV) from an E1/partial E3 deleted replication defective recombinant adenovirus, and Ad.sflk-1 expression was determined by Western blotting. We have shown that conditioned media from Ad.sflk-1-infected ARPE-19 cells significantly reduced VEGF-induced human umbilical vein endothelial cells (HUVEC) and murine endothelial cells (SVEC) proliferation in vitro compared with the control vector. In vivo, adenoviral vectors expressing green fluorescent protein alone (Ad.GFP) were utilized to monitor gene transfer to the cornea. Moreover, in the models of corneal neovascularization, the injection of Ad.sflk-1 (108 PFU) into the anterior chamber could significantly inhibit angiogenic changes compared with Ad.null-injected and vehicle-injected models. Immunohistochemical analysis showed that corneal endothelial cells and corneal stroma of cauterized rat eyes were efficiently transduced and expressed sFlk-1. These results not only support that adenoviral vectors are capable of high-level transgene expression but also demonstrate that Ad.sflk-1 gene therapy might be a feasible approach for inhibiting the development of corneal neovascularization

  6. Suppression of mutagenesis by Rad51D-mediated homologous recombination

    Hinz, J M; Tebbs, R S; Wilson, P F; Nham, P B; Salazar, E P; Nagasawa, H; Urbin, S S; Thompson, L H

    2005-11-15

    Homologous recombinational repair (HRR) restores chromatid breaks arising during DNA replication and prevents chromosomal rearrangements that can occur from the misrepair of such breaks. In vertebrates, five Rad51 paralogs are identified that contribute in a nonessential but critical manner to HRR efficiency. We constructed and characterized a Rad51D knockout cell line in widely studied CHO cells. The rad51d mutant (51D1) displays sensitivity to a wide spectrum of induced DNA damage, indicating the broad relevance of HRR to genotoxicity. Untreated 51D1 cells exhibit {approx}5-fold elevated chromosomal breaks, a 12-fold increased rate of hprt mutation, and 4- to 10-fold increased rates of gene amplification at the dhfr and CAD loci, respectively. These results explicitly show the quantitative importance of HHR in preventing these types genetic alterations, which are associated with carcinogenesis. Thus, HRR copes in an error-free manner with spontaneous DNA damage encountered during DNA replication, and Rad51D is essential for this fidelity.

  7. Current Challenges and Future Directions in Recombinant AAV-Mediated Gene Therapy of Duchenne Muscular Dystrophy

    Shin'ichi Takeda

    2013-06-01

    Full Text Available Various characteristics of adeno-associated virus (AAV-based vectors with long-term safe expression have made it an exciting transduction tool for clinical gene therapy of Duchenne muscular dystrophy (DMD. Although host immune reactions against the vector as well as transgene products were detected in some instances of the clinical studies, there have been promising observations. Methods of producing AAV vectors for considerable in vivo experimentation and clinical investigations have been developed and a number of studies with AAV vector-mediated muscle transduction were attempted. Notably, an intravenous limb perfusion transduction technique enables extensive transgene expression in the skeletal muscles without noticeable adverse events. Furthermore, cardiac transduction by the rAAV9-microdystrophin would be promising to prevent development of cardiac dysfunction. Recent achievements in transduction technology suggest that long-term transgene expression with therapeutic benefits in DMD treatment would be achieved by the rAAV-mediated transduction strategy with an adequate regimen to regulate host immune response.

  8. A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination

    Holt Robert A

    2006-04-01

    Full Text Available Abstract Background Cre-loxP recombination refers to the process of site-specific recombination mediated by two loxP sequences and the Cre recombinase protein. Transgenic experiments exploit integrative recombination, where a donor plasmid carrying a loxP site and DNA of interest integrate into a recipient loxP site in a target genome. Unfortunately, integrative recombination is highly inefficient because the insert is flanked by two loxP sites, which themselves become targets for Cre and lead to subsequent excision of the insert. A small number of mutations have been discovered in parts of the loxP sequence, specifically the spacer and inverted repeat segments, that increase the efficiency of integrative recombination. In this study we introduce a high-throughput in vitro assay to rapidly detect novel loxP spacer mutants and describe the sequence characteristics of successful recombinants. Results We created synthetic loxP oligonucleotides that contained a combination of inverted repeat mutations (the lox66 and lox71 mutations and mutant spacer sequences, degenerate at 6 of the 8 positions. After in vitro Cre recombination, 3,124 recombinant clones were identified by sequencing. Included in this set were 31 unique, novel, self-recombining sequences. Using network visualization tools, we recognized 12 spacer sets with restricted promiscuity. We observed that increased guanine content at all spacer positions save for position 8 resulted in increased recombination. Interestingly, recombination between identical spacers was not preferred over non-identical spacers. We also identified a set of 16 pairs of loxP spacers that reacted at least twice with another spacer, but not themselves. Further, neither the wild-type P1 phage loxP sequence nor any of the known loxP spacer mutants appeared to be kinetically favoured by Cre recombinase. Conclusion This study approached loxP spacer mutant screening in an unbiased manner, assuming nothing about candidate loxP sites save for the conserved 4 and 5 spacer positions. Candidate sites were free to recombine with any other sequence in the pool of all possible sites. The subset of loxP sites identified here are candidates for in vivo serial recombination as they have already demonstrated limited promiscuity with other loxP spacer and stability in the presence of Cre.

  9. Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells

    Dimitrov, Lazar; Pedersen, Darlene; Ching, Kathryn H.; Yi, Henry; Collarini, Ellen J.; Izquierdo, Shelley; van de Lavoir, Marie-Cecile; Leighton, Philip A.

    2016-01-01

    The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds. PMID:27099923

  10. Imaging of human sodium-iodide symporter gene expression mediated by recombinant adenovirus in skeletal muscle of living rats

    We evaluated the feasibility of non-invasive imaging of recombinant adenovirus-mediated human sodium-iodide symporter (hNIS) gene expression by 99mTcO4- scintigraphy in skeletal muscle of rats. Replication-defective recombinant adenovirus encoding hNIS gene [Rad-CMV-hNIS 5 x 107, 2 x 108 or 1 x 109 plaque forming units (pfu)] or β-galactosidase gene (Rad-CMV-LacZ 1 x 109 pfu) was injected into the right biceps femoris muscle of rats (n=5-6 for each group). Three days after gene transfer, scintigraphy was performed using a gamma camera 30 min after injection of 99mTcO4- (1.85 MBq). An additional two rats injected with 1 x 109 pfu of Rad-CMV-hNIS underwent 99mTcO4- scintigraphy with sodium perchlorate. After the imaging studies, rats were sacrificed for assessment of the biodistribution of 99mTcO4- and measurement of hNIS mRNA expression. In all the rats injected with 1 x 109 pfu of Rad-CMV-hNIS, hNIS expression was successfully imaged by 99mTcO4- scintigraphy, while rats injected with Rad-CMV-LacZ or lower doses of Rad-CMV-hNIS failed to show uptake. The biodistribution studies indicated that a significantly different amount of 99mTcO4- was retained in the liver (p9 pfu of Rad-CMV-hNIS. The muscular hNIS mRNA level quantified by real-time reverse transcription-polymerase chain reaction was significantly higher in rats injected with 1 x 109 pfu of Rad-CMV-hNIS (p9 pfu of Rad-CMV-hNIS were specifically inhibited by sodium perchlorate. This study illustrated that 99mTcO4- scintigraphy can monitor Rad-CMV-hNIS-mediated gene expression in skeletal muscle of rats, non-invasively and quantitatively. (orig.)

  11. Protective immunity to Listeria monocytogenes infection mediated by recombinant Listeria innocua harboring the VGC locus.

    Mohamed, Walid; Sethi, Shneh; Tchatalbachev, Svetlin; Darji, Ayub; Chakraborty, Trinad

    2012-01-01

    In this study we propose a novel bacterial vaccine strategy where non-pathogenic bacteria are complemented with traits desirable for the induction of protective immunity. To illustrate the proof of principle of this novel vaccination strategy, we use the model organism of intracellular immunity Listeria. We introduced a, low copy number BAC-plasmid harbouring the virulence gene cluster (vgc) of L. monocytogenes (Lm) into the non-pathogenic L. innocua (L.inn) strain and examined for its ability to induce protective cellular immunity. The resulting strain (L.inn::vgc) was attenuated for virulence in vivo and showed a strongly reduced host detrimental inflammatory response compared to Lm. Like Lm, L.inn::vgc induced the production of Type I Interferon's and protection was mediated by Listeria-specific CD8(+) T cells. Rational vaccine design whereby avirulent strains are equipped with the capabilities to induce protection but lack detrimental inflammatory effects offer great promise towards future studies using non-pathogenic bacteria as vectors for vaccination. PMID:22536395

  12. Quantal Brownian Motion from RPA dynamics: The master and Fokker-Planck equations

    From the purely quantal RPA description of the damped harmonic oscillator and of the corresponding Brownian Motion within the full space (phonon subspace plus reservoir), a master equation (as well as a Fokker-Planck equation) for the reduced density matrix (for the reduced Wigner function, respectively) within the phonon subspace is extracted. The RPA master equation agrees with the master equation derived by the time-dependent perturbative approaches which utilize Tamm-Dancoff Hilbert spaces and invoke the rotating wave approximation. Since the RPA yields a full, as well as a contracted description, it can account for both the kinetic and the unperturbed oscillator momenta. The RPA description of the quantal Brownian Motion contrasts with the descriptions provided by the time perturbative approaches whether they invoke or not the rotating wave approximation. The RPA description also contrasts with the phenomenological phase space quantization. (orig.)

  13. Microscopic nuclear-dissipation mechanism as damping of collective motion in the second RPA

    A microscopic model for the damping of the one-phonon RPA collective state, absolute value c > = Q/sub c/ 0 > /sub S//sub R/, has been previously described. This one-phonon RPA collective state is defined within a restricted subspace, S/sub R/, of the discrete 1p-1h structure. Its damping is described within an extended subspace, S = S/sub R/ + S/sub A/, by the time evolution of a wave packet according to the RPA and the Second RPA approximations of the complete Schroedinger equation when initialized with the one-phonon state. The one-phonon state, however, is unable to describe time-varying oscillations of the mean field. Such oscillations require wave packets formed by linear superposition of the RPA many-phonon eigenstates. Coherent time-varying oscillations of the mean field (multi-phonon initial states) are discussed

  14. Targeted Integration of Single-Copy Transgenes in Drosophila melanogaster Tissue-Culture Cells Using Recombination-Mediated Cassette Exchange.

    Manivannan, Sathiya N; Jacobsen, Thomas L; Lyon, Peter; Selvaraj, Bhavani; Halpin, Peter; Simcox, Amanda

    2015-12-01

    Transfection of transgenes into Drosophila cultured cells is a standard approach for studying gene function. However, the number of transgenes present in the cell following transient transfection or stable random integration varies, and the resulting differences in expression level affect interpretation. Here we developed a system for Drosophila cell lines that allows selection of cells with a single-copy transgene inserted at a specific genomic site using recombination-mediated cassette exchange (RMCE). We used the ?C31 integrase and its target sites attP and attB for RMCE. Cell lines with an attP-flanked genomic cassette were transfected with donor plasmids containing a transgene of interest (UAS-x), a dihydrofolate reductase (UAS-DHFR) gene flanked by attB sequences, and a thymidine kinase (UAS-TK) gene in the plasmid backbone outside the attB sequences. In cells undergoing RMCE, UAS-x and UAS-DHFR were exchanged for the attP-flanked genomic cassette, and UAS-TK was excluded. These cells were selected using methotrexate, which requires DHFR expression, and ganciclovir, which causes death in cells expressing TK. Pure populations of cells with one copy of a stably integrated transgene were efficiently selected by cloning or mass culture in ?6 weeks. Our results show that RMCE avoids the problems associated with current methods, where transgene number is not controlled, and facilitates the rapid generation of Drosophila cell lines in which expression from a single transgene can be studied. PMID:26500255

  15. Thermal nuclear pairing within the self-consistent quasiparticle RPA

    Dang, N Dinh

    2010-01-01

    The self-consistent quasiparticle RPA (SCQRPA) is constructed to study the effects of fluctuations on pairing properties in nuclei at finite temperature and z-projection M of angular momentum. Particle-number projection (PNP) is taken into account within the Lipkin-Nogami method. Several issues such as the smoothing of superfluid-normal phase transition, thermally assisted pairing in hot rotating nuclei, extraction of the nuclear pairing gap using an improved odd-even mass difference are discussed. A novel approach of embedding the PNP SCQRPA eigenvalues in the canonical and microcanonical ensembles is proposed and applied to describe the recent empirical thermodynamic quantities for iron, molybdenum, dysprosium, and ytterbium isotopes.

  16. Escherichia coli MW005: lambda Red-mediated recombineering and copy-number induction of oriV-equipped constructs in a single host

    Hope Ian A

    2010-03-01

    Full Text Available Abstract Background Escherichia coli strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2 copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.g. pCC1FOS, have been constructed that contain the additional replication origin, oriV, which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate trans-replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer oriV-equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone. To eliminate these intervening DNA isolation and transformation steps we retrofitted EL350 with a PBAD-driven trfA gene generating strain MW005 that supports, independently, both recombineering and copy-number induction. Results The PBAD-driven copy of cre in EL350 was replaced seamlessly with a copy of trfA, PCR-amplified from EPI300 chromosomal DNA, to generate MW005. This new strain has been used to both generate, via recombineering, a number of reporter gene fusions directly from pCC1FOS-based Caenorhabditis elegans genomic clones and to transiently induce copy-number of fosmid and BAC clones prior to DNA preparation. Conclusions By retrofitting EL350, an established 'recombineering' E. coli strain, with a tightly regulated copy of trfA we have produced a new strain, MW005, which combines recombineering capacity with the useful ability to transiently induce copy-number of oriV-equipped clones. By coupling these two steps in a single strain, use of MW005 will enable the more rapid recombineering-mediated production of recombinant clones in the yield and quality necessary for many downstream purposes.

  17. In vitro and in vivo activities of recombinant anthrax protective antigen co-expressed with thioredoxin in Escherichia coli.

    Ma, Yao; Yu, Yun-Zhou; Zhu, Yu-Feng; Xu, Qing; Sun, Zhi-Wei

    2013-11-01

    Because of the central role it plays in the formation of lethal toxin and edema toxin, protective antigen (PA) is the principal target for the development of vaccines against anthrax. In the present study, we explored and compared the in vitro and in vivo activities of recombinant anthrax protective antigen (rPA) and receptor binding domain of protective antigen (PA4). As a result, the fully soluble rPA and PA4 proteins were successfully expressed in Escherichia coli by co-expression with thioredoxin (Trx), and the rPA was active in forming cytotoxic lethal toxins, indicating that the rPA protein retains a functionally biological activity. Furthermore, immunization with rPA protein induced stronger PA-specific immune responses in mice than PA4 protein. The protection elicited by immunization with PA4 suggests the presence of common neutralizing epitopes between rPA and PA4, but the immunization with rPA protein induced stronger neutralizing antibodies and protective levels against challenge with the B. anthracis strain A16R than the PA4 protein. The sera neutralizing antibodies titers correlated well with anti-PA group ELISA antibodies titers and the in vivo protective potency. Based on the results of cell cytotoxicity assays and the observed immune responses and protective potency, we concluded that the soluble rPA protein retains the in vitro and in vivo functionally biological activity and can be developed into a highly effective human subunit vaccine candidate against anthrax. PMID:23880942

  18. Evaluation of the effect of RFCA in patients with WPW syndrome using RPA

    Whether radionuclide phase analysis (RPA) could evaluate the effect of radiofrequency current ablation (RFCA) in patients with Wolff-Parkinson-White (WPW) syndrome was evaluated. 18 patients with WPW syndrome were studied using RPA pre- and post-RFCA. RPA identified the sites of pre-excitation in all patients before RFCA. Compared with the pre-RFCA study, the sites of pre-excitation disappeared in 12 cases, disappeared gradually in 4 cases and unchanged in 2 cases. 50 RFCA was successful in the former two patterns, but failed in the last pattern. RPA can evaluate the changes of pre-excitation sites in patients with WPW syndrome before and after RFCA. It was a noninvasive and reliable method for assessing and monitoring the effect of RFCA in patients with WPW syndrome

  19. Validation of the RTOG recursive partitioning analysis (RPA) classification for brain metastases

    Purpose: The Radiation Therapy Oncology Group (RTOG) previously developed three prognostic classes for brain metastases using recursive partitioning analysis (RPA) of a large database. These classes were based on Karnofsky performance status (KPS), primary tumor status, presence of extracranial system metastases, and age. An analysis of RTOG 91-04, a randomized study comparing two dose-fractionation schemes with a comparison to the established RTOG database, was considered important to validate the RPA classes. Methods and Materials: A total of 445 patients were randomized on RTOG 91-04, a Phase III study of accelerated hyperfractionation versus accelerated fractionation. No difference was observed between the two treatment arms with respect to survival. Four hundred thirty-two patients were included in this analysis. The majority of the patients were under age 65, had KPS 70-80, primary tumor controlled, and brain-only metastases. The initial RPA had three classes, but only patients in RPA Classes I and II were eligible for RTOG 91-04. Results: For RPA Class I, the median survival time was 6.2 months and 7.1 months for 91-04 and the database, respectively. The 1-year survival was 29% for 91-04 versus 32% for the database. There was no significant difference in the two survival distributions (p = 0.72). For RPA Class II, the median survival time was 3.8 months for 91-04 versus 4.2 months for the database. The 1-year survival was 12% and 16% for 91-04 and the database, respectively (p = 0.22). Conclusion: This analysis indicates that the RPA classes are valid and reliable for historical comparisons. Both the RTOG and other clinical trial organizers should currently utilize this RPA classification as a stratification factor for clinical trials

  20. Oxidative Damage to RPA Limits the Nucleotide Excision Repair Capacity of Human Cells.

    Guven, Melisa; Brem, Reto; Macpherson, Peter; Peacock, Matthew; Karran, Peter

    2015-11-01

    Nucleotide excision repair (NER) protects against sunlight-induced skin cancer. Defective NER is associated with photosensitivity and a high skin cancer incidence. Some clinical treatments that cause photosensitivity can also increase skin cancer risk. Among these, the immunosuppressant azathioprine and the fluoroquinolone antibiotics ciprofloxacin and ofloxacin interact with UVA radiation to generate reactive oxygen species that diminish NER capacity by causing protein damage. The replication protein A (RPA) DNA-binding protein has a pivotal role in DNA metabolism and is an essential component of NER. The relationship between protein oxidation and NER inhibition was investigated in cultured human cells expressing different levels of RPA. We show here that RPA is limiting for NER and that oxidative damage to RPA compromises NER capability. Our findings reveal that cellular RPA is surprisingly vulnerable to oxidation, and we identify oxidized forms of RPA that are associated with impaired NER. The vulnerability of NER to inhibition by oxidation provides a connection between cutaneous photosensitivity, protein damage, and increased skin cancer risk. Our findings emphasize that damage to DNA repair proteins, as well as to DNA itself, is likely to be an important contributor to skin cancer risk. PMID:26134950

  1. Metalaxyl Resistance in Phytophthora infestans: Assessing Role of RPA190 Gene and Diversity Within Clonal Lineages.

    Matson, Michael E H; Small, Ian M; Fry, William E; Judelson, Howard S

    2015-12-01

    Prior work has shown that the inheritance of resistance to metalaxyl, an oomycete-specific fungicide, is complex and may involve multiple genes. Recent research indicated that a single nucleotide polymorphism (SNP) in the gene encoding RPA190, the largest subunit of RNA polymerase I, confers resistance to metalaxyl (or mefenoxam) in some isolates of the potato late blight pathogen Phytophthora infestans. Using both DNA sequencing and high resolution melt assays for distinguishing RPA190 alleles, we show here that the SNP is absent from certain resistant isolates of P. infestans from North America, Europe, and Mexico. The SNP is present in some members of the US-23 and US-24 clonal lineages, but these tend to be fairly sensitive to the fungicide based on artificial media and field test data. Diversity in the level of sensitivity, RPA190 genotype, and RPA190 copy number was observed in these lineages but were uncorrelated. Controlled laboratory crosses demonstrated that RPA190 did not cosegregate with metalaxyl resistance from a Mexican and British isolate. We conclude that while metalaxyl may be used to control many contemporary strains of P. infestans, an assay based on RPA190 will not be sufficient to diagnose the sensitivity levels of isolates. PMID:26551315

  2. Inhibition of highly pathogenic porcine reproductive and respiratory syndrome virus replication by recombinant pseudorabies virus-mediated RNA interference in piglets.

    Cao, Su-Fang; Guo, Qing-Yong; Wang, Yan

    2015-12-31

    Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) is a variant of porcine reproductive and respiratory syndrome virus (PRRSV) which, in recent years, has caused heavy economic losses to swine-producing areas. Although current vaccines are somewhat prophylactic, they provide only limited protection. Furthermore, there are currently no effective anti-HP-PRRSV drugs. Consequently, it is necessary to develop novel antiviral strategies. In the present study, three recombinant pseudorabies viruses (PRV) expressing siRNAs against the ORF7 of HP-PRRSV strain HN1 (PRV gG-/siRNAN1, PRV gG-/siRNAN2, and PRV gG-/siRNAN3) were evaluated for the inhibition of HP-PRRSV replication. The results indicated that recombinant PRV-mediated siRNA could significantly decrease the replication of traditional PRRSV strain H1 at mRNA and protein levels in Marc-145 cells. Moreover, one recombinant PRV (PRV gG-/siRNAN2) was found to be inhibit the multiplication of HP-PRRSV strain HN1 effectively in Marc-145 cells at both the protein and ORF7 mRNA level. Twenty 21-day-old healthy weaned piglets were divided into four groups of five piglets each. Groups 1 and 2 were injected i.m. with PRV gG-/siRNAN2 and PRV gG-/siRNANeg individually. The piglets in group 3 were challenged with the HP-PRRSV control. After 24h, the piglets in groups 1-3 were challenged i.m. with HP-PRRSV strain HN1, while those in group 4 were i.m. administered with PBS as a negative control. The results showed that HP-PRRSV in serum and lung samples from piglets was effectively inhibited by PRV gG-/siRNAN2. The clinical signs and gross lesions of piglets inoculated with PRV gG-/siRNAN2 were significantly less invasive than those of the PRV gG-/siRNANeg group and HP-PRRSV control group. These results showed that siRNAs mediated by recombinant PRV could effectively suppress HP-PRRSV replication in vitro as well as in vivo. RNAi mediated by recombinant PRV presents a potential novel method to prevent HP-PRRSV infections in swine. However, the protective efficiency of PRV gG-/siRNAN2 should be assessed in a larger number of piglets in future studies. PMID:26560709

  3. Opportunistic DNA Recombination With Epstein-Barr Virus at Sites of Control Region Rearrangements Mediating JC Virus Neurovirulence.

    Wortman, Margaret J; Lundberg, Patric S; Dagdanova, Ayuna V; Venkataraman, Pranav; Daniel, Dianne C; Johnson, Edward M

    2016-05-01

    We document a unique DNA recombination between polyomavirus JC (JC virus [JCV]) and Epstein-Barr virus (EBV) at sequences of JCV found infecting the brain. Archetype JCV is present in bone marrow and uroepithelial cells of most adults. During immunosuppression, JCV can infect the brain, causing a demyelinating disease, progressive multifocal leukoencephalopathy. Rearrangements in the archetype noncoding control region are necessary for neurovirulence. Two NCCR deletions and a duplication occur at sequences of homology with EBV, present latently in B cells, which may be coinfected with both viruses. Recombination between JCV and EBV occurs in B lymphoblasts at a sequence essential for JCV neurovirulence and in cerebrospinal fluid of immunosuppressed patients with multiple sclerosis, those susceptible to progressive multifocal leukoencephalopathy. Interviral recombination is a model for conferring advantages on JCV in the brain. It can alter a critical noncoding control region sequence and potentially facilitate use of EBV DNA abilities to transfer among different cell types. PMID:26690342

  4. Cultured mast cells from asthmatic patients and controls respond with similar sensitivity to recombinant Der P2 induced, IgE-mediated activation

    Krohn, Inge Jacoba Maria Kortekaas; Sverrild, Asger; Lund, Gitte; Dahl, Ronald; Erjeflt, Jonas S; Backer, Vibeke; Hoffmann, Hans Jrgen

    2013-01-01

    The function of cultured mast cells may depend on genetic or environmental influence on the stem cell donor. This study investigates whether asthma or atopy in the donor influenced the growth and sensitivity of mast cells cultured from asthma patients and healthy controls under identical conditions...... allergen Der p2. The sensitivity of IgE-mediated activation of mast cells was investigated as Fc?RI-mediated up-regulation of CD63. Ten subjects were atopic, defined as a positive skin prick test (>3 mm) to at least one of ten common allergens. After activation with recombinant Der p2, the maximum CD63...... appears to depend on culture conditions (IL-4, IgE) rather than on donor status as atopy and asthma. This article is protected by copyright. All rights reserved....

  5. Bacillus subtilis RecO and SsbA are crucial for RecA-mediated recombinational DNA repair

    Carrasco, Begoña; Yadav, Tribhuwan; Serrano, Ester; Alonso, Juan C.

    2015-01-01

    Genetic data have revealed that the absence of Bacillus subtilis RecO and one of the end-processing avenues (AddAB or RecJ) renders cells as sensitive to DNA damaging agents as the null recA, suggesting that both end-resection pathways require RecO for recombination. RecA, in the rATP·Mg2+ bound form (RecA·ATP), is inactive to catalyze DNA recombination between linear double-stranded (ds) DNA and naked complementary circular single-stranded (ss) DNA. We showed that RecA·ATP could not nucleate...

  6. Sum-rules and Goldstone modes from extended RPA theories in Fermi systems with spontaneously broken symmetries

    Delion, D S; Tohyama, M

    2016-01-01

    The Self-Consistent RPA (SCRPA) approach is elaborated for cases with a continuously broken symmetry, this being the main focus of the present article. Correlations beyond standard RPA are summed up correcting for the quasi-boson approximation in standard RPA. Desirable properties of standard RPA such as fullfillment of energy weighted sum rule and appearance of Goldstone (zero) modes are kept. We show theoretically and, for a model case, numerically that, indeed, SCRPA maintains all properties of standard RPA for practically all situations of spontaneously broken symmetries. A simpler approximate form of SCRPA, the so-called renormalised RPA, also has these properties. The SCRPA equations are first outlined as an eigenvalue problem, but it is also shown how an equivalent many body Green's function approach can be formulated.

  7. GENE INSERTION AND REPLACEMENT IN SCHIZOSACCHAROMYCES POMBE MEDIATED BY THE STREPTOMYCES BACTERIOPHAGE C31 SITE-SPECIFIC RECOMBINATION SYSTEM

    The Streptomyces bacteriophage íC31 site-specific recombination system was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to...

  8. Generation of Fibroblasts Lacking the Sal-like 1 Gene by Using Transcription Activator-like Effector Nuclease-mediated Homologous Recombination.

    Kim, Se Eun; Kim, Ji Woo; Kim, Yeong Ji; Kwon, Deug-Nam; Kim, Jin-Hoi; Kang, Man-Jong

    2016-04-01

    The Sal-like 1 gene (Sall1) is essential for kidney development, and mutations in this gene result in abnormalities in the kidneys. Mice lacking Sall1 show agenesis or severe dysgenesis of the kidneys. In a recent study, blastocyst complementation was used to develop mice and pigs with exogenic organs. In the present study, transcription activator-like effector nuclease (TALEN)-mediated homologous recombination was used to produce Sall1-knockout porcine fibroblasts for developing knockout pigs. The vector targeting the Sall1 locus included a 5.5-kb 5' arm, 1.8-kb 3' arm, and a neomycin resistance gene as a positive selection marker. The knockout vector and TALEN were introduced into porcine fibroblasts by electroporation. Antibiotic selection was performed over 11 days by using 300 μg/mL G418. DNA of cells from G418-resistant colonies was amplified using polymerase chain reaction (PCR) to confirm the presence of fragments corresponding to the 3' and 5' arms of Sall1. Further, mono- and bi-allelic knockout cells were isolated and analyzed using PCR-restriction fragment length polymorphism. The results of our study indicated that TALEN-mediated homologous recombination induced bi-allelic knockout of the endogenous gene. PMID:26949958

  9. Fowlpox virus recombinants expressing HPV-16 E6 and E7 oncogenes for the therapy of cervical carcinoma elicit humoral and cell-mediated responses in rabbits

    Pacchioni Sole

    2010-04-01

    Full Text Available Abstract Background Around half million new cases of cervical cancer arise each year, making the development of an effective therapeutic vaccine against HPV a high priority. As the E6 and E7 oncoproteins are expressed in all HPV-16 tumour cells, vaccines expressing these proteins might clear an already established tumour and support the treatment of HPV-related precancerous lesions. Methods Three different immunisation regimens were tested in a pre-clinical trial in rabbits to evaluate the humoral and cell-mediated responses of a putative HPV-16 vaccine. Fowlpoxvirus (FP recombinants separately expressing the HPV-16 E6 (FPE6 and E7 (FPE7 transgenes were used for priming, followed by E7 protein boosting. Results All of the protocols were effective in eliciting a high antibody response. This was also confirmed by interleukin-4 production, which increased after simultaneous priming with both FPE6 and FPE7 and after E7 protein boost. A cell-mediated immune response was also detected in most of the animals. Conclusion These results establish a preliminary profile for the therapy with the combined use of avipox recombinants, which may represent safer immunogens than vaccinia-based vectors in immuno-compromised individuals, as they express the transgenes in most mammalian cells in the absence of a productive replication.

  10. Dielectric matrix formulation of correlation energies in the Random Phase Approximation (RPA): inclusion of exchange effects

    Mussard, Bastien; Jansen, Georg; Angyan, Janos

    2016-01-01

    Starting from the general expression for the ground state correlation energy in the adiabatic connection fluctuation dissipation theorem (ACFDT) framework, it is shown that the dielectric matrix formulation, which is usually applied to calculate the direct random phase approximation (dRPA) correlation energy, can be used for alternative RPA expressions including exchange effects. Within this famework, the ACFDT analog of the second order screened exchange (SOSEX) approximation leads to a logarithmic formula for the correlation energy similar to the direct RPA expression. Alternatively, the contribution of the exchange can be included in the kernel used to evaluate the response functions. In this case the use of an approximate kernel is crucial to simplify the formalism and to obtain a correlation energy in logarithmic form. Technical details of the implementation of these methods are discussed and it is shown that one can take advantage of density fitting or Cholesky decomposition techniques to improve the co...

  11. Use of Aspirin in normalization of recombinant human erythropoietin-mediated hyper-reactivity of platelets in rats

    Hitesh M Soni; Vekaria, Amit M.; Rath, Akshyaya C.; Sateesh Belemkar; Mukul R Jain

    2014-01-01

    Objectives: The cytokine erythropoietin is the primary stimulator of erythropoiesis and recombinant human erythropoietin (rHuEPO), which is widely used in the treatment of anemia associated with advanced chronic kidney disease (CKD). Adverse cardiovascular outcomes have been observed during clinical trials of anemia correction with rHuEPO in CKD patients. We investigated the effects of short-term, high-dose treatment with rHuEPO on platelet reactivity and effects of aspirin on platelet reacti...

  12. Eri silkworm (Samia ricini), a non-mulberry host system for AcMNPV mediated expression of recombinant proteins.

    Hosamani, Madhusudan; Basagoudanavar, Suresh H; Sreenivasa, B P; Inumaru, Shigeki; Ballal, Chandish R; Venkataramanan, Ramamurthy

    2015-12-20

    The baculovirus expression system (BVES) based on Autographa californica nucleopolyhedrovirus (AcMNPV) is widely used for the expression of eukaryotic proteins. Several insect cells/larvae that are permissive to AcMNPV have been routinely used as hosts to express heterologous proteins. Domesticated Eri silkworm (Samia ricini), reared in many parts of India, Japan and China, is a non-mulberry silkworm. The present study shows that the Eri silkworm larvae are susceptible to intra-haemocoelical inoculation of AcMNPV. The virus replicates in the larva, as indicated by an increased viral loads in the haemolymph upon injection of a recombinant AcMNPV carrying green fluorescent protein gene. The virus showed localized replication in different tissues including the fat body, haemocytes, tracheal matrix and in the Malphigian tubules. The larval system was successfully used to express heterologous protein, by infecting with a recombinant AcMNPV carrying the 3ABC coding sequence of foot-and-mouth disease virus (FMDV). The study shows that the Eri silkworm larva can be a potential alternative bioreactor, for scaling up of the recombinant proteins employing the baculovirus system. PMID:26467714

  13. PTEN microdeletions in T-cell acute lymphoblastic leukemia are caused by illegitimate RAG-mediated recombination events.

    Mendes, Rui D; Sarmento, Leonor M; Canté-Barrett, Kirsten; Zuurbier, Linda; Buijs-Gladdines, Jessica G C A M; Póvoa, Vanda; Smits, Willem K; Abecasis, Miguel; Yunes, J Andres; Sonneveld, Edwin; Horstmann, Martin A; Pieters, Rob; Barata, João T; Meijerink, Jules P P

    2014-07-24

    Phosphatase and tensin homolog (PTEN)-inactivating mutations and/or deletions are an independent risk factor for relapse of T-cell acute lymphoblastic leukemia (T-ALL) patients treated on Dutch Childhood Oncology Group or German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia protocols. Some monoallelic mutated or PTEN wild-type patients lack PTEN protein, implying that additional PTEN inactivation mechanisms exist. We show that PTEN is inactivated by small deletions affecting a few exons in 8% of pediatric T-ALL patients. These microdeletions were clonal in 3% and subclonal in 5% of patients. Conserved deletion breakpoints are flanked by cryptic recombination signal sequences (cRSSs) and frequently have non-template-derived nucleotides inserted in between breakpoints, pointing to an illegitimate RAG recombination-driven activity. Identified cRSSs drive RAG-dependent recombination in a reporter system as efficiently as bona fide RSSs that flank gene segments of the T-cell receptor locus. Remarkably, equivalent microdeletions were detected in thymocytes of healthy individuals. Microdeletions strongly associate with the TALLMO subtype characterized by TAL1 or LMO2 rearrangements. Primary and secondary xenotransplantation of TAL1-rearranged leukemia allowed development of leukemic subclones with newly acquired PTEN microdeletions. Ongoing RAG activity may therefore actively contribute to the acquisition of preleukemic hits, clonal diversification, and disease progression. PMID:24904117

  14. IS2-mediated re-arrangement of the promoter sequence suppresses metabolic burden of the recombinant plasmid.

    Valesová, R; Stepánek, V; Vecerek, B; Kyslík, P

    2005-01-01

    Recombinant plasmid pKA18 of the high expression bacterial system for penicillin amidase ('penicillin G acylase') bears the 3' end region of IS2 element. The IS2 sequence replaces the -35 region of promoter of pga and extends up to TAGTAT box at position -10 of the promoter region. It therefore forms a hybrid promoter of pga ppgaHT. A natural promoter ppgaWT was not detected on any recombinant plasmid isolated from recombinant strains of Escherichia coli constitutively producing penicillin amidase. PCR fragments carrying both types of promoters were cloned into the promoter-probe vector pET2 to compare their transcriptional activity: the activity of ppgaWT was 5x higher than that of ppgaHT. The same nucleotide "G" localized 28 nucleotides upstream of the translation start point was identified as the respective transcription start point of both mRNAs. An attempt was made to place the pga gene cloned on a plasmid under the control of the natural promoter: not a single clone expressing penicillin amidase was found among 150 transformants. High transcriptional activity of the natural promoter together with high pga gene dosage could result in a deleterious metabolic burden of the periplasmic enzyme. PMID:16408844

  15. Effects of recombinant adenovirus mediated retinoblastoma gene 94 combined with γ-ray on growth of esophageal carcinoma cells

    Objective: To study the combined effect of exogenous recombinant adenovirus-medicated retinoblastoma gene 94 (Ad-Rb94) combined with γ-ray on the growth of esophageal carcinoma cells. Methods: Cell culture were randomly divided into 5 groups: control group, recombinant adenovirus vector containing β-galactosidase gene (Ad-LacZ) group, Ad-Rb94 group, γ-ray radiation group and Ad-Rb94 combined with γ-ray radiation group. EC109 cells were transfected by Ad-Rb94 and exposed to 4 Gy 137Cs γ-ray irradiation 6 hours after transfection. The inhibition rate of EC109 cells were detected by MTT assay. Results: EC109 cells transfected with Ad-Rb94 group, γ-ray radiation group and Ad-Rb94 combined with γ-ray radiation group were all inhibited. The inhibition rate of Ad-Rb94 combined with γray radiation group reached (40.30%±4.2%), significantly higher than Ad-Rb94 group (18.3±0.4%) and γ-ray radiation group (27.40%±2.9%) (χ2=7.91, χ2=5.82, P2=5.12, P<0.05). Conclusion: The recombinant Ad-Rb94 gene transfection combined with γ-ray shows the synergism for the inhibition of the growth of EC109 cells. (authors)

  16. RPA classification has prognostic significance for surgically resected single brain metastasis

    Purpose: To retrospectively evaluate prognostic factors that correlate with overall survival among patients with a surgically resected single brain metastasis. Methods and Materials: An Institutional Review Board-approved database of Cleveland Clinic Brain Tumor Institute was queried for patients with a single brain metastasis treated by surgical resection between February 1984 and January 2004. The primary endpoint was overall survival from the date of surgery by the Kaplan-Meier method. Results: A total of 271 patients were included. Statistically significant variables for improved survival on multivariate analysis included age <65 years, lack of extracranial metastases, control of primary tumor, histology (non-small-cell lung carcinoma), and use of stereotactic radiosurgery. The median survival for all patients was 10.2 months. Survival of patients in recursive partitioning analysis (RPA) class 1 was better (21.4 months) than those in RPA class 2 (9.0 months, p < 0.001), RPA class 3 (8.9 months, p = 0.15), or the combined group of RPA classes 2 and 3 (9.0 months, p < 0.001). Patients had a median survival of 10.6 months after documented gross total resection and 8.7 months after subtotal resection, which approached statistical significance (p 0.07). Those who were treated with stereotactic radiosurgery had a median survival of 17.1 months, which was greater than patients who were not treated with stereotactic radiosurgery (8.9 months, p = 0.006). Conclusions: This analysis supports the prognostic significance of the RPA classification in patients with a single brain metastasis who undergo surgical resection and adjuvant therapy. RPA class 1 patients have a very favorable prognosis with a median survival of 21.4 months

  17. A complex integration technique to remove spurious states associated with spontaneously broken symmetries in RPA calculations

    The removal of the spurious states associated with the violation of rotational and gauge invariance by mean field approximation from the RPA solutions describing the low-energy spectrum of nuclei is a notoriously difficult task, in particular in a rotating frame. A novel, numerically accurate yet technically simple method to accomplish this aim has been developed based on linear response theory. The results of calculations carried out for the deformed, superfluid, rotating nucleus 168Yb testify to the power of the technique, the associated solutions respecting the variety of sum rules required to be fulfilled by the RPA solutions because of conservation laws

  18. A complex integration technique to remove spurious states associated with spontaneously broken symmetries in RPA calculations

    Donati, P; Shimizu, Y R; Bortignon, P F; Broglia, R A

    1999-01-01

    The removal of the spurious states associated with the violation of rotational and gauge invariance by mean field approximation from the RPA solutions describing the low-energy spectrum of nuclei is a notoriously difficult task, in particular in a rotating frame. A novel, numerically accurate yet technically simple method to accomplish this aim has been developed based on linear response theory. The results of calculations carried out for the deformed, superfluid, rotating nucleus sup 1 sup 6 sup 8 Yb testify to the power of the technique, the associated solutions respecting the variety of sum rules required to be fulfilled by the RPA solutions because of conservation laws.

  19. Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling

    Choi, Jun-Hyuk; Lindsey-Boltz, Laura A.; Kemp, Michael; Mason, Aaron C.; Wold, Marc S.; Sancar, Aziz

    2010-01-01

    ATR kinase is a critical upstream regulator of the checkpoint response to various forms of DNA damage. Previous studies have shown that ATR is recruited via its binding partner ATR-interacting protein (ATRIP) to replication protein A (RPA)-covered single-stranded DNA (RPA-ssDNA) generated at sites of DNA damage where ATR is then activated by TopBP1 to phosphorylate downstream targets including the Chk1 signal transducing kinase. However, this critical feature of the human ATR-initiated DNA da...

  20. [Production of l-ephedrine and d-pseudoephedrine in recombined yeasts obtained by argon ion implantation mediated Ephedra genome DNA transformation].

    Mao, Pei-Hong; Ma, Xiang-Dong; Jin, Xiang; Yang, Hong-Mei; Lou, Kai

    2007-10-01

    The genome DNA from Ephedra glauca was randomly transferred into Hansenula anomala, respectively, by argon ion bombardment. Then, after screening by the motheds of bromothymol blue indicator selection, slant cultivation, l-ephedrine and d-pseudoephedrine copper chromic salt qualitative test and RP-HPLC determination, 3 strains, producing recombined yeasts were obtained, which can use glucose as a carbon source, NaNO3 as nitrogen source and be genetically stable. After cultivated in liquid medium for 72 hours and analyzed by the RP-HPLC, the recombined strains can produce l-ephedrine 11.87 mg/L and d-pseudoephedrine 4.11 mg/L excellular, d-pseudoephedrine 294.86 mg/g dry cell incellular, but l-ephedrine not detected incellular. The transformation efficiency of Ephedra genome DNA transferred into yeasts via argon ion bombardment was 0.65%. The effects of Ephedra genome DNA macromolecule integrity on yeast transformation system were discussed. The results shown that DNA macromolecule with integrated structure used as exogenous donor can obtain higher transformation efficiency than DNA macromolecule random fragments by ion implantation mediated DNA transformation. It was inferred that biosynthesis of l-ephedrine and the d-pseudoephedrin were controlled by linked together genes or gene clusters. PMID:18062271

  1. Characterization of transferrin receptor-mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (Oryza sativa L.

    Zhang Deshui

    2012-11-01

    Full Text Available Abstract Background Transferrin (TF plays a critical physiological role in cellular iron delivery via the transferrin receptor (TFR-mediated endocytosis pathway in nearly all eukaryotic organisms. Human serum TF (hTF is extensively used as an iron-delivery vehicle in various mammalian cell cultures for production of therapeutic proteins, and is also being explored for use as a drug carrier to treat a number of diseases by employing its unique TFR-mediated endocytosis pathway. With the increasing concerns over the risk of transmission of infectious pathogenic agents of human plasma-derived TF, recombinant hTF is preferred to use for these applications. Here, we carry out comparative studies of the TFR binding, TFR-mediated endocytosis and cellular iron delivery of recombinant hTF from rice (rhTF, and evaluate its suitability for biopharmaceutical applications. Result Through a TFR competition binding affinity assay with HeLa human cervic carcinoma cells (CCL-2 and Caco-2 human colon carcinoma cells (HTB-37, we show that rhTF competes similarly as hTF to bind TFR, and both the TFR binding capacity and dissociation constant of rhTF are comparable to that of hTF. The endocytosis assay confirms that rhTF behaves similarly as hTF in the slow accumulation in enterocyte-like Caco-2 cells and the rapid recycling pathway in HeLa cells. The pulse-chase assay of rhTF in Caco-2 and HeLa cells further illustrates that rice-derived rhTF possesses the similar endocytosis and intracellular processing compared to hTF. The cell culture assays show that rhTF is functionally similar to hTF in the delivery of iron to two diverse mammalian cell lines, HL-60 human promyelocytic leukemia cells (CCL-240 and murine hybridoma cells derived from a Sp2/0-Ag14 myeloma fusion partner (HB-72, for supporting their proliferation, differentiation, and physiological function of antibody production. Conclusion The functional similarity between rice derived rhTF and native hTF in their cellular iron delivery, TFR binding, and TFR-mediated endocytosis and intracellular processing support that rice-derived rhTF can be used as a safe and animal-free alternative to serum hTF for bioprocessing and biopharmaceutical applications.

  2. Experimental study on the effects of recombinant adenoviral-mediated mI?B? gene combined with irradiation on the treatment of hepatocarcinoma

    Objective: To explore the effect of recombinant adenovirus vector mediated mutant I?B? (mI?B?) combined with radiation on the hepatocarcinoma. Methods: Limited dilution method was used to test the virus titer in 293 cells. The HCC9204 cells were infected with MOI 10,20,30 and 50 for 48 h, respectively. The expression of p65 and mI?B? protein was analyzed by Western blot. Transfected HCC9204 cells and controls were treated with 4 Gy ? rays. The inhibition rate of HCC9204 cells was examined by MTT. Rat models of HCC9204 was constructed. AdmI?B? plasmids were injected into tumor tissue and the tumors were administered with 6 Gy ? irradiation 48 hours later. Tumor growth at different time points was recorded during 28 days. Results: The titer of AdmI?B? is 1.252 x 109 pfu/ml. The expression of mI?B? protein was increased with titer of AdmI?B?, and p65 protein began to decrease when MOI was 10, and reached the lowest when MOI was 50, they were all dose-dependent. The proliferation of HCC9204 cell lines were suppressed, as was more significant combined with radiation, and the effect was in a viral dose-dependent manner. From days 7 to 28 after AdmI?B? gene and radiotherapy, the tumor growth was significantly slower than after irradiation or gene therapy alone. Conclusions: Recombinant adenoviral-mediated mI?B? gene, combined with irradiation, can increase the cell-killing effect. It is better than that of either one alone. (authors)

  3. Detection and Characterization of Viral Species/Subspecies Using Isothermal Recombinase Polymerase Amplification (RPA) Assays.

    Glais, Laurent; Jacquot, Emmanuel

    2015-01-01

    Numerous molecular-based detection protocols include an amplification step of the targeted nucleic acids. This step is important to reach the expected sensitive detection of pathogens in diagnostic procedures. Amplifications of nucleic acid sequences are generally performed, in the presence of appropriate primers, using thermocyclers. However, the time requested to amplify molecular targets and the cost of the thermocycler machines could impair the use of these methods in routine diagnostics. Recombinase polymerase amplification (RPA) technique allows rapid (short-term incubation of sample and primers in an enzymatic mixture) and simple (isothermal) amplification of molecular targets. RPA protocol requires only basic molecular steps such as extraction procedures and agarose gel electrophoresis. Thus, RPA can be considered as an interesting alternative to standard molecular-based diagnostic tools. In this paper, the complete procedures to set up an RPA assay, applied to detection of RNA (Potato virus Y, Potyvirus) and DNA (Wheat dwarf virus, Mastrevirus) viruses, are described. The proposed procedure allows developing species- or subspecies-specific detection assay. PMID:25981257

  4. On the dynamics of polymer mixtures in solution using the RPA

    The dynamics of polymer mixtures and copolymers in solution is investigated using the Random Phase Approximation (RPA). It is shown that the known results for the intermediate scattering functions are recovered in the Rouse limit only. If hydrodynamic interaction is not negligible, a discrepancy appears. This discrepancy can be observed by combining static and dynamic scattering experiments. (author). 10 refs

  5. Lyapunov stability and Poisson structure of the thermal TDHF and RPA equations

    The thermal TDHF equation is analyzed in the Liouville representation of quantum mechanics, where the matrix elements of the single-particle (s.p.) density ρ behave as classical dynamical variables. By introducing the Lie-Poisson bracket associated with the unitary group of the s.p. Hilbert space, we show that TDHF has a hamiltonian, but non-canonical, classical form. Within this Poisson structure, either the s.p. energy or the s.p. grand potential Ω(ρ) act as a Hamilton function. The Lyapunov stability of both the TDHF and RPA equations around a HF state then follows, since the HF approximation for thermal equilibrium is determined by minimizing Ω(ρ). The RPA matrix in the Liouville space is expressed as the product of the Poisson tensor with the HF stability matrix, interpreted as a metric tensor generated by the entropy. This factorization displays the roles of the energy and entropy terms arising from Ω(ρ) in the RPA dynamics, and it helps to construct the RPA modes. Several extensions are considered

  6. Identification of transglutaminase-mediated deamidation sites in a recombinant α-gliadin by advanced mass-spectrometric methodologies

    Mazzeo, Maria Fiorella; De Giulio, Beatrice; Senger, Stefania; Rossi, Mauro; Malorni, Antonio; Siciliano, Rosa Anna

    2003-01-01

    Celiac disease is a permanent immune-mediated food intolerance triggered by ingestion of wheat gliadins in genetically susceptible individuals. It has been reported that tissue transglutaminase plays an important role in the onset of celiac disease by converting specific glutamine residues within gliadin fragments into glutamic acid residues. This process increases binding affinity of gliadin peptides to HLA-DQ2/DQ8 molecules, thus enhancing the immune response. The aim of the present study w...

  7. Evaluation in mice of the capillary leak syndrome (CLS) mediated by the systemic administration of recombinant interleukin-2 (IL-2)

    Since a CLS with interstitial pulmonary edema has been the major toxicity of IL-2 administration in humans, the authors studied this CLS in mice by quantitating the IL-2 mediated, tissue extravasation (Ex) of intravenously injected 125I-bovine serum albumin (BSA). Mice received saline (HBSS) or 200,000 U of IL-2 intraperitoneally thrice daily from day 0-6 before tissues were counted. A permeability index (PI) was calculated by dividing the mean counts per minute (CPM) from tissues of treated mice by those from controls. In a representative experiment, increased BSA Ex was noted in the lungs of mice treated with IL-2 when compared with HBSS (6187 +/- 141 and 638 +/- 64 CPM +/- SEM, respectively; p2 < .001; PI = 9.7). Other tissues with increased BSA Ex included the liver, spleen, kidneys and mesenteric lymph nodes (PI = 6.7, 10.0, 6.3, 6.0, respectively). BSA Ex, which did not occur with the excipient control, was dependent upon the dose and the duration of IL-2. Serial lung weights showed dramatic increases in water weight induced by IL-2. Treatment of mice with radiation (500R), cyclophosphamide, or cortisone acetate significantly reduced IL-2 mediated BSA Ex. Thus, IL-2 induced a generalized CLS which is mediated directly or indirectly by cellular mechanisms

  8. Evaluation in mice of the capillary leak syndrome (CLS) mediated by the systemic administration of recombinant interleukin-2 (IL-2)

    Ettinghausen, S.E.; Rosenstein, M.; Rosenberg, S.A.

    1986-03-01

    Since a CLS with interstitial pulmonary edema has been the major toxicity of IL-2 administration in humans, the authors studied this CLS in mice by quantitating the IL-2 mediated, tissue extravasation (Ex) of intravenously injected /sup 125/I-bovine serum albumin (BSA). Mice received saline (HBSS) or 200,000 U of IL-2 intraperitoneally thrice daily from day 0-6 before tissues were counted. A permeability index (PI) was calculated by dividing the mean counts per minute (CPM) from tissues of treated mice by those from controls. In a representative experiment, increased BSA Ex was noted in the lungs of mice treated with IL-2 when compared with HBSS (6187 +/- 141 and 638 +/- 64 CPM +/- SEM, respectively; p2 < .001; PI = 9.7). Other tissues with increased BSA Ex included the liver, spleen, kidneys and mesenteric lymph nodes (PI = 6.7, 10.0, 6.3, 6.0, respectively). BSA Ex, which did not occur with the excipient control, was dependent upon the dose and the duration of IL-2. Serial lung weights showed dramatic increases in water weight induced by IL-2. Treatment of mice with radiation (500R), cyclophosphamide, or cortisone acetate significantly reduced IL-2 mediated BSA Ex. Thus, IL-2 induced a generalized CLS which is mediated directly or indirectly by cellular mechanisms.

  9. Protection of erythrocytes from human complement-mediated lysis by membrane-targeted recombinant soluble CD59: a new approach to PNH therapy.

    Hill, Anita; Ridley, Simon H; Esser, Dirk; Oldroyd, Rodney G; Cullen, Matthew J; Kareclas, Paula; Gallagher, Sen; Smith, Geoffrey P; Richards, Stephen J; White, Jennifer; Smith, Richard A G; Hillmen, Peter

    2006-03-01

    Paroxysmal nocturnal hemoglobinuria (PNH) results from the expansion of a hematopoietic clone that is deficient in glycosylphosphatidylinositol-anchored molecules. PNH is characterized by chronic hemolysis with acute exacerbations due to the uncontrolled activity of complement on PNH cells, which lack the inhibitor of homologous complement, CD59. Symptoms include severe fatigue, hemoglobinuria, esophageal spasm, erectile dysfunction, and thrombosis. We report the use of a novel synthetically modified recombinant human CD59, rhCD59-P, a soluble protein that attaches to cell membranes. In vitro treatment of PNH erythrocytes with rhCD59-P resulted in levels of CD59 equivalent to normal erythrocytes and effectively protected erythrocytes from complement-mediated hemolysis. The administration of rhCD59-P to CD1 mice resulted in levels of CD59 on erythrocytes, which protected them from complement-mediated lysis. Thus, rhCD59-P corrects the CD59 deficiency in vitro and can bind to erythrocytes in an in vivo murine model, protecting the cells from the activity of human complement, and represents a potential therapeutic strategy in PNH. PMID:16322479

  10. Targeted gene addition in human epithelial stem cells by zinc-finger nuclease-mediated homologous recombination.

    Coluccio, Andrea; Miselli, Francesca; Lombardo, Angelo; Marconi, Alessandra; Malagoli Tagliazucchi, Guidantonio; Gonalves, Manuel A; Pincelli, Carlo; Maruggi, Giulietta; Del Rio, Marcela; Naldini, Luigi; Larcher, Fernando; Mavilio, Fulvio; Recchia, Alessandra

    2013-09-01

    Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a "safe harbor" locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of > 20% in a human keratinocyte cell line, > 10% in immortalized keratinocytes, and integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs. PMID:23760447

  11. Self-Consistent Separable Rpa Approach for Skyrme Forces: Axial Nuclei

    Nesterenko, V O; Kleinig, W; Kvasil, J; Reinhard, P G

    2005-01-01

    The self-consistent separable RPA (random phase approximation) method is formulated for Skyrme forces with pairing. The method is based on a general self-consistent procedure for factorization of the two-body interaction. It is relevant for various density- and current-dependent functionals. The contributions of the time-even and time-odd Skyrme terms as well as of the Coulomb and pairing terms to the residual interaction are taken self-consistently into account. Most of the expression have a transparent analytical form, which makes the method convenient for the treatment and analysis. The separable character of the residual interaction allows to avoid diagonalization of high-rank RPA matrices and thus to minimize the calculation effort. The previous studies have demonstrated high numerical accuracy and efficiency of the method for spherical nuclei. In this contribution, the method is specified for axial nuclei. We provide systematic and detailed presentation of formalism and discuss different aspects of the ...

  12. The Bogolubov representation of the polaron model and its completely integrable RPA-approximation

    N.N. Bogolubov (Jr.); Prykarpatsky, Ya. A.; A.A. Ghazaryan

    2010-01-01

    The polaron model in ionic crystal is studied in the Bogolubov representation using a special RPA-approximation. A new exactly solvable approximated polaron model is derived and described in detail. Its free energy at finite temperature is calculated analytically. The polaron free energy in the constant magnetic field at finite temperature is also discussed. Based on the structure of the Bogolubov unitary transformed polaron Hamiltonian there is stated a very important new result: the full po...

  13. Sequential binding of DNA repair proteins RPA and ERCC1 to XPA in vitro.

    Saijo, M.; Kuraoka, I.; Masutani, C.; Hanaoka, F; Tanaka, K.

    1996-01-01

    Recent studies have shown that many proteins are involved in the early steps of nucleotide excision repair and that there are some interactions between nucleotide excision repair proteins, suggesting that these interactions are important in the reaction mechanism. The xeroderma pigmentosum group A protein (XPA) was shown to bind to the replication protein A (RPA) or the excision repair cross complementing rodent repair deficiency group 1 protein (ERCC1), and these interactions might be involv...

  14. Self-consistent quasi-particle RPA for the description of superfluid Fermi systems

    Self-Consistent Quasi-Particle RPA (SCQRPA) is for the first time applied to a more level pairing case. Various filling situation and values for the coupling constant are considered. Very encouraging results in comparison with the exact solution of the model are obtaining. The nature of the low lying mode in SCQRPA is identified. The strong reduction of the number fluctuation in SCQRPA vs BCS is pointed out. The transition from superfluidity to the normal fluid case is carefully investigated. (authors)

  15. The Bogolubov Representation of the Polaron Model and Its Completely Integrable RPA-Approximation

    The polaron model in ionic crystal is studied in the N. Bogolubov representation using a special RPA-approximation. A new exactly solvable approximated polaron model is derived and described in detail. Its free energy at finite temperature is calculated analytically. The polaron free energy in the constant magnetic field at finite temperature is also discussed. Based on the structure of the N. Bogolubov unitary transformed polaron Hamiltonian a very important new result is stated: the full polaron model is exactly solvable. (author)

  16. Self-Consistent RPA from a Coupled Cluster Wave Function Perspective

    Jemai, M.; Delion, D. S.; Schuck, P.

    2013-01-01

    Self-Consistent RPA is rederived in a consistent way with the help of the Coupled Cluster ground state wave function truncated at the two body level. An exact killing operator for this wave function is introduced allowing for a detailed discussion of the approximation scheme. Several exactly solvable models are reanalyzed under this new perspective giving raise to a quantitative evaluation of the performances of this many body method.

  17. The nature of excess electrons in anatase and rutile from hybrid DFT and RPA.

    Spreafico, Clelia; VandeVondele, Joost

    2014-12-21

    The behavior of excess electrons in undoped and defect free bulk anatase and rutile TiO2 has been investigated by state-of-the-art electronic structure methods including hybrid density functional theory (DFT) and the random phase approximation (RPA). Consistent with experiment, charge trapping and polaron formation is observed in both anatase and rutile. The difference in the anisotropic shape of the polarons is characterized, confirming for anatase the large polaron picture. For anatase, where polaron formation energies are small, charge trapping is observed also with standard hybrid functionals, provided the simulation cell is sufficiently large (864 atoms) to accommodate the lattice relaxation. Even though hybrid orbitals are required as a starting point for RPA in this system, the obtained polaron formation energies are relatively insensitive to the amount of Hartree-Fock exchange employed. The difference in trapping energy between rutile and anatase can be obtained accurately with both hybrid functionals and RPA. Computed activation energies for polaron hopping and delocalization clearly show that anatase and rutile might have different charge transport mechanisms. In rutile, only hopping is likely, whereas in anatase hopping and delocalization are competing. Delocalization will result in conduction-band-like and thus enhanced transport. Anisotropic conduction, in agreement with experimental data, is observed, and results from the tendency to delocalize in the [001] direction in rutile and the (001) plane in anatase. For future work, our calculations serve as a benchmark and suggest RPA on top on hybrid orbitals (PBE0 with 30% Hartree-Fock exchange), as a suitable method to study the rich chemistry and physics of TiO2. PMID:25360624

  18. Self-consistent HF-RPA description of electron and photon nuclear reactions with Skyrme forces

    A mean-field nuclear dynamics is investigated in the analysis of nuclear electromagnetic processes at low and intermediate (q, ω) transfers. The theoretical framework is a self-consistent HF-RPA theory with Skyrme forces formulated in the one-nucleon energy continuum. We review the results obtained in the Skyrme HF-RPA model by focusing on some specific aspects of the theoretical frame and discussing their incidence in the prediction of data. Main points of interest are: (i) the quasiparticle formulation of the Skyrme HF-RPA nuclear dynamics with the identification of a quasi-particle effective mass, (ii) the gauge-invariance of the Skyrme Hamiltonian which produces nuclear electromagnetic currents satisfying the continuity equation, (iii) the excitation and decay properties of the one-nucleon energy continuum. The problems dealt with in the discussion of experimental data are the following: the quasi-deuteron effect in (γ, p) and (γ, n) reactions of closed shell nuclei at energies Eγ≤300 MeV, Giant Multipole Resonances versus momentum transfer in inclusive (e, e' x) responses, the reaction mechanism in polarized ( vector e, e' x) angular distributions at q≤200 MeV/c, the evaluation of the missing strength in inclusive (e, e') longitudinal responses at high momentum transfer, final state interactions and missing momentum distributions in coincidence (e, e' p) reactions in the quasi-elastic region. (orig.)

  19. Recombinant AAV-mediated HSVtk gene transfer with direct intratumoral injections and Tet-On regulation for implanted human breast cancer

    HSVtk/ganciclovir (GCV) gene therapy has been extensively studied in tumors and relies largely on the gene expression of HSVtk. Most studies, however, have failed to demonstrate any significant benefit of a controlled gene expression strategy in cancer treatment. The Tet-On system is commonly used to regulate gene expression following Dox induction. We have evaluated the antitumor effect of HSVtk/ganciclovir gene therapy under Tet-On regulation by means of adeno-associated virus-2 (AAV-2)-mediated HSVtk gene transfer with direct intratumoral injections in mice bearing breast cancer tumors. Recombinant adeno-associated virus-2 (rAAV) was constructed and transduced into MCF-7 cell line. GCV treatment to the rAAV infected MCF-7 cells was performed by MTT assay under the doxycycline (Dox) induction or without Dox induction at a vp (viral particle) number of ≥104 /cell. The virus was administered intratumorally to nude mice that had also received GCV intraperitoneally. The antitumor effects were evaluated by measuring tumor regression and histological analysis. We have demonstrated that GCV treatment to the infected MCF-7 cells under the Dox induction was of more inhibited effects than those without Dox induction at ≥104 vp/cell. In ex vivo experiments, tumor growth of BALB/C nude mice breast cancer was retarded after rAAV-2/HSVtk/Tet-On was injected into the tumors under the Dox induction. Infiltrating cells were also observed in tumors after Dox induction followed by GCV treatment and cells were profoundly damaged. The expression of HSVtk gene in MCF-7 cells and BALB/C nude mice tumors was up-regulated by Tet-On under Dox induction with reverse transcription-PCR (RT-PCR) analysis. The antitumor effect of rAAV-mediated HSVtk/GCV gene therapy under the Dox induction with direct intratumoral injections may be a useful treatment for breast cancer and other solid tumors

  20. Recombinant adeno-associated virus-mediated microRNA delivery into the postnatal mouse brain reveals a role for miR-134 in dendritogenesis in vivo

    Mette Christensen

    2010-01-01

    Full Text Available Recent studies using primary neuronal cultures have revealed important roles of the microRNA pathway in the regulation of neuronal development and morphology. For example, miR-134 is involved in dendritogenesis and spine development in hippocampal neurons by regulating local mRNA translation in dendrites. The in vivo roles of microRNAs in these processes are still uninvestigated, partly due to the lack of tools enabling stable in vivo delivery of microRNAs or microRNA inhibitors into neurons of the mammalian brain. Here we describe the construction and validation of a vector-based tool for stable delivery of microRNAs in vivo by use of recombinant adeno-associated virus (rAAV. rAAV-mediated overexpression of miR-134 in neurons of the postnatal mouse brain provided evidence for a negative role of miR-134 in dendritic arborization of cortical layer V pyramidal neurons in vivo, thereby confirming previous findings obtained with cultured neurons. Our system provides researchers with a unique tool to study the role of any candidate microRNA in vivo and can easily be adapted to microRNA loss-of-function studies. This platform should therefore greatly facilitate investigations on the role of microRNAs in synapse development, plasticity and behavior in vivo.

  1. Recombinant Kluyveromyces lactis expressing highly pathogenic porcine reproductive and respiratory syndrome virus GP5 elicits mucosal and cell-mediated immune responses in mice

    Zhao, Haiyan; Wang, Yalan; Ma, Zhitao; Wang, Yongqiang

    2014-01-01

    Currently, killed-virus and modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccines are used to control porcine reproductive and respiratory syndrome. However, both types of vaccines have inherent drawbacks; accordingly, the development of novel PRRSV vaccines is urgently needed. Previous studies have suggested that yeast possesses adjuvant activities, and it has been used as an expression vehicle to elicit immune responses to foreign antigens. In this report, recombinant Kluyveromyces lactis expressing GP5 of HP-PRRSV (Yeast-GP5) was generated and immune responses to this construct were analyzed in mice. Intestinal mucosal PRRSV-specific sIgA antibody and higher levels of IFN-? in spleen CD4+ and CD8+ T cells were induced by oral administration of Yeast-GP5. Additionally, Yeast-GP5 administered subcutaneously evoked vigorous cell-mediated immunity, and PRRSV-specific lymphocyte proliferation and IFN-? secretion were detected in the splenocytes of mice. These results suggest that Yeast-GP5 has the potential for use as a vaccine for PRRSV in the future. PMID:24378591

  2. Effects of biosurfactants on assays of PCB congeners in transgenic arabidopsis plants carrying a recombinant guinea pig AhR-mediated GUS reporter gene expression system.

    Shimazu, Sayuri; Ohta, Masaya; Inui, Hideyuki; Nanasato, Yoshihiko; Ashida, Hitoshi; Ohkawa, Hideo

    2010-11-01

    The transgenic Arabidopsis plants carrying a recombinant guinea pig (g) aryl hydrocarbon receptor (AhR)-mediated β-glucuronidase (GUS) reporter gene expression system were generated for assays of polychlorinated biphenyl (PCB) congeners. The selected transgenic Arabidopsis plant XgD2V11-6 exhibited a correlation between uptake of PCB126 and PCB126-induced GUS activity. Also, the plants showed induced GUS activity towards the supplemental indole 3-acetic acid (IAA). Thus, the GUS assay may reflect induction by both endogenous and exogenous AhR ligands. When biosurfactants, MEL-B, produced in the culture of yeast isolated from plants were used for assays of PCB congeners in the transgenic Arabidopsis plants, they showed marked PCB126 dose-dependent and toxic equivalency factor (TEF) dependent GUS activities. The effects of biosurfactants were clearer when the plants were cultivated on soils containing PCB congeners for 7 days as compared with on soils for 3 days as well as in the medium for 3 days. Therefore, it was estimated that biosurfactants form micellae with PCB congeners, which are easily uptaken by the plants in a mode of passive diffusion, transport into the aerial parts and then induce GUS activity. PMID:20936563

  3. Perfluorochemical (PFC liquid enhances recombinant adenovirus vector-mediated viral interleukin-10 (AdvIL-10 expression in rodent lung

    Zimmerman Jerry J

    2007-05-01

    Full Text Available Abstract Adenovirus and cationic liposome mediated transfer of Interleukin-10 (IL-10, a potent anti-inflammatory cytokine, has been shown to decrease pro-inflammatory cytokine levels and overall lung inflammation in models of lung transplantation and injury. Limitations to current approaches of IL-10 gene therapy include poor vector delivery methods and pro-inflammatory properties of human IL-10 under certain conditions. We hypothesize that using perfluorochemical (PFC liquid to deliver the highly homologous viral IL-10 (vIL-10, which is predominantly anti-inflammatory with minimal pro-inflammatory activities, can potentially be a more effective strategy to combat inflammatory lung diseases. In this study, we compare the use of PFC liquid versus aerosolized method to deliver adenovirus encoding the vIL-10 gene (AdvIL-10 in C57Bl6 mice. Detectable vIL-10 levels were measured from bronchoalveolar lavage fluid and lung homogenates at one, four, ten and thirty days after AdvIL-10. Furthermore, we determined if use of PFC liquid could allow for the use of a lower dose of AdvIL-10 by comparing the levels of detectable vIL-10 at different doses of AdvIL-10 delivered +/- PFC liquid. Results showed that PFC liquid enhanced detectable vIL-10 by up to ten fold and that PFC liquid allowed the use of ten-fold less vector. PFC liquid increased detectable vIL-10 in lung homogenates at all time points; however, the increase in detectable vIL-10 in BAL fluid peaked at four days and was no longer evident by thirty days after intratracheal instillation. In summary, this is the first report utilizing PFC liquid to enhance the delivery of a potentially therapeutic molecule, vIL-10. We believe this strategy can be used to perform future studies on the use of the predominantly anti-inflammatory vIL-10 to treat inflammatory lung diseases.

  4. Benefits of Recombinant Adeno-Associated Virus (rAAV)-Mediated Insulinlike Growth Factor I (IGF-I) Overexpression for the Long-Term Reconstruction of Human Osteoarthritic Cartilage by Modulation of the IGF-I Axis

    Weimer, Anja; Madry, Henning; Venkatesan, Jagadeesh K.; Schmitt, Gertrud; Frisch, Janina; Wezel, Anna; Jung, Jochen; Kohn, Dieter; Terwilliger, Ernest F.; TRIPPEL, STEPHEN B.; Cucchiarini, Magali

    2011-01-01

    Administration of therapeutic genes to human osteoarthritic (OA) cartilage is a potential approach to generate effective, durable treatments against this slow, progressive disorder. Here, we tested the ability of recombinant adeno-associated virus (rAAV)-mediated overexpression of human insulinlike growth factor (hIGF)-I to reproduce an original surface in human OA cartilage in light of the pleiotropic activities of the factor. We examined the proliferative, survival and anabolic effects of t...

  5. Recombinant AAV-mediated HSVtk gene transfer with direct intratumoral injections and Tet-On regulation for implanted human breast cancer

    Sai-Qun LUO

    2006-03-01

    Full Text Available Abstract Background HSVtk/ganciclovir (GCV gene therapy has been extensively studied in tumors and relies largely on the gene expression of HSVtk. Most studies, however, have failed to demonstrate any significant benefit of a controlled gene expression strategy in cancer treatment. The Tet-On system is commonly used to regulate gene expression following Dox induction. We have evaluated the antitumor effect of HSVtk/ganciclovir gene therapy under Tet-On regulation by means of adeno-associated virus-2 (AAV-2-mediated HSVtk gene transfer with direct intratumoral injections in mice bearing breast cancer tumors. Methods Recombinant adeno-associated virus-2 (rAAV was constructed and transduced into MCF-7 cell line. GCV treatment to the rAAV infected MCF-7 cells was performed by MTT assay under the doxycycline (Dox induction or without Dox induction at a vp (viral particle number of ?104 /cell. The virus was administered intratumorally to nude mice that had also received GCV intraperitoneally. The antitumor effects were evaluated by measuring tumor regression and histological analysis. Results We have demonstrated that GCV treatment to the infected MCF-7 cells under the Dox induction was of more inhibited effects than those without Dox induction at ?104 vp/cell. In ex vivo experiments, tumor growth of BALB/C nude mice breast cancer was retarded after rAAV-2/HSVtk/Tet-On was injected into the tumors under the Dox induction. Infiltrating cells were also observed in tumors after Dox induction followed by GCV treatment and cells were profoundly damaged. The expression of HSVtk gene in MCF-7 cells and BALB/C nude mice tumors was up-regulated by Tet-On under Dox induction with reverse transcription-PCR (RT-PCR analysis. Conclusion The antitumor effect of rAAV-mediated HSVtk/GCV gene therapy under the Dox induction with direct intratumoral injections may be a useful treatment for breast cancer and other solid tumors.

  6. Cell type-specific conditional regulation of the c-myc proto-oncogene by combining Cre/loxP recombination and tamoxifen-mediated activation.

    Jäger, Richard; Maurer, Jochen; Jacob, Andrea; Schorle, Hubert

    2004-03-01

    Development of inducible genetic switches for in vivo use with transgenic mice has revolutionized many areas in modern molecular biology. Combining two techniques, Cre/loxP-based genetic recombination and ligand-dependent activation of a chimeric protein, we generated transgenic mice which allow for the spatiotemporal control of expression and of activity of the proto-oncogene c-myc. To these ends, the gene encoding the tamoxifen-inducible c-mycER(T) fusion protein (mycER(T)) was inserted in the ubiquitously active ROSA 26 gene locus by gene targeting. In the resulting ROSAMER allele, generalized transcription of the mycER(T) gene is prevented by a preceding transcriptional stop sequence which is flanked by loxP sites. Crosses of ROSAMER transgenic mice with Mox2 cre transgenic mice revealed tight control of mycER(T) transcription in various tissues unless the transcriptional stop sequence was removed by cre-mediated excision. Furthermore, we were able to demonstrate tamoxifen-dependent activation of the MycER(T) protein in embryonic fibroblasts derived from such mice. As a proof of principle, we demonstrate that primary neural crest cultures established from ROSAMER mice maintain their proliferative capacity in a 4-OHT-dependent manner. Furthermore, we demonstrate that such neural crest cells retain their differentiation potential as shown by expression of NF 160, a marker of neuronal differentiation upon 4-OHT withdrawal. The transgenic mice produced may thus be valuable tools for studying the cell type-specific effects of c-myc activity in development and disease. PMID:15048812

  7. Recombinant adeno-associated virus 2-mediated transfer of the human superoxide-dismutase gene does not confer radioresistance on HeLa cervical carcinoma cells

    Background and purpose: The success rate of any therapeutic approach depends on the therapeutic window, which can be increased by either raising the resistance of the normal tissue without protecting the tumor cells or by sensitizing the tumor cells but not the normal cells. Two promising candidate genes for normal tissue protection against radiation-induced damage may be the copper-zinc (CuZnSOD) and manganese superoxide-dismutase genes (MnSOD). The recombinant adeno-associated virus 2 (rAAV-2) offers attractive advantages over other vector systems: low immunogenicity, ability to infect dividing and non-dividing tissues and a low chance of insertional mutagenesis, due to extra-chromosomal localization. We report the production of novel rAAV-2-SOD vectors and the investigation of their modulating effects on HeLa-RC cells after irradiation. Material and methods: rAAV-2 vectors were cloned containing the human CuZnSOD or MnSOD as transgene and vector stocks were produced. In the initial experiments human cervix carcinoma (HeLa-RC) cells were chosen for their susceptibility to rAAV-2. On day 0, cells were seeded and transduced with the rAAV-2-SOD vectors. On day 3, cells were harvested, irradiated (0.5-8 Gy) and reseeded in different assays (FACS, SOD, MTT and colony assays). Results: Although >70% of all cells expressed SOD and significant amounts of functional SOD protein were detected, no radioprotective effect of SOD was observed after transduction of HeLa-RC cells. Conclusions: Novel rAAV-2-SOD vectors that could be produced at high titer, were able to efficiently infect cells and express the SOD genes. The absence of a radioprotective effect in HeLa-RC cancer cells indicates an additional safety feature and suggests that rAAV-mediated MnSOD overexpression might contribute to increasing the therapeutic index when applied for normal tissue protection

  8. The Bogolubov representation of the polaron model and its completely integrable RPA-approximation

    N.N.Bogolubov (jr.

    2010-01-01

    Full Text Available The polaron model in ionic crystal is studied in the Bogolubov representation using a special RPA-approximation. A new exactly solvable approximated polaron model is derived and described in detail. Its free energy at finite temperature is calculated analytically. The polaron free energy in the constant magnetic field at finite temperature is also discussed. Based on the structure of the Bogolubov unitary transformed polaron Hamiltonian there is stated a very important new result: the full polaron model is exactly solvable.

  9. Electromagnetic transitions between giant resonances within a continuum-RPA approach

    A general continuum-RPA approach is developed to describe electromagnetic transitions between giant resonances. Using a diagrammatic representation for the three-point Green's function, an expression for the transition amplitude is derived which allows one to incorporate effects of mixing of single and double giant resonances as well as to take the entire basis of particle-hole states into consideration. The radiative widths for E1 transition between the charge-exchange spin-dipole giant resonance and Gamow-Teller states are calculated for 90Nb and 208Bi. (orig.)

  10. An effective interaction derived from HJ potential for use in TDA and RPA calculations

    An effective interaction is derived by fitting the matrix elements of a sum of Yukawa terms to the G-matrix elements of a sum of Yukawa terms to the G-matrix elements derived from the Hamada-Johnston potential. Central, spin-orbit and tensor components are taken into account. Numerical results are given and compared with those obtained from the Paris and Reid potentials. As an application, the excitation spectra in 16O are investigated in the framework of the TDA and RPA. (author). 12 refs, 3 tabs

  11. A Remotely Piloted Aircraft (RPA) as a Measurement Tool for Wind-Energy Research

    Wildmann, Norman; Bange, Jens

    2014-05-01

    In wind energy meteorology, RPA have the clear advantage compared to manned aircraft that they allow to fly very close to the ground and even in between individual wind turbines in a wind farm. Compared to meteorological towers and lidar systems, the advantage is the flexibility of the system, which makes it possible to measure at the desired site on short notice and not only in main wind direction. At the Center of Applied Geoscience at the University of Tübingen, the research RPA MASC (Multi-purpose Airborne Sensor Carrier) was developed. RPA of type MASC have a wingspan of about 3 m and a maximum take-off weight of 7.5 kg, including payload. The standard meteorological payload includes instruments for temperature, humidity, barometric pressure and wind measurement. It is possible to resolve turbulence fluctuations of wind and temperature up to 20 Hz. The autopilot ROCS (Research Onboard Computer System), which is developed at the Institute of Flight Mechanics and Control, University of Stuttgart, makes it possible to automatically follow predefined waypoints at constant altitude and airspeed. At a cruising speed of 24 m/s and a battery life of approx. one hour, a range of 80 km is feasible. The project 'Lidar Complex', funded by the German Federal Ministry for the Environment, Nature Conservation and Nuclear Safety, is part of the research network 'WindForS', based in Southern Germany. The goal of the project is to establish lidar technology for wind energy plant site evaluation in complex terrain. Additional goals are the comparison of different measurement techniques and the validation of wind-field models in not IEC 61400 conform terrain. It is planned to design a turbulent wind-field generator, fed by real measurement data, which can be used to analyse WEC behaviour. Two test sites were defined for the 'Lidar Complex' project, one in IEC-conform terrain about 15 km from the Baltic Sea, the other in the Swabian Alb, only 2 km downstream of a 100 m steep escarpment. At both sites, flight measurements were performed in 2013 with the RPA MASC. The data that was collected allows to investigate the influence of thermal stability of the atmosphere at the test site and turbulence intensity around individual wind energy converters (WECs). Several measurement flights were done to investigate the wake structure downstream a running WEC. Preliminary results will be presented as well as an outlook for future research with the instrument.

  12. Tensor hypercontracted ppRPA: Reducing the cost of the particle-particle random phase approximation from O(r {sup 6}) to O(r {sup 4})

    Shenvi, Neil; Yang, Yang; Yang, Weitao [Department of Chemistry, Duke University, Durham, NC 27708 (United States); Aggelen, Helen van [Department of Chemistry, Princeton University, Princeton, New Jersey 08544 (United States)

    2014-07-14

    In recent years, interest in the random-phase approximation (RPA) has grown rapidly. At the same time, tensor hypercontraction has emerged as an intriguing method to reduce the computational cost of electronic structure algorithms. In this paper, we combine the particle-particle random phase approximation with tensor hypercontraction to produce the tensor-hypercontracted particle-particle RPA (THC-ppRPA) algorithm. Unlike previous implementations of ppRPA which scale as O(r{sup 6}), the THC-ppRPA algorithm scales asymptotically as only O(r{sup 4}), albeit with a much larger prefactor than the traditional algorithm. We apply THC-ppRPA to several model systems and show that it yields the same results as traditional ppRPA to within mH accuracy. Our method opens the door to the development of post-Kohn Sham functionals based on ppRPA without the excessive asymptotic cost of traditional ppRPA implementations.

  13. Generation of Modified Pestiviruses by Targeted Recombination

    Rasmussen, Thomas Bruun; Friis, Martin Barfred; Risager, Peter Christian; Reimann, Ilona; Beer, Martin

    involves targeted modification of viral cDNA genomes, cloned within BACs, by Red/ET recombination-mediated mutagenesis in E.coli DH10B cells. Using recombination-mediated mutagenesis for the targeted design, the work can be expedited and focused in principal on any sequence within the viral genome and...

  14. Function of Rad51 paralogs in eukaryotic homologous recombinational repair

    Full text: Homologous recombinational repair (HRR) is an important mechanism for maintaining genetic integrity and cancer prevention by accurately repair of DNA double strand breaks induced by environmental insults or occurred in DNA replication. A critical step in HRR is the polymerization of Rad51 on single stranded DNA to form nuclear protein filaments, the later conduct DNA strand paring and exchange between homologous strands. A number of proteins, including replication protein A (RPA), Rad52 and Rad51 paralogs, are suggested to modulate or facilitate the process of Rad51 filament formation. Five Rad51 paralogs, namely XRCC2, XRCC3, Rad51B, Rad51C and Rad51D have been identified in eucaryotic cells. These proteins show distant protein sequence identity to Rad51, to yeast Rad51 paralogs (Rad55 and Rad57) and to each other. Hamster or chicken mutants of Rad51 paralogs exhibit hypersensitivity to a variety of DNA damaging agents, especially cross-linking agents, and are defective in assembly of Rad51 onto HRR site after DNA damage. Recent data from our and other labs showed that Rad51 paralogs constitute two distinct complexes in cell extracts, one contains XRCC2, Rad51B, Rad51C and Rad51D, and the other contains Rad51C and XRCC3. Rad51C is involved in both complexes. Our results also showed that XRCC3-Rad51C complex interacts with Rad51 in vivo. Furthermore, overexpression of Rad52 can partially suppress the hypersensitivity of XRCC2 mutant irs1 to ionizing radiation and corrected the defects in Rad51 focus formation. These results suggest that XRCC2 and other Rad51 paralogs play a mediator function to Rad51 in the early stage of HRR

  15. Code-switching to Know a TL Equivalent of an L1 Word: Request-Provision-Acknowledgement (RPA Sequence

    Edgar Lucero

    2011-12-01

    Full Text Available This article focuses on the learner’s use of Code-switching to learn the TL (Target Language equivalent of an L1 word. The interactional pattern that this situation creates defines the Request-Provision-Acknowledgement (RPA sequence. The article explains each of the turns of the sequence under the combination of the Ethnomethodological Conversation Analysis and the Speech Act Analysis. The RPA sequence emerges from the analysis of a set of observations of EFL learners at university level. The insights of this study suggest that this RPA sequence presents pedagogical implications in the dynamics of classroom interaction and the way language learners and teachers negotiate meaning in class.

  16. Recombination instability

    D'Angelo, N.

    1967-01-01

    A recombination instability is considered which may arise in a plasma if the temperature dependence of the volume recombination coefficient, alpha, is sufficiently strong. Two cases are analyzed: (a) a steady-state plasma produced in a neutral gas by X-rays or high energy electrons; and (b) an...

  17. Validation of the RTOG recursive partitioning analysis (RPA) classification for small-cell lung cancer-only brain metastases

    Purpose: Radiation Therapy Oncology Group (RTOG) developed a prognostic classification based on a recursive partitioning analysis (RPA) of patient pretreatment characteristics from three completed brain metastases randomized trials. Clinical trials for patients with brain metastases generally exclude small-cell lung cancer (SCLC) cases. We hypothesize that the RPA classes are valid in the setting of SCLC brain metastases. Methods and Materials: A retrospective review of 154 SCLC patients with brain metastases treated between April 1983 and May 2005 was performed. RPA criteria used for class assignment were Karnofsky performance status (KPS), primary tumor status (PT), presence of extracranial metastases (ED), and age. Results: Median survival was 4.9 months, with 4 patients (2.6%) alive at analysis. Median follow-up was 4.7 months (range, 0.3-40.3 months). Median age was 65 (range, 42-85 years). Median KPS was 70 (range, 40-100). Number of patients with controlled PT and no ED was 20 (13%) and with ED, 27 (18%); without controlled PT and ED, 34 (22%) and with ED, 73 (47%). RPA class distribution was: Class I: 8 (5%); Class II: 96 (62%); Class III: 51 (33%). Median survivals (in months) by RPA class were: Class I: 8.6; Class II: 4.2; Class III: 2.3 (p = 0.0023). Conclusions: Survivals for SCLC-only brain metastases replicate the results from the RTOG RPA classification. These classes are therefore valid for brain metastases from SCLC, support the inclusion of SCLC patients in future brain metastases trials, and may also serve as a basis for historical comparisons

  18. Prognostic factors in brain metastases: should patients be selected for aggressive treatment according to recursive partitioning analysis (RPA) classes?

    Purpose: To determine whether or not Radiation Therapy Oncology Group (RTOG) recursive partitioning analysis (RPA) derived prognostic classes for patients with brain metastases are generally applicable and can be recommended as rational strategy for patient selection for future clinical trials. Inclusion of time to non-CNS death as additional endpoint besides death from any cause might result in further valuable information, as survival limitation due to uncontrolled extracranial disease can be explored. Methods: We performed a retrospective analysis of prognostic factors for survival and time to non-CNS death in 528 patients treated at a single institution with radiotherapy or surgery plus radiotherapy for brain metastases. For this purpose, patients were divided into groups with Karnofsky performance status (KPS) 0.05 for RPA class II versus III). However, it was 8.5 months in RPA class II patients with controlled primary tumor, which was found to be the only prognostic factor for time to non-CNS death in patients with KPS ≥70%. In patients with KPS <70%, no statistically significant prognostic factors were identified for this endpoint. Conclusions: Despite some differences, this analysis essentially confirmed the value of RPA-derived prognostic classes, as published by the RTOG, when survival was chosen as endpoint. RPA class I patients seem to be most likely to profit from aggressive treatment strategies and should be included in appropriate clinical trials. However, their number appears to be very limited. Considering time to non-CNS death, our results suggest that certain patients in RPA class II also might benefit from increased local control of brain metastases

  19. Optimization of Recombinant Adeno-Associated Virus-Mediated Expression for Large Transgenes, Using a Synthetic Promoter and Tandem Array Enhancers.

    Yan, Ziying; Sun, Xingshen; Feng, Zehua; Li, Guiying; Fisher, John T; Stewart, Zoe A; Engelhardt, John F

    2015-06-01

    The packaging capacity of recombinant adeno-associated viral (rAAV) vectors limits the size of the promoter that can be used to express the 4.43-kb cystic fibrosis transmembrane conductance regulator (CFTR) cDNA. To circumvent this limitation, we screened a set of 100-mer synthetic enhancer elements, composed of ten 10-bp repeats, for their ability to augment CFTR transgene expression from a short 83-bp synthetic promoter in the context of an rAAV vector designed for use in the cystic fibrosis (CF) ferret model. Our initial studies assessing transcriptional activity in monolayer (nonpolarized) cultures of human airway cell lines and primary ferret airway cells revealed that three of these synthetic enhancers (F1, F5, and F10) significantly promoted transcription of a luciferase transgene in the context of plasmid transfection. Further analysis in polarized cultures of human and ferret airway epithelia at an air-liquid interface (ALI), as well as in the ferret airway in vivo, demonstrated that the F5 enhancer produced the highest level of transgene expression in the context of an AAV vector. Furthermore, we demonstrated that increasing the size of the viral genome from 4.94 to 5.04 kb did not significantly affect particle yield of the vectors, but dramatically reduced the functionality of rAAV-CFTR vectors because of small terminal deletions that extended into the CFTR expression cassette of the 5.04-kb oversized genome. Because rAAV-CFTR vectors greater than 5 kb in size are dramatically impaired with respect to vector efficacy, we used a shortened ferret CFTR minigene with a 159-bp deletion in the R domain to construct an rAAV vector (AV2/2.F5tg83-fCFTR?R). This vector yielded an ?17-fold increase in expression of CFTR and significantly improved Cl(-) currents in CF ALI cultures. Our study has identified a small enhancer/promoter combination that may have broad usefulness for rAAV-mediated CF gene therapy to the airway. PMID:25763813

  20. Mutant Telomeric Repeats in Yeast Can Disrupt the Negative Regulation of Recombination-Mediated Telomere Maintenance and Create an Alternative Lengthening of Telomeres-Like Phenotype▿

    Bechard, Laura H.; Butuner, Bilge D.; Peterson, George J.; McRae, Will; TOPCU, Zeki; McEachern, Michael J.

    2008-01-01

    Some human cancers maintain telomeres using alternative lengthening of telomeres (ALT), a process thought to be due to recombination. In Kluyveromyces lactis mutants lacking telomerase, recombinational telomere elongation (RTE) is induced at short telomeres but is suppressed once telomeres are moderately elongated by RTE. Recent work has shown that certain telomere capping defects can trigger a different type of RTE that results in much more extensive telomere elongation that is reminiscent o...

  1. Comparative genome analysis of "Candidatus Phytoplasma australiense" (subgroup tuf-Australia I; rp-A) and "Ca. Phytoplasma asteris" Strains OY-M and AY-WB.

    Tran-Nguyen, L T T; Kube, M; Schneider, B; Reinhardt, R; Gibb, K S

    2008-06-01

    The chromosome sequence of "Candidatus Phytoplasma australiense" (subgroup tuf-Australia I; rp-A), associated with dieback in papaya, Australian grapevine yellows in grapevine, and several other important plant diseases, was determined. The circular chromosome is represented by 879,324 nucleotides, a GC content of 27%, and 839 protein-coding genes. Five hundred two of these protein-coding genes were functionally assigned, while 337 genes were hypothetical proteins with unknown function. Potential mobile units (PMUs) containing clusters of DNA repeats comprised 12.1% of the genome. These PMUs encoded genes involved in DNA replication, repair, and recombination; nucleotide transport and metabolism; translation; and ribosomal structure. Elements with similarities to phage integrases found in these mobile units were difficult to classify, as they were similar to both insertion sequences and bacteriophages. Comparative analysis of "Ca. Phytoplasma australiense" with "Ca. Phytoplasma asteris" strains OY-M and AY-WB showed that the gene order was more conserved between the closely related "Ca. Phytoplasma asteris" strains than to "Ca. Phytoplasma australiense." Differences observed between "Ca. Phytoplasma australiense" and "Ca. Phytoplasma asteris" strains included the chromosome size (18,693 bp larger than OY-M), a larger number of genes with assigned function, and hypothetical proteins with unknown function. PMID:18359806

  2. Comparative Genome Analysis of Candidatus Phytoplasma australiense (Subgroup tuf-Australia I; rp-A) and Ca. Phytoplasma asteris Strains OY-M and AY-WB?

    Tran-Nguyen, L. T. T.; Kube, M.; Schneider, B.; Reinhardt, R.; Gibb, K. S.

    2008-01-01

    The chromosome sequence of Candidatus Phytoplasma australiense (subgroup tuf-Australia I; rp-A), associated with dieback in papaya, Australian grapevine yellows in grapevine, and several other important plant diseases, was determined. The circular chromosome is represented by 879,324 nucleotides, a GC content of 27%, and 839 protein-coding genes. Five hundred two of these protein-coding genes were functionally assigned, while 337 genes were hypothetical proteins with unknown function. Potential mobile units (PMUs) containing clusters of DNA repeats comprised 12.1% of the genome. These PMUs encoded genes involved in DNA replication, repair, and recombination; nucleotide transport and metabolism; translation; and ribosomal structure. Elements with similarities to phage integrases found in these mobile units were difficult to classify, as they were similar to both insertion sequences and bacteriophages. Comparative analysis of Ca. Phytoplasma australiense with Ca. Phytoplasma asteris strains OY-M and AY-WB showed that the gene order was more conserved between the closely related Ca. Phytoplasma asteris strains than to Ca. Phytoplasma australiense. Differences observed between Ca. Phytoplasma australiense and Ca. Phytoplasma asteris strains included the chromosome size (18,693 bp larger than OY-M), a larger number of genes with assigned function, and hypothetical proteins with unknown function. PMID:18359806

  3. RPA and QRPA calculations for low-lying states in 96Zr

    The spectroscopic properties of the doubly closed-subshell nucleus 96Zr have been investigated within the framework of the RPA and QRPA. For the residual interaction a Landau-Migdal density-dependent zero-range force has been used in the particle-hole and particle-particle channels. The full level scheme of 96Zr up to 5 MeV has been reproduced, including both the predominantly collective levels and the more pure particle-hole and particle-particle states. It is shown that the state-dependent pairing gaps, which result from a density-dependent δ-interaction as well as the inclusion of the particle-particle interaction, are essential in the description of the level structure of this nucleus. (orig.)

  4. Precise all-electron dynamical response functions: Application to COHSEX and the RPA correlation energy

    Betzinger, Markus; Friedrich, Christoph; Görling, Andreas; Blügel, Stefan

    2015-12-01

    We present a methodology to calculate frequency and momentum dependent all-electron response functions determined within Kohn-Sham density functional theory. It overcomes the main obstacle in calculating response functions in practice, which is the slow convergence with respect to the number of unoccupied states and the basis-set size. In this approach, the usual sum-over-states expression of perturbation theory is complemented by the response of the orbital basis functions, explicitly constructed by radial integrations of frequency-dependent Sternheimer equations. To an essential extent an infinite number of unoccupied states are included in this way. Furthermore, the response of the core electrons is treated virtually exactly, which is out of reach otherwise. The method is an extension of the recently introduced incomplete-basis-set correction (IBC) [Betzinger et al., Phys. Rev. B 85, 245124 (2012), 10.1103/PhysRevB.85.245124; Phys. Rev. B 88, 075130 (2013), 10.1103/PhysRevB.88.075130] to the frequency and momentum domain. We have implemented the generalized IBC within the all-electron full-potential linearized augmented-plane-wave method and demonstrate for rocksalt BaO the improved convergence of the dynamical Kohn-Sham polarizability. We apply this technique to compute (a) quasiparticle energies employing the COHSEX approximation for the self-energy of many-body perturbation theory and (b) all-electron RPA correlation energies. It is shown that the favorable convergence of the polarizability is passed over to the COHSEX and RPA calculation.

  5. Spectra of nuclei in the lead region in the framework of the RPA with OBE-G-matrix interactions

    On the base of an existing Computer program for the calculation of particle-hole RPA matrix elements (especially) for finite-range interactions as well for the solution of the particle-hole RPA equations the corresponding matrix elements for two-particle respectively two-hole nuclei were calculated (and tested) and the corresponding RPA equations solved. For the calculation of the nuclear spectra meson-exchange G-matrix interactions were used instead of phenomenological approaches. The former constitute only a part of the effective NN interaction. A direct comparison with the experimental spectra is therefore just as little convenient as the calculation of transition probabilities or an evaluating comparison of TDA and RPA. The results let nevertheless recognize following: (1) The density dependence of the effective NN interaction. (2) For the states of two-particle respectively two-hole nuclei the (π+rho) exchange plays no such dominating role as for unnatural-parity states in double-magic nuclei. (3) An approximation of the missing part of the effective NN interaction by delta-function interactions is not sufficient. (orig.)

  6. Extended minus-strand DNA as template for R-U5-mediated second-strand transfer in recombinational rescue of primer binding site-modified retroviral vectors

    Mikkelsen, J G; Lund, Anders Henrik; Dybkaer, K; Duch, M; Pedersen, F S

    1998-01-01

    We have previously demonstrated recombinational rescue of primer binding site (PBS)-impaired Akv murine leukemia virus-based vectors involving initial priming on endogenous viral sequences and template switching during cDNA synthesis to obtain PBS complementarity in second-strand transfer of reve...

  7. SETD2-Dependent Histone H3K36 Trimethylation Is Required for Homologous Recombination Repair and Genome Stability

    SophiaX. Pfister

    2014-06-01

    Full Text Available Modulating chromatin through histone methylation orchestrates numerous cellular processes. SETD2-dependent trimethylation of histone H3K36 is associated with active transcription. Here, we define a role for H3K36 trimethylation in homologous recombination (HR repair in human cells. We find that depleting SETD2 generates a mutation signature resembling RAD51 depletion at I-SceI-induced DNA double-strand break (DSB sites, with significantly increased deletions arising through microhomology-mediated end-joining. We establish a presynaptic role for SETD2 methyltransferase in HR, where it facilitates the recruitment of C-terminal binding protein interacting protein (CtIP and promotes DSB resection, allowing Replication Protein A (RPA and RAD51 binding to DNA damage sites. Furthermore, reducing H3K36me3 levels by overexpressing KDM4A/JMJD2A, an oncogene and H3K36me3/2 demethylase, or an H3.3K36M transgene also reduces HR repair events. We propose that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions promotes cell homeostasis. Moreover, these findings provide insights as to why oncogenic mutations cluster within the H3K36me3 axis.

  8. Surface reengineering of RPA70N enables cocrystallization with an inhibitor of the replication protein A interaction motif of ATR interacting protein.

    Feldkamp, Michael D; Frank, Andreas O; Kennedy, J Phillip; Patrone, James D; Vangamudi, Bhavatarini; Waterson, Alex G; Fesik, Stephen W; Chazin, Walter J

    2013-09-17

    Replication protein A (RPA) is the primary single-stranded DNA (ssDNA) binding protein in eukaryotes. The N-terminal domain of the RPA70 subunit (RPA70N) interacts via a basic cleft with a wide range of DNA processing proteins, including several that regulate DNA damage response and repair. Small molecule inhibitors that disrupt these protein-protein interactions are therefore of interest as chemical probes of these critical DNA processing pathways and as inhibitors to counter the upregulation of DNA damage response and repair associated with treatment of cancer patients with radiation or DNA-damaging agents. Determination of three-dimensional structures of protein-ligand complexes is an important step for elaboration of small molecule inhibitors. However, although crystal structures of free RPA70N and an RPA70N-peptide fusion construct have been reported, RPA70N-inhibitor complexes have been recalcitrant to crystallization. Analysis of the P61 lattice of RPA70N crystals led us to hypothesize that the ligand-binding surface was occluded. Surface reengineering to alter key crystal lattice contacts led to the design of RPA70N E7R, E100R, and E7R/E100R mutants. These mutants crystallized in a P212121 lattice that clearly had significant solvent channels open to the critical basic cleft. Analysis of X-ray crystal structures, target peptide binding affinities, and (15)N-(1)H heteronuclear single-quantum coherence nuclear magnetic resonance spectra showed that the mutations do not result in perturbations of the RPA70N ligand-binding surface. The success of the design was demonstrated by determining the structure of RPA70N E7R soaked with a ligand discovered in a previously reported molecular fragment screen. A fluorescence anisotropy competition binding assay revealed this compound can inhibit the interaction of RPA70N with the peptide binding motif from the DNA damage response protein ATRIP. The implications of the results are discussed in the context of ongoing efforts to design RPA70N inhibitors. PMID:23962067

  9. DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli

    Willson Richard C

    2010-12-01

    Full Text Available Abstract Background Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis. Results Sequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli, the chimeric RNA (3×pen aRNA was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse. Conclusions The feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use in clinical and biomedical research.

  10. [Two recombinant adenovirus vaccine candidates containing neuraminidase Gene of H5N1 influenza virus (A/Anhui/1/2005) elicited effective cell-mediated immunity in mice].

    Ma, Jing; Zhang, Xiao-Guang; Chen, Hong; Li, Kui-Biao; Zhang, Xiao-Mei; Zhang, Ke; Yang, Liang; Xu, Hong; Shu, Yue-Long; Tan, Wen-Jie; Zeng, Yi

    2009-09-01

    The aim of this study is to develop the recombinant adenovirus vaccine (rAdV) candidates containing neuraminidase (NA) gene of H5N1 influenza virus and test in BALB/c mice the effect of cell-mediated immunity. In this study, two kind of NA gene (WtNA gene, the wild type; Mod. NA gene, the codon-modified type) derived from H5N1 influenza virus (A/Anhui/1/2005) were cloned and inserted respectively into plasmid of adenovirus vector, then the rAdV vaccines candidates (rAdV-WtNA and rAdV-Mod. NA) were developed and purified, followed by immunization intramuscularly (10(9) TCID50 per dose, double injection at 0 and 4th week) in BALB/c mice, the effect of cell-mediated immunity were analysed at 5th week. Results indicated that: (i) NA protein expression was detected in two rAdV vaccines candidates by Western blotting; (ii) the rAdV-Mod. NA vaccine could elicit more robust NA specific cell-mediated immunity in mice than that of rAdV-WtNA vaccine (P = 0. 016) by IFN-gamma ELIspot assay. These findings suggested rAdV-Mod. NA vaccine was a potential vaccine candidate against H5N1 influenza and worthy of further investigation. PMID:19954107

  11. The interacting boson model, BSC-RPA theory and the theory of pairing interactions applied to Ge nuclei

    The low lying states and especialy the extraordinary behaviour of the first excited O+ state as a function of neutron number in the sequence of nuclei, 68Ge, 70Ge, 72Ge, 74Ge, 76Ge, are described (except for 76Ge) by introducing a s'-boson into the interacting boson model (IBM) which usually involves only s- and d-bosons. This introduction could be understood by considering results from BCS-RPA calculations in the neutron configuration only. BCS-RPA theory is, however, shown to be a poor approximation in the case under consideration. Results from the exact diagonalisation of the Hamiltonian for the charge independant pairing interaction in both the proton and neutron configuration are discussed. 7 figs., 2 tabs., 21 refs

  12. Centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet RPA.

    Schuler, Friedrich; Schwemmer, Frank; Trotter, Martin; Wadle, Simon; Zengerle, Roland; von Stetten, Felix; Paust, Nils

    2015-07-01

    Aqueous microdroplets provide miniaturized reaction compartments for numerous chemical, biochemical or pharmaceutical applications. We introduce centrifugal step emulsification for the fast and easy production of monodisperse droplets. Homogenous droplets with pre-selectable diameters in a range from 120 ?m to 170 ?m were generated with coefficients of variation of 2-4% and zero run-in time or dead volume. The droplet diameter depends on the nozzle geometry (depth, width, and step size) and interfacial tensions only. Droplet size is demonstrated to be independent of the dispersed phase flow rate between 0.01 and 1 ?l s(-1), proving the robustness of the centrifugal approach. Centrifugal step emulsification can easily be combined with existing centrifugal microfluidic unit operations, is compatible to scalable manufacturing technologies such as thermoforming or injection moulding and enables fast emulsification (>500 droplets per second and nozzle) with minimal handling effort (2-3 pipetting steps). The centrifugal microfluidic droplet generation was used to perform the first digital droplet recombinase polymerase amplification (ddRPA). It was used for absolute quantification of Listeria monocytogenes DNA concentration standards with a total analysis time below 30 min. Compared to digital droplet polymerase chain reaction (ddPCR), with processing times of about 2 hours, the overall processing time of digital analysis was reduced by more than a factor of 4. PMID:25947077

  13. The Wigner cusp revisited: The Strutinskij smoothing of the RPA part of the isovector pair correlation energy

    Neergård, K.

    2016-01-01

    Previously published expressions for a smooth counterterm in the sense of the Strutinskij theory to the correction to the Bardeen-Cooper-Schrieffer isovector pair correlation energy obtained in the Random Phase Approximation (RPA) are derived in detail and extended. In particular the counterterm to the neutron-proton pair correlation energy, whose variation contributes to the so-called Wigner cusp in plots of masses along isobaric chains, is analyzed rigorously and found to be less attenuated...

  14. Photon - A Fortran programme for the calculation of photoreaction cross-sections and polarizations in RPA theory with a Skyrme interaction

    Description is given for the Photon programme written for the Ibm 370/168 computer in Fortran 4. language. The programme calculates the photoreaction cross-sections, polarization and asymmetries for closed shell nuclei in RPA theory

  15. High-resolution terrain and landcover mapping with a lightweight, semi-autonomous, remotely-piloted aircraft (RPA): a case study and accuracy assessment

    Hugenholtz, C.; Whitehead, K.; Moorman, B.; Brown, O.; Hamilton, T.; Barchyn, T.; Riddell, K.; LeClair, A.

    2012-04-01

    Remotely-piloted aircraft (RPA) have evolved into a viable research tool for a range of Earth science applications. Significant technological advances driven by military and surveillance programs have steadily become mainstream and affordable. Thus, RPA technology has the potential to reinvigorate various aspects of geomorphological research, especially at the landform scale. In this presentation we will report results and experiences using a lightweight, semi-autonomous RPA for high-resolution terrain and landcover mapping. The goal was to test the accuracy of the photogrammetrically-derived terrain model and assess the overall performance of the RPA system for landform characterization. The test site was comprised an area of semi-vegetated sand dunes in the Canadian Prairies. The RPA survey was conducted with a RQ-84Z AreoHawk (Hawkeye UAV Ltd) and a low-cost digital camera. During the survey the RPA acquired images semi-autonomously with the aid of proprietary mission planning software developed by Accuas Inc. A total of 44 GCPs were used in the block adjustment to create the terrain model, while an additional 400 independent GPS check points were used for accuracy assessment. The 1 m resolution terrain model developed with Trimble's INPHO photogrammetric software was compared to the independent check points, yielding a RMS error comparable to airborne LiDAR data. The resulting orthophoto mosaic had a resolution of 0.1 m, revealing a number of geomorphic features beyond the resolution of airborne and QuickBird imagery. Overall, this case study highlights the potential of RPA technology for resolving terrain and landcover attributes at the landform scale. We believe one of the most significant and emerging applications of RPA in geomorphology is their potential to quantify rates of landform erosion/deposition in an affordable and flexible manner, allowing investigators to reduce the gap between recorded and natural morphodynamics.

  16. Results of the whole-brain radiotherapy for patients with brain metastases from lung cancer: The RTOG RPA intra-classes analysis

    Kepka, Lucyna; Bujko, Krzysztof; Fijuth, Jacek; Wierzchowski, Marek [Memorial M. Sklodowska-Curie Cancer Center and Inst., Warsaw (Poland). Dept. of Radiation Oncology; Cieslak, Ewa [Public Hospital of Pneumology and Oncology, Olsztyn (Poland)

    2005-06-01

    We evaluated the overall survival with respect to prognostic factors in patients with brain metastases (BM) from lung cancer in order to assess the RTOG RPA (Recursive Partitioning Analysis) classification value and to perform intra-classes analyses including pretreatment and treatment-related variables. Between 1986 and 1997, 322 consecutive patients with BM from lung cancer were treated with whole-brain radiotherapy. Patients' distribution according to the RTOG RPA classes was: Class 1 - 13%, Class 2 - 67% and Class 3 - 20%. Prognostic value of the following variables was tested: RTOG RPA classes, performance status, age, extracranial metastases, control of the primary tumour, gender, histology, number of BM and interval from diagnosis to the development of BM. Intra-classes analyses were performed including radiation dose and surgery of BM. Median survival was 4.0 months. Median survival for RTOG RPA classes 1, 2 and 3 were 5.2, 4.0 and 2.5 months, respectively (p=0.003). Extracranial metastases, performance status, control of the primary and RTOG RPA classes were prognostic for survival. Within class 2 higher radiation dose, female, no extracranial metastases and surgery of BM were related to the improved survival. RTOG RPA classes maintain their prognostic significance for patients with BM from lung cancer not participating in clinical trials.

  17. Applications of the Remotely Piloted Aircraft (RPA) 'MASC' in Atmospheric Boundary Layer Research

    Wildmann, Norman; Platis, Andreas; Tupman, David-James; Bange, Jens

    2015-04-01

    The remotely piloted aircraft (RPA) MASC (Multipurpose Airborne Sensor Carrier) was developed at the University of Tübingen in cooperation with the University of Stuttgart, University of Applied Sciences Ostwestfalen-Lippe and 'ROKE-Modelle'. Its purpose is the investigation of thermodynamic processes in the atmospheric boundary layer (ABL), including observations of temperature, humidity and wind profiles, as well as the measurement of turbulent heat, moisture and momentum fluxes. The aircraft is electrically powered, has a maximum wingspan of 3.40~m and a total weight of 5-8~kg, depending on the battery- and payload. The standard meteorological payload consists of two temperature sensors, a humidity sensor, a flow probe, an inertial measurement unit and a GNSS. The sensors were optimized for the resolution of small-scale turbulence down to length scales in the sub-meter range. In normal operation, the aircraft is automatically controlled by the ROCS (Research Onboard Computer System) autopilot to be able to fly predefined paths at constant altitude and airspeed. Only take-off and landing are carried out by a human RC pilot. Since 2012, the system is operational and has since then been deployed in more than ten measurement campaigns, with more than 100 measurement flights. The fields of research that were tackled in these campaigns include sensor validation, fundamental boundary-layer research and wind-energy research. In 2014, for the first time, two MASC have been operated at the same time within a distance of a few kilometres, in order to investigate the wind field over an escarpment in the Swabian Alb. Furthermore, MASC was first deployed off-shore in October 2014, starting from the German island Heligoland in the North Sea, for the purpose of characterization of the marine boundary layer for offshore wind parks. Detailed descriptions of the experimental setup and first preliminary results will be presented.

  18. Replication protein A: directing traffic at the intersection of replication and repair

    Oakley, Greg G.; Patrick, Steve M.

    2010-01-01

    Since the initial discovery of replication protein A (RPA) as a DNA replication factor, much progress has been made on elucidating critical roles for RPA in other DNA metabolic pathways. RPA has been shown to be required for DNA replication, DNA repair, DNA recombination, and the DNA damage response pathway with roles in checkpoint activation. This review summarizes the current understanding of RPA structure, phosphorylation and protein-protein interactions in mediating these DNA metabolic pr...

  19. Protective Effect of Recombinant Adeno-Associated Virus 2/8-Mediated Gene Therapy from the Maternal Hyperphenylalaninemia in Offsprings of a Mouse Model of Phenylketonuria

    Jung, Sung-Chul; PARK, JOO-WON; Oh, Hyun-Jeong; Choi, Jin-Ok; Seo, Kyung-In; Park, Eun-Sook; PARK, HAE-YOUNG

    2008-01-01

    Phenylketonuria (PKU) is an autosomal recessively inherited metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH). The accumulation of phenylalanine leads to severe mental and psychomotor retardation, and the fetus of an uncontrolled pregnant female patient presents with maternal PKU syndrome. We have reported previously on the cognitive outcome of biochemical and phenotypic reversal of PKU in a mouse model, Pahenu2, by the AAV serotype 2-mediated gene delivery of a hum...

  20. Efficient generation of recombinant adenoviral vectors by Cre-lox recombination in vitro.

    Aoki, K.; Barker, C; Danthinne, X; Imperiale, M. J.; Nabel, G. J.

    1999-01-01

    BACKGROUND: Although recombinant adenovirus vectors are attractive for use in gene expression studies and therapeutic applications, the construction of these vectors remains relatively time-consuming. We report here a strategy that simplifies the production of adenoviruses using the Cre-loxP system. MATERIALS AND METHODS: Full-length recombinant adenovirus DNA was generated in vitro by Cre-mediated recombination between loxP sites in a linearized shuttle plasmid containing a transgene and ade...

  1. Recombinant adeno-associated virus-mediated gene transfer for the potential therapy of adenosine deaminase-deficient severe combined immune deficiency.

    Silver, Jared N; Elder, Melissa; Conlon, Thomas; Cruz, Pedro; Wright, Amy J; Srivastava, Arun; Flotte, Terence R

    2011-08-01

    Severe combined immune deficiency due to adenosine deaminase (ADA) deficiency is a rare, potentially fatal pediatric disease, which results from mutations within the ADA gene, leading to metabolic abnormalities and ultimately profound immunologic and nonimmunologic defects. In this study, recombinant adeno-associated virus (rAAV) vectors based on serotypes 1 and 9 were used to deliver a secretory version of the human ADA (hADA) gene to various tissues to promote immune reconstitution following enzyme expression in a mouse model of ADA deficiency. Here, we report that a single-stranded rAAV vector, pTR2-CB-Ig?-hADA, (1) facilitated successful gene delivery to multiple tissues, including heart, skeletal muscle, and kidney, (2) promoted ectopic expression of hADA, and (3) allowed enhanced serum-based enzyme activity over time. Moreover, the rAAV-hADA vector packaged in serotype 9 capsid drove partial, prolonged, and progressive immune reconstitution in ADA-deficient mice. Overview Summary Gene therapies for severe combined immune deficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) over two decades have exclusively involved retroviral vectors targeted to lymphocytes and hematopoietic progenitor cells. These groundbreaking gene therapies represented an unprecedented revolution in clinical medicine but in most cases did not fully correct the immune deficiency and came with the potential risk of insertional mutagenesis. Alternatively, recombinant adeno-associated virus (rAAV) vectors have gained attention as valuable tools for gene transfer, having demonstrated no pathogenicity in humans, minimal immunogenicity, long-term efficacy, ease of administration, and broad tissue tropism (Muzyczka, 1992 ; Flotte et al., 1993 ; Kessler et al., 1996 ; McCown et al., 1996 ; Lipkowitz et al., 1999 ; Marshall, 2001 ; Chen et al., 2003 ; Conlon and Flotte, 2004 ; Griffey et al., 2005 ; Pacak et al., 2006 ; Stone et al., 2008 ; Liu et al., 2009 ; Choi et al., 2010 ). Currently, rAAV vectors are being utilized in phase I/II clinical trials for cystic fibrosis, ?-1 antitrypsin deficiency, Canavan's disease, Parkinson's disease, hemophilia, limb-girdle muscular dystrophy, arthritis, Batten's disease, and Leber's congenital amaurosis (Flotte et al., 1996 , 2004 ; Kay et al., 2000 ; Aitken et al., 2001 ; Wagner et al., 2002 ; Manno et al., 2003 ; Snyder and Francis, 2005 ; Maguire et al., 2008 ; Cideciyan et al., 2009 ). In this study, we present preclinical data to support the viability of an rAAV-based gene transfer strategy for cure of ADA-SCID. We report efficient transduction of a variety of postmitotic target tissues in vivo, subsequent human ADA (hADA) expression, and enhanced hADA secretion in tissues and blood, with increasing peripheral lymphocyte populations over time. PMID:21142972

  2. Generation and Characterization of a MYF5 Reporter Human iPS Cell Line Using CRISPR/Cas9 Mediated Homologous Recombination.

    Wu, Jianbo; Hunt, Samuel D; Xue, Haipeng; Liu, Ying; Darabi, Radbod

    2016-01-01

    Human iPS cells hold great promise for disease modeling and treatment of degenerative disorders including muscular dystrophies. Although a few research groups have used them for skeletal muscle differentiation, most were based on gene over-expression or long-term mesenchymal differentiation and retrospective identification of myogenic cells. Therefore, this study was aimed to generate a knock-in reporter human iPS cell line for MYF5, as an early myogenic specification gene, to allow prospective identification and purification of myogenic progenitors from human iPS cells. By using a CRISPR/Cas9 double nickase strategy, a 2A-GFP reporter was inserted before the stop codon of the MYF5 gene using homologous recombination. This approach allowed for highly efficient in-frame targeting of MYF5 in human iPS cells. Furthermore, in order to prove the reporter function, endogenous MYF5 expression was induced using a novel dead Cas9-VP160 transcriptional activator. Induced clones demonstrated appropriate MYF5-GFP co-expression. Finally, to confirm the differentiation potential, reporter human iPS clones were differentiated through embryoid body method and MYF5-GFP(+) myogenic cells were sorted and characterized. These data provides valuable guidelines for generation of knock-in reporter human iPS cell lines for myogenic genes which can be used for disease modeling, drug screening, gene correction and future in vivo applications. PMID:26729410

  3. The SAGA Deubiquitination Module Promotes DNA Repair and Class Switch Recombination through ATM and DNAPK-Mediated γH2AX Formation.

    Ramachandran, Shaliny; Haddad, Dania; Li, Conglei; Le, Michael X; Ling, Alexanda K; So, Clare C; Nepal, Rajeev M; Gommerman, Jennifer L; Yu, Kefei; Ketela, Troy; Moffat, Jason; Martin, Alberto

    2016-05-17

    Class switch recombination (CSR) requires activation-induced deaminase (AID) to instigate double-stranded DNA breaks at the immunoglobulin locus. DNA breaks activate the DNA damage response (DDR) by inducing phosphorylation of histone H2AX followed by non-homologous end joining (NHEJ) repair. We carried out a genome-wide screen to identify CSR factors. We found that Usp22, Eny2, and Atxn7, members of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitination module, are required for deubiquitination of H2BK120ub following DNA damage, are critical for CSR, and function downstream of AID. The SAGA deubiquitinase activity was required for optimal irradiation-induced γH2AX formation, and failure to remove H2BK120ub inhibits ATM- and DNAPK-induced γH2AX formation. Consistent with this effect, these proteins were found to function upstream of various double-stranded DNA repair pathways. This report demonstrates that deubiquitination of histone H2B impacts the early stages of the DDR and is required for the DNA repair phase of CSR. PMID:27160905

  4. Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1)

    Tomono, Taro; Hirai, Yukihiko; Okada, Hironori; Adachi, Kumi; Ishii, Akiko; Shimada, Takashi; Onodera, Masafumi; Tamaoka, Akira; Okada, Takashi

    2016-01-01

    Recombinant adeno-associated virus (rAAV) is an attractive tool for gene transfer and shows potential for use in human gene therapies. The current methods for the production and purification of rAAV from the transfected cell lysate are mainly based on cesium chloride and iodixanol density ultracentrifugation, although those are not scalable. Meanwhile, chromatography-based systems are more scalable. Therefore, in this study, we developed a novel method for the production and purification of rAAV serotype 1 (rAAV1) from serum-free culture supernatant based on ion-exchange and gel-filtration chromatography to obtain highly purified products with an ultracentrifugation-free technique towards Good Manufacturing Practice (GMP) production. The purified rAAV1 displayed three clear and sharp bands (VP1, VP2, and VP3) following sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and more than 90% of rAAV1 particles contained fully packaged viral genomes according to negative-stain electron micrographic analysis. Consequently, the resultant genomic titer of the purified rAAV1 was 3.63 × 1013 v.g./ml (the total titer was 4.17 × 1013 v.g.) from the 4 × 109 HEK293 cells. This novel chromatography-based method will facilitate scale-up of manufacturing for clinical applications in gene therapy. PMID:26913289

  5. DNA RECOMBINATION IN EUCARYOTIC CELLS BY THE BACTERIOPHAGE PHIC31 RECOMBINATION SYSTEM",

    This invention provides methods for obtaining specific and stable integration of nucleic acids into eukaryotic cells. The invention makes use of site-specific recombination systems that use prokaryotic recombinase polypeptides, such as the ph:C31 integrase, that can mediate recombination between th...

  6. Synthesis, characterization and immunological properties of LPS-based conjugate vaccine composed of O-polysaccharide from pseudomonas aeruginosa IATS 10 bound to recombinant exoprotein A

    Pseudomonas aeruginosa is an improtant opportunistic pathogen that can cause infection in immunocompromised patient. Lipopolysaccharide (LPS), the major surface antigen of P. aeruginosa, is immunogenic and elieits protective antibodies in animals. The O-polysaccharids (O-PS) from international Antigenic typing Scheme (IATS) 10, the antigenic determinant of LPS, was coupled to recombinant exoprotein A (rPA) through adipic acid dihydrazide (ADH) mediated by carbodiimide condensation reaction. Mice were immunized with the conjugate emulsifield with monophosphoryl lipid A-trehalose dicorynomycolate (MPL-T) and freund's adjuvants. The conjiugate emulsified with MPL-T adjuvant elicited the highest level of IgG and IgM followed by freuns's adjuvant. IgG titers using both MPL-T and freund's adjuvants were recorded to be higher than IgM titers after the second post of the immunization. Immunization of mice with the prepared conjugates emulsified with MPL-T and freund's adjvaided provide high level of protection (100%) against ten times the LD50 of homologous strain of P. aeruginsoa. the elicited high IgG level and the in vivo protection test results provided good evidences for the possible protection of the conjugate aginst subsequent infection with the pathogen. These findings will enable us to use it as protective vaccine candidate (authors).

  7. Study of recombineering technology in Salmonella and its applications

    Yu, Bin; 于斌

    2012-01-01

    In the past few years, in vivo recombination technologies have emerged to improve the efficiency and simplicity of genetic engineering in Escherichia coli, Salmonella enterica serovar, and other gram-negative bacteria. Phage λ Red homologous recombination system is used to mediate the accurate replacement of target DNA with PCR-generated ?targeting cassettes? that contain flanking regions of shared homologous DNA sequence. However, the efficiency of λ Red-mediated recombineering in Salmonella...

  8. The Wigner cusp revisited: The Strutinskij-smoothing of the RPA part of the isovector pair correlation energy

    Neergård, K

    2016-01-01

    Previously published expressions for a smooth counterterm in the sense of the Strutinskij theory to the correction to the Bardeen-Cooper-Schrieffer isovector pair correlation energy obtained in the Random Phase Approximation (RPA) are derived in detail and extended. In particular the counterterm to the neutron-proton pair correlation energy, whose variation contributes to the so-called Wigner cusp in plots of masses along isobaric chains, is analyzed rigorously and found to be less attenuated at large excesses of neutrons or protons than suggested by the previous estimate.

  9. Recombinant Bivalent Fusion Protein rVE Induces CD4+ and CD8+ T-Cell Mediated Memory Immune Response for Protection Against Yersinia enterocolitica Infection.

    Singh, Amit K; Kingston, Joseph J; Gupta, Shishir K; Batra, Harsh V

    2015-01-01

    Studies investigating the correlates of immune protection against Yersinia infection have established that both humoral and cell mediated immune responses are required for the comprehensive protection. In our previous study, we established that the bivalent fusion protein (rVE) comprising immunologically active regions of Y. pestis LcrV (100-270 aa) and YopE (50-213 aa) proteins conferred complete passive and active protection against lethal Y. enterocolitica 8081 challenge. In the present study, cohort of BALB/c mice immunized with rVE or its component proteins rV, rE were assessed for cell mediated immune responses and memory immune protection against Y. enterocolitica 8081. rVE immunization resulted in extensive proliferation of both CD4 and CD8 T cell subsets; significantly high antibody titer with balanced IgG1: IgG2a/IgG2b isotypes (1:1 ratio) and up-regulation of both Th1 (TNF-α, IFN-γ, IL-2, and IL-12) and Th2 (IL-4) cytokines. On the other hand, rV immunization resulted in Th2 biased IgG response (11:1 ratio) and proliferation of CD4+ T-cell; rE group of mice exhibited considerably lower serum antibody titer with predominant Th1 response (1:3 ratio) and CD8+ T-cell proliferation. Comprehensive protection with superior survival (100%) was observed among rVE immunized mice when compared to the significantly lower survival rates among rE (37.5%) and rV (25%) groups when IP challenged with Y. enterocolitica 8081 after 120 days of immunization. Findings in this and our earlier studies define the bivalent fusion protein rVE as a potent candidate vaccine molecule with the capability to concurrently stimulate humoral and cell mediated immune responses and a proof of concept for developing efficient subunit vaccines against Gram negative facultative intracellular bacterial pathogens. PMID:26733956

  10. Recombinant Bivalent Fusion Protein rVE Induces CD4+ and CD8+ T-Cell Mediated Memory Immune Response for Protection Against Yersinia enterocolitica Infection

    Singh, Amit K.; Kingston, Joseph J.; Gupta, Shishir K.; Batra, Harsh V.

    2015-01-01

    Studies investigating the correlates of immune protection against Yersinia infection have established that both humoral and cell mediated immune responses are required for the comprehensive protection. In our previous study, we established that the bivalent fusion protein (rVE) comprising immunologically active regions of Y. pestis LcrV (100270 aa) and YopE (50213 aa) proteins conferred complete passive and active protection against lethal Y. enterocolitica 8081 challenge. In the present study, cohort of BALB/c mice immunized with rVE or its component proteins rV, rE were assessed for cell mediated immune responses and memory immune protection against Y. enterocolitica 8081. rVE immunization resulted in extensive proliferation of both CD4 and CD8 T cell subsets; significantly high antibody titer with balanced IgG1: IgG2a/IgG2b isotypes (1:1 ratio) and up-regulation of both Th1 (TNF-?, IFN-?, IL-2, and IL-12) and Th2 (IL-4) cytokines. On the other hand, rV immunization resulted in Th2 biased IgG response (11:1 ratio) and proliferation of CD4+ T-cell; rE group of mice exhibited considerably lower serum antibody titer with predominant Th1 response (1:3 ratio) and CD8+ T-cell proliferation. Comprehensive protection with superior survival (100%) was observed among rVE immunized mice when compared to the significantly lower survival rates among rE (37.5%) and rV (25%) groups when IP challenged with Y. enterocolitica 8081 after 120 days of immunization. Findings in this and our earlier studies define the bivalent fusion protein rVE as a potent candidate vaccine molecule with the capability to concurrently stimulate humoral and cell mediated immune responses and a proof of concept for developing efficient subunit vaccines against Gram negative facultative intracellular bacterial pathogens. PMID:26733956

  11. Non-small lung cancer cells are prime targets for p53 gene transfer mediated by a recombinant adeno-associated virus type-2 vector.

    Rohr, Ulrich-Peter; Wulf, Marc-Andre; Stahn, Susanne; Heyd, Florian; Steidl, Ulrich; Fenk, Roland; Opalka, Bertram; Pitschke, Gerald; Prisack, Hans-Bernd; Bojar, Hans; Haas, Rainer; Kronenwett, Ralf

    2003-12-01

    In this study, we elucidated the potential of recombinant adeno-associated virus type-2 (rAAV-2) vectors for lung cancer gene therapy. Cell lines of the three major histological subtypes of non-small cell lung cancer (NSCLC) were highly susceptible for rAAV-2 showing transduction rates between 63.4 and 98.9%. In contrast, cell lines of small cell carcinomas were resistant to rAAV-2 infection. For restoration of p53 function in p53 deficient NSCLC, a rAAV-2 vector was constructed containing wt p53 cDNA. Following transduction with rAAV-p53, cell growth of all NSCLC cell lines was significantly reduced in a dose-dependent manner between 44 and 71.7% in comparison with rAAV-GFP transduced cells. The reduction of tumor cell growth was associated with increased apoptosis. Adding cisplatin to rAAV-p53-infected cells led to a significant growth inhibition between 81 and 91% indicating a synergistic effect between cisplatin and rAAV-p53. Interestingly, the tumor cells surviving cisplatin and rAAV-p53 treatment were inhibited in their ability to form colonies as reflected by a reduction of colony growth between 57 and 90.4%. In conclusion, rAAV-2 vectors exhibit a strong tropism for NSCLC. Successful inhibition of tumor cell growth following transduction with a rAAV-p53 vector underlines the potential role of rAAV-2 in cancer gene therapy. PMID:14712316

  12. Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus

    Hill-Cawthorne, Grant A.

    2014-06-27

    Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (?4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates. © 2014 Hill-Cawthorne et al.

  13. ATRIP Deacetylation by SIRT2 Drives ATR Checkpoint Activation by Promoting Binding to RPA-ssDNA.

    Zhang, Hui; Head, PamelaSara E; Daddacha, Waaqo; Park, Seong-Hoon; Li, Xingzhe; Pan, Yunfeng; Madden, Matthew Z; Duong, Duc M; Xie, Maohua; Yu, Bing; Warren, Matthew D; Liu, Elaine A; Dhere, Vishal R; Li, Chunyang; Pradilla, Ivan; Torres, Mylin A; Wang, Ya; Dynan, William S; Doetsch, Paul W; Deng, Xingming; Seyfried, Nicholas T; Gius, David; Yu, David S

    2016-02-16

    The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase checkpoint pathway maintains genome integrity; however, the role of the sirtuin 2 (SIRT2) acetylome in regulating this pathway is not clear. We found that deacetylation of ATR-interacting protein (ATRIP), a regulatory partner of ATR, by SIRT2 potentiates the ATR checkpoint. SIRT2 interacts with and deacetylates ATRIP at lysine 32 (K32) in response to replication stress. SIRT2 deacetylation of ATRIP at K32 drives ATR autophosphorylation and signaling and facilitates DNA replication fork progression and recovery of stalled replication forks. K32 deacetylation by SIRT2 further promotes ATRIP accumulation to DNA damage sites and binding to replication protein A-coated single-stranded DNA (RPA-ssDNA). Collectively, these results support a model in which ATRIP deacetylation by SIRT2 promotes ATR-ATRIP binding to RPA-ssDNA to drive ATR activation and thus facilitate recovery from replication stress, outlining a mechanism by which the ATR checkpoint is regulated by SIRT2 through deacetylation. PMID:26854234

  14. Nuclear dissipation as damping of collective motion in the time-dependent RPA and extensions of it

    We have formulated a nonperturbative, microscopic dissipative process in the limit of an infinite mean free path which does not require any statistical assumptions. It attributes the damping of the collective motion to real transitions from the collective state to degenerate, more complicated nucelar states. The dissipation is described through wave packets which solve an approximate Schroedinger equation within extended subspaces, larger than the original subspace of the undamped motion. When the simple RPA is used, this process associates the dissipation with the escape width for direct particle emission. When the Second RPA is used, it associates the dissipation with the spreading width for transitions to the 2p-2h components of the nuclear compound states. The energy loss rate for sharp n-phonon initial states is proportional to the total collective energy. The classical dissipation, however, is obtained for coherent, multiphonon, initial packets which describe the damping of the mean field oscillations, and allow a theoretical connection with the Vibrating Potential Model, and thereby with models of one-body dissipation. The present model contrasts with linear response theories. Canonical coordinates for the collective degree of freedom are explicitly introduced. This allows the construction of a nonlinear frictional Hamiltonian which provides a connection with quantal friction. The dissipation process developed here is properly reversible rather than irreversible, in the sense that it is described by an approximate Schroedinger equation which honors time reversibility, rather than by a coarse grained master equation which violates it. Thus, the present theory contrasts with transport theories

  15. Two fast temperature sensors for probing of the atmospheric boundary layer using small remotely piloted aircraft (RPA

    N. Wildmann

    2013-08-01

    Full Text Available Two types of temperature sensors are designed and tested: a thermocouple and a fine wire resistance thermometer. The intention of this study is to figure out which kind of measurement principle is in general more suited for atmospheric boundary layer meteorology with small remotely piloted aircraft (RPA. The sensors are calibrated in a NIST traceable climate chamber and validated in flight against tower measurements, radiosondes and remote sensing. The sensors have a measurement range of at least −10–50 °C, an absolute RMS error of less than ±0.2 K which is stable over the lifetime of the sensors, and a resolution of about 0.01 K. Both devices are tested for typical errors like radiation error and adiabatic heating, as well as for their dynamic response. Spectral resolutions of up to approximately 10 Hz can be obtained with both sensors, which makes them suitable for turbulence measurement. Their low cost of less than 100 EUR in pure hardware is a major advantage for research with small RPA.

  16. Static correlation and electron localization in molecular dimers from the self-consistent RPA and GW approximation

    Hellgren, Maria; Rohr, Daniel R; Ren, Xinguo; Rubio, Angel; Scheffler, Matthias; Rinke, Patrick

    2014-01-01

    We investigate static correlation and delocalization errors in the self-consistent GW and random-phase approximation (RPA) by studying molecular dissociation of the H_2 and LiH molecules. Although both approximations are diagrammatically identical, the non-locality and frequency dependence of the GW self-energy crucially influence the different energy contributions to the total energy as compared to the use of a static local potential in the RPA. The latter leads to significantly larger correlation energies which allows for a better description of static correlation at intermediate bond distances. The substantial error found in GW is further analyzed by comparing spin-restricted and spin-unrestricted calculations. At large but finite nuclear separation their difference gives an estimate of the so-called fractional spin error normally determined only in the dissociation limit. Furthermore, a calculation of the dipole moment of the LiH molecule at dissociation reveals a large delocalization error in GW making t...

  17. Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination.

    Baude, Annika; Aaes, Tania Løve; Zhai, Beibei; Al-Nakouzi, Nader; Oo, Htoo Zarni; Daugaard, Mads; Rohde, Mikkel; Jäättelä, Marja

    2016-03-18

    We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage. PMID:26721387

  18. Recombinant hTRBP and hPACT Modulate hAgo2-Catalyzed siRNA-Mediated Target RNA Cleavage In Vitro

    Willkomm, Sarah; Deerberg, Andrea; Heidemann, Johannes; Flgge, Friedemann; Meine, Janica; Hu, Rui; Kretschmer-Kazemi Far, Rosel; Restle, Tobias

    2016-01-01

    The human TAR RNA-binding protein (hTRBP) and protein activator of protein kinase R (hPACT) are important players in RNA interference (RNAi). Together with hArgonaute2 (hAgo2) and hDicer they have been reported to form the RISC-loading complex (RLC). Among other functions, hTRBP was suggested to assist the loading of hAgo2 with small interfering RNAs (siRNAs) within the RLC. Although several studies have been conducted to evaluate the specific functions of hTRBP and hPACT in RNAi, exact mechanisms and modes of action are still unknown. Here, we present a biochemical study further evaluating the role of hTRBP and hPACT in hAgo2-loading. We found that both proteins enhance hAgo2-mediated RNA cleavage significantly; even a hAgo2 mutant impaired in siRNA binding shows full cleavage activity in the presence of hTRBP or hPACT. Pre-steady state binding studies reveal that the assembly of wildtype-hAgo2 (wt-hAgo2) and siRNAs remains largely unaffected, whereas the binding of mutant hAgo2-PAZ9 to siRNA is restored by adding either hTRBP or hPACT. We conclude that both proteins assist in positioning the siRNA within hAgo2 to ensure optimal binding and cleavage. Overall, our data indicate that hTRBP and hPACT are part of a regulative system of RNAi that is important for efficient target RNA cleavage. PMID:26784517

  19. Arabidopsis thaliana mutants altered in homologous recombination.

    Masson, J E; Paszkowski, J

    1997-10-14

    Homologous recombination contributes both to the generation of allelic diversity and to the preservation of genetic information. In plants, a lack of suitable experimental material has prevented studies of the regulatory and enzymatic aspects of recombination in somatic and meiotic cells. We have isolated nine Arabidopsis thaliana mutants hypersensitive to x-ray irradiation (xrs) and examined their recombination properties. For the three xrs loci described here, single recessive mutations were found to confer simultaneous hypersensitivities to the DNA-damaging chemicals mitomycin C (MMCs) and/or methyl methanesulfonate (MMSs) and alterations in homologous recombination. Mutant xrs9 (Xrays, MMSs) is reduced in both somatic and meiotic recombination and resembles yeast mutants of the rad52 epistatic group. xrs11 (Xrays, MMCs) is deficient in the x-ray-mediated stimulation of homologous recombination in somatic cells in a manner suggesting a specific signaling defect. xrs4 (Xrays, MMSs, MMCs) has a significant deficiency in somatic recombination, but this is accompanied by meiotic hyper-recombination. A corresponding phenotype has not been reported in other systems and thus this indicates a novel, plant-specific regulatory circuit linking mitotic and meiotic recombination. PMID:9326679

  20. Recombinant Technology and Probiotics

    Icy D’Silva

    2011-09-01

    Full Text Available Recombinant technology has led the way to monumental advances in the development of useful molecules, including the development of safe probiotics. The development of novel approaches using recombinant technology and probiotics that allow accurate targeting of therapeutics to the mucosa is an interesting area of research. The creation and use of recombinant probiotics expressing recombinantovalbumin, recombinant ovalbumin mutants and yet-to-be-designed recombinant hypo/non-allergenic molecules offer the opportunity to further investigate their effects for food, nutrition, environment andhealth. This review highlights advances in native probiotics and recombinant probiotics expressing native and recombinant molecules for food, nutrition, environment and health.

  1. Recombinant Technology and Probiotics

    Icy DSilva

    2011-01-01

    Recombinant technology has led the way to monumental advances in the development of useful molecules, including the development of safe probiotics. The development of novel approaches using recombinant technology and probiotics that allow accurate targeting of therapeutics to the mucosa is an interesting area of research. The creation and use of recombinant probiotics expressing recombinantovalbumin, recombinant ovalbumin mutants and yet-to-be-designed recombinant hypo/non-allergenic molecule...

  2. EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair

    Wu, Yuehan; Lee, Suk-Hee; Williamson, Elizabeth A.; Reinert, Brian L.; Cho, Ju Hwan; Xia, Fen; Jaiswal, Aruna Shanker; Srinivasan, Gayathri; Patel, Bhavita; Brantley, Alexis; Zhou, Daohong; Shao, Lijian; Pathak, Rupak; Hauer-Jensen, Martin; Singh, Sudha; Kong, Kimi; Wu, Xaiohua; Kim, Hyun-Suk; Beissbarth, Timothy; Gaedcke, Jochen; Burma, Sandeep; Nickoloff, Jac A.; Hromas, Robert A.

    2015-01-01

    Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5’ end resection near the fork junction, which permits 3’ single strand invasion of a homologous template for fork restart. This 5’ end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5’ DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5’ overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ. PMID:26684013

  3. Changes in primary metabolism under light and dark conditions in response to overproduction of a response regulator RpaA in the unicellular cyanobacterium Synechocystis sp. PCC 6803.

    Iijima, Hiroko; Shirai, Tomokazu; Okamoto, Mami; Kondo, Akihiko; Hirai, Masami Yokota; Osanai, Takashi

    2015-01-01

    The study of the primary metabolism of cyanobacteria in response to light conditions is important for environmental biology because cyanobacteria are widely distributed among various ecological niches. Cyanobacteria uniquely possess circadian rhythms, with central oscillators consisting from three proteins, KaiA, KaiB, and KaiC. The two-component histidine kinase SasA/Hik8 and response regulator RpaA transduce the circadian signal from KaiABC to control gene expression. Here, we generated a strain overexpressing rpaA in a unicellular cyanobacterium Synechocystis sp. PCC 6803. The rpaA-overexpressing strain showed pleiotropic phenotypes, including slower growth, aberrant degradation of an RNA polymerase sigma factor SigE after the light-to-dark transition, and higher accumulation of sugar catabolic enzyme transcripts under dark conditions. Metabolome analysis revealed delayed glycogen degradation, decreased sugar phosphates and organic acids in the tricarboxylic acid cycle, and increased amino acids under dark conditions. The current results demonstrate that in this cyanobacterium, RpaA is a regulator of primary metabolism and involved in adaptation to changes in light conditions. PMID:26379657

  4. On the evolutionary advantage of fitness-associated recombination.

    Hadany, Lilach; Beker, Tuvik

    2003-01-01

    The adaptive value of recombination remains something of a puzzle. One of the basic problems is that recombination not only creates new and advantageous genetic combinations, but also breaks down existing good ones. A negative correlation between the fitness of an individual and its recombination rate would result in prolonged integrity of fitter genetic combinations while enabling less fit ones to produce new combinations. Such a correlation could be mediated by various factors, including st...

  5. Recombinant DNA in Medicine

    Cederbaum, Stephen D.; Fareed, George C.; Lovett, Michael A.; Shapiro, Larry J.

    1984-01-01

    Studies in bacteria and bacterial viruses have led to methods to manipulate and recombine DNA in unique and reproducible ways and to amplify these recombined molecules millions of times. Once properly identified, the recombinant DNA molecules can be used in various ways useful in medicine and human biology. There are many applications for recombinant DNA technology. Cloned complementary DNA has been used to produce various human proteins in microorganisms. Insulin and growth hormone have been...

  6. Tipin-Replication Protein A Interaction Mediates Chk1 Phosphorylation by ATR in Response to Genotoxic Stress*

    Kemp, Michael G.; Akan, Zafer; Yilmaz, Seçil; Grillo, Mary; Smith-Roe, Stephanie L.; Kang, Tae-Hong; Cordeiro-Stone, Marila; Kaufmann, William K.; Abraham, Robert T.; Sancar, Aziz; Ünsal-Kaçmaz, Keziban

    2010-01-01

    Mammalian Timeless is a multifunctional protein that performs essential roles in the circadian clock, chromosome cohesion, DNA replication fork protection, and DNA replication/DNA damage checkpoint pathways. The human Timeless exists in a tight complex with a smaller protein called Tipin (Timeless-interacting protein). Here we investigated the mechanism by which the Timeless-Tipin complex functions as a mediator in the ATR-Chk1 DNA damage checkpoint pathway. We find that the Timeless-Tipin complex specifically mediates Chk1 phosphorylation by ATR in response to DNA damage and replication stress through interaction of Tipin with the 34-kDa subunit of replication protein A (RPA). The Tipin-RPA interaction stabilizes Timeless-Tipin and Tipin-Claspin complexes on RPA-coated ssDNA and in doing so promotes Claspin-mediated phosphorylation of Chk1 by ATR. Our results therefore indicate that RPA-covered ssDNA not only supports recruitment and activation of ATR but also, through Tipin and Claspin, it plays an important role in the action of ATR on its critical downstream target Chk1. PMID:20233725

  7. Therapeutic Recombinant Monoclonal Antibodies

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  8. Therapeutic Recombinant Monoclonal Antibodies

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to

  9. Progress in Many Body Theory with the Equation of Motion method. Time dependent Density Matrix meets Self-Consistent RPA. Applications to solvable Models

    Schuck, Peter

    2016-01-01

    The Bogoliubov-Born-Green-Kirkwood-Yvon or Time-Dependent Density Matrix (TDDM) hierarchy of equations for higher density matrices is truncated at the three body level in approximating the three body correlation function by a quadratic form of two body ones, closing the equations in this way. The procedure is discussed in detail and it is shown in non-trivial model cases that the approximate inclusion of three body correlation functions is very important to obtain precise results. A small amplitude approximation of this time dependent nonlinear equation for the two body correlation function is performed (STDDM*-b) and it is shown that the one body sector of this generalised non-linear second RPA equation is equivalent to the Self-Consistent RPA (SCRPA) approach which had been derived previously by different techniques. It is discussed in which way SCRPA also contains the three body correlations. TDDM and SCRPA are tested versus exactly solvable model cases.

  10. Experimental investigation of hole boring and light sail regimes of RPA by varying laser and target parameters

    Temporal evolution of plasma jets from micrometre-scale thick foils following the interaction of intense (3 × 1020 W cm−2) laser pulses is studied systematically by time resolved optical interferometry. The fluid velocity in the plasma jets is determined by comparing the data with 2D hydrodynamic simulation, which agrees with the expected hole-boring (HB) velocity due to the laser radiation pressure. The homogeneity of the plasma density across the jets has been found to be improved substantially when irradiating the laser at circular polarization compared to linear polarization. While overdense plasma jets were formed efficiently for micrometre thick targets, decreasing the target areal density and/or increasing the irradiance on the target have provided indication of transition from the ‘HB’ to the ‘light sail (LS)’ regime of RPA, characterized by the appearance of narrow-band spectral features at several MeV/nucleon in proton and carbon spectra. (paper)

  11. Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination

    Gao, Min; Wei, Wei; Li, Ming Hua; Wu, Yong-Sheng; Ba, Zhaoqing; Jin, Kang-Xuan; Li, Miao-Miao; Liao, You-Qi; Adhikari, Samir; Chong, Zechen; Zhang, Ting; Guo, Cai-Xia; Tang, Tie-Shan; Zhu, Bing-Tao; Xu, Xing-Zhi; Mailand, Niels; Yang, Yun-Gui; Qi, Yijun; Danielsen, Jannie Michaela Rendtlew

    2014-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic lesions and pose a major threat to genome stability if not properly repaired. We and others have previously shown that a class of DSB-induced small RNAs (diRNAs) is produced from sequences around DSB sites. DiRNAs are associated with Argonaute...... resection as well as RPA and Mre11 loading is unaffected by Ago2 or Dicer depletion, suggesting that Ago2 very likely functions directly in mediating Rad51 accumulation at DSBs. Taken together, our findings suggest that guided by diRNAs, Ago2 can promote Rad51 recruitment and/or retention at DSBs to...

  12. Kinetic theory of spin-polarized systems in electric and magnetic fields with spin-orbit coupling: II. RPA response functions and collective modes

    Morawetz, K.

    2015-01-01

    The spin and density response functions in the random phase approximation (RPA) are derived by linearizing the kinetic equation including a magnetic field, the spin-orbit coupling, and mean fields with respect to an external electric field. Different polarization functions appear describing various precession motions showing Rabi satellites due to an effective Zeeman field. The latter turns out to consist of the mean-field magnetization, the magnetic field, and the spin-orbit vector. The coll...

  13. Recombination and chromosome segregation.

    Sherratt, David J.; Søballe, Britta; Barre, François-Xavier; Filipe, Sergio; Lau, Ivy; Massey, Thomas; Yates, James

    2004-01-01

    The duplication of DNA and faithful segregation of newly replicated chromosomes at cell division is frequently dependent on recombinational processes. The rebuilding of broken or stalled replication forks is universally dependent on homologous recombination proteins. In bacteria with circular chromosomes, crossing over by homologous recombination can generate dimeric chromosomes, which cannot be segregated to daughter cells unless they are converted to monomers before cell division by the con...

  14. Minutes and group memories from all NERBC/USGS-RPA power plant siting task force meetings through October, 1980. Appendix

    The New England River Basins Commission/United States Geological Survey-Resource Planning Analysis Office (NERBC/USGS-RPA) Power Plant Siting Task Force has formerly met seven times between July 1979 and August 1980. At the first meeting on July 13, 1979, the members agreed that there were many problems with the current process of selecting sites for power plants in New England, and that they would work by consensus to find solutions for these problems. At the second meeting on October 19, 1979, NERBC staff presented information on the site selection and approval processes in New England. The Task Force began a preliminary discussion of problems in these processes, and agreed that the initial scope of work of the Task Force would focus on issues in site selection. At the third meeting on January 18, 1980, the Task Force began initial discussions in three areas: imperfections in the site selection process, stakeholders in the site selection process, and principles to guide solutions to the problems in site selection. On March 7, 1980, at the fourth meeting, the Task Force continued discussions on imperfections, stakeholders, and principles. At the fifth meeting on May 2, 1980, the Task Force reached a wide range of agreements on the difficulties encountered in the site selection process and on the principles guiding problem solving in site selection. At the sixth meeting on May 29, 1980, the Task Force focused on solutions to the problems identified at earlier meetings. Groups of Task Force members constructed eight different scenarios describing alternative power plant siting processes. In July 1980, the Task Force met for the seventh time and refined the eight scenarios, paring them down to five. An attempt was made to develop two scenarios using the common elements from the five. One of these two graphic models was based on government involvement in the site selection process, and the other was based on stakeholder involvement in the process

  15. DNA rearrangement mediated by inverted repeats.

    Bi, X.; Liu, L. F.

    1996-01-01

    Inverted repeats of DNA are widespread in the genomes of eukaryotes and prokaryotes and can mediate genome rearrangement. We studied rearrangement mediated by plasmid-borne inverted repeats in Escherichia coli. We show that inverted repeats can mediate an efficient and recA-independent recombination event. Surprisingly, the product of this recombination is not that of simple inversion between the inverted repeats, but almost exclusively an unusual head-to-head dimer with complex DNA rearrange...

  16. Do avian mitochondria recombine?

    Berlin, Sofia; Smith, Nick G C; Ellegren, Hans

    2004-02-01

    The dogma of strict maternal inheritance of mitochondria is now being tested with population genetics methods on sequence data from many species. In this study we investigated whether recombination occurs in the mitochondria of the blue tit ( Parus caeruleus) by studying polymorphisms in the mitochondrial control region and in a recently identified (A)(n) microsatellite on the W chromosome. The female heterogamety of avian sex chromosomes allows a test of whether mitochondrial recombination affects genealogical inference by comparison of mitochondrial and W-linked sequence variation. There is no discrepancy between mitochondrial and W-linked genealogies in blue tits, consistent with no recombination. We also analyzed mitochondrial sequence variation in both blue tits and peregrine falcons ( Falco peregrinus) using a coalescent-based approach which accounts for recurrent mutation; in neither bird species did we find evidence of recombination. We conclude that it is unlikely that mitochondrial recombination has large effects on mitochondrial genetic variability in birds. PMID:15042336

  17. Regulation of Homologous Recombination by SUMOylation

    Pinela da Silva, Sonia Cristina

    , deletions, and genome rearrangements that can lead to cell death or cancer in humans. The post-translational modification by SUMO (small ubiquitinlike modifier) has proven to be an important regulator of HR and genome integrity, but the molecular mechanisms responsible for these roles are still unclear. In...... this study I present new insights for the role of SUMOylation in regulating HR by dissecting the role of SUMO in the interaction between the central HR-mediator protein Rad52 and its paralogue Rad59 and the outcome of recombination. This data provides evidence for the importance of SUMO in promoting...... protein-protein interactions at the sites of repair, enabling effective Rad51-mediated recombination through the concerted action of the Rad52-Rad59 complex and the helicase Srs2. In addition, I also peer into the role of Rad52 SUMOylation in the context of persistent DSBs and telomere homeostasis...

  18. Lambda Red Mediated Gap Repair Utilizes a Novel Replicative Intermediate in Escherichia coli

    Reddy, Thimma R.; Fevat, Léna M. S.; Munson, Sarah E.; Stewart, A. Francis; Cowley, Shaun M.

    2015-01-01

    The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed recombineering. Red mediated insertion of DNA requires DNA replication, involves a single-stranded DNA intermediate and is more efficient on the lagging strand of the replication fork. Lagging strand recombination has also been postulated to explain the Red mediated repair of gapped plasmids by an Okazaki fragment gap filling ...

  19. Resolution-of-identity approach to Hartree-Fock, hybrid density functionals, RPA, MP2 and GW with numeric atom-centered orbital basis functions

    The efficient implementation of electronic structure methods is essential for first principles modeling of molecules and solids. We present here a particularly efficient common framework for methods beyond semilocal density-functional theory (DFT), including Hartree-Fock (HF), hybrid density functionals, random-phase approximation (RPA), second-order Møller-Plesset perturbation theory (MP2) and the GW method. This computational framework allows us to use compact and accurate numeric atom-centered orbitals (NAOs), popular in many implementations of semilocal DFT, as basis functions. The essence of our framework is to employ the ‘resolution of identity (RI)’ technique to facilitate the treatment of both the two-electron Coulomb repulsion integrals (required in all these approaches) and the linear density-response function (required for RPA and GW). This is possible because these quantities can be expressed in terms of the products of single-particle basis functions, which can in turn be expanded in a set of auxiliary basis functions (ABFs). The construction of ABFs lies at the heart of the RI technique, and we propose here a simple prescription for constructing ABFs which can be applied regardless of whether the underlying radial functions have a specific analytical shape (e.g. Gaussian) or are numerically tabulated. We demonstrate the accuracy of our RI implementation for Gaussian and NAO basis functions, as well as the convergence behavior of our NAO basis sets for the above-mentioned methods. Benchmark results are presented for the ionization energies of 50 selected atoms and molecules from the G2 ion test set obtained with the GW and MP2 self-energy methods, and the G2-I atomization energies as well as the S22 molecular interaction energies obtained with the RPA method. (paper)

  20. Human XPC-hHR23B interacts with XPA-RPA in the recognition of triplex-directed psoralen DNA interstrand crosslinks

    Thoma, Brian S; Wakasugi, Mitsuo; Christensen, Jesper; Reddy, Madhava C; Vasquez, Karen M

    2005-01-01

    DNA interstrand crosslinks (ICLs) represent a severe form of damage that blocks DNA metabolic processes and can lead to cell death or carcinogenesis. The repair of DNA ICLs in mammals is not well characterized. We have reported previously that a key protein complex of nucleotide excision repair...... recognizes psoralen ICLs, which have a structure fundamentally different from other lesions that XPC-hHR23B is known to bind, with high affinity and specificity. XPC-hHR23B and XPA-RPA protein complexes were also observed to bind psoralen ICLs simultaneously, demonstrating not only that psoralen ICLs are...

  1. Arabidopsis thaliana mutants altered in homologous?recombination

    Masson, Jean E.; Paszkowski, Jerzy

    1997-01-01

    Homologous recombination contributes both to the generation of allelic diversity and to the preservation of genetic information. In plants, a lack of suitable experimental material has prevented studies of the regulatory and enzymatic aspects of recombination in somatic and meiotic cells. We have isolated nine Arabidopsis thaliana mutants hypersensitive to x-ray irradiation (xrs) and examined their recombination properties. For the three xrs loci described here, single recessive mutations were found to confer simultaneous hypersensitivities to the DNA-damaging chemicals mitomycin C (MMCs) and/or methyl methanesulfonate (MMSs) and alterations in homologous recombination. Mutant xrs9 (Xrays, MMSs) is reduced in both somatic and meiotic recombination and resembles yeast mutants of the rad52 epistatic group. xrs11 (Xrays, MMCs) is deficient in the x-ray-mediated stimulation of homologous recombination in somatic cells in a manner suggesting a specific signaling defect. xrs4 (Xrays, MMSs, MMCs) has a significant deficiency in somatic recombination, but this is accompanied by meiotic hyper-recombination. A corresponding phenotype has not been reported in other systems and thus this indicates a novel, plant-specific regulatory circuit linking mitotic and meiotic recombination. PMID:9326679

  2. Recombinant Baculovirus Isolation.

    King, Linda A; Hitchman, Richard; Possee, Robert D

    2016-01-01

    Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3 ), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac(®) or BaculoDirect™, Invitrogen) or virus genome and transfer vector. In the latter case, this allows the natural process of homologous recombination to transfer the foreign gene, under control of the polyhedrin or other baculovirus gene promoter, from the transfer vector to the virus genome to create the recombinant virus. Previously, many methods required a plaque-assay to separate parental and recombinant virus prior to amplification and use of the recombinant virus. Fortunately, this step is no longer required for most systems currently available. This chapter provides an overview of the historical development of increasingly more efficient systems for the isolation of recombinant baculoviruses (Chapter 3 provides a full account of the different systems and transfer vectors available). The practical details cover: transfection of insect cells with either virus DNA or virus DNA and plasmid transfer vector; a reliable plaque-assay method that can be used to separate recombinant virus from parental (nonrecombinant) virus where this is necessary; methods for the small-scale amplification of recombinant virus; and subsequent titration by plaque-assay or real-time polymerase chain reaction (PCR). Methods unique to the Bac-to-Bac(®) system are also covered and include the transformation of bacterial cells and isolation of bacmid DNA ready for transfection of insect cells. PMID:26820854

  3. Excitation energy and angular momentum dependence of nuclear level densities and spin cut-off factor in SPA and SPA + RPA approaches

    We investigate the excitation energy (E*) and angular momentum (J) dependence of nuclear level density and spin cut-off factor (σ) within microscopic approaches based on SPA and its extension SPA+RPA representation of the grand partition function for quadrupole-quadrupole interaction model Hamiltonian. For 110Sn, we find that excitation energy dependence of the total level density obtained within these approaches is significantly different. On the other hand, these approaches yield similar behaviour for J-dependence of the level density at fixed values of E*. Values of σSPA+RPA at low E* are found to be slightly smaller than σSPA but they tend to become almost the same at higher E* (>30 MeV). We also find that Bethe's formula for fixed-J level density based on the spin cut-off approximation can be used to compute ρ(E*,J) near the yrast line provided one uses an appropriate value of the spin cut-off factor. (orig.)

  4. Regulation of Meiotic Recombination

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system for assaying recombination using tetrad analysis in a higher eukaryotic system (6). This system enabled the measurement of the frequency and distribution of recombination events at a genome wide level in wild type Arabidopsis (7), construction of genetic linkage maps which include positions for each centromere (8), and modeling of the strength and pattern of interference (9). This proposal extends the use of tetrad analysis in Arabidopsis by using it as the basis for assessing the phenotypes of mutants in genes important for recombination and the regulation of crossover interference and performing a novel genetic screen. In addition to broadening our knowledge of a classic genetic problem - the regulation of recombination by crossover interference - this proposal also provides broader impact by: generating pedagogical tools for use in hands-on classroom experience with genetics, building interdisciplinary collegial partnerships, and creating a platform for participation by junior scientists from underrepresented groups. There are three specific aims: (1) Isolate mutants in Arabidopsis MUS81 homologs using T-DNA and TILLING (2) Characterize recombination levels and interference in mus81 mutants (3) Execute a novel genetic screen, based on tetrad analysis, for genes that regulate meiotic recombination

  5. A Feed-Forward Loop Consisting of the Response Regulator RpaB and the Small RNA PsrR1 Controls Light Acclimation of Photosystem I Gene Expression in the Cyanobacterium Synechocystis sp. PCC 6803.

    Kadowaki, Taro; Nagayama, Ryuta; Georg, Jens; Nishiyama, Yoshitaka; Wilde, Annegret; Hess, Wolfgang R; Hihara, Yukako

    2016-04-01

    Since cyanobacteria need to decrease PSI content to avoid absorption of excess light energy, down-regulation of PSI gene expression is one of the key characteristics of the high-light (HL) acclimation response. The transcriptional regulator RpaB and the small RNA PsrR1 (photosynthesis regulatory RNA1) have been suggested to be the two most critical factors for this response inSynechocystissp. PCC 6803. In this study, we found that the HLR1 DNA-binding motif, the recognition sequence for RpaB, is highly conserved in the core promoter region of thepsrR1gene among cyanobacterial species. Gel mobility shift assay revealed that RpaB binds to the HLR1 sequence ofpsrR1in vitro. RNA gel blot analysis together with chromatin affinity purification (ChAP) analysis suggested that PSI genes are activated and thepsrR1gene is repressed by the binding of RpaB under low-light (LL) conditions. A decrease in DNA binding affinity of RpaB occurs within 5 min after the shift from LL to HL conditions, leading to the prompt decrease in PSI promoter activity together with derepression ofpsrR1gene expression. Accumulating PsrR1 molecules then prevent translation from pre-existing PSI transcripts. By this dual repression at transcriptional and post-transcriptional levels, rapid and strict down-regulation of PSI expression under HL is secured. Our findings suggest that RpaB and PsrR1 constitute a feed-forward loop for the regulation of PSI gene expression to achieve a rapid acclimation response to the damaging HL conditions. PMID:26872833

  6. Generation and Characterization of Human Monoclonal Antibodies Targeting Anthrax Protective Antigen following Vaccination with a Recombinant Protective Antigen Vaccine.

    Chi, Xiangyang; Li, Jianmin; Liu, Weicen; Wang, Xiaolin; Yin, Kexin; Liu, Ju; Zai, Xiaodong; Li, Liangliang; Song, Xiaohong; Zhang, Jun; Zhang, Xiaopeng; Yin, Ying; Fu, Ling; Xu, Junjie; Yu, Changming; Chen, Wei

    2015-05-01

    The anthrax protective antigen (PA) is the central component of the three-part anthrax toxin, and it is the primary immunogenic component in the approved AVA anthrax vaccine and the "next-generation" recombinant PA (rPA) anthrax vaccines. Animal models have indicated that PA-specific antibodies (AB) are sufficient to protect against infection with Bacillus anthracis. In this study, we investigated the PA domain specificity, affinity, mechanisms of neutralization, and synergistic effects of PA-specific antibodies from a single donor following vaccination with the rPA vaccine. Antibody-secreting cells were isolated 7 days after the donor received a boost vaccination, and 34 fully human monoclonal antibodies (hMAb) were identified. Clones 8H6, 4A3, and 22F1 were able to neutralize lethal toxin (LeTx) both in vitro and in vivo. Clone 8H6 neutralized LeTx by preventing furin cleavage of PA in a dose-dependent manner. Clone 4A3 enhanced degradation of nicked PA, thereby interfering with PA oligomerization. The mechanism of 22F1 is still unclear. A fourth clone, 2A6, that was protective only in vitro was found to be neutralizing in vivo in combination with a toxin-enhancing antibody, 8A7, which binds to domain 3 of PA and PA oligomers. These results provide novel insights into the antibody response elicited by the rPA vaccine and may be useful for PA-based vaccine and immunotherapeutic cocktail design. PMID:25787135

  7. The 32 kDa subunit of replication protein A (RPA) participates in the DNA replication of Mung bean yellow mosaic India virus (MYMIV) by interacting with the viral Rep protein

    Singh, Dharmendra Kumar; Islam, Mohammad Nurul; Choudhury, Nirupam Roy; Karjee, Sumona; Mukherjee, Sunil Kumar

    2006-01-01

    Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stemloop structure of the replication origin is crucial. Hence the role of host single-stranded DNA-binding protein, Replication protein A (RPA), in the RCR of MYMIV was examined. Two RPA...

  8. Illegitimate V(D)J recombination-mediated deletions in Notch1 and Bcl11b are not sufficient for extensive clonal expansion and show minimal age or sex bias in frequency or junctional processing

    Champagne, Devin P., E-mail: devin.champagne@uvm.edu; Shockett, Penny E., E-mail: pshockett@selu.edu

    2014-03-15

    Highlights: • Examines illegitimate V(D)J deletion junctions in Notch1 and Bcl11b. • Suggests little influence of deletions alone on clonal outgrowth in wild-type mice. • No age or sex biases in frequency, clonality, or junctional processing observed. • Contrasts with previous results at TCRβ and HPRT1 loci. • Deletions in Bcl11b may be tolerated more easily than those in Notch1. - Abstract: Illegitimate V(D)J recombination at oncogenes and tumor suppressor genes is implicated in formation of several T cell malignancies. Notch1 and Bcl11b, genes involved in developing T cell specification, selection, proliferation, and survival, were previously shown to contain hotspots for deletional illegitimate V(D)J recombination associated with radiation-induced thymic lymphoma. Interestingly, these deletions were also observed in wild-type animals. In this study, we conducted frequency, clonality, and junctional processing analyses of Notch1 and Bcl11b deletions during mouse development and compared results to published analyses of authentic V(D)J rearrangements at the T cell receptor beta (TCRβ) locus and illegitimate V(D)J deletions observed at the human, nonimmune HPRT1 locus not involved in T cell malignancies. We detect deletions in Notch1 and Bcl11b in thymic and splenic T cell populations, consistent with cells bearing deletions in the circulating lymphocyte pool. Deletions in thymus can occur in utero, increase in frequency between fetal and postnatal stages, are detected at all ages examined between fetal and 7 months, exhibit only limited clonality (contrasting with previous results in radiation-sensitive mouse strains), and consistent with previous reports are more frequent in Bcl11b, partially explained by relatively high Recombination Signal Information Content (RIC) scores. Deletion junctions in Bcl11b exhibit greater germline nucleotide loss, while in Notch1 palindromic (P) nucleotides are more abundant, although average P nucleotide length is similar for both genes and consistent with results at the TCRβ locus. Non-templated (N) nucleotide insertions appear to increase between fetal and postnatal stages for Notch1, consistent with normal terminal deoxynucleotidyl transferase (TdT) activity; however, neonatal Bcl11b junctions contain elevated levels of N insertions. Finally, contrasting with results at the HPRT1 locus, we find no obvious age or gender bias in junctional processing, and inverted repeats at recessed coding ends (P{sub r} nucleotides) correspond mostly to single-base additions consistent with normal TdT activity.

  9. Illegitimate V(D)J recombination-mediated deletions in Notch1 and Bcl11b are not sufficient for extensive clonal expansion and show minimal age or sex bias in frequency or junctional processing

    Highlights: • Examines illegitimate V(D)J deletion junctions in Notch1 and Bcl11b. • Suggests little influence of deletions alone on clonal outgrowth in wild-type mice. • No age or sex biases in frequency, clonality, or junctional processing observed. • Contrasts with previous results at TCRβ and HPRT1 loci. • Deletions in Bcl11b may be tolerated more easily than those in Notch1. - Abstract: Illegitimate V(D)J recombination at oncogenes and tumor suppressor genes is implicated in formation of several T cell malignancies. Notch1 and Bcl11b, genes involved in developing T cell specification, selection, proliferation, and survival, were previously shown to contain hotspots for deletional illegitimate V(D)J recombination associated with radiation-induced thymic lymphoma. Interestingly, these deletions were also observed in wild-type animals. In this study, we conducted frequency, clonality, and junctional processing analyses of Notch1 and Bcl11b deletions during mouse development and compared results to published analyses of authentic V(D)J rearrangements at the T cell receptor beta (TCRβ) locus and illegitimate V(D)J deletions observed at the human, nonimmune HPRT1 locus not involved in T cell malignancies. We detect deletions in Notch1 and Bcl11b in thymic and splenic T cell populations, consistent with cells bearing deletions in the circulating lymphocyte pool. Deletions in thymus can occur in utero, increase in frequency between fetal and postnatal stages, are detected at all ages examined between fetal and 7 months, exhibit only limited clonality (contrasting with previous results in radiation-sensitive mouse strains), and consistent with previous reports are more frequent in Bcl11b, partially explained by relatively high Recombination Signal Information Content (RIC) scores. Deletion junctions in Bcl11b exhibit greater germline nucleotide loss, while in Notch1 palindromic (P) nucleotides are more abundant, although average P nucleotide length is similar for both genes and consistent with results at the TCRβ locus. Non-templated (N) nucleotide insertions appear to increase between fetal and postnatal stages for Notch1, consistent with normal terminal deoxynucleotidyl transferase (TdT) activity; however, neonatal Bcl11b junctions contain elevated levels of N insertions. Finally, contrasting with results at the HPRT1 locus, we find no obvious age or gender bias in junctional processing, and inverted repeats at recessed coding ends (Pr nucleotides) correspond mostly to single-base additions consistent with normal TdT activity

  10. Dielectronic recombination at nebular temperatures

    Rate coefficients for dielectronic recombination via low-lying resonance states are calculated for the recombined ions C+, C2+, N2+, N3+ and O4+ at the temperatures and densities appropriate to planetary nebulae. The total dielectronic recombination coefficients obtained are substantially larger than the corresponding radiative recombination coefficients, calculated neglecting resonances, and differ considerably from the dielectronic recombination coefficients generally in use. Effective recombination coefficients are given for spectrum lines that are formed during the cascade process. Some of these lines have been observed in IUE spectra of planetary nebulae. (author)

  11. Cre-/IoxP-Mediated Recombination between the SIL and SCL Genes Leads to a Block in T-Cell Development at the CD4-CD8- to CD4+CD8+ Transition

    Yue Cheng

    2007-04-01

    Full Text Available In the most common form of stem cell leukemia (SCL gene rearrangement, an interstitial deletion of 82 kb brings SCL under the control of regulatory elements that normally govern expression of the ubiquitously expressed SCL interrupting locus (SIL gene, which is located directly upstream of SCL. To investigate the effect of this fusion in a mouse model, a bacterial artificial chromosome (BAC clone containing both human SIL and SCL genes was isolated, and IoxP sites were inserted into intron 1 of both the SIL and SCL genes, corresponding to the sites at which recombination occurs in human T-cell acute lymphocytic leukemia patients. This BAC clone was used to generate transgenic SILIoxloxSCL mice. These transgenic mice were subsequently bred to Lck-Cre mice that express the Cre recombinase specifically in the thymus. The BAC transgene was recombined between the two IoxP sites in over 50% of the thymocytes from SILIoxloxSCL/Cre double-transgenic mice, bringing the SCL gene under the direct control of SIL regulatory elements. Aberrant SCL gene expression in the thymus was verified by reverse transcription- polymerase chain reaction. Using FACS analysis, we found that mice carrying both SILIoxloxSCL and Cre transgenes have increased CD4-/CD8- thymocytes compared with transgenenegative mice. In the spleen, these transgenic mice show a marked reduction in the number of mature CD4+ or CD8+ cells. These results demonstrate that conditional activation of SCL under control of SIL regulatory elements can impair normal T-cell development.

  12. Dissociative recombination of NH+

    We have experimentally investigated dissociative recombination of NH+ with electrons using a merged ion and electron beam configuration in a storage ring. A fast counting and position sensitive imaging detector enabled us to perform fragment imaging measurements over relative electron-ion collision energies from 0 to 12 eV. The results show unprecedented details on product excitation and on the reaction dynamics.

  13. Recombination Phenotypes of Escherichia coli greA Mutants

    Poteete Anthony R

    2011-03-01

    Full Text Available Abstract Background The elongation factor GreA binds to RNA polymerase and modulates transcriptional pausing. Some recent research suggests that the primary role of GreA may not be to regulate gene expression, but rather, to promote the progression of replication forks which collide with RNA polymerase, and which might otherwise collapse. Replication fork collapse is known to generate dsDNA breaks, which can be recombinogenic. It follows that GreA malfunction could have consequences affecting homologous recombination. Results Escherichia coli mutants bearing substitutions of the active site acidic residues of the transcription elongation factor GreA, D41N and E44K, were isolated as suppressors of growth inhibition by a toxic variant of the bacteriophage lambda Red-beta recombination protein. These mutants, as well as a D41A greA mutant and a greA deletion, were tested for proficiency in recombination events. The mutations were found to increase the efficiency of RecA-RecBCD-mediated and RecA-Red-mediated recombination, which are replication-independent, and to decrease the efficiency of replication-dependent Red-mediated recombination. Conclusion These observations provide new evidence for a role of GreA in resolving conflicts between replication and transcription.

  14. RNA-RNA recombination in plant virus replication and evolution.

    Sztuba-Soli?ska, Joanna; Urbanowicz, Anna; Figlerowicz, Marek; Bujarski, Jozef J

    2011-01-01

    RNA-RNA recombination is one of the strongest forces shaping the genomes of plant RNA viruses. The detection of recombination is a challenging task that prompted the development of both in vitro and in vivo experimental systems. In the divided genome of Brome mosaic virus system, both inter- and intrasegmental crossovers are described. Other systems utilize satellite or defective interfering RNAs (DI-RNAs) of Turnip crinkle virus, Tomato bushy stunt virus, Cucumber necrosis virus, and Potato virus X. These assays identified the mechanistic details of the recombination process, revealing the role of RNA structure and proteins in the replicase-mediated copy-choice mechanism. In copy choice, the polymerase and the nascent RNA chain from which it is synthesized switch from one RNA template to another. RNA recombination was found to mediate the rearrangement of viral genes, the repair of deleterious mutations, and the acquisition of nonself sequences influencing the phylogenetics of viral taxa. The evidence for recombination, not only between related viruses but also among distantly related viruses, and even with host RNAs, suggests that plant viruses unabashedly test recombination with any genetic material at hand. PMID:21529157

  15. Kinetic theory of spin-polarized systems in electric and magnetic fields with spin-orbit coupling: II. RPA response functions and collective modes

    Morawetz, K

    2015-01-01

    The spin and density response functions in the random phase approximation (RPA) are derived by linearizing the kinetic equation including a magnetic field, the spin-orbit coupling, and mean fields with respect to an external electric field. Different polarization functions appear describing various precession motions showing Rabi satellites due to an effective Zeeman field. The latter turns out to consist of the mean-field magnetization, the magnetic field, and the spin-orbit vector. The collective modes for charged and neutral systems are derived and a threefold splitting of the spin waves dependent on the polarization and spin-orbit coupling is shown. The dielectric function including spin-orbit coupling, polarization and magnetic fields is presented analytically for long wave lengths and in the static limit. The dynamical screening length as well as the long-wavelength dielectric function shows an instability in charge modes, which are interpreted as spin segregation and domain formation. The spin response...

  16. TDDFT and RPA for mesoscopic systems with thousands to millions of electrons: understating the red-shift in silver clusters absorption around 5 nm

    Neuhauser, Daniel

    2014-03-01

    Two quantum approaches for describing mesoscopic quantum systems with TDDFT will be described: The first, in collaboration with G. Lu and colleagues, is a single-orbital Madelung-like TDDFT propagation incorporating the correct homogenous electron gas dependence of the susceptibility on frequency, wavevector and density. We used this approach to understand the red-shift in the absorption of silver clusters around 5nm diameters. The second, in collaboration with R. Baer and E. Rabani, obtains the exact TDDFT and RPA results by stochastic averaging where the system's time-dependent density and potential is obtained by propagating small set of randomly chosen stochastic-orbitals, each of which is initially a random combination of the system's occupied orbitals. For large systems ~ 10-40 orbitals are sufficient to get the correct dynamics regardless of the number of electrons.

  17. Recombinational DNA repair and human disease

    We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in the ataxia telangiectasia mutated (ATM), NBS, and Mre11 genes, respectively. These genes are critical in maintaining cellular resistance to ionizing radiation (IR), which kills largely by the production of double-strand breaks (DSBs). Bloom syndrome involves a defect in the BLM helicase, which seems to play a role in restarting DNA replication forks that are blocked at lesions, thereby promoting chromosome stability. The Werner syndrome gene (WRN) helicase, another member of the RecQ family like BLM, has very recently been found to help mediate homologous recombination. Fanconi anemia (FA) is a genetically complex chromosomal instability disorder involving seven or more genes, one of which is BRCA2. FA may be at least partially caused by the aberrant production of reactive oxidative species. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in HRR; BRCA2 associates with Rad51 and appears to regulate its activity. We discuss in detail the phenotypes of the various mutant cell lines and the signaling pathways mediated by the ATM kinase. ATM's phosphorylation targets can be grouped into oxidative stress-mediated transcriptional changes, cell cycle checkpoints, and recombinational repair. We present the DNA damage response pathways by using the DSB as the prototype lesion, whose incorrect repair can initiate and augment karyotypic abnormalities

  18. 3D Radiation Therapy Boost Improves the Outcome of Whole Brain Radiation Therapy Treated RPA II Patients with One or Two Brain Metastases

    Delphine Antoni

    2014-05-01

    Full Text Available Purpose: to evaluate the role of whole brain radiotherapy (WBRT and radiation boost (RB for 208 patients recursive partitioning analysis (RPA II with 1 or 2 brain metastases (BM at a single institution. Methods and Materials: the dose of WBRT was 30 Gy (10 fractions of 3 Gy. One hundred thirty-two patients (63.5% benefited from RB of 9 Gy in 3 fractions of 3 Gy at the metastatic site. Patients had 1 or 2 BM in 122 (58.7% and 86 cases (41.3%, respectively. Results: patients with one or two metastases had similar survival (4.6 and 5.1 months, respectively (p = 0.4. Median overall survival (OS for patients treated with WBRT and RB, and with WBRT alone was 5.9 and 3.7 months, respectively (p = 0.03. The 6-, 12- and 24-month OS rates after WBRT and RB were 48.5%, 25% and 10.6%, respectively, while WBRT alone resulted in OS rates of 34%, 22.4% and 3.2%, respectively (p = 0.03. After WBRT and RB, the 6-, 12- and 24-month local control rates were 92%, 82% and 67%, respectively, while they were 81.2%, 75% and 37.5%, respectively, after WBRT alone (p = 0.03. The 6-, 12- and 24-month brain control rates after WBRT and RB were 88.7%, 75.8% and 62%, respectively, and after WBRT alone they were 78.5%, 59% and 37.7%, respectively (p = 0.03. Conclusion: additional boost delivered with 3D conformal radiotherapy improves local and brain control rates significantly as well as overall survival for RPA II patients with 1 or 2 unresectable BM.

  19. Recombinant Protein Production by In Vivo Polymer Inclusion Display ?

    Grage, Katrin; Peters, Verena; Rehm, Bernd H A

    2011-01-01

    A novel approach to produce purified recombinant proteins was established. The target protein is produced as polyhydroxyalkanoate (PHA) synthase fusion protein, which mediates intracellular formation of PHA inclusions displaying the target protein. After isolation of the PHA inclusions, the pure target protein was released by simple enterokinase digestion.

  20. Auger recombination in sodium-iodide scintillators from first principles

    McAllister, Andrew [Applied Physics Program, University of Michigan, Ann Arbor, Michigan 48109 (United States); berg, Daniel [Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California 94550 (United States); Schleife, Andr [Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Kioupakis, Emmanouil, E-mail: kioup@umich.edu [Department of Materials Science and Engineering, University of Michigan, Ann Arbor, Michigan 48109 (United States)

    2015-04-06

    Scintillator radiation detectors suffer from low energy resolution that has been attributed to non-linear light yield response to the energy of the incident gamma rays. Auger recombination is a key non-radiative recombination channel that scales with the third power of the excitation density and may play a role in the non-proportionality problem of scintillators. In this work, we study direct and phonon-assisted Auger recombination in NaI using first-principles calculations. Our results show that phonon-assisted Auger recombination, mediated primarily by short-range phonon scattering, dominates at room temperature. We discuss our findings in light of the much larger values obtained by numerical fits to z-scan experiments.

  1. Recombinant influenza vaccines.

    Sedova, E S; Shcherbinin, D N; Migunov, A I; Smirnov, Iu A; Logunov, D Iu; Shmarov, M M; Tsybalova, L M; Naroditskiĭ, B S; Kiselev, O I; Gintsburg, A L

    2012-10-01

    This review covers the problems encountered in the construction and production of new recombinant influenza vaccines. New approaches to the development of influenza vaccines are investigated; they include reverse genetics methods, production of virus-like particles, and DNA- and viral vector-based vaccines. Such approaches as the delivery of foreign genes by DNA- and viral vector-based vaccines can preserve the native structure of antigens. Adenoviral vectors are a promising gene-delivery platform for a variety of genetic vaccines. Adenoviruses can efficiently penetrate the human organism through mucosal epithelium, thus providing long-term antigen persistence and induction of the innate immune response. This review provides an overview of the practicability of the production of new recombinant influenza cross-protective vaccines on the basis of adenoviral vectors expressing hemagglutinin genes of different influenza strains. PMID:23346377

  2. Recombinant Human Enterovirus 71

    2004-01-01

    Two human enterovirus 71 (HEV71) isolates were identified from hand, foot and mouth disease patients with genome sequences that had high similarity to HEV71 (>93%) at 5´ UTR, P1, and P2 and coxsackievirus A16 (CV-A16, >85%) at P3 and 3´UTR. Intertypic recombination is likely to have occurred between HEV71 and CV-A16 or an as-yet to be described CV-A16-like virus.

  3. Relativistic dielectronic recombination theory

    Dielectronic recombination (DR) is an inverse Auger process in which a free electron is captured by a recombining ion to form a doubly excited autoionizing state. The subsequent decay of the autoionizing state to a stabilized bound state by emitting photons completes the recombination process. DR is an important recombination process for high temperature plasmas. It can affect the ionization balance and level kinetics of the hot plasmas. In addition, the dielectronic satellite lines observed in the emission spectra are frequently used as plasmas diagnostic tools. In the past decade, intense theoretical and experimental studies on the DR process have been carried out. Most of the earlier theoretical calculations on the DR rate coefficients were done either by using a term average approximation or in LS coupling without including the effects of relativity and configuration interaction. The early experimental investigations were concentrated on few times ionized low-Z ions. Recently, the development of electron beam ion trap (EBIT), electron beam ion source (EBIS) and heavy ion storage ring has become possible to produce very highly-charged heavy ions (e.g. U82+ and Xe53+)and to study the interaction between electrons and these ions. For highly-charged heavy ions, one excepts that the nonrelativistic method would be inadequate and a relativistic treatment is necessary. To meet this challenge we have developed a relativistic package based on the multiconfiguration Dirac-Fock method and have carried out systematic relativistic calculations of DR cross sections and rate coefficients and resonant transfer and excitation cross sections in ion-atom collisions. In this paper, we will briefly discuss the relativistic calculations of atomic structure and transition rates and will focus for attention on the effects of relativity and intermediate coupling on the DR cross sections and rate coefficients

  4. Nonradiative recombination in semiconductors

    Abakumov, VN; Yassievich, IN

    1991-01-01

    In recent years, great progress has been made in the understandingof recombination processes controlling the number of excessfree carriers in semiconductors under nonequilibrium conditions. As a result, it is now possible to give a comprehensivetheoretical description of these processes. The authors haveselected a number of experimental results which elucidate theunderlying physical problems and enable a test of theoreticalmodels. The following topics are dealt with: phenomenological theory ofrecombination, theoretical models of shallow and deep localizedstates, cascade model of carrier captu

  5. Hydrogen--oxygen recombiner

    An apparatus is disclosed for efficiently and safely recombining hydrogen and oxygen gas to form water vapor, the apparatus being particularly adapted for use with a nuclear reactor system in which potentially dangerous hydrogen gas, evolved within the containment vessel during certain postulated accident conditions, can be eliminated. Further, this apparatus also aids in the removal of certain radioactive contaminents from the gases in a containment vessel

  6. Intrachromosomal recombination in plants.

    Peterhans, A; Schlpmann, H; Basse, C; Paszkowski, J

    1990-01-01

    Molecular evidence for intrachromosomal recombination between closely linked DNA repeats within the plant genome is presented. The non-overlapping complementary deletion derivatives of the selectable neomycin phosphotransferase gene (nptII), when intact conferring kanamycin resistance, were inserted into the genome of Nicotiana tabacum. The functional marker gene was restored with frequencies between 10(-4) and 10(-6) per proliferating cell clone. Prolonged tissue culture prior to kanamycin s...

  7. Dielectronic recombination theory

    A theory now in wide use for the calculation of dielectronic recombination cross sections (?DR) and rate coefficients (?DR) was one introduced originally by Feshbach for nuclear physics applications, and then later adapted for atomic scattering problems by Hahn. In the following, we briefly review this theory in a very general form, which allows one to account for the effects of overlapping and interacting resonances, as well as continuum-continuum coupling. An extension of our notation will then also allow for the inclusion of the effects of direct radiative recombination, along with a treatment of the interference between radiative and dielectronic recombination. Other approaches to the calculation of ?DR have been described by Fano and by Seaton. We will not consider those theories here. Calculations of ?DR have progressed considerably over the last 25 years, since the early work of Burgess. Advances in the reliability of theoretical predictions have also been promoted recently b a variety of direct laboratory measurements of ?DR. While the measurements of ?DR for ?n ? 0 excitations have tended to agree very well with calculations, the case of ?n = 0 has been much problematic. However, by invoking a mechanism originally proposed by Jacobs, which takes into account the effect of stray electric fields on high Rydberg states (HRS) participating in the DR process, new calculations have improved the agreement between theory and experiment for these cases. Nevertheless, certain discrepancies still remain

  8. Recombinant Collagenlike Proteins

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  9. Novel Heterotypic Rox Sites for Combinatorial Dre Recombination Strategies

    Katherine Chuang

    2016-03-01

    Full Text Available Site-specific recombinases (SSRs such as Cre are widely used in gene targeting and genetic approaches for cell labeling and manipulation. They mediate DNA strand exchange between two DNA molecules at dedicated recognition sites. Precise understanding of the Cre recombination mechanism, including the role of individual base pairs in its loxP target site, guided the generation of mutant lox sites that specifically recombine with themselves but not with the wild type loxP. This has led to the development of a variety of combinatorial Cre-dependent genetic strategies, such as multicolor reporters, irreversible inversions, or recombination-mediated cassette exchange. Dre, a Cre-related phage integrase that recognizes roxP sites, does not cross-react with the Cre-loxP system, but has similar recombination efficiency. We have previously described intersectional genetic strategies combining Dre and Cre. We now report a mutagenesis screen aimed at identifying roxP base pairs critical for self-recognition. We describe several rox variant sites that are incompatible with roxP, but are able to efficiently recombine with themselves in either purified systems or bacterial and eukaryotic tissue culture systems. These newly identified rox sites are not recognized by Cre, thus enabling potential combinatorial strategies involving Cre, Dre, and target loci including multiple loxP and roxP variants.

  10. Primordial magnetogenesis before recombination

    Fabre, Ophélia

    2015-01-01

    The origin of large magnetic fields in the Universe remains currently unknown. We investigate here a mechanism before recombination based on known physics. The source of the vorticity is due to the changes in the photon distribution function caused by the fluctuations in the background photons. We show that the magnetic field generated in the MHD limit, due to the Coulomb scattering, is of the order $10^{-49}$ G. We explicitly show that the magnetic fields generated from this process are sustainable and are not erased by resistive diffusion. We compare the results with current observations and discuss the implications.

  11. Brh2-Dss1 interplay enables properly controlled recombination in Ustilago maydis

    Kojic, Milorad; Zhou, Qingwen; Lisby, Michael; Holloman, William K

    2005-01-01

    (GFP)-Rad51 foci following DNA damage by gamma radiation. To understand more about the interplay between Brh2 and Dss1, we isolated mutant variants of Brh2 able to bypass the requirement for Dss1. These variants were found to lack the entire C-terminal DNA-Dss1 binding domain but to maintain the N......Brh2, the BRCA2 homolog in Ustilago maydis, functions in recombinational repair of DNA damage by regulating Rad51 and is, in turn, regulated by Dss1. Dss1 is not required for Brh2 stability in vivo, nor for Brh2 to associate with Rad51, but is required for formation of green fluorescent protein...... after DNA damage was almost fully restored by a chimeric form of Brh2 having a DNA-binding domain from RPA70 fused to the Brh2 N-terminal domain, but Rad51 focus formation and mitotic recombination were elevated above wild-type levels. The results provide evidence for a mechanism in which Dss1 activates...

  12. Enteric Immunization of Mice Against Influenza with Recombinant Vaccinia

    Meitin, Catherine A.; Bender, Bradley S.; Small, Parker A., Jr.

    1994-11-01

    Intrajejunal administration to mice of a recombinant vaccinia virus containing the influenza virus hemagglutinin gene induced IgA antibody in nasal, gut, and vaginal secretions. It also induced IgG antibody in serum and cell-mediated immunity. The immunization provided significant protection against an influenza virus challenge. This work suggests that enteric-coated recombinant vaccinia could be an orally administered, inexpensive, multivalent, temperature-stable, safe, and effective vaccine for children that could be particularly useful in developing nations, where multiple injections are not easily administered. Oral administration of vaccines should also reduce children's fear of shots at the doctor's office.

  13. Cell biology of mitotic recombination

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as...... well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this...

  14. Spin dependent recombination

    The spin dependent recombination (SDR) technique is used to observe the 29Si hyperfine spectra of radiation-induced Pb centers at the Si/SiO2 interface in a MOSFET. The Pb center is a paramagnetic, trivalent silicon defect that is the dominant radiation-induced interface state. The 29Si hyperfine spectra give detailed atomic scale information about the Pb center. The authors' SDR results show that the 29Si hyperfine spectra vary with surface potential. This result indicates that differences in the defect's local geometry lead to substantial differences in the defect's energy level. However, the 29Si hyperfine spectra are found to be relatively independent of the ionizing radiation dosage

  15. Intercultural Mediation

    Dragos Marian Radulescu; Denisa Mitrut

    2012-01-01

    The Intercultural Mediator facilitates exchanges between people of different socio-cultural backgrounds and acts as a bridge between immigrants and national and local associations, health organizations, services and offices in order to foster integration of every single individual. As the use mediation increases, mediators are more likely to be involved in cross-cultural mediation, but only the best mediators have the opportunity to mediate cross border business disputes or international poli...

  16. Night-time electron temperature troughs in the equatorial topside ionosphere revealed from RPA experiments on the ISS-b satellite

    Night-time electron temperature (Tsub(e)), electron density (Nsub(e)) and the mean ion mass (Msub(i)) of the topside ionosphere at altitudes of about 1100 km have been obtained from the RPA experiments on ISS-b. Global maps of these quantities are derived by adopting the spherical surface harmonic functions fitted to the satellite data by means of a least-square method. The night-time map of Tsub(e) shows a marked equatorial trough which is limited to a 1000 longitudinal sector and a 200 latitudinal extent. The center of the trough is located a few degrees 'summerward' of the dip equator. The longitude of the trough is strongly controlled by the magnetic declination. This longitude changes rapidly around autumnal equinox from the region of maximum westward declination to that of eastward declination, the opposite change occurs around the time of vernal equinox. The maps of Msub(i) and Nsub(e) in low latitudes have features apparently related to the Tsub(e) trough. These features can be explained in terms of field-aligned interhemispheric plasma flow driven by the neutral air wind. (author)

  17. Detecting variable (V, diversity (D and joining (J gene segment recombination using a two-colour fluorescence system

    Scott Gina B

    2010-03-01

    Full Text Available Abstract Background Diversity of immunoglobulins and the T cell antigen receptors is achieved via the recombination activating gene (RAG-mediated rearrangement of variable (V, diversity (D and joining (J gene segments, and this underpins the efficient recognition of a seemingly limitless array of antigens. Analysis of V(DJ recombination activity is typically performed using extrachromosomal recombination substrates that are recovered from transfected cells and selected using bacterial transformation. We have developed a two-colour fluorescence-based system that simplifies detection of both deletion and inversion joining events mediated by RAG proteins. Results This system employs two fluorescent reporter genes that differentially mark unrearranged substrates and those that have undergone RAG-mediated deletion or inversion events. The recombination products bear the hallmarks of true V(DJ recombination and activity can be detected using fluorescence microscopy or flow cytometry. Recombination events can be detected without the need for cytotoxic selection of recombination products and the system allows analysis of recombination activity using substrates integrated into the genome. Conclusions This system will be useful in the analysis and exploitation of the V(DJ recombination machinery and suggests that similar approaches could be used to replace expression of one gene with another during lymphocyte development.

  18. Delayed recombination and standard rulers

    Measurements of baryonic acoustic oscillations (BAOs) in galaxy surveys have been recognized as a powerful tool for constraining dark energy. However, this method relies on the knowledge of the size of the acoustic horizon at recombination derived from cosmic microwave background (CMB) anisotropy measurements. This estimate is typically derived assuming a standard recombination scheme; additional radiation sources can delay recombination altering the cosmic ionization history and the cosmological inferences drawn from CMB and BAO data. In this paper we quantify the effect of delayed recombination on the determination of dark energy parameters from future BAO surveys such as the Baryon Oscillation Spectroscopic Survey and the Wide-Field Multi-Object Spectrograph. We find the impact to be small but still not negligible. In particular, if recombination is nonstandard (to a level still allowed by CMB data), but this is ignored, future surveys may incorrectly suggest the presence of a redshift-dependent dark energy component. On the other hand, in the case of delayed recombination, adding to the analysis one extra parameter describing deviations from standard recombination does not significantly degrade the error bars on dark energy parameters and yields unbiased estimates. This is due to the CMB-BAO complementarity.

  19. Intercultural Mediation

    Dragos Marian Radulescu

    2012-11-01

    Full Text Available The Intercultural Mediator facilitates exchanges between people of different socio-cultural backgrounds and acts as a bridge between immigrants and national and local associations, health organizations, services and offices in order to foster integration of every single individual. As the use mediation increases, mediators are more likely to be involved in cross-cultural mediation, but only the best mediators have the opportunity to mediate cross border business disputes or international politics conflicts. This article attempts to provide a new perspective about the intercultural mediation.

  20. Iodine chemistry in hydrogen recombiners

    Sabroux, J.C. [IRSN/DSU/SERAC, B.P. No. 68, 91192 Gif-sur-Yvette cedex (France); Deschamps, F. [APTUS, 77 rue des Chantiers, 78000 Versailles (France)

    2005-07-01

    Hydrogen recombiners, recently introduced in the French nuclear reactor buildings, display high temperature (up to about 900 deg. C) and several thousands square meters of a very reactive surface when operating during a severe accident scenario. Small scale analytical experiments show that cesium and cadmium iodides are unstable, and generate volatile iodine, when heated in an oven that reproduces most physico-chemical parameters of recombiner operation. Based on these results, and due to its potential with regard to the environmental source term of a severe accident, iodine chemistry in hydrogen recombiners deserves close and careful scrutiny. (authors)

  1. Iodine chemistry in hydrogen recombiners

    Hydrogen recombiners, recently introduced in the French nuclear reactor buildings, display high temperature (up to about 900 deg. C) and several thousands square meters of a very reactive surface when operating during a severe accident scenario. Small scale analytical experiments show that cesium and cadmium iodides are unstable, and generate volatile iodine, when heated in an oven that reproduces most physico-chemical parameters of recombiner operation. Based on these results, and due to its potential with regard to the environmental source term of a severe accident, iodine chemistry in hydrogen recombiners deserves close and careful scrutiny. (authors)

  2. CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri

    Oh, Jee-Hwan; van Pijkeren, Jan-Peter

    2014-01-01

    Clustered regularly interspaced palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) nuclease protect bacteria and archeae from foreign DNA by site-specific cleavage of incoming DNA. Type-II CRISPR–Cas systems, such as the Streptococcus pyogenes CRISPR–Cas9 system, can be adapted such that Cas9 can be guided to a user-defined site in the chromosome to introduce double-stranded breaks. Here we have developed and optimized CRISPR–Cas9 function in the lactic acid bacterium Lactobacillus reuteri ATCC PTA 6475. We established proof-of-concept showing that CRISPR–Cas9 selection combined with single-stranded DNA (ssDNA) recombineering is a realistic approach to identify at high efficiencies edited cells in a lactic acid bacterium. We show for three independent targets that subtle changes in the bacterial genome can be recovered at efficiencies ranging from 90 to 100%. By combining CRISPR–Cas9 and recombineering, we successfully applied codon saturation mutagenesis in the L. reuteri chromosome. Also, CRISPR–Cas9 selection is critical to identify low-efficiency events such as oligonucleotide-mediated chromosome deletions. This also means that CRISPR–Cas9 selection will allow identification of recombinant cells in bacteria with low recombineering efficiencies, eliminating the need for ssDNA recombineering optimization procedures. We envision that CRISPR–Cas genome editing has the potential to change the landscape of genome editing in lactic acid bacteria, and other Gram-positive bacteria. PMID:25074379

  3. CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri.

    Oh, Jee-Hwan; van Pijkeren, Jan-Peter

    2014-01-01

    Clustered regularly interspaced palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) nuclease protect bacteria and archeae from foreign DNA by site-specific cleavage of incoming DNA. Type-II CRISPR-Cas systems, such as the Streptococcus pyogenes CRISPR-Cas9 system, can be adapted such that Cas9 can be guided to a user-defined site in the chromosome to introduce double-stranded breaks. Here we have developed and optimized CRISPR-Cas9 function in the lactic acid bacterium Lactobacillus reuteri ATCC PTA 6475. We established proof-of-concept showing that CRISPR-Cas9 selection combined with single-stranded DNA (ssDNA) recombineering is a realistic approach to identify at high efficiencies edited cells in a lactic acid bacterium. We show for three independent targets that subtle changes in the bacterial genome can be recovered at efficiencies ranging from 90 to 100%. By combining CRISPR-Cas9 and recombineering, we successfully applied codon saturation mutagenesis in the L. reuteri chromosome. Also, CRISPR-Cas9 selection is critical to identify low-efficiency events such as oligonucleotide-mediated chromosome deletions. This also means that CRISPR-Cas9 selection will allow identification of recombinant cells in bacteria with low recombineering efficiencies, eliminating the need for ssDNA recombineering optimization procedures. We envision that CRISPR-Cas genome editing has the potential to change the landscape of genome editing in lactic acid bacteria, and other Gram-positive bacteria. PMID:25074379

  4. On the evolutionary advantage of fitness-associated recombination.

    Hadany, Lilach; Beker, Tuvik

    2003-12-01

    The adaptive value of recombination remains something of a puzzle. One of the basic problems is that recombination not only creates new and advantageous genetic combinations, but also breaks down existing good ones. A negative correlation between the fitness of an individual and its recombination rate would result in prolonged integrity of fitter genetic combinations while enabling less fit ones to produce new combinations. Such a correlation could be mediated by various factors, including stress responses, age, or direct DNA damage. For haploid population models, we show that an allele for such fitness-associated recombination (FAR) can spread both in asexual populations and in populations reproducing sexually at any uniform recombination rate. FAR also carries an advantage for the population as a whole, resulting in a higher average fitness at mutation-selection balance. These results are demonstrated in populations adapting to new environments as well as in well-adapted populations coping with deleterious mutations. Current experimental results providing evidence for the existence of FAR in nature are discussed. PMID:14704195

  5. Calculation of Gamow-Teller #betta#-strength functions in the Rubidium region in the RPA approximation with Nilsson model wave functions

    We calculate allowed Gamow-Teller and, in a few cases, Fermi #betta#strength functions in a model that is applicable to studies of nuclei throughout the periodic system. For our first study we have selected a sequence of rubidium isotopes, namely 8937Rb - 9937Rb. We develop a model that use calculated Nilsson model wave functions, spherical or deformed, as the case may be, as the starting point for determining the wave functions of the mother and daughter nuclei in the #betta# decay. Pairing is treated in the BCS approximation. To account for the retardation of low-energy GT-decay rates we add, as is customarily done, a simple residual interaction specific to GT decay, namely V sub (GT)=:#betta#-1 x #betta#1:, to the Hamiltonian. This residual interaction is treated in the RPA approximation. The strength of the interaction is adjusted to get agreement between the calculated and experimental energy of the giant Gamow-Teller resonance for 208Pb and 144Sm. Since the present model is based on calculated wave functions and single-particle levels, studies of nuclei far from stability, where little experimental information is available, are more straightforward relative to calculations where experimental levels are used. The model can treat deformed nuclei employing wave functions calculated to desired accuracy, within the framework of the model, for the deformed single-particle well. The calcualtions show that use of single-particle parameters appropriate to the region studied and taking deformation into account is important. We find good agreement between calculated and experimental spectra over the region studied, provided an appropriate choice of single-particle parameters and deformation is made. (Authors)

  6. Stable recombination hotspots in birds.

    Singhal, Sonal; Leffler, Ellen M; Sannareddy, Keerthi; Turner, Isaac; Venn, Oliver; Hooper, Daniel M; Strand, Alva I; Li, Qiye; Raney, Brian; Balakrishnan, Christopher N; Griffith, Simon C; McVean, Gil; Przeworski, Molly

    2015-11-20

    The DNA-binding protein PRDM9 has a critical role in specifying meiotic recombination hotspots in mice and apes, but it appears to be absent from other vertebrate species, including birds. To study the evolution and determinants of recombination in species lacking the gene that encodes PRDM9, we inferred fine-scale genetic maps from population resequencing data for two bird species: the zebra finch, Taeniopygia guttata, and the long-tailed finch, Poephila acuticauda. We found that both species have recombination hotspots, which are enriched near functional genomic elements. Unlike in mice and apes, most hotspots are shared between the two species, and their conservation seems to extend over tens of millions of years. These observations suggest that in the absence of PRDM9, recombination targets functional features that both enable access to the genome and constrain its evolution. PMID:26586757

  7. Mutations affecting RNA polymerase I-stimulated exchange and rDNA recombination in yeast

    HOT1 is a cis-acting recombination-stimulatory sequence isolated from the rDNA repeat unit of yeast. The ability of HOT1 to stimulate mitotic exchange appears to depend on its ability to promote high levels of RNA polymerase I transcription. A qualitative colony color sectoring assay was developed to screen for trans-acting mutations that alter the activity of HOT1. Both hypo-recombination and hyper-recombination mutants were isolated. Genetic analysis of seven HOT1 recombination mutants (hrm) that decrease HOT1 activity shows that they behave as recessive nuclear mutations and belong to five linkage groups. Three of these mutations, hrm1, hrm2, and hrm3, also decrease rDNA exchange but do not alter recombination in the absence of HOT1. Another mutation, hrm4, decreases HOT1-stimulated recombination but does not affect rDNA recombination or exchange in the absence of HOT1. Two new alleles of RAD52 were also isolated using this screen. With regard to HOT1 activity, rad52 is epistatic to all four hrm mutations indicating that the products of the HRM genes and of RAD52 mediate steps in the same recombination pathway. Finding mutations that decrease both the activity of HOT1 and exchange in the rDNA supports the hypothesis that HOT1 plays a role in rDNA recombination

  8. Progenitors of Recombining Supernova Remnants

    Moriya, Takashi J.

    2012-01-01

    Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with the ionization temperature higher than the electron temperature, is recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the time of the superno...

  9. Hydrogen Recombination with Multilevel atoms

    De, Soma; Baron, E.; Peter H. Hauschildt

    2010-01-01

    Hydrogen recombination is one of the most important atomic processes in many astrophysical objects such as Type II supernova (SN~II) atmospheres, the high redshift universe during the cosmological recombination era, and H II regions in the interstellar medium. Accurate predictions of the ionization fraction can be quite different from those given by a simple solution if one takes into account many angular momentum sub-states, non-resonant processes, and calculates the rates of all atomic proc...

  10. Combinatorics in Recombinational Population Genomics

    Parida, Laxmi

    The work that I will discuss is motivated by the need for understanding, and processing, the manifestations of recombination events in chromosome sequences. In this talk, we focus on two related problems. First, we explore the very general problem of reconstructability of pedigree history. How plausible is it to unravel the history of a complete unit (chromosome) of inheritance? The second problem deals with reconstructing the recombinational history of a collection of chromosomes.

  11. Do mitochondria recombine in humans?

    Eyre-Walker, A

    2000-01-01

    Until very recently, mitochondria were thought to be clonally inherited through the maternal line in most higher animals. However, three papers published in 2000 claimed population-genetic evidence of recombination in human mitochondrial DNA. Here I review the current state of the debate. I review the evidence for the two main pathways by which recombination might occur: through paternal leakage and via a mitochondrial DNA sequence in the nuclear genome. There is no strong evidence for either...

  12. Eukaryotes arose after genetic recombination

    Stupar Milanko R.

    2006-01-01

    Full Text Available Division of ancestral prokaryotic pragenome into two circular double-stranded DNA molecules by genetic recombination, is a base for future separate evolution of nuclear and mitochondrial gene compartment. This suggests monophyletic origin of both, mitochondrion and nucleus. Presumed organism which genome undergoes genetic recombination has to be searched among an aerobic, oxygen nonproducing, archaeon with no rigid cell wall, but a plasma membrane. Plastid evolves from an aerobic, oxygen producing protoeukaryote, after mitoplastid genome duplication and subsequent functional segregation.

  13. Delayed recombination and cosmic parameters

    Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, ns, and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z*=1078±11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1σ to R=1.734±0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: εαi<0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.

  14. Targeting the site of RNA-RNA recombination in brome mosaic virus with antisense sequences.

    Nagy, P. D.; Bujarski, J J

    1993-01-01

    It has been postulated that local hybridizations between viral RNAs can mediate recombination in brome mosaic virus (BMV) and in poliovirus. To test this model, a 3' fragment of BMV RNA1 was inserted into the 3' noncoding sequence of BMV RNA3 in an antisense orientation. This resulted in high-frequency nonhomologous crossovers at or near the hybridized region. Insertion of the same RNA1 fragment in a positive-sense orientation did not promote recombination. Modification of the antisense inser...

  15. Double strand interaction is the predominant pathway for intermolecular recombination of adeno-associated viral genomes

    Intermolecular recombination is the foundation for dual vector mediated larger gene transfer by recombinant adeno-associated virus (rAAV). To identify precursors for intermolecular recombination, we sequentially infected skeletal muscle with AAV LacZ trans-splicing viruses. At 1 month postinfection, nearly all inputting single-strand (ss) AAV genomes were cleared out in muscle. If ss-ss interaction is absolutely required for intermolecular recombination, LacZ expression from sequential infection will be negligible to that from coinfection. Interestingly, expression from sequential infection reached ?50% of that from coinfection at the 1-month time-point in BL6 mice. In immune deficient SCID mice, expression from sequential infection was comparable to that from coinfection at the 4- and 13-month time points. Our results suggest that ds interaction represents the predominant pathway for AAV intermolecular recombination

  16. The Slx5-Slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination

    Burgess, Rebecca C; Rahman, Sadia; Lisby, Michael; Rothstein, Rodney; Zhao, Xiaolan

    2007-01-01

    Recombination is important for repairing DNA lesions, yet it can also lead to genomic rearrangements. This process must be regulated, and recently, sumoylation-mediated mechanisms were found to inhibit Rad51-dependent recombination. Here, we report that the absence of the Slx5-Slx8 complex, a newly...... identified player in the SUMO (small ubiquitin-like modifier) pathway, led to increased Rad51-dependent and Rad51-independent recombination. The increases were most striking during S phase, suggesting an accumulation of DNA lesions during replication. Consistent with this view, Slx8 protein localized to...... propose that, during replication, the Slx5-Slx8 complex helps prevent DNA lesions that are acted upon by recombination. In addition, the complex inhibits Rad51-independent recombination via modulating the sumoylation of DNA repair proteins....

  17. Recombining WMAP: Constraints on ionizing and resonance radiation at recombination

    We place new constraints on sources of ionizing and resonance radiation at the epoch of the recombination process using the recent cosmic microwave background temperature and polarization spectra coming from the Wilkinson Microwave Anisotropy Probe (WMAP). We find that non-standard recombination scenarios are still consistent with the current data. In light of this we study the impact that such models can have on the determination of several cosmological parameters. In particular, the constraints on curvature and baryon density appear to be weakly affected by a modified recombination scheme. However, it may affect the current WMAP constraints on inflationary parameters such as the spectral index ns and its running. Physically motivated models, such as those based on primordial black holes or super heavy dark matter decay, are able to provide a good fit to the current data. Future observations in both temperature and polarization will be needed to more stringently test these models

  18. Failure patterns by prognostic group determined by recursive partitioning analysis (RPA) of 1547 patients on four radiation therapy oncology group (RTOG) studies in inoperable nonsmall-cell lung cancer (NSCLC)

    Purpose: To identify groups of patients who might benefit from more aggressive systemic or local treatment, based on failure patterns when unresectable NSCLC was treated by radiation therapy (RT) alone. Methods: From 4 RTOG trials, 1547 patients treated by RT alone were analyzed for patterns of first failure by RPA class defined by prognostic factors, including KPS, weight loss, nodal stage, pleural effusion, age and radiation therapy dose. All patients had NSCLC AJCC Stage II, IIIA, or IIIB, KPS > 50, with no previous RT or chemotherapy. Progressions in the primary (within irradiated fields), thorax (outside irradiated area, but within thorax), brain and distant metastasis other than brain were compared (2-sided) for each failure category by RPA. Results: The RPA classes were 4 distinct subgroups that had significantly different median survivals of 12.6, 8.3, 6.3 and 3.3 months for Classes I, II, III and IV, respectively, (all groups, p = 0.0002). There were 583, 667, 249 and 48 patients in Classes I, II, III and IV, respectively. Primary failure was seen in 27%, 25%, 21% and 10% for Classes I, II, III, and IV, respectively (I vs. IV, p = 0.014; II vs. IV, p = 0.022). Distant metastasis, including brain metastasis, occurred at significantly higher rates among Classes I and II (58% and 54%) than in Classes III and IV (42% and 27%). A higher rate (58%) of death without an identifiable site of failure was found in Class IV than in Classes I, II and III (27%, 28% and 36%, respectively). Conclusions: The data suggest that physiologic compromise from the intrathoracic disease in Class IV patients is sufficient to cause death before specific sites of failure became evident. Clinical investigations using treatments directed at specific sites of failure could lead to improved outcome for Class I, II and, possibly, Class III patients. Inclusion of Class IV patients in clinical trials may obscure outcomes

  19. Failure patterns by prognostic group as determined by recursive partitioning analysis (RPA) of 1547 on four radiation therapy oncology group studies in operable non-small cell lung cancer (NSCLC)

    Purpose: To identify groups of patients who might benefit from more aggressive systemic or local treatment based on failure patterns when unresectable NSCLC was treated by radiation therapy alone. Methods: 1547 patients from 4 RTOG trials treated by RT alone were analyzed for the patterns of first failure by PRA class which was defined by prognostic factors, e.g., stage, KPS, weight loss, pleural effusion, age. All patients were AJCC stage II, IIIA or IIIB with KPS of at least 50 and n previous radiotherapy or chemotherapy for their NSCLC. Progressions in the primary (within irradiated fields), thorax (outside irradiated area), brain and distant metastasis other than brain were compared (two-sided) for each failure category by RPA. Results: The RPA classes are four distinct subgroups that had significantly different median survivals of 12.6, 8.3, 6.2 and 3.3 months for classes I, II, III and IV respectively (all groups p=0.0002). Pair comparison showed that RPA I vs IV p<0.0001, I vs III p=0.006, II vs IV p<0.0001, and III vs IV p=0.06. Conclusions: These results suggest the burden of disease and physiologic compromise in class IV patients are sufficient to cause death before specific sites of failure can be discerned. Site specific treatment strategies (intensive local therapy, combination chemotherapy, prophylactic cranial irradiation) may lead to improved outcome in class I and II, but are unlikely to alter outcome in class III and IV

  20. Inhomogeneous recombinations during cosmic reionization

    Sobacchi, Emanuele

    2014-01-01

    By depleting the ionizing photon budget available to expand cosmic HII regions, recombining systems (or Lyman limit systems) can have a large impact during (and following) cosmic reionization. Unfortunately, directly resolving such structures in large-scale reionization simulations is computationally impractical. Instead, here we implement a sub-grid prescription for tracking inhomogeneous recombinations in the intergalactic medium. Building on previous work parameterizing photo-heating feedback on star-formation, we present large-scale, semi-numeric reionization simulations which self-consistently track the local (sub-grid) evolution of both sources and sinks of ionizing photons. Our simple, single-parameter model naturally results in both an extended reionization and a modest, slowly-evolving emissivity, consistent with observations. Recombinations are instrumental in slowing the growth of large HII regions, and damping the rapid rise of the ionizing background in the late stages of (and following) reioniza...

  1. Dissociative recombination of protonated methanol

    Geppert, Wolf; Hamberg, Mathias; Thomas, Richard D; Österdahl, Fabian; Hellberg, Fredrik; Zhaunerchyk, Vitali; Ehlerding, Anneli; Millar, Tom; Roberts, Helen; Semaniak, Jacek; af Ugglas, Magnus; Källberg, Anders; Simonsson, Ansgar; Kaminska, Magdalena; Larsson, Mats

    2006-01-01

    The branching ratios of the different reaction pathways and the overall rate coefficients of the dissociative recombination reactions of CH3OH2+ and CD3OD2+ have been measured at the CRYRING storage ring located in Stockholm, Sweden. Analysis of the data yielded the result that formation of methanol or deuterated methanol accounted for only 3 and 6% of the total rate in CH3OH2+ and CD3OD2+, respectively. Dissociative recombination of both isotopomeres mainly involves fragmentation of the C–O ...

  2. Selenium incorporation using recombinant techniques

    An overview of techniques for recombinant incorporation of selenium and subsequent purification and crystallization of the resulting labelled protein. Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in prokaryal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included

  3. Lambda red mediated gap repair utilizes a novel replicative intermediate in Escherichia coli.

    Reddy, Thimma R; Fevat, Léna M S; Munson, Sarah E; Stewart, A Francis; Cowley, Shaun M

    2015-01-01

    The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed recombineering. Red mediated insertion of DNA requires DNA replication, involves a single-stranded DNA intermediate and is more efficient on the lagging strand of the replication fork. Lagging strand recombination has also been postulated to explain the Red mediated repair of gapped plasmids by an Okazaki fragment gap filling model. Here, we demonstrate that gap repair involves a different strand independent mechanism. Gap repair assays examining the strand asymmetry of recombination did not show a lagging strand bias. Directly testing an ssDNA plasmid showed lagging strand recombination is possible but dsDNA plasmids did not employ this mechanism. Insertional recombination combined with gap repair also did not demonstrate preferential lagging strand bias, supporting a different gap repair mechanism. The predominant recombination route involved concerted insertion and subcloning though other routes also operated at lower frequencies. Simultaneous insertion of DNA resulted in modification of both strands and was unaffected by mutations to DNA polymerase I, responsible for Okazaki fragment maturation. The lower efficiency of an alternate Red mediated ends-in recombination pathway and the apparent lack of a Holliday junction intermediate suggested that gap repair does not involve a different Red recombination pathway. Our results may be explained by a novel replicative intermediate in gap repair that does not involve a replication fork. We exploited these observations by developing a new recombineering application based on concerted insertion and gap repair, termed SPI (subcloning plus insertion). SPI selected against empty vector background and selected for correct gap repair recombinants. We used SPI to simultaneously insert up to four different gene cassettes in a single recombineering reaction. Consequently, our findings have important implications for the understanding of E. coli replication and Red recombination. PMID:25803509

  4. CRMAGE: CRISPR Optimized MAGE Recombineering.

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Sommer, Morten O A; Nielsen, Alex Toftgaard

    2016-01-01

    A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and ? Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli. Using CRMAGE, the recombineering efficiency was between 96.5% and 99.7% for gene recoding of three genomic targets, compared to between 0.68% and 5.4% using traditional recombineering. For modulation of protein synthesis (small insertion/RBS substitution) the efficiency was increased from 6% to 70%. CRMAGE can be multiplexed and enables introduction of at least two mutations in a single round of recombineering with similar efficiencies. PAM-independent loci were targeted using degenerate codons, thereby making it possible to modify any site in the genome. CRMAGE is based on two plasmids that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the ? Red oligos and the gRNAs. The CRMAGE platform enables highly efficient and fast genome editing and may open up promising prospective for automation of genome-scale engineering. PMID:26797514

  5. KEGG PATHWAY / Homologous recombination [KEGG

    Full Text Available PATHWAY: map03440 Entry map03440Pathway Name Homologous recombination Description Homologous rec ... drome mutated) are tumor suppressors that maintain genome ... integrity, at least in part, through HR. Class Gen ... 2MRX complex [PATH:map03440]M00295BRCA1-associated genome ... surveillance complex (BASC) [PATH:map03440] Diseas ...

  6. Recombination in immunoglobulin gene loci

    Komisarenko S. V.

    2009-02-01

    Full Text Available Gene network of the lymphoid cell differentiation coordinates precisely the recombination process in immunoglobulin gene loci. In our opinion, cellular microRNAs can contribute to the allelic exclusion through microRNA-directed DNA methylation and participate in retargeting recombinases activity from the gene loci of heavy immunoglobulin chains to the gene loci of light chains

  7. Recombination in immunoglobulin gene loci

    Komisarenko S. V.; Halytskiy V. A.

    2009-01-01

    Gene network of the lymphoid cell differentiation coordinates precisely the recombination process in immunoglobulin gene loci. In our opinion, cellular microRNAs can contribute to the allelic exclusion through microRNA-directed DNA methylation and participate in retargeting recombinases activity from the gene loci of heavy immunoglobulin chains to the gene loci of light chains

  8. Multifaceted regulation of V(D)J recombination

    Wang, Guannan

    V(D)J recombination is responsible for generating an enormous repertoire of immunoglobulins and T cell receptors, therefore it is a centerpiece to the formation of the adaptive immune system. The V(D)J recombination process proceeds through two steps, site-specific cleavage at RSS (Recombination Signal Sequence) site mediated by the RAG recombinase (RAG1/2) and the subsequent imprecise resolution of the DNA ends, which is carried out by the ubiquitous non-homologous end joining pathway (NHEJ). The V(D)J recombination reaction is obliged to be tightly controlled under all circumstances, as it involves generations of DNA double strand breaks, which are considered the most dangerous lesion to a cell. Multifaceted regulatory mechanisms have been evolved to create great diversity of the antigen receptor repertoire while ensuring genome stability. The RAG-mediated cleavage reaction is stringently regulated at both the pre-cleavage stage and the post-cleavage stage. Specifically, RAG1/2 first forms a pre-cleavage complex assembled at the boarder of RSS and coding flank, which ensures the appropriate DNA targeting. Subsequently, this complex initiates site-specific cleavage, generating two types of double stranded DNA breaks, hairpin-ended coding ends (HP-CEs) and blunt signal ends (SEs). After the cleavage, RAG1/2 proteins bind and retain the recombination ends to form post-cleavage complexes (PCC), which collaborates with the NHEJ machinery for appropriate transfer of recombination ends to NHEJ for proper end resolution. However, little is known about the molecular basis of this collaboration, partly attributed to the lack of sensitive assays to reveal the interaction of PCC with HP-CEs. Here, for the first time, by using two complementary fluorescence-based techniques, fluorescence anisotropy and fluorescence resonance energy transfer (FRET), I managed to monitor the RAG1/2-catalyzed cleavage reaction in real time, from the pre-cleavage to the post-cleavage stages. By examining the dynamic fluorescence changes during the RAG-mediated cleavage reactions, and by manipulating the reaction conditions, I was able to characterize some fundamental properties of RAG-DNA interactions before and after cleavage. Firstly, Mg 2+, known as a physiological cofactor at the excision step, also promotes the HP-CEs retention in the RAG complex after cleavage. Secondly, the structure of pre-cleavage complex may affect the subsequent collaborations with NHEJ for end resolution. Thirdly, the non-core region of RAG2 may have differential influences on the PCC retention of HP-CEs and SEs. Furthermore, I also provide the first evidence of RAG1-mediated regulation of RAG2. Our study provides important insights into the multilayered regulatory mechanisms, in modulating recombination events in developing lymphocytes and paves the way for possible development of detection and diagnotic markers for defective recombination events that are often associated immunodeficiency and/or lymphoid malignancy.

  9. Genetically Engineered Poxviruses for Recombinant Gene Expression, Vaccination, and Safety

    Moss, Bernard

    1996-10-01

    Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure--function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.

  10. Protective Effector Cells of the Recombinant Asp f3 Anti-Aspergillosis Vaccine

    MarkusKalkum; JamesI.Ito

    2012-01-01

    An Aspergillus fumigatus vaccine based on recombinant Asp f3 protein has the potential to prevent aspergillosis in humans, a devastating fungal disease that is the prime obstacle to the success of hematopoietic cell transplantation. This vaccine protects cortisone acetate (CA)-immunosuppressed mice from invasive pulmonary aspergillosis via CD4+ T cell mediators. Aside from these mediators, the nature of downstream fungicidal effectors is not well understood. Neutrophils and macrophages protec...

  11. Protective Effector Cells of the Recombinant Asp f3 Anti-Aspergillosis Vaccine

    Diaz-Arevalo, Diana; Ito, James I.; Kalkum, Markus

    2012-01-01

    An Aspergillus fumigatus vaccine based on recombinant Asp f3-protein has the potential to prevent aspergillosis in humans, a devastating fungal disease that is the prime obstacle to the success of hematopoietic cell transplantation. This vaccine protects cortisone acetate (CA)-immunosuppressed mice from invasive pulmonary aspergillosis via CD4+ T cell mediators. Aside from these mediators, the nature of downstream fungicidal effectors is not well understood. Neutrophils and macrophages protec...

  12. Recombinant λ-phage nanobioparticles for tumor therapy in mice models

    Ghaemi, Amir; Soleimanjahi, Hoorieh; Gill, Pooria; Hassan, Zuhair; Jahromi, Soodeh Razeghi M; Roohvand, Farzin

    2010-01-01

    Lambda phages have considerable potential as gene delivery vehicles due to their genetic tractability, low cost, safety and physical characteristics in comparison to other nanocarriers and gene porters. Little is known concerning lambda phage-mediated gene transfer and expression in mammalian hosts. We therefore performed experiments to evaluate lambda-ZAP bacteriophage-mediated gene transfer and expression in vitro. For this purpose, we constructed recombinant λ-phage nanobioparticles contai...

  13. Repair and recombination of X-irradiated plasmids in Xenopus laevis oocytes

    Plasmid DNA substrates were X-irradiated and injected into the nuclei of Xenopus laevis oocytes. After incubation for 20 h, DNA was recovered from the oocytes and analyzed simultaneously for repair and for intermolecular homologous recombination by electrophoresis and bacterial transformation. Oocyte-mediated repair of DNA strand breaks was observed with both methods. Using a repair-deficient mutant Escherichia coli strain and its repair-proficient parent as hosts for the transformation assay, we also demonstrated that oocytes repaired oxidative-type DNA base damage induced by X-rays. X-irradiation of a circular DNA stimulated its potential to recombine with a homologous linear partner. Recombination products were detected directly by Southern blot hybridization and as bacterial transformant clones expressing two antibiotic resistance markers originally carried separately on the two substrates. The increase in recombination was dependent on X-ray dose. There is some suggestion that lesions other than double-strand breaks contribute to the stimulation of oocyte-mediated homologous recombination. In summary, oocytes have considerable capacity to repair X-ray-induced damage, and some X-ray lesions stimulate homologous recombination in these cells

  14. Genetics Home Reference: recombinant 8 syndrome

    ... copy of the recombinant chromosome 8 in each cell is sufficient to cause the disorder. Most people with recombinant 8 ... SL, Sample T, Bleskan J, Sujansky E, Patterson D. Cloning, sequencing, and analysis of inv8 chromosome breakpoints associated ...

  15. [Recombination in Drosophila in space flight].

    Filatova, L P; Vaulina, E N; Lapteva, N Sh; Grozdova, T Ia

    1988-04-01

    An experiment with Drosophila melanogaster males was performed aboard the Artificial Satellite "Kosmos-1667". Mutagenic effects of a 7-day space flight on intergene recombination in chromosome 2 were studied. The space flight factors decreased the frequency of recombination. A model experiment on a laboratory centrifuge demonstrated insignificant increase in recombination frequency caused by acceleration. PMID:3135244

  16. Recombinational substrates designed to study recombination between unique and repetitive sequences in vivo

    Three recombination events, reciprocal recombination, sister-chromatid recombination, and gene conversion, were studied using substrates designed in vitro. Each type of recombination event can be monitored at any chromosomal location. The authors have shown that sister-chromatid recombination is induced mitotically by DNA damaging agents, such as methyl methanesulfonate and γ-rays, but is decreased mitotically in strains defective in rad52. Reciprocal recombination by which circular plasmids integrate into the genome is unaffected by rad52 defective alleles and occurs by a different recombination pathway. Mechanisms are suggested by which gene conversion between sister chromatids can generate chromosome rearrangements

  17. Dissecting the Requirement for Subgenomic Promoter Sequences by RNA Recombination of Brome Mosaic Virus In Vivo: Evidence for Functional Separation of Transcription and Recombination

    Wierzchoslawski, Rafal; Dzianott, Aleksandra; Bujarski, Jozef

    2004-01-01

    Previously, we and others mapped an increased homologous recombination activity within the subgenomic promoter (sgp) region in brome mosaic virus (BMV) RNA3 (A. Bruyere et al., J. Virol. 74:4214-4219, 2000; R. Wierzchoslawski et al., J. Virol. 77:6769-6776, 2003). In order to correlate sgp-mediated recombination and transcription, in the present work we used BMV RNA3 constructs that carried altered sgp repeats. We observed that the removal or extension of the poly(U) tract reduced or increase...

  18. Mechanisms of sister chromatid recombination

    Studies using T948 as a model system have been carried out aimed at elucidating the mechanism of sister chromatid recombination (SCR). Characterization of U.V. light- and x-ray-induced SCR, the relationiship between SCR induction and DNA repair using rad mutations, and the relationship between SCR induction and the time of cell division using cdc mutations are presented. It has been supposed that SCR is induced at the phase of S-G2 following DNA replication, that postreplication break of DNA strands is strongly involved in the induction of SCR, and that induction type of SCR, i.e., conversion type or recombination type, is dependent upon the type of molecular damage of DNA. (Namekawa, K.)

  19. Microbial factories for recombinant pharmaceuticals

    Domingo-Espn Joan; Ferrer-Miralles Neus; Corchero Jos; Vzquez Esther; Villaverde Antonio

    2009-01-01

    Abstract Most of the hosts used to produce the 151 recombinant pharmaceuticals so far approved for human use by the Food and Drug Administration (FDA) and/or by the European Medicines Agency (EMEA) are microbial cells, either bacteria or yeast. This fact indicates that despite the diverse bottlenecks and obstacles that microbial systems pose to the efficient production of functional mammalian proteins, namely lack or unconventional post-translational modifications, proteolytic instability, po...

  20. Detection of Quantitative Trait Loci Influencing Recombination Using Recombinant Inbred Lines

    Dole, Jefferey; Weber, David F.

    2007-01-01

    The genetic basis of variation in recombination in higher plants is polygenic and poorly understood, despite its theoretical and practical importance. Here a method of detecting quantitative trait loci (QTL) influencing recombination in recombinant inbred lines (RILs) is proposed that relies upon the fact that genotype data within RILs carry the signature of past recombination. Behavior of the segregational genetic variance in numbers of chromosomal crossovers (recombination) over generations...

  1. Workshop on Radio Recombination Lines

    1980-01-01

    Since their first detection 15 years ago, radio recombination lines from several elements have been observed in a wide variety of objects including HII regions, planetary nebulae, molecular clouds, the diffuse interstellar medium, and recently, other galaxies. The observations span almost the entire range from 0.1 to 100 GHz, and employ both single­ djsh and aperture synthesis techniques. The theory of radio recombination lines has also advanced strongly, to the point where it is perhaps one of the best-understood in astro­ physics. In a parallel development, it has become possible over the last decade to study these same highly-excited atoms in the laboratory; this work provides further confirmation of the theoretical framework. However there has been continuing controversy over the astrophysical interpre­ tation of radio recombination line observations, especially regarding the role of stimulated emission. A workshop was held in Ottawa on 24-25 August, 1979, bringing together many of the active scientist...

  2. Nondisjunction of chromosome 15: Origin and recombination

    Robinson, W.P.; Bernasconi, F.; Schinzel, A.A.; Mutirangura, A.; Ledbetter, D.H. (Baylor College of Medicine, Houston, TX (United States)); Langlois, S. (Univ. of Britisch Columbia, Vancouver (Canada)); Morris, M.A.; Malcolm, S.

    1993-09-01

    Thirty-two cases of uniparental disomy (UPD), ascertained from Prader-Willi syndrome patients (N=27) and Angelman syndrome patients (N-5), are used to investigate the pattern of recombination associated with nondisjunction of chromosome 15. In addition, the meiotic stage of nondisjunction is inferred by using markers mapping near the centromere. Two basic approaches to the analysis of recombination in specific pairwise intervals along the chromosome. This method shows a significant reduction in recombination for two of five intervals examined. Second, the observed frequency of each recombinant class (i.e., zero, one, two, three, or more observable crossovers) is compared with expected values. This is useful for testing whether the reduction in recombination can be attributed solely to a proportion of cases with no recombination at all (because of asynapsis), with the remaining groups showing normal recombination (or even excess recombination), or whether recombination is uniformly reduced. Analysis of maternal UPD(15) data shows a slight reduction in the multiple-recombinant classes, with a corresponding increase in both the zero- and one-recombinant classes over expected values. The majority, more than 82%, of the extra chromosomes in maternal UPD(15) cases are due to meiotic I nondisjunction events. In contrast, more paternal UPD(15) cases so far examined appear to have a postzygotic origin of the extra paternal chromosome. 33 refs., 1 fig., 7 tabs.

  3. Homologous Recombination in Negative Sense RNA Viruses

    Michael Worobey

    2011-08-01

    Full Text Available Recombination is an important process that influences biological evolution at many different levels. More and more homologous recombination events have been reported among negative sense RNA viruses recently. While sporadic authentic examples indicate that homologous recombination does occur, recombination seems to be generally rare or even absent in most negative sense RNA viruses, and most of the homologous recombination events reported in the literature were likely generated artificially due to lab contamination or inappropriate bioinformatics methods. Homologous recombination in negative sense RNA viruses should be reported with caution in the future, and only after stringent quality control efforts. Moreover, co-infection experiments should be performed to confirm whether recombination can occur.

  4. Accelerated homologous recombination and subsequent genome modification in Drosophila.

    Baena-Lopez, Luis Alberto; Alexandre, Cyrille; Mitchell, Alice; Pasakarnis, Laurynas; Vincent, Jean-Paul

    2013-12-01

    Gene targeting by 'ends-out' homologous recombination enables the deletion of genomic sequences and concurrent introduction of exogenous DNA with base-pair precision without sequence constraint. In Drosophila, this powerful technique has remained laborious and hence seldom implemented. We describe a targeting vector and protocols that achieve this at high frequency and with very few false positives in Drosophila, either with a two-generation crossing scheme or by direct injection in embryos. The frequency of injection-mediated gene targeting can be further increased with CRISPR-induced double-strand breaks within the region to be deleted, thus making homologous recombination almost as easy as conventional transgenesis. Our targeting vector replaces genomic sequences with a multifunctional fragment comprising an easy-to-select genetic marker, a fluorescent reporter, as well as an attP site, which acts as a landing platform for reintegration vectors. These vectors allow the insertion of a variety of transcription reporters or cDNAs to express tagged or mutant isoforms at endogenous levels. In addition, they pave the way for difficult experiments such as tissue-specific allele switching and functional analysis in post-mitotic or polyploid cells. Therefore, our method retains the advantages of homologous recombination while capitalising on the mutagenic power of CRISPR. PMID:24154526

  5. Site-specific recombination of P2-like phages; possible tools for safe gene therapy : A focus on phage ΦD145

    Mandali, Sridhar

    2010-01-01

    P2-like bacteriophages integrate their genome into the E. coli host cell by a site-specific recombination event upon lysogenization. The integrative recombination occurs between a specific sequence in the phage genome, attP, and a specific sequence in the host genome, attB, generating the host-phage junctions attL and attR. The integration is mediated by the phage enzyme integrase (Int) and the host factor IHF. The excisive recombination takes place between attL and attR, and is mediated by I...

  6. Phase II Radiation Therapy Oncology Group trial of conventional radiation therapy followed by treatment with recombinant interferon-? for supratentorial glioblastoma: Results of RTOG 9710

    Purpose: The aim of this study was to determine whether recombinant human interferon ?-1a (rhIFN-?), when given after radiation therapy, improves survival in glioblastoma. Methods and Materials: After surgery, 109 patients with newly diagnosed supratentorial glioblastoma were enrolled and treated with radiation therapy (60 Gy). A total of 55 patients remained stable after radiation and were treated with rhIFN-? (6 MU/day i.m., 3 times/week). Outcomes were compared with Radiation Therapy Oncology Group glioma historical database. Results: RhIFN-? was well tolerated, with 1 Grade 4 toxicity and 8 other patients experiencing Grade 3 toxicity. Median survival time (MST) of the 55 rhIFN-?-treated patients was 13.4 months. MST for the 34 rhIFN-?-treated in RPA Classes III and IV was 16.9 vs. 12.4 months for historical controls (hazard ratio [HR] = 1.27, 95% confidence interval [CI] = 0.89-1.81). There was also a trend toward improved survival across all RPA Classes comparing the 55 rhIFN-? treated patients and 1,658 historical controls (HR = 1.24, 95% CI = 0.94-1.63). The high rate of early failures (54/109) after radiation and before initiation of rhIFN-? was likely caused by stricter interpretation of early radiographic changes in the current study. Matched-pair and intent-to-treat analyses performed to try to address this bias showed no difference in survival between study patients and controls. Conclusion: RhIFN-? given after conventional radiation therapy was well tolerated, with a trend toward survival benefit in patients who remained stable after radiation therapy. These data suggest that rhIFN-? warrants further evaluation in additional studies, possibly in combination with current temozolomide-based regimens

  7. Current trends of HIV recombination worldwide

    Katherine A. Lau

    2013-06-01

    Full Text Available One of the major characteristics of HIV-1 is its high genetic variability and extensive heterogeneity. This characteristic is due to its molecular traits, which in turn allows it to vary, recombine, and diversify at a high frequency. As such, it generates complex molecular forms, termed recombinants, which evade the human immune system and so survive. There is no sequence constraint to the recombination pattern as it appears to occur at inter-group (between groups M and O, as well as inter- and intra-subtype within group M. Rapid emergence and active global transmission of HIV-1 recombinants, known as circulating recombinant forms (CRFs and unique recombinant forms (URFs, requires urgent attention. To date, 55 CRFs have been reported around the world. The first CRF01_AE originated from Central Africa but spread widely in Asia. The most recent CRF; CRF55_01B is a recombinant form of CRF01_AE and subtype B, although its origin is yet to be publicly disclosed. HIV-1 recombination is an ongoing event and plays an indispensable role in HIV epidemics in different regions. Africa, Asia and South America are identified as recombination hot-spots. They are affected by continual emergence and co-circulation of newly emerging CRFs and URFs, which are now responsible for almost 20% of HIV-1 infections worldwide. Better understanding of recombinants is necessary to determine their biological and molecular attributes.

  8. Dissociative Recombination of He_2^+

    Coman, L.; Guna, M.; Simons, L.; Hardy, Kenneth A.

    1999-10-01

    The final product states of the dissociative recombination (DR) of He_2^+ have been studied by time of flight (TOF) spectroscopy. DR final product state atoms were seen in six final product states, and from the V = 2,3,4, and 5 vibrational levels of the molecular ion. The energy resolution of the TOF method is sufficient to separate the rotational levels built of these vibrational states. Rotational states up to J=15 were observed. From the rotational states observed, we made the first measurements of the rotational constants of the molecule, and find that the vibrational constants are in reasonable agreement with those predicted by theory.

  9. CRMAGE: CRISPR Optimized MAGE Recombineering

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Sommer, Morten Otto Alexander; Nielsen, Alex Toftgaard

    2016-01-01

    A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli...... assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red...

  10. Dielectronic recombination for average ions

    Dielectronic recombination (DR) in the average-atom (AA) model has been studied. The reasonableness of directly applying the Burgess-Merts (BM) formula to the AA model has been examined. We have noticed that it is inappropriate to use the BM formula to calculate DR rate coefficients for average ions. We have developed a self-consistent-field theoretical method to treat the DR for average ions with electrons. As an example, the DR rate coefficients for argon have been calculated at a defined electron population, and have been compared with the rate-coefficient average via detailed configuration accounting. copyright 1997 The American Physical Society

  11. Dissociative recombination in interstellar clouds

    Black, John H.; Van Dishoeck, Ewine F.

    1989-01-01

    Molecular ions play a significant role in the chemistry and evolution of interstellar molecular clouds, even though these regions are overwhelmingly neutral. The dissociative recombination (DR) process governs the abundances of many of these ions and of related neutral species. The gas-phase ion-molecule chemistry of the simplest species is summarized, with emphasis on those problems which are most sensitive to uncertain rates or product branching ratios of DR processes. Examples of the kinds of information needed about DR processes are presented. The importance of the H3(+) ion and prospects for its direct observation are discussed.

  12. Effects of nuclear mutations for recombination and repair functions and of caffeine on mitochondrial recombination

    Studies of both prokaryotic and eukaryotic organisms indicate that pathways governing repair of damage to nuclear DNA caused by x-ray or ultraviolet irradiation overlap with those controlling recombination. Fourteen nuclear mutants of Saccharomyces cerevisiae were tested in order to determine whether these mutant genes affected mitochondrial recombination. None of the mutations studied significantly affected mitochondrial recombination. The nuclear recombination and repair pathways studied do not overlap with the nuclear pathway which controls recombination of mitochondrial DNA. A second set of experiments was designed to test the effect of caffeine on both nuclear and mitochondrial recombination in Saccharomyces cerevisiae. (U.S.)

  13. Rad52/Rad59-dependent recombination as a means to rectify faulty Okazaki fragment processing.

    Lee, Miju; Lee, Chul-Hwan; Demin, Annie Albert; Munashingha, Palinda Ruvan; Amangyeld, Tamir; Kwon, Buki; Formosa, Tim; Seo, Yeon-Soo

    2014-05-23

    The correct removal of 5'-flap structures by Rad27 and Dna2 during Okazaki fragment maturation is crucial for the stable maintenance of genetic materials and cell viability. In this study, we identified RAD52, a key recombination protein, as a multicopy suppressor of dna2-K1080E, a lethal helicase-negative mutant allele of DNA2 in yeasts. In contrast, the overexpression of Rad51, which works conjointly with Rad52 in canonical homologous recombination, failed to suppress the growth defect of the dna2-K1080E mutation, indicating that Rad52 plays a unique and distinct role in Okazaki fragment metabolism. We found that the recombination-defective Rad52-QDDD/AAAA mutant did not rescue dna2-K1080E, suggesting that Rad52-mediated recombination is important for suppression. The Rad52-mediated enzymatic stimulation of Dna2 or Rad27 is not a direct cause of suppression observed in vivo, as both Rad52 and Rad52-QDDD/AAAA proteins stimulated the endonuclease activities of both Dna2 and Rad27 to a similar extent. The recombination mediator activity of Rad52 was dispensable for the suppression, whereas both the DNA annealing activity and its ability to interact with Rad59 were essential. In addition, we found that several cohesion establishment factors, including Rsc2 and Elg1, were required for the Rad52-dependent suppression of dna2-K1080E. Our findings suggest a novel Rad52/Rad59-dependent, but Rad51-independent recombination pathway that could ultimately lead to the removal of faulty flaps in conjunction with cohesion establishment factors. PMID:24711454

  14. A cruciform-dumbbell model for inverted dimer formation mediated by inverted repeats.

    Lin, C.T.; Lyu, Y L; Liu, L. F.

    1997-01-01

    Small inverted repeats (small palindromes) on plasmids have been shown to mediate a recombinational rearrangement event in Escherichia coli leading to the formation of inverted dimers (giant palindromes). This recombinational rearrangement event is efficient and independent of RecA and RecBCD. In this report, we propose a cruciform-dumbbell model to explain the inverted dimer formation mediated by inverted repeats. In this model, the inverted repeats promote the formation of a DNA cruciform w...

  15. Detecting the cosmological recombination signal from space

    Desjacques, Vincent; Silk, Joseph; de Bernardis, Francesco; Doré, Olivier

    2015-01-01

    Spectral distortions of the CMB have recently experienced an increased interest. One of the inevitable distortion signals of our cosmological concordance model is created by the cosmological recombination process, just a little before photons last scatter at redshift $z\\simeq 1100$. These cosmological recombination lines, emitted by the hydrogen and helium plasma, should still be observable as tiny deviation from the CMB blackbody spectrum in the cm--dm spectral bands. In this paper, we present a forecast for the detectability of the recombination signal with future satellite experiments. We argue that serious consideration for future CMB experiments in space should be given to probing spectral distortions and, in particular, the recombination line signals. The cosmological recombination radiation not only allows determination of standard cosmological parameters, but also provides a direct observational confirmation for one of the key ingredients of our cosmological model: the cosmological recombination histo...

  16. Microbial factories for recombinant pharmaceuticals

    Domingo-Espín Joan

    2009-03-01

    Full Text Available Abstract Most of the hosts used to produce the 151 recombinant pharmaceuticals so far approved for human use by the Food and Drug Administration (FDA and/or by the European Medicines Agency (EMEA are microbial cells, either bacteria or yeast. This fact indicates that despite the diverse bottlenecks and obstacles that microbial systems pose to the efficient production of functional mammalian proteins, namely lack or unconventional post-translational modifications, proteolytic instability, poor solubility and activation of cell stress responses, among others, they represent convenient and powerful tools for recombinant protein production. The entering into the market of a progressively increasing number of protein drugs produced in non-microbial systems has not impaired the development of products obtained in microbial cells, proving the robustness of the microbial set of cellular systems (so far Escherichia coli and Saccharomyces cerevisae developed for protein drug production. We summarize here the nature, properties and applications of all those pharmaceuticals and the relevant features of the current and potential producing hosts, in a comparative way.

  17. Charge recombination in undoped cuprates

    Lenar?i?, Zala; Prelovek, Peter

    2014-12-01

    We theoretically analyze the process of charge recombination in the planar Mott-Hubbard insulators with the aim to explain the short picosecond-range lifetimes of photoexcited carriers, experimentally studied via pump-probe experiments on the undoped cuprates. The recombination mechanism consists of two essential ingredients: the formation of a metastable s -type bound holon-doublon pair, i.e., the Mott exciton, and the decay of such an excitonic state via the multimagnon emission. In spite of the large gap that requires many bosons to be emitted, the latter process is fast due to a large exchange scale and strong charge-spin coupling in planar systems. As the starting microscopic model we consider the single-band Hubbard model and then a more realistic three-band model for cuprates, both leading to the same minimal one. The decay rate of the exciton is evaluated numerically via the Fermi golden rule, having consistency also with the direct time-evolution calculation. The decay rate reveals exponential dependence on the ratio of the Mott-Hubbard gap and the exchange coupling, the result qualitatively reproduced also within a toy exciton-boson model.

  18. Microbial factories for recombinant pharmaceuticals.

    Ferrer-Miralles, Neus; Domingo-Espn, Joan; Corchero, Jos Luis; Vzquez, Esther; Villaverde, Antonio

    2009-01-01

    Most of the hosts used to produce the 151 recombinant pharmaceuticals so far approved for human use by the Food and Drug Administration (FDA) and/or by the European Medicines Agency (EMEA) are microbial cells, either bacteria or yeast. This fact indicates that despite the diverse bottlenecks and obstacles that microbial systems pose to the efficient production of functional mammalian proteins, namely lack or unconventional post-translational modifications, proteolytic instability, poor solubility and activation of cell stress responses, among others, they represent convenient and powerful tools for recombinant protein production. The entering into the market of a progressively increasing number of protein drugs produced in non-microbial systems has not impaired the development of products obtained in microbial cells, proving the robustness of the microbial set of cellular systems (so far Escherichia coli and Saccharomyces cerevisae) developed for protein drug production. We summarize here the nature, properties and applications of all those pharmaceuticals and the relevant features of the current and potential producing hosts, in a comparative way. PMID:19317892

  19. Silicon Solar Cells: Recombination and Electrical Parameters

    Sadou,; Madougou,; Kaka, Mohamadou; Sissoko, Gregoire

    2010-01-01

    This study shows that most of silicon solar cell is configured in N-P junctions. In these solar cells, recombination mechanisms occur in five regions and are mainly two types: recombination mechanisms in bulk and surface recombination. In practice, the solar cells can be illuminated using several illumination modes: under monochromatic light, under multispectral light, under intense light concentration or under other modes. Basic equations describe the behaviour of the excess minority carrier...

  20. Mediatized play

    Johansen, Stine Liv

    Children’s play must nowadays be understood as a mediatized field in society and culture. Media – understood in a very broad sense - holds severe explanatory power in describing and understanding the practice of play, since play happens both with, through and inspired by media of different sorts........ In this presentation the case of ‘playing soccer’ will be outlined through its different mediated manifestations, including soccer games and programs on TV, computer games, magazines, books, YouTube videos and soccer trading cards....

  1. Mediating Business

    "Mediating Business" is a study of the expansion of business journalism. Building on evidence from Denmark, Finland, Norway and Sweden, "Mediating Business" is a comparative and multidisciplinary study of one of the major transformations of the mass media and the realm of business - nationally and...... globally. The book explores the history of key innovations and innovators in the business press. It analyzes changes in the discourse of business journalism associated with the growth in business news and the development of new ways of framing business issues and events. Finally, it examines the...... organizational implications of the increased media visibility of business and, in particular, the development of corporate governance and media relations....

  2. Complex Mediation

    Bødker, Susanne; Andersen, Peter Bøgh

    2005-01-01

    This article has its starting point in a large number of empirical findings regarding computer-mediated work. These empirical findings have challenged our understanding of the role of mediation in such work; on the one hand as an aspect of communication and cooperation at work and on the other hand...... as an aspect of human engagement with instruments of work. On the basis of previous work in activity-theoretical and semiotic human—computer interaction, we propose a model to encompass both of these aspects. In a dialogue with our empirical findings we move on to propose a number of types of...

  3. Human Insulin from Recombinant DNA Technology

    Johnson, Irving S.

    1983-02-01

    Human insulin produced by recombinant DNA technology is the first commercial health care product derived from this technology. Work on this product was initiated before there were federal guidelines for large-scale recombinant DNA work or commercial development of recombinant DNA products. The steps taken to facilitate acceptance of large-scale work and proof of the identity and safety of such a product are described. While basic studies in recombinant DNA technology will continue to have a profound impact on research in the life sciences, commercial applications may well be controlled by economic conditions and the availability of investment capital.

  4. Experimental recombination rates for highly charged ions

    Recent studies of recombination between free electrons and highly charged ions using electron coolers of heavy-ion storage rings have produced accurate rate coefficients of interest for plasma modeling and diagnostics. Some surprises were discovered which can lead to revisions of recombination models. With bare ions one finds at low energy a strong and puzzling deviation from radiative recombination theory. Dielectronic recombination with C3+, N4+) show that jj coupling gives essential contributions to the cross section also for light ions. (author)

  5. Fine-Scale Population Recombination Rates, Hotspots, and Correlates of Recombination in the Medicago truncatula Genome

    Paape, Timothy; ZHOU, Peng; Branca, Antoine; Briskine, Roman; Young, Nevin; Tiffin, Peter

    2012-01-01

    Recombination rates vary across the genome and in many species show significant relationships with several genomic features, including distance to the centromere, gene density, and GC content. Studies of fine-scale recombination rates have also revealed that in several species, there are recombination hotspots, that is, short regions with recombination rates 10100 greater than those in surrounding regions. In this study, we analyzed whole-genome resequence data from 26 accessions of the mode...

  6. Mediatized Humanitarianism

    Vestergaard, Anne

    2014-01-01

    The article investigates the implications of mediatization for the legitimation strategies of humanitarian organizations. Based on a (full population) corpus of ~400 pages of brochure material from 1970 to 2007, the micro-textual processes involved in humanitarian organizations' efforts to legiti...

  7. Heterogeneity in recombinant protein production

    Schalén, Martin; Johanson, Ted; Lundin, Luisa; Sørensen, Søren; Eliasson Lantz, Anna

    bioreactors. For this purpose, a Saccharomyces cerevisiae strain, that functions as a protein production reporter, has been developed. A heterologous protein has been tagged with a fluorescent protein providing a way to measure the amount of heterologous protein produced by the cells on single cell level....... Gradients are simulated in small bioreactors and the population heterogeneity can be visualised by analysing single cells with flow cytometry. This can give new insights to cell physiology and recombinant protein production at the industrial scale.......A crucial step in biotechnology is the scale-up process. Normally, lab scale verification and optimization of production processes and strains are performed in small reactors with perfect mixing and hence the cells experience a homogenous environment. The gradients that occur in industrial scale...

  8. Recombinant viral vaccines for cancer.

    Cawood, Ryan; Hills, Thomas; Wong, Suet Ling; Alamoudi, Aliaa A; Beadle, Storm; Fisher, Kerry D; Seymour, Leonard W

    2012-09-01

    Cancer arises from 'self' in a series of steps that are all subject to immunoediting. Therefore, therapeutic cancer vaccines must stimulate an immune response against tumour antigens that have already evaded the body's immune defences. Vaccines presenting a tumour antigen in the context of obvious danger signals seem more likely to stimulate a response. This approach can be facilitated by genetic engineering using recombinant viral vectors expressing tumour antigens, cytokines, or both, from an immunogenic virus particle. We overview clinical attempts to use these agents for systemic immunisation and contrast the results with strategies employing direct intratumoural administration. We focus on the challenge of producing an effective response within the immune-suppressive tumour microenvironment, and discuss how the technology can overcome these obstacles. PMID:22917663

  9. Fundamental Studies of Recombinant Hydrogenases

    Adams, Michael W

    2014-01-25

    This research addressed the long term goals of understanding the assembly and organization of hydrogenase enzymes, of reducing them in size and complexity, of determining structure/function relationships, including energy conservation via charge separation across membranes, and in screening for novel H2 catalysts. A key overall goal of the proposed research was to define and characterize minimal hydrogenases that are produced in high yields and are oxygen-resistant. Remarkably, in spite of decades of research carried out on hydrogenases, it is not possible to readily manipulate or design the enzyme using molecular biology approaches since a recombinant form produced in a suitable host is not available. Such resources are essential if we are to understand what constitutes a “minimal” hydrogenase and design such catalysts with certain properties, such as resistance to oxygen, extreme stability and specificity for a given electron donor. The model system for our studies is Pyrococcus furiosus, a hyperthermophile that grows optimally at 100°C, which contains three different nickel-iron [NiFe-] containing hydrogenases. Hydrogenases I and II are cytoplasmic while the other, MBH, is an integral membrane protein that functions to both evolve H2 and pump protons. Three important breakthroughs were made during the funding period with P. furiosus soluble hydrogenase I (SHI). First, we produced an active recombinant form of SHI in E. coli by the co-expression of sixteen genes using anaerobically-induced promoters. Second, we genetically-engineered P. furiosus to overexpress SHI by an order of magnitude compared to the wild type strain. Third, we generated the first ‘minimal’ form of SHI, one that contained two rather than four subunits. This dimeric form was stable and active, and directly interacted with a pyruvate-oxidizing enzyme with any intermediate electron carrier. The research resulted in five peer-reviewed publications.

  10. Cell biology of homologous recombination in yeast

    Eckert-Boulet, Nadine Valerie; Rothstein, Rodney; Lisby, Michael

    Homologous recombination is an important pathway for error-free repair of DNA lesions, such as single- and double-strand breaks, and for rescue of collapsed replication forks. Here, we describe protocols for live cell imaging of single-lesion recombination events in the yeast Saccharomyces...

  11. Electron-ion recombination at low energy

    The work is based on results obtained with a merged-beams experiment. A beam of electronics with a well characterized density and energy distribution was merged with a fast, monoenergetic ion beam. Results have been obtained for radiative recombination and dielectronic recombination at low relative energies (0 to ?70eV). The obtained energy resolution was improved by about a factor of 30. High vacuum technology was used to suppress interactions with electrons from the environments. The velocity distribution of the electron beam was determined. State-selective dielectronic-recombination measurements were performable. Recombination processes were studied. The theoretical background for radiative recombination and Kramers' theory are reviewed. The quantum mechanical result and its relation to the semiclassical theory is discussed. Radiative recombination was also measured with several different non-bare ions, and the applicability of the semiclassical theory to non-bare ions was investigated. The use of an effective charge is discussed. For dielectronic recombination, the standard theoretical approach in the isolated resonance and independent-processes approximation is debated. The applicability of this method was tested. The theory was able to reproduce most of the experimental data except when the recombination process was sensitive to couplings between different electronic configurations. The influence of external perturbing electrostatic fields is discussed. (AB) (31 refs.)

  12. Titania Photocatalysis beyond Recombination: A Critical Review

    Bunsho Ohtani

    2013-01-01

    This short review paper shows the significance of recombination of a photoexcited electron and a hole in conduction and valence bands, respectively, of a titania photocatalyst, since recombination has not yet been fully understood and has not been evaluated adequately during the past several decades of research on heterogeneous photocatalysis.

  13. RNAi and heterochromatin repress centromeric meiotic recombination.

    Ellermeier, Chad; Higuchi, Emily C; Phadnis, Naina; Holm, Laerke; Geelhood, Jennifer L; Thon, Genevieve; Smith, Gerald R

    2010-05-11

    During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essential in most species for proper homologue segregation. Nevertheless, recombination is repressed specifically in and around the centromeres of chromosomes, apparently because rare centromeric (or pericentromeric) recombination events, when they do occur, can disrupt proper segregation and lead to genetic disabilities, including birth defects. The basis by which centromeric meiotic recombination is repressed has been largely unknown. We report here that, in fission yeast, RNAi functions and Clr4-Rik1 (histone H3 lysine 9 methyltransferase) are required for repression of centromeric recombination. Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis. PMID:20421495

  14. Recombinant Vaccinia Virus: Immunization against Multiple Pathogens

    Perkus, Marion E.; Piccini, Antonia; Lipinskas, Bernard R.; Paoletti, Enzo

    1985-09-01

    The coding sequences for the hepatitis B virus surface antigen, the herpes simplex virus glycoprotein D, and the influenza virus hemagglutinin were inserted into a single vaccinia virus genome. Rabbits inoculated intravenously or intradermally with this polyvalent vaccinia virus recombinant produced antibodies reactive to all three authentic foreign antigens. In addition, the feasibility of multiple rounds of vaccination with recombinant vaccinia virus was demonstrated.

  15. Consequences of recombination on traditional phylogenetic analysis

    Schierup, M H; Hein, J

    2000-01-01

    We investigate the shape of a phylogenetic tree reconstructed from sequences evolving under the coalescent with recombination. The motivation is that evolutionary inferences are often made from phylogenetic trees reconstructed from population data even though recombination may well occur (mtDNA o...

  16. Recombinant organisms for production of industrial products

    Adrio, Jose-Luis; Demain, Arnold L.

    2009-01-01

    A revolution in industrial microbiology was sparked by the discoveries of ther double-stranded structure of DNA and the development of recombinant DNA technology. Traditional industrial microbiology was merged with molecular biology to yield improved recombinant processes for the industrial production of primary and secondary metabolites, protein biopharmaceuticals and industrial enzymes. Novel genetic techniques such as metabolic engineering, combinatorial biosynthesis and molecular breeding...

  17. V(D)J recombination deficiencies.

    de Villartay, Jean-Pierre

    2009-01-01

    V(D)J recombination not only comprises the molecular mechanism that insures diversity of the immune system but also constitutes a critical checkpoint in the developmental program of B- and T-lymphocytes. The analysis of human patients with Severe Combined Immune Deficiency (SCID) has contributed to the understanding of the biochemistry of the V(D)J recombination reaction. The molecular study V(D)J recombination settings in humans, mice and in cellular mutants has allowed to unravel the process of Non Homologous End Joining (NHEJ), one of the key pathway that insure proper repair of DNA double strand breaks (dsb), whether they occur during V(D)J recombination or secondary to other DNA injuries. Two NHEJ factors, Artemis and Cernunnos, were indeed discovered through the study of human V(D)J recombination defective human SCID patients. PMID:19731800

  18. Stress analysis of passive hydrogen autocatalytic recombiner

    Background: Passive hydrogen autocatalytic recombiner is a device for eliminating hydrogen in the containment of the nuclear power plant when severe accident occurs, avoiding hydrogen explosion. After the Fukushima nuclear accident, the nuclear power plants pay more attention to the role of Passive hydrogen autocatalytic recombiner. Purpose: This paper studies the stresses of passive hydrogen autocatalytic recombiner under the seismic and LOCA conditions, Methods: Modeling by using the finite element software ANSYS, the impacts of airflow load under the LOCA conditions are considered reasonably and the strength of passive hydrogen autocatalytic recombiner is also evaluated according RCC-M, Results: The results show that the model can meet the requirement of the standard document. Conclusions: This paper will provide technical support for stress analysis and evaluation of passive hydrogen autocatalytic recombiner. (authors)

  19. Identifying the nature of charge recombination in organic solar cells from charge-transfer state electroluminescence

    Wetzelaer, Gert-Jan A.H. [Molecular Electronics, Zernike Institute for Advanced Materials, University of Groningen (Netherlands); Dutch Polymer Institute, Eindhoven (Netherlands); Kuik, Martijn [Molecular Electronics, Zernike Institute for Advanced Materials, University of Groningen (Netherlands); Blom, Paul W.M. [Molecular Electronics, Zernike Institute for Advanced Materials, University of Groningen (Netherlands); TNO/Holst Centre, Eindhoven (Netherlands)

    2012-10-15

    Charge-transfer (CT) state electroluminescence is investigated in several polymer:fullerene bulk heterojunction solar cells. The ideality factor of the electroluminescence reveals that the CT emission in polymer:fullerene solar cells originates from free-carrier bimolecular recombination at the donor-acceptor interface, rather than a charge-trap-mediated process. The fingerprint of the presence of nonradiative trap-assisted recombination, a voltage-dependent CT electroluminescence quantum efficiency, is only observed for the P3HT:PCBM system, which is explained by a reduction of the competing bimolecular recombination rate. These results are in agreement with measurements of the illumination-intensity dependence of the open-circuit voltage. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  20. Containment air circulation for optimal hydrogen recombination

    An accepted first-line defense for hydrogen mitigation is to design for the hydrogen to be rapidly mixed with the containment atmosphere and diluted to below flammability concentrations. Then, as hydrogen continues to be produced in the longer term, recombiners can be used to remove hydrogen: recombiners can be located in forced-air ducts or passive recombiners can be distributed within containment and the heat of recombination used to promote local air circulation. However, this principle does not eliminate the possibility of high hydrogen concentrations at locations removed from the recombiners. An improvement on this strategy is to arrange for a specific, buoyancy-driven, overall circulation of the containment atmosphere such that the recombiners can be located within the recirculation flow, immediately downstream of the hydrogen source. This would make the mixing process more predictable and solve the mass-transfer problem associated with distributed recombiners. Ideally, the recombiners would be located just above the hydrogen source so that the heat of recombination would assist the overall circulation. In this way, the hydrogen would be removed as close as possible to the source, thereby minimizing the amount of hydrogen immediately downstream of the source and reducing the hydrogen concentration to acceptable levels at other locations. Such a strategy requires the containment volume to be divided into an upflow path, past the hydrogen source and the recombiner, and a downflow path to complete the circuit. The flow could be generated actively using fans or passively using buoyancy forces arising from the difference in density of gases in the upfiow and downflow paths; the gases in the downflow path being cooled at an elevated heat sink. (author)

  1. Origins of the machinery of recombination and sex.

    Cavalier-Smith, T

    2002-02-01

    Mutation plays the primary role in evolution that Weismann mistakenly attributed to sex. Homologous recombination, as in sex, is important for population genetics--shuffling of minor variants, but relatively insignificant for large-scale evolution. Major evolutionary innovations depend much more on illegitimate recombination, which makes novel genes by gene duplication and by gene chimaerisation--essentially mutational forces. The machinery of recombination and sex evolved in two distinct bouts of quantum evolution separated by nearly 3 Gy of stasis; I discuss their nature and causes. The dominant selective force in the evolution of recombination and sex has been selection for replicational fidelity and viability; without the recombination machinery, accurate reproduction, stasis, resistance to radical deleterious evolutionary change and preservation of evolutionary innovations would be impossible. Recombination proteins betray in their phylogeny and domain structure a key role for gene duplication and chimaerisation in their own origin. They arose about 3.8 Gy ago to enable faithful replication and segregation of the first circular DNA genomes in precellular ancestors of Gram-negative eubacteria. Then they were recruited and modified by selfish genetic parasites (viruses; transposons) to help them spread from host to host. Bacteria differ fundamentally from eukaryotes in that gene transfer between cells, whether incidental to their absorptive feeding on DNA and virus infection or directly by plasmids, involves only genomic fragments. This was radically changed by the neomuran revolution about 850 million years ago when a posibacterium evolved into the thermophilic cenancestor of eukaryotes and archaebacteria (jointly called neomurans), radically modifying or substituting its DNA-handling enzymes (those responsible for transcription as well as for replication, repair and recombination) as a coadaptive consequence of the origin of core histones to stabilise its chromosome. Substitution of glycoprotein for peptidoglycan walls in the neomuran ancestor and the evolution of an endoskeleton and endomembrane system in eukaryotes alone required the origin of nuclei, mitosis and novel cell cycle controls and enabled them to evolve cell fusion and thereby the combination of whole genomes from different cells. Meiosis evolved because of resulting selection for periodic ploidy reduction, with incidental consequences for intrapopulation genetic exchange. Little modification was needed to recombination enzymes or to the ancient bacterial catalysts of homology search by spontaneous base pairing to mediate chromosome pairing. The key innovation was the origin of meiotic cohesins delaying centromere splitting to allow two successive divisions before reversion to vegetative growth and replication, necessarily yielding two-step meiosis. Also significant was the evolution of synaptonemal complexes to stabilise bivalents and of monopolins to orient sister centromeres to one spindle pole. The primary significance of sex was not to promote evolutionary change but to limit it by facilitating ploidy cycles to balance the conflicting selective forces acting on rapidly growing phagotrophic protozoa and starved dormant cysts subject to radiation and other damage. PMID:11932771

  2. Recent Developments in Dissociative Recombination

    There have been a number of recent developments in dissociative recombination research as it relates to ITER, that should be highlighted. These concern primarily experimental and modelling issues and this document will not touch upon the topics of the other scientists involved in DR studies that are present at the meeting. The topic of branching ratios in general is a topic fundamental to DR especially how it influences the formation of radical and stable neutral molecules that again might play a role in particle formation. It should be remembered that the reactions of neutral radicals to form cyclic compounds are responsible for the formation of soot in combustion, though the role played by ions in flames is at best uncertain. In the near wall plasma environment, ion processes may well be more important since neutral species are rarer. Modelling studies by Pernot and collaborators at the Universite de Paris-Sud have shown that if one compares the yields of individual neutral species in ion-chemistry models (in this particular case, the ionosphere of Titan), and if one assumes that DR reactions of hydrocarbon ions primarily decay via the ejection of a hydrogen ion (which is assumed by most Titan ionospheric models) and if one compares these predictions with those coming from a model where actual measured branching ratios are used, differences of up to 5 orders of magnitude are found. This shows very clearly the need for branching ratio studies. In early merged beam studies of DR performed in Canada in the 1970's, it was noticed that cross sections for polyatomic species typically displayed a sharp fall-off above 0.1 eV. This has since been seen in many storage ring studies and clearly this has important consequences for ITER chemistry where plasma temperatures are likely to be well above ambient. In a recent analysis, Jungen and Pratt have explained this phenomenon on the basis that the recombination is dominated by the indirect process (initial capture into a vibrationally excited, neutral Rydberg state) in which the propensity rule (+?v=1) dominates the capture. When the electron energy exceeds that between the v'=0 and v'=1 levels of the ion, where the capture must now involve a ?v=2 transition, this will be much less effective and so the cross section drops precipitously. This assumes of course that the recombining ion is primarily in the ground v=0 level. H3+ continues to be an active subject of research and a very recent experiment at the TSR ring in Heidelberg has examined the influence of rotational excitation on the rate of the recombination. This is a very beautiful study but an important outcome is that even though a cryogenically cooled storage trap was used to produce the ions, the internal rotational temperature of the ions was never found to be below 150K. This suggests that ion cooling by storage in the ring leads eventually to an equilibrium value for the internal energy of the ions as they are de-excited/re-excited by passage through the electron cooler. As observed in earlier merged beam experiments in Canada, the extraction field in the ion source plays an important role in determining the excitation state of the ions as collisions outside the source can lead to re-heating. Indeed in the TSR experiments using a conventional Penning source and a normal extraction field, the ions were found to have a rotational temperature of several thousands of degrees. This clearly has important significance for earlier measurements taken in storage rings. Finally, the world will soon have a new storage ring facility for dissociative recombination research and this will be in Langzhou in China. This machine will have a higher magnetic rigidity that previous rings used for DR and so heavier ions and higher mass resolution experiments can be performed there. Experimental operation of this new ring is expected to commence in 2012/2013. (author)

  3. Recombination of cold electrons with cooled ions

    Recombination, one of the possible reactions of cold electrons with ions, has several important applications, besides being of fundamental interest. Astrophysical objects are studied through their radiation spectra emitted from electron-ion recombination. Plasma modeling and diagnostics are based on the knowledge of recombination cross sections. It is the proposed mechanism for anti-hydrogen production in a trap filled with antiprotons and positrons. The most fundamental process of recombination is radiative recombination (RR): Zq+ + e→ Z(q-1)+ + hν. Here, we discuss measurements of recombination rate coefficients in absolute scale between free electrons and ions at the electron cooler of the CRYRING storage ring at MSL, Stockholm. Surprisingly, there is a consistent disagreement between the measured rates and the rates obtained from theoretical descriptions of RR. It could be shown that this deviation depends on external fields, such as a weak magnetic field in the interaction region. Another effect presented is the enhancement by two orders of magnitude of the recombination rates into a certain quantum state of the atom in a strong laser field. We will discuss these results with respect to their implications for the formation of anti-hydrogen atoms in a Penning trap. (authors)

  4. Off-gas system recombining system

    In a BWR type reactor, exhaust gases are accumulated in the upper portion of a condensator which contain hydrogen, oxygen, nirogen, etc. and, accordingly, can not be released as they are into the atmosphere. An off-gas system is disposed for processing the exhaust gases safely and releasing them into the atmosphere. The exhaust gases are sucked by a steam type extract or, sent to a preheater, warmed to about 160degC and then sent to a recombiner. The recombiner is adapted to recombine hydrogen and oxygen into steams while keeping them out of a detonating range to prevent an accidental explosion. Then, a hydrogen densitometer is disposed in the exhaust gases guided to the recombiner and oxygen is injected such that the oxygen density in the exhaust gases flowing into the recombiner is made higher thanb 1/2 of the hydrogen concentration depending on the signals from the densitometer. Since the inside of the recombiner is in an oxygen excess region, the catalyst performance is improved outstandingly to increase the hydrogen conversion ratio. Thus, it is possible to reduce the volume and the size of the catalyst and the recombiner. (N.H.)

  5. Recombinant fowlpox virus elicits transient cytotoxic T cell responses due to suboptimal innate recognition and recruitment of T cell help.

    Diener, Kerrilyn R; Lousberg, Erin L; Beukema, Emma L; Yu, Anastasia; Howley, Paul M; Brown, Michael P; Hayball, John D

    2008-07-01

    Recombinant fowlpox viruses (FPVs) have been used in a variety of vaccine strategies; however strong data clearly demonstrating the characteristics of the strength and nature of the resultant immune response elicited by these vectors are lacking. By utilising a recombinant variant of FPV which expresses the nominal antigen chicken ovalbumin (OVA), and assessing innate FPV- and OVA-specific adaptive immune responses, we show that recombinant FPV induces a rapid type I interferon (IFN) response, mediated primarily by plasmacytoid dendritic cells (pDCs). These cells are necessary for the development of a strong but transient CD8(+) T cell effector response directed against OVA-expressing target cells. We propose that a combination of suboptimal type I IFN production, poor CD4(+) T cell helper function and inefficient DC licensing likely contribute to this transient response. These findings now provide a sound basis for rational modifications to be made to recombinant FPV, designed to improve subsequent vaccine responses. PMID:18541346

  6. Immunogenicity of therapeutic recombinant immunotoxins.

    Mazor, Ronit; Onda, Masanori; Pastan, Ira

    2016-03-01

    Recombinant immunotoxins (RITs) are chimeric proteins designed to treat cancer. They are made up of an Fv or Fab that targets an antigen on a cancer cell fused to a 38-kDa portion of Pseudomonas exotoxin A (PE38). Because PE38 is a bacterial protein, it is highly immunogenic in patients with solid tumors that have normal immune systems, but much less immunogenic in patients with hematologic malignancies where the immune system is suppressed. RITs have shown efficacy in refractory hairy cell leukemia and in some children with acute lymphoblastic leukemia, but have been much less effective in solid tumors, because neutralizing antibodies develop and prevent additional treatment cycles. In this paper we will (i) review data from clinical trials describing the immunogenicity of PE38 in different patient populations; (ii) review results from clinical trials using different immunosuppressive drugs; and (iii) describe our efforts to make new less-immunogenic RITs by identifying and removing T- and B-cell epitopes to hide the RIT from the immune system. PMID:26864110

  7. Intra-chromosomal rearrangements generated by Cre-lox site-specific recombination.

    Medberry, S L; Dale, E; Qin, M.; Ow, D W

    1995-01-01

    Chromosomal rearrangements are useful genetic and breeding tools but are often difficult to detect and characterize. To more easily identify and define chromosome deletions and inversions, we have used the bacteriophage P1 Cre-lox site-specific recombination system to generate these events in plants. This involves three steps: (i) the introduction of two lox sites into one locus in a plant genome, including one site within a modified Ds transposon; (ii) Ac transposase-mediated transposition o...

  8. Construction and expression of recombinant plasmids encoding type 1 fimbriae of a urinary Klebsiella pneumoniae isolate.

    Purcell, B K; Clegg, S.

    1983-01-01

    The type 1 fimbriae of Klebsiella pneumoniae have been implicated as important virulence factors in mediating Klebsiella urinary infections. The chromosomally encoded fimbrial genes were cloned by a cosmid cloning technique. Further subcloning was performed with the cloning vehicles pBR322 and pACYC184, and a recombinant plasmid containing the fimbrial genes was constructed. After transformation by this plasmid, both Escherichia coli and Salmonella typhimurium were shown to express fimbriae w...

  9. Systemic and Mucosal T-Lymphocyte Activation Induced by Recombinant Adenovirus Vaccines in Rhesus Monkeys▿

    Sun, Yue; Bailer, Robert T.; Rao, Srinivas S.; Mascola, John R.; Nabel, Gary J.; Koup, Richard A.; Letvin, Norman L.

    2009-01-01

    The administration of vectors designed to elicited cell-mediated immune responses may have other consequences that are clinically significant. To explore this possibility, we evaluated T-cell activation during the first 2 months after recombinant adenovirus serotype 5 (rAd5) prime or boost immunizations in rhesus monkeys. We also evaluated the kinetics of T-lymphocyte activation in both the systemic and the mucosal compartments after rAd5 administration in monkeys with preexisting immunity to...

  10. Construction and analysis of the transgenic carrot and celery plants expressing the recombinant thaumatin II protein

    Luchakivska Yu. S.; Komarnytskii I. K.; Kurchenko I. M.; Yurieva O. M.; Zhytkevich N. V.; Kuchuk M. V.

    2015-01-01

    Aim To obtain the transgenic carrot and celery plants able to express recombinant thaumatin II in order to increase plant stress tolerance. Methods. Agrobacterium-mediated transformation of the carrot and celery seedlings was used for obtaining the transgenic plants. Presence and transcription of the transgene in plant tissues were proved by PCR and RT-PCR analysis. The plants were tested for the biotic stress tolerance by in vitro antifungal and antibacterial activity assays and for the sali...

  11. Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins

    Mamat, Uwe; Wilke, Kathleen; Bramhill, David; Schromm, Andra Beate; Lindner, Buko; Kohl, Thomas Andreas; Corchero Nieto, José Luis; Villaverde Corrales, Antonio; Schaffer, Lana; Head, Steven Robert; Souvignier, Chad; Meredith, Timothy Charles; Woodard, Ronald Wesley

    2015-01-01

    Background Lipopolysaccharide (LPS), also referred to as endotoxin, is the major constituent of the outer leaflet of the outer membrane of virtually all Gram-negative bacteria. The lipid A moiety, which anchors the LPS molecule to the outer membrane, acts as a potent agonist for Toll-like receptor 4/myeloid differentiation factor 2-mediated pro-inflammatory activity in mammals and, thus, represents the endotoxic principle of LPS. Recombinant proteins, commonly manufactured in Escherichia coli...

  12. RNAi and heterochromatin repress centromeric meiotic recombination

    Ellermeier, Chad; Higuchi, Emily C; Phadnis, Naina; Holm, Laerke; Geelhood, Jennifer L; Thon, Genevieve; Smith, Gerald R

    2010-01-01

    genetic disabilities, including birth defects. The basis by which centromeric meiotic recombination is repressed has been largely unknown. We report here that, in fission yeast, RNAi functions and Clr4-Rik1 (histone H3 lysine 9 methyltransferase) are required for repression of centromeric recombination......During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is...

  13. Recombination in narrow-gapped semiconductors

    In narrow-gapped semiconductors of the type Hgsub(1-x)Cdsub(x)Te as well as in lead chalcogenides and their mixed crystals with energy gaps of some tenths of eV, the band-band recombination processes dominate if the samples are sufficiently perfect in their crystal lattices. The relative importance of the radiative or Auger recombination depends on the width of the energy gap and the charge carrier concentration. In the extreme case of very narrow energy gaps plasmon and one-electron recombination occurs additionally

  14. Enhancement of antibody class-switch recombination by the cumulative activity of four separate elements.

    Dunnick, Wesley A; Shi, Jian; Zerbato, Jennifer M; Fontaine, Clinton A; Collins, John T

    2011-11-01

    Class-switch recombination of Ab isotype is mediated by a recombinational DNA deletion event and must be robustly upregulated during Ag-driven differentiation of B cells. The enhancer region 3' of the Cα gene is important for the upregulation of switch recombination. Using a transgene of the entire H chain C region locus, we demonstrate in this study that it is the four 3' enhancer elements themselves (a total of 4.7 kb) that are responsible for the upregulation rather than the 24 kb of DNA in between them. Neither allelic exclusion nor transgenic μ expression is reduced by deletion of the four 3' enhancers. We also test deletions of two or three of the 3' enhancers and show that deletion of more 3' enhancers results in a progressive reduction in both switch recombination and germline transcription of all H chain genes. Nevertheless, we find evidence for special roles for some 3' enhancers; different H chain genes are affected by different 3' enhancer deletions. Thus, we find that the dramatic induction of class-switch recombination during Ag-driven differentiation is the result of an interaction among four separated regulatory elements. PMID:21949022

  15. Construction and characterization of recombinant Japanese encephalitis virus carrying brainspecific miRNA target sequences

    Wen-yuan CAO

    2014-08-01

    Full Text Available Objective To construct the recombinant Japanese encephalitis virus ( JEV carrying brain-specific miRNA targeting sequences. Methods The target sequences of brain-specific miR-124 and miR-125 were introduced into the infectious cDNA clone of JEV to generate recombinant plasmids based on reverse genetics technology. The recombinant plasmids were linearized with Xho Ⅰ and served as templates of transcription with SP6 RNA polymerase to generate infectious viral RNA. The RNA transcripts were then transfected into BHK-21 cells, and the supernatant was obtained after incubated at 37℃, 5% CO2 for 3 days. The cytopathic changes of BHK-21 cells inoculated with the supernatant were observed after one passage. The rescued viruses carrying miRNA target sequences were validated by RT-PCR, standard plaque forming test on BHK-21 cells and growth curves analysis. Results Two recombinant viruses carrying miR-124 or miR-125 target sequence were rescued, respectively. The insertion of miRNA target sequences was confirmed by DNA sequencing. The rescued viruses yielded similar plaque morphology and replication efficiency compared with wild type JEV. Conclusion The recombinant JEV containing brain-specific miRNA target sequences can be obtained by reverse genetics technique, which could be used in further studies of miRNA-mediated tissue-specific attenuation mechanism of JEV. DOI: 10.11855/j.issn.0577-7402.2014.06.01

  16. Recombinant IκBα-loaded curcumin nanoparticles for improved cancer therapeutics

    The field of recombinant protein therapeutics has been evolving rapidly, making significant impact on clinical applications for several diseases, including cancer. However, the functional aspects of proteins rely exclusively on their structural integrity, in which nanoparticle mediated delivery offers unique advantages over free proteins. In the present work, a novel strategy has been developed where the nanoparticles (NPs) used for the delivery of the recombinant protein could contribute to enhancing the therapeutic efficacy of the recombinant protein. The transcription factor, NFκB, involved in cell growth and its inhibitor, IκBα, regulates its proliferation. Another similar naturally available molecule, which inhibits the function of NFκB, is curcumin. Hence, we have developed a ‘green synthesis’ method for preparing water-soluble curcumin nanoparticles to stabilize recombinant IκBα protein. The NPs were characterized by UV–vis and fluorescence spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering before administration into human cervical carcinoma (HeLa) and glioblastoma (U87MG) cells. Experimental results demonstrated that this combined module had enhanced therapeutic efficacy, causing apoptotic cell death, which was confirmed by cytotoxicity assay and flowcytometry analyses. The expression of apoptotic genes studied by semi-quantitative reverse transcription PCR delineated the molecular pathways involved in cell death. Thus, our study revealed that the functional delivery of recombinant IκBα-loaded curcumin NPs has promise as a natural-product-based protein therapeutics against cancer cells. (paper)

  17. A new rabies vaccine based on a recombinant ORF virus (parapoxvirus) expressing the rabies virus glycoprotein.

    Amann, Ralf; Rohde, Jörg; Wulle, Ulrich; Conlee, Douglas; Raue, Rudiger; Martinon, Olivier; Rziha, Hanns-Joachim

    2013-02-01

    The present study describes the generation of a new Orf virus (ORFV) recombinant, D1701-V-RabG, expressing the rabies virus (RABV) glycoprotein that is correctly presented on the surface of infected cells without the need of replication or production of infectious recombinant virus. One single immunization with recombinant ORFV can stimulate high RABV-specific virus-neutralizing antibody (VNA) titers in mice, cats, and dogs, representing all nonpermissive hosts for the ORFV vector. The protective immune response against severe lethal challenge infection was analyzed in detail in mice using different dosages, numbers, and routes for immunization with the ORFV recombinant. Long-term levels of VNA could be elicited that remained greater than 0.5 IU per ml serum, indicative for the protective status. Single applications of higher doses (10(7) PFU) can be sufficient to confer complete protection against intracranial (i.c.) challenge, whereas booster immunization was needed for protection by the application of lower dosages. Anamnestic immune responses were achieved by each of the seven tested routes of inoculation, including oral application. Finally, in vivo antibody-mediated depletion of CD4-positive and/or CD8-posititve T cell subpopulations during immunization and/or challenge infection attested the importance of CD4 T cells for the induction of protective immunity by D1701-V-RabG. This report demonstrates another example of the potential of the ORFV vector and also indicates the capability of the new recombinant for vaccination of animals. PMID:23175365

  18. The Study of the Inhibition of the Recombinant TACE Prodomain to Endotoxemia in Mice

    Yuzhen Yang

    2009-12-01

    Full Text Available Objective: To demonstrate the inhibitory function of the prodomain of tumor necrosis factor-α (TNF-α converting enzyme (TACE on TACE activity and to develop an approach to interfere with inflammation processes. Methods: The cDNA encoding the fulllength ectodomain (T1300 and prodomain (T591 of TACE were amplified by RT-PCR. The expression plasmids (pET-28a (+-T1300 and pET-28a (+-T591 were constructed and transformed into E. coli BL21. After Ni2+-NTA resin affinity chromatography, the recombinant T591 protein was obtained and assayed. In order to detect its inhibiton of TACE activity, the mice in the LPS-induced endotoxemia model group were treated with the recombinant TACE prodomain protein prior to the injection of LPS. Murine peritoneal macrophages were isolated from mice abdominal cavity for FCM and the liver, kidney and lung were removed for traditionally histopathology sectioning. Results: The FCM results showed that the recombinant prodomain protein decreased the release of the sTNF-α, which mediated the accumulation of TNF-α on the surface of macrophage cells. HE staining proved that the recombinant protein can decrease the inflammatory response in internal organs of endotoxaemia mice. Conclusions: The recombinant prodomain of TACE has the ability to inhibit sTNF-α release, which indicates that prodomain is an effective antagonist of TACE and might be useful in the molecular design of anti-inflammatory drugs.

  19. Emergence of distinct brome mosaic virus recombinants is determined by the polarity of the inoculum RNA.

    Kwon, Sun-Jung; Rao, A L N

    2012-05-01

    Despite overwhelming interest in the impact exerted by recombination during evolution of RNA viruses, the relative contribution of the polarity of inoculum templates remains poorly understood. Here, by agroinfiltrating Nicotiana benthamiana leaves, we show that brome mosaic virus (BMV) replicase is competent to initiate positive-strand [(+)-strand] synthesis on an ectopically expressed RNA3 negative strand [(-) strand] and faithfully complete the replication cycle. Consequently, we sought to examine the role of RNA polarity in BMV recombination by expressing a series of replication-defective mutants of BMV RNA3 in (+) or (-) polarity. Temporal analysis of progeny sequences revealed that the genetic makeup of the primary recombinant pool is determined by the polarity of the inoculum template. When the polarity of the inoculum template was (+), the recombinant pool that accumulated during early phases of replication was a mixture of nonhomologous recombinants. These are longer than the inoculum template length, and a nascent 3' untranslated region (UTR) of wild-type (WT) RNA1 or RNA2 was added to the input mutant RNA3 3' UTR due to end-to-end template switching by BMV replicase during (-)-strand synthesis. In contrast, when the polarity of the inoculum was (-), the progeny contained a pool of native-length homologous recombinants generated by template switching of BMV replicase with a nascent UTR from WT RNA1 or RNA2 during (+)-strand synthesis. Repair of a point mutation caused by polymerase error occurred only when the polarity of the inoculum template was (+). These results contribute to the explanation of the functional role of RNA polarity in recombination mediated by copy choice mechanisms. PMID:22357282

  20. Recombinant activated factor VII: 30 years of research and innovation.

    Hedner, Ulla

    2015-06-01

    Recombinant activated factor VII (rFVIIa) was initially developed to treat bleeding episodes in patients with congenital haemophilia and inhibitors. The story of its development began in the 1970s, when FVIIa was identified as one of the activated coagulation factors that has minimal potential for inducing thromboembolic side-effects. Extensive research over the last 30 years has greatly increased our knowledge of the characteristics of FVII, its activation, and the mechanisms by which rFVIIa restores haemostasis. In haemophilia, the haemostatic effect of rFVIIa is mediated via binding to thrombin-activated platelets at the site of injury, thereby enhancing thrombin generation also in the absence of factor (F) VIII or FIX. The mechanism of action of rFVIIa has also allowed its successful use in other clinical scenarios characterised by impaired thrombin generation, and its licensed uses have now been extended to acquired haemophilia, congenital FVII deficiency and Glanzmann's thrombasthenia. PMID:26073368

  1. Crystallization of recombinant Haemophilus influenzaee (P4) acid phosphatase

    Ou, Zhonghui [Department of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211 (United States); Felts, Richard L. [Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211 (United States); Reilly, Thomas J. [Department of Veterinary Pathobiology and Veterinary Medical Diagnostic Laboratory, University of Missouri-Columbia, Columbia, MO 65211 (United States); Nix, Jay C. [Molecular Biology Consortium, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Tanner, John J., E-mail: tannerjj@missouri.edu [Department of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211 (United States); Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211 (United States)

    2006-05-01

    Lipoprotein e (P4) is a class C acid phosphatase and a potential vaccine candidate for nontypeable H. influenzae infections. This paper reports the crystallization of recombinant e (P4) and the acquisition of a 1.7 Å resolution native X-ray diffraction data set. Haemophilus influenzae infects the upper respiratory tract of humans and can cause infections of the middle ear, sinuses and bronchi. The virulence of the pathogen is thought to involve a group of surface-localized macromolecular components that mediate interactions at the host–pathogen interface. One of these components is lipoprotein e (P4), which is a class C acid phosphatase and a potential vaccine candidate for nontypeable H. influenzae infections. This paper reports the crystallization of recombinant e (P4) and the acquisition of a 1.7 Å resolution native X-ray diffraction data set. The space group is P4{sub 2}2{sub 1}2, with unit-cell parameters a = 65.6, c = 101.4 Å, one protein molecule per asymmetric unit and 37% solvent content. This is the first report of the crystallization of a class C acid phosphatase.

  2. Crystallization of recombinant Haemophilus influenzaee (P4) acid phosphatase

    Lipoprotein e (P4) is a class C acid phosphatase and a potential vaccine candidate for nontypeable H. influenzae infections. This paper reports the crystallization of recombinant e (P4) and the acquisition of a 1.7 Å resolution native X-ray diffraction data set. Haemophilus influenzae infects the upper respiratory tract of humans and can cause infections of the middle ear, sinuses and bronchi. The virulence of the pathogen is thought to involve a group of surface-localized macromolecular components that mediate interactions at the host–pathogen interface. One of these components is lipoprotein e (P4), which is a class C acid phosphatase and a potential vaccine candidate for nontypeable H. influenzae infections. This paper reports the crystallization of recombinant e (P4) and the acquisition of a 1.7 Å resolution native X-ray diffraction data set. The space group is P42212, with unit-cell parameters a = 65.6, c = 101.4 Å, one protein molecule per asymmetric unit and 37% solvent content. This is the first report of the crystallization of a class C acid phosphatase

  3. Paroxetine suppresses recombinant human P2X7 responses.

    Dao-Ung, Phuong; Skarratt, Kristen K; Fuller, Stephen J; Stokes, Leanne

    2015-12-01

    P2X7 receptor (P2X7) activity may link inflammation to depressive disorders. Genetic variants of human P2X7 have been linked with major depression and bipolar disorders, and the P2X7 knockout mouse has been shown to exhibit anti-depressive-like behaviour. P2X7 is an ATP-gated ion channel and is a major regulator of the pro-inflammatory cytokine interleukin 1β (IL-1β) secretion from monocytes and microglia. We hypothesised that antidepressants may elicit their mood enhancing effects in part via modulating P2X7 activity and reducing inflammatory responses. In this study, we determined whether common psychoactive drugs could affect recombinant and native human P2X7 responses in vitro. Common antidepressants demonstrated opposing effects on human P2X7-mediated responses; paroxetine inhibited while fluoxetine and clomipramine mildly potentiated ATP-induced dye uptake in HEK-293 cells stably expressing recombinant human P2X7. Paroxetine inhibited dye uptake mediated by human P2X7 in a concentration-dependent manner with an IC(50) of 24 μM and significantly reduces ATP-induced inward currents. We confirmed that trifluoperazine hydrochloride suppressed human P2X7 responses (IC(50) of 6.4 μM). Both paroxetine and trifluoperazine did not inhibit rodent P2X7 responses, and mutation of a known residue (F 95L) did not alter the effect of either drug, suggesting neither drug binds at this site. Finally, we demonstrate that P2X7-induced IL-1β secretion from lipopolysaccharide (LPS)-primed human CD14(+) monocytes was suppressed with trifluoperazine and paroxetine. PMID:26341077

  4. Recombination Lines from two Distant HII Regions

    Jones, Courtney; Dickey, John; Anderson, Loren; Bania, Thomas

    2012-04-01

    This proposal uses the CABB system in the newly developed CFB64M-32k mode to observe 15 sets of Hnalpha recombination lines simultaneously in two HII regions that are particularly interesting from a Galactic structure perspective. Their HI absorption spectra indicate that they are on the far side of the Galactic centre, beyond the solar circle. G337 has no reported recombination lines, while G334 has two recombination line detections at quite different velocities. These may be among the most distant HII regions known, with relatively high luminosities and dramatic structure in the infrared continuum. In this project we will detect and map the recombination line and continuum emission from the ionized gas, and measure much more accurate 21-cm absorption spectra.

  5. Recombinant vaccines: experimental and applied aspects

    Lorenzen, Niels

    1999-01-01

    Development of vaccines for aquaculture fish represent an important applied functional aspect of fish immunology research. Particularly in the case of recombinant vaccines, where a single antigen is usually expected to induce immunity to a specific pathogen, knowledge of mechanisms involved in...... induction of a protective immune response may become vital. The few recombinant vaccines licensd so far, despite much research during the last decade, illustrate that this is not a straightforward matter. However, as vaccine technology as well as our knowledge of the fish immune system is steadily improved......, these fields will open up a number of interesting research objectives of mutual benefit. Recent aspects of recombinant protein vaccines, live recombinant vaccines and DNA vaccines are discussed....

  6. Potential energy curves for dissociative recombination

    This paper presents a discussion of ab inito calculations of potential energy curves. The direct dissociative recombination (DR) of a diatomic molecular ion is identified. Atomic and molecular recombination are briefly compared. The DR mechanisms which will proceed with shock rate if there is a favorable crossing between the potential energy curves for Ds* and Ds+ in (1) are studied. The nature of dissociative states is considered. Wave functions, configuration of orbital, orbital optimization, configuration interaction, are surveyed. The dissociative recombination of 02+ to O ('S), to O ('D), to O (3p) + O (3p) are calculated. The dissociative recombination of H2+ and He2+, and potential energy curves for all the dissociations, are graphed

  7. Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine

    This page contains brief information about recombinant human papillomavirus (HPV) nonavalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  8. Recombinant Human Papillomavirus (HPV) Bivalent Vaccine

    This page contains brief information about recombinant human papillomavirus (HPV) bivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  9. Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine

    This page contains brief information about recombinant human papillomavirus (HPV) quadrivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  10. Constraints from jet calculus on quark recombination

    Within the quantum-chromodynamic jet-calculus formalism, we deduce an equation describing recombination of quarks and antiquarks into mesons within a quark or gluon jet. This equation relates the recombination function R(x1,x2,x) used in current literature to the fragmentation function for producing that same meson out of the parton initiating the jet. We submit currently used recombination functions to our consistency test, taking as input mainly the u-quark fragmentation ''data'' into π+ mesons. The qq-bar→π recombination functions popular in the literature are consistent with measured fragmentation functions, but they must be supplemented by other contributions to provide the full Dπ+/sub u/. We also discuss the Q2 dependence of the resulting fragmentation functions

  11. Recombination Effects on Supernovae Light-Curves

    Goldfriend, Tomer; Sari, Re'em

    2014-01-01

    Supernovae of type IIP are marked by the long plateau seen in their optical light curves. The plateau is believed to be the result of a recombination wave that propagates through the outflowing massive hydrogen envelope. Here, we analytically investigate the transition from a fully ionized envelope to a partially recombined one and its effects on the SN light curve. The motivation is to establish the underlying processes which dominate the evolution at late times when recombination takes place in the envelope, yet early enough so that $^{56}$Ni decay is a negligible source of energy. We assume a simple, yet adequate, hydrodynamic profile of the envelope and study the mechanisms which dominate the energy emission and the observed temperature. We consider the diffusion of photons through the envelope while analyzing the ionization fraction and the coupling between radiation and gas. We find that once recombination starts, the observed temperature decreases slowly in time. However, in a typical red supergiant (R...

  12. Recombination-deficient mutant of Streptococcus faecalis

    An ultraviolet radiation-sensitive derivative of Streptococcus faecalis strain JH2-2 was isolated and found to be deficient in recombination, using a plasmid-plasmid recombination system. The strain was sensitive to chemical agents which interact with deoxyribonucleic acid and also underwent deoxyribonucleic acid degradation after ultraviolet irradiation. Thus, the mutant has properties similar to those of recA strains of Escherichia coli

  13. Recombination-generation currents in degenerate semiconductors

    Von Roos, O.

    1978-01-01

    The classical Shockley-Read-Hall theory of free carrier recombination and generation via traps is extended to degenerate semiconductors. A concise and simple expression is found which avoids completely the concept of a Fermi level, a concept which is alien to nonequilibrium situations. Assumptions made in deriving the recombination generation current are carefully delineated and are found to be basically identical to those made in the original theory applicable to nondegenerate semiconductors.

  14. Recombination-deficient mutant of Streptococcus faecalis.

    Yagi, Y; Clewell, D B

    1980-01-01

    An ultraviolet radiation-sensitive derivative of Streptococcus faecalis strain JH2-2 was isolated and found to be deficient in recombination, using a plasmid-plasmid recombination system. The strain was sensitive to chemical agents which interact with deoxyribonucleic acid and also underwent deoxyribonucleic acid degradation after ultraviolet irradiation. Thus, the mutant has properties similar to those of recA strains of Escherichia coli.

  15. Dissociative Recombination of Astrochemically Interesting Ions

    Hamberg, Mathias

    2010-01-01

    In this thesis the major work described concerns experimental determination of the dissociative recombination (DR) reaction for several molecular ions of astrochemical interest. DR is the process where an electron recombines with a molecular ion to form an excited neutral that disintegrates into two or more neutral fragments to release the gained excess energy. It is very efficient under cold conditions and therefore ubiquitously occurring in interstellar environments such as dark clouds and ...

  16. Dielectronic recombination of hydrogen-like ions

    Decay dynamics of dielectronic recombination (DR) processes of H-like titanium ions was investigated with an electron beam ion trap. In the DR of H-like ions a K-shell vacancy is available even after the decay of the doubly excited state produced by the recombination. Therefore secondary X-ray emission is possible. An observed X-ray spectrum of DR obtained in the present experiment was well reproduced theoretically by taking into account the secondary X-rays

  17. Detecting the cosmological recombination signal from space

    Desjacques, Vincent; Chluba, Jens; Silk, Joseph; de Bernardis, Francesco; Dor, Olivier

    2015-08-01

    Spectral distortions of the cosmic microwave background (CMB) have recently experienced an increased interest. One of the inevitable distortion signals of our cosmological concordance model is created by the cosmological recombination process, just a little before photons last scatter at redshift z ? 1100. These cosmological recombination lines, emitted by the hydrogen and helium plasma, should still be observable as tiny deviation from the CMB blackbody spectrum in the cm-dm spectral bands. In this paper, we present a forecast for the detectability of the recombination signal with future satellite experiments. We argue that serious consideration for future CMB experiments in space should be given to probing spectral distortions and, in particular, the recombination line signals. The cosmological recombination radiation not only allows determination of standard cosmological parameters, but also provides a direct observational confirmation for one of the key ingredients of our cosmological model: the cosmological recombination history. We show that, with present technology, such experiments are futuristic but feasible. The potential rewards won by opening this new window to the very early universe could be considerable.

  18. The kissing-loop motif is a preferred site of 5' leader recombination during replication of SL3-3 murine leukemia viruses in mice

    Lund, Anders Henrik; Mikkelsen, J G; Schmidt, J; Duch, M; Pedersen, F S

    1999-01-01

    exogenous input virus and endogenous MLV-like sequences within the 5' leader region. Evidence of recombination within the region studied was found in 14 of 52 tumors analyzed. Sequence analysis of a approximately 330-bp fragment of 44 chimeric proviruses, encompassing the U5, the primer binding site, and...... loop, presumably via a role in RNA dimer formation, constitutes a hot spot for reverse transcriptase-mediated recombination in MLV....

  19. A recombinase-mediated transcriptional induction system in transgenic plants

    Hoff, T; Schnorr, K M; Mundy, J

    2001-01-01

    We constructed and tested a Cre-loxP recombination-mediated vector system termed pCrox for use in transgenic plants. In this system, treatment of Arabidopsis under inducing conditions mediates an excision event that removes an intervening piece of DNA between a promoter and the gene to be expressed......-mediated GUS activation. Induction was shown to be possible at essentially any stage of plant growth. This single vector system circumvents the need for genetic crosses required by other, dual recombinase vector systems. The pCrox system may prove particularly useful in instances where transgene over...

  20. Recombination analysis based on the complete genome of bocavirus

    Chen Shengxia

    2011-04-01

    Full Text Available Abstract Bocavirus include bovine parvovirus, minute virus of canine, porcine bocavirus, gorilla bocavirus, and Human bocaviruses 1-4 (HBoVs. Although recent reports showed that recombination happened in bocavirus, no systematical study investigated the recombination of bocavirus. The present study performed the phylogenetic and recombination analysis of bocavirus over the complete genomes available in GenBank. Results confirmed that recombination existed among bocavirus, including the likely inter-genotype recombination between HBoV1 and HBoV4, and intra-genotype recombination among HBoV2 variants. Moreover, it is the first report revealing the recombination that occurred between minute viruses of canine.

  1. Graded Recombination Layers for Multijunction Photovoltaics

    Koleilat, Ghada I.

    2012-06-13

    Multijunction devices consist of a stack of semiconductor junctions having bandgaps tuned across a broad spectrum. In solar cells this concept is used to increase the efficiency of photovoltaic harvesting, while light emitters and detectors use it to achieve multicolor and spectrally tunable behavior. In series-connected current-matched multijunction devices, the recombination layers must allow the hole current from one cell to recombine, with high efficiency and low voltage loss, with the electron current from the next cell. We recently reported a tandem solar cell in which the recombination layer was implemented using a progression of n-type oxides whose doping densities and work functions serve to connect, with negligible resistive loss at solar current densities, the constituent cells. Here we present the generalized conditions for design of efficient graded recombination layer solar devices. We report the number of interlayers and the requirements on work function and doping of each interlayer, to bridge an work function difference as high as 1.6 eV. We also find solutions that minimize the doping required of the interlayers in order to minimize optical absorption due to free carriers in the graded recombination layer (GRL). We demonstrate a family of new GRL designs experimentally and highlight the benefits of the progression of dopings and work functions in the interlayers. © 2012 American Chemical Society.

  2. Human recombinant lysosomal enzymes produced in microorganisms.

    Espejo-Mojica, ngela J; Almciga-Daz, Carlos J; Rodrguez, Alexander; Mosquera, ngela; Daz, Dennis; Beltrn, Laura; Daz, Sergio; Pimentel, Natalia; Moreno, Jefferson; Snchez, Jhonnathan; Snchez, Oscar F; Crdoba, Henry; Poutou-Piales, Ral A; Barrera, Luis A

    2015-01-01

    Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD. PMID:26071627

  3. Metallurgy of recycled lead for recombinant batteries

    David Prengaman, R.

    Recombinant and stationary lead/acid batteries generally use leadcalciumtin alloys for grids, leadtin alloys for strap and top lead, and high purity lead for oxide. In many cases, primary lead has been specified for these materials in recombinant batteries because both recycled lead and recycled lead alloys were not thought to be of sufficient purity. With improvements in analytical instruments, it is now possible to determine impurity elements at much lower levels. Because problem elements can be analyzed, refining procedures have now been developed to remove gas-producing impurity elements from recycled lead to levels as low as, or lower than, those in primary lead. These new refinery practices and analytical tests have increased the purity of refined recycled lead, and have permitted the use of recycled lead in recombinant and stand-by batteries. Some elements, deemed to be impurities that must be removed, may in fact be beneficial in alloys for recombinant batteries. This paper examines the purity requirements of lead and lead alloys for recombinant and stand-by batteries, indicates the problem elements, and shows the effects of beneficial elements.

  4. Graded recombination layers for multijunction photovoltaics.

    Koleilat, Ghada I; Wang, Xihua; Sargent, Edward H

    2012-06-13

    Multijunction devices consist of a stack of semiconductor junctions having bandgaps tuned across a broad spectrum. In solar cells this concept is used to increase the efficiency of photovoltaic harvesting, while light emitters and detectors use it to achieve multicolor and spectrally tunable behavior. In series-connected current-matched multijunction devices, the recombination layers must allow the hole current from one cell to recombine, with high efficiency and low voltage loss, with the electron current from the next cell. We recently reported a tandem solar cell in which the recombination layer was implemented using a progression of n-type oxides whose doping densities and work functions serve to connect, with negligible resistive loss at solar current densities, the constituent cells. Here we present the generalized conditions for design of efficient graded recombination layer solar devices. We report the number of interlayers and the requirements on work function and doping of each interlayer, to bridge an work function difference as high as 1.6 eV. We also find solutions that minimize the doping required of the interlayers in order to minimize optical absorption due to free carriers in the graded recombination layer (GRL). We demonstrate a family of new GRL designs experimentally and highlight the benefits of the progression of dopings and work functions in the interlayers. PMID:22554234

  5. Polyploidization increases meiotic recombination frequency in Arabidopsis

    Rehmsmeier Marc

    2011-04-01

    Full Text Available Abstract Background Polyploidization is the multiplication of the whole chromosome complement and has occurred frequently in vascular plants. Maintenance of stable polyploid state over generations requires special mechanisms to control pairing and distribution of more than two homologous chromosomes during meiosis. Since a minimal number of crossover events is essential for correct chromosome segregation, we investigated whether polyploidy has an influence on the frequency of meiotic recombination. Results Using two genetically linked transgenes providing seed-specific fluorescence, we compared a high number of progeny from diploid and tetraploid Arabidopsis plants. We show that rates of meiotic recombination in reciprocal crosses of genetically identical diploid and autotetraploid Arabidopsis plants were significantly higher in tetraploids compared to diploids. Although male and female gametogenesis differ substantially in meiotic recombination frequency, both rates were equally increased in tetraploids. To investigate whether multivalent formation in autotetraploids was responsible for the increased recombination rates, we also performed corresponding experiments with allotetraploid plants showing strict bivalent pairing. We found similarly increased rates in auto- and allotetraploids, suggesting that the ploidy effect is independent of chromosome pairing configurations. Conclusions The evolutionary success of polyploid plants in nature and under domestication has been attributed to buffering of mutations and sub- and neo-functionalization of duplicated genes. Should the data described here be representative for polyploid plants, enhanced meiotic recombination, and the resulting rapid creation of genetic diversity, could have also contributed to their prevalence.

  6. Development of non-defective recombinant densovirus vectors for microRNA delivery in the invasive vector mosquito, Aedes albopictus.

    Liu, Peiwen; Li, Xiaocong; Gu, Jinbao; Dong, Yunqiao; Liu, Yan; Santhosh, Puthiyakunnon; Chen, Xiaoguang

    2016-01-01

    We previously reported that mosquito densoviruses (MDVs) are potential vectors for delivering foreign nucleic acids into mosquito cells. However, considering existing expression strategies, recombinant viruses would inevitably become replication-defective viruses and lose their ability for secondary transmission. The packaging limitations of the virion represent a barrier for the development of MDVs for viral paratransgenesis or as high-efficiency bioinsecticides. Herein, we report the development of a non-defective recombinant Aedes aegypti densovirus (AaeDV) miRNA expression system, mediated by an artificial intron, using an intronic miRNA expression strategy. We demonstrated that this recombinant vector could be used to overexpress endogenous miRNAs or to decrease endogenous miRNAs by generating antisense sponges to explore the biological functions of miRNAs. In addition, the vector could express antisense-miRNAs to induce efficient gene silencing in vivo and in vitro. The recombinant virus effectively self-replicated and retained its secondary transmission ability, similar to the wild-type virus. The recombinant virus was also genetically stable. This study demonstrated the first construction of a non-defective recombinant MDV miRNA expression system, which represents a tool for the functional analysis of mosquito genes and lays the foundation for the application of viral paratransgenesis for dengue virus control. PMID:26879823

  7. Development of non-defective recombinant densovirus vectors for microRNA delivery in the invasive vector mosquito, Aedes albopictus

    Liu, Peiwen; Li, Xiaocong; Gu, Jinbao; Dong, Yunqiao; Liu, Yan; Santhosh, Puthiyakunnon; Chen, Xiaoguang

    2016-01-01

    We previously reported that mosquito densoviruses (MDVs) are potential vectors for delivering foreign nucleic acids into mosquito cells. However, considering existing expression strategies, recombinant viruses would inevitably become replication-defective viruses and lose their ability for secondary transmission. The packaging limitations of the virion represent a barrier for the development of MDVs for viral paratransgenesis or as high-efficiency bioinsecticides. Herein, we report the development of a non-defective recombinant Aedes aegypti densovirus (AaeDV) miRNA expression system, mediated by an artificial intron, using an intronic miRNA expression strategy. We demonstrated that this recombinant vector could be used to overexpress endogenous miRNAs or to decrease endogenous miRNAs by generating antisense sponges to explore the biological functions of miRNAs. In addition, the vector could express antisense-miRNAs to induce efficient gene silencing in vivo and in vitro. The recombinant virus effectively self-replicated and retained its secondary transmission ability, similar to the wild-type virus. The recombinant virus was also genetically stable. This study demonstrated the first construction of a non-defective recombinant MDV miRNA expression system, which represents a tool for the functional analysis of mosquito genes and lays the foundation for the application of viral paratransgenesis for dengue virus control. PMID:26879823

  8. Mechanism Analysis Indicates That Recombination Events In HIV-1 Initiate And Complete Over Short Distances, Explaining Why Recombination Frequency is Similar in Different Sections of the Genome

    Rigby, Sean T.; Rose, April E.; Hanson, Mark N.; Bambara, Robert A.

    2009-01-01

    Strand transfer drives recombination between the co-packaged genomes of HIV-1, a process that allows rapid viral evolution. The proposed invasion-mediated mechanism of strand transfer during HIV-1 reverse transcription has three steps: invasion of the initial or donor primer-template by the second or acceptor template, propagation of the primer-acceptor hybrid, and then primer terminus transfer. Invasion occurs at a site at which the RT RNase has created a nick or short gap in the donor templ...

  9. Homologous Recombination and Its Role in Carcinogenesis

    Bishop Alexander J. R.

    2002-01-01

    Full Text Available Cancer develops when cells no longer follow their normal pattern of controlled growth. In the absence or disregard of such regulation, resulting from changes in their genetic makeup, these errant cells acquire a growth advantage, expanding into precancerous clones. Over the last decade, many studies have revealed the relevance of genomic mutation in this process, be it by misreplication, environmental damage, or a deficiency in repairing endogenous and exogenous damage. Here, we discuss homologous recombination as another mechanism that can result in a loss of heterozygosity or genetic rearrangements. Some of these genetic alterations may play a primary role in carcinogenesis, but they are more likely to be involved in secondary and subsequent steps of carcinogenesis by which recessive oncogenic mutations are revealed. Patients, whose cells display an increased frequency of recombination, also have an elevated frequency of cancer, further supporting the link between recombination and carcinogenesis.

  10. Jet Fragmentation via Recombination of Parton Showers

    Han, Kyong Chol; Ko, Che Ming

    2016-01-01

    We propose to model hadronization of parton showers in QCD jets through a hybrid approach involving quark recombination and string fragmentation. This is achieved by allowing gluons at the end of the perturbative shower evolution to undergo a non-perturbative splitting into quark and antiquark pairs, then applying a Monte-Carlo version of instantaneous quark recombination, and finally subjecting remnant quarks (those which have not found a recombination partner) to Lund string fragmentation. When applied to parton showers from the PYTHIA Monte Carlo event generator, the final hadron spectra from our calculation compare quite well to PYTHIA jets that have been hadronized with the default Lund string fragmentation. Our new approach opens up the possibility to generalize hadronization to jets embedded in a quark gluon plasma.

  11. Constraints from jet calculus on quark recombination

    Within the QCD jet calculus formalism, we deduce an equation describing recombination of quarks and antiquarks into mesons within a quark or gluon jet. This equation relates the recombination function R(x1,x2,x) used in current literature to the fragmentation function for producing that same meson out of the parton initiating the jet. We submit currently used recombination functions to our consistency test, taking as input mainly the u-quark fragmentation data into π+ mesons, but also s-quark fragmentation into K- mesons. The constraint is well satisfied at large Q2 for large moments. Our results depend on one parameter, Q02, the constraint equation being satisfied for small values of this parameter

  12. Jet fragmentation via recombination of parton showers

    Han, Kyong Chol; Fries, Rainer J.; Ko, Che Ming

    2016-04-01

    We propose to model hadronization of parton showers in QCD jets through a hybrid approach involving quark recombination and string fragmentation. This is achieved by allowing gluons at the end of the perturbative shower evolution to undergo a nonperturbative splitting into quark and antiquark pairs, then applying a Monte Carlo version of instantaneous quark recombination, and finally subjecting remnant quarks (those which have not found a recombination partner) to Lund string fragmentation. When applied to parton showers from the pythia Monte Carlo event generator, the final hadron spectra from our calculation compare quite well to pythia jets that have been hadronized with the default Lund string fragmentation. Our new approach opens up the possibility to generalize hadronization to jets embedded in a quark gluon plasma.

  13. Dielectronic recombination of xenonlike tungsten ions

    Schippers, S; Mller, A; Krantz, C; Grieser, M; Repnow, R; Wolf, A; Lestinsky, M; Hahn, M; Novotn, O; Savin, D W

    2011-01-01

    Dielectronic recombination (DR) of xenonlike W20+ forming W19+ has been studied experimentally at a heavy-ion storage-ring. A merged-beams method has been employed for obtaining absolute rate coefficients for electron-ion recombination in the collision energy range 0-140 eV. The measured rate coefficient is dominated by strong DR resonances even at the lowest experimental energies. At plasma temperatures where the fractional abundance of W20+ is expected to peak in a fusion plasma, the experimentally derived plasma recombination rate coefficient is over a factor of 4 larger than the theoretically-calculated rate coefficient which is currently used in fusion plasma modeling. The largest part of this discrepancy stems most probably from the neglect in the theoretical calculations of DR associated with fine-structure excitations of the W20+([Kr] 4d10 4f8) ion core.

  14. Comparison of recombination models in organic bulk heterojunction solar cells

    Recombination in bulk-heterojunction (BHJ) organic solar cells is the key loss mechanism, and it directly affects characteristic parameters such as power conversion efficiency, short-circuit current, open-circuit voltage, and fill factor. However, which recombination mechanism dominates the loss in organic materials is unclear at present. In this work, we simulate state-of-art BHJ solar cells using five recombination models, including direct recombination, Langevin recombination, charge transfer state recombination, trap-assisted recombination, and recombination via tail. All processes are strongly dependent on charge carrier mobility and exhibit a similar recombination distribution in active layer. For high mobilities, all models present a similar behavior along with the increased mobilities, whereas, there are slight differences in open-circuit voltage between trap/tail model and other ones at lower mobilities, resulting from the interaction between photo-carriers and dark-carriers

  15. Chromosomal transformation in Bacillus subtilis is a non-polar recombination reaction.

    Carrasco, Begoña; Serrano, Ester; Sánchez, Humberto; Wyman, Claire; Alonso, Juan C

    2016-04-01

    Natural chromosomal transformation is one of the primary driving forces of bacterial evolution. This reaction involves the recombination of the internalized linear single-stranded (ss) DNA with the homologous resident duplex via RecA-mediated integration in concert with SsbA and DprA or RecO. We show that sequence divergence preventsBacillus subtilischromosomal transformation in a log-linear fashion, but it exerts a minor effect when the divergence is localized at a discrete end. In the nucleotide bound form, RecA shows no apparent preference to initiate recombination at the 3'- or 5'-complementary end of the linear duplex with circular ssDNA, but nucleotide hydrolysis is required when heterology is present at both ends. RecA·dATP initiates pairing of the linear 5' and 3' complementary ends, but only initiation at the 5'-end remains stably paired in the absence of SsbA. Our results suggest that during gene transfer RecA·ATP, in concert with SsbA and DprA or RecO, shows a moderate preference for the 3'-end of the duplex. We show that RecA-mediated recombination initiated at the 3'- or 5'-complementary end might have significant implication on the ecological diversification of bacterial species with natural transformation. PMID:26786319

  16. Dielectronic recombination of xenonlike tungsten ions

    Schippers, S.; Bernhardt, D; Mller, A.; Krantz, C.; Grieser, M.; Repnow, R.; Wolf, A.; Lestinsky, M; Hahn, M. (Matthias); Novotn, O.; Savin, D. W.

    2011-01-01

    Dielectronic recombination (DR) of xenonlike W20+ forming W19+ has been studied experimentally at a heavy-ion storage-ring. A merged-beams method has been employed for obtaining absolute rate coefficients for electron-ion recombination in the collision energy range 0-140 eV. The measured rate coefficient is dominated by strong DR resonances even at the lowest experimental energies. At plasma temperatures where the fractional abundance of W20+ is expected to peak in a fusion plasma, the experi...

  17. Dielectronic recombination measurements of multicharged ions

    Dielectronic recombination rates have been measured for several charge states of isoelectronic Li-like, Be-like, B-like, and Na-like ions. The amount of electron capture attending the passage of MeV/nucleon ion beams through a collinear, magnetically confined space-charge-limited electron beam as a function of relative energy has been observed. The experimental rates are consistent in magnitude and shape with rates determined from distorted-wave calculations of the dielectronic-recombination cross sections. 72 refs., 6 figs

  18. DNA origami based visualization system for studying site-specific recombination events.

    Suzuki, Yuki; Endo, Masayuki; Katsuda, Yousuke; Ou, Keiyu; Hidaka, Kumi; Sugiyama, Hiroshi

    2014-01-01

    Site-specific recombination involves reciprocal exchange between defined DNA sites. The reaction initiates from the formation of a recombinase-DNA synaptic complex, in which two recombination sites arrange in an appropriate configuration. However, there is incomplete information about how the topological state of the substrate influences the synapsis and outcome of the reaction. Here, we show that Cre-mediated recombination can be regulated by controlling the orientation and topology of the loxP substrate in a DNA frame nanoscaffold. High-speed atomic force microscopy analyses revealed that the loxP-containing substrate strands in the antiparallel orientation can be recombined only through formation of synaptic complexes. By tethering Holliday junction (HJ) intermediates to DNA frames in different connection patterns and using them as a starting substrate, we found that the topological state of the HJ intermediates dictates the outcome of the resolution. Our approach should provide a new platform for structural-functional studies of various DNA targeting enzymes, especially which require formation of synaptic complexes. PMID:24328161

  19. Recombination-activating gene 1 and 2 (RAG1 and RAG2) in flounder (Paralichthys olivaceus)

    Xianlei Wang; Xungang Tan; Pei-Jun Zhang; Yuqing Zhang; Peng Xu

    2014-12-01

    During the development of B and T lymphocytes, Ig and TCR variable region genes are assembled from germline V, D, and J gene segments by a site-specific recombination reaction known as V(D)J recombination. The process of somatic V(D)J recombination, mediated by the recombination-activating gene (RAG) products, is the most significant characteristic of adaptive immunity in jawed vertebrates. Flounder (Paralichthys olivaceus) RAG1 and RAG2 were isolated by Genome Walker and RT-PCR, and their expression patterns were analysed by RT-PCR and in situ hybridization on sections. RAG1 spans over 7.0 kb, containing 4 exons and 3 introns, and the full-length ORF is 3207 bp, encoding a peptide of 1068 amino acids. The first exon lies in the 5′-UTR, which is an alternative exon. RAG2 full-length ORF is 1062 bp, encodes a peptide of 533 amino acids, and lacks introns in the coding region. In 6-month-old flounders, the expression of RAG1 and RAG2 was essentially restricted to the pronephros (head kidney) and mesonephros (truck kidney). Additionally, both of them were mainly expressed in the thymus. These results revealed that the thymus and kidney most likely serve as the primary lymphoid tissues in the flounder.

  20. Construction and analysis of the transgenic carrot and celery plants expressing the recombinant thaumatin II protein

    Luchakivska Yu. S.

    2015-08-01

    Full Text Available Aim To obtain the transgenic carrot and celery plants able to express recombinant thaumatin II in order to increase plant stress tolerance. Methods. Agrobacterium-mediated transformation of the carrot and celery seedlings was used for obtaining the transgenic plants. Presence and transcription of the transgene in plant tissues were proved by PCR and RT-PCR analysis. The plants were tested for the biotic stress tolerance by in vitro antifungal and antibacterial activity assays and for the salinity and osmotic stress tolerance by plant survival test in presence of NaCl and PEG in different concentrations. Results. Transgenic plants able to express recombinant thaumatin II gene (transcription proved for 60–100 % were obtained by agrobacterial transformation. The transgenic carrot plant extracts inhibited the growth of the studied phytopathogenic bacteria strains but exhibited no antifungal activity. Survival level of transgenic plants under the salinity and osmotic stress effect was definitely higher comparing to the untransgenic ones. The analysis of the photosynthetic pigment content in the transgenic carrot plants showed no significant difference of this parameter under salinity stress that may indicate a possible protective activity of the recombinant protein. Conclusions. The obtained in our study transgenic carrot and celery plants able to express the recombinant thaumatin II gene were characterized by antibacterial activity and increased tolerance to salinity and osmotic stress factors.

  1. Immune responses of pigs immunized with a recombinant porcine reproductive and respiratory syndrome virus expressing porcine GM-CSF.

    Li, Zhijun; Wang, Gang; Wang, Yan; Zhang, Chong; Huang, Baicheng; Li, Qiongyi; Li, Liangliang; Xue, Biyun; Ding, Peiyang; Cai, Xuehui; Wang, Chengbao; Zhou, En-Min

    2015-11-15

    Porcine reproductive and respiratory syndrome virus (PRRSV) has spread worldwide, causing huge economic losses to the swine industry. The current PRRSV vaccines have failed to provide broad protection against various strains. Granulocyte macrophage colony-stimulating factor (GM-CSF), an efficacious adjuvant, has been shown to enhance the immunogenicity of various vaccines. The purpose of this study was to construct a recombinant live attenuated PRRSV that expresses porcine GM-CSF (pGM-CSF) and evaluate the immune responses of pigs immunized with the recombinant virus. The results showed that the recombinant PRRSV was successfully rescued and had similar growth properties to parental virus grown in Marc-145 cells. The recombinant virus was stable for 10 passages in cell culture. Pigs intramuscularly immunized with the recombinant virus produced a similar humoral response to that elicited using parental virus. With regard to cell-mediated immunity assessed in peripheral blood, the recombinant virus induced higher proportion of CD4(+)CD8(+) double-positive T cells (DPT), higher IFN-? level at 0 and 7 days post-challenge (DPC), and lower viremia at 21 DPC than pigs immunized with parental virus. These results indicate that recombinant PRRSV expressing pGM-CSF can induce a significant higher cellular immune response and reduce the persistent infection compared pigs vaccinated with the parental virus. This is first report of evaluation of immune response in pigs elicited by a recombinant live attenuated PRRSV expressing porcine GM-CSF. It may represent a novel strategy for future development of genetic engineered vaccines against PRRSV infection. PMID:26300317

  2. Anomalous Abundances in Gaseous Nebulae From Recombination and Collisional Lines: Improved Photoionization and Recombination Studies

    Pradhan, Anil Kumar; Nahar, S. N.; Eissner, W. B.; Montenegro, M.

    2011-01-01

    A perplexing anomaly arises in the determination of abundances of common elements in gaseous nebulae, as derived from collisionally excited lines (CEL) as opposed to those from Recombination Lines (RCL). The "abundance discrepancy factors" can range from a factor of 2 to an order of magnitude or more. That has led to quite different interpretation of the physical structure and processes in gaseous nebulae, such as temperature fluctuations across the object, or metal-rich concentrations leading to a dual-abundnace scenario. We show that the problem may lie in inaccuracies in photoionization and recombination models neglecting low-energy resonance phenomena due to fine structure. Whereas the atomic physics of electron impact excitation of forbidden lines is well understood, and accurate collision strengths have long been available, that is not generally the case for electron-ion recombination cross sections. A major problem is the inclusion of relativisitic effects as it pertains to the existence of very low-energy fine structure resonances in photoionization cross sections. We carry out new relativistic calculations for photoionization and recombination cross sections using a recently extended version of the Breit-Pauli R-matrix codes, and the unified electron-ion recombination method that subsumes both the radiative and the dielectronic recombination (RR and DR) processes in an ab initio and self-consistent manner. We find that near-thresold resonances manifest themselves within fine structure levels of the ground state of ions, enhancing low-temperature recombination rate coefficients at 1000-10,000 K. The resulting enahncement in level-specific and total recombination rate coefficients should therefore lead to reduced abundances derived from RCL, and in accordance with those from CEL. We present results for photoionization of O II into, and recombination from, O III. Theoretical cross sections are benchmarked against high-resolution measurements from synchrotron based light sources. Work on other atomic species is in progress.

  3. Recombination coefficients in extrinsic n-InSb

    The bulk recombination coefficients for linear recombination via recombination centers as well as for direct recombination have been determined measuring the conductivity decay after two-photon absorption with a CO2 laser. The Suhl effect was applied to measure the surface recombination velocity. The corresponding literature is discussed and compared with our results. We conclude that two different kinds of recombination centers are possible in n-InSb, with energy levels (0.1-0.12)eV above the valence band, or (0.14-0.2)eV respectively. (orig.)

  4. Influence of exogenous phytohormones, methyl jasmonate and suppressors of jasmonate biosynthesis on Agrobacterium-mediated transient expression in Nicotiana excelsior

    Kuchuk M. V.; Gerasymenko I. M.; Sindarovska Y. R.; Sheludko Y. V.

    2012-01-01

    Our aim was to investigate the influence of some exogenous agents on the recombinant protein accumulation in plants via Agrobacterium-mediated transient expression. Methods. Agrobacterium-mediated transient expression method, spectrophotometric methods for protein analysis, statistical calculations. Results. It was shown that the tested compounds in different concentrations (namely, auxins, cytokinin, methyl jasmonate and suppressors of jasmonate biosynthesis (phenidon and diethyldithiocarbam...

  5. Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

    Penn Charles W

    2009-12-01

    Full Text Available Abstract Background Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains. Results Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the λ-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6 × His, 3 × FLAG, 4 × ProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the λ-Red system, which can lead to unwanted secondary alterations to the chromosome. Conclusion We have developed a counter-selective recombineering technique for epitope tagging or for deleting genes in E. coli. We have demonstrated the versatility of the technique by modifying the chromosome of the enterohaemorrhagic O157:H7 (EHEC, uropathogenic CFT073 (UPEC, enteroaggregative O42 (EAEC and enterotoxigenic H10407 (ETEC E. coli strains as well as in K-12 laboratory strains.

  6. Recombinant Bovine Growth Hormone Criticism Grows.

    Gaard, Greta

    1995-01-01

    Discusses concerns related to the use of recombinant bovine growth hormone in the United States and other countries. Analyses the issue from the perspectives of animal rights, human health, world hunger, concerns of small and organic farmers, costs to the taxpayer, and environmental questions. A sidebar discusses Canadian review of the hormone.…

  7. Algae-based oral recombinant vaccines.

    Specht, Elizabeth A; Mayfield, Stephen P

    2014-01-01

    Recombinant subunit vaccines are some of the safest and most effective vaccines available, but their high cost and the requirement of advanced medical infrastructure for administration make them impractical for many developing world diseases. Plant-based vaccines have shifted that paradigm by paving the way for recombinant vaccine production at agricultural scale using an edible host. However, enthusiasm for "molecular pharming" in food crops has waned in the last decade due to difficulty in developing transgenic crop plants and concerns of contaminating the food supply. Microalgae could be poised to become the next candidate in recombinant subunit vaccine production, as they present several advantages over terrestrial crop plant-based platforms including scalable and contained growth, rapid transformation, easily obtained stable cell lines, and consistent transgene expression levels. Algae have been shown to accumulate and properly fold several vaccine antigens, and efforts are underway to create recombinant algal fusion proteins that can enhance antigenicity for effective orally delivered vaccines. These approaches have the potential to revolutionize the way subunit vaccines are made and delivered - from costly parenteral administration of purified protein, to an inexpensive oral algae tablet with effective mucosal and systemic immune reactivity. PMID:24596570

  8. Recombinative plasma in electron runaway discharge

    Kuznetsov, Yu. K.; Galvão, R. M. O.; Usuriaga, O. C.; Krasheninnikov, S. I.; Soboleva, T. K.; Tsypin, V. S.; Fonseca, A. M. M.; Ruchko, L. F.; Sanada, E. K.

    2005-07-01

    Cold recombinative plasma is the basic feature of the new regime of runaway discharges recently discovered in the Tokamak Chauffage Alfvén Brésilien tokamak [R. M. O. Galvão et al., Plasma Phys. Controlled Fusion 43, 1181 (2001)]. With low plasma temperature, the resistive plasma current and primary Dreicer process of runaway generation are strongly suppressed at the stationary phase of the discharge. In this case, the runaway avalanche, which has been recently recognized as a novel important mechanism for runaway electron generation in large tokamaks, such as International Thermonuclear Experimental Reactor, during disruptions, and for electric breakdown in matter, is the only mechanism responsible for toroidal current generation and can be easily observed. The measurement of plasma temperature by the usual methods is a difficult task in fully runaway discharges. In the present work, various indirect evidences for low-temperature recombinative plasma are presented. The direct observation of recombinative plasma is obtained as plasma detachment from the limiter. The model of cold recombinative plasma is also supported by measurements of plasma density and Hα emission radial profiles, analysis of time variations of these parameters due to the relaxation instability, estimations of plasma resistivity from voltage spikes, and energy and particle balance calculations.

  9. Elimination kinetic of recombinant somatotropin in bovine.

    Le Breton, Marie-Hélène; Rochereau-Roulet, Sandrine; Pinel, Gaud; Cesbron, Nora; Le Bizec, Bruno

    2009-04-01

    Bovine somatotropin (bST), also called growth hormone is a protein hormone produced by the pituitary gland and responsible directly or indirectly for various effects on growth, development and reproductive functions. Its recombinant bovine somatotropin form (rbST) is used in dairy cattle to enhance milk production. Even if the effects of treatment with rbST have been largely studied, until now analytical methods able to detect rbST were limited to immunoassays, which suffer from the impossibility to distinguish between the endogenous and the recombinant form. In this study, a sample preparation procedure based on different precipitation steps, extraction on solid phase and enzymatic digestion was used to purify rbST from serum. The detection was performed by liquid chromatography coupled to tandem mass spectrometry in positive electrospray ionization mode (LC-ESI(+)-MS/MS) allowing the unambiguous identification and quantification of rbST in serum. Samples collected from a cow treated with recombinant bovine somatotropin were analysed and for the first time, the elimination kinetic specific to recombinant somatotropin has been characterized in serum. Detection of rbST was possible from 4h 30min to 4 days after administration and concentration was found up to 10ngmL(-1) during the kinetic. PMID:19286020

  10. Precise genotyping and recombination detection of Enterovirus.

    Lin, Chieh-Hua; Wang, Yu-Bin; Chen, Shu-Hwa; Hsiung, Chao; Lin, Chung-Yen

    2015-12-01

    Enteroviruses (EV) with different genotypes cause diverse infectious diseases in humans and mammals. A correct EV typing result is crucial for effective medical treatment and disease control; however, the emergence of novel viral strains has impaired the performance of available diagnostic tools. Here, we present a web-based tool, named EVIDENCE (EnteroVirus In DEep conception, http://symbiont.iis.sinica.edu.tw/evidence), for EV genotyping and recombination detection. We introduce the idea of using mixed-ranking scores to evaluate the fitness of prototypes based on relatedness and on the genome regions of interest. Using phylogenetic methods, the most possible genotype is determined based on the closest neighbor among the selected references. To detect possible recombination events, EVIDENCE calculates the sequence distance and phylogenetic relationship among sequences of all sliding windows scanning over the whole genome. Detected recombination events are plotted in an interactive figure for viewing of fine details. In addition, all EV sequences available in GenBank were collected and revised using the latest classification and nomenclature of EV in EVIDENCE. These sequences are built into the database and are retrieved in an indexed catalog, or can be searched for by keywords or by sequence similarity. EVIDENCE is the first web-based tool containing pipelines for genotyping and recombination detection, with updated, built-in, and complete reference sequences to improve sensitivity and specificity. The use of EVIDENCE can accelerate genotype identification, aiding clinical diagnosis and enhancing our understanding of EV evolution. PMID:26678286

  11. Recombination times in germanium under high pressure

    The influence of pressure on a well defined recombination process was studied. The centres were introduced by γirradiation and the lifetime determined by the decay time of photoconductivity. An optical pressure vessel is described which allows for a hydrostatic variation of 3000 bars. The diffusion constant and lifetime measurements are presented and analysed. (V.J.C.)

  12. Radiative recombination of excitons in amorphous semiconductors

    A theory for calculating the radiative lifetime of excitons in amorphous semiconductors is presented. Four possibilities of excitonic radiative recombination are considered and the corresponding rates are derived at thermal equilibrium. The radiative lifetime is calculated from the inverse of the maximum rate for all the four possibilities. Results agree very well with experiments

  13. Algae-based oral recombinant vaccines

    ElizabethASpecht

    2014-02-01

    Full Text Available Recombinant subunit vaccines are some of the safest and most effective vaccines available, but their high cost and the requirement of advanced medical infrastructure for administration make them impractical for many developing world diseases. Plant-based vaccines have shifted that paradigm by paving the way for recombinant vaccine production at agricultural scale using an edible host. However, enthusiasm for “molecular pharming” in food crops has waned in the last decade due to difficulty in developing transgenic crop plants and concerns of contaminating the food supply. Microalgae are poised to become the next candidate in recombinant subunit vaccine production, and they present several advantages over terrestrial crop plant-based platforms including scalable and contained growth, rapid transformation, easily obtained stable cell lines, and consistent transgene expression levels. Algae have been shown to accumulate and properly fold several vaccine antigens, and efforts are underway to create recombinant algal fusion proteins that can enhance antigenicity for effective orally-delivered vaccines. These approaches have the potential to revolutionize the way subunit vaccines are made and delivered – from costly parenteral administration of purified protein, to an inexpensive oral algae tablet with effective mucosal and system immune reactivity.

  14. Limiting efficiency calculation of silicon single-nanowire solar cells with considering Auger recombination

    Single-nanowire solar cells (SNSCs) have attracted considerable attention due to their unique light-harvesting capability mediated by the optical antenna effect and the high photoconversion efficiency due to the orthogonalization of the carrier collection to the photon incidence. We present a detailed prediction of the light-conversion efficiency of Si SNSCs based on finite-element simulation and thermodynamic balance analysis, with especially focusing on the comparison between SNSCs and film systems. Carrier losses due to radiative and Auger recombinations are introduced in the analysis of the limiting efficiency, which show that the Auger recombination plays a key role in accurately predicting the efficiency of Si SNSCs, otherwise, the device performance would be strongly overestimated. The study paves a more realistic way to evaluate the nanostructured solar cells based on indirect-band photoactive materials

  15. Enhanced wound healing by recombinant Escherichia coli Nissle 1917 via human epidermal growth factor receptor in human intestinal epithelial cells: therapeutic implication using recombinant probiotics.

    Choi, Hye Jin; Ahn, Jung Hoon; Park, Seong-Hwan; Do, Kee Hun; Kim, Juil; Moon, Yuseok

    2012-03-01

    The gastrointestinal mucosa has a remarkable ability to repair damage with the support of epidermal growth factor (EGF), which stimulates epithelial migration and proliferative reepithelialization. For the treatment of mucosal injuries, it is important to develop efficient methods for the localized delivery of mucoactive biotherapeutics. The basic idea in the present study came from the assumption that an intestinal probiotic vehicle can carry and deliver key recombinant medicinal proteins to the injured epithelial target in patients with intestinal ulcerative diseases, including inflammatory bowel disease. The study was focused on the use of the safe probiotic E. coli Nissle 1917, which was constructed to secrete human EGF in conjunction with the lipase ABC transporter recognition domain (LARD). Using the in vitro physically wounded monolayer model, ABC transporter-mediated EGF secretion by probiotic E. coli Nissle 1917 was demonstrated to enhance the wound-healing migration of human enterocytes. Moreover, the epithelial wound closure was dependent on EGF receptor-linked activation, which exclusively involved the subsequent signaling pathway of the mitogen-activated protein kinase kinase (MEK) extracellular-related kinases 1 and 2 (ERK1/2). In particular, the migrating frontier of the wounded edge displayed the strongest EGF receptor-linked signaling activation in the presence of the recombinant probiotic. The present study provides a basis for the clinical application of human recombinant biotherapeutics via an efficient, safe probiotic vehicle. PMID:22184415

  16. Some recent developments in the recombination model

    A critical review of the recombination model for hadron production at low P/sub T/ is first given, emphasizing not so much the successes as unanswered questions that the model faces. A systematic program to answer some of the basic questions is then developed. The theoretical framework is quantum chromodynamics. First, in what may appear as a digression, the possibility of formation of valence quark clusters (called valons) in a nucleon due to gluon bremsstrahlung and quark-pair creation is considered. Evidences are found not only for the valons in neutrino scattering data, but also indications for their momentum distribution in a nucleon. When similar considerations are applied to a meson, the meaning of the recombination function is discussed and its normalization as well as its shape are determined. Next, the problem of quark decay in a hard scattering process (e.g., pion production in e+e- annihilation) is considered. The joint distribution of partons in a quark jet is determined in QCD. The quark decay function for pions in the recombination model is then obtained with excellent fit to the data. Similar investigation is applied to the problem of photoproduction of pions in the fragmentation region; again good agreement with data is achieved. The results indicate the reliability of the recombination model when the two-parton distributions can be calculated in QCD. Finally, hadron initiated reactions are considered. A duality between quark recombination and valon fragmentation is suggested. The picture is consistent with dual Regge model. A possible way to determine the inclusive distribution in the context of QCD is suggested

  17. A molecular recombination map of Antirrhinum majus

    Hudson Andrew

    2010-12-01

    Full Text Available Abstract Background Genetic recombination maps provide important frameworks for comparative genomics, identifying gene functions, assembling genome sequences and for breeding. The molecular recombination map currently available for the model eudicot Antirrhinum majus is the result of a cross with Antirrhinum molle, limiting its usefulness within A. majus. Results We created a molecular linkage map of A. majus based on segregation of markers in the F2 population of two inbred lab strains of A. majus. The resulting map consisted of over 300 markers in eight linkage groups, which could be aligned with a classical recombination map and the A. majus karyotype. The distribution of recombination frequencies and distorted transmission of parental alleles differed from those of a previous inter-species hybrid. The differences varied in magnitude and direction between chromosomes, suggesting that they had multiple causes. The map, which covered an estimated of 95% of the genome with an average interval of 2 cM, was used to analyze the distribution of a newly discovered family of MITE transposons and tested for its utility in positioning seven mutations that affect aspects of plant size. Conclusions The current map has an estimated interval of 1.28 Mb between markers. It shows a lower level of transmission ratio distortion and a longer length than the previous inter-species map, making it potentially more useful. The molecular recombination map further indicates that the IDLE MITE transposons are distributed throughout the genome and are relatively stable. The map proved effective in mapping classical morphological mutations of A. majus.

  18. Radiative transfer effects in primordial hydrogen recombination

    The calculation of a highly accurate cosmological recombination history has been the object of particular attention recently, as it constitutes the major theoretical uncertainty when predicting the angular power spectrum of cosmic microwave background anisotropies. Lyman transitions, in particular the Lyman-? line, have long been recognized as one of the bottlenecks of recombination, due to their very low escape probabilities. The Sobolev approximation does not describe radiative transfer in the vicinity of Lyman lines to a sufficient degree of accuracy, and several corrections have already been computed in other works. In this paper, we compute the impact of some radiative transfer effects that were previously ignored, or for which previous treatments were incomplete. First, the effect of Thomson scattering in the vicinity of the Lyman-? line is evaluated, using a full redistribution kernel incorporated into a radiative transfer code. The effect of feedback of distortions generated by the optically thick deuterium Lyman-? line blueward of the hydrogen line is investigated with an analytic approximation. It is shown that both effects are negligible during cosmological hydrogen recombination. Second, the importance of high-lying, nonoverlapping Lyman transitions is assessed. It is shown that escape from lines above Ly? and frequency diffusion in Ly? and higher lines can be neglected without loss of accuracy. Third, a formalism generalizing the Sobolev approximation is developed to account for the overlap of the high-lying Lyman lines, which is shown to lead to negligible changes to the recombination history. Finally, the possibility of a cosmological hydrogen recombination maser is investigated. It is shown that there is no such maser in the purely radiative treatment presented here.

  19. Annexin-Mediated Calcium Signalling in Plants

    Davies, Julia M.

    2014-01-01

    Calcium-permeable channels underpin elevations of free calcium that encode specific signals in stress adaptation, development and immunity. Identifying the genes encoding these channels remains a central goal of plant signalling research. Evidence now suggests that members of the plant annexin family function as unconventional calcium-permeable channels, with roles in development and stress signalling. Arabidopsis annexin 1 mediates a plasma membrane calcium-permeable conductance in roots that is activated by reactive oxygen species. Recombinant annexin 1 forms a very similar conductance in planar lipid bilayers, indicating that this protein could facilitate the in vivo conductance directly. The annexin 1 mutant is impaired in salinity-induced calcium signalling. Protein–protein interactions, post-translational modification and dynamic association with membranes could all influence annexin-mediated calcium signalling and are reviewed here. The prospect of annexins playing roles in calcium signalling events in symbiosis and immunity are considered.

  20. Naturally occurring radiation - an RPA's perspective

    The occurrence of radioactive scales on oil platforms in the North Sea and the associated health hazard are considered. Generally the problem is linked with secondary production by means of sea water injection. The radioactive isotopes involved are Ra226, Ra228 and their progeny which coprecipitate as radium sulphate with barium sulphate when the injection water breaks through and mixes with the formation water. Scales are first deposited on the tubulars in the perforation zones and can then be found anywhere through to the produced water discharge system. The hazard arises principally from the ingestion, particularly by inhalation, of scale particles. Appropriate handling and monitoring procedures are described. Scaled items of equipment, even those going for scrap, have to be sent for onshore cleaning and long-term storage. Chemical scale inhibitors are now being injected into most wells but there is some evidence that occasionally almost pure radium sulphate plates out when they are being used. (UK)

  1. The Λ0 polarization and the recombination mechanism

    We use the recombination and the Thomas Precession Model to obtain a prediction for the Λ0 polarization in the p+p→Λ0+X reaction. We study the effect of the recombination function on the Λ0 polarization

  2. Pervasive genomic recombination of HIV-1 in vivo.

    SHRINER, DANIEL; Rodrigo, Allen G; Nickle, David C.; Mullins, James I.

    2004-01-01

    Recombinants of preexisting human immunodeficiency virus type 1 (HIV-1) strains are now circulating globally. To increase our understanding of the importance of these recombinants, we assessed recombination within an individual infected from a single source by studying the linkage patterns of the auxiliary genes of HIV-1 subtype B. Maximum-likelihood phylogenetic techniques revealed evidence for recombination from topological incongruence among adjacent genes. Coalescent methods were then use...

  3. Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenza

    Moleres, Javier; Sinha, Sunita; Fernández-Calvet, Ariadna; Porsch, Eric A.; St. Geme, Joseph W.; Nislow, Corey; Redfield, Rosemary J.; Garmendia, Junkal

    2016-01-01

    Many bacterial species actively take up and recombine homologous DNA into their genomes, called natural competence, a trait that offers a means to identify the genetic basis of naturally occurring phenotypic variation. Here, we describe “transformed recombinant enrichment profiling” (TREP), in which natural transformation is used to generate complex pools of recombinants, phenotypic selection is used to enrich for specific recombinants, and deep sequencing is used to survey for the genetic variation responsible. We applied TREP to investigate the genetic architecture of intracellular invasion by the human pathogen Haemophilus influenzae, a trait implicated in persistence during chronic infection. TREP identified the HMW1 adhesin as a crucial factor. Natural transformation of the hmw1 operon from a clinical isolate (86-028NP) into a laboratory isolate that lacks it (Rd KW20) resulted in ~1,000-fold increased invasion into airway epithelial cells. When a distinct recipient (Hi375, already possessing hmw1 and its paralog hmw2) was transformed by the same donor, allelic replacement of hmw2AHi375 by hmw1A86-028NP resulted in a ~100-fold increased intracellular invasion rate. The specific role of hmw1A86-028NP was confirmed by mutant and western blot analyses. Bacterial self-aggregation and adherence to airway cells were also increased in recombinants, suggesting that the high invasiveness induced by hmw1A86-028NP might be a consequence of these phenotypes. However, immunofluorescence results found that intracellular hmw1A86-028NP bacteria likely invaded as groups, instead of as individual bacterial cells, indicating an emergent invasion-specific consequence of hmw1A-mediated self-aggregation. PMID:27124727

  4. Ecological genomics in Xanthomonas: the nature of genetic adaptation with homologous recombination and host shifts

    Huang, Chao-Li

    2015-03-15

    Background: Comparative genomics provides insights into the diversification of bacterial species. Bacterial speciation usually takes place with lasting homologous recombination, which not only acts as a cohering force between diverging lineages but brings advantageous alleles favored by natural selection, and results in ecologically distinct species, e.g., frequent host shift in Xanthomonas pathogenic to various plants. Results: Using whole-genome sequences, we examined the genetic divergence in Xanthomonas campestris that infected Brassicaceae, and X. citri, pathogenic to a wider host range. Genetic differentiation between two incipient races of X. citri pv. mangiferaeindicae was attributable to a DNA fragment introduced by phages. In contrast to most portions of the genome that had nearly equivalent levels of genetic divergence between subspecies as a result of the accumulation of point mutations, 10% of the core genome involving with homologous recombination contributed to the diversification in Xanthomonas, as revealed by the correlation between homologous recombination and genomic divergence. Interestingly, 179 genes were under positive selection; 98 (54.7%) of these genes were involved in homologous recombination, indicating that foreign genetic fragments may have caused the adaptive diversification, especially in lineages with nutritional transitions. Homologous recombination may have provided genetic materials for the natural selection, and host shifts likely triggered ecological adaptation in Xanthomonas. To a certain extent, we observed positive selection nevertheless contributed to ecological divergence beyond host shifting. Conclusion: Altogether, mediated with lasting gene flow, species formation in Xanthomonas was likely governed by natural selection that played a key role in helping the deviating populations to explore novel niches (hosts) or respond to environmental cues, subsequently triggering species diversification. © Huang et al.

  5. Plasmid-to-plasmid recombination in Haemophilus influenzae.

    Balganesh, M; Setlow, J K

    1986-01-01

    No recombination between plasmids was observed after conjugal transfer of a plasmid into a cell carrying another plasmid. Two types of such recombination took place after transformation, one type being Rec+ dependent and suggesting a preferred site of recombination. The other much rarer type was at least partially Rec+ independent.

  6. Molecular Evolution and Functional Diversification of Replication Protein A1 in Plants.

    Aklilu, Behailu B; Culligan, Kevin M

    2016-01-01

    Replication protein A (RPA) is a heterotrimeric, single-stranded DNA binding complex required for eukaryotic DNA replication, repair, and recombination. RPA is composed of three subunits, RPA1, RPA2, and RPA3. In contrast to single RPA subunit genes generally found in animals and yeast, plants encode multiple paralogs of RPA subunits, suggesting subfunctionalization. Genetic analysis demonstrates that five Arabidopsis thaliana RPA1 paralogs (RPA1A to RPA1E) have unique and overlapping functions in DNA replication, repair, and meiosis. We hypothesize here that RPA1 subfunctionalities will be reflected in major structural and sequence differences among the paralogs. To address this, we analyzed amino acid and nucleotide sequences of RPA1 paralogs from 25 complete genomes representing a wide spectrum of plants and unicellular green algae. We find here that the plant RPA1 gene family is divided into three general groups termed RPA1A, RPA1B, and RPA1C, which likely arose from two progenitor groups in unicellular green algae. In the family Brassicaceae the RPA1B and RPA1C groups have further expanded to include two unique sub-functional paralogs RPA1D and RPA1E, respectively. In addition, RPA1 groups have unique domains, motifs, cis-elements, gene expression profiles, and pattern of conservation that are consistent with proposed functions in monocot and dicot species, including a novel C-terminal zinc-finger domain found only in plant RPA1C-like sequences. These results allow for improved prediction of RPA1 subunit functions in newly sequenced plant genomes, and potentially provide a unique molecular tool to improve classification of Brassicaceae species. PMID:26858742

  7. The role of BRCA1 in homologous recombination repair in response to replication stress: significance in tumorigenesis and cancer therapy

    Zhang, Junran

    2013-01-01

    Germ line mutations in breast cancer gene 1 (BRCA1) predispose women to breast and ovarian cancers. Although BRCA1 is involved in many important biological processes, the function of BRCA1 in homologous recombination (HR) mediated repair is considered one of the major mechanisms contributing to its tumor suppression activity, and the cause of hypersensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors when BRCA1 is defective. Mounting evidence suggests that the mechanism of repairing DN...

  8. Akt1 inhibits homologous recombination in Brca1-deficient cells by blocking the Chk1-Rad51 pathway

    Jia, Y.; Song, W.; Zhang, F; Yan, J; Yang, Q.

    2012-01-01

    Brca1 deficiency leads to the development of breast cancer. We previously found that Brca1 deficiency activates the Akt oncogenic pathway. Reduced expression of Brca1 was highly correlated with increased activated Akt in human breast cancer samples. Furthermore, activation of Akt1 was involved in Brca1-deficiency-mediated tumorigenesis in mice. Defective homologous recombination (HR) is thought to be a major contributor to tumorigenesis in Brca1 deficiency. Here, we show that Akt1 promotes ch...

  9. Recombinant production of peptide C-terminal ?-amides using an engineered intein

    Albertsen, Louise; Shaw, Allan C; Norrild, Jens Chr.; Strmgaard, Kristian

    2013-01-01

    Peptides are of increasing interest as therapeutics in a wide range of diseases, including metabolic diseases such as diabetes and obesity. In the latter, peptide hormones such as peptide YY (PYY) and pancreatic peptide (PP) are important templates for drug design. Characteristic for these peptides...... is that they contain a C-terminal that is ?-amidated, and this amidation is crucial for biological function. A challenge is to generate such peptides by recombinant means and particularly in a production scale. Here, we have examined an intein-mediated approach to generate a PYY derivative in a...

  10. Hα diagnostic in a recombining plasma

    Wenzel, U.; Goto, M.

    2016-05-01

    In fusion devices the hydrogen Balmer lines are used to measure the neutral flux from the walls into the plasma using the atomic physics factor S/XB. This is a standard diagnostic which can be applied in ionizing plasma using {{H}α} , {{H}β} or {{H}γ} without knowledge of the electron density. We will extend this method to a recombining plasma in front of a surface. {{H}α} can be used in an analogous way to measure the plasma flow to this surface which can be e.g. a divertor target. The other Balmer lines are not suitable because the corresponding atomic physics factor R/YB depends on density due to three-body recombination. An application of this diagnostic method is provided.

  11. Dielectronic recombination of lithium-like gold

    Spies, W.; Mueller, A.; Linkemann, J.; Frank, A.; Wagner, M. (Institut fuer Kernphysik, Universitaet Giessen, D-6300 Giessen (Germany)); Kozhuharov, C.; Franzke, B.; Beckert, K.; Bosch, F.; Eickhoff, H.; Jung, M.; Klepper, O.; Koenig, W.; Mokler, P.H.; Moshammer, R.; Nolden, F.; Schaaf, U.; Spaedtke, P.; Steck, M. (Gesellschaft fuer Schwerionenforschung (GSI), D-6100 Darmstadt (Germany)); Zimmerer, P.; Gruen, N.; Scheid, W. (Institut fuer Theoretische Physik, Universitaet Giessen, D-6300 Giessen (Germany)); Pindzola, M.S.; Badnell, N.R. (Department of Phsyics, Auburn University, Auburn, AL 36849 (United States))

    1993-06-05

    We report measurements on radiative and dielectronic recombination (RR and DR) of very highly charged ions at energies [ital E][sub cm]=0 to 50 [ital eV]. Novel techniques were employed at the heavy ion storage ring ESR to obtain absolute recombination rates of [ital Au][sup 76+](1[ital s][sup 2]2[ital s]). The increase of the RR rate for [ital E][sub [ital c]m][r arrow]0 could be recorded and single DR resonances in [ital Au][sup 75+](1[ital s][sup 2]2[ital p][sub 3/2]6[ital l][sub [ital j

  12. Recombination and dissociative recombination of H2+ and H3+ ions on surfaces with application to hydrogen negative ion sources

    A four-step model for recombination and dissociative recombination of H2+ and H3+ ions on metal surfaces is discussed. Vibrationally excited molecules, H2(v''), from H3+ recombination are produced in a broad spectrum that enhances the excited level distribution. The application of this latter process to hydrogen negative ion discharges is discussed. 5 refs., 3 figs., 1 tab

  13. Precise genotyping and recombination detection of Enterovirus

    Lin, Chieh-Hua; Wang, Yu-bin; Chen, Shu-Hwa; Hsiung, Chao Agnes; Lin, Chung-Yen

    2015-01-01

    Enteroviruses (EV) with different genotypes cause diverse infectious diseases in humans and mammals. A correct EV typing result is crucial for effective medical treatment and disease control; however, the emergence of novel viral strains has impaired the performance of available diagnostic tools. Here, we present a web-based tool, named EVIDENCE (EnteroVirus In DEep conception, http://symbiont.iis.sinica.edu.tw/evidence), for EV genotyping and recombination detection. We introduce the idea of...

  14. Soluble Variants of Human Recombinant Glutaminyl Cyclase

    Castaldo, Cristiana; Ciambellotti, Silvia; de Pablo-Latorre, Raquel; Lalli, Daniela; Porcari, Valentina; Turano, Paola

    2013-01-01

    Recombinant human Glutaminyl Cyclase expressed in E. coli is produced as inclusion bodies. Lack of glycosylation is the main origin of its accumulation in insoluble aggregates. Mutation of single isolated hydrophobic amino acids into negative amino acids was not able to circumvent inclusion bodies formation. On the contrary, substitution with carboxyl-terminal residues of two or three aromatic residues belonging to extended hydrophobic patches on the protein surface provided soluble but still...

  15. Contributions of gluon recombination to saturation phenomena

    Zhu, Wei; Ruan, Jian-hong; Yang, Ji-feng; Shen, Zhen-qi

    2003-01-01

    Parton distributions in the small $x$ region are numerically predicted by using a modified DGLAP equation with the GRV-like input distributions. We find that gluon recombination at twist-4 level obviously suppresses the rapid growth of parton densities with $x$ decrease. We show that before the saturation scale $Q^2_s$ is reached, saturation and partial saturation appear in the small $x$ behavior of parton distributions in nucleus and free proton, respectively. The antishadowing contributions...

  16. Size effects on generation recombination noise

    Gomila, G.; Reggiani, L.

    2002-01-01

    We carry out an analytical theory of generation-recombination noise for a two level resistor model which goes beyond those presently available by including the effects of both space charge fluctuations and diffusion current. Finite size effects are found responsible for the saturation of the low frequency current spectral density at high enough applied voltages. The saturation behaviour is controlled essentially by the correlations coming from the long range Coulomb interaction. It is suggest...

  17. Relativistic effects in dielectronic recombination probabilities

    The method of the relativistic parametric potential was used to compute autoionization and radiative transition probabilities for Li-like Mg, Fe, Ni and Mo (Dielectronic Recombination on He-like). Computations were performed with different values of c, enabling analysis of the behaviour of relativistic corrections. It is shown that for atoms up to Fe, the dominant effect is the spin orbit interaction. The effects of the wave-functions being relativistic contribute only a few percent

  18. Recombination and DNA Repair in Helicobacter pylori

    Dorer, Marion S.; Sessler, Tate H.; Salama, Nina R.

    2011-01-01

    All organisms have pathways that repair the genome, ensuring their survival and that of their progeny. But these pathways also serve to diversify the genome, causing changes on the level of nucleotide, whole gene, and genome structure. Sequencing of bacteria has revealed wide allelic diversity and differences in gene content within the same species, highlighting the importance of understanding pathways of recombination and DNA repair. The human stomach pathogen Helicobacter pylori is an excel...

  19. A Holliday recombination intermediate is twofold symmetric.

    Churchill, M E; Tullius, T. D.; Kallenbach, N. R.; Seeman, N. C.

    1988-01-01

    Four-arm Holliday structures are ephemeral intermediates in genetic recombination. We have used an oligodeoxynucleotide system to form immobile DNA junctions, which are stable analogs of Holliday structures. We have probed the equilibrium structure of a junction by means of hydroxyl radicals generated by the reaction of iron(II)EDTA with hydrogen peroxide. The hydroxyl radical cleavage pattern shows twofold symmetry throughout the molecule. Strong protection from hydroxyl radical attack is ev...

  20. Cultivating Insect Cells To Produce Recombinant Proteins

    Spaulding, Glenn; Goodwin, Thomas; Prewett, Tacey; Andrews, Angela; Francis, Karen; O'Connor, Kim

    1996-01-01

    Method of producing recombinant proteins involves growth of insect cells in nutrient solution in cylindrical bioreactor rotating about cylindrical axis, oriented horizontally and infecting cells with viruses into which genes of selected type cloned. Genes in question those encoding production of desired proteins. Horizontal rotating bioreactor preferred for use in method, denoted by acronym "HARV", described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662).

  1. Modelling of procecces in catalytic recombiners

    In order to achieve a high degree of safety in nuclear power plants and prevent possible accident scenarios, their consequences are calculated and analysed with numeric codes. One of the most important part of nuclear safety research of hazardous incidents are development and validation of these numeric models, which are implemented into accident codes. The severe hydrogen release during a core meltdown is one of the considered scenario of performed accident analyses. One of the most important measure for the elimination of the hydrogen is catalytic recombiners. Converting the hydrogen with the atmospheric oxygen to water vapor in an exothermic reaction will prevent possible detonation of the hydrogen/air atmosphere. Within the dissertation the recombiner simulation REKO-DIREKT was developed and validated by an extensive experimental database. The performance of recombiners with regard to the conversion of the hydrogen and the temperature development is modelled. The REKO-DIREKT program is unique and has made significant revolution in research of hydrogen safety. For the first time it has been possible to show the performance of the recombiner so great in detail by using REKO-DIREKT. In the future engineers of nuclear power plants will have opportunity to have precise forecasts about the process of the possible accidents with hydrogen release. Also with presence of water vapor or with oxygen depletion which are included in the model. The major discussion of the hydrogen ignition at hot catalyst steel plates can be evaluated in the future with REKO-DIREKT more reliably than the existing used models. (orig.)

  2. Recombination-assisted megaprimer (RAM) cloning

    Jacques Mathieu; Emilia Alvarez; Alvarez, Pedro J.J.

    2014-01-01

    No molecular cloning technique is considered universally reliable, and many suffer from being too laborious, complex, or expensive. Restriction-free cloning is among the simplest, most rapid, and cost-effective methods, but does not always provide successful results. We modified this method to enhance its success rate through the use of exponential amplification coupled with homologous end-joining. This new method, recombination-assisted megaprimer (RAM) cloning, significantly extends the app...

  3. Dielectronic recombination of Xe8+ ions

    Based on the fully relativistic configuration interaction method, theoretical calculations are carried out for the dielectronic recombination (DR) rate coefficients of Xe8+ ions in the temperature region from 0.1 to 1 650 eV. The comparison of the DR rate coefficients from 4s, 4p and 4d subshell excitations shows that 4d subshell excitation dominates in the whole temperature region. The contribution from 4p subshell excitation is very important at temperature above 10 eV and the contributions from 4s subshell ex- citation is lower than 7.5% in the whole temperature region. Similarly, the comparison of the DR rate coefficients through △n= 0, I and 2 core excitation shows that the contribution from △n= 2 core excitation can not be neglected, the contributions from n'>15 can also not be neglected. The DR rate coefficients of △n=0, 1 and 2 core excitation and the total DR rate coefficients are fitted with some parameters, which are in good agreement with theoretical calculations values (within 1 % difference)The total DR rate coefficients are greater than radiative recombination (RR) and three-body recombination (TBR) rate coefficients at temperature above 1 eV. Therefore, the DR process can strongly influence the ionization balance of laser produced xenon plasmas. (authors)

  4. Experimental studies of electron-ion recombination

    A short history of electron-ion recombination research, discussing dielectronic combination, begins the study. Two classes of measurement techniques--those which determine rate coefficient of recombination by observing decay of plasma species, and those where cross sections are obtained by studying the formation of neutrals in intersecting beam experiments--are reviewed. Of the former, Biondi's microwave afterglow apparatus is detailed. Shock tubes and flame sampling are touched upon. Of the latter, intersecting beams, McGowan's Meide I apparatus, its advantages and disadvantages, are discussed. Hybrid techniques, quadropole traps (schematicized) and hollow electron beam traps are studied. Table II summarizes all measurements of rate coefficients and cross sections for dissociative recombination made thus far. The diatomics, A2+, with examination of isotope effects, and ion-pair formation; the rare gas ions, He2+, Ne2+, Ar2+; the atmospheric gas ions, 02+, N2+; the polytomics, H3+; and the cluster ions, all are listed as to rate coefficient and cross section

  5. Recombination studies using flowing afterglow plasmas

    Using the FALP apparatus the first reliable data have been obtained on the pure binary ionic recombination coefficients (or mutual neutralization coefficients), ?2, for the reactions of mass-identified ground-state reactant ions at thermal energies. Thus values of ?2 have been obtained for several reactions involving simple molecular ions and for several reactions involving cluster ions. The values of ?2 at 300 K for all of these reactions are within the limited range (4 - 10) x 10-8cm3s-1 and are consistent with theoretical expectations for such reactions. The study of the temperature dependence of ?2 for two reactions has shown that they also vary in accordance with theoretical predictions. Upper limits to ?2 have been obtained for a few reactions involving atomic ions only; the very small values for ?2 for these particular reactions are consistent with expectations although this must not be taken as a generalisation for all atomic ion reactions since others are expected to have larger values of ?2 (this has been demonstrated in merged beam experiments). The neutral products and the energy partition in these reactions have not been seriously studied to date; the products of only one reaction (NO+] + NO2-) have been studied spectroscopically in the FALP apparatus. A start has also been made in the study of collision enhanced ionic recombination in the FALP. This very versatile apparatus can be exploited much more in the study of ionic and electronic recombination reactions at thermal energies

  6. CFD modeling of passive autocatalytic recombiners*

    Orszulik Magdalena

    2015-06-01

    Full Text Available This study deals with numerical modeling of passive autocatalytic hydrogen recombiners (PARs. Such devices are installed within containments of many nuclear reactors in order to remove hydrogen and convert it to steam. The main purpose of this work is to develop a numerical model of passive autocatalytic recombiner (PAR using the commercial computational fluid dynamics (CFD software ANSYS-FLUENT and tuning the model using experimental results. The REKO 3 experiment was used for this purpose. Experiment was made in the Institute for Safety Research and Reactor Technology in Julich (Germany. It has been performed for different hydrogen concentrations, different flow rates, the presence of steam, and different initial temperatures of the inlet mixture. The model of this experimental recombiner was elaborated within the framework of this work. The influence of mesh, gas thermal conductivity coefficient, mass diffusivity coefficients, and turbulence model was investigated. The best results with a good agreement with REKO 3 data were received for k-ɛ model of turbulence, gas thermal conductivity dependent on the temperature and mass diffusivity coefficients taken from CHEMKIN program. The validated model of the PAR was next implemented into simple two-dimensional simulations of hydrogen behavior within a subcompartment of a containment building.

  7. The Role of Recombination in Evolutionary Rescue.

    Uecker, Hildegard; Hermisson, Joachim

    2016-02-01

    How likely is it that a population escapes extinction through adaptive evolution? The answer to this question is of great relevance in conservation biology, where we aim at species' rescue and the maintenance of biodiversity, and in agriculture and medicine, where we seek to hamper the emergence of pesticide or drug resistance. By reshuffling the genome, recombination has two antagonistic effects on the probability of evolutionary rescue: it generates and it breaks up favorable gene combinations. Which of the two effects prevails depends on the fitness effects of mutations and on the impact of stochasticity on the allele frequencies. In this article, we analyze a mathematical model for rescue after a sudden environmental change when adaptation is contingent on mutations at two loci. The analysis reveals a complex nonlinear dependence of population survival on recombination. We moreover find that, counterintuitively, a fast eradication of the wild type can promote rescue in the presence of recombination. The model also shows that two-step rescue is not unlikely to happen and can even be more likely than single-step rescue (where adaptation relies on a single mutation), depending on the circumstances. PMID:26627842

  8. Dissociative recombination of small molecular ions

    In this thesis an analysis is given of merged electron-ion beam experiment and work on dissociative recombination of molecular ions and electrons is described. Chapter II covers a brief introduction of the theory of dissociative recombination. In chapter III, a description is given of the merged electron-ion beam experiment and a method is described which allows the determination of the mean angle between the electron and ion trajectories in a merged electron-ion beam experiment. In chapter IV a paper on the three dominant atmospheric diatomic ions NO+, O2+ and N2+ is presented and in chapter V the dissociative recombination for N2H+ and N2D+ is discussed. In chapter VI two papers on the polyatomic ions of the carbon-containing molecular ions are presented, and in chapter VII a letter with some results of the work presented in more detail in the chapters IV, V and VI is presented. The magnitude and the energy dependence of the cross-section measured by the merged beam technique and by other techniques is compared and discussed. (Auth.)

  9. Use of Helical Transport Channels for Bunch Recombination

    Neuffer, David; Yonehara, Katsuya; /Fermilab; Yoshikawa, Cary; /MUONS Inc., Batavia

    2010-03-01

    Cooling scenarios for a high-luminosity Muon Collider require bunch recombination for optimal luminosity. In this report we note that the tunable chronicity property of a helical transport channel (HTC) makes it a desirable component of a bunch recombiner. A large chronicity HTC is desirable for the bunch recombining transport, while more isochronous transport may be preferred for rf manipulations. Scenarios for bunch recombination are presented, with initial 1-D simulations, in order to set the stage for future 3-D simulation and optimization. HTC transports may enable a very compact bunch recombiner.

  10. Mechanisms and factors that influence high frequency retroviral recombination

    Delviks-Frankenberry, Krista; Galli, Andrea; Nikolaitchik, Olga; Mens, Helene; Pathak, Vinay K; Hu, Wei-Shau

    2011-01-01

    transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity of...... the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug...

  11. Genome modifications and cloning using a conjugally transferable recombineering system

    Mohammad J Hossain

    2015-12-01

    Full Text Available The genetic modification of primary bacterial disease isolates is challenging due to the lack of highly efficient genetic tools. Herein we describe the development of a modified PCR-based, λ Red-mediated recombineering system for efficient deletion of genes in Gram-negative bacteria. A series of conjugally transferrable plasmids were constructed by cloning an oriT sequence and different antibiotic resistance genes into recombinogenic plasmid pKD46. Using this system we deleted ten different genes from the genomes of Edwardsiella ictaluri and Aeromonas hydrophila. A temperature sensitive and conjugally transferable flp recombinase plasmid was developed to generate markerless gene deletion mutants. We also developed an efficient cloning system to capture larger bacterial genetic elements and clone them into a conjugally transferrable plasmid for facile transferring to Gram-negative bacteria. This system should be applicable in diverse Gram-negative bacteria to modify and complement genomic elements in bacteria that cannot be manipulated using available genetic tools.

  12. Gene therapy: recombinant adeno-associated virus vectors.

    Smith-Arica, J R; Bartlett, J S

    2001-01-01

    Gene transfer using recombinant adeno-associated virus (rAAV) vectors shows great promise for human gene therapy. The broad host range, low level of immune response, and longevity of gene expression observed with these vectors in numerous disease paradigms has enabled the initiation of a number of clinical trials using this gene delivery system. This review presents an overview of the current developments in the field of AAV-mediated gene delivery. Such developments include the establishment of new production methods allowing the generation of high titer preparations, improved purification methods, the use of alternative AAV serotypes, and the generation of trans-splicing rAAV genomes. Together, these developments have improved results interpretation, host range, and the coding capacity of rAAV vectors. Furthermore, the recent identification of regions within the viral capsid that are amenable to modification has begun to address the issue of direct rAAV vector targeting, which could potentially allow targeted gene delivery to specific cell populations. The versatility shown by this vector has enabled new diseases to be realistically considered for therapeutic intervention and considerably broadened the scope of gene therapy. PMID:11139798

  13. FASEB Summer Research Conference. Genetic Recombination and Chromosome Rearrangements

    Jinks-Robertson, Sue

    2002-02-01

    The 2001 meeting entitled ''Genetic Recombination and Genome Rearrangements'' was held July 21-26 in Snowmass, Colorado. The goal of the meeting was to bring together scientists using diverse approaches to study all aspects of genetic recombination. This goal was achieved by integrating talks covering the genetics, biochemistry and structural biology of homologous recombination, site-specific recombination, and nonhomologous recombination. The format of the meeting consisted of a keynote address on the opening evening, two formal plenary sessions on each of the four full meeting days, a single afternoon workshop consisting of short talks chosen from among submitted abstracts, and afternoon poster sessions on each of the four full meeting days. The eight plenary session were entitled: (1) Recombination Mechanisms, (2) Prokaryotic Recombination, (3) Repair and Recombination, (4) Site-specific Recombination and Transposition, (5) Eukaryotic Recombination I, (6) Genome Rearrangements, (7) Meiosis, and (8) Eukaryotic Recombination II. Each session included a mix of genetic, biochemical and structural talks; talks were limited to 20 minutes, followed by 10 minutes of very lively, general discussion. Much of the data presented in the plenary sessions was unpublished, thus providing attendees with the most up-to-date knowledge of this rapidly-moving field.

  14. Monitoring homologous recombination in rice (Oryza sativa L.)

    Yang Zhuanying; Tang Li [Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, South China Normal University, Guangzhou 510631 (China); Li Meiru [South China Botanic Garden, Chinese Academy of Sciences, Guangzhou 510650 (China); Chen Lei; Xu Jie [Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, South China Normal University, Guangzhou 510631 (China); Wu Goujiang [South China Botanic Garden, Chinese Academy of Sciences, Guangzhou 510650 (China); Li Hongqing, E-mail: hqli@scnu.edu.cn [Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, South China Normal University, Guangzhou 510631 (China)

    2010-09-10

    Here we describe a system to assay homologous recombination during the complete life cycle of rice (Oryza sativa L.). Rice plants were transformed with two copies of non-functional GUS reporter overlap fragments as recombination substrate. Recombination was observed in all plant organs examined, from the seed stage until the flowering stage of somatic plant development. Embryogenic cells exhibited the highest recombination ability with an average of 3 x 10{sup -5} recombination events per genome, which is about 10-fold of that observed in root cells, and two orders of that observed in leaf cells. Histological analysis revealed that recombination events occurred in diverse cell types, but preferentially in cells with small size. Examples of this included embryogenic cells in callus, phloem cells in the leaf vein, and cells located in the root apical meristem. Steady state RNA analysis revealed that the expression levels of rice Rad51 homologs are positively correlated with increased recombination rates in embryogenic calli, roots and anthers. Finally, radiation treatment of plantlets from distinct recombination lines increased the recombination frequency to different extents. These results showed that homologous recombination frequency can be effectively measured in rice using a transgene reporter assay. This system will facilitate the study of DNA damage signaling and homologous recombination in rice, a model monocot.

  15. Antagonistic experimental coevolution with a parasite increases host recombination frequency

    Kerstes Niels AG

    2012-02-01

    Full Text Available Abstract Background One of the big remaining challenges in evolutionary biology is to understand the evolution and maintenance of meiotic recombination. As recombination breaks down successful genotypes, it should be selected for only under very limited conditions. Yet, recombination is very common and phylogenetically widespread. The Red Queen Hypothesis is one of the most prominent hypotheses for the adaptive value of recombination and sexual reproduction. The Red Queen Hypothesis predicts an advantage of recombination for hosts that are coevolving with their parasites. We tested predictions of the hypothesis with experimental coevolution using the red flour beetle, Tribolium castaneum, and its microsporidian parasite, Nosema whitei. Results By measuring recombination directly in the individuals under selection, we found that recombination in the host population was increased after 11 generations of coevolution. Detailed insights into genotypic and phenotypic changes occurring during the coevolution experiment furthermore helped us to reconstruct the coevolutionary dynamics that were associated with this increase in recombination frequency. As coevolved lines maintained higher genetic diversity than control lines, and because there was no evidence for heterozygote advantage or for a plastic response of recombination to infection, the observed increase in recombination most likely represented an adaptive host response under Red Queen dynamics. Conclusions This study provides direct, experimental evidence for an increase in recombination frequency under host-parasite coevolution in an obligatory outcrossing species. Combined with earlier results, the Red Queen process is the most likely explanation for this observation.

  16. Mediation and Conflict Management

    Gerald Eisenkopf

    2009-01-01

    Mediation is a popular process to manage conflicts, but there is little systematic insight into its mechanisms. This paper discusses the results from an experiment in which a mediator can induce two conflict parties to behave cooperatively. If the mediator recommends cooperative behavior and threatens to punish deviations, she achieves the efficient solution. Similar results even obtain if the mediator is biased towards one party or has no incentive to prevent the conflict. Communication betw...

  17. Molecular mechanisms of DNA recombination: testing mitotic and meiotic models

    A hyperhaploid n + 1 strain of Saccharomyces cerevisiae (LBL1) disomic for chromosome VII was employed to isolate hyper-rec and hypo-rec mutations affecting spontaneous mitotic gene conversion and intergenic recombination. The genotype of LBL1 permits simultaneous and independent identification of rec mutations that enhance or diminish gene conversion and those that enhance or diminish intergenic recombination. Five phenotypic groups of rec mutants were isolated following ultraviolet light mutagenesis. Rec mutations that simultaneously abolish or enhance both classes of recombinational events were detected. These results demonstrate that gene conversion and intergenic recombination are under joint genetic control in mitotic cells. Conversion-specific and intergenic recombination-specific rec mutants were also recovered. Their properties indicate that conversion and intergenic recombination are separable pheonomena dependent upon discrete REC genes. The rec mutants isolated in LBL1 provide a method to test molecular models of mitotic and meiotic recombination

  18. Mechanisms and Factors that Influence High Frequency Retroviral Recombination

    Krista Delviks-Frankenberry

    2011-09-01

    Full Text Available With constantly changing environmental selection pressures, retroviruses rely upon recombination to reassort polymorphisms in their genomes and increase genetic diversity, which improves the chances for the survival of their population. Recombination occurs during DNA synthesis, whereby reverse transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity of the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug treatment, and vaccine development.

  19. Recombinations to the Rydberg States of Hydrogen and Their Effect During the Cosmological Recombination Epoch

    Chluba, J; Dursi, L J

    2010-01-01

    In this paper we discuss the effect of recombinations to highly excited states (n > 100) in hydrogen during the cosmological recombination epoch. For this purpose, we developed a new ODE solver for the recombination problem, based on an implicit Gear's method. This solver allows us to include up to 350 l-resolved shells or ~61 000 separate levels in the hydrogen model and to solve the recombination problem for one cosmology in ~27 hours. This is a huge improvement in performance over our previous recombination code, for which a 100-shell computation (5050 separate states) already required ~150 hours on a single processor. We show that for 350 shells down to redshift z ~200 the results for the free electron fraction have practically converged. The final modification in the free electron fraction at z ~200 decreases from about \\DeltaNe/Ne ~2.8% for 100 shells to \\DeltaNe/Ne ~1.6% for 350 shells. However, the associated changes in the CMB power spectra at large multipoles l are rather small, so that for accurate...

  20. Evolution of recombination in eutherian mammals: insights into mechanisms that affect recombination rates and crossover interference.

    Segura, Joana; Ferretti, Luca; Ramos-Onsins, Sebastin; Capilla, Laia; Farr, Marta; Reis, Fernanda; Oliver-Bonet, Maria; Fernndez-Belln, Hugo; Garcia, Francisca; Garcia-Calds, Montserrat; Robinson, Terence J; Ruiz-Herrera, Aurora

    2013-11-22

    Recombination allows faithful chromosomal segregation during meiosis and contributes to the production of new heritable allelic variants that are essential for the maintenance of genetic diversity. Therefore, an appreciation of how this variation is created and maintained is of critical importance to our understanding of biodiversity and evolutionary change. Here, we analysed the recombination features from species representing the major eutherian taxonomic groups Afrotheria, Rodentia, Primates and Carnivora to better understand the dynamics of mammalian recombination. Our results suggest a phylogenetic component in recombination rates (RRs), which appears to be directional, strongly punctuated and subject to selection. Species that diversified earlier in the evolutionary tree have lower RRs than those from more derived phylogenetic branches. Furthermore, chromosome-specific recombination maps in distantly related taxa show that crossover interference is especially weak in the species with highest RRs detected thus far, the tiger. This is the first example of a mammalian species exhibiting such low levels of crossover interference, highlighting the uniqueness of this species and its relevance for the study of the mechanisms controlling crossover formation, distribution and resolution. PMID:24068360

  1. Bayesian Mediation Analysis

    Yuan, Ying; MacKinnon, David P.

    2009-01-01

    In this article, we propose Bayesian analysis of mediation effects. Compared with conventional frequentist mediation analysis, the Bayesian approach has several advantages. First, it allows researchers to incorporate prior information into the mediation analysis, thus potentially improving the efficiency of estimates. Second, under the Bayesian

  2. mediation: R Package for Causal Mediation Analysis

    Dustin Tingley

    2014-09-01

    Full Text Available In this paper, we describe the R package mediation for conducting causal mediation analysis in applied empirical research. In many scientific disciplines, the goal of researchers is not only estimating causal effects of a treatment but also understanding the process in which the treatment causally affects the outcome. Causal mediation analysis is frequently used to assess potential causal mechanisms. The mediation package implements a comprehensive suite of statistical tools for conducting such an analysis. The package is organized into two distinct approaches. Using the model-based approach, researchers can estimate causal mediation effects and conduct sensitivity analysis under the standard research design. Furthermore, the design-based approach provides several analysis tools that are applicable under different experimental designs. This approach requires weaker assumptions than the model-based approach. We also implement a statistical method for dealing with multiple (causally dependent mediators, which are often encountered in practice. Finally, the package also offers a methodology for assessing causal mediation in the presence of treatment noncompliance, a common problem in randomized trials.

  3. Mek1 stabilizes Hop1-Thr318 phosphorylation to promote interhomolog recombination and checkpoint responses during yeast meiosis.

    Chuang, Chi-Ning; Cheng, Yun-Hsin; Wang, Ting-Fang

    2012-12-01

    Red1, Hop1 and Mek1 are three yeast meiosis-specific chromosomal proteins that uphold the interhomolog (IH) bias of meiotic recombination. Mek1 is also an effector protein kinase in a checkpoint that responds to aberrant DNA and/or axis structure. The activation of Mek1 requires Red1-dependent Hop1-Thr(T)318 phosphorylation, which is mediated by Mec1 and Tel1, the yeast homologs of the mammalian DNA damage sensor kinases ATR and ATM. As the ectopic expression of Mek1-glutathione S-transferase (GST) was shown to promote IH recombination in the absence of Mec1/Tel1-dependent checkpoint function, it was proposed that Mek1 might play dual roles during meiosis by directly phosphorylating targets that are involved in the recombination checkpoint. Here, we report that Mek1 has a positive feedback activity in the stabilization of Mec1/Tel1-mediated Hop1-T318 phosphorylation against the dephosphorylation mediated by protein phosphatase 4. Our results also reveal that GST-Mek1 or Mek1-GST further increases Hop1-T318 phosphorylation. This positive feedback function of Mek1 is independent of Mek1's kinase activity, but dependent on Mek1's forkhead-associated (FHA) domain and its arginine 51 residue. Arginine 51 directly mediates the interaction of Mek1-FHA and phosphorylated Hop1-T318. We suggest that the Hop1-Mek1 interaction is similar to the Rad53-Dun1 signaling pathway, which is mediated through the interaction of phosphorylated Rad53 and Dun1-FHA. PMID:23047948

  4. Phenomenological Implications of Deflected Mirage Mediation: Comparison with Mirage Mediation

    Altunkaynak, Baris; Everett, Lisa L.; Kim, Ian-Woo; Nelson, Brent D; Rao, Yongyan

    2010-01-01

    We compare the collider phenomenology of mirage mediation and deflected mirage mediation, which are two recently proposed "mixed" supersymmetry breaking scenarios motivated from string compactifications. The scenarios differ in that deflected mirage mediation includes contributions from gauge mediation in addition to the contributions from gravity mediation and anomaly mediation also present in mirage mediation. The threshold effects from gauge mediation can drastically alter the low energy s...

  5. General gauge mediation

    We give a general definition of gauge mediated supersymmetry breaking which encompasses all the known gauge mediation models. In particular, it includes both models with messengers as well as direct mediation models. A formalism for computing the soft terms in the generic model is presented. Such a formalism is necessary in strongly-coupled direct mediation models where perturbation theory cannot be used. It allows us to identify features of the entire class of gauge mediation models and to distinguish them from specific signatures of various subclasses. (author)

  6. Recombinant preparation and functional studies of EspI ATP binding domain from Mycobacterium tuberculosis.

    Chen, Hanyu; Wang, Huilin; Sun, Tao; Tian, Shuangliang; Lin, Donghai; Guo, Chenyun

    2016-07-01

    The ESX-1 secretion system of Mycobacterium tuberculosis is required for the virulence of tubercle bacillus. EspI, the ESX-1 secretion-associated protein in Mycobacterium tuberculosis (MtEspI), is involved in repressing the activity of ESX-1-mediated secretion when the cellular ATP level is low. The ATP binding domain of MtEspI plays a crucial role in this regulatory process. However, further structural and functional studies of MtEspI are hindered due to the bottleneck of obtaining stable and pure recombinant protein. In this study, we systematically analyzed the structure and function of MtEspI using bioinformatics tools and tried various expression constructs to recombinantly express full-length and truncated MtEspI ATP binding domain. Finally, we prepared pure and stable MtEspI ATP binding domain, MtEspI415-493, in Escherichia coli by fusion expression and purification with dual tag, Glutathione S-transferase (GST) tag and (His)6 tag. (31)P NMR titration assay indicated that MtEspI415-493 possessed a moderate affinity (∼μM) for ATP and the residue K425 was located at the binding site. The protocol described here may provide a train of thought for recombinant preparation of other ESX-1 secretion-associated proteins. PMID:27017992

  7. Mechanism of Regulation of Intrachromatid Recombination and Long-Range Chromosome Interactions in Saccharomyces cerevisiae.

    Zaman, Shamsu; Choudhury, Malay; Jiang, James C; Srivastava, Pankaj; Mohanty, Bidyut K; Danielson, Christopher; Humphrey, Sean J; Jazwinski, S Michal; Bastia, Deepak

    2016-05-15

    The NAD-dependent histone deacetylase Sir2 controls ribosomal DNA (rDNA) silencing by inhibiting recombination and RNA polymerase II-catalyzed transcription in the rDNA of Saccharomyces cerevisiae Sir2 is recruited to nontranscribed spacer 1 (NTS1) of the rDNA array by interaction between the RENT ( RE: gulation of N: ucleolar S: ilencing and T: elophase exit) complex and the replication terminator protein Fob1. The latter binds to its cognate sites, called replication termini (Ter) or replication fork barriers (RFB), that are located in each copy of NTS1. This work provides new mechanistic insights into the regulation of rDNA silencing and intrachromatid recombination by showing that Sir2 recruitment is stringently regulated by Fob1 phosphorylation at specific sites in its C-terminal domain (C-Fob1), which also regulates long-range Ter-Ter interactions. We show further that long-range Fob1-mediated Ter-Ter interactions in trans are downregulated by Sir2. These regulatory mechanisms control intrachromatid recombination and the replicative life span (RLS). PMID:26951198

  8. New Strategies for Expression and Purification of Recombinant Human RNASET2 Protein in Pichia pastoris.

    Lualdi, Marta; Pedrini, Edoardo; Petroni, Francesca; Nsman, Johnny; Lindqvist, Christer; Scaldaferri, Debora; Taramelli, Roberto; Inforzato, Antonio; Acquati, Francesco

    2015-06-01

    Ribonucleases form a large family of enzymes involved in RNA metabolism and are endowed with a broad range of biological functions. Among the different RNase proteins described in the last decades, those belonging to the Rh/T2/S subfamily show the highest degree of evolutionary conservation, suggesting the occurrence of a key critical ancestral role for this protein family. We have recently defined the human RNASET2 gene as a novel member of a group of oncosuppressors called "tumor antagonizing genes," whose activity in the control of cancer growth is carried out mainly in vivo. However, to better define the molecular pathways underlying the oncosuppressive properties of this protein, further structural and functional investigations are necessary, and availability of high-quality recombinant RNASET2 is of paramount importance. Here, we describe a multi-step strategy that allows production of highly pure, catalytically competent recombinant RNASET2 in both wild-type and mutant forms. The recombinant proteins that were produced with our purification strategy will be instrumental to perform a wide range of functional assays aimed at dissecting the molecular mechanisms of RNASET2-mediated tumor suppression. PMID:25663099

  9. In utero recombinant adeno-associated virus gene transfer in mice, rats, and primates

    Marrero Luis

    2003-09-01

    Full Text Available Abstract Background Gene transfer into the amniotic fluid using recombinant adenovirus vectors was shown previously to result in high efficiency transfer of transgenes into the lungs and intestines. Adenovirus mediated in utero gene therapy, however, resulted in expression of the transgene for less than 30 days. Recombinant adenovirus associated viruses (rAAV have the advantage of maintaining the viral genome in daughter cells thus providing for long-term expression of transgenes. Methods Recombinant AAV2 carrying green fluorescent protein (GFP was introduced into the amniotic sac of fetal rodents and nonhuman primates. Transgene maintenance and expression was monitor. Results Gene transfer resulted in rapid uptake and long-term gene expression in mice, rats, and non-human primates. Expression and secretion of the reporter gene, GFP, was readily demonstrated within 72 hours post-therapy. In long-term studies in rats and nonhuman primates, maintenance of GFP DNA, protein expression, and reporter gene secretion was documented for over one year. Conclusions Because only multipotential stem cells are present at the time of therapy, these data demonstrated that in utero gene transfer with AAV2 into stem cells resulted in long-term systemic expression of active transgene roducts. Thus, in utero gene transfer via the amniotic fluid may be useful in treatment of gene disorders.

  10. Competitive repair by naturally dispersed repetitive DNA during non-allelic homologous recombination

    Hoang, Margaret L.; Tan, Frederick J.; Lai, David C.; Celniker, Sue E.; Hoskins, Roger A.; Dunham, Maitreya J.; Zheng, Yixian; Koshland, Douglas

    2010-08-27

    Genome rearrangements often result from non-allelic homologous recombination (NAHR) between repetitive DNA elements dispersed throughout the genome. Here we systematically analyze NAHR between Ty retrotransposons using a genome-wide approach that exploits unique features of Saccharomyces cerevisiae purebred and Saccharomyces cerevisiae/Saccharomyces bayanus hybrid diploids. We find that DNA double-strand breaks (DSBs) induce NAHR-dependent rearrangements using Ty elements located 12 to 48 kilobases distal to the break site. This break-distal recombination (BDR) occurs frequently, even when allelic recombination can repair the break using the homolog. Robust BDR-dependent NAHR demonstrates that sequences very distal to DSBs can effectively compete with proximal sequences for repair of the break. In addition, our analysis of NAHR partner choice between Ty repeats shows that intrachromosomal Ty partners are preferred despite the abundance of potential interchromosomal Ty partners that share higher sequence identity. This competitive advantage of intrachromosomal Tys results from the relative efficiencies of different NAHR repair pathways. Finally, NAHR generates deleterious rearrangements more frequently when DSBs occur outside rather than within a Ty repeat. These findings yield insights into mechanisms of repeat-mediated genome rearrangements associated with evolution and cancer.

  11. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    Jeremy A. Kroemer

    2015-01-01

    Full Text Available Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification.

  12. Creating Porcine Biomedical Models Through Recombineering

    Lawrence B. Schook

    2006-03-01

    Full Text Available Recent advances in genomics provide genetic information from humans and other mammals (mouse, rat, dog and primates traditionally used as models as well as new candidates (pigs and cattle. In addition, linked enabling technologies, such as transgenesis and animal cloning, provide innovative ways to design and perform experiments to dissect complex biological systems. Exploitation of genomic information overcomes the traditional need to choose naturally occurring models. Thus, investigators can utilize emerging genomic knowledge and tools to create relevant animal models. This approach is referred to as reverse genetics. In contrast to ‘forward genetics’, in which gene(s responsible for a particular phenotype are identified by positional cloning (phenotype to genotype, the ‘reverse genetics’ approach determines the function of a gene and predicts the phenotype of a cell, tissue, or organism (genotype to phenotype. The convergence of classical and reverse genetics, along with genomics, provides a working definition of a ‘genetic model’ organism (3. The recent construction of phenotypic maps defining quantitative trait loci (QTL in various domesticated species provides insights into how allelic variations contribute to phenotypic diversity. Targeted chromosomal regions are characterized by the construction of bacterial artificial chromosome (BAC contigs to isolate and characterize genes contributing towards phenotypic variation. Recombineering provides a powerful methodology to harvest genetic information responsible for phenotype. Linking recombineering with gene-targeted homologous recombination, coupled with nuclear transfer (NT technology can provide ‘clones’ of genetically modified animals.

  13. Recombination-deficient mutants of Bacillus subtilis

    Two mutant strains of Bacillus subtilis Marburg, NIG43 and NIG45, were isolated. They showed high sensitivities to gamma rays, ultraviolet light (uv), and chemicals. Deficiencies in genetic recombination of these two mutants were shown by the experiments on their capacity in transformation, SPO2 transfection, and PBS1 phage transduction, as well as on their radiation and drug sensitivities and their Hcr+ capacity for uv-exposed phage M2. Some of these characteristics were compared with those of the known strains possessing the recA1 or recB2 alleles. Mapping studies revealed that the mutation rec-43 of strain NIG43 lies in the region of chromosome replication origin. The order was purA dna-8132 rec-43. Another mutation, rec-45, of strain NIG45 was found to be tightly linked to recA1. The mutation rec-43 reduced mainly the frequency of PBS1 transduction. On the other hand, the mutation rec-45 reduced the frequency of recombination involved both in transformation and PBS1 tranduction. The mutation rec-43 of strain NIG43 is conditional, but rec-45 of strain NIG45 is not. The uv impairment in cellular survival of strain NIG43 was gradually reverted at higher salt or sucrose concentrations, suggesting cellular possession of a mutated gene product whose function is conditional. In contrast to several other recombination-deficient strains, SPO2 lysogens of strains NIG43 and NIG45 were not inducible, indicating involvement of rec-43+ or rec-45+ gene product in the development of SPO2 prophage to a vegetative form. The uv-induced deoxyribonucleic acid degradation in vegetative cells was higher in rec-43 and rec-45 strains

  14. Tobacco - a producer of recombinant interferons.

    Budzianowski, Jaromir

    2014-01-01

    The approved therapeutic interferons, which are chiefly indicated for a treatment of hepatitis C or hairy cell leukaemia (IFN-?), relapsingl remitting sclerosis multiplex (IFN-?) and chronic granulomatous disease (IFN-?), are commercially produced by recombinant DNA technology, mainly in bacteria Escherichia coli (IFN-?, IFN-?1b, IFN-?), rarely in a mammalian cell line CHO (IFN-?la). A serum half-life time of some non-glycosylated IFN-? products was extended by a chemical attachment of a branched polyethylene glycol (PEG) to give PEGylated IFN-?. The therapy with recombinant interferons proves expensive and hence much hope is concerned with their production in other platforms assumed to be cheaper, like transgenic plants. Currently, tobacco, botanically species Nicotiana tabacum, its cultivars and some related species, especially N. benthamiana, is one of the most important plant expression systems tested for the production of therapeutical polypeptides and proteins (so-called biopharmaceuticals or biologics), especially vaccines, by using either greenhouse or field cultivated plants or cell suspension culture. IFN-? subtypes were expressed in tobacco nuclear genom e (IFN-?2a and 2b), chloroplast genome (IFN-?2b) and by transient expression (IFN-?2b). The IF?-a2b chimera fusions with O-glycosylated protein with O-a-rabinogalactans expressed in tobacco BY-2 cell culture showed increased half-life time similar to that obtained by PEGylation. The production of IFN-?2b (non-glycosydated) in tobacco glasshouse or field cultivation has been also elaborated. One report concerned expression of IFN-? but with low yield. N-glycosylated IFN-? could be efficiently expressed in tobacco protoplast infected with recombinant brome mosaic virus (BMV) with the yield of 5-10% of total extracted protein. This type interferon (non-glycosylated), when expressed in chloroplast genome, proved unstable and could be obtained with reasonable yield as a fusion with GUS (?-glucuronidase). PMID:25799861

  15. Functions and structures of eukaryotic recombination proteins

    We have found that Rad51 and RecA Proteins form strikingly similar structures together with dsDNA and ATP. Their right handed helical nucleoprotein filaments extend the B-form DNA double helixes to 1.5 times in length and wind the helix. The similarity and uniqueness of their structures must reflect functional homologies between these proteins. Therefore, it is highly probable that similar recombination proteins are present in various organisms of different evolutional states. We have succeeded to clone RAD51 genes from human, mouse, chicken and fission yeast genes, and found that the homologues are widely distributed in eukaryotes. The HsRad51 and MmRad51 or ChRad51 proteins consist of 339 amino acids differing only by 4 or 12 amino acids, respectively, and highly homologous to both yeast proteins, but less so to Dmcl. All of these proteins are homologous to the region from residues 33 to 240 of RecA which was named ''homologous core. The homologous core is likely to be responsible for functions common for all of them, such as the formation of helical nucleoprotein filament that is considered to be involved in homologous pairing in the recombination reaction. The mouse gene is transcribed at a high level in thymus, spleen, testis, and ovary, at lower level in brain and at a further lower level in some other tissues. It is transcribed efficiently in recombination active tissues. A clear functional difference of Rad51 homologues from RecA was suggested by the failure of heterologous genes to complement the deficiency of Scrad51 mutants. This failure seems to reflect the absence of a compatible partner, such as ScRad52 protein in the case of ScRad51 protein, between different species. Thus, these discoveries play a role of the starting point to understand the fundamental gene targeting in mammalian cells and in gene therapy. (J.P.N.)

  16. Mode and recombination control in semiconductor lasers

    A self consistent mathematical description of a semiconductor GaAlAs heterostructure laser has been developed. It includes excess hole and electron injection, diffusion, drift and recombination. The excess carrier motion is described by the ambipolar diffusion equation. The boundary conditions at each heterojunction are found by imposing electron and hole current continuity as well as quasi-Fermi level continuity. The Fermi-level continuity condition is applied using Kane's non-parabolicity for the GAMMA1/sub C/ minimum and a parabolic expression for both the GAMMA15 /sub v/ and the X1/sub C/ minima. The effect of aluminum concentration on carrier mobility, effective mass and gap is included. The volume recombination rate is related to the optical gain of the laser in linear fashion using coefficients obtained by integrating over parabolic bands without quasi-momentum conservation. The recombination lifetime is found in a similar manner. The diffusion coefficients are calculated using the generalized Einstein equation. The dependence of the index of refraction in the device upon carrier injection is included. The E-M field is calculated by solving Maxwell's equations for a general five layer dielectric waveguide. Above threshold the gain is assumed to be pinned at the threshold value. For each solution, the E--M field distribution is determined self-consistently for a particular excess carrier distribution. The theory has been applied extensively to many contemporary laser structures, including double heterostructure, single heterostructure and symmetrical optical cavities. Design curves including injection efficiencies, peak power at catastrophic degradation, threshold current density and I--V characteristics have been developed for various cavity geometries and dopants

  17. Recombinant production of Streptococcus equisimilis streptokinase by Streptomyces lividans

    Vallín Carlos

    2007-07-01

    Full Text Available Abstract Background Streptokinase (SK is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. It is naturally secreted by several strains of beta-haemolytic streptococci. The low yields obtained in SK production, lack of developed gene transfer methodology and the pathogenesis of its natural host have been the principal reasons to search for a recombinant source for this important therapeutic protein. We report here the expression and secretion of SK by the Gram-positive bacterium Streptomyces lividans. The structural gene encoding SK was fused to the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi signal sequence or to the Streptomyces lividans xylanase C (xlnC signal sequence. The native Vsi protein is translocated via the Sec pathway while the native XlnC protein uses the twin-arginine translocation (Tat pathway. Results SK yield in the spent culture medium of S. lividans was higher when the Sec-dependent signal peptide mediates the SK translocation. Using a 1.5 L fermentor, the secretory production of the Vsi-SK fusion protein reached up to 15 mg SK/l. SK was partially purified from the culture supernatant by DEAE-Sephacel chromatography. A 44-kDa degradation product co-eluted with the 47-kDa mature SK. The first amino acid residues of the S. lividans-produced SK were identical with those of the expected N-terminal sequence. The Vsi signal peptide was thus correctly cleaved off and the N-terminus of mature Vsi-SK fusion protein released by S. lividans remained intact. This result also implicates that the processing of the recombinant SK secreted by Streptomyces probably occurred at its C-terminal end, as in its native host Streptococcus equisimilis. The specific activity of the partially purified Streptomyces-derived SK was determined at 2661 IU/mg protein. Conclusion Heterologous expression of Streptococcus equisimilis ATCC9542 skc-2 in Streptomyces lividans was successfully achieved. SK can be translocated via both the Sec and the Tat pathway in S. lividans, but yield was about 30 times higher when the SK was fused to the Sec-dependent Vsi signal peptide compared to the fusion with the Tat-dependent signal peptide of S. lividans xylanase C. Small-scale fermentation led to a fourfold improvement of secretory SK yield in S. lividans compared to lab-scale conditions. The partially purified SK showed biological activity. Streptomyces lividans was shown to be a valuable host for the production of a world-wide important, biopharmaceutical product in a bio-active form.

  18. Recombinant DNA. Rifkin's regulatory revivalism runs riot.

    David, P

    Jeremy Rifkin, activist opponent of genetic engineering, has adopted tactics of litigation, persuasion, and confrontation in his campaign to halt genetic experimentation. The Recombinant DNA Advisory Committee of the National Institutes of Health has often been the target of his criticism, most recently for its failure to prepare an environmental risk assessment for some DNA tests it approved. Rifkin has won support for his position from religious organizations in the United States, and in June 1983 persuaded an ecumenical group of religious leaders to ask Congress to ban genetic experiments that would affect the human germ line. PMID:6578418

  19. Hydrogen recombiner catalyst test supporting data

    Britton, M.D.

    1995-01-19

    This is a data package supporting the Hydrogen Recombiner Catalyst Performance and Carbon Monoxide Sorption Capacity Test Report, WHC-SD-WM-TRP-211, Rev 0. This report contains 10 appendices which consist of the following: Mass spectrometer analysis reports: HRC samples 93-001 through 93-157; Gas spectrometry analysis reports: HRC samples 93-141 through 93-658; Mass spectrometer procedure PNL-MA-299 ALO-284; Alternate analytical method for ammonia and water vapor; Sample log sheets; Job Safety analysis; Certificate of mixture analysis for feed gases; Flow controller calibration check; Westinghouse Standards Laboratory report on Bois flow calibrator; and Sorption capacity test data, tables, and graphs.

  20. Hydrogen recombiner catalyst test supporting data

    This is a data package supporting the Hydrogen Recombiner Catalyst Performance and Carbon Monoxide Sorption Capacity Test Report, WHC-SD-WM-TRP-211, Rev 0. This report contains 10 appendices which consist of the following: Mass spectrometer analysis reports: HRC samples 93-001 through 93-157; Gas spectrometry analysis reports: HRC samples 93-141 through 93-658; Mass spectrometer procedure PNL-MA-299 ALO-284; Alternate analytical method for ammonia and water vapor; Sample log sheets; Job Safety analysis; Certificate of mixture analysis for feed gases; Flow controller calibration check; Westinghouse Standards Laboratory report on Bois flow calibrator; and Sorption capacity test data, tables, and graphs

  1. Liaisons dangereuses: sexual recombination among pathogenic trypanosomes.

    Gibson, Wendy

    2015-01-01

    Sexual recombination between pathogenic microbes has the potential to mobilise genes for harmful traits into new genetic backgrounds creating new pathogen strains. Since 1986 we have known that genetic exchange can occur in trypanosomes, but we are only now starting to unravel details of the process. In Trypanosoma brucei genetic exchange occurs in the tsetse vector, but is not an obligatory part of the life cycle. The process involves meiosis and production of haploid gametes, and thus appears to be true sexual reproduction. This review looks at the experimental evidence concerning genetic exchange and identifies current gaps in our knowledge. PMID:26027775

  2. The cell pole: the site of cross talk between the DNA uptake and genetic recombination machinery.

    Kidane, Dawit; Ayora, Silvia; Sweasy, Joann B; Graumann, Peter L; Alonso, Juan C

    2012-01-01

    Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as "guardians", protects ssDNA from degradation and limit the RecA recombinase loading. Then, the "mediators" overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by "modulators", catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or "resolver" cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the "rescuers" will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective. PMID:23046409

  3. Precise pattern of recombination in serotonergic and hypothalamic neurons in a Pdx1-cre transgenic mouse line

    Liou Angela

    2010-10-01

    Full Text Available Abstract Background Multicellular organisms are characterized by a remarkable diversity of morphologically distinct and functionally specialized cell types. Transgenic techniques for the manipulation of gene expression in specific cellular populations are highly useful for elucidating the development and function of these cellular populations. Given notable similarities in developmental gene expression between pancreatic β-cells and serotonergic neurons, we examined the pattern of Cre-mediated recombination in the nervous system of a widely used mouse line, Pdx1-cre (formal designation, Tg(Ipf1-cre89.1Dam, in which the expression of Cre recombinase is driven by regulatory elements upstream of the pdx1 (pancreatic-duodenal homeobox 1 gene. Methods Single (hemizygous transgenic mice of the pdx1-creCre/0 genotype were bred to single (hemizygous transgenic reporter mice (Z/EG and rosa26R lines. Recombination pattern was examined in offspring using whole-mount and sectioned histological preparations at e9.5, e10.5, e11.5, e16.5 and adult developmental stages. Results In addition to the previously reported pancreatic recombination, recombination in the developing nervous system and inner ear formation was observed. In the central nervous system, we observed a highly specific pattern of recombination in neuronal progenitors in the ventral brainstem and diencephalon. In the rostral brainstem (r1-r2, recombination occurred in newborn serotonergic neurons. In the caudal brainstem, recombination occurred in non-serotonergic cells. In the adult, this resulted in reporter expression in the vast majority of forebrain-projecting serotonergic neurons (located in the dorsal and median raphe nuclei but in none of the spinal cord-projecting serotonergic neurons of the caudal raphe nuclei. In the adult caudal brainstem, reporter expression was widespread in the inferior olive nucleus. In the adult hypothalamus, recombination was observed in the arcuate nucleus and dorsomedial hypothalamus. Recombination was not observed in any other region of the central nervous system. Neuronal expression of endogenous pdx1 was not observed. Conclusions The Pdx1-cre mouse line, and the regulatory elements contained in the corresponding transgene, could be a valuable tool for targeted genetic manipulation of developing forebrain-projecting serotonergic neurons and several other unique neuronal sub-populations. These results suggest that investigators employing this mouse line for studies of pancreatic function should consider the possible contributions of central nervous system effects towards resulting phenotypes.

  4. Induction of intrachromosomal homologous recombination in whole plants

    The influence of different factors on frequencies of intrachromosomal homologous recombination in whole Arabidopsis thaliana and tobacco plants was analyzed using a disrupted β-glucuronidase marker gene. Recombination frequencies were enhanced several fold by DNA damaging agents like UV-light or MMS (methyl methanesulfonate). Applying 3-methoxybenzamide (3-MB), an inhibitor of poly(ADP)ribose polymerase (PARP), an enzyme that is postulated to be involved in DNA repair, enhanced homologous recombination frequencies strongly. These findings indicate that homologous recombination is involved in DNA repair and can (at least partially) compensate for other DNA repair pathways. Indications that recombination in plants can be induced by environmental stress factors that are not likely to be involved in DNA metabolism were also found; Arabidopsis plants growing in a medium containing 0.1 M NaCl exhibited elevated recombination frequencies. The possible general effects of ‘environmental’ challenges on genome flexibility are discussed. (author)

  5. Trap-limited recombination in dye-sensitized nanocrystalline metal oxide electrodes

    Nelson, Jenny; Haque, Saif A.; Klug, David R.; Durrant, James R.

    2001-05-01

    We use transient and steady-state optical spectroscopies to study the recombination reaction between electrons and dye cations in a dye-sensitized nanocrystalline TiO2 electrode in several different chemical environments. Kinetic decay curves are approximately stretched exponential, and the cation half-life, t50% varies with electron density n as t50%~n-1/α, where α is a constant in the range 0.2-0.5. We have developed a model of electron transport in the presence of an energetic distribution of trap states and consider two regimes. In the first, the continuous-time random-walk (CTRW) electrons are free to diffuse through the lattice, by means of multiple trapping events mediated by the conduction band. In the second, the hopping regime, trapped electrons are allowed to tunnel to other, vacant trap sites, or to the dye cation, according to a Miller-Abrahams model for the transition rate. We carry out Monte Carlo simulations of the recombination kinetics as a function of electron density, trap state distributions and other parameters. The CTRW reproduces both the dependence of t50% on n and the shape of the kinetic curves with only one free fitting parameter, for the case of an exponential density of trap states. The hopping model is ruled out by subnanosecond measurements. We conclude that multiple trapping with a broad energetic distribution of electron traps is responsible for the slow recombination kinetics. When applied to recombination in a nanocrystalline photovoltaic junction at open circuit, the model predicts a sublinear power-law variation of electron density with light intensity G, n~Gα, compatible with the observed behavior.

  6. Recombinant human bone morphogenetic protein-9 potently induces osteogenic differentiation of human periodontal ligament fibroblasts.

    Fuchigami, Sawako; Nakamura, Toshiaki; Furue, Kirara; Sena, Kotaro; Shinohara, Yukiya; Noguchi, Kazuyuki

    2016-04-01

    To accomplish effective periodontal regeneration for periodontal defects, several regenerative methods using growth and differentiation factors, including bone morphogenetic proteins (BMPs), have been developed. Bone morphogenetic protein-9 exhibits the most potent osteogenic activity of this growth factor family. However, it is unclear whether exogenous BMP-9 can induce osteogenic differentiation in human periodontal ligament (PDL) fibroblasts. Here, we examined the effects of recombinant human (rh) BMP-9 on osteoblastic differentiation in human PDL fibroblasts in vitro, compared with rhBMP-2. Recombinant human BMP-9 potently induced alkaline phosphatase (ALP) activity, mineralization, and increased expression of runt-related transcription factor-2/core binding factor alpha 1 (RUNX2/CBFA1), osterix, inhibitor of DNA binding/differentiation-1 (ID1), osteopontin, and bone sialoprotein genes, compared with rhBMP-2. The levels of rhBMP-9-induced osterix and ALP mRNA were significantly reduced in activin receptor-like kinase-1 and -2 small interfering RNA (siRNA)-transfected human PDL fibroblasts. Recombinant human BMP-9-induced ALP activity was not inhibited by noggin, in contrast to rhBMP-2 induced ALP activity, which was. Phosphorylation of SMAD1/5/8 in human PDL fibroblasts was induced by addition of rhBMP-9. Recombinant human BMP-9-induced ALP activity was suppressed by SB203580, SP600125, and U0126, which are inhibitors of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2), respectively. Our data suggest that rhBMP-9 is a potent inducer of the differentiation of human PDL fibroblasts into osteoblast-like cells and that this may be mediated by the SMAD and mitogen-activated protein kinase (p38, ERK1/2, and JNK) pathways. PMID:26879145

  7. Sorbitol production using recombinant Zymomonas mobilis strain.

    Liu, Changjun; Dong, Hongwei; Zhong, Jianjiang; Ryu, Dewey D Y; Bao, Jie

    2010-07-20

    A recombinant Zymomonas mobilis strain harboring the plasmid pHW20a-gfo for over-expression of glucose-fructose oxidoreductase (GFOR) was constructed. The specific activity of GFOR enzyme in the new recombinant strain was at least two folds greater than that in the wild strain. The maximum GFOR activity achieved in terms of the volumetric, and the cellular were 2.59 U ml(-1), and 0.70 U mg(-1), respectively, in the batch cultures. A significant improvement of the bioconversion process for the production of sorbitol and gluconic acid from glucose and fructose was made using divalent metal ions which drastically reduced the ethanol yield and significantly increased the yield of target product. Among several divalent metal ions evaluated, Zn(2+) was found to be most effective by inhibiting the Entner-Doudoroff pathway enzymes. The yield of the byproduct ethanol was reduced from 16.7 to 1.8 gl(-1) and the sorbitol yield was increased to almost 100% from 89%. The Ca(2+) enhanced the sorbitol yield and the formation of calcium gluconate salt made the separation of gluconate from the reaction system easier. PMID:20438775

  8. Numerical and experimental investigations on catalytic recombiners

    Numerous containments of European light water reactors (LWR) are equipped with passive auto-catalytic recombiners (PAR). These devices are designed for the removal of hydrogen generated during a severe accident in order to avoid serious damage caused by a detonation. PARs make use of the fact that hydrogen and oxygen react exothermally on catalytic surfaces generating steam and heat even below conventional ignition concentrations and temperatures. Activities at ISR aim at overcoming existing limitations of today's systems. These are e.g. limited conversion capacity or unintended ignition of the gaseous mixture due to overheating of the catalyst elements caused by strong reaction heat generation. Experiments at the REKO facilities are conducted in order to achieve a profound understanding of the processes inside a recombiner, such as reaction kinetics or heat and mass transfer. Innovative PAR designs which may overcome existing limitations can be developed based on the knowledge obtained from these experiments. For the analysis of the processes inside a PAR the numerical code REKO-DIREKT is being developed. The code calculates the local catalyst temperatures and the concentration regression along the catalyst plates. For the validation of the model numerous experiments have been performed with different types of coating and different plate arrangements. The first calculations fit well with the experimental results indicating a proper understanding of the fundamental processes. The paper describes the experiments as well as the numerical model and presents model calculations in comparison with experimental results. (authors)

  9. Monitoring recombinant human erythropoietin abuse among athletes.

    Citartan, Marimuthu; Gopinath, Subash C B; Chen, Yeng; Lakshmipriya, Thangavel; Tang, Thean-Hock

    2015-01-15

    The illegal administration of recombinant human erythropoietin (rHuEPO) among athletes is largely preferred over blood doping to enhance stamina. The advent of recombinant DNA technology allowed the expression of EPO-encoding genes in several eukaryotic hosts to produce rHuEPO, and today these performance-enhancing drugs are readily available. As a mimetic of endogenous EPO (eEPO), rHuEPO augments the oxygen carrying capacity of blood. Thus, monitoring the illicit use of rHuEPO among athletes is crucial in ensuring an even playing field and maintaining the welfare of athletes. A number of rHuEPO detection methods currently exist, including measurement of hematologic parameters, gene-based detection methods, glycomics, use of peptide markers, electrophoresis, isoelectric focusing (IEF)-double immunoblotting, aptamer/antibody-based methods, and lateral flow tests. This review gleans these different strategies and highlights the leading molecular recognition elements that have potential roles in rHuEPO doping detection. PMID:25058943

  10. Selenomethionyl analog of recombinant human choriogonadotropin.

    Chen, W Y; Bahl, O P

    1991-05-25

    Selenomethionyl and high mannose type analog of recombinant human choriogonadotropin (hCG) to solve the crystallization and phase problems has been obtained by gene transfer methodology. SF9 insect cells were infected with the recombinant viruses containing hCG alpha and hCG beta cDNAs in selenomethionine containing methionine-free Grace's medium. The selenomethionyl hCG (SehCG) was purified from the culture medium by one step immunoaffinity chromatography using an immobilized monoclonal antibody against hCG beta. The presence of selenomethionine was demonstrated by amino acid analysis of SehCG. The amino acid composition indicated that more than 84% of methionine residues were substituted by selenomethionine. Its sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis yielded a single 38-kDa protein band under nonreducing conditions. The carbohydrate analysis of SehCG was consistent with the presence of four N-linked high mannose type carbohydrates and four O-linked simple disaccharide chains. The in vitro immunological and biological studies of SehCG indicated that selenomethionine substitution had no effect on the immunopotency, receptor binding, and steroidogenic activities of the hormone. PMID:2033036

  11. Recombinant antigens for immunodiagnosis of cystic echinococcosis

    Li Jun

    2004-01-01

    Full Text Available Three cDNAs, termed EpC1, TPxEg and EgG5, were isolated by immunoscreening from an Echinococcus granulosus cDNA library. The recombinant phages exhibited strong reactivity with sera from humans with confirmed cystic echinococcosis (CE and with sera from mice infected with E. granulosus oncospheres. The cDNAs were subcloned into a pET vector, expressed as fusion proteins tagged with GST and affinity purified against the GST tag. Of the three recombinant proteins, EpC1 achieved the highest performance for serodiagnosis of CE in Western blot analysis using a panel of clinically defined human sera to initially address the sensitivity and specificity of the molecules. The protein yielded an overall sensitivity of 92.2% and specificity of 95.6%, levels unprecedented taking into account the large panel of 896 human sera that were tested. The strategy used may also prove suitable for improved immunodiagnosis of other parasitic infections.

  12. Pre-dimensioning the catalytic recombiners

    Severe accident calculations for Belgian PWRs have confirmed that the production of hydrogen and carbon monoxide is a concern for the containment integrity. Therefore several preventive solutions were examined, among which the catalytic recombiners offer decisive advantages. This paper briefly describes the calculation method developed to determine the required surface area. The release rates are provided by STCP/MARCH3 runs and are used as input data of a post processor developed in Belgatom. This program computes the modified atmosphere composition for a given catalyst surface area. In addition to this, it calculates the adiabatic combustion pressure with and without recombiners, which allows to assess the risk of containment failure in both situations. Calculations were performed for two 900 MW plants with different concrete compositions. One of them is of the siliceous type which releases limited quantities of carbon monoxide, while the second is made of limestone common sand and produces large quantities of that gas during the decomposition process. Because the decomposition process is governed by a time constant, the pre-dimensioning was based on accident scenarios with the highest computed instantaneous release rates of flammable gases. Hence, attention was focused on scenarios initiated by a small break LOCA because they lead to high release rates of hydrogen at vessel breach. The calculation results show that a catalyst surface area of 250 m2 prevents the deflagration pressure to exceed the ultimate containment capability

  13. Dielectronic recombination of lithium-like gold

    We report measurements on radiative and dielectronic recombination (RR and DR) of very highly charged ions at energies Ecm=0 to 50 eV. Novel techniques were employed at the heavy ion storage ring ESR to obtain absolute recombination rates of Au76+(1s22s). The increase of the RR rate for Ecm?0 could be recorded and single DR resonances in Au75+(1s22p3/26lj) resolved. The experimental data are compared to theoretical results for RR and DR. The calculations for RR are based on the Bethe-Salpeter approach for completely stripped ions corrected to match Stobbe's theory and corrected for the presence of several core electrons in the ions studied here. The theoretical RR rates are combined with results for DR from perturbative-relativistic, semi-relavistic and fully relativistic theories. There is remarkable agreement between theory and experiment. Nevertheless the high quality of the experimental data allows one to distinguish between the different theoretical approaches to DR

  14. Recombinant shark natural antibodies to thyroglobulin.

    Schluter, Samuel F; Jensen, Ingvill; Ramsland, Paul A; Marchalonis, John J

    2005-01-01

    As cartilaginous fish are the vertebrates most distal from man to produce antibodies, fundamental information regarding conservation and variation of the antigen binding site should be gained by comparing the properties of antibodies directed against the same antigen from the two species. Since monoclonal cell lines cannot be generated using shark B cells, we isolated antigen binding recombinant single chain Fv antibodies (scFv) comprising of the complete variable regions from shark light and heavy chains. Thyroglobulin was used as the selecting antigen as both sharks and humans express natural antibodies to mammalian thyroglobulin in the absence of purposeful immunization. We report that recombinant sandbar shark (Carcharhinus plumbeus) scFvs that bind bovine thyroglobulin consist of heavy chain variable regions (VH) homologous to those of the human VHIII subset and light chain variable regions (VL) homologous to those of the human Vlambda6 subgroup. The homology within the frameworks is sufficient to enable the building of three-dimensional models of the shark VH/VL structure using established human structures as templates. In natural antibodies of both species, the major variability lies in the third complementarity determining region (CDR3) of both VH and VL. PMID:15954089

  15. Recombinant protein scaffolds for tissue engineering

    New biological materials for tissue engineering are now being developed using common genetic engineering capabilities to clone and express a variety of genetic elements that allow cost-effective purification and scaffold fabrication from these recombinant proteins, peptides or from chimeric combinations of these. The field is limitless as long as the gene sequences are known. The utility is dependent on the ease, product yield and adaptability of these protein products to the biomedical field. The development of recombinant proteins as scaffolds, while still an emerging technology with respect to commercial products, is scientifically superior to current use of natural materials or synthetic polymer scaffolds, in terms of designing specific structures with desired degrees of biological complexities and motifs. In the field of tissue engineering, next generation scaffolds will be the key to directing appropriate tissue regeneration. The initial period of biodegradable synthetic scaffolds that provided shape and mechanical integrity, but no biological information, is phasing out. The era of protein scaffolds offers distinct advantages, particularly with the combination of powerful tools of molecular biology. These include, for example, the production of human proteins of uniform quality that are free of infectious agents and the ability to make suitable quantities of proteins that are found in low quantity or are hard to isolate from tissue. For the particular needs of tissue engineering scaffolds, fibrous proteins like collagens, elastin, silks and combinations of these offer further advantages of natural well-defined structural scaffolds as well as endless possibilities of controlling functionality by genetic manipulation. (topical review)

  16. Reconstructing the modular recombination history of Staphylococcus aureus phages

    Swenson, Krister M; Guertin, Paul; Deschênes, Hugo; Bergeron, Anne

    2013-01-01

    Background Viruses that infect bacteria, called phages, are well-known for their extreme mosaicism, in which an individual genome shares many different parts with many others. The mechanisms for creating these mosaics are largely unknown but are believed to be recombinations, either illegitimate, or partly homologous. In order to reconstruct the history of these recombinations, we need to identify the positions where recombinations may have occurred, and develop algorithms to generate and exp...

  17. Intermediate bands versus levels in non-radiative recombination

    There is a practical interest in developing semiconductors with levels situated within their band gap while preventing the non-radiative recombination that these levels promote. In this paper, the physical causes of this non-radiative recombination are analyzed and the increase in the density of the impurities responsible for the mid-gap levels to the point of forming bands is suggested as the means of suppressing the recombination. Simple models supporting this recommendation and helping in its quantification are presented

  18. Advanced Three Level Approximation for Numerical Treatment of Cosmological Recombination

    Kholupenko, Evgeny E.; Ivanchik, Alexandre V.; Balashev, Sergey A.; Varshalovich, Dmitry A.

    2011-01-01

    New public numerical code for fast calculations of the cosmological recombination of primordial hydrogen-helium plasma is presented. The code is based on the three-level approximation (TLA) model of recombination and allows us to take into account some fine physical effects of cosmological recombination simultaneously with using fudge factors. The code can be found at http://www.ioffe.ru/astro/QC/CMBR/atlant/atlant.html

  19. Branching innovation, recombinant innovation, and endogenous technological transitions

    Frenken, K.; Izquierdo, L; Zeppini, P.

    2012-01-01

    We propose a model of technological transitions based on two different types of innovations. Branching innovations refer to technological improvements along a particular path, while recombinant innovations represent fusions of multiple paths. Recombinant innovations create "short-cuts" which reduce switching costs allowing agents to escape a technological lock-in. As a result, recombinant innovations speed up technological progress allowing transitions that are impossible with only branching ...

  20. A novel and simple method for construction of recombinant adenoviruses

    Tan, Rong; Li, Chunhua; Jiang, Sijing; Ma, Lixin

    2006-01-01

    Recombinant adenoviruses have been widely used for various applications, including protein expression and gene therapy. We herein report a new and simple cloning approach to an efficient and robust construction of recombinant adenoviral genomes based on the mating-assisted genetically integrated cloning (MAGIC) strategy. The production of recombinant adenovirus serotype 5-based vectors was greatly facilitated by the use of the MAGIC procedure and the development of the Adeasy adenoviral vect...