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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

    DEFF Research Database (Denmark)

    Sleeth, Kate M; Sørensen, 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 direct...

  2. RPA Accumulation during Class Switch Recombination Represents 5?–3? DNA-End Resection during the S–G2/M Phase of the Cell Cycle

    Directory of Open Access Journals (Sweden)

    Arito Yamane

    2013-01-01

    Full Text Available Activation-induced cytidine deaminase (AID promotes chromosomal translocations by inducing DNA double-strand breaks (DSBs at immunoglobulin (Ig genes and oncogenes in the G1 phase. RPA is a single-stranded DNA (ssDNA-binding protein that associates with resected DSBs in the S phase and facilitates the assembly of factors involved in homologous repair (HR, such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage, but its role in Ig gene recombination remains unclear. Here, we demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, recombination-activating genes (RAG, or other nucleases. Small amounts of RPA are deposited at AID targets in G1 in an ATM-dependent manner. In contrast, recruitment in the S–G2/M phase is extensive, ATM independent, and associated with Rad51 accumulation. In the S–G2/M phase, RPA increases in nonhomologous-end-joining-deficient lymphocytes, where there is more extensive DNA-end resection. Thus, most RPA recruitment during class switch recombination represents salvage of unrepaired breaks by homology-based pathways during the S–G2/M phase of the cell cycle.

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

    DEFF Research Database (Denmark)

    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 recombinati...

  4. The sensitivity of R_pA to color recombination effects

    CERN Document Server

    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 ...

  5. RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks.

    Science.gov (United States)

    Elia, Andrew E H; Wang, David C; Willis, Nicholas A; Boardman, Alexander P; Hajdu, Ildiko; Adeyemi, Richard O; Lowry, Elizabeth; Gygi, Steven P; Scully, Ralph; Elledge, Stephen J

    2015-10-15

    We have used quantitative proteomics to profile ubiquitination in the DNA damage response (DDR). We demonstrate that RPA, which functions as a protein scaffold in the replication stress response, is multiply ubiquitinated upon replication fork stalling. Ubiquitination of RPA occurs on chromatin, involves sites outside its DNA binding channel, does not cause proteasomal degradation, and increases under conditions of fork collapse, suggesting a role in repair at stalled forks. We demonstrate that the E3 ligase RFWD3 mediates RPA ubiquitination. RFWD3 is necessary for replication fork restart, normal repair kinetics during replication stress, and homologous recombination (HR) at stalled replication forks. Mutational analysis suggests that multisite ubiquitination of the entire RPA complex is responsible for repair at stalled forks. Multisite protein group sumoylation is known to promote HR in yeast. Our findings reveal a similar requirement for multisite protein group ubiquitination during HR at stalled forks in mammalian cells. PMID:26474068

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

    OpenAIRE

    Schild, David; Wiese, Claudia

    2009-01-01

    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 an...

  7. Human DNA helicase B functions in cellular homologous recombination and stimulates Rad51-mediated 5'-3' heteroduplex extension in vitro.

    Science.gov (United States)

    Liu, Hanjian; Yan, Peijun; Fanning, Ellen

    2015-01-01

    Homologous recombination is involved in the repair of DNA damage and collapsed replication fork, and is critical for the maintenance of genomic stability. Its process involves a network of proteins with different enzymatic activities. Human DNA helicase B (HDHB) is a robust 5'-3' DNA helicase which accumulates on chromatin in cells exposed to DNA damage. HDHB facilitates cellular recovery from replication stress, but its role in DNA damage response remains unclear. Here we report that HDHB silencing results in reduced sister chromatid exchange, impaired homologous recombination repair, and delayed RPA late-stage foci formation induced by ionizing radiation. Ectopically expressed HDHB colocalizes with Rad51, Rad52, RPA, and ssDNA. In vitro, HDHB stimulates Rad51-mediated heteroduplex extension in 5'-3' direction. A helicase-defective mutant HDHB failed to promote this reaction. Our studies implicate HDHB promotes homologous recombination in vivo and stimulates 5'-3' heteroduplex extension during Rad51-mediated strand exchange in vitro. PMID:25617833

  8. Anthrax Sub-unit Vaccine: the structural consequences of binding rPA83 to Alhydrogel®

    OpenAIRE

    Soliakov, Andrei; Kelly, Ian. F.; Lakey, Jeremy H.; Watkinson, Allan

    2011-01-01

    An anthrax sub-unit vaccine, comprising recombinant Protective Antigen (rPA83) and aluminium hydroxide adjuvant (Alhydrogel®) is currently being developed. Here a series of biophysical techniques have been applied to free and adjuvant bound antigen. Limited proteolysis and fluorescence identified no changes in rPA83 tertiary structure following binding to Alhydrogel and the bound rPA83 retained two structurally-important calcium ions. For adsorbed rPA83, differential scanning calorimetry reve...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. Cre-Mediated Recombination in Rhombic Lip Derivatives

    OpenAIRE

    Fünfschilling, Ursula; Reichardt, Louis F.

    2002-01-01

    To study the development of the cerebellum, we generated a transgenicmouse line Tg(m?6-cre)B1LFR that expresses CRE recombinase under the GABAA receptor ?6 subunit promoter. In this line, recombination of an R26R reporter allele occurred postnatally in granule cells of the cerebellum and dorsal cochlear nucleus, as well as in a subset of precerebellar nuclei in the brainstem. All neurons in which recombination occurred originated during embryogenesis from the rhombic lip. This might be explai...

  11. Chain-Bias of Escherichia Coli Rec-Mediated ? Patch Recombinants Is Independent of the Orientation of ? Cos

    OpenAIRE

    Rosenberg, S M

    1988-01-01

    Chi is a hotspot for homologous recombination mediated by the RecBCD (Rec) pathway of Escherichia coli. For Rec-mediated recombination of phage ?, the orientation of ? cos in the ? chromosome dictates the direction of travel of RecBCD enzyme through DNA and dictates which orientation of Chi or Chi-like sequences will be active in stimulating recombination. I previously found that Rec-mediated ? patch heteroduplexes, stimulated by Chi or not, are chain-biased; at the ? P locus, recombinant inf...

  12. Mechanochemical regulations of RPA's binding to ssDNA

    Science.gov (United States)

    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.

  13. PCR-mediated recombination of the amplification products of the Hibiscus tiliaceus cytosolic glyceraldehyde-3-phosphate dehydrogenase gene.

    Science.gov (United States)

    Wu, Linghui; Tang, Tian; Zhou, Renchao; Shi, Suhua

    2007-03-31

    PCR-mediated recombination describes the process of in vitro chimera formation from related template sequences present in a single PCR amplification. The high levels of genetic redundancy in eukaryotic genomes should make recombination artifacts occur readily. However, few evolutionary biologists adequately consider this phenomenon when studying gene lineages. The cytosolic glyceraldehyde-3-phosphate dehydrogenase gene (GapC), which encodes a NADP-dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase in the cytosol, is a classical low-copy nuclear gene marker and is commonly used in molecular evolutionary studies. Here, we report on the occurrence of PCR-mediated recombination in the GapC gene family of Hibiscus tiliaceus. The study suggests that recombinant areas appear to be correlated with DNA template secondary structures. Our observations highlight that recombination artifacts should be considered when studying specific and allelic phylogenies. The authors suggest that nested PCR be used to suppress PCR-mediated recombination. PMID:17394766

  14. Recombinant adenovirus-mediated gene transfer suppresses experimental arthritis

    Directory of Open Access Journals (Sweden)

    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 ?-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 ?-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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. A Stabilized Respiratory Syncytial Virus Reverse Genetics System Amenable to Recombination Mediated Mutagenesis

    OpenAIRE

    Hotard, Anne L.; Shaikh, Fyza Y; Lee, Sujin; YAN, DAN; Teng, Michael N.; Plemper, Richard K; Crowe, James E.; Moore, Martin L

    2012-01-01

    We describe the first example of combining bacterial artificial chromosome (BAC) recombination-mediated mutagenesis with reverse genetics for a negative strand RNA virus. A BAC-based respiratory syncytial virus (RSV) rescue system was established. An important advantage of this system is that RSV antigenomic cDNA was stabilized in the BAC vector. The RSV genotype chosen was A2-line19F, a chimeric strain previously shown to recapitulate in mice key features of RSV pathogenesis. We recovered tw...

  18. Tamoxifen-Containing Eye Drops Successfully Trigger Cre-Mediated Recombination in the Entire Eye.

    Science.gov (United States)

    Schlecht, Anja; Leimbeck, Sarah V; Tamm, Ernst R; Braunger, Barbara M

    2016-01-01

    Embryonic lethality in mice with targeted gene deletion is a major issue that can be circumvented by using Cre-loxP-based animal models. Various inducible Cre systems are available, e.g. such that are activated following tamoxifen treatment, and allow deletion of a specific target gene at any desired time point during the life span of the animal. In this study, we describe the efficiency of topical tamoxifen administration by eye drops using a Cre- reporter mouse strain (R26R). We report that tamoxifen-responsive CAGGCre-ER (TM) mice show a robust Cre- mediated recombination throughout the entire eye. PMID:26427451

  19. BRG1 promotes the repair of DNA double-strand breaks by facilitating the replacement of RPA with RAD51.

    Science.gov (United States)

    Qi, Wenjing; Wang, Ruoxi; Chen, Hongyu; Wang, Xiaolin; Xiao, Ting; Boldogh, Istvan; Ba, Xueqing; Han, Liping; Zeng, Xianlu

    2015-01-15

    DNA double-strand breaks (DSBs) are a type of lethal DNA damage. The repair of DSBs requires tight coordination between the factors modulating chromatin structure and the DNA repair machinery. BRG1, the ATPase subunit of the chromatin remodelling complex Switch/Sucrose non-fermentable (SWI/SNF), is often linked to tumorigenesis and genome instability, and its role in DSB repair remains largely unclear. In the present study, we show that BRG1 is recruited to DSB sites and enhances DSB repair. Using DR-GFP and EJ5-GFP reporter systems, we demonstrate that BRG1 facilitates homologous recombination repair rather than nonhomologous end-joining (NHEJ) repair. Moreover, the BRG1-RAD52 complex mediates the replacement of RPA with RAD51 on single-stranded DNA (ssDNA) to initiate DNA strand invasion. Loss of BRG1 results in a failure of RAD51 loading onto ssDNA, abnormal homologous recombination repair and enhanced DSB-induced lethality. Our present study provides a mechanistic insight into how BRG1, which is known to be involved in chromatin remodelling, plays a substantial role in the homologous recombination repair pathway in mammalian cells. PMID:25395584

  20. Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Risager, Peter Christian; Fahnøe, Ulrik; Friis, Martin Barfred; Belsham, Graham; Höper, Dirk; Reimann, Ilona; Beer, Martin

    2013-01-01

    Background Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. Results A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and ...

  1. Homologous recombination-mediated gene targeting in the liverwort Marchantia polymorpha L.

    Science.gov (United States)

    Ishizaki, Kimitsune; Johzuka-Hisatomi, Yasuyo; Ishida, Sakiko; Iida, Shigeru; Kohchi, Takayuki

    2013-01-01

    The liverwort Marchantia polymorpha is an emerging model organism on account of its ideal characteristics for molecular genetics in addition to occupying a crucial position in the evolution of land plants. Here we describe a method for gene targeting by applying a positive/negative selection system for reduction of non-homologous random integration to an efficient Agrobacterium-mediated transformation system using M. polymorpha sporelings. The targeting efficiency was evaluated by knocking out the NOP1 gene, which impaired air-chamber formation. Homologous recombination was observed in about 2% of the thalli that passed the positive/negative selection. With the advantage of utilizing the haploid gametophytic generation, this strategy should facilitate further molecular genetic analysis of M. polymorpha, in which many of the mechanisms found in land plants are conserved, yet in a less complex form. PMID:23524944

  2. Regression of established pulmonary metastases and subcutaneous tumor mediated by the systemic administration of high-dose recombinant interleukin 2

    International Nuclear Information System (INIS)

    Incubation of resting lymphoid cells with recombinant interleukin 2 (IL-2) in vitro leads to the generation of lymphokine activated killer (LAK) cells capable of lysing fresh tumor cell suspensions in short-term chromium-release assays. Previous studies have demonstrated that the injection of LAK cells plus low doses of recombinant IL-2 were capable of inhibiting the growth of pulmonary metastases. The authors have now explored the ability of high doses of recombinant IL-2, administered systemically, to generate LAK cells in vivo, and to mediate antitumor effects directly. Doses of 100,000 U recombinant IL-2 administered intraperitoneally approximately every 8 h for 5 d were capable of dramatically inhibiting established 3-d pulmonary metastases from the MCA-105 and MCA-106 syngeneic sarcomas and the syngeneic B16 melanoma in C57BL/6 mice. Grossly visible metastases present at 10 d after tumor injection also underwent regression following IL-2 therapy. All antitumor effects of the systemic administration of recombinant IL-2 were eliminated if mice received prior treatment with 500 rad total body irradiation. The administration of high doses of recombinant IL-2 was also capable of inhibiting the growth of 3-d established subcutaneous tumors from the MCA-105 sarcoma, and of mediating the inhibition of growth and regression of established palpable subcutaneous MCA-105 sarcomas. The ready availability of high doses of recombinant human IL-2, and the demonstration of antitumor effects seen in animal models have led us to the initiation of the clinical trials of recombinant IL-2 in humans

  3. Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Risager, Peter Christian

    2013-01-01

    Background Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. Results A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence. Conclusions These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses.

  4. Enhanced levels of ? Red-mediated recombinants in mismatch repair mutants

    OpenAIRE

    Costantino, Nina; Court, Donald L.

    2003-01-01

    Homologous recombination can be used to generate recombinants on episomes or directly on the Escherichia coli chromosome with PCR products or synthetic single-stranded DNA (ssDNA) oligonucleotides (oligos). Such recombination is possible because bacteriophage ?-encoded functions, called Red, efficiently recombine linear DNA with homologies as short as 20–70 bases. This technology, termed recombineering, provides ways to modify genes and segments of the chromosome as well as to study homologou...

  5. Wilms' tumor gene WT1 promotes homologous recombination-mediated DNA damage repair.

    Science.gov (United States)

    Oji, Yusuke; Tatsumi, Naoya; Kobayashi, Junya; Fukuda, Mari; Ueda, Tazu; Nakano, Eri; Saito, Chisae; Shibata, Syohei; Sumikawa, Mihoko; Fukushima, Hisashi; Saito, Akari; Hojo, Nozomi; Suzuki, Miyu; Hoshikawa, Tomoko; Shimura, Tsutomu; Morii, Eiichi; Oka, Yoshihiro; Hosen, Naoki; Komatsu, Kenshi; Sugiyama, Haruo

    2015-12-01

    The Wilms' tumor gene WT1 is overexpressed in leukemia and various types of solid tumors and plays an oncogenic role in these malignancies. Alternative splicing at two sites yields four major isoforms, 17AA(+)KTS(+), 17AA(+)KTS(-), 17AA(-)KTS(+), and 17AA(-)KTS(-), and all the isoforms are expressed in the malignancies. However, among the four isoforms, function of WT1[17AA(-)KTS(+)] isoform still remains undetermined. In the present study, we showed that forced expression of WT1[17AA(-)KTS(+)] isoform significantly inhibited apoptosis by DNA-damaging agents such as Doxorubicin, Mitomycin, Camptothesisn, and Bleomycin in immortalized fibroblast MRC5SV and cervical cancer HeLa cells. Knockdown of Rad51, an essential factor for homologous recombination (HR)-mediated DNA repair canceled the resistance to Doxorubicin induced by WT1[17AA(-)KTS(+)] isoform. GFP recombination assay showed that WT1[17AA(-)KTS(+)] isoform alone promoted HR, but that three other WT1 isoforms did not. WT1[17AA(-)KTS(+)] isoform significantly upregulated the expression of HR genes, XRCC2, Rad51D, and Rad54. Knockdown of XRCC2, Rad51D, and Rad54 inhibited the HR activity and canceled resistance to Doxorubicin in MRC5SV cells with forced expression of WT1[17AA(-)KTS(+)] isoform. Furthermore, chromatin immunoprecipitation (ChIP) assay showed the binding of WT1[17AA(-)KTS(+)] isoform protein to promoters of XRCC2 and Rad51D. Immunohistochemical study showed that Rad54 and XRCC2 proteins were highly expressed in the majority of non-small-cell lung cancer (NSCLC) and gastric cancer, and that expression of these two proteins was significantly correlated with that of WT1 protein in NSCLCs. Our results presented here showed that WT1[17AA(-)KTS(+)] isoform had a function to promote HR-mediated DNA repair. © 2014 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc. PMID:25418835

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

    Science.gov (United States)

    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...

  7. CtIP promotes microhomology-mediated alternative end joining during class-switch recombination.

    Science.gov (United States)

    Lee-Theilen, Mieun; Matthews, Allysia J; Kelly, Dierdre; Zheng, Simin; Chaudhuri, Jayanta

    2011-01-01

    Immunoglobulin heavy chain (Igh locus) class-switch recombination (CSR) requires targeted introduction of DNA double strand breaks (DSBs) into repetitive 'switch'-region DNA elements in the Igh locus and subsequent ligation between distal DSBs. Both canonical nonhomologous end joining (C-NHEJ) that seals DNA ends with little or no homology and a poorly defined alternative end joining (A-NHEJ, also known as alt-NHEJ) process that requires microhomology ends for ligation have been implicated in CSR. Here, we show that the DNA end-processing factor CtIP is required for microhomology-directed A-NHEJ during CSR. Additionally, we demonstrate that microhomology joins that are enriched upon depletion of the C-NHEJ component Ku70 require CtIP. Finally, we show that CtIP binds to switch-region DNA in a fashion dependent on activation-induced cytidine deaminase. Our results establish CtIP as a bona fide component of microhomology-dependent A-NHEJ and unmask a hitherto unrecognized physiological role of microhomology-mediated end joining in a C-NHEJ-proficient environment. PMID:21131982

  8. RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia

    OpenAIRE

    Papaemmanuil, Elli; Rapado, Inmaculada; Li, Yilong; Potter, Nicola E.; Wedge, David C; Tubio, Jose; Alexandrov, Ludmil B.; Loo, Peter; Cooke, Susanna L; Marshall, John; Martincorena, Inigo; Hinton, Jonathan; Gundem, Gunes; van Delft, Frederik W; Nik-Zainal, Serena

    2014-01-01

    The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL), is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near the breakpoints; incorporation of non-templated sequence at the juncti...

  9. Extended GIPQ description of the RPA excitations

    International Nuclear Information System (INIS)

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

    Science.gov (United States)

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

  11. The IRG Mouse: A Two-Color Fluorescent Reporter for Assessing Cre-Mediated Recombination and Imaging Complex Cellular Relationships In Situ

    OpenAIRE

    Gasperi, Rita De; Rocher, Anne B.; Sosa, Miguel A Gama; Wearne, Susan L; Perez, Gissel M.; Friedrich, Victor L.; Hof, Patrick R; Elder, Gregory A.

    2008-01-01

    The Cre-loxP system is widely used for making conditional alterations to the mouse genome. Cre-mediated recombination is frequently monitored using reporter lines in which Cre expression activates a reporter gene driven by a ubiquitous promoter. Given the distinct advantages of fluorescent reporters, we developed a transgenic reporter line, termed IRG, in which DsRed-Express, a red fluorescent protein (RFP) is expressed ubiquitously prior to Cre-mediated recombination and an enhanced green fl...

  12. Copy-choice recombination by reverse transcriptases: Reshuffling of genetic markers mediated by RNA chaperones

    OpenAIRE

    Negroni, Matteo; Buc, Henri

    2000-01-01

    Copy-choice recombination efficiently reshuffles genetic markers in retroviruses. In vivo, the folding of the genomic RNA is controlled by the nucleocapsid protein (NC). We show that binding of NC onto the acceptor RNA molecule is sufficient to enhance recombination, providing evidence for a mechanism where the structure of the acceptor template determines the template switch. NC as well as another RNA chaperone (StpA) converts recombination into a widespread process no longer restricted to r...

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

    Science.gov (United States)

    Modi, Souvik; Halder, Saheli; Nizak, Clément; 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

  14. Generation of Cell Lines to Complement Adenovirus Vectors using Recombination-Mediated Cassette Exchange

    Directory of Open Access Journals (Sweden)

    Farley Daniel C

    2010-12-01

    Full Text Available Abstract Background Adenovirus serotype 5 (Ad5 has many favourable characteristics for development as a gene therapy vector. However, the utility of current Ad5 vectors is limited by transient transgene expression, toxicity and immunogenicity. The most promising form of vector is the high capacity type, which is deleted for all viral genes. However, these vectors can only be produced to relatively low titres and with the aid of helper virus. Therefore a continuing challenge is the generation of more effective Ad5 vectors that can still be grown to high titres. Our approach is to generate complementing cell lines to support the growth of Ad5 vectors with novel late gene deficiencies. Results We have used LoxP/Cre recombination mediated cassette exchange (RMCE to generate cell lines expressing Ad5 proteins encoded by the L4 region of the genome, the products of which play a pivotal role in the expression of Ad5 structural proteins. A panel of LoxP parent 293 cell lines was generated, each containing a GFP expression cassette under the control of a tetracycline-regulated promoter inserted at a random genome location; the cassette also contained a LoxP site between the promoter and GFP sequence. Clones displayed a variety of patterns of regulation, stability and level of GFP expression. Clone A1 was identified as a suitable parent for creation of inducible cell lines because of the tight inducibility and stability of its GFP expression. Using LoxP-targeted, Cre recombinase-mediated insertion of an L4 cassette to displace GFP from the regulated promoter in this parent clone, cell line A1-L4 was generated. This cell line expressed L4 100K, 22K and 33K proteins at levels sufficient to complement L4-33K mutant and L4-deleted viruses. Conclusions RMCE provides a method for rapid generation of Ad5 complementing cell lines from a pre-selected parental cell line, chosen for its desirable transgene expression characteristics. Parent cell lines can be selected for high or low gene expression, and for tight regulation, allowing viral protein expression to mirror that found during infection. Cell lines derived from a single parent will allow the growth of different vectors to be assessed without the complication of varying complementing protein expression.

  15. Generation of Nkx2.2:lacZ mice using Recombination-Mediated Cassette Exchange Technology

    OpenAIRE

    Arnes, Luis; Leclerc, Kevin; Friel, Jessica M.; Hipkens, Susan B.; Magnuson, Mark A; Sussel, Lori

    2012-01-01

    Nkx2.2 encodes a homeodomain transcription factor required for the correct specification and/or differentiation of cells in the pancreas, intestine and CNS. To follow the fate of cells deleted for Nkx2.2 within these tissues, we generated Nkx2.2:lacZ knockin mice using a recombination-mediated cassette exchange (RMCE) approach. Expression analysis of lacZ and/or ?-galactosidase in Nkx2.2lacZ/+ heterozygote embryos and adults demonstrates that lacZ faithfully recapitulates endogenous Nkx2.2 ex...

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

    DEFF Research Database (Denmark)

    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 ...

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

    Directory of Open Access Journals (Sweden)

    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 R6K? 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.

  18. Quantization in the high-spin RPA

    International Nuclear Information System (INIS)

    The problem of how to quantize the angular momentum of the self-consistent cranking model at high spin when small oscillations (RPA) about the steady rotation are included is reexamined, in view of a recent criticism by Reinhardt of an earlier treatment. This criticism is shown to be unfounded. On the other hand, it is shown that Reinhardt's quantization procedure leads to some serious problems, and the result that the vibrational frequencies differ in the rotating and lab frames is called into question. (orig.)

  19. Multiple Regulation of Rad51-Mediated Homologous Recombination by Fission Yeast Fbh1

    OpenAIRE

    Tsutsui, Yasuhiro; Kurokawa, Yumiko; Ito, Kentaro; Siddique, Md. Shahjahan P.; Kawano, Yumiko; Yamao, Fumiaki; IWASAKI, HIROSHI

    2014-01-01

    Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent ma...

  20. CtIP promotes microhomology-mediated alternative end-joining during class switch recombination

    OpenAIRE

    Lee-Theilen, Mieun; Matthews, Allysia J.; Kelly, Dierdre; Zheng, Simin; Chaudhuri, Jayanta

    2010-01-01

    Immunoglobulin heavy chain (Igh) class switch recombination (CSR) requires targeted introduction of DNA double strand breaks (DSBs) into repetitive “switch” region DNA elements in the Igh locus and subsequent ligation between distal DSBs. Both canonical non-homologous end-joining (C-NHEJ) that seals DNA ends with little or no homology, and a poorly defined alternative-end joining (A-NHEJ) process that requires microhomology ends for ligation have been implicated in CSR. Here we show that the ...

  1. Generation of TALEN-Mediated GRdim Knock-In Rats by Homologous Recombination

    OpenAIRE

    Ponce de León, Verónica; Mérillat, Anne-Marie; Tesson, Laurent; Anegón, Ignacio; Hummler, Edith

    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 GRdim, that prevents receptor homodimerization in the mouse. TALEN mRN...

  2. Srs2 promotes Mus81–Mms4-mediated resolution of recombination intermediates

    OpenAIRE

    Chavdarova, Melita; Marini, Victoria; Sisakova, Alexandra; Sedlackova, Hana; Vigasova, Dana; Brill, Steven J; Lisby, Michael; Krejci, Lumir

    2015-01-01

    A variety of DNA lesions, secondary DNA structures or topological stress within the DNA template may lead to stalling of the replication fork. Recovery of such forks is essential for the maintenance of genomic stability. The structure-specific endonuclease Mus81–Mms4 has been implicated in processing DNA intermediates that arise from collapsed forks and homologous recombination. According to previous genetic studies, the Srs2 helicase may play a role in the repair of double-strand breaks and ...

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

    Scientific Electronic Library Online (English)

    A., Di Pietro; G., Dayan; G., Conseil; E., Steinfels; T., Krell; D., Trompier; H., Baubichon-Cortay; J.-M., Jault.

    1999-08-01

    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 dom [...] ain 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.

  4. The therapeutic potential of the recombinant antigen from Dirofilaria immitis (rDiAg) for immune-mediated pregnancy loss.

    Science.gov (United States)

    Komine-Aizawa, Shihoko; Izumi, Yasuyuki; Imai, Shinjiro; Fujita, Koichiro; Hayakawa, Satoshi

    2011-12-01

    The mammalian fetuses are semi-allograft for mothers. Therefore the failure of immunological tolerance often causes pregnancy loss. Recently, the effects of helminthes therapy for immune mediated diseases have been reported. In the present study we employed the murine model to examine the therapeutic potential of the recombinant antigen from a nematoda parasite, Dirofilaria immitis for immune mediated pregnancy loss. Recombinant D. immitis polyproteins (rDiAg) had been cloned and selected by us for the strongest immuno-regulatory activities in parasite antigens. Female CBA/J mice were injected with sterilized rDiAg or PBS solution using micro-osmotic pumps before mating. Pregnant CBA/J mice were sacrificed on day 13.5 for scoring the number of resorbed and viable fetuses for histological and immunological analysis. The serum cytokine concentrations were measured using suspension array system. The resorption rate of mock-treated mice was 42.9% (resorbed fetus 12/total fetus 28). The resorption rate was decreased to 11.1% (resorbed fetus 3/total fetus 27) with rDiAg treatments. The IL-4, IL-23 and TNF-? concentrations in serum were significantly lower in rDiAg-treated mice than mock-treated mice. The serum IL-17 level was also reduced in rDiAg-treated mice but the difference was not significant. The rDiAg treatment reduced the resorption rates of CBA/J×DBA/2J mouse model, which mimic human pregnancy failures with allo-immune backgrounds. Our observations suggest as the first time of therapeutic potentials of the rDiAg for pregnancy loss. PMID:21983343

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

    Directory of Open Access Journals (Sweden)

    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.

  6. MODEST: a web-based design tool for oligonucleotide-mediated genome engineering and recombineering

    DEFF Research Database (Denmark)

    Bonde, Mads; Klausen, Michael S.

    2014-01-01

    Recombineering and multiplex automated genome engineering (MAGE) offer the possibility to rapidly modify multiple genomic or plasmid sites at high efficiencies. This enables efficient creation of genetic variants including both single mutants with specifically targeted modifications as well as combinatorial cell libraries. Manual design of oligonucleotides for these approaches can be tedious, time-consuming, and may not be practical for larger projects targeting many genomic sites. At present, the change from a desired phenotype (e.g. altered expression of a specific protein) to a designed MAGE oligo, which confers the corresponding genetic change, is performed manually. To address these challenges, we have developed the MAGE Oligo Design Tool (MODEST). This web-based tool allows designing of MAGE oligos for (i) tuning translation rates by modifying the ribosomal binding site, (ii) generating translational gene knockouts and (iii) introducing other coding or non-coding mutations, including amino acid substitutions, insertions, deletions and point mutations. The tool automatically designs oligos based on desired genotypic or phenotypic changes defined by the user, which can be used for high efficiency recombineering and MAGE. MODEST is available for free and is open to all users at http://modest.biosustain.dtu.dk.

  7. Photoinduced molecular dissociation and photoinduced recombination mediated by superfluid helium nanodroplets.

    Science.gov (United States)

    Kautsch, Andreas; Koch, Markus; Ernst, Wolfgang E

    2015-05-14

    We have investigated photoinduced chemical reaction dynamics of cold, isolated Cr2 molecules in helium nanodroplets (HeN), exploiting the quantum state specific spatial separation of solvated and surface locations on the droplet. The molecules are excited to achieve dissociation to a ground state (a(7)S3) and a metastable state (a(5)S2) atom. State specific spatial separation, in combination with efficient translational cooling to avoid ejection, causes the ground state atom to be solvated inside the droplet while the metastable atom migrates to the surface. A barrier between the two reactants formed by the HeN prevents recombination. We apply a resonance-enhanced multiphoton ionization scheme including the y(5)P°(1,2,3) laser scheme including the y(7)P°(2,3,4) <-- a(7)S(3) transition of the solvated atom in order to verify the locations and separation of the dissociation products. Furthermore, ionization of the a(5)S2 surface atom triggers solvation followed by geminate recombination with the a(7)S3 atom, which is verified by the detection of Cr2(+) molecular ions. For small Cr clusters, our results indicate that they may be composed of chromium dimers that exhibit the same dissociation behavior. PMID:25894482

  8. Homologous recombination-mediated gene targeting in the liverwort Marchantia polymorpha L.

    OpenAIRE

    Kimitsune Ishizaki; Yasuyo Johzuka-Hisatomi; Sakiko Ishida; Shigeru Iida; Takayuki Kohchi

    2013-01-01

    The liverwort Marchantia polymorpha is an emerging model organism on account of its ideal characteristics for molecular genetics in addition to occupying a crucial position in the evolution of land plants. Here we describe a method for gene targeting by applying a positive/negative selection system for reduction of non-homologous random integration to an efficient Agrobacterium-mediated transformation system using M. polymorpha sporelings. The targeting efficiency was evaluated by knocking ou...

  9. The crystal structure of the complex of replication protein A subunits RPA32 and RPA14 reveals a mechanism for single-stranded DNA binding.

    OpenAIRE

    Bochkarev, A.; Bochkareva, E; Frappier, L.; A.M. Edwards

    1999-01-01

    Replication protein A (RPA), the eukaryote single-stranded DNA-binding protein (SSB), is a heterotrimer. The largest subunit, RPA70, which harbours the major DNA-binding activity, has two DNA-binding domains that each adopt an OB-fold. The complex of the two smaller subunits, RPA32 and RPA14, has weak DNA-binding activity but the mechanism of DNA binding is unknown. We have determined the crystal structure of the proteolytic core of RPA32 and RPA14, which consists of the central two-thirds of...

  10. Surface reengineering of RPA70N enables co-crystallization with an inhibitor of the RPA interaction motif of ATRIP

    OpenAIRE

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

    2013-01-01

    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 up-regulati...

  11. Attenuated Nontoxinogenic and Nonencapsulated Recombinant Bacillus anthracis Spore Vaccines Protect against Anthrax

    OpenAIRE

    Cohen, S; Mendelson, I.; Altboum, Z.; Kobiler, D.; Elhanany, E.; Bino, T; Leitner, M; Inbar, I.; Rosenberg, H.; Gozes, Y.; Barak, R.; Fisher, M; Kronman, C.; Velan, B.; Shafferman, A

    2000-01-01

    Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinant protective antigen (rPA) were constructed. Biochemical analyses (including electrospray mass spectroscopy and N terminus amino acid sequencing) as well as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs with 5 × 107 spores of one of these recombinant st...

  12. Suppression of mutagenesis by Rad51D-mediated homologous recombination

    Energy Technology Data Exchange (ETDEWEB)

    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.

  13. Interethnic polymorphism of EWS intron 6: genome plasticity mediated by Alu retroposition and recombination.

    Science.gov (United States)

    Zucman-Rossi, J; Batzer, M A; Stoneking, M; Delattre, O; Thomas, G

    1997-03-01

    The EWS gene has been identified as being systematically translocated in Ewing's sarcoma. In order to ascertain the basis of a marked interethnic difference in the incidence of Ewing's sarcoma, intron 6 of EWS, which is located near the translocation breakpoint region (EWSR1), was characterized. Sequence analysis of the entire intron 6 region revealed a very high density of Alu elements. Most of these Alu sequences could be classified in previously described subfamilies, facilitating delineation of an evolutionary model that involves successive retroposition events. According to this model, the EWS intron 6 region progressively expanded until about 5 million years ago. More recently (10(5) years ago), in part of the human population, the size of this region decreased by over 50% as the result of a homeologous recombination between two Alu sequences, which removed 2480 bp. This rare allele has only been observed in individuals of African origin, a population that is characterized by the lowest incidence of Ewing's sarcoma. PMID:9050923

  14. Peptide nucleic acid-mediated recombination for targeted genomic repair and modification.

    Science.gov (United States)

    Schleifman, Erica B; Glazer, Peter M

    2014-01-01

    The ability to directly manipulate the human genome to correct a disease-related mutation, introduce a sequence change that would lead to site-specific gene knockout, or increase gene expression is a very powerful tool with tremendous clinical value. Triplex formation by synthetic DNA-binding molecules such as peptide nucleic acids (PNAs) has been studied for over 20 years and much of the work in the last 10 years has shown its great promise in its use to direct site-specific gene modification for the use in gene therapy. In this chapter, detailed protocols are described for the design and use of triplex-forming PNAs to bind and mediate gene modification at specific chromosomal targets. Target site identification, PNA and donor oligonucleotide design, in vitro characterization of binding, optimization with reporter systems, as well as various methods to assess gene modification and isolate modified cells are described. PMID:24297362

  15. RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia.

    Science.gov (United States)

    Papaemmanuil, Elli; Rapado, Inmaculada; Li, Yilong; Potter, Nicola E; Wedge, David C; Tubio, Jose; Alexandrov, Ludmil B; Van Loo, Peter; Cooke, Susanna L; Marshall, John; Martincorena, Inigo; Hinton, Jonathan; Gundem, Gunes; van Delft, Frederik W; Nik-Zainal, Serena; Jones, David R; Ramakrishna, Manasa; Titley, Ian; Stebbings, Lucy; Leroy, Catherine; Menzies, Andrew; Gamble, John; Robinson, Ben; Mudie, Laura; Raine, Keiran; O'Meara, Sarah; Teague, Jon W; Butler, Adam P; Cazzaniga, Giovanni; Biondi, Andrea; Zuna, Jan; Kempski, Helena; Muschen, Markus; Ford, Anthony M; Stratton, Michael R; Greaves, Mel; Campbell, Peter J

    2014-02-01

    The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL) cases, is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near breakpoints, incorporation of non-templated sequence at junctions, ?30-fold enrichment at promoters and enhancers of genes actively transcribed in B cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single-cell tracking shows that this mechanism is active throughout leukemic evolution, with evidence of localized clustering and reiterated deletions. Integration of data on point mutations and rearrangements identifies ATF7IP and MGA as two new tumor-suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1-positive lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B cell differentiation. PMID:24413735

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

    Directory of Open Access Journals (Sweden)

    Choi Jin-Ok

    2010-04-01

    Full Text Available 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 compared it with that of ERT. Methods A pseudotyped rAAV2/8 vector encoding ?-Gal A cDNA (rAAV2/8-hAGA was prepared and injected into 18-week-old male Fabry mice through the tail vein. The ?-Gal A expression level and globotriaosylceramide (Gb3 levels in the Fabry mice were examined and compared with Fabry mice with ERT. Immunohistochemical and ultrastructural studies were conducted. Results Treatment of Fabry mice with rAAV2/8-hAGA resulted in the clearance of accumulated Gb3 in tissues such as liver, spleen, kidney, heart, and brain with concomitant elevation of ?-Gal A enzyme activity. Enzyme activity was elevated for up to 60 weeks. In addition, expression of the ?-Gal A protein was identified in the presence of rAAV2/8-hAGA at 6, 12, and 24 weeks after treatment. ?-Gal A activity was significantly higher in the mice treated with rAAV2/8-hAGA than in Fabry mice that received ERT. Along with higher ?-Gal A activity in the kidney of the Fabry mice treated with gene therapy, immunohistochemical studies showed more ?-Gal A expression in the proximal tubules and glomerulus, and less Gb3 deposition in Fabry mice treated with this gene therapy than in mice given ERT. The ?-gal A gene transfer significantly reduced the accumulation of Gb3 in the tubules and podocytes of the kidney. Electron microscopic analysis of the kidneys of Fabry mice also showed that gene therapy was more effective than ERT. Conclusions The rAAV2/8-hAGA mediated ?-Gal A gene therapy provided improved efficiency over ERT in the Fabry disease mouse model. Furthermore, rAAV2/8-hAGA-mediated expression showed a greater effect in the kidney than ERT.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Comparison of the Structural Stability and Dynamic Properties of Recombinant Anthrax Protective Antigen and its 2-Fluorohistidine Labeled Analogue

    OpenAIRE

    Hu, Lei; Joshi, Sangeeta B.; Kiran K. Andra; Thakkar, Santosh V; Volkin, David B; James G. Bann; Middaugh, C. Russell

    2012-01-01

    Protective antigen (PA) is the primary protein antigenic component of both the currently used anthrax vaccine and related recombinant vaccines under development. An analogue of recombinant PA (2-FHis rPA) has been recently shown to block the key steps of pore formation in the process of inducing cytotoxicity in cells, and thus can potentially be used as an antitoxin or a vaccine. This rPA analogue was produced by fermentation to incorporate the unnatural amino acid 2-fluorohistidine (2-FHis)....

  19. Structure Factor in the Presence of Shear - an RPA Calculation

    CERN Document Server

    Vinograd, G; Vinograd, Guy; Schwartz, Moshe

    2002-01-01

    We consider the structure factor of a system of colloidal particles immersed in a host liquid. Each particle is assumed to be affected by forces due to other particles, a drag force proportional to the velocity of the particle relative to the local velocity of the fluid and a fluctuating random noise. The effect of the particles on the velocity field of the host liquid is neglected. The problem is treated within the RPA approximation which is a widely used tool in many body theory. The validity of the RPA result is discussed.

  20. Pauli principle corrections to RPA within a boson formalism

    International Nuclear Information System (INIS)

    The Random Phase Approximation (RPA) is examined in a boson formalism and special attention is focused on the problem of the violation of the Pauli Principle affecting this theory. A mapping technique is discussed and the boson image of a two-body fermion Hamiltonian is constructed. Within the boson space so defined, an extension of RPA is proposed where structure and energy of the ground state, as well as of the first excited one, are analyzed on the basis of the Thouless Theorem for bosons and with regard to the role of the spurious components. Numerical tests are performed within the Lipkin-Meshkov-Glick model. (author). 17 refs., 2 figs

  1. Small molecule inhibitor of the RPA70 N-terminal protein interaction domain discovered using in silico and in vitro methods

    OpenAIRE

    Glanzer, Jason G.; Liu, Shengqin; Oakley, Gregory G.

    2011-01-01

    The pharmacological suppression of the DNA damage response and DNA repair can increase the therapeutic indices of conventional chemotherapeutics. Replication Protein A (RPA), the major single-stranded DNA binding protein in eukaryotes, is required for DNA replication, DNA repair, DNA recombination, and DNA damage response signaling. Through the use of high-throughput screening of 1500 compounds, we have identified a small molecule inhibitor, 15-carboxy-13-isopropylatis-13-ene-17,18-dioic acid...

  2. Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

    Science.gov (United States)

    Dulal, Kalpana; Silver, Benjamin; Zhu, Hua

    2012-01-01

    Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning. PMID:22500089

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    RPA is a single-stranded DNA binding protein that physically associates with the BLM complex. RPA stimulates BLM helicase activity as well as the double Holliday junction dissolution activity of the BLM-topoisomerase IIIa complex. We investigated the effect of RPA on the ssDNA decatenase activity 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...

  4. The partial tandem duplication of ALL1 (MLL) is consistently generated by Alu-mediated homologous recombination in acute myeloid leukemia.

    Science.gov (United States)

    Strout, M P; Marcucci, G; Bloomfield, C D; Caligiuri, M A

    1998-03-01

    Chromosome abnormalities resulting in gene fusions are commonly associated with acute myeloid leukemia (AML), however, the molecular mechanism(s) responsible for these defects are not well understood. The partial tandem duplication of the ALL1 (MLL) gene is found in patients with AML and trisomy 11 as a sole cytogenetic abnormality and in 11% of patients with AML and normal cytogenetics. This defect results from the genomic fusion of ALL1 intron 6 or intron 8 to ALL1 intron 1. Here, we examined the DNA sequence at the genomic fusion in nine cases of AML with a tandem duplication of ALL1 spanning exons 2-6. Each breakpoint occurred within intron 6 of the ALL1 breakpoint cluster region and within a discrete 3.8-kb region near the 3' end of intron 1. In seven cases, a distinct point of fusion of intron 6 with intron 1 could not be identified. Instead, the sequence gradually diverged from an Alu element in intron 6 to an Alu element in intron 1 through a heteroduplex fusion. Thus, these rearrangements appear to be the result of a recombination event between homologous Alu sequences in introns 6 and 1. In two cases, the genomic junction was distinct and involved the fusion of a portion of an Alu element in intron 6 with non-Alu sequence in intron 1. These data support the hypothesis that a recombination event between homologous Alu sequences is responsible for the partial tandem duplication of ALL1 in the majority of AML cases with this genetic defect. Although Alu element-mediated homologous recombination events in germline cells are thought to be responsible for partial gene duplications or deletions in many inherited diseases, this appears to be the first demonstration identifying Alu element-mediated recombination as a consistent mechanism for gene rearrangement in somatic tissue. PMID:9482895

  5. Cell cycle-specific UNG2 phosphorylations regulate protein turnover, activity and association with RPA

    DEFF Research Database (Denmark)

    Hagen, Lars; Kavli, Bodil

    2008-01-01

    Human UNG2 is a multifunctional glycosylase that removes uracil near replication forks and in non-replicating DNA, and is important for affinity maturation of antibodies in B cells. How these diverse functions are regulated remains obscure. Here, we report three new phosphoforms of the non-catalytic domain that confer distinct functional properties to UNG2. These are apparently generated by cyclin-dependent kinases through stepwise phosphorylation of S23, T60 and S64 in the cell cycle. Phosphorylation of S23 in late G1/early S confers increased association with replication protein A (RPA) and replicating chromatin and markedly increases the catalytic turnover of UNG2. Conversely, progressive phosphorylation of T60 and S64 throughout S phase mediates reduced binding to RPA and flag UNG2 for breakdown in G2 by forming a cyclin E/c-myc-like phosphodegron. The enhanced catalytic turnover of UNG2 p-S23 likely optimises the protein to excise uracil along with rapidly moving replication forks. Our findings may aid further studies of how UNG2 initiates mutagenic rather than repair processing of activation-induced deaminase-generated uracil at Ig loci in B cells.

  6. IS2-mediated re-arrangement of the promoter sequence supresses metabolic burden of the recombinant plasmid.

    Czech Academy of Sciences Publication Activity Database

    Valešová, Renata; Št?pánek, Václav; Ve?erek, Branislav; Kyslík, Pavel

    2005-01-01

    Ro?. 50, ?. 4 (2005), s. 275-282. ISSN 0015-5632 Institutional research plan: CEZ:AV0Z50200510 Keywords : is2 * recombinant plasmid * escherichia coli Subject RIV: EE - Microbiology, Virology Impact factor: 0.918, year: 2005

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Immunogenicity of Recombinant Protective Antigen and Efficacy against Aerosol Challenge with Anthrax

    OpenAIRE

    Williamson, E. D.; Hodgson, I; Walker, N. J.; Topping, A. W.; Duchars, M. G.; Mott, J. M.; Estep, J.; LeButt, C.; Flick-Smith, H. C.; Jones, H E; LI, H.; Quinn, C. P.

    2005-01-01

    Immunization with a recombinant form of the protective antigen (rPA) from Bacillus anthracis has been carried out with rhesus macaques. Rhesus macaques immunized with 25 ?g or more of B. subtilis-expressed rPA bound to alhydrogel had a significantly increased immunoglobulin G (IgG) response to rPA compared with macaques receiving the existing licensed vaccine from the United Kingdom (anthrax vaccine precipitated [AVP]), although the isotype profile was unchanged, with bias towards the IgG1 an...

  9. Structural and Immunological Analysis of Anthrax Recombinant Protective Antigen Adsorbed to Aluminum Hydroxide Adjuvant

    OpenAIRE

    Wagner, Leslie; Verma, Anita; Meade, Bruce D.; Reiter, Karine; NARUM, DAVID L.; Brady, Rebecca A.; Little, Stephen F.; Burns, Drusilla L.

    2012-01-01

    New anthrax vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored f...

  10. Assessment of toxicity and biodistribution of recombinant AAV8 vector–mediated immunomodulatory gene therapy in mice with Pompe disease

    OpenAIRE

    Wang, Gensheng; Young, Sarah P; Bali, Deeksha; Hutt, Julie; Li, Songtao; Benson, Janet; Koeberl, Dwight D

    2014-01-01

    A preclinical safety study was conducted to evaluate the short- and long-term toxicity of a recombinant adeno-associated virus serotype 8 (AAV2/8) vector that has been developed as an immune-modulatory adjunctive therapy to recombinant human acid ?-glucosidase (rhGAA, Myozyme) enzyme replacement treatment (ERT) for patients with Pompe disease (AAV2/8-LSPhGAApA). The AAV2/8-LSPhGAApA vector at 1.6?×?1013 vector particles/kg, after intravenous injection, did not cause significant short- or long...

  11. Skyrme RPA for spherical and axially symmetric nuclei

    CERN Document Server

    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...

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

    CERN Document Server

    Avancini, Sidney S; Pinto, Marcus B

    2015-01-01

    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.

  13. COM-1 promotes homologous recombination during Caenorhabditis elegans meiosis by antagonizing Ku-mediated non-homologous end joining.

    OpenAIRE

    Lemmens BB1; Johnson NM; Tijsterman M.

    2013-01-01

    Successful completion of meiosis requires the induction and faithful repair of DNA double-strand breaks (DSBs). DSBs can be repaired via homologous recombination (HR) or non-homologous end joining (NHEJ), yet only repair via HR can generate the interhomolog crossovers (COs) needed for meiotic chromosome segregation. Here we identify COM-1, the homolog of CtIP/Sae2/Ctp1, as a crucial regulator of DSB repair pathway choice during Caenorhabditis elegans gametogenesis. COM-1–deficient germ cells ...

  14. Engineering neonatal Fc receptor-mediated recycling and transcytosis in recombinant proteins by short terminal peptide extensions

    OpenAIRE

    Sockolosky, Jonathan T.; Tiffany, Matthew R.; Szoka, Francis C

    2012-01-01

    The importance of therapeutic recombinant proteins in medicine has led to a variety of tactics to increase their circulation time or to enable routes of administration other than injection. One clinically successful tactic to improve both protein circulation and delivery is to fuse the Fc domain of IgG to therapeutic proteins so that the resulting fusion proteins interact with the human neonatal Fc receptor (FcRn). As an alternative to grafting the high molecular weight Fc domain to therapeut...

  15. Stable transgene expression in rod photoreceptors after recombinant adeno-associated virus-mediated gene transfer to monkey retina

    OpenAIRE

    Bennett, Jean; Maguire, Albert M.; Cideciyan, Artur V; Schnell, Michael; Glover, Ernest; Anand, Vibha; Aleman, Tomas S.; Chirmule, Narendra; Gupta, Abha R; Huang, Yijun; Gao, Guang-Ping; Nyberg, William C.; Tazelaar, John; Hughes, Joseph; Wilson, James M

    1999-01-01

    Recombinant adeno-associated virus (rAAV) is a promising vector for therapy of retinal degenerative diseases. We evaluated the efficiency, cellular specificity, and safety of retinal cell transduction in nonhuman primates after subretinal delivery of an rAAV carrying a cDNA encoding green fluorescent protein (EGFP), rAAV.CMV.EGFP. The treatment results in efficient and stable EGFP expression lasting >1 year. Transgene expression in the neural retina is limited exclusively to rod photoreceptor...

  16. TRESK gene recombinant adenovirus vector inhibits capsaicin-mediated substance P release from cultured rat dorsal root ganglion neurons

    OpenAIRE

    zhou, Jun; YAO, SHANG-LONG; YANG, CHENG-XIANG; ZHONG, JI-YING; WANG, HAN-BING; Zhang, Yan

    2012-01-01

    The present study was conducted to determine whether the activation of TRESK in the dorsal root ganglion (DRG) by the TRESK gene recombinant adenovirus vector inhibits the capsaicin-evoked substance P (SP) release using a radioimmunoassay. TRESK is an outwardly rectifying K+ current channel that contributes to the resting potential and is the most important background potassium channel in DRG. Previous studies have shown that neuropathic pain (NP) is closely related to the regulation of certa...

  17. Adeno-associated-virus-mediated transduction of the mammary gland enables sustained production of recombinant proteins in milk.

    Science.gov (United States)

    Wagner, Stefan; Thresher, Rosemary; Bland, Ross; Laible, Götz

    2015-01-01

    Biopharming for the production of recombinant pharmaceutical proteins in the mammary gland of transgenic animals is an attractive but laborious alternative compared to mammalian cell fermentation. The disadvantage of the lengthy process of genetically modifying an entire animal could be circumvented with somatic transduction of only the mammary epithelium with recombinant, replication-defective viruses. While other viral vectors offer very limited scope for this approach, vectors based on adeno-associated virus (AAV) appear to be ideal candidates because AAV is helper-dependent, does not induce a strong immune response and has no association with disease. Here, we sought to test the suitability of recombinant AAV (rAAV) for biopharming. Using reporter genes, we showed that injected rAAV efficiently transduced mouse mammary cells. When rAAV encoding human myelin basic protein (hMBP) was injected into the mammary glands of mice and rabbits, this resulted in the expression of readily detectable protein levels of up to 0.5?g/L in the milk. Furthermore we demonstrated that production of hMBP persisted over extended periods and that protein expression could be renewed in a subsequent lactation by re-injection of rAAV into a previously injected mouse gland. PMID:26463440

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

    Directory of Open Access Journals (Sweden)

    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.

  19. An Alternative Method to Facilitate cDNA Cloning for Expression Studies in Mammalian Cells by Introducing Positive Blue White Selection in Vaccinia Topoisomerase I-Mediated Recombination.

    Science.gov (United States)

    Udo, Hiroshi

    2015-01-01

    One of the most basic techniques in biomedical research is cDNA cloning for expression studies in mammalian cells. Vaccinia topoisomerase I-mediated cloning (TOPO cloning by Invitrogen) allows fast and efficient recombination of PCR-amplified DNAs. Among TOPO vectors, a pcDNA3.1 directional cloning vector is particularly convenient, since it can be used for expression analysis immediately after cloning. However, I found that the cloning efficiency was reduced when RT-PCR products were used as inserts (about one-quarter). Since TOPO vectors accept any PCR products, contaminating fragments in the insert DNA create negative clones. Therefore, I designed a new mammalian expression vector enabling positive blue white selection in Vaccinia topoisomerase I-mediated cloning. The method utilized a short nontoxic LacZ? peptide as a linker for GFP fusion. When cDNAs were properly inserted into the vector, minimal expression of the fusion proteins in E. coli (harboring lacZ?M15) resulted in formation of blue colonies on X-gal plates. This method improved both cloning efficiency (75%) and directional cloning (99%) by distinguishing some of the negative clones having non-cording sequences, since these inserts often disturbed translation of lacZ?. Recombinant plasmids were directly applied to expression studies using GFP as a reporter. Utilization of the P2A peptide allowed for separate expression of GFP. In addition, the preparation of Vaccinia topoisomerase I-linked vectors was streamlined, which consisted of successive enzymatic reactions with a single precipitation step, completing in 3 hr. The arrangement of unique restriction sites enabled further modification of vector components for specific applications. This system provides an alternative method for cDNA cloning and expression in mammalian cells. PMID:26422141

  20. An Alternative Method to Facilitate cDNA Cloning for Expression Studies in Mammalian Cells by Introducing Positive Blue White Selection in Vaccinia Topoisomerase I-Mediated Recombination

    Science.gov (United States)

    Udo, Hiroshi

    2015-01-01

    One of the most basic techniques in biomedical research is cDNA cloning for expression studies in mammalian cells. Vaccinia topoisomerase I-mediated cloning (TOPO cloning by Invitrogen) allows fast and efficient recombination of PCR-amplified DNAs. Among TOPO vectors, a pcDNA3.1 directional cloning vector is particularly convenient, since it can be used for expression analysis immediately after cloning. However, I found that the cloning efficiency was reduced when RT-PCR products were used as inserts (about one-quarter). Since TOPO vectors accept any PCR products, contaminating fragments in the insert DNA create negative clones. Therefore, I designed a new mammalian expression vector enabling positive blue white selection in Vaccinia topoisomerase I–mediated cloning. The method utilized a short nontoxic LacZ? peptide as a linker for GFP fusion. When cDNAs were properly inserted into the vector, minimal expression of the fusion proteins in E. coli (harboring lacZ?M15) resulted in formation of blue colonies on X-gal plates. This method improved both cloning efficiency (75%) and directional cloning (99%) by distinguishing some of the negative clones having non-cording sequences, since these inserts often disturbed translation of lacZ?. Recombinant plasmids were directly applied to expression studies using GFP as a reporter. Utilization of the P2A peptide allowed for separate expression of GFP. In addition, the preparation of Vaccinia topoisomerase I-linked vectors was streamlined, which consisted of successive enzymatic reactions with a single precipitation step, completing in 3 hr. The arrangement of unique restriction sites enabled further modification of vector components for specific applications. This system provides an alternative method for cDNA cloning and expression in mammalian cells. PMID:26422141

  1. Cultured mast cells from patients with asthma and controls respond with similar sensitivity to recombinant Der p2-induced, IgE-mediated activation

    DEFF Research Database (Denmark)

    Krohn, I K; Sverrild, A

    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 patients with asthma and healthy controls under identical conditions. Mast cells were cultured from peripheral blood from twelve patients with an objectively confirmed asthma diagnosis and eight healthy subjects. During the last 2 weeks of culture, mast cells were incubated with IL-4 and 80 kU/l recombinant human IgE containing two clones (7% + 7%) specific for mite allergen Der p2. The sensitivity of IgE-mediated activation of mast cells was investigated as Fc?RI-mediated upregulation 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 median fluorescence intensity was 20 456 ± 1640 (SE) for patients with asthma and 22,275 ± 1971 (SE) for controls (ns). The fraction of CD63 positive cells was 54.4% in patients with asthma and 48.4% in controls (ns). The allergen concentration inducing 50% of the maximal CD63 response was similar in patients with asthma [-0.4795 log ng/ml ± 0.092 (SE)] and controls (-0.6351 log ng/ml ± 0.083, ns) and in atopic and non-atopic subjects. When cultured, sensitized and activated under identical conditions, mast cells from allergic asthmatics and healthy controls respond similar. Activation of cultured mast cells appears to depend on culture conditions (IL-4, IgE) rather than on donor status as atopy and asthma.

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

    DEFF Research Database (Denmark)

    Krohn, Inge Jacoba Maria Kortekaas; Sverrild, Asger

    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. Mast cells were cultured from peripheral blood from twelve patients with an objectively confirmed asthma diagnosis and eight healthy subjects. During the last 2 weeks of culture, mast cells were incubated with IL-4 and 80 kU/L recombinant human IgE containing two clones (7%+7%) specific for mite 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 Median Fluorescence Intensity was 20 456 ± 1640 (SE) for asthma patients and 22 275 ± 1971 (SE) for controls (ns). The fraction of CD63 positive cells was 54.4% in asthma patients and 48.4% in controls (ns). The allergen concentration inducing 50% of the maximal CD63 response was similar in asthma patients (-0.4795 log ng/mL ± 0.092 (SE)) and controls (-0.6351 log ng/mL ± 0.083, ns) and in atopic and non-atopic subjects. When cultured, sensitised and activated under identical conditions, mast cells from allergic asthmatics and healthy controls respond similar. Activation of cultured mast cells 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.

  3. Human RPA (hSSB) interacts with EBNA1, the latent origin binding protein of Epstein-Barr virus.

    OpenAIRE

    ZHANG, D; Frappier, L.; Gibbs, E; Hurwitz, J; O'Donnell, M

    1998-01-01

    RPA is the replicative single-strand DNA (ssDNA) binding protein of eukaryotic chromosomes. This report shows that human RPA interacts with EBNA1, the latent origin binding protein of Epstein-Barr virus (EBV). RPA binds to EBNA1 both in solution, and when EBNA1 is bound to the EBV origin. RPA is a heterotrimer, and the main contact with EBNA1 is formed through the 70 kDa subunit of RPA, the subunit which binds to ssDNA. We propose that this interaction between RPA and EBNA1 is an early step i...

  4. Recombinant Adeno-associated virus (rAAV)-mediated transduction and optogenetic manipulation of cortical neurons in vitro

    Science.gov (United States)

    Lange, Wienke; Jin, Lei; Maybeck, Vanessa; Meisenberg, Annika; Baumann, Arnd; Offenhäusser, Andreas

    2014-03-01

    Genetically encoded light-sensitive proteins can be used to manipulate and observe cellular functions. According to different modes of action, these proteins are divided into actuators like the blue-light gated cation channel Channelrhodopsin-2 (ChR2) and detectors like the calcium sensor GCaMP. In order to optogenetically control and study the activity of rat primary cortical neurons, we established a transduction procedure using recombinant Adeno-associated viruses (rAAVs) as gene-ferries. Thereby, we achieved high transduction rates of these neurons with ChR2. In ChR2 expressing neurons, action potentials could be repeatedly and precisely elicited with laser pulses and measured via patch clamp recording.

  5. The lactoferrin receptor may mediate the reduction of eosinophils in the duodenum of pigs consuming milk containing recombinant human lactoferrin.

    Science.gov (United States)

    Cooper, Caitlin; Nonnecke, Eric; Lönnerdal, Bo; Murray, James

    2014-10-01

    Lactoferrin is part of the immune system and multiple tissues including the gastrointestinal (GI) tract, liver, and lung contain receptors for lactoferrin. Lactoferrin has many functions, including antimicrobial, immunomodulatory, and iron binding. Additionally, lactoferrin inhibits the migration of eosinophils, which are constitutively present in the GI tract, and increase during inflammation. Lactoferrin suppresses eosinophil infiltration into the lungs and eosinophil migration in -vitro. Healthy pigs have a large population of eosinophils in their small intestine and like humans, pigs have small intestinal lactoferrin receptors (LFR); thus, pigs were chosen to investigate the effects of consumption of milk containing recombinant human lactoferrin (rhLF-milk) on small intestinal eosinophils and expression of eosinophilic cytokines. In addition, LFR localization was analyzed in duodenum and circulating eosinophils to determine if the LFR could play a role in lactoferrin's ability to inhibit eosinophil migration. In the duodenum there were significantly fewer eosinophils/unit area in pigs fed rhLF-milk compared to pigs fed control milk (p = 0.025); this was not seen in the ileum (p = 0.669). In the duodenum, no differences were observed in expression of the LFR, or any eosinophil migratory cytokines, and the amount of LFR protein was not different (p = 0.386). Immunohistochemistry (IHC) showed that within the duodenum the LFR localized on the brush border of villi, crypts, and within the lamina propria. Circulating eosinophils also contained LFRs, which may be a mechanism allowing lactoferrin to directly inhibit eosinophil migration. PMID:25085595

  6. Inhibition of DNA replication factor RPA by p53.

    Science.gov (United States)

    Dutta, A; Ruppert, J M; Aster, J C; Winchester, E

    1993-09-01

    The tumour suppressor p53 specifically interferes with the onset of S phase. The mechanism of the growth suppression action of the protein is unclear, though recent evidence points to transcriptional activation and repression functions of the protein. A competing hypothesis suggests that p53 interacts with the DNA replication apparatus and directly interferes with DNA replication. The major evidence for this hypothesis is that p53 interacts with the simian virus 40 (SV40)-encoded protein T antigen and interferes with the ability of T antigen to unwind the SV40 origin of DNA replication, and recruit DNA polymerase alpha to the replication initiation complex. Here we report that p53 physically interacts with and inhibits the function of a cellular DNA replication factor, the single-stranded DNA-binding protein complex RPA. PMID:8361542

  7. Inhibition of tumor growth in xenograft nude mice model by recombinant adenovirus-mediated human endostatin gene therapy

    International Nuclear Information System (INIS)

    Objective: To investigate the expression efficiency of adenovirus-mediated human endostatin gene (Ad/hEndo) in vitro and in vivo, and to observe its inhibition of tumor growth in xenografted nude mice model. Methods: The expression efficiency of endostatin gene was examined during the infection of Ad/hEndo in nasopharyngeal carcinoma (NPC) CNE-2 cell and human umbilical vein endothelial cells (ECV304) by Western blot and ELISA. The effect on inhibition of growth of NPC CNE-2 xenografted tumors in Balb/c nude mice was observed after administration with Ad/hEndo. The serum endostatin levels were measured by ELISA, and intratumoral microvessel density (MVD) was analyzed. Results: Western blot and ELISA analysis demonstrated high level of endostatin expression in CNE2 and ECV304 cells infected with Ad/ hEndo. The highest concentration of endostatin in supernatant reached 588.34 ng/ml after 72 h of Ad/hEndo infection at a MOI of 20. Ad/hEndo significantly inhibited growth of xenografted CNE-2 (nasopharyngeal carcinoma) tumors with inhibition rate of 46.43% (Ad/ hEndo group versus Ad/LacZ group, t=2.226, P<0.05) and 49.70% (Ad/ hEndo group versus DMEM group, t=2.254, P<0.05), respectively. In the study group, serum levels of endostatin in treated group were much higher than that of control groups for 3- and 7- day term. The intratumoral MVD also decreased significantly in the treated tumors (9.95±2.20 versus 18.54±1.80, t=7.158, P< 0.05). Conclusion: Adenovirus-mediated human endostatin gene had obtained high level of expression in vitro and in vivo, and significantly inhibited the angiogenesis and growth of CNE-2 xenografted tumors in nude mice

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

    International Nuclear Information System (INIS)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Radiation protection authorized persons (RPA) in Germany; Der Strahlenschutzbevollmaechtigte in Deutschland

    Energy Technology Data Exchange (ETDEWEB)

    Sahre, P.; Beutmann, A.; Lorenz, J. [Verein fuer Kernverfahrenstechnik und Analytik Rossendorf e.V., Dresden (Germany); Leder, F. [Saechsisches Staatsministerium fuer Umwelt und Landwirtschaft, Dresden (Germany); Philipp, T. [Saechsisches Landesamt fuer Umwelt, Landwirtschaft und Geologie, Dresden (Germany)

    2013-07-01

    The radiation protection authorized person (RPA) is playing an important role in the fields of organization, realization and checking the radiation protection in Germany, first of all in big institutions like research centers, facilities and medical centers. The paper deals with the legal status of the RPA especially the clear dividing line between his tasks and the tasks of the radiation protection supervisor and the radiation protection commissioner. The paper shows that the embodiment of the RPA in the radiation protection law has advantages also in coordinating the tasks of radiation protection officer and radiation protection expert recommended by the European Union. (orig.)

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Accuracy of basis-set extrapolation schemes for DFT-RPA correlation energies in molecular calculations

    OpenAIRE

    Fabiano, E.; della Sala, F.

    2012-01-01

    We construct a reference benchmark set for atomic and molecular random-phase-approximation (RPA) correlation energies in a density functional theory (DFT) framework at the complete basis set limit. This set is used to evaluate the accuracy of some popular extrapolation schemes for RPA all-electron molecular calculations. The results indicate that for absolute energies accurate results, clearly outperforming raw data, are achievable with two-point extrapolation schemes based ...

  14. Cre-Mediated Recombination in Tas2r131 Cells-A Unique Way to Explore Bitter Taste Receptor Function Inside and Outside of the Taste System.

    Science.gov (United States)

    Voigt, Anja; Hübner, Sandra; Döring, Linda; Perlach, Nathalie; Hermans-Borgmeyer, Irm; Boehm, Ulrich; Meyerhof, Wolfgang

    2015-11-01

    The type 2 taste receptors (Tas2rs) comprise a large family of G protein-coupled receptors that recognize compounds bitter to humans and aversive to vertebrates. Tas2rs are expressed in both gustatory and nongustatory tissues, however, identification and functional analyses of T2R-expressing cells have been difficult in most tissues. To overcome these limitations and to be able to manipulate Tas2r-expressing cells in vivo, we used gene-targeting to generate a Tas2r131-specific Cre knock-in mouse strain. We then employed a binary genetic approach to characterize Cre-mediated recombination in these animals and to investigate Tas2r131 expression during postnatal development. We demonstrate that a Cre-activated fluorescent reporter reliably visualizes Tas2r131-cells in gustatory tissue. We show that the onset of Tas2r131 as well as of ?-Gustducin expression is initiated at different developmental stages depending on the type of taste bud. Furthermore, the number of Tas2r131- and ?-Gustducin-expressing cells increased during postnatal development. Our results demonstrate that the Tas2r131-expressing cells constitute a subpopulation of ?-Gustducin positive cells at all stages. We detected Tas2r131-expressing cells in several nongustatory tissues including lung, trachea, ovary, ganglia, and brain. Thus, the Tas2r131-Cre strain will help to dissect the functional role of Tas2r131 cells in both gustatory and nongustatory tissues in the future. PMID:26377344

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

    International Nuclear Information System (INIS)

    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

  16. Tensor hypercontracted ppRPA: Reducing the cost of the particle-particle random phase approximation from O(r?6) to O(r?4)

    International Nuclear Information System (INIS)

    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(r6), the THC-ppRPA algorithm scales asymptotically as only O(r4), 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

  17. Encapsidation of adeno-associated virus type 2 Rep proteins in wild-type and recombinant progeny virions: Rep-mediated growth inhibition of primary human cells.

    OpenAIRE

    Kube, D M; Ponnazhagan, S; Srivastava, A.

    1997-01-01

    The adeno-associated virus type 2 (AAV) arrests the growth of primary human fibroblasts in vitro at high particle-to-cell ratios. To test the role of AAV gene expression in the observed growth inhibition, primary human cells were infected, under identical conditions, with wild-type (wt) AAV or with recombinant AAV that lacked all viral promoters and coding sequences. Significant, dose-dependent growth inhibition of primary human cells was observed with both wt and recombinant AAV at particle-...

  18. A novel third type of recurrent NF1 microdeletion mediated by non-allelic homologous recombination between LRRC37B-containing low-copy repeats in 17q11.2

    OpenAIRE

    Bengesser, Kathrin; Cooper, David N.; Steinmann, Katharina; Kluwe, Lan; Chuzhanova, Nadia; Wimmer, Katharina; Tatagiba, Marcos; Tinschert, Sigrid; Mautner, Victor; Kehrer-Sawatzki, Hildegard

    2010-01-01

    Abstract Large microdeletions encompassing the neurofibromatosis type-1 (NF1) gene and its flanking regions at 17q11.2 belong to the group of genomic disorders caused by aberrant recombination between segmental duplications. The most common NF1 microdeletions (type-1) span 1.4-Mb and have breakpoints located within NF1-REPs A and C, low-copy repeats (LCRs) containing LRRC37-core duplicons. We have identified a novel type of recurrent NF1 deletion mediated by non-allelic homologous ...

  19. Function of Rad51 paralogs in eukaryotic homologous recombinational repair

    International Nuclear Information System (INIS)

    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

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

    OpenAIRE

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

    2013-01-01

    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 thi...

  1. Transformation of tobacco cpDNA with fusion E7GGG/GUS gene and homologous recombination mediated elimination of the marker gene.

    Czech Academy of Sciences Publication Activity Database

    B?íza, Jind?ich; Vlasák, Josef; Ryba, Š.; Ludvíková, V.; Niedermeierová, Hana

    2013-01-01

    Ro?. 27, ?. 2 (2013), s. 3644-3648. ISSN 1310-2818 R&D Projects: GA AV ?R IAA500960903 Institutional support: RVO:60077344 Keywords : E7GGG oncogene * chloroplast transformation * marker-free plant * homologous recombination Subject RIV: EB - Gene tics ; Molecular Biology Impact factor: 0.379, year: 2013

  2. Second RPA dynamics at finite temperature: time-evolutions of dynamical operators

    International Nuclear Information System (INIS)

    Time-evolutions of dynamical operators, in particular the generalized density matrix comprising both diagonal and off-diagonal elements, are investigated within the framework of second RPA dynamics at finite temperature. The calculation of the density matrix previously carried out through the appliance of the second RPA master equation by retaining only the slowly oscillating coupling terms is extended to include in the interaction Hamiltonian both the rapidly and slowly oscillating coupling terms. The extended second RPA master equation, thereby formulated without making use of the so-called resonant approximation, is analytically solved and a closed expression for the generalized density matrix is extracted. We provide illustrative examples of the generalized density matrix for various specific initial conditions. We turn particularly our attention to the Poisson distribution type of initial condition for which we deduce specifically a particular form of the density matrix from the solution of the Fokker-Planck equation for the coherent state representation. The relation of the Fokker-Planck equation to the second RPA master equation and its properties are briefly discussed. The oversight incurred in the time-evolution of operators by the resonant approximation is elucidated. The first and second moments of collective coordinates are also computed in relation to the expectation value of various dynamical operators involved in the extended master equation

  3. G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding

    Science.gov (United States)

    Ray, Sujay; Bandaria, Jigar N.; Qureshi, Mohammad H.; Yildiz, Ahmet; Balci, Hamza

    2014-01-01

    Human telomeres terminate with a single-stranded 3? G overhang, which can be recognized as a DNA damage site by replication protein A (RPA). The protection of telomeres (POT1)/POT1-interacting protein 1 (TPP1) heterodimer binds specifically to single-stranded telomeric DNA (ssTEL) and protects G overhangs against RPA binding. The G overhang spontaneously folds into various G-quadruplex (GQ) conformations. It remains unclear whether GQ formation affects the ability of POT1/TPP1 to compete against RPA to access ssTEL. Using single-molecule Förster resonance energy transfer, we showed that POT1 stably loads to a minimal DNA sequence adjacent to a folded GQ. At 150 mM K+, POT1 loading unfolds the antiparallel GQ, as the parallel conformation remains folded. POT1/TPP1 loading blocks RPA’s access to both folded and unfolded telomeres by two orders of magnitude. This protection is not observed at 150 mM Na+, in which ssTEL forms only a less-stable antiparallel GQ. These results suggest that GQ formation of telomeric overhangs may contribute to suppression of DNA damage signals. PMID:24516170

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

    International Nuclear Information System (INIS)

    The thermal TDHF equation is analyzed in the Liouville representation of quantum mechanics, where the matrix elements of the single-particle (s.p.) density ? 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 ?(?) 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 ?(?). 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 ?(?) in the RPA dynamics, and it helps to construct the RPA modes. Several extensions are considered

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

    Science.gov (United States)

    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

  6. Human immunodeficiency virus type 1 replication in the absence of integrase-mediated dna recombination: definition of permissive and nonpermissive T-cell lines.

    Science.gov (United States)

    Nakajima, N; Lu, R; Engelman, A

    2001-09-01

    Functional retroviral integrase protein is thought to be essential for productive viral replication. Yet, previous studies differed on the extent to which integrase mutant viruses expressed human immunodeficiency virus type 1 (HIV-1) genes from unintegrated DNA. Although one reason for this difference was that class II integrase mutations pleiotropically affected the viral life cycle, another reason apparently depended on the identity of the infected cell. Here, we analyzed integrase mutant viral infectivities in a variety of cell types. Single-round infectivity of class I integration-specific mutant HIV-1 ranged from culture infectious doses, however, was only 0.0006 to 0.005% of that of WT. Since the frequencies of mutant DNA recombination in these lines ranged from 0.023 to <0.093% of the WT, we conclude that productive replication in the absence of integrase function most likely required the illegitimate integration of HIV-1 into host chromosomes by cellular DNA recombination enzymes. PMID:11483739

  7. I-SceI-Mediated Double-Strand Break Does Not Increase the Frequency of Homologous Recombination at the Dct Locus in Mouse Embryonic Stem Cells

    OpenAIRE

    Fenina, Myriam; Simon-Chazottes, Dominique; Vandormael-Pournin, Sandrine; Soueid, Jihane; Langa, Francina; Cohen-Tannoudji, Michel; Bernard, Bruno A.; Panthier, Jean-Jacques

    2012-01-01

    Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embed...

  8. Oral Administration of Recombinant Adeno-associated Virus-mediated Bone Morphogenetic Protein-7 Suppresses CCl4-induced Hepatic Fibrosis in Mice

    OpenAIRE

    Hao, Zhi-Ming; Cai, Min; Lv, Yi-Fei; Huang, Yan-Hua; Li, Hong-Hong

    2012-01-01

    Fibrogenesis and hepatocyte degeneration are the main pathological processes in chronic liver diseases. Transforming growth factor-?1 (TGF-?1) is the key profibrotic cytokine in hepatic fibrosis. Bone morphogenetic protein-7 (BMP-7) is a potent antagonist of TGF-?1 and an antifibrotic factor. In this study, we generated a recombinant adeno-associated virus carrying BMP-7 (AAV–BMP-7) and tested its ability to suppress carbon tetrachloride (CCl4)-induced hepatic fibrosis when orally administere...

  9. Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery

    OpenAIRE

    Beggah Ahmed T; Pertin Marie; Towne Chris; Aebischer Patrick; Decosterd Isabelle

    2009-01-01

    Abstract Background Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the ...

  10. A Limited Number of Transducible Hepatocytes Restricts a Wide-Range Linear Vector Dose Response in Recombinant Adeno-Associated Virus-Mediated Liver Transduction

    OpenAIRE

    Nakai, Hiroyuki; Thomas, Clare E.; Storm, Theresa A.; Fuess, Sally; Powell, Sharon; Wright, J Fraser; KAY, MARK A.

    2002-01-01

    Recombinant adeno-associated virus (rAAV) vectors are promising vehicles for achieving stable liver transduction in vivo. However, the mechanisms of liver transduction are not fully understood, and furthermore, the relationships between rAAV dose and levels of transgene expression, total number of hepatocytes transduced, and proportion of integrated vector genomes have not been well established. To begin to elucidate the liver transduction dose response with rAAV vectors, we injected mice wit...

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

    OpenAIRE

    Zhang Deshui; Lee Hsin-Fang; Pettit Steven C; Zaro Jennica L; Huang Ning; Shen Wei-Chiang

    2012-01-01

    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 incr...

  12. The addition of recombinant vaccinia HER2/neu to oncolytic vaccinia-GMCSF given into the tumor microenvironment overcomes MDSC-mediated immune escape and systemic anergy.

    Science.gov (United States)

    de Vries, C R; Monken, C E; Lattime, E C

    2015-04-01

    Effective immunotherapeutic strategies require the ability to generate a systemic antigen-specific response capable of impacting both primary and metastatic disease. We have built on our oncolytic vaccinia a granulocyte-macrophage colony-stimulating factor (GM-CSF) strategy by adding recombinant tumor antigen to increase the response in the tumor microenvironment and systemically. In the present study, orthotopic growth of a syngeneic HER2/neu-overexpressing mammary carcinoma in FVB/N mice (NBT1) was associated with increased Gr1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs) both systemically and in the tumor microenvironment. This MDSC population had inhibitory effects on the HER2/neu-specific Th1 immune response. VVneu and VVGMCSF are recombinant oncolytic vaccinia viruses that encode HER2/neu and GM-CSF, respectively. Naive FVB mice vaccinated with combined VVneu and VVGMCSF given systemically developed systemic HER2/neu-specific immunity. NBT1-bearing mice became anergic to systemic immunization with combined VVneu and VVGMCSF. Intratumoral VVGMCSF failed to result in systemic antitumor immunity until combined with intratumoral VVneu. Infection/transfection of the tumor microenvironment with combined VVGMCSF and VVneu resulted in development of systemic tumor-specific immunity, reduction in splenic and tumor MDSC and therapeutic efficacy against tumors. These studies demonstrate the enhanced efficacy of oncolytic vaccinia virus recombinants encoding combined tumor antigen and GM-CSF in modulating the microenvironment of MDSC-rich tumors. PMID:25633483

  13. Recombinant AAV-mediated in vivo long-term expression and antitumour activity of an anti-ganglioside GM3(Neu5Gc) antibody.

    Science.gov (United States)

    Piperno, G M; López-Requena, A; Predonzani, A; Dorvignit, D; Labrada, M; Zentilin, L; Burrone, O R; Cesco-Gaspere, M

    2015-12-01

    The ganglioside GM3(Neu5Gc) has gained increasing attention as therapeutic target because of its selective expression in various human tumours, such as melanoma, breast and lung cancer. 14F7 is a mouse IgG1 with specific reactivity to GM3(Neu5Gc)-positive tumours. The therapeutic activity of 14F7 has also been demonstrated in vivo, through its repetitive passive administration in tumour-bearing animals. In this work we used an alternative strategy to deliver recombinant 14F7 in vivo and analysed the therapeutic efficacy of this approach. We engineered a recombinant adeno-associated vector to direct the expression of secretable recombinant 14F7 in BALB/c animals. A single administration of the rAAV induced efficient production and secretion of the antibody in the bloodstream, with an expression level reaching plateau at ?3 weeks after injection and persisting for almost a year. Strikingly, upon challenge with GM3(Neu5Gc)-positive X63-AG8.653 myeloma cells, tumour development was significantly delayed in animals treated with rAAV-14F7 with respect to animals treated with a control rAAV codifying for an irrelevant antibody. Finally, no significant differences in survival proportion were detected in animals injected with rAAV-14F7 or treated by standard administration of repetitive doses of purified monoclonal antibody 14F7. PMID:26181624

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria

    OpenAIRE

    Harding, CO; Gillingham, MB; Hamman, K; Clark, H; Goebel-Daghighi, E; Bird, A.; Koeberl, DD

    2006-01-01

    Novel recombinant adeno-associated virus vectors pseudo-typed with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have produced a novel liver-specific rAAV2/8 vector expressing the mouse phenylalanine hydroxylase (Pah) cDNA and have administered this vector to hyperphenylalaninemic PAH-deficient Pahenu2 mice, a model of human phenylketonuria (PKU). Our hypothesis was that this vector would produce sufficient hepatocyte t...

  16. Self-Consistent Quasi-Particle RPA for the Description of Superfluid Fermi Systems

    CERN Document Server

    Rabhi, A; Chanfray, G; Schuck, P

    2002-01-01

    Self-Consistent Quasi-Particle RPA (SCQRPA) is for the first time applied to a more level pairing case. Various filling situations and values for the coupling constant are considered. Very encouraging results in comparison with the exact solution of the model are obtained. 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.

  17. Vibronic contributions to the n ? ? ? absorption band of cyclopentanone. An ab initio SCF-RPA calculation

    Science.gov (United States)

    Flament, J. P.; Gervais, H. P.

    The SCF-RPA scheme is applied to the n ? ? ? absorption band of cyclopentanone in its ( C2) ground-state nuclear configuration. Using the Cederbaum-Domcke algorithm, the effects of the ( ?3) C=0 stretching, ( ?18) ring puckering and ( ?25) C=0 out-of-plane vibrations are investigated. The calculated spectrum shows both allowed and vibronic components, in agreement with the experimental observations of Howard-Lock and King.

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

    International Nuclear Information System (INIS)

    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)

  19. The correlation energy functional within the GW-RPA approximation: exact forms, approximate forms and challenges

    OpenAIRE

    Ismail-Beigi, Sohrab

    2010-01-01

    In principle, the Luttinger-Ward Green's function formalism allows one to compute simultaneously the total energy and the quasiparticle band structure of a many-body electronic system from first principles. We present approximate and exact expressions for the correlation energy within the GW-RPA approximation that are more amenable to computation and allow for developing efficient approximations to the self-energy operator and correlation energy. The exact form is a sum over...

  20. Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells

    OpenAIRE

    Truong, Lan N.; Li, Yongjiang; Shi, Linda Z; Hwang, Patty Yi-Hwa; He, Jing; WANG, HAILONG; Razavian, Niema; Berns, Michael W.; WU, XIAOHUA

    2013-01-01

    Microhomology-mediated end joining (MMEJ) is a major pathway for Ku-independent alternative nonhomologous end joining, which contributes to chromosomal translocations and telomere fusions, but the underlying mechanism of MMEJ in mammalian cells is not well understood. In this study, we demonstrated that, distinct from Ku-dependent classical nonhomologous end joining, MMEJ—even with very limited end resection—requires cyclin-dependent kinase activities and increases significantly when cells en...

  1. Manipulating Mitotic Recombination in the Zebrafish Embryo Through RecQ Helicases

    OpenAIRE

    Xie, Jing; Bessling, Seneca L.; Cooper, Timothy K.; Harry C Dietz; McCallion, Andrew S; Fisher, Shannon

    2007-01-01

    RecQ DNA helicases resolve Rad-51-mediated recombination and suppress aberrant homologous recombination. RecQ gene loss is associated with cancer susceptibility and increased mitotic recombination. We have developed an in vivo assay based on a zebrafish pigment mutant for suppression of RecQ activity, and demonstrate that zebrafish RecQ genes have conserved function in suppressing mitotic recombination.

  2. Cell-Mediated and Humoral Immune Responses after Immunization of Calves with a Recombinant Multiantigenic Mycobacterium avium subsp. paratuberculosis Subunit Vaccine at Different Ages

    DEFF Research Database (Denmark)

    Thakur, Aneesh; Aagaard, Claus

    2013-01-01

    Neonates and juvenile ruminants are very susceptible to paratuberculosis infection. This is likely due to a high degree of exposure from their dams and an immature immune system. To test the influence of age on vaccine-induced responses, a cocktail of recombinant Mycobacterium avium subsp. paratuberculosis proteins (MAP0217, MAP1508, MAP3701c, MAP3783, and MAP1609c/Ag85B) was formulated in a cationic liposome adjuvant (CAF01) and used to vaccinate animals of different ages. Male jersey calves were divided into three groups that were vaccinated at 2, 8, or 16 weeks of age and boosted twice at weeks 4 and 12 relative to the first vaccination. Vaccine-induced immune responses, the gamma interferon (IFN-?) cytokine secretion and antibody responses, were followed for 20 weeks. In general, the specific responses were significantly elevated in all three vaccination groups after the first booster vaccination with no or only a minor effect from the second booster. However, significant differences were observed in the immunogenicity levels of the different proteins, and it appears that the older age group produced a more consistent IFN-? response. In contrast, the humoral immune response is seemingly independent of vaccination age as we found no difference in the IgG1 responses when we compared the three vaccination groups. Combined, our results suggest that an appropriate age of vaccination should be considered in vaccination protocols and that there is a possible interference of vaccine-induced immune responses with weaning (week 8).

  3. Anti-prostate cancer effects of CTL cell induction in vitro by recombinant adenovirus mediated PSMA/4-1BBL dendritic cells: an immunotherapy study.

    Science.gov (United States)

    Sui, C-G; Wu, D; Meng, F-D; Yang, M-H; Jiang, Y-H

    2015-01-01

    This study aimed to examine anti-prostate cancer immune response induced by dendritic cells (DCs) transduced with PSMA/4-1BBL recombinant adenoviruses in vitro. Ad-PSMA, Ad-4-1BBL, and Ad-GFP were transfected into DCs derived from peripheral blood of healthy volunteers. Ad-PSMA/4-1BBL-DC, Ad-PSMA-DC, Ad-4-1BBL-DC, Ad-GFP-DC, and normal-DC, PSMA and 4-1BBL protein levels in DCs were detected by western blot. IL-12, IFN-? and IL-10 were measured by ELISA. Mixed lymphocyte reaction and the cytotoxicity of each group targeted to LNCap, Du145, and 22RV prostate cancer cells were determined by CCK-8 assay. PSMA and 4-1BBL protein could express on DC successfully, the IL-12 supernatant content (134.29 ± 2.22 pg) was higher than others (P PSMA/4-1BBL-DC-CTL group showed the highest content of IFN-? (1176.10 ± 14.37pg/5 x 10(6) cells), but the lowest IL-10 content (75.14 ± 2.01 pg/5 x 10(6) cells) (P PSMA/4- 1BBL not only showed high secretion of IL-12, but also induced CTLs to stimulate and enhance the killing effect of PSMA specific effector cells to PSMA positively expressing prostate cancer cells. Furthermore, the DCs infected with two kinds of tumor-associated antigens would induce more effective tumor-specific CTL induction. PMID:26125931

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. Superallowed Fermi transitions in RPA with a relativistic point-coupling energy functional

    CERN Document Server

    Li, Z X; Chen, H; 10.1007/s11433-011-4320-2

    2011-01-01

    The self-consistent random phase approximation (RPA) approach with the residual interaction derived from a relativistic point-coupling energy functional is applied to evaluate the isospin symmetry-breaking corrections {\\delta}c for the 0+\\to0+ superallowed Fermi transitions. With these {\\delta}c values, together with the available experimental ft values and the improved radiative corrections, the unitarity of the Cabibbo-Kobayashi-Maskawa (CKM) matrix is examined. Even with the consideration of uncertainty, the sum of squared top-row elements has been shown to deviate from the unitarity condition by 0.1% for all the employed relativistic energy functionals.

  6. Gimeracil, an inhibitor of dihydropyrimidine dehydrogenase, inhibits the early step in homologous recombination

    International Nuclear Information System (INIS)

    Gimeracil (5-chloro-2, 4-dihydroxypyridine) is an inhibitor of dihydropyrimidine dehydrogenase (DPYD), which degrades pyrimidine including 5-fluorouracil in the blood. Gimeracil was originally added to an oral fluoropyrimidine derivative S-1 to yield prolonged 5-fluorouracil concentrations in serum and tumor tissues. We have already reported that gimeracil had radiosensitizing effects by partially inhibiting homologous recombination (HR) in the repair of DNA double strand breaks. We investigated the mechanisms of gimeracil radiosensitization. Comet assay and radiation-induced focus formation of various kinds of proteins involved in HR was carried out. Small interfering RNA (siRNA) for DPYD were transfected to HeLa cells to investigate the target protein for radiosensitization with gimeracil. SCneo assay was carried out to examine whether DPYD depletion by siRNA inhibited HR repair of DNA double strand breaks. Tail moments in neutral comet assay increased in gimeracil-treated cells. Gimeracil restrained the formation of foci of Rad51 and replication protein A (RPA), whereas it increased the number of foci of Nbs1, Mre11, Rad50, and FancD2. When HeLa cells were transfected with the DPYD siRNA before irradiation, the cells became more radiosensitive. The degree of radiosensitization by transfection of DPYD siRNA was similar to that of gimeracil. Gimeracil did not sensitize DPYD-depleted cells. Depletion of DPYD by siRNA significantly reduced the frequency of neopositive clones in SCneo assay. Gimeracil partially inhibits the early step in HR. It was found that DPYD is the target protein for radiosensitization by gimeracil. The inhibitors of DPYD, such as gimeracil, could enhance the efficacy of radiotherapy through partial suppression of HR-mediated DNA repair. (author)

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

    International Nuclear Information System (INIS)

    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).

  8. Recombinant Technology and Probiotics

    Directory of Open Access Journals (Sweden)

    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.

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

    OpenAIRE

    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...

  10. Recombinant Technology and Probiotics

    OpenAIRE

    Icy D’Silva

    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...

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

    International Nuclear Information System (INIS)

    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 (?+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.)

  12. Generation of Modified Pestiviruses by Targeted Recombination

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Friis, Martin Barfred

    Infectious cDNA clones are a prerequisite for directed genetic manipulation of pestivirus RNA genomes. We have developed a novel strategy to facilitate manipulation and rescue of modified pestiviruses from infectious cDNA clones based on bacterial artificial chromosomes (BACs). The strategy 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 hence is not limited to the use of internal restriction sites. Rescue of modified pestiviruses can be obtained by electroporation of cell cultures with full-length RNA transcripts in vitro transcribed from the recombined BAC clones. We have used this approach to generate a series of new pestivirus BACs modified within different genomic regions and infectious pestiviruses have been rescued from several of these new constructs,demonstrating that recombination-mediated mutagenesis of pestivirus BACs provides a useful tool for expediting the construction of recombinant pestiviruses.

  13. Effect of isospin mixing on superallowed Fermi beta decay in self-consistent relativistic RPA approaches

    CERN Document Server

    Liang, Haozhao; Meng, Jie

    2009-01-01

    Self-consistent Random Phase Approximation (RPA) approaches in the relativistic framework are applied to calculate the isospin symmetry-breaking corrections $\\delta_c$ for the $0^+\\to0^+$ superallowed transitions. It is found that the corrections $\\delta_c$ are sensitive to the proper treatments of the Coulomb mean field, but not so much to specific effective interactions. With these corrections $\\delta_c$, the nucleus-independent $\\mathcal{F}t$ values are obtained in combination the experimental $ft$ values in the most recent survey and the improved radiative corrections. It is found that the constancy of the $\\mathcal{F}t$ values is satisfied for all effective interactions employed. Furthermore, the element $V_{ud}$ and unitarity of the Cabibbo-Kobayashi-Maskawa matrix are discussed.

  14. Nuclear vorticity in isoscalar E1 modes: Skyrme-RPA analysis

    CERN Document Server

    Reinhard, P -G; Repko, A; Kvasil, J

    2013-01-01

    Two basic concepts of nuclear vorticity, hydrodynamical (HD) and Rawenthall-Wambach (RW), are critically inspected. As a test case, we consider the interplay of irrotational and vortical motion in isoscalar electric dipole E1(T=0) modes in $^{208}$Pb, namely the toroidal and compression modes. The modes are described in a self-consistent random-phase-approximation (RPA) with the Skyrme force SLy6. They are examined in terms of strength functions, transition densities, current fields, and formfactors. It is shown that the RW conception (suggesting the upper component of the nuclear current as the vorticity indicator) is not robust. The HD vorticity is not easily applicable either because the definition of a velocity field is too involved in nuclear systems. Instead, the vorticity is better characterized by the toroidal strength which closely corresponds to HD treatment and is approximately decoupled from the continuity equation.

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

    KAUST Repository

    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.

  16. PRODUCTION OF RECOMBINANT PROTEINS IN INSECT CELLS

    Directory of Open Access Journals (Sweden)

    Christian Kollewe

    2013-01-01

    Full Text Available Among the wide range of methods and expression hosts available for the heterologous production of recombinant proteins, insect cells are ideal for the production of complex proteins requiring extensive post-translational modification. This review article provides an overview of the available insect-cell expression systems and their properties, focusing on the widely-used Baculovirus Expression Vector System (BEVS. We discuss the different strategies used to generate recombinant baculovirus vectors and show how advanced techniques for virus titer determination can accelerate the production of recombinant proteins. The stable transfection of insect cells is an alternative to BEVS which has recently been augmented with recombinase-mediated cassette exchange for site-specific gene integration. We consider the advantages and limitations of these techniques for the production of recombinant proteins in insect cells and compare them to other expression platforms.

  17. Single-stranded heteroduplex intermediates in ? Red homologous recombination

    Directory of Open Access Journals (Sweden)

    Zhang Youming

    2010-07-01

    Full Text Available Abstract Background The Red proteins of lambda phage mediate probably the simplest and most efficient homologous recombination reactions yet described. However the mechanism of dsDNA recombination remains undefined. Results Here we show that the Red proteins can act via full length single stranded intermediates to establish single stranded heteroduplexes at the replication fork. We created asymmetrically digestible dsDNA substrates by exploiting the fact that Red? exonuclease activity requires a 5' phosphorylated end, or is blocked by phosphothioates. Using these substrates, we found that the most efficient configuration for dsDNA recombination occurred when the strand that can prime Okazaki-like synthesis contained both homology regions on the same ssDNA molecule. Furthermore, we show that Red recombination requires replication of the target molecule. Conclusions Hence we propose a new model for dsDNA recombination, termed 'beta' recombination, based on the formation of ssDNA heteroduplexes at the replication fork. Implications of the model were tested using (i an in situ assay for recombination, which showed that recombination generated mixed wild type and recombinant colonies; and (ii the predicted asymmetries of the homology arms, which showed that recombination is more sensitive to non-homologies attached to 5' than 3' ends. Whereas beta recombination can generate deletions in target BACs of at least 50 kb at about the same efficiency as small deletions, the converse event of insertion is very sensitive to increasing size. Insertions up to 3 kb are most efficiently achieved using beta recombination, however at greater sizes, an alternative Red-mediated mechanism(s appears to be equally efficient. These findings define a new intermediate in homologous recombination, which also has practical implications for recombineering with the Red proteins.

  18. Recombinant DNA in Medicine

    OpenAIRE

    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...

  19. Improving baculovirus recombination

    OpenAIRE

    Zhao, Yuguang; Chapman, David A.G.; Jones, Ian M

    2003-01-01

    Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins. The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments. Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination. Viral DNA prepared from the knockout fails to initiate an infecti...

  20. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    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…

  1. Recombination in RNA.

    Science.gov (United States)

    King, A M; McCahon, D; Slade, W R; Newman, J W

    1982-07-01

    The aphthovirus genome consists of a single molecule of single-stranded RNA that encodes all the virus-induced proteins. We isolated recombinant aphthoviruses from cells simultaneously infected with temperature-sensitive mutants of two different subtype strains. Analysis of the proteins induced by 16 independently generated recombinants revealed two types of protein pattern, which were consistent with single genetic crossovers on the 5' side and 3' side, respectively, of the central P34-coding region. Recombinants invariably inherited all four coat proteins from the same parent, and novel recombinant proteins were not observed. RNAase T1 fingerprints of virus RNA, prepared from representatives of each recombinant type, confirmed the approximate crossover sites that had been deduced from the inheritance of proteins. These fingerprints provide molecular evidence of recombination at the level of RNA and demonstrate the potential of RNA recombination for producing genetic diversity among picornaviruses. PMID:6295637

  2. Range-Separated Approach to the RPA Correlation Applied to the van der Waals Bond and to Diffusion of Defects

    Science.gov (United States)

    Bruneval, Fabien

    2012-06-01

    The random-phase approximation (RPA) is a promising approximation to the exchange-correlation energy of density functional theory, since it contains the van der Waals (vdW) interaction and yields a potential with the correct band gap. However, its calculation is computationally very demanding. We apply a range-separation concept to RPA and demonstrate how it drastically speeds up the calculations without loss of accuracy. The scheme is then successfully applied to a layered system subjected to weak vdW attraction and is used to address the controversy of the self-diffusion in silicon. We calculate the formation and migration energies of self-interstitials and vacancies taking into account atomic relaxations. The obtained activation energies deviate significantly from the earlier calculations and challenge some of the experimental interpretations: the diffusion of vacancies and interstitials has almost the same activation energy.

  3. Range-separated approach to the RPA correlation applied to van der Waals bond and to diffusion of defects

    Science.gov (United States)

    Bruneval, Fabien

    2013-03-01

    The Random Phase Approximation (RPA) is a promising approximation to the exchange-correlation energy of Density Functional Theory (DFT), since it contains the van der Waals (vdW) interaction and yields a potential with the correct band gap. However, its calculation is computationally very demanding. We apply a range separation concept to RPA and demonstrate how it drastically speeds up the calculations without loss of accuracy. The scheme is succesfully applied to a layered system subjected to weak vdW attraction and to address the controversy of the self-diffusion in silicon. We calculate the formation and migration energies of self-interstitials and vacancies taking into account atomic relaxations. The obtained activation energies deviate significantly from the earlier calculations that were affected by the band gap problem and challenge some of the experimental interpretations: the diffusion of vacancies and interstitials have almost the same activation energy.

  4. Detection of a Rickettsia Closely Related to Rickettsia aeschlimannii, “Rickettsia heilongjiangensis,” Rickettsia sp. Strain RpA4, and Ehrlichia muris in Ticks Collected in Russia and Kazakhstan

    OpenAIRE

    Shpynov, Stanislav; Fournier, Pierre-Edouard; Rudakov, Nikolay; Tankibaev, Marat; Tarasevich, Irina; Raoult, Didier

    2004-01-01

    Using PCR, we screened 411 ticks from four genera collected in Russia and Kazakhstan for the presence of rickettsiae and ehrlichiae. In Russia, we detected “Rickettsia heilongjiangensis,” Rickettsia sp. strain RpA4, and Ehrlichia muris. In Kazakhstan, we detected Rickettsia sp. strain RpA4 and a rickettsia closely related to Rickettsia aeschlimannii. These agents should be considered in a differential diagnosis of tick-borne infections in these areas.

  5. Static correlation and electron localization in molecular dimers from the self-consistent RPA and G W approximation

    Science.gov (United States)

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

    2015-04-01

    We investigate static correlation and delocalization errors in the self-consistent G W and random-phase approximation (RPA) by studying molecular dissociation of the H2 and LiH molecules. Although both approximations contain topologically identical diagrams, the nonlocality and frequency dependence of the G W 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 allow for a better description of static correlation at intermediate bond distances. The substantial error found in G W 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 G W making the fractional charge error comparable to the RPA. The analyses are supplemented by explicit formulas for the G W Green's function and total energy of a simplified two-level model providing additional insights into the dissociation limit.

  6. Recombination and Genetic Diversity

    Scientific Electronic Library Online (English)

    T. C., Coutinho; T.T.da, Silva; G.L., Toledo.

    2012-12-01

    Full Text Available In this paper we present a spatial stochastic model for genetic recombination, that answers if diversity is preserved in an infinite population of recombinat-ing individuals distributed spatially. We show that, for finite times, recombination may maintain all the various potential different types, b [...] ut when time grows infinitely, the diversity of individuals extinguishes off. So under the model premisses, recombination and spatial localization alone are not enough to explain diversity in a population. Further we discuss an application of the model to a controversy regarding the diversity of "Major Histocompatibility Complex" (MHC).

  7. DNA Detection Using Recombination Proteins

    OpenAIRE

    Piepenburg, Olaf; Williams, Colin H; Stemple, Derek L; Armes, Niall A

    2006-01-01

    DNA amplification is essential to most nucleic acid testing strategies, but established techniques require sophisticated equipment or complex experimental procedures, and their uptake outside specialised laboratories has been limited. Our novel approach, recombinase polymerase amplification (RPA), couples isothermal recombinase-driven primer targeting of template material with strand-displacement DNA synthesis. It achieves exponential amplification with no need for pretreatment of sample DNA....

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

    Science.gov (United States)

    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.

  9. Phenylbutyrate inhibits homologous recombination induced by camptothecin and methyl methanesulfonate

    DEFF Research Database (Denmark)

    Kaiser, Gitte Schalck; Germann, Susanne Manuela; Westergaard, Tine; Lisby, Michael

    2011-01-01

    Homologous recombination is accompanied by extensive changes to chromatin organization at the site of DNA damage. Some of these changes are mediated through acetylation/deacetylation of histones. Here, we show that recombinational repair of DNA damage induced by the anti-cancer drug camptothecin (CPT) and the alkylating agent methyl methanesulfonate (MMS) is blocked by sodium phenylbutyrate (PBA) in the budding yeast Saccharomyces cerevisiae. In particular, PBA suppresses CPT- and MMS-induced ge...

  10. The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart

    OpenAIRE

    Ciccia, Alberto; Bredemeyer, Andrea L; Sowa, Mathew E.; Terret, Marie-Emilie; Jallepalli, Prasad V.; Harper, J.Wade; Elledge, Stephen J

    2009-01-01

    The integrity of genomic DNA is continuously challenged by the presence of DNA base lesions or DNA strand breaks. Here we report the identification of a new DNA damage response protein, SMARCAL1 (SWI/SNF-related, matrix associated, actin-dependent regulator of chromatin, subfamily a-like 1), which is a member of the SNF2 family and is mutated in Schimke immunoosseous dysplasia (SIOD). We demonstrate that SMARCAL1 directly interacts with Replication protein A (RPA) and is recruited to sites of...

  11. The use of recombinant DNA plasmids for the determination of DNA-repair and recombination in cultured mammalian cells.

    OpenAIRE

    Cox, R; Masson, W. K.; Debenham, P G; Webb, M. B.

    1984-01-01

    Using the recombinant plasmid pSV2gpt and DNA transfer techniques, cell mediated DNA ligation and recombination of plasmid DNA have been demonstrated in four human cell lines. Data suggesting the involvement of a possible defect in the cellular equilibrium between ligation and exonuclease digestion of double strand DNA scissions in an ataxia-telangiectasia (A-T) cell line is discussed. The same A-T line was grossly proficient in DNA recombination but it will be necessary to distinguish betwee...

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

    Directory of Open Access Journals (Sweden)

    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.

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

    International Nuclear Information System (INIS)

    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

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

    CERN Document Server

    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...

  15. Multiphoton Assisted Recombination

    International Nuclear Information System (INIS)

    We have observed multiphoton assisted recombination in the presence of a 38.8 GHz microwave field. Stimulated emission of up to ten microwave photons results in energy transfer from continuum electrons, enabling recombination. The maximum electron energy loss is far greater than the 2Up predicted by the standard 'simpleman's' model. The data are well reproduced by both an approximate analytic expression and numerical simulations in which the combined Coulomb and radiation fields are taken into account

  16. Activated recombinant adenovirus proteinases

    Science.gov (United States)

    Anderson, Carl W. (Stony Brook, NY); Mangel, Walter F. (Shoreham, NY)

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  17. Cytotoxic T cells specific for a single peptide on the M2 protein of respiratory syncytial virus are the sole mediators of resistance induced by immunization with M2 encoded by a recombinant vaccinia virus.

    OpenAIRE

    Kulkarni, A. B.; Collins, P L; Bacik, I; Yewdell, J. W.; Bennink, J R; Crowe, J.E.; Murphy, B R

    1995-01-01

    We have studied the immunobiology of respiratory syncytial virus (RSV), a major cause of respiratory tract morbidity in children. As part of these studies, it was previously found that immunization of BALB/c (H-2d) mice with a recombinant vaccinia virus (rVV) which encoded the M2 protein of RSV provided complete protection against infection with RSV. This protection was transient and associated with M2-specific CD8+ T-cell (TCD8+) responses. In this study, we used two approaches to demonstrat...

  18. Recombination Phenotypes of Escherichia coli greA Mutants

    Directory of Open Access Journals (Sweden)

    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.

  19. Regulation of Meiotic Recombination

    Energy Technology Data Exchange (ETDEWEB)

    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

  20. Phenylbutyrate inhibits homologous recombination induced by camptothecin and methyl methanesulfonate

    DEFF Research Database (Denmark)

    Kaiser, Gitte Schalck; Germann, Susanne Manuela

    2011-01-01

    Homologous recombination is accompanied by extensive changes to chromatin organization at the site of DNA damage. Some of these changes are mediated through acetylation/deacetylation of histones. Here, we show that recombinational repair of DNA damage induced by the anti-cancer drug camptothecin (CPT) and the alkylating agent methyl methanesulfonate (MMS) is blocked by sodium phenylbutyrate (PBA) in the budding yeast Saccharomyces cerevisiae. In particular, PBA suppresses CPT- and MMS-induced genetic recombination as well as DNA double-strand break repair during mating-type interconversion. Treatment with PBA is accompanied by a dramatic reduction in histone H4 lysine 8 acetylation. Live cell imaging of homologous recombination proteins indicates that repair of CPT-induced DNA damage is redirected to a non-recombinogenic pathway in the presence of PBA without loss in cell viability. In contrast, the suppression of MMS-induced recombination by PBA is accompanied by a dramatic loss in cell viability. Taken together, our results demonstrate that PBA inhibits DNA damage-induced homologous recombination likely by mediating changes in chromatin acetylation. Moreover, the combination of PBA with genotoxic agents can lead to different cell fates depending on the type of DNA damage inflicted.

  1. 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

    Energy Technology Data Exchange (ETDEWEB)

    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 TCR? 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 (TCR?) 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 TCR? 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.

  2. 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

    International Nuclear Information System (INIS)

    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 TCR? 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 (TCR?) 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 TCR? 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

  3. 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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    McAllister, Andrew; Åberg, Daniel; Schleife, André; Kioupakis, Emmanouil

    2015-04-01

    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.

  5. Recombinational DNA repair and human disease

    International Nuclear Information System (INIS)

    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

  6. Recombineering: A Homologous Recombination-Based Method of Genetic Engineering

    OpenAIRE

    Sharan, Shyam K.; Thomason, Lynn C.; Kuznetsov, Sergey G; Court, Donald L.

    2009-01-01

    Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in E. coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these recombineering reactions using double- and single-strand linear DNA sub...

  7. Recombineering linear BACs.

    Science.gov (United States)

    Chen, Qingwen; Narayanan, Kumaran

    2015-01-01

    Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells. PMID:25239740

  8. Recombineering Pseudomonas syringae

    Science.gov (United States)

    Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

  9. Recombinant hormones in osteoporosis

    DEFF Research Database (Denmark)

    Rejnmark, Lars

    2013-01-01

    For the last 10 years, bone anabolic therapy with the recombinant human parathyroid hormone (rhPTH) analogue, teriparatide (rhPTH[1 - 34]), or full-length rhPTH(1 - 84) has been an option in the treatment of osteoporosis. Both drugs are given as a daily subcutaneous injection. In the USA, only teriparatide is marketed.

  10. Cell biology of mitotic recombination

    DEFF Research Database (Denmark)

    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 ...

  11. Degradation of RPA 202248 [U-14C-phenyl]alpha(-(cyclopropylcarbonyl)-2-(methylsulfonyl)-beta-oxo-4-(trifluromethyl)benzenepropanenitrile), the primary degradation product of isoxaflutole, in an outdoor aquatic microcosm system.

    Science.gov (United States)

    Rupprecht, J Kent; Liu, Anne; Kelly, Iain; Allen, Richard

    2004-01-01

    Isoxaflutole, the active ingredient in BALANCE WDG and BALANCE PRO corn herbicides and a co-formulant with the herbicide flufenacet in the product EPIC, is readily degraded in soil and water to RPA 202248 alpha(-(cyclopropylcarbonyl)-2-(methyvlsulfonyl)-beta-oxo-4-(trifluromethyl)benzenepropanenitrile). Because RPA 202248 is responsible at the molecular level for isoxaflutole's herbicidal activity it is important to understand the environmental behavior of the degradation product. Laboratory studies suggest that RPA 202248 is stable to hydrolysis and photolysis in aqueous systems and hence poses a possible environmental concern. As part of a program of work towards understanding the actual field situation, an outdoor microcosm study was carried out. Over the course of the 29-day study, residues remained predominantly in the aqueous phase. A slow but steady degradation of RPA 202248 was observed leading to the formation of RPA 203328 (2-methylsulfonyl-4-trifluoromethylbenzoic acid), which has no herbicidal activity. The half-life of RPA 202248 was calculated to be 103 days. These findings indicate that aqueous degradation should be considered as a potential route of dissipation when assessing the fate of RPA 202248 in large scale impounded water bodies, such as ponds, lakes, or reservoirs in the Mid-West Corn Belt. PMID:15620081

  12. Radiative recombination rate coefficients

    International Nuclear Information System (INIS)

    Radiative recombination rate coefficients obtained using the nonrelativistic dipole result of Stobbe for the RR cross section are compared to other nonrelativistic approaches. A good agreement is found with widely used formulas. The RR rate coefficients can be calculated for different beam temperatures and for arbitrary nl sublevels up to high Rydberg states. The computation technique used allows us a fast evaluation of the dipole matrix elements without any additional approximation. (orig.)

  13. Radiative Recombination Rate Coefficients

    Science.gov (United States)

    Brinzanescu, O.; Brinzanescu, O.; Stöhlker, Th.; Stöhlker, Th.

    Radiative recombination rate coefficients obtained using the nonrelativistic dipole result of Stobbe for the RR cross section are compared to other nonrelativistic approaches. A good agreement is found with widely used formulas. The RR rate coefficients can be calculated for different beam temperatures and for arbitrary nl sublevels up to high Rydberg states. The computation technique used allows us a fast evaluation of the dipole matrix elements without any additional approximation.

  14. Radiative recombination rate coefficients

    Energy Technology Data Exchange (ETDEWEB)

    Brinzanescu, O. [Gesellschaft fuer Schwerionenforschung mbH, Darmstadt (Germany); National Inst. for Laser, Plasma and Radiation Physics, Bucharest Magurele (Romania); Stoehlker, T. [Gesellschaft fuer Schwerionenforschung mbH, Darmstadt (Germany); Frankfurt Univ. (Germany). Inst. fuer Kernphysik

    2001-07-01

    Radiative recombination rate coefficients obtained using the nonrelativistic dipole result of Stobbe for the RR cross section are compared to other nonrelativistic approaches. A good agreement is found with widely used formulas. The RR rate coefficients can be calculated for different beam temperatures and for arbitrary nl sublevels up to high Rydberg states. The computation technique used allows us a fast evaluation of the dipole matrix elements without any additional approximation. (orig.)

  15. Radiative recombination rate coefficients

    Energy Technology Data Exchange (ETDEWEB)

    Brinzanescu, O. [Gesellschaft fuer Schwerionenforschung mbH, Darmstadt (Germany); National Inst. for Laser, Plasma and Radiation Physics, Bucharest (Romania); Stoehlker, T. [Gesellschaft fuer Schwerionenforschung mbH, Darmstadt (Germany); Frankfurt Univ. (Germany). Inst. fuer Kernphysik

    2000-10-01

    Radiative recombination rate coefficients obtained using the nonrelativistic dipole result of Stobbe for the RR cross section are compared to other nonrelativistic approaches. A good agreement is found with widely used formulas. The RR rate coefficients can be calculated for different beam temperatures and for arbitrary nl sublevels up to high Rydberg states. The computation technique used allows us a fast evaluation of the dipole matrix elements without any additional approximation. (orig.)

  16. Intrachromosomal recombination in plants.

    OpenAIRE

    Peterhans, A; Schlüpmann, 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...

  17. Relativistic dielectronic recombination theory

    International Nuclear Information System (INIS)

    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

  18. Nonradiative recombination in semiconductors

    CERN Document Server

    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

  19. A Simplified Recombinant PSO

    OpenAIRE

    Bratton, Daniel; Blackwell, Tim M.

    2008-01-01

    Simplified forms of the particle swarm algorithm are very beneficial in contributing to understanding how a particle swarm optimization (PSO) swarm functions. One of these forms, PSO with discrete recombination, is extended and analyzed, demonstrating not just improvements in performance relative to a standard PSO algorithm, but also significantly different behavior, namely, a reduction in bursting patterns due to the removal of stochastic components from the update equations.

  20. Recombinant human milk proteins.

    Science.gov (United States)

    Lönnerdal, Bo

    2006-01-01

    Human milk provides proteins that benefit newborn infants. They not only provide amino acids, but also facilitate the absorption of nutrients, stimulate growth and development of the intestine, modulate immune function, and aid in the digestion of other nutrients. Breastfed infants have a lower prevalence of infections than formula-fed infants. Since many women in industrialized countries choose not to breastfeed, and an increasing proportion of women in developing countries are advised not to breastfeed because of the risk of HIV transmission, incorporation of recombinant human milk proteins into infant foods is likely to be beneficial. We are expressing human milk proteins known to have anti-infective activity in rice. Since rice is a normal constituent of the diet of infants and children, limited purification of the proteins is required. Lactoferrin has antimicrobial and iron-binding activities. Lysozyme is an enzyme that is bactericidal and also acts synergistically with lactoferrin. These recombinant proteins have biological activities identical to their native counterparts. They are equally resistant to heat processing, which is necessary for food applications, and to acid and proteolytic enzymes which are needed to maintain their biological activity in the gastrointestinal tract of infants. These recombinant human milk proteins may be incorporated into infant formulas, baby foods and complementary foods, and used with the goal to reduce infectious diseases. PMID:16902336

  1. Improved generation of recombinant baculovirus genomes in Escherichia coli

    OpenAIRE

    Airenne, Kari J.; Peltomaa, Erik; Hytönen, Vesa P; Laitinen, Olli H; Ylä-Herttuala, Seppo

    2003-01-01

    An improved method for the generation of recombinant baculoviruses by Tn7-mediated transposition is described. The method is based on the modified donor vector (pBVboost) and an improved selection scheme of the baculovirus bacmids in Escherichia coli with a mutated SacB gene. Recombinant bacmids can be generated at a frequency of ?107/µg of donor vector with a negligible background. This easy-to-use and efficient pBVboost system provides the basis for a high-throughput generation of recombina...

  2. Schizosaccharomyces pombe Mms1 channels repair of perturbed replication into Rhp51 independent homologous recombination

    DEFF Research Database (Denmark)

    Vejrup-Hansen, Rasmus; Mizuno, Ken'Ichi

    2011-01-01

    In both Schizosaccharomyces pombe and Saccharomyces cerevisiae, Mms22 and Mms1 form a complex with important functions in the response to DNA damage, loss of which leads to perturbations during replication. Furthermore, in S. cerevisiae, Mms1 has been suggested to function in concert with a Cullin-like protein, Rtt101/Cul8, a potential paralog of Cullin 4. We performed epistasis analysis between ¿mms1 and mutants of pathways with known functions in genome integrity, and measured the recruitment of homologous recombination proteins to blocked replication forks and recombination frequencies. We show that, in S. pombe, the functions of Mms1 and the conserved components of the Cullin 4 ubiquitin ligase, Pcu4 and Ddb1, do not significantly overlap. Furthermore, unlike in S. cerevisiae, the function of the H3K56 acetylase Rtt109 is not essential for Mms1 function. We provide evidence that Mms1 function is particularly important when a single strand break is converted into a double strand break during replication. Genetic data connect Mms1 to a Mus81 and Rad22(Rad52) dependent, but Rhp51 independent, branch of homologous recombination. This is supported by results demonstrating that Mms1 is recruited to a site-specific replication fork barrier and that, in a ¿mms1 strain, Rad22(Rad52) and RPA recruitment to blocked forks are reduced, whereas Rhp51 recruitment is unaffected. In addition, Mms1 appears to specifically promote chromosomal rearrangements in a recombination assay. These observations suggest that Mms1 acts to channel repair of perturbed replication into a particular sub-pathway of homologous recombination.

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

    Science.gov (United States)

    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

  4. Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination.

    Science.gov (United States)

    Pines, Gur; Freed, Emily F; Winkler, James D; Gill, Ryan T

    2015-11-20

    The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing. PMID:25856528

  5. Calculation of the RPA response function of nuclei to quasi-elastic electron scattering with a density-dependent NN interaction

    International Nuclear Information System (INIS)

    So far, the non-relativistic longitudinal and transverse functions in electron quasi-elastic scattering on the nuclei failed in reproducing satisfactorily the existent experimental data. The calculations including relativistic RPA correlations utilize until now the relativistic Hartree approximation to describe the nuclear matter. But, this provides an incompressibility module two times higher than its experimental value what is an important drawback for the calculation of realistic relativistic RPA correlations. Hence, we have determined the RPA response functions of nuclei by utilising a description of the relativistic nuclear matter leading to an incompressibility module in agreement with the empirical value. To do that we have utilized an interaction in the relativistic Hartree approximation in which we have determined the coupling constants ?-N and ?-N as a function of the density in order to reproduce the saturation curve obtained by a Dirac-Brueckner calculation. The results which we have obtained show that the longitudinal response function and the Coulomb sum generally overestimated when one utilizes the pure relativistic Hartree approximation, are here in good agreement with the experimental data for several nuclei

  6. Monitoring and Evaluation of Smolt Migration in the Columbia Basin Volume VIII : Comparison of the RPA Testing Rules, Technical Report 2002.

    Energy Technology Data Exchange (ETDEWEB)

    Skalski, John; Ngouenet, Roger

    2002-08-01

    The 2000 FCRPS Biological Opinion (BO) suggested two statistical hypothesis tests to assess the RPA compliance by the years 2005 and 2008. With the decision rules proposed in the BO, Skalski and Ngouenet (2001) developed a compliance framework based on classical t-tests and used Monte-Carlo simulations to calculate power curves. Unfortunately, the two-sample t tests proposed in the BO only have moderate-to-low probability of correctly assessing the true status of the smolt survival recovery. We have developed a superior two-phase regression statistical model for testing the RPA compliance. The two-phase regression model improves the statistical power over the standard two-sample t-tests. In addition, the two-phase regression model has a higher probability of correctly assessing the true status of the smolt survival recovery. These classical statistical power curve approaches do not incorporate prior knowledge into the decision process. Therefore, we propose to examine Bayesian methods that complement classical statistics in situations where uncertainty must be taken into account. The Bayesian analysis will incorporate scientific/biological knowledge/expertise to thoroughly assess the RPA compliance in 2005 and 2008.

  7. Intercultural Mediation

    Directory of Open Access Journals (Sweden)

    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.

  8. Primordial magnetogenesis before recombination

    CERN Document Server

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Unraveling recombination rate evolution using ancestral recombination maps

    DEFF Research Database (Denmark)

    Munch, Kasper; Schierup, Mikkel H

    2014-01-01

    Recombination maps of ancestral species can be constructed from comparative analyses of genomes from closely related species, exemplified by a recently published map of the human-chimpanzee ancestor. Such maps resolve differences in recombination rate between species into changes along individual branches in the speciation tree, and allow identification of associated changes in the genomic sequences. We describe how coalescent hidden Markov models are able to call individual recombination events in ancestral species through inference of incomplete lineage sorting along a genomic alignment. In the great apes, speciation events are sufficiently close in time that a map can be inferred for the ancestral species at each internal branch - allowing evolution of recombination rate to be tracked over evolutionary time scales from speciation event to speciation event. We see this approach as a way of characterizing the evolution of recombination rate and the genomic properties that influence it.

  11. Transposon-facilitated recombination in classical biotypes of Vibrio cholerae.

    OpenAIRE

    Sublett, R D; Romig, W. R.

    1981-01-01

    Transposon-facilitated recombination (Tfr) donors of classical Vibrio cholerae strain 162 were constructed by introducing the ampicillin transposon Tn1 into the P conjugative plasmid and the bacterial chromosome. The improved donors mediated high-frequency, polarized transfer of chromosomal genes from origins to confirm the gene orders of the previous classical strain 162 genetic map and to establish its circularity. Significant transfer of linked genes from E1 Tor Tfr donors to classical rec...

  12. Structural basis for broad DNA-specificity in integron recombination.

    OpenAIRE

    MacDonald, Douglas; Demarre, Gaëlle; Bouvier, Marie; Mazel, Didier; Gopaul, Deshmukh N.

    2006-01-01

    Lateral DNA transfer--the movement of genetic traits between bacteria--has a profound impact on genomic evolution and speciation. The efficiency with which bacteria incorporate genetic information reflects their capacity to adapt to changing environmental conditions. Integron integrases are proteins that mediate site-specific DNA recombination between a proximal primary site (attI) and a secondary target site (attC) found within mobile gene cassettes encoding resistance or virulence factors. ...

  13. Similarity of Recombinant Human Perlecan Domain 1 by Alternative Expression Systems Bioactive Heterogenous Recombinant Human Perlecan D1

    Directory of Open Access Journals (Sweden)

    Ellis April L

    2010-09-01

    Full Text Available Abstract Background Heparan sulfate glycosaminoglycans are diverse components of certain proteoglycans and are known to interact with growth factors as a co-receptor necessary to induce signalling and growth factor activity. In this report we characterize heterogeneously glycosylated recombinant human perlecan domain 1 (HSPG2 abbreviated as rhPln.D1 synthesized in either HEK 293 cells or HUVECs by transient gene delivery using either adenoviral or expression plasmid technology. Results By SDS-PAGE analysis following anion exchange chromatography, the recombinant proteoglycans appeared to possess glycosaminoglycan chains ranging, in total, from 6 kDa to >90 kDa per recombinant. Immunoblot analysis of enzyme-digested high Mr rhPln.D1 demonstrated that the rhPln.D1 was synthesized as either a chondroitin sulfate or heparan sulfate proteoglycan, in an approximately 2:1 ratio, with negligible hybrids. Secondary structure analysis suggested helices and sheets in both recombinant species. rhPln.D1 demonstrated binding to rhFGF-2 with an apparent kD of 2 ± 0.2 nM with almost complete susceptibility to digestion by heparinase III in ligand blot analysis but not to chondroitinase digestion. Additionally, we demonstrate HS-mediated binding of both rhPln.D1 species to several other GFs. Finally, we corroborate the augmentation of FGF-mediated cell activation by rhPln.D1 and demonstrate mitogenic signalling through the FGFR1c receptor. Conclusions With importance especially to the emerging field of DNA-based therapeutics, we have shown here that proteoglycan synthesis, in different cell lines where GAG profiles typically differ, can be directed by recombinant technology to produce populations of bioactive recombinants with highly similar GAG profiles.

  14. Hadron Correlations and Parton Recombination

    OpenAIRE

    Fries, Rainer J.(Cyclotron Institute, Department of Physics & Astronomy, Texas A&M University, College Station, TX, 77843-3366, USA)

    2007-01-01

    Parton recombination has been found to be an extremely useful model to understand hadron production at the Relativistic Heavy Ion Collider. It is particularly important to explore its connections with hard processes. This article reviews some of the aspects of the quark recombination model and places particular emphasis on hadron correlations.

  15. CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  17. Telomeric transcripts stimulate telomere recombination to suppress senescence in cells lacking telomerase

    OpenAIRE

    Yu, Tai-Yuan; Kao, Yu-wen; Lin, Jing-Jer

    2014-01-01

    Telomerase expression is essential for the long-term proliferation of most cancer cells. In cancer cells lacking telomerase, an alternative lengthening of telomeres (ALT) pathway is activated to maintain telomere length through telomere recombination. Using yeast as a model system, we found that the noncoding telomeric repeat-containing RNA (TERRA) plays a major role in recombination-mediated maintenance of telomere in telomerase-deficient cells. Increased levels of telomere-associated TERRA ...

  18. Immunization of cattle with recombinant Babesia bovis merozoite surface antigen-1.

    OpenAIRE

    Hines, S.A.; Palmer, G H; JASMER, D. P.; Goff, W L; McElwain, T F

    1995-01-01

    Cattle immunized with a recombinant merozoite surface antigen-1 molecule (MSA-1) produced high-titered antibody that reacted with the surface of the parasite and neutralized merozoite infectivity in vitro. However, recombinant MSA-1 immunization did not confer protection against challenge with virulent Babesia bovis. These results indicate that antibody-mediated neutralization of merozoite infectivity in vitro, at least for MSA-1-specific antibody, does not reflect in vivo protective immunity...

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

    International Nuclear Information System (INIS)

    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

  20. Recombinant Goat VEGF164 Increases Hair Growth by Painting Process on the Skin of Shaved Mouse

    OpenAIRE

    Bao, Wenlei; Yin, Jianxin; Liang, Yan; Guo, Zhixin; WANG, YANFENG; Liu, Dongjun; WANG, XIAO; Wang, ZhiGang

    2014-01-01

    To detect goat vascular endothelial growth factor (VEGF)-mediated regrowth of hair, full-length VEGF164 cDNA was cloned from Inner Mongolia cashmere goat (Capra hircus) into the pET-his prokaryotic expression vector, and the recombinant plasmid was transferred into E. coli BL21 cells. The expression of recombinant 6×his-gVEGF164 protein was induced by 0.5 mM isopropyl thio-?-D-galactoside at 32°C. Recombinant goat VEGF164 (rgVEGF164) was purified and identi ed by western blot using monoclonal...

  1. Delayed recombination and standard rulers

    International Nuclear Information System (INIS)

    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.

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

    International Nuclear Information System (INIS)

    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)

  3. Iodine chemistry in hydrogen recombiners

    International Nuclear Information System (INIS)

    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)

  4. Recombining WMAP: constraints on ionizing and resonance radiation at recombination

    OpenAIRE

    Bean, Rachel; Melchiorri, Alessandro; Silk, Joe

    2003-01-01

    We place new constraints on sources of ionizing and resonance radiation at the epoch of the recombination process using the recent CMB temperature and polarization spectra coming from 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...

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

    DEFF Research Database (Denmark)

    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 (NER), XPA-RPA, recognizes DNA ICLs. We now report the use of triplex technology to direct a site-specific psoralen ICL to a target DNA substrate to determine whether the human global genome NER damage re...

  6. Recombineering: genetic engineering in bacteria using homologous recombination.

    Science.gov (United States)

    Thomason, Lynn C; Sawitzke, James A; Li, Xintian; Costantino, Nina; Court, Donald L

    2014-01-01

    The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. It then presents support protocols that describe several two-step selection/counter-selection methods of making genetic alterations without leaving any unwanted changes in the targeted DNA, and a method for retrieving onto a plasmid a genetic marker (cloning by retrieval) from the Escherichia coli chromosome or a co-electroporated DNA fragment. Additional protocols describe methods to screen for unselected mutations, removal of the defective prophage from recombineering strains, and other useful techniques. Curr. Protoc. Mol. Biol. 106:1.16.1-1.16.39. © 2014 by John Wiley & Sons, Inc. PMID:24733238

  7. Gravitino Dark Matter in Tree Level Gauge Mediation with and without R-parity

    DEFF Research Database (Denmark)

    Arcadi, G.; Di Luzio, L.; Nardecchia, M.

    2011-01-01

    We investigate the cosmological aspects of Tree Level Gauge Mediation, a recently proposed mechanism in which the breaking of supersymmetry is communicated to the soft scalar masses by extra gauge interactions at the tree level. Embedding the mechanism in a Grand Unified Theory and requiring the observability of sfermion masses at the Large Hadron Collider, it follows that the Lightest Supersymmetric Particle is a gravitino with a mass of the order of 10 GeV. The analysis in the presence of R-pa...

  8. Hydrogen recombiner development at AECL

    International Nuclear Information System (INIS)

    Catalytic recombiners have been developed at AECL for the purpose of hydrogen removal in post-accident nuclear containment buildings. The recombiners are based on a particular catalyst designed by AECL which has extraordinary resistance to fouling from water and water vapour and a large thermodynamic range of operation. The catalysts were developed, originally, for the purpose of heavy water manufacturing by way of a catalytic exchange process. Application of these catalyst materials in recombiners for containment applications began in the late 1980's. The first application was a passive recombiner, qualified for use in control of radiolytic hydrogen in the headspace of a pool-type experimental reactor of AECL design in 1988. The passive, or natural convection recombiner concept has continued development to commercial stage for application in power reactor containments. This paper reviews the AECL recombiner development, describes the current model and shows results from tests of full-scale recombiners in the Large Scale Vented Combustion Test Facility at AECL-WL. The AECL recombiner is designed for compactness and ease of engineering into containment. The design is a simple, open-ended rectangular enclosure with catalyst elements arranged inside to promote optimum convective flow driven by heat of recombination at the catalyst surface. Self start, as evidenced by catalyst heating and initiation of flow, is achieved in less than 1% hydrogen, with available oxygen, at room temperature and 100% relative humidity. This low temperature start-up in condensing atmospheres is viewed as the most challenging condition for wet-proofing effectiveness. Cold start-up is a vital performance requirement in containments, such as CANDU, where engineered air-cooling systems are operating and where long-term hydrogen control is required, after containment atmospheres have cooled. Once started, the removal capacity scales linearly with the inlet cross-section area and the partial pressure of hydrogen. The recombiner also reacts carbon monoxide, in the presence of hydrogen, at approximately the same rate as the hydrogen. The catalyst materials and wet-proofing are unaffected by radiation or high temperatures. Large scale tests confirm self-start behavior and demonstrate strong mixing, irrespective of recombiner placement. (author)

  9. Progenitors of Recombining Supernova Remnants

    OpenAIRE

    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 t...

  10. Rapid Purification of Recombinant Histones

    OpenAIRE

    Klinker, Henrike; Haas, Caroline; Harrer, Nadine; Peter B. Becker; Mueller-Planitz, Felix

    2014-01-01

    The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method “rapid histone purification” (RHP) as it circumvents isolation of inclusion bodies and thereby cuts out the mos...

  11. Construction of Gene Targeting Vectors by Recombineering

    OpenAIRE

    Lee, Song-Choon; Wang, Wei; Liu, Pentao

    2009-01-01

    Recombineering is a technology that utilizes the efficient homologous recombination functions encoded by ? phage to manipulate DNA in E.coli. Construction of knockout vectors has been greatly facilitated by recombineering as it allows one to choose any genomic region to manipulate. We describe here an efficient recombineering-based protocol for making mouse conditional knockout targeting vectors.

  12. Delayed recombination and cosmic parameters

    International Nuclear Information System (INIS)

    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 1? to R=1.734±0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: ??i<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.

  13. In vivo effects of anti-inflammatory agents on arachidonic acid (AA) metabolites in the pleural fluid of rats injured in a reverse passive Arthus (RPA) reaction

    International Nuclear Information System (INIS)

    Leukotriene (LT)C4-D4, prostaglandin (PG)E2, thromboxane B2 (TXB), and 6-ketoprostaglandin F1? (6-KP) were measured by radioimmunoassay in pleural fluid of rats immunologically injured in an RPA paradigm. Rats given intravenous BSA were injected intrapleurally 20 min. later with anti-BSA. Phenidone (30 mg/kg), indomethacin (0.3-100 mg/kg), dazoxiben (100 mg/kg), AA-861 (2,3,5 trimethyl-6(12 hydroxy-5,10-dodecadinyl)-1,4-benzoquinone; 100 mg/kg) or vehicle was given intragastrically 1 hr prior to injury. Pleural fluid samples were collected 1 hr after injury. Statistically significant (P 4-D4 after phenidone and AA-861 treatment, in PGE2 and 6-KP after phenidone and indomethacin treatment and in TXB after dazoxiben and indomethacin treatment. Dazoxiben significantly (p < 0.05) increased 6-KP. These data suggest that anti-inflammatory agents given in this in vivo RPA paradigm inhibited AA metabolism in a predictable manner. Also, drug administration was associated with changes in metabolite concentrations at additional pathway sites. Consequently, this paradigm may be a useful model in evaluating shifts in AA metabolism brought about by inflammatory responses and treatments

  14. Randomized Phase II Trial of High-Dose Melatonin and Radiation Therapy for RPA Class 2 Patients With Brain Metastases (RTOG 0119)

    International Nuclear Information System (INIS)

    Purpose: To determine if high-dose melatonin for Radiation Therapy Oncology Group (RTOG) recursive partitioning analysis (RPA) Class 2 patients with brain metastases improved survival over historical controls, and to determine if the time of day melatonin was given affected its toxicity or efficacy. RTOG 0119 was a phase II randomized trial for this group of patients. Methods and Materials: RTOG RPA Class 2 patients with brain metastases were randomized to 20 mg of melatonin, given either in the morning (8-9 AM) or in the evening (8-9 PM). All patients received radiation therapy (30 Gy in 10 fractions) in the afternoon. Melatonin was continued until neurologic deterioration or death. The primary endpoint was overall survival time. Neurologic deterioration, as reflected by the Mini-Mental Status Examination, was also measured. Results: Neither of the randomized groups had survival distributions that differed significantly from the historic controls of patients treated with whole-brain radiotherapy. The median survivals of the morning and evening melatonin treatments were 3.4 and 2.8 months, while the RTOG historical control survival was 4.1 months. Conclusions: High-dose melatonin did not show any beneficial effect in this group of patients

  15. Infectious Molecular Clones with the Nonhomologous Dimer Initiation Sequences Found in Different Subtypes of Human Immunodeficiency Virus Type 1 Can Recombine and Initiate a Spreading Infection In Vitro

    OpenAIRE

    St. Louis, Daniel C.; Gotte, Deanna; Sanders-Buell, Eric; Ritchey, David W.; Salminen, Mika O; Carr, Jean K.; McCutchan, Francine E

    1998-01-01

    Recombinant forms of human immunodeficiency virus type 1 (HIV-1) have been shown to be of major importance in the global AIDS pandemic. Viral RNA dimer formation mediated by the dimerization initiation sequence (DIS) is believed to be essential for viral genomic RNA packaging and therefore for RNA recombination. Here, we demonstrate that HIV-1 recombination and replication are not restricted by variant DIS loop sequences. Three DIS loop forms found among HIV-1 isolates, DIS (CG), DIS (TA), an...

  16. Investigations for designing catalytic recombiners

    International Nuclear Information System (INIS)

    In case of a severe accident in pressurised water reactors (PWR) a high amount of hydrogen up to about 20,000 m3 might be generated and released into the containments. The mixture consisting of hydrogen and oxygen may either burn or detonate, if ignited. In case of detonation the generated shock wave may endanger the components of the plant or the plant itself. Consequently, effective removal of hydrogen is required. The fact that hydrogen and oxygen react exo-thermally on catalytically acting surfaces already at low temperatures generating steam and heat is made use of in catalytic recombiners. They consist of substrates coated with catalyst (mainly platinum or palladium) which are arranged inside a casing. Being passively acting measures, recombiners do not need any additional energy supply. Experimental investigations on catalytic hydrogen recombination are conducted at FZJ (Forschungszentrum Juelich) using three test facilities. The results yield insight in the development potential of contemporary recombiner systems as well as of innovative systems. Detailed investigations on a recombiner section show strong temperature gradients over the surface of a catalytically coated sample. Dependent on the flow velocity, ignition temperature may be reached at the leading edge already at an inlet hydrogen concentration of about 5 vol.-%. The thermal strain of the substrate leads to considerable detachment of catalyst particles probably causing unintended ignition of the flammable mixture. Temperature peaks can be prevented effectively by leaving the first part of the plate uncoated. In order to avoid overheating of the catalyst elements of a recombiner even at high hydrogen concentrations a modular system of porous substrates is proposed. The metallic substrates are coated with platinum at low catalyst densities thus limiting the activity of the single specimen. A modular arrangement of these elements provides high recombination rates over a large hydrogen concentration range without igniting the mixture

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

    International Nuclear Information System (INIS)

    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. Workshop on Radio Recombination Lines

    CERN Document Server

    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...

  19. AAV-mediated gene transfer to the mouse CNS

    OpenAIRE

    Stoica, Lorelei; Ahmed, Seemin S.; Gao, Guangping; Esteves, Miguel Sena

    2013-01-01

    Recombinant adeno associated virus (rAAV) vectors are great tools for gene transfer due to their ability to mediate long-term gene expression. Recombinant AAVs have been used at various ages of development with no apparent toxicity. There are multiple ways of delivering AAV vectors to the CNS, depending on the stage of development of the mouse. In neonates, intravascular injections into the facial vein are often used. In adults, direct injections into target regions of the brain are achieved ...

  20. Global divergence of microbial genome sequences mediated by propagating fronts

    OpenAIRE

    Vetsigian, Kalin; Goldenfeld, Nigel

    2005-01-01

    We model the competition between homologous recombination and point mutation in microbial genomes, and present evidence for two distinct phases, one uniform, the other genetically diverse. Depending on the specifics of homologous recombination, we find that global sequence divergence can be mediated by fronts propagating along the genome, whose characteristic signature on genome structure is elucidated, and apparently observed in closely related Bacillus strains. Front propagation provides an...

  1. Selenium incorporation using recombinant techniques

    International Nuclear Information System (INIS)

    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

  2. Recombination in the human Pseudoautosomal region PAR1.

    Science.gov (United States)

    Hinch, Anjali G; Altemose, Nicolas; Noor, Nudrat; Donnelly, Peter; Myers, Simon R

    2014-07-01

    The pseudoautosomal region (PAR) is a short region of homology between the mammalian X and Y chromosomes, which has undergone rapid evolution. A crossover in the PAR is essential for the proper disjunction of X and Y chromosomes in male meiosis, and PAR deletion results in male sterility. This leads the human PAR with the obligatory crossover, PAR1, to having an exceptionally high male crossover rate, which is 17-fold higher than the genome-wide average. However, the mechanism by which this obligatory crossover occurs remains unknown, as does the fine-scale positioning of crossovers across this region. Recent research in mice has suggested that crossovers in PAR may be mediated independently of the protein PRDM9, which localises virtually all crossovers in the autosomes. To investigate recombination in this region, we construct the most fine-scale genetic map containing directly observed crossovers to date using African-American pedigrees. We leverage recombination rates inferred from the breakdown of linkage disequilibrium in human populations and investigate the signatures of DNA evolution due to recombination. Further, we identify direct PRDM9 binding sites using ChIP-seq in human cells. Using these independent lines of evidence, we show that, in contrast with mouse, PRDM9 does localise peaks of recombination in the human PAR1. We find that recombination is a far more rapid and intense driver of sequence evolution in PAR1 than it is on the autosomes. We also show that PAR1 hotspot activities differ significantly among human populations. Finally, we find evidence that PAR1 hotspot positions have changed between human and chimpanzee, with no evidence of sharing among the hottest hotspots. We anticipate that the genetic maps built and validated in this work will aid research on this vital and fascinating region of the genome. PMID:25033397

  3. Mediating Business

    DEFF Research Database (Denmark)

    2007-01-01

    "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 ...

  4. Mediatized play

    DEFF Research Database (Denmark)

    Johansen, Stine Liv

    2011-01-01

    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 vid...

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

    CERN Document Server

    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...

  6. KEGG PATHWAY / Homologous recombination [KEGG

    Lifescience Database Archive (English)

    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 ...

  7. Improving recombinant protein purification yield

    Science.gov (United States)

    Production of adequate amounts of recombinant proteins is essential for antibody production, biochemical activity study, and structural determination during the post-genomic era. It’s technologically challenging and a limiting factor for tung oil research because analytical reagents such as high qua...

  8. Recombination luminescence in rigid media

    International Nuclear Information System (INIS)

    When separated ion-pairs result from the ?-irradiation of pure or doped matrices or from solute photoionization, some of the photoejected electrons undergo spontaneous recombination, giving rise to the so-called isothermal luminescence (ITL). Cation-anion recombination takes place when molecular diffusion is possible; that is, it appears mostly in thermoluminescence (TL), upon warming irradiated samples. Electrons are photoextracted from matrix traps or from anions, and the neutralization luminescence is designated as stimulated luminescence (SL). ITL, TL, and SL all constitute very sensitive test for the presence of charged species. ITL decay kinetics throw some light on the recombination mechanism. In TL studies, the maxima of the glow curves correlate with phase transition temperatures (crystal ? crystal or glass ? crystal), providing insight into matrix molecular dynamics. From SL spectra, positive and negative charges can be discriminated, and various characteristics of the negative ions - electrons or anions - can be attained. The global SL irrespective of its spectrum composition, SL emission spectra, and SL excitation spectra or stimulation spectra are reviewed. The SL spectra, being specific of negative charged species, complement optical absorption spectroscopy when cations and anions have undistinguishable spectra. Even though radiative charge recombination constitutes the final step of ion-pair existence, it may serve to track back the successive stages of photoelectron life: electron ejection, matrix trapping or the attachments of electrons to molecules or radicals, and the release of trapped electrons. (Yamashita, S.)

  9. Recombination of Physical Unclonable Functions

    OpenAIRE

    Devadas, Srinivas; Yu, Meng-Day (Mandel)

    2010-01-01

    A new Physical Unclonable Function (PUF) construction is described, by treating silicon unique features extracted from PUF circuits as “genetic material” unique to each silicon, and recombining this chip-unique material in a way to obtain a combination of advantages not possible with the original PUF circuits, including altering PUF output statistics to better suit PUF-based key generation and authentication.

  10. The ?0 polarization and the recombination mechanism

    International Nuclear Information System (INIS)

    We use the recombination and the Thomas Precession Model to obtain a prediction for the ?0 polarization in the p+p ? ?0 + X reaction. We study the effect of the recombination function on the ?0 polarization. (author)

  11. Population inversion in recombining hydrogen plasma

    International Nuclear Information System (INIS)

    The collisional-radiative model is applied to a recombining hydrogen plasma in order to investigate plasma conditions in which a population inversion between the energy levels of hydrogen can be generated. Population inversion is expected in plasmas for which three-body recombination makes a large contribution to the recombining processes and the effective recombination rate is larger than a critical value for a given electron density and temperature. Calculated results are presented in figures and tables. (author)

  12. Dielectronic recombination in laser generated plasmas

    International Nuclear Information System (INIS)

    Dielectronic recombination coefficients have been computed for hydrogenic ions from HeII to FeXVI over a range of conditions typical of laser generated plasma. The results are displayed in a set a graphs together with the corresponding collisional radiative recombination coefficients. A comparison of these results indicates plasma conditions where dielectronic recombination is a significant process. (author)

  13. Pairing symmetry of the one-band Hubbard model in the paramagnetic weak-coupling limit: A numerical RPA study

    Science.gov (United States)

    Rømer, A. T.; Kreisel, A.; Eremin, I.; Malakhov, M. A.; Maier, T. A.; Hirschfeld, P. J.; Andersen, B. M.

    2015-09-01

    We study the spin-fluctuation-mediated superconducting pairing gap in a weak-coupling approach to the Hubbard model for a two-dimensional square lattice in the paramagnetic state. Performing a comprehensive theoretical study of the phase diagram as a function of filling, we find that the superconducting gap exhibits transitions from p -wave at very low electron fillings to dx2-y2-wave symmetry close to half filling in agreement with previous reports. At intermediate filling levels, different gap symmetries appear as a consequence of the changes in the Fermi surface topology and the associated structure of the spin susceptibility. In particular, the vicinity of a Van Hove singularity in the electronic structure close to the Fermi level has important consequences for the gap structure in favoring the otherwise subdominant triplet solution over the singlet d -wave solution. By solving the full gap equation, we find that the energetically favorable triplet solutions are chiral and break time-reversal symmetry. Finally, we also calculate the detailed angular gap structure of the quasiparticle spectrum, and show how spin-fluctuation-mediated pairing leads to significant deviations from the first harmonics both in the singlet dx2-y2 gap as well as the chiral triplet gap solution.

  14. Osmoregulation of Dimer Resolution at the Plasmid pJHCMW1 mwr Locus by Escherichia coli XerCD Recombination

    OpenAIRE

    Pham, Van H.; Dery, KJ; Sherratt, DJ; Tolmasky, ME

    2002-01-01

    Xer-mediated dimer resolution at the mwr site of plasmid pJHCMW1 is osmoregulated in Escherichia coli. Whereas under low-salt conditions, the site-specific recombination reaction is efficient, under high-salt conditions, it proceeds inefficiently. Regulation of dimer resolution is independent of H-NS and is mediated by changes in osmolarity rather than ionic effects. The low level of recombination at high salt concentrations can be overcome by high levels of PepA or by mutating the ARG box to...

  15. Complex Mediation

    DEFF Research Database (Denmark)

    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 ...

  16. Nondisjunction of chromosome 15: Origin and recombination

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. Mechanisms of sister chromatid recombination

    International Nuclear Information System (INIS)

    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.)

  18. Inhibited Recombination of Charged Magnetoexcitons

    OpenAIRE

    Okamura, H; Heiman, D; Sundaram, M.; Gossard, A. C.

    1998-01-01

    Time-resolved photoluminescence measurements show that the decay time for charged excitons in a GaAs two-dimensional electron gas increases by an order of magnitude at high magnetic fields. Unlike neutral excitons, the charged exciton center-of-mass is spatially confined in a ``magnetically-adjustable quantum dot'' by the cyclotron orbit and the quantum well. The inhibited recombination is explained by a reduced phase coherence volume of the magnetically-confined charged exc...

  19. Recombinant bacteriophage lysins as antibacterials

    OpenAIRE

    Fenton, Mark; Ross, Paul; McAuliffe, Olivia; O'Mahony, Jim; Coffey, Aidan

    2010-01-01

    With the increasing worldwide prevalence of antibiotic resistant bacteria, bacteriophage endolysins (lysins) represent a very promising novel alternative class of antibacterial in the fight against infectious disease. Lysins are phage-encoded peptidoglycan hydrolases which, when applied exogenously (as purified recombinant proteins) to Gram-positive bacteria, bring about rapid lysis and death of the bacterial cell. A number of studies have recently demonstrated the strong potential of these e...

  20. Asymmetric DNA bending in the Cre-loxP site-specific recombination synapse

    OpenAIRE

    Guo, Feng; Gopaul, Deshmukh N.; Van Duyne, Gregory D.

    1999-01-01

    Cre recombinase catalyzes site-specific recombination between two 34-bp loxP sites in a variety of DNA substrates. At the start of the recombination pathway, the loxP sites are each bound by two recombinase molecules, and synapsis of the sites is mediated by Cre–Cre interactions. We describe the structures of synaptic complexes formed between a symmetrized loxP site and two Cre mutants that are defective in strand cleavage. The DNA in these complexes is bent sharply at a single base pair step...

  1. Functional Interactions of Meiotic Recombination Factors Rdh54 and Dmc1

    OpenAIRE

    Chi, Peter; Kwon, YoungHo; Moses, Dana N; Seong, Changhyun; Sehorn, Michael G.; Singh, Akhilesh K.; Tsubouchi, Hideo; Greene, Eric C.; Klein, Hannah L.; Sung, Patrick

    2008-01-01

    Genetic studies in budding and fission yeasts have provided evidence that Rdh54, a Swi2/Snf2-like factor, synergizes with the Dmc1 recombinase to mediate inter-homologue recombination during meiosis. Rdh54 associates with Dmc1 in the yeast two-hybrid assay, but whether the Rdh54-Dmc1 interaction is direct and the manner in which these two recombination factors may functionally co-operate to accomplish their biological task have not yet been defined. Here, using purified Schizosaccharomyces po...

  2. Bacteriophage recombination systems and biotechnical applications.

    Science.gov (United States)

    Nafissi, Nafiseh; Slavcev, Roderick

    2014-04-01

    Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, ?, and N15, the integrase from the Streptomyces phage, ?C31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed. PMID:24442504

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

    International Nuclear Information System (INIS)

    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.)

  4. Heterogeneity in recombinant protein production

    DEFF Research Database (Denmark)

    Schalén, Martin; Johanson, Ted

    2012-01-01

    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 bioreactors are often not taken into consideration in these experiments. Gradients occur due to insufficient mixing in the reactor, and affect the process in a variety of ways. When cells travel through the reactor and encounter different substrate concentrations, oxygen availability, pH, temperature, etc. the cell physiology is affected. Cells are stressed, and this may severely affect growth, by-product accumulation, biomass yield and recombinant product yield. The stress caused by exposure to divergent microenvironments, genetic differences of individual cells, differing cell cycle stage and cell age, all contribute to make a population in a fermenter heterogeneous, resulting in cell-to-cell variation in physiological parameters of the microbial culture. Our study aims at investigating how population heterogeneity and recombinant protein production is affected by environmental gradients in 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.

  5. Mediatized Humanitarianism

    DEFF Research Database (Denmark)

    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 legitimate themselves and their moral claim were examined. A time trend analysis of the prioritization of actors in the material indicates that marked shifts in legitimation loci have taken place during the pa...

  6. Trap-limited charge recombination in intrinsic perovskite film and meso-superstructured perovskite solar cells and the passivation effect of the hole-transport material on trap states.

    Science.gov (United States)

    Wang, Yi; Wang, Hao-Yi; Yu, Man; Fu, Li-Min; Qin, Yujun; Zhang, Jian-Ping; Ai, Xi-Cheng

    2015-11-28

    Charge recombination dynamics in intrinsic perovskite film and in meso-superstructured perovskite solar cells have been systematically studied, which are found to be mediated by the energetic distribution of intra-gap trap states as described by the trap-limited recombination theory. Besides, the passivation effect of the hole-transport material on trap states is discussed. PMID:26497590

  7. Recombinant DNA production of spider silk proteins.

    Science.gov (United States)

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-11-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. PMID:24119078

  8. Recombination activity enhancement by stress in silicon

    OpenAIRE

    Gundel, P.; Schubert, M.C.; Heinz, F.D.; Warta, W.

    2010-01-01

    The recombination activity of grain boundaries and precipitate colonies is analyzed with submicron spatial resolution and compared to the surrounding stress field. This analysis reveals a positive correlation between tensile stress and recombination activity and a negative correlation between compressive stress and recombination activity. This correlation can be explained by the stress induced mobility enhancement due to the strong piezoresistance of silicon. This observation could lead to a ...

  9. Consequences of recombination on traditional phylogenetic analysis.

    DEFF Research Database (Denmark)

    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 or viral sequences) or does occur (nuclear sequences). We investigate the size and direction of biases when a single tree is reconstructed ignoring recombination. Standard software (PHYLIP) was used to construct the best phylogenetic tree from sequences simulated under the coalescent with recombination. With recombination present, the length of terminal branches and the total branch length are larger, and the time to the most recent common ancestor smaller, than for a tree reconstructed from sequences evolving with no recombination. The effects are pronounced even for small levels of recombination that may not be immediately detectable in a data set. The phylogenies when recombination is present superficially resemble phylogenies for sequences from an exponentially growing population. However, exponential growth has a different effect on statistics such as Tajima's D. Furthermore, ignoring recombination leads to a large overestimation of the substitution rate heterogeneity and the loss of the molecular clock. These results are discussed in relation to viral and mtDNA data sets. Udgivelsesdato: 2000-Oct

  10. Human Insulin from Recombinant DNA Technology

    Science.gov (United States)

    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.

  11. p53 suppresses hyper-recombination by modulating BRCA1 function.

    Science.gov (United States)

    Dong, Chao; Zhang, Fengmei; Luo, Yue; Wang, Hui; Zhao, Xipeng; Guo, Gongshe; Powell, Simon N; Feng, Zhihui

    2015-09-01

    Both p53 and BRCA1 are tumor suppressors and are involved in a number of cellular processes including cell cycle arrest, apoptosis, transcriptional regulation, and DNA damage repair. Some studies have suggested that the association of BRCA1 and p53 is required for transcriptional regulation of genes involved in cell replication and DNA repair pathways. However, the relationship between the two proteins in molecular mechanisms of DNA repair is still not clear. Therefore, we sought to determine whether there is a functional link between p53 and BRCA1 in DNA repair. Firstly, using a plasmid recombination substrate, pDR-GFP, integrated into the genome of breast cancer cell line MCF7, we have demonstrated that p53 suppressed Rad51-mediated hyper-recombinational repair by two independent cell models of HPV-E6 induced p53 inactivation and p53 knockdown assay. Our study further indicated that p53 mediated homologous recombination (HR) through inhibiting BRCA1 over-function via mechanism of transcription regulation in response to DNA repair. Since it was found p53 and BRCA1 existed in a protein complex, indicating both proteins may be associated at post-transcriptional level. Moreover, defective p53-induced hyper-recombination was associated with cell radioresistance and chromosomal stability, strongly supporting the involvement of p53 in the inhibition of hyper-recombination, which led to genetic stability and cellular function in response to DNA damage. In addition, it was found that p53 loss rescued BRCA1 deficiency via recovering HR and chromosomal stability, suggesting that p53 is also involved in the HR-inhibition independently of BRCA1. Thus, our data indicated that p53 was involved in inhibiting recombination by both BRCA1-dependent and -independent mechanisms, and there is a functional link between p53-suppression and BRCA1-promotion in regulation of HR activity at transcription level and possible post-transcription level. PMID:26162908

  12. Endoplasmic reticulum–mitochondria crosstalk in NIX-mediated murine cell death

    OpenAIRE

    Diwan, Abhinav; Matkovich, Scot J; Yuan, Qunying; Zhao, Wen; Yatani, Atsuko; Brown, Joan Heller; Molkentin, Jeffery D; Kranias, Evangelia G.; Dorn, Gerald W., II

    2008-01-01

    Transcriptional upregulation of the proapoptotic BCL2 family protein NIX limits red blood cell formation and can cause heart failure by inducing cell death, but the requisite molecular events are poorly defined. Here, we show complementary mechanisms for NIX-mediated cell death involving direct and ER/sarcoplasmic reticulum–mediated (ER/SR-mediated) mitochondria disruption. Endogenous cardiac NIX and recombinant NIX localize both to the mitochondria and to the ER/SR. In genetic mouse models, ...

  13. The Purified and Recombinant Legionella pneumophila Chaperonin Alters Mitochondrial Trafficking and Microfilament Organization?

    OpenAIRE

    Chong, Audrey; Lima, Celia A.; Allan, David S.; Nasrallah, Gheyath K.; Garduño, Rafael A.

    2009-01-01

    A portion of the total cellular pool of the Legionella pneumophila chaperonin, HtpB, is found on the bacterial cell surface, where it can mediate invasion of nonphagocytic cells. HtpB continues to be abundantly produced and released by internalized L. pneumophila and may thus have postinvasion functions. We used here two functional models (protein-coated beads and expression of recombinant proteins in CHO cells) to investigate the competence of HtpB in mimicking early intracellular traffickin...

  14. Molecular engineering of bacterial natural product biosynthetic pathways via Red/ET recombineering

    OpenAIRE

    Bian, Xiaoying

    2012-01-01

    Bacterial genome-sequencing projects have revealed that a large number of natural product biosynthetic pathways presented in genomes are cryptic. Cloning, engineering and expression of a cryptic biosynthetic pathway in a well-characterized heterologous host to discover and optimize its product is an effective alternate to traditional approaches. We developed RecE/RecT mediated linear-linear homologous recombination (LLHR) for direct cloning of ten unknown NRPS/PKS gene clusters from the genom...

  15. Recombinant allergen-specific antibody fragments: tools for diagnosis, prevention and therapy of type I allergy.

    OpenAIRE

    VALENTA, R; Flicker, S.; Eibensteiner, PB; Steinberger, P.; Laffer, S; Dolecek, C; Kraft, D

    1997-01-01

    Type I allergy represents a hypersensitivity occurring in almost 20% of the population that is based on the recognition of innocuous airborn antigens (pollen, mite, mould and pet allergens) by specific immunoglobulin E. Allergic symptoms (e.g. allergic rhinitis, conjunctivitis, asthma) are caused by the release of biological mediators from effector-cells after allergen-induced crosslink of receptor-bound IgE. Here we discuss strategies to obtain recombinant allergen-specific antibody fragment...

  16. Rad52 forms DMA repair and recombination centers during S phase

    DEFF Research Database (Denmark)

    Lisby, M.; Rothstein, R.; Mortensen, Uffe Hasbro

    2001-01-01

    Maintenance of genomic integrity and stable transmission of genetic information depend on a number of DNA repair processes. Failure to faithfully perform these processes can result in genetic alterations and subsequent development of cancer and other genetic diseases. In the eukaryote Saccharomyces cerevisiae, homologous recombination is the major pathway for repairing DNA double-strand breaks. The key role played by Rad52 in this pathway has been attributed to its ability to seek out and mediat...

  17. Expression, purification, and characterization of recombinant human transferrin from rice (Oryza sativa L.)

    OpenAIRE

    Zhang, Deshui; Nandi, Somen; Bryan, Paula; Pettit, Steve; Nguyen, Diane; Santos, Mary Ann; Huang, Ning

    2010-01-01

    Transferrin is an essential ingredient used in cell culture media due to its crucial role in regulating cellular iron uptake, transport, and utilization. It is also a promising drug carrier used to increase a drug’s therapeutic index via the unique transferrin receptor-mediated endocytosis pathway. Due to the high risk of contamination with blood-borne pathogens from the use of human- or animal plasma-derived transferrin, recombinant transferrin is preferred for use as a replacement for nativ...

  18. Mediation of mouse natural cytotoxic activity by tumour necrosis factor

    Science.gov (United States)

    Ortaldo, John R.; Mason, Llewellyn H.; Mathieson, Bonnie J.; Liang, Shu-Mei; Flick, David A.; Herberman, Ronald B.

    1986-06-01

    Natural cell-mediated cytotoxic activity in the mouse has been associated with two types of effector cells, the natural killer (NK) cell and the natural cytotoxic (NC) cell, which seem to differ with regard to their patterns of target selectivity, cell surface characteristics and susceptibility to regulatory factors1. During studies on the mechanism of action of cytotoxic molecules, it became evident that WEHI-164, the prototype NC target cell, was highly susceptible to direct lysis by both human and mouse recombinant tumour necrosis factor (TNF). Here we show that NC, but not NK activity mediated by normal splenocytes, is abrogated by rabbit antibodies to recombinant and natural TNF, respectively. Thus, the cell-mediated activity defined as NC is due to release of TNF by normal spleen cells and does not represent a unique natural effector mechanism.

  19. Telomeric recombination induced by dysfunctional telomeres

    OpenAIRE

    Brault, Marie Eve; AUTEXIER, CHANTAL

    2011-01-01

    Telomeric recombination has been observed in telomerase-negative alternative lengthening of telomeres in human cancer cells and following telomerase inhibition or gene deletion. This study shows that telomeric recombination mechanisms can also be activated by dysfunctional telomeres without telomerase inhibition in telomerase-positive cells.

  20. Recombinant Swinepox Virus for Veterinary Vaccine Development.

    Science.gov (United States)

    Fan, Hong-Jie; Lin, Hui-Xing

    2016-01-01

    Poxvirus-vectors have been widely used in vaccine development for several important human and animal diseases; some of these vaccines have been licensed and used extensively. Swinepox virus (SPV) is well suited to develop recombinant vaccines because of its large packaging capacity for recombinant DNA, its host range specificity, and its ability to induce appropriate immune responses. PMID:26458836

  1. Titania Photocatalysis beyond Recombination: A Critical Review

    OpenAIRE

    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.

  2. Titania Photocatalysis beyond Recombination: A Critical Review

    Directory of Open Access Journals (Sweden)

    Bunsho Ohtani

    2013-11-01

    Full Text Available 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.

  3. Cell biology of homologous recombination in yeast

    DEFF Research Database (Denmark)

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

    2011-01-01

    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 cerevisiae using fluorescence microscopy.

  4. RNAi and heterochromatin repress centromeric meiotic recombination

    DEFF Research Database (Denmark)

    Ellermeier, Chad; Higuchi, Emily C

    2010-01-01

    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.

  5. Recombinant organisms for production of industrial products

    OpenAIRE

    Adrio, Jose-Luis; Arnold L. Demain

    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...

  6. Glycosylation of recombinant antibody therapeutics.

    Science.gov (United States)

    Jefferis, Royston

    2005-01-01

    The adaptive immune system has the capacity to produce antibodies with a virtually infinite repertoire of specificities. Recombinant antibodies specific for human targets are established in the clinic as therapeutics and represent a major new class of drug. Therapeutic efficacy depends on the formation of complexes with target molecules and subsequent activation of downstream biologic effector mechanisms that result in elimination of the target. The activation of effector mechanisms is dependent on structural characteristics of the antibody molecule that result from posttranslational modifications, in particular, glycosylation. The production of therapeutic antibody with a consistent human glycoform profile has been and remains a considerable challenge to the biopharmaceutical industry. Recent research has shown that individual glycoforms of antibody may provide optimal efficacy for selected outcomes. Thus a further challenge will be the production of a second generation of antibody therapeutics customized for their clinical indication. PMID:15903235

  7. Recombination at silicon dangling bonds

    International Nuclear Information System (INIS)

    In the past, pulsed electrically detected magnetic resonance experiments (pEDMR) with silicon dangling bonds (db) in hydrogenated microcrystalline silicon (?c-Si:H) showed that at low temperatures, two db recombination mechanisms exist where electrons are captured (i) by dbs directly (db-dc) or (ii) via band-tail states (tail-db). Here, similar experiments on hydrogenated amorphous silicon (a-Si:H) and crystalline silicon/silicondioxide interfaces (c-Si/SiO2) are presented. They show that at low temperatures, only the db-dc is detectable at dbs in the c-Si/SiO2 interface (Pb centers) while in a-Si:H, only tail-db processes are observed

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

    Directory of Open Access Journals (Sweden)

    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 ? 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

  9. Recombinant I?B?-loaded curcumin nanoparticles for improved cancer therapeutics

    International Nuclear Information System (INIS)

    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, NF?B, involved in cell growth and its inhibitor, I?B?, regulates its proliferation. Another similar naturally available molecule, which inhibits the function of NF?B, is curcumin. Hence, we have developed a ‘green synthesis’ method for preparing water-soluble curcumin nanoparticles to stabilize recombinant I?B? 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 I?B?-loaded curcumin NPs has promise as a natural-product-based protein therapeutics against cancer cells. (paper)

  10. Recombinant I?B?-loaded curcumin nanoparticles for improved cancer therapeutics

    Science.gov (United States)

    Banerjee, Subhamoy; Sahoo, Amaresh Kumar; Chattopadhyay, Arun; Sankar Ghosh, Siddhartha

    2014-08-01

    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, NF?B, involved in cell growth and its inhibitor, I?B?, regulates its proliferation. Another similar naturally available molecule, which inhibits the function of NF?B, is curcumin. Hence, we have developed a ‘green synthesis’ method for preparing water-soluble curcumin nanoparticles to stabilize recombinant I?B? 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 I?B?-loaded curcumin NPs has promise as a natural-product-based protein therapeutics against cancer cells.

  11. Fundamental Studies of Recombinant Hydrogenases

    Energy Technology Data Exchange (ETDEWEB)

    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.

  12. Containment air circulation for optimal hydrogen recombination

    International Nuclear Information System (INIS)

    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)

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

    Science.gov (United States)

    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

  14. Mediated homogenization

    International Nuclear Information System (INIS)

    Homogenization protocols model the quantum mechanical evolution of a system to a fixed state independently from its initial configuration by repeatedly coupling it with a collection of identical ancillas. Here we analyze these protocols within the formalism of ''relaxing'' channels providing an easy-to-check sufficient condition for homogenization. In this context we describe mediated homogenization schemes where a network of connected qudits relaxes to a fixed state by only partially interacting with a bath. We also study configurations which allow us to introduce entanglement among the elements of the network. Finally we analyze the effect of having competitive configurations with two different baths and we prove the convergence to dynamical equilibrium for Heisenberg chains

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

    International Nuclear Information System (INIS)

    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)

  16. Cervical cancer isolate PT3, super-permissive for adeno-associated virus replication, over-expresses DNA polymerase ?, PCNA, RFC and RPA

    Directory of Open Access Journals (Sweden)

    Melchert Russell B

    2009-04-01

    Full Text Available Abstract Background Adeno-associated virus (AAV type 2 is an important virus due to its use as a safe and effective human gene therapy vector and its negative association with certain malignancies. AAV, a dependo-parvovirus, autonomously replicates in stratified squamous epithelium. Such tissue occurs in the nasopharynx and anogenitals, from which AAV has been clinically isolated. Related autonomous parvoviruses also demonstrate cell tropism and preferentially replicate in oncogenically transformed cells. Combining these two attributes of parvovirus tropism, squamous and malignant, we assayed if AAV might replicate in squamous cervical carcinoma cell isolates. Results Three primary isolates (PT1-3 and two established cervical cancer cell lines were compared to normal keratinocytes (NK for their ability to replicate AAV. One isolate, PT3, allowed for high levels of AAV DNA replication and virion production compared to others. In research by others, four cellular components are known required for in vitro AAV DNA replication: replication protein A (RPA, replication factor C (RFC, proliferating cell nuclear antigen (PCNA, and DNA polymerase delta (POLD1. Thus, we examined PT3 cells for expression of these components by DNA microarray and real-time quantitative PCR. All four components were over-expressed in PT3 over two representative low-permissive cell isolates (NK and PT1. However, this super-permissiveness did not result in PT3 cell death by AAV infection. Conclusion These data, for the first time, provide evidence that these four cellular components are likely important for AAV in vivo DNA replication as well as in vitro. These data also suggest that PT3 will be a useful reagent for investigating the AAV-permissive transcriptome and AAV anti-cancer effect.

  17. Modifiers of (CAG)(n) instability in Machado-Joseph disease (MJD/SCA3) transmissions: an association study with DNA replication, repair and recombination genes.

    Science.gov (United States)

    Martins, Sandra; Pearson, Christopher E; Coutinho, Paula; Provost, Sylvie; Amorim, António; Dubé, Marie-Pierre; Sequeiros, Jorge; Rouleau, Guy A

    2014-10-01

    Twelve neurological disorders are caused by gene-specific CAG/CTG repeat expansions that are highly unstable upon transmission to offspring. This intergenerational repeat instability is clinically relevant since disease onset, progression and severity are associated with repeat size. Studies of model organisms revealed the involvement of some DNA replication and repair genes in the process of repeat instability, however, little is known about their role in patients. Here, we used an association study to search for genetic modifiers of (CAG)n instability in 137 parent-child transmissions in Machado-Joseph disease (MJD/SCA3). With the hypothesis that variants in genes involved in DNA replication, repair or recombination might alter the MJD CAG instability patterns, we screened 768 SNPs from 93 of these genes. We found a variant in ERCC6 (rs2228528) associated with an expansion bias of MJD alleles. When using a gene-gene interaction model, the allele combination G-A (rs4140804-rs2972388) of RPA3-CDK7 is also associated with MJD instability in a direction-dependent manner. Interestingly, the transcription-coupled repair factor ERCC6 (aka CSB), the single-strand binding protein RPA, and the CDK7 kinase part of the TFIIH transcription repair complex, have all been linked to transcription-coupled repair. This is the first study performed in patient samples to implicate specific modifiers of CAG instability in humans. In summary, we found variants in three transcription-coupled repair genes associated with the MJD mutation that points to distinct mechanisms of (CAG)n instability. PMID:25026993

  18. Electron-ion recombination rates for merged-beams experiments

    International Nuclear Information System (INIS)

    Energy dependence of the electron-ion recombination rates are studied for different recombination processes (radiative recombination, three-body recombination, dissociative recombination) for Maxwellian relative velocity distribution of arbitrary asymmetry. The results are discussed in context of the electron-ion merged beams experiments in cooling ion storage rings. The question of indication of a possible contribution of the three-body recombination to the measured recombination rates versus relative energy is particularly addressed. Its influence on the electron beam temperature derived from the energy dependence of recombination rate is discussed

  19. Paroxetine suppresses recombinant human P2X7 responses.

    Science.gov (United States)

    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 1? (IL-1?) 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 IC50 of 24 ?M and significantly reduces ATP-induced inward currents. We confirmed that trifluoperazine hydrochloride suppressed human P2X7 responses (IC50 of 6.4 ?M). 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-1? secretion from lipopolysaccharide (LPS)-primed human CD14(+) monocytes was suppressed with trifluoperazine and paroxetine. PMID:26341077

  20. V(D)J recombination frequency is affected by the sequence interposed between a pair of recombination signals: sequence comparison reveals a putative recombinational enhancer element.

    OpenAIRE

    Roch, F A; Hobi, R; Berchtold, M W; Kuenzle, C C

    1997-01-01

    The immunoglobulin heavy chain intron enhancer (Emu) not only stimulates transcription but also V(D)J recombination of chromosomally integrated recombination substrates. We aimed at reproducing this effect in recombination competent cells by transient transfection of extrachromosomal substrates. These we prepared by interposing between the recombination signal sequences (RSS) of the plasmid pBlueRec various fragments, including Emu, possibly affecting V(D)J recombination. Our work shows that ...

  1. Recombination Every Day: Abundant Recombination in a Virus during a Single Multi-Cellular Host Infection

    Directory of Open Access Journals (Sweden)

    Froissart Remy

    2005-01-01

    Full Text Available Viral recombination can dramatically impact evolution and epidemiology. In viruses, the recombination rate depends on the frequency of genetic exchange between different viral genomes within an infected host cell and on the frequency at which such co-infections occur. While the recombination rate has been recently evaluated in experimentally co-infected cell cultures for several viruses, direct quantification at the most biologically significant level, that of a host infection, is still lacking. This study fills this gap using the cauliflower mosaic virus as a model. We distributed four neutral markers along the viral genome, and co-inoculated host plants with marker-containing and wild-type viruses. The frequency of recombinant genomes was evaluated 21 d post-inoculation. On average, over 50% of viral genomes recovered after a single host infection were recombinants, clearly indicating that recombination is very frequent in this virus. Estimates of the recombination rate show that all regions of the genome are equally affected by this process. Assuming that ten viral replication cycles occurred during our experiment-based on data on the timing of coat protein detection-the per base and replication cycle recombination rate was on the order of 2 x 10-5 to 4 x 10-5. This first determination of a virus recombination rate during a single multi-cellular host infection indicates that recombination is very frequent in the everyday life of this virus.

  2. Micro dynamics in mediation

    OpenAIRE

    Boserup, Hans

    2014-01-01

    The author has identified a number of styles in mediation, which lead to different processes and different outcomes. Through discourse and conversation analysis he examines the micro dynamics in three of these, the postmodern styles: systemic, transformative and narrative mediation. The differences between the three mediation ideologies and practice is illustrated through role play scripts enacted in each style. Mediator and providers of mediation and trainers in mediation are encouraged to a...

  3. Recombinant vaccine for canine parvovirus in dogs.

    OpenAIRE

    López de Turiso, J A; Cortés, E; Martínez, C.; Ruiz de Ybáñez, R; Simarro, I; Vela, C.; Casal, I

    1992-01-01

    VP2 is the major component of canine parvovirus (CPV) capsids. The VP2-coding gene was engineered to be expressed by a recombinant baculovirus under the control of the polyhedrin promoter. A transfer vector that contains the lacZ gene under the control of the p10 promoter was used in order to facilitate the selection of recombinants. The expressed VP2 was found to be structurally and immunologically indistinguishable from authentic VP2. The recombinant VP2 shows also the capability to self-as...

  4. Recombination in narrow-gapped semiconductors

    International Nuclear Information System (INIS)

    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

  5. Influence of exogenous phytohormones, methyl jasmonate and suppressors of jasmonate biosynthesis on Agrobacterium-mediated transient expression in Nicotiana excelsior

    OpenAIRE

    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...

  6. A MEDIATOR METHYLATION MYSTERY: JMJD1C DEMETHYLATES MDC1 TO REGULATE DNA DAMAGE REPAIR

    OpenAIRE

    Jian LU; Matunis, Michael J.

    2013-01-01

    Mediator of DNA-Damage Checkpoint 1 (MDC1) has a central role in repair of DNA double-strand breaks (DSBs) by both homologous recombination and nonhomologous end joining, and its function is regulated by post-translational phosphorylation, ubiquitylation, and SUMOylation. In this issue, a new study by Watanabe et al. reveals that methylation of MDC1 is also critical for its function in DSB repair and specifically affects repair through BRCA1-dependent homologous recombination.

  7. Dielectronic recombination of boronlike argon

    International Nuclear Information System (INIS)

    We present measurements and calculations of ?n=0 dielectronic recombination resonances of boronlike argon between 0.2 and 6 eV. A storage ring equipped with an electron cooler was used for the measurements. Methods employed to reduce the electron energy distribution and improve the accuracy of resonance energy measurements have yielded an energy resolution of 30 meV full width at half maximum at low energies, and an energy uncertainty better than 30 meV. The high energy resolution results from the use of an adiabatic expansion technique to reduce the transverse electron energy distribution. The improved accuracy in energy determinations is achieved through the inclusion of variations in the ion velocity, which occur during scans of the electron velocity, in the relative velocity transformations. Calculations of the resonance strengths and energies were made using two different methods, multiconfiguration Dirac-Fock and multiconfiguration Breit-Pauli approximations. A comparison of the experimental data to the calculations shows fair agreement in both the spectral features and integrated intensities above 3 eV. However, poor agreement is found below 3 eV. copyright 1996 The American Physical Society

  8. Recombinant vaccines: experimental and applied aspects

    DEFF Research Database (Denmark)

    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.

  9. DNA Helicase Activity of PcrA Is Not Required for the Displacement of RecA Protein from DNA or Inhibition of RecA-Mediated Strand Exchange?

    OpenAIRE

    Anand, Syam P.; Zheng, Haocheng; Bianco, Piero R.; Leuba, Sanford H; Khan, Saleem A

    2007-01-01

    PcrA is a conserved DNA helicase present in all gram-positive bacteria. Bacteria lacking PcrA show high levels of recombination. Lethality induced by PcrA depletion can be overcome by suppressor mutations in the recombination genes recFOR. RecFOR proteins load RecA onto single-stranded DNA during recombination. Here we test whether an essential function of PcrA is to interfere with RecA-mediated DNA recombination in vitro. We demonstrate that PcrA can inhibit the RecA-mediated DNA strand exch...

  10. Dielectronic recombination of hydrogen-like ions

    International Nuclear Information System (INIS)

    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

  11. Competition between ion recombination and scavenging

    International Nuclear Information System (INIS)

    Complete text of publication follows. In low permittivity solvents ion recombination is dominated by the effects of the relative drift of the ions caused by the Coulomb attraction. However, such systems are frequently investigated by scavenging methods. Since the work of Tachiya on electric field effects, drift has been known to affect the steady-state scavenging rate constant. However, during the recombination drift depends on the instantaneous distance between the ions, and is therefore inherently transient. This paper describes an investigation of this problem using simulation methods. It is found that, within the constraint of the diffusion approximation, there are conditions where the Smoluchowski time-dependent rate constant underestimates the degree to which scavenging intercepts geminate recombination. For this to be a substantial effect the initial distance between the ions must be relatively small (e.g. 4 nm) compared to the typical thermalisation distance of an electron (e.g. 8 nm). Simulations have been used to generate numerical time-dependent rate constants for scavenging. But these proved barely more successful than the Smoluchowski theory, in spite of having been calculated from the simulation results. Stratification of results by recombination time shows that there is a strong correlation between the recombination time and the scavenging time. It was hypothesised that this correlation arises through the strong transient drift as the ions approach one another. This hypothesis was confirmed by the application of a novel simulation method in which the ion trajectories are simulated conditional on the recombination time. It was found that in every case the scavenging rate increases sharply just prior to recombination. This dependence of scavenging rate on recombination time is a fundamental breakdown of the assumptions underlying both the theory of diffusion kinetics and the IRT method. Nonetheless, a path decomposition method has been devised that allows IRT simulations to be corrected for this effect with good accuracy.

  12. Mechanisms of nonhomologous recombination in mammalian cells.

    OpenAIRE

    Roth, D. B.; Porter, T N; Wilson, J H

    1985-01-01

    The primary mechanism of nonhomologous recombination in transfected DNA involves breakage followed by end joining. To probe the joining step in more detail, linear simian virus 40 genomes with mismatched ends were transfected into cultured monkey cells, and individual viable recombinants were analyzed. The transfected genomes carried mismatched ends as a result of cleavage with two restriction enzymes, the recognition sites of which are located in the intron of the gene encoding the T antigen...

  13. Breaking the sound barrier in recombination fronts

    OpenAIRE

    Williams, R. J. R.; J. E. Dyson

    1995-01-01

    We exploit a generic instability in the integration of steady, sonic, near-isothermal flows to find the complete transition diagram for recombination fronts (for a model system of equations). The instability requires the integration of the flow equations for speeds between the isothermal and adiabatic sound speeds to be performed with particular care. As a result of this, the previous work of Newman & Axford on the structure of recombination fronts neglected an important cla...

  14. Recombination energy in double white dwarf formation

    OpenAIRE

    Nandez, Jose L. A.; Ivanova, Natalia; Lombardi Jr., James C.

    2015-01-01

    In this Letter we investigate the role of recombination energy during a common envelope event. We confirm that taking this energy into account helps to avoid the formation of the circumbinary envelope commonly found in previous studies. For the first time, we can model a complete common envelope event, with a clean compact double white dwarf binary system formed at the end. The resulting binary orbit is almost perfectly circular. In addition to considering recombination ener...

  15. Recombinant DNA production of spider silk proteins

    OpenAIRE

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L.

    2013-01-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce...

  16. Co-factor activated recombinant adenovirus proteinases

    Science.gov (United States)

    Anderson, Carl W. (Stony Brook, NY); Mangel, Walter F. (Shoreham, NY)

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  17. Persistence and Loss of Meiotic Recombination Hotspots

    OpenAIRE

    Pineda-Krch, Mario; Redfield, Rosemary J

    2005-01-01

    The contradiction between the long-term persistence of the chromosomal hotspots that initiate meiotic recombination and the self-destructive mechanism by which they act strongly suggests that our understanding of recombination is incomplete. This “hotspot paradox” has been reinforced by the finding that biased gene conversion also removes active hotspots from human sperm. To investigate the requirements for hotspot persistence, we developed a detailed computer simulation model of their activi...

  18. Determination of recombination in Mycoplasma hominis

    DEFF Research Database (Denmark)

    Jacobsen, Iben SØgaard; Boesen, Thomas

    2002-01-01

    Mycoplasma hominis has been previously described as a heterogeneous species, and in the present study intraspecies diversity of 20 M. hominis isolates from different individuals was analyzed using parts of the unlinked gyrase B (gyrB), elongation factor Tu (tuf), SRalpha homolog (ftsY), hitB-hitL, excinuclease ABC subunit A (uvrA) and glyceraldehyde-3-phosphate dehydrogenase (gap) genes. The level of variability of these M. hominis genes was low compared with the housekeeping genes from Helicobacter pylori and Neisseria meningitidis, but only few M. hominis isolates had identical sequences in all genes indicating the presence of recombination. In order to test for intergenic recombination, phylogenetic trees were reconstructed for each of the genes but no well-supported bifurcating phylogenetic trees could be obtained. The genes were tested for intragenic recombination using the correlation between linkage disequilibrium and distance between the segregating sites, by the homoplasy ratio (H ratio), and by compatibility matrices. The gap gene showed well-supported evidence for high levels of recombination, whereas recombination was less frequent and not significant within the other genes. The analysis revealed intergenic and intragenic recombination in M. hominis and this may explain the high intraspecies variability. The results obtained in the present study may be of importance for future population studies of Mycoplasma species.

  19. Transcription-coupled eviction of histones H2A/H2B governs V(D)J recombination.

    Science.gov (United States)

    Bevington, Sarah; Boyes, Joan

    2013-05-15

    Initiation of V(D)J recombination critically relies on the formation of an accessible chromatin structure at recombination signal sequences (RSSs) but how this accessibility is generated is poorly understood. Immunoglobulin light-chain loci normally undergo recombination in pre-B cells. We show here that equipping (earlier) pro-B cells with the increased pre-B-cell levels of just one transcription factor, IRF4, triggers the entire cascade of events leading to premature light-chain recombination. We then used this finding to dissect the critical events that generate RSS accessibility and show that the chromatin modifications previously associated with recombination are insufficient. Instead, we establish that non-coding transcription triggers IgL RSS accessibility and find that the accessibility is transient. Transcription transiently evicts H2A/H2B dimers, releasing 35-40 bp of nucleosomal DNA, and we demonstrate that H2A/H2B loss can explain the RSS accessibility observed in vivo. We therefore propose that the transcription-mediated eviction of H2A/H2B dimers is an important mechanism that makes RSSs accessible for the initiation of recombination. PMID:23463099

  20. Electron - ion recombination processes - an overview

    International Nuclear Information System (INIS)

    Extensive theoretical and experimental studies have been carried out for the past 20 years on electron - ion recombination processes, as they are applied to the analysis of astrophysical and laboratory plasmas. We review the basic understanding gained through these efforts, with emphasis on some of the more recent progress made in recombination theory as the recombining system is affected by time-dependent electric fields and plasma particles at low temperature. Together with collisional ionization and excitation processes, recombination is important in determining ionization balance and excited-state population in non-equilibrium plasmas. The radiation emitted by plasmas is usually the principal medium with which to study the plasma condition, as it is produced mainly during the recombination and decay of excited states of ions inside the plasma. This is especially true when the plasma under study is not readily accessible by direct probes, as in astrophysical plasmas. Moreover, external probes may sometimes cause undesirable disturbances of the plasma. Electron-ion recombination proceeds in several different modes. The direct modes include three-body recombination (TBR) and one-step radiative recombination (RR), all to the ground- and singly-excited states of the target ions. By contrast, the indirect resonant mode is a two-step dielectronic recombination (DR), which proceeds first with the formation of doubly-excited states by radiationless excitation/capture. The resonant states thus formed may relax by autoionization and/or radiative cascades. For more exotic modes of recombination, we consider off-shell dielectronic recombination (radiative DR = RDR), in which an electron capture is accompanied by simultaneous radiative emission and excitation of the target ion. Some discussion on attachment of electrons to neutral atoms, resulting in the formation of negative ions, is also given. When resonance states involve one or more electrons in high Rydberg states, presence of an external or intrinsic electric field in the vicinity of the target ions can seriously affect the ionic states involved and the resulting reaction rates. Such perturbative fields can be intrinsic, as in the case of the plasma ion field, or externally imposed. A proper theoretical treatment of this difficult problem is crucial in understanding the recombination process which takes place in a field contaminated environment. The simple off-shell dressing procedure of high Rydberg states by a time-dependent field is reviewed, and the possibility of an anomalously large enhancement in the rates, due to the momentum coherence effect (MCE), is discussed. The presently available data on recombination rates are summarized, and several important deficiencies and future directions for further research are pointed out. Based on the detailed calculations for a number of cases, several empirical rate formulae for RR and DR processes have been generated to summarize the data for ready applications. As the collection of atoms is cooled to very low temperatures, T 8 Ryd, and the bound electrons are ionized by laser irradiation to states of very precisely controlled energies, the prospect for accurate experimental measurements of very-low-energy recombination rates is considered, where the electron temperature can be very low. Therefore, it is of interest to reconsider theoretically some new phenomena which may occur at such cold environments, in which the electron de Broglie wavelength can be very large, and both the density and coherent effects, as well as possible field effects, must be properly taken into account. Finally, a broader understanding of the various recombination processes may be achieved by studying their relationships to other reactions initiated by electron, ion and photon impact. (author)

  1. Dual regulation of Dmc1-driven DNA strand exchange by Swi5–Sfr1 activation and Rad22 inhibition

    OpenAIRE

    Murayama, Yasuto; Kurokawa, Yumiko; Tsutsui, Yasuhiro; IWASAKI, HIROSHI

    2013-01-01

    Meiotic recombination requires two key recombinases: the ubiquitously expressed Rad51 and the meiosis-specific Dmc1. Rad52 and its fission yeast ortholog, Rad22, are mediators that help load Rad51 onto ssDNA coated with replication protein A (RPA). Here, Iwasaki and colleagues reveal how the Swi5–Sfr1 complex functions as both a mediator (loading DMC1 onto ssDNA) and an activator (stimulating Dmc1-driven strand exchange). In contrast, Rad22 inhibits Dmc1 by competing for binding to RPA-coated...

  2. Role of attP in Integrase-Mediated Integration of the Shigella Resistance Locus Pathogenicity Island of Shigella flexneri

    OpenAIRE

    Turner, Sally A.; Luck, Shelley N.; Sakellaris, Harry; Rajakumar, Kumar; Adler, Ben

    2004-01-01

    The Shigella resistance locus (SRL) pathogenicity island (PAI) in Shigella spp. mediates resistance to streptomycin, ampicillin, chloramphenicol, and tetracycline. It can be excised from the chromosome via site-specific recombination mediated by the P4-related int gene. Here, we show that SRL PAI attP is capable of RecA-independent, site-specific, int-mediated integration into two bacterial tRNA attB sites.

  3. Annexin-Mediated Calcium Signalling in Plants

    Directory of Open Access Journals (Sweden)

    Julia M. Davies

    2014-02-01

    Full Text Available 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.

  4. Distortion of the CMB Spectrum by Primeval Hydrogen Recombination

    OpenAIRE

    Boschan, Peter; Biltzinger, Peter

    1996-01-01

    We solve the recombination equation by taking into account the induced recombinations and a physical cut off in the hydrogen spectrum. The effective recombination coefficient is parametrized as a function of temperature and free electron density and is about a factor of four larger than without the induced recombination. This accelerates the last stage of the recombination processes and diminishes the residual ionization by a factor of about 2.6. The number and energy distri...

  5. Performance testing of passive autocatalytic recombiners (PARs)

    Energy Technology Data Exchange (ETDEWEB)

    Blanchat, T. [Sandia National Laboratories, Albuquerque, NM (United States); Malliakos, A. [U.S. Nuclear Regulatory Commission, Washington, DC (United States)

    1997-03-01

    Passive autocatalytic recombiners (PARs) have been under consideration in the U.S. as a combustible gas control system in advanced light water reactor (ALWR) containments for design basis and severe accidents. PARs do not require a source of power. Instead they use palladium or platinum as a catalyst to recombine hydrogen and oxygen gases into water vapor upon contact with the catalyst. Energy from the recombination of hydrogen with oxygen is released at a relatively slow but continuous rate into the containment which prevents the pressure from becoming too high. The heat produced creates strong buoyancy effects which increases the influx of the surrounding gases to the recombiner. These natural convective flow currents promote mixing of combustible gases in the containment. PARs are self-starting and self-feeding under a very wide range of conditions. The recombination rate of the PAR system needs to be great enough to keep the concentration of hydrogen (or oxygen) below acceptable limits. There are several catalytic recombiner concepts under development worldwide. The USNRC is evaluating a specific design of a PAR which is in an advanced stage of engineering development and has been proposed for ALWR designs. Sandia National laboratories (SNL), under the sponsorship and the direction of the USNRC, is conducting an experimental program to evaluate the performance of PARs. The PAR will be tested at the SURTSEY facility at SNL. The test plan currently includes the following experiments: experiments will be conducted to define the startup characteristics of PARs (i.e., to define what is the lowest hydrogen concentration that the PAR starts recombining the hydrogen with oxygen); experiments will be used to define the hydrogen depletion rate of PARs as a function of hydrogen concentration; and experiments will be used to define the PAR performance in the presence of high concentrations of steam. (author)

  6. Performance testing of passive autocatalytic recombiners (PARs)

    International Nuclear Information System (INIS)

    Passive autocatalytic recombiners (PARs) have been under consideration in the U.S. as a combustible gas control system in advanced light water reactor (ALWR) containments for design basis and severe accidents. PARs do not require a source of power. Instead they use palladium or platinum as a catalyst to recombine hydrogen and oxygen gases into water vapor upon contact with the catalyst. Energy from the recombination of hydrogen with oxygen is released at a relatively slow but continuous rate into the containment which prevents the pressure from becoming too high. The heat produced creates strong buoyancy effects which increases the influx of the surrounding gases to the recombiner. These natural convective flow currents promote mixing of combustible gases in the containment. PARs are self-starting and self-feeding under a very wide range of conditions. The recombination rate of the PAR system needs to be great enough to keep the concentration of hydrogen (or oxygen) below acceptable limits. There are several catalytic recombiner concepts under development worldwide. The USNRC is evaluating a specific design of a PAR which is in an advanced stage of engineering development and has been proposed for ALWR designs. Sandia National laboratories (SNL), under the sponsorship and the direction of the USNRC, is conducting an experimental program to evaluate the performance of PARs. The PAR will be tested at the SURTSEY facility at SNL. The test plan currently includes the following experiments: experiments will be conducted to define the startup characteristics of PARs (i.e., to define what is the lowest hydrogen concentration that the PAR starts recombining the hydrogen with oxygen); experiments will be used to define the hydrogen depletion rate of PARs as a function of hydrogen concentration; and experiments will be used to define the PAR performance in the presence of high concentrations of steam. (author)

  7. Three-dimensional structure of recombinant type 1 inositol 1,4,5-trisphosphate receptor

    Science.gov (United States)

    Wolfram, Francis; Morris, Edward; Taylor, Colin W.

    2010-01-01

    IP3Rs (inositol 1,4,5-trisphosphate receptors) are the intracellular channels that mediate release of Ca2+ from the endoplasmic reticulum in response to the many stimuli that evoke Ins(1,4,5)P3 formation. We characterized and purified type 1 IP3R heterologously expressed in Sf9 insect cells, and used the purified IP3R1 to determine its three-dimensional structure by electron microscopy and single-particle analysis. Recombinant IP3R1 has 4-fold symmetry with overall dimensions of approx. 19.5 nm×19.5 nm×17.5 nm. It comprises a small domain, which is likely to include the pore, linked by slender bridges to a large cytoplasmic domain with four petal-like regions. Our structures of recombinant IP3R1 and native cerebellar IP3R have similar appearances and dimensions. The only notable difference is the absence of a central stigma-like domain from the cytoplasmic region of recombinant IP3R1. The first structure of a recombinant IP3R is an important step towards developing three-dimensional structures of IP3R that better contribute to our understanding of the structural basis of IP3R activation. PMID:20377523

  8. Recombination-activating gene 1 and 2 (RAG1 and RAG2) in flounder (Paralichthys olivaceus)

    Indian Academy of Sciences (India)

    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.

  9. Immune responses of pigs immunized with a recombinant porcine reproductive and respiratory syndrome virus expressing porcine GM-CSF.

    Science.gov (United States)

    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

  10. Monitoring and Evaluation of Smolt Migration in the Columbia Basin : Volume VII : Evaluation of the Compliance Testing Framework for RPA Improvement as Stated in the 2000 Federal Columbia River Power System (FCRPS) Biological Opinion.

    Energy Technology Data Exchange (ETDEWEB)

    Skalski, John R.; Ngouenet, Roger F.

    2001-05-01

    Using the pre-2000 reach survival probabilities reported in the 2000 FCRPS Biological Opinion (BO) for three selected stocks: yearling and sub-yearling chinook and steelhead, power curves were constructed for each of the two statistical hypothesis tests suggested in the BO. These power calculation results were interpreted in terms of the ability of the statistical tests to correctly identify the true states of recovery (i.e., fail or succeed in fulfilling RPA expectations). The proposed one-sided tests have a moderate to low probability of correctly assessing the true status of the recovery by the years 2005 and 2008. The relatively poor odds of making the correct decision with the BO proposed Tests 1 and 2 suggest alternative decision rules need to be investigated and developed for assessing RPA compliance. Therefore, we propose to immediately examine alternative decision rules that might maximize the likelihood of correct decisions while minimizing the prospect of incorrect decisions. The Bayesian analysis will incorporate scientific/biological knowledge/expertise.

  11. H-Ras regulation of TRAIL death receptor mediated apoptosis

    OpenAIRE

    Chen, Jun-Jie; Bozza, William P.; Di, Xu; Zhang, Yaqin; Hallett, William; Zhang, Baolin

    2014-01-01

    TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through the death receptors (DRs) 4 and/or 5 expressed on the cell surface. Multiple clinical trials are underway to evaluate the antitumor activity of recombinant human TRAIL and agonistic antibodies to DR4 or DR5. However, their therapeutic potential is limited by the high frequency of cancer resistance. Here we provide evidence demonstrating the role of H-Ras in TRAIL receptor mediated apoptosis. By analyzing the genome wide m...

  12. Microarrayed recombinant allergens for diagnosis of allergy.

    Science.gov (United States)

    Harwanegg, C; Laffer, S; Hiller, R; Mueller, M W; Kraft, D; Spitzauer, S; Valenta, R

    2003-01-01

    We suggest that the coapplication of recombinant allergens and microarray technology can lead to the development of new forms of multi-allergen tests which allow the determining and monitoring of complex sensitization profiles of allergic patients in single assays. The allergen extracts which have so far been used for diagnosis only allowed the determining of whether an allergic patient is sensitized against a particular allergen source, but the disease-eliciting allergens could not be identified. Through the application of recombinant DNA technology a rapidly growing panel of recombinant allergen molecules has become available which meanwhile comprises the epitope spectrum of most of the important allergen sources. We demonstrate that microarray technology can be used to establish multi-allergen tests consisting of microarrayed recombinant allergen molecules. Microarrayed recombinant allergens can be used to determine and monitor the profile of disease-eliciting allergens using single tests that require minute amounts of serum from allergic patients. The wealth of diagnostic information gained through microarray-based allergy testing will likely improve diagnosis, prevention and treatment of allergy. PMID:12534543

  13. Recombination in Eukaryotic Single Stranded DNA Viruses

    Directory of Open Access Journals (Sweden)

    Philippe Roumagnac

    2011-09-01

    Full Text Available Although single stranded (ss DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution.

  14. Human recombinant lysosomal enzymes produced in microorganisms.

    Science.gov (United States)

    Espejo-Mojica, Ángela J; Alméciga-Díaz, Carlos J; Rodríguez, Alexander; Mosquera, Ángela; Díaz, Dennis; Beltrán, Laura; Díaz, Sergio; Pimentel, Natalia; Moreno, Jefferson; Sánchez, Jhonnathan; Sánchez, Oscar F; Córdoba, Henry; Poutou-Piñales, Raúl 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

  15. Polyploidization increases meiotic recombination frequency in Arabidopsis

    Directory of Open Access Journals (Sweden)

    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.

  16. Towards universal systems for recombinant gene expression

    Directory of Open Access Journals (Sweden)

    Sørensen Hans

    2010-04-01

    Full Text Available Abstract Recombinant gene expression is among the most important techniques used both in molecular and medical research and in industrial settings. Today, two recombinant expression systems are particularly well represented in the literature reporting on recombinant expression of specific genes. According to searches in the PubMed citation database, during the last 15 years 80% of all recombinant genes reported on in the literature were expressed in either the enterobacterium Escherichia coli or the methylotropic yeast Pichia pastoris. Nevertheless, some eukaryotic proteins are misfolded or inadequately posttranslationally modified in these expression systems. This situation demands identification of other recombinant expression systems that enable the proper expression of the remaining eukaryotic genes. As of now, a single universal system allowing expression of all target genes is still a distant goal. In this light, thorough experimental screening for systems that can yield satisfying quantity and quality of target protein is required. In recent years, a number of new expression systems have been described and used for protein production. Two systems, namely Drosophila melanogaster S2 insect cells and human embryonic kidney 293 (HEK293 cells stably expressing the EBNA-1 gene, show exceptional promise. The time has come to identify a few well-performing systems that will allow us to express, purify, and characterize entire eukaryotic genomes.

  17. Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

    Directory of Open Access Journals (Sweden)

    Penn Charles W

    2009-12-01

    Full Text Available Abstract Background Homologous recombination mediated by the ?-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 ?-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 ?-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 ?-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 ?-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.

  18. Triplex-mediated genome targeting and editing.

    Science.gov (United States)

    Reza, Faisal; Glazer, Peter M

    2014-01-01

    Genome targeting and editing in vitro and in vivo can be achieved through an interplay of exogenously introduced molecules and the induction of endogenous recombination machinery. The former includes a repertoire of sequence-specific binding molecules for targeted induction and appropriation of this machinery, such as by triplex-forming oligonucleotides (TFOs) or triplex-forming peptide nucleic acids (PNAs) and recombinagenic donor DNA, respectively. This versatile targeting and editing via recombination approach facilitates high-fidelity and low-off-target genome mutagenesis, repair, expression, and regulation. Herein, we describe the current state-of-the-art in triplex-mediated genome targeting and editing with a perspective towards potential translational and therapeutic applications. We detail several materials and methods for the design, delivery, and use of triplex-forming and recombinagenic molecules for mediating and introducing specific, heritable, and safe genomic modifications. Furthermore we denote some guidelines for endogenous genome targeting and editing site identification and techniques to test targeting and editing efficiency. PMID:24557900

  19. Limiting efficiency calculation of silicon single-nanowire solar cells with considering Auger recombination

    International Nuclear Information System (INIS)

    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

  20. Limiting efficiency calculation of silicon single-nanowire solar cells with considering Auger recombination

    Science.gov (United States)

    Zhai, Xiongfei; Wu, Shaolong; Shang, Aixue; Li, Xiaofeng

    2015-02-01

    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.

  1. Comparison of recombination models in organic bulk heterojunction solar cells

    International Nuclear Information System (INIS)

    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

  2. LIMITED EFFECT OF RECOMBINANT PORCINE INTERLEUKIN 12 ON PORCINE LYMPHOCYTES DUE TO A LOW LEVEL OF IL-12 BETA 2 RECEPTOR

    Science.gov (United States)

    The cytokine interleukin-12 (IL-12) is a key molecule in the regulation of CD4+ T cell development and specifically potentiates T helper 1 responses in mouse and man. However, biological effects mediated by IL-12 have not been well defined in pigs. Herein, recombinant porcine IL-12 (rPoIL-12) was ...

  3. Recombinant human erythropoietin in sports: a review

    Directory of Open Access Journals (Sweden)

    Bento Rafael Maia de Almeida

    2003-01-01

    Full Text Available Erythropoietin is an endogenous hormone of glicoproteic nature secreted by the kidneys and is the main regulator of the erythropoiesis. An alteration in its production generates a disturbance in the plasmatic concentration giving rise to several types of pathologies related to the hematopoietic system. The recombinant forms of erythropoietin have indiscriminately been used by athletes, mainly in endurance sports, by increasing the erythrocytes concentration, generating a better delivery of oxygen to the muscle tissue. The administration of recombinant erythropoietin was prohibited by the International Olympic Committee and its use considered as doping. This review has the intention to describe the physical, biological and pharmacokinetic properties of the endogenous erythropoietin, as well as its recombinant form, describing also its use in sports and the process of searching methodologies for its detection in doping control.

  4. Jet Fragmentation via Recombination of Parton Showers

    CERN Document Server

    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.

  5. Directed Evolution of RecA Variants with Enhanced Capacity for Conjugational Recombination

    Science.gov (United States)

    Kim, Taejin; Chitteni-Pattu, Sindhu; Cox, Benjamin L.; Wood, Elizabeth A.; Sandler, Steven J.; Cox, Michael M.

    2015-01-01

    The recombination activity of Escherichia coli (E. coli) RecA protein reflects an evolutionary balance between the positive and potentially deleterious effects of recombination. We have perturbed that balance, generating RecA variants exhibiting improved recombination functionality via random mutagenesis followed by directed evolution for enhanced function in conjugation. A recA gene segment encoding a 59 residue segment of the protein (Val79-Ala137), encompassing an extensive subunit-subunit interface region, was subjected to degenerate oligonucleotide-mediated mutagenesis. An iterative selection process generated at least 18 recA gene variants capable of producing a higher yield of transconjugants. Three of the variant proteins, RecA I102L, RecA V79L and RecA E86G/C90G were characterized based on their prominence. Relative to wild type RecA, the selected RecA variants exhibited faster rates of ATP hydrolysis, more rapid displacement of SSB, decreased inhibition by the RecX regulator protein, and in general displayed a greater persistence on DNA. The enhancement in conjugational function comes at the price of a measurable RecA-mediated cellular growth deficiency. Persistent DNA binding represents a barrier to other processes of DNA metabolism in vivo. The growth deficiency is alleviated by expression of the functionally robust RecX protein from Neisseria gonorrhoeae. RecA filaments can be a barrier to processes like replication and transcription. RecA regulation by RecX protein is important in maintaining an optimal balance between recombination and other aspects of DNA metabolism. PMID:26047498

  6. Regulation of Homologous Recombination by SUMOylation : SUMO, DNA Repair and New Insights into Telomere Maintenance

    DEFF Research Database (Denmark)

    Pinela da Silva, Sonia Cristina

    2014-01-01

    Double-strand breaks (DSBs) are one of the most deleterious types of DNA lesions challenging genome integrity. The DNA damage response (DDR) promotes fast and effective detection and repair of the damaged DNA, leading to cell cycle arrest through checkpoint activation and the recruitment of repair factors such as the homologous recombination (HR) machinery. HR constitutes the main DSB repair pathway in Saccharomyces cerevisiae and despite being largely considered an error-free process and essential for genome stability, uncontrolled recombination can lead to loss of heterozygosity, translocations, 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. Furthermore, I characterize Mte1, a novel protein involved in DDR that associates with the helicase Mph1 and Rad52. Moreover, I find that Mte1 associates with dysfunctional single-stranded telomeric DNA, constituting a novel factor in telomere homeostasis, potentially associated with replication-stress relief.

  7. Recombinant microorganisms for increased production of organic acids

    Science.gov (United States)

    Yi, Jian; Kleff, Susanne; Guettler, Michael V

    2013-04-30

    Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

  8. MicroRNAs down-regulate homologous recombination in the G1 phase of cycling cells to maintain genomic stability

    OpenAIRE

    Choi, Young Eun; Pan, Yunfeng; Park, Eunmi; Konstantinopoulos, Panagiotis; De, Subhajyoti; D'Andrea, Alan; Chowdhury, Dipanjan

    2014-01-01

    Homologous recombination (HR)-mediated repair of DNA double-strand break (DSB)s is restricted to the post-replicative phases of the cell cycle. Initiation of HR in the G1 phase blocks non-homologous end joining (NHEJ) impairing DSB repair. Completion of HR in G1 cells can lead to the loss-of-heterozygosity (LOH), which is potentially carcinogenic. We conducted a gain-of-function screen to identify miRNAs that regulate HR-mediated DSB repair, and of these miRNAs, miR-1255b, miR-148b*, and miR-...

  9. Breaking the sound barrier in recombination fronts

    CERN Document Server

    Williams, R J R

    1995-01-01

    We exploit a generic instability in the integration of steady, sonic near-isothermal flows to find the complete transition diagram for recombination fronts (for a model system of equations). The instability requires the integration of the flow equations for speeds between the isothermal and adiabatic sound speeds to be performed with particular care. As a result of this, the previous work of Newman & Axford on the structure of recombination fronts neglected an important class of solution, that of transonic fronts; our method is readily extensible to a more complete treatment of the ionization structure. Future papers will apply these results in models of the structure of ultracompact HII regions.

  10. Role of Cellular FKBP52 Protein in Intracellular Trafficking of Recombinant Adeno-associated Virus 2 Vectors

    OpenAIRE

    Zhao, Weihong; Zhong, Li; Wu, Jianqing; Chen, Linyuan; Qing, Keyun; Weigel-Kelley, Kirsten A.; Larsen, Steven H.; Shou, Weinian; Warrington, Kenneth H.; Srivastava, Arun

    2006-01-01

    We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and...

  11. Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors.

    OpenAIRE

    Ferrari, F K; Samulski, T; Shenk, T; Samulski, R. J.

    1996-01-01

    The ability of recombinant adeno-associated virus (AAV) to transduce cells with a marker gene in vitro was found to be substantially increased by the presence of adenovirus. Transfection experiments with adenovirus genomic DNA suggest that this increase is not facilitated by adenovirus-mediated viral uptake but is instead dependent on adenovirus gene expression. Using various adenovirus mutants, we were able to map this function to early-region E4 open reading frame 6. Plasmid expression of o...

  12. Technology-Use Mediation

    DEFF Research Database (Denmark)

    Bansler, Jørgen P.; Havn, Erling C.

    2003-01-01

    This study analyzes how a group of ‘mediators’ in a large, multinational company adapted a computer-mediated communication technology (a ‘virtual workspace’) to the organizational context (and vice versa) by modifying features of the technology, providing ongoing support for users, and promoting appropriate conventions of use. Our findings corroborate earlier research on technology-use mediation, which suggests that such mediators can exert considerable influence on how a particular technology w...

  13. Illegitimate V(D)J recombination involving nonantigen receptor loci in lymphoid malignancy.

    Science.gov (United States)

    Onozawa, Masahiro; Aplan, Peter D

    2012-06-01

    V(D)J recombination of antigen receptor loci (IGH, IGK, IGL, TCRA, TCRB, TCRG, and TCRD) is an essential mechanism that confers enormous diversity to the mammalian immune system. However, there are now at least six examples of intrachromosomal interstitial deletions caused by aberrant V(D)J recombination between nonantigen receptor loci; five of out these six are associated with lymphoid malignancy. The SIL-SCL fusion and deletions of CDKN2A, IKZF1, Notch1, and Bcl11b are all associated with lymphoid malignancy. These interstitial deletions seem to be species specific, as the deletions seen in mice are not seen in humans; the converse is true as well. Nucleotide sequence analysis of these rearrangements reveals the hallmarks of V(D)J recombination, including site specificity near cryptic heptamer signal sequences, exonucleolytic "nibbling" at the junction site, and nontemplated "N"-region nucleotide insertion at the junction site. Two of these interstitial deletions (murine Notch1 and Bcl11b deletions) have been detected, at low frequency, in tissues from healthy mice with no evidence of malignancy, similar to the finding of chromosomal translocations in the peripheral blood or tonsils of healthy individuals. The contention that these are mediated via V(D)J recombination is strengthened by in vivo assays using extrachromosomal substrates, and chromatin immunoprecipitation-sequence analysis which shows Rag2 binding at the sites of rearrangement. Although the efficiency of these "illegitimate" recombination events is several orders of magnitude less than that at bona fide antigen receptor loci, the consequence of such deletions, namely activation of proto-oncogenes or deletion of tumor suppressor genes, is devastating, and a major cause for lymphoid malignancy. PMID:22334400

  14. Ecological genomics in Xanthomonas: the nature of genetic adaptation with homologous recombination and host shifts

    KAUST Repository

    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.

  15. Bayesian Mediation Analysis

    Science.gov (United States)

    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…

  16. Stromelysin 1, neutrophil collagenase, and collagenase 3 do not play major roles in a model of chondrocyte mediated cartilage breakdown.

    OpenAIRE

    Kozaci, L D; Brown, C. J.; Adcocks, C; Galloway, A.; Hollander, A.P.; Buttle, D J

    1998-01-01

    AIMS: To determine the collective roles of stromelysin 1, neutrophil collagenase, and collagenase 3 in chondrocyte mediated cartilage proteoglycan and type II collagen degradation in tissue culture model systems. METHODS: Bovine nasal cartilage explants were cultured with and without recombinant human interleukin 1 alpha (IL-1 alpha), recombinant human tumour necrosis factor alpha, or retinoic acid. Proteoglycan and type II collagen release were determined by colorimetric assay and immunoassa...

  17. Identification of the MMS22L-TONSL complex that promotes homologous recombination.

    OpenAIRE

    Duro, E; Lundin, C.; Ask, K; Sanchez-Pulido, L.; Macartney, TJ; Toth, R; Ponting, CP; Groth, A; Helleday, T; Rouse, J

    2010-01-01

    Budding yeast Mms22 is required for homologous recombination (HR)-mediated repair of stalled or broken DNA replication forks. Here we identify a human Mms22-like protein (MMS22L) and an MMS22L-interacting protein, NF?BIL2/TONSL. Depletion of MMS22L or TONSL from human cells causes a high level of double-strand breaks (DSBs) during DNA replication. Both proteins accumulate at stressed replication forks, and depletion of MMS22L or TONSL from cells causes hypersensitivity to agents that cause S ...

  18. Recombinant human VEGF165b protein is an effective anti-cancer agent in mice

    OpenAIRE

    Rennel, Emma S; Hamdollah-Zadeh, Maryam A.; Wheatley, Edward R.; MAGNUSSEN, ANETTE; Schüler, Yvonne; Kelly, Sara P.; Finucane, Ciara; Ellison, David; Cebe-Suarez, Stephanie; Ballmer-Hofer, Kurt; Mather, Stephen; Stewart, Lorna; Bates, David O.; HARPER, STEVEN J.

    2008-01-01

    Tumour growth is dependent on angiogenesis, the key mediator of which is vascular endothelial growth factor-A (VEGF-A). VEGF-A exists as two families of alternatively spliced isoforms - pro-angiogenic VEGFxxx generated by proximal, and anti-angiogenic VEGFxxxb by distal splicing of exon 8. VEGF165b inhibits angiogenesis and is downregulated in tumours. Here, we show for the first time that administration of recombinant human VEGF165b inhibits colon carcinoma tumour growth and tumour vessel de...

  19. [The formation of an immune response in volunteers inoculated with a live recombinant influenza vaccine].

    Science.gov (United States)

    Vorob'ev, K V; Rudenko, L G; Islamova, I T; Grigor'eva, E P; Egorov, A Iu; Ermachenko, T A; Chepik, E B; Zakharova, N G; Karachentsev, A M

    1991-01-01

    The capacity of a live influenza vaccine (LIV) to stimulate cytotoxic cells (ADCMC and NK) was studied in 49 volunteers and 56 patients with influenza. Experimental batches of LIV from influenza A and B viruses prepared by genetic recombination on the basis of cold-adapted attenuation donors were used. Type A and B LIV were shown to stimulate the cytotoxic cell-mediated and humoral immunity; the intensity of immune response, however, depended on the molecular genetic characteristics of the vaccine (genome structure, properties of the donor of attenuation), its biological activity and capacity of reproduction in tissues. PMID:1785182

  20. Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells

    Directory of Open Access Journals (Sweden)

    Isobe Masaharu

    2011-04-01

    Full Text Available Abstract Background Molecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies. Results We developed a single-step cloning method, target-selective homologous recombination (TS-HR, in which PCR-amplified immunoglobulin variable genes were selectively inserted into vectors, even in the presence of nonspecifically amplified DNA. TS-HR utilizes Red/ET-mediated homologous recombination with a target-selective vector (TS-vector with unique homology arms on its termini. Using TS-HR, immunoglobulin variable genes were cloned directly into expression vectors by co-transforming unpurified PCR products and the TS-vector into E. coli. Furthermore, the high cloning specificity of TS-HR allowed plasmids to be extracted from pools of transformed bacteria without screening single colonies for correct clones. We present a one-week protocol for the production of recombinant mouse monoclonal antibodies from large numbers of single plasma cells. Conclusion The time requirements and limitations of traditional cloning procedures for the production of recombinant immunoglobulins have been significantly reduced with the development of the TS-HR cloning technique.

  1. The recombinant globular head domain of the measles virus hemagglutinin protein as a subunit vaccine against measles.

    Science.gov (United States)

    Lobanova, Liubov M; Eng, Nelson F; Satkunarajah, Malathy; Mutwiri, George K; Rini, James M; Zakhartchouk, Alexander N

    2012-04-26

    Despite the availability of live attenuated measles virus (MV) vaccines, a large number of measles-associated deaths occur among infants in developing countries. The development of a measles subunit vaccine may circumvent the limitations associated with the current live attenuated vaccines and eventually contribute to global measles eradication. Therefore, the goal of this study was to test the feasibility of producing the recombinant globular head domain of the MV hemagglutinin (H) protein by stably transfected human cells and to examine the ability of this recombinant protein to elicit MV-specific immune responses. The recombinant protein was purified from the culture supernatant of stably transfected HEK293T cells secreting a tagged version of the protein. Two subcutaneous immunizations with the purified recombinant protein alone resulted in the production of MV-specific serum IgG and neutralizing antibodies in mice. Formulation of the protein with adjuvants (polyphosphazene or alum) further enhanced the humoral immune response and in addition resulted in the induction of cell-mediated immunity as measured by the production of MV H-specific interferon gamma (IFN-?) and interleukin 5 (IL-5) by in vitro re-stimulated splenocytes. Furthermore, the inclusion of polyphosphazene into the vaccine formulation induced a mixed Th1/Th2-type immune response. In addition, the purified recombinant protein retained its immunogenicity even after storage at 37°C for 2 weeks. PMID:22406109

  2. Measuring Diffusion and Recombination in Polycrystalline Silicon

    Science.gov (United States)

    Zook, J. D.

    1983-01-01

    Light-beam-induced currents yield information about solar cell material. Apparatus measures short-circuit current generated when spot of concentrated light is scanned across grains and grain boundaries in material under test. Technique used to evaluate SOC samples for diffusion and recombination effects of cell processing and chemical and structural defects.

  3. Algae-based oral recombinant vaccines

    Directory of Open Access Journals (Sweden)

    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.

  4. Precise genotyping and recombination detection of Enterovirus.

    Science.gov (United States)

    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

  5. Recombinant Bovine Growth Hormone Criticism Grows.

    Science.gov (United States)

    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.…

  6. Different strategies for the recombinant production.

    Czech Academy of Sciences Publication Activity Database

    Leitner, C.; Ertl, S.; Volc, Jind?ich; Haltrich, D.; Ludwig, R.

    Praha : Verlag, 2006, s. 15-15. [International Symposium on the Genetics of Industrial Microorganisms /10./. Praha (CZ), 24.06.2006-28.06.2006] R&D Projects: GA MŠk ME 395 Institutional research plan: CEZ:AV0Z50200510 Keywords : recombinant pyranose 2-oxidase * heterologous expression Subject RIV: EE - Microbiology, Virology

  7. Radiative recombination of excitons in amorphous semiconductors

    International Nuclear Information System (INIS)

    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

  8. Different strategies for the recombinant production.

    Czech Academy of Sciences Publication Activity Database

    Leitner, C.; Ertl, S.; Volc, Jind?ich; Haltrich, D.; Ludwig, R.

    Prague : Springer, 2007, s. 460-460. [International Symposium on the Genetics of Industrial Microorganisms /10./. Praha (CZ), 24.06.2006-28.06.2006] Grant ostatní: CZ(CZ) KONTAKT ?R–Rakousko Institutional research plan: CEZ:AV0Z50200510 Source of funding: V - iné verejné zdroje Keywords : recombinant production Subject RIV: EE - Microbiology, Virology

  9. Magnetic affinity separation of recombinant fusion proteins.

    Czech Academy of Sciences Publication Activity Database

    Šafa?ík, Ivo; Šafa?íková, Miroslava

    2010-01-01

    Ro?. 38, ?. 1 (2010), s. 1-7. ISSN 1303-5002 R&D Projects: GA MŠk(CZ) OC 157; GA MPO(CZ) 2A-1TP1/094 Institutional research plan: CEZ:AV0Z60870520 Keywords : recombinant fusion proteins * affinity tags * magnetic separation Subject RIV: CE - Biochemistry

  10. Recombinant CBM-fusion technology - Applications overview.

    Science.gov (United States)

    Oliveira, Carla; Carvalho, Vera; Domingues, Lucília; Gama, Francisco M

    2015-01-01

    Carbohydrate-binding modules (CBMs) are small components of several enzymes, which present an independent fold and function, and specific carbohydrate-binding activity. Their major function is to bind the enzyme to the substrate enhancing its catalytic activity, especially in the case of insoluble substrates. The immense diversity of CBMs, together with their unique properties, has long raised their attention for many biotechnological applications. Recombinant DNA technology has been used for cloning and characterizing new CBMs. In addition, it has been employed to improve the purity and availability of many CBMs, but mainly, to construct bi-functional CBM-fused proteins for specific applications. This review presents a comprehensive summary of the uses of CBMs recombinantly produced from heterologous organisms, or by the original host, along with the latest advances. Emphasis is given particularly to the applications of recombinant CBM-fusions in: (a) modification of fibers, (b) production, purification and immobilization of recombinant proteins, (c) functionalization of biomaterials and (d) development of microarrays and probes. PMID:25689072

  11. Vaccine development using recombinant DNA technology

    Science.gov (United States)

    Vaccines induce an immune response in the host that subsequently recognizes infectious agents and helps fight off the disease; vaccines must do this without causing the disease. This paper reviews the development of recombinant DNA technologies as a means of providing new ways for attenuating diseas...

  12. Selected techniques in recombinant DNA technology

    International Nuclear Information System (INIS)

    Recombined DNA technology comprises a complex of techniques in the fields of nucleic acid biochemistry and molecular biology. This presentation gives an introduction, a brief description and example of the procedures of some of the basic techniques in the DNA cloning work currently used. 8 refs

  13. Genetic Recombination as a Chemical Reaction Network

    OpenAIRE

    Müller, Stefan; Hofbauer, Josef

    2015-01-01

    The process of genetic recombination can be seen as a chemical reaction network with mass-action kinetics. We review the known results on existence, uniqueness, and global stability of an equilibrium in every compatibility class and for all rate constants, from both the population genetics and the reaction networks point of view.

  14. The efficacy of recombinant versus urinary HCG in ART outcome

    OpenAIRE

    Eftekhar, Maryam; KHALILI, Mohammad Ali; Rahmani, Elham

    2012-01-01

    Background: Human chorionic gonadotropin (HCG) has been used as a replacement for the mid-cycle luteinizing hormone (LH) surge for several years. The recent arrival of recombinant DNA technology has made recombinant HCG (rHCG) accessible.

  15. Positive Selection Can Create False Hotspots of Recombination

    OpenAIRE

    Reed, Floyd A.; Tishkoff, Sarah A.

    2006-01-01

    Simulations of positive directional selection, under parameter values appropriate for approximating human genetic diversity and rates of recombination, reveal that the effects of strong selective sweeps on patterns of linkage disequilibrium (LD) mimic the pattern expected with recombinant hotspots.

  16. Multiple barriers to recombination between divergent HIV-1 variants revealed by a dual-marker recombination assay

    DEFF Research Database (Denmark)

    Nikolaitchik, Olga A; Galli, Andrea

    2011-01-01

    Recombination is a major force for generating human immunodeficiency virus type 1 (HIV-1) diversity and produces numerous recombinants circulating in the human population. We previously established a cell-based system using green fluorescent protein gene (gfp) as a reporter to study the mechanisms of HIV-1 recombination. We now report an improved system capable of detecting recombination using authentic viral sequences. Frameshift mutations were introduced into the gag gene so that parental viruses do not express full-length Gag; however, recombination can generate a progeny virus that expresses a functional Gag. We demonstrate that this Gag reconstitution assay can be used to detect recombination between two group M HIV-1 variants of the same or of different subtypes. Using both gfp and gag assays, we found that, similar to group M viruses, group O viruses also recombine frequently. When recombination between a group M virus and a group O virus was examined, we found three distinct barriers for intergroup recombination. First, similar to recombination within group M viruses, intergroup recombination is affected by the identity of the dimerization initiation signal (DIS); variants with the same DIS recombined at a higher rate than those with different DIS. Second, using the gfp recombination assay, we showed that intergroup recombination occurs much less frequently than intragroup recombination, even though the gfp target sequence is identical in all viruses. Finally, Gag reconstitution between variants from different groups is further reduced compared with green fluorescent protein, indicating that sequence divergence interferes with recombination efficiency in the gag gene. Compared with identical sequences, we estimate that recombination rates are reduced by 3-fold and by 10- to 13-fold when the target regions in gag contain 91% and 72-73% sequence identities, respectively. These results show that there are at least three distinct mechanisms preventing exchange of genetic information between divergent HIV-1 variants from different groups.

  17. Markov jump processes in modeling coalescent with recombination

    OpenAIRE

    Chen, Xian; Ma, Zhi-Ming; Wang, Ying

    2014-01-01

    Genetic recombination is one of the most important mechanisms that can generate and maintain diversity, and recombination information plays an important role in population genetic studies. However, the phenomenon of recombination is extremely complex, and hence simulation methods are indispensable in the statistical inference of recombination. So far there are mainly two classes of simulation models practically in wide use: back-in-time models and spatially moving models. Ho...

  18. Evolution of the Genomic Recombination Rate in Murid Rodents

    OpenAIRE

    Dumont, Beth L.; Payseur, Bret A.

    2011-01-01

    Although very closely related species can differ in their fine-scale patterns of recombination hotspots, variation in the average genomic recombination rate among recently diverged taxa has rarely been surveyed. We measured recombination rates in eight species that collectively represent several temporal scales of divergence within a single rodent family, Muridae. We used a cytological approach that enables in situ visualization of crossovers at meiosis to quantify recombination rates in mult...

  19. Genetic Analysis of Meiotic Recombination in Schizosaccharomyces pombe

    OpenAIRE

    Smith, Gerald R.

    2009-01-01

    The fission yeast Schizosaccharomyces pombe is well-suited for studying meiotic recombination. Methods are described here for culturing S. pombe and for genetic assays of intragenic recombination (gene conversion), intergenic recombination (crossing-over), and spore viability. Both random spore and tetrad analyses are described.

  20. Characterization of recombinant tetanus toxin derivatives suitable for vaccine development.

    OpenAIRE

    Figueiredo, D.; Turcotte, C; Frankel, G; Li, Y.; Dolly, O; Wilkin, G.; Marriott, D.; FAIRWEATHER, N; Dougan, G

    1995-01-01

    Recombinant derivatives of tetanus toxin (TeTx) were isolated and used to immunize mice. Recombinant TeTx light chain, a derivative of fragment C that had lost the ability to bind neurons, and a recombinant TeTx holotoxoid that could protect mice against TeTx challenge were identified.

  1. Gimeracil sensitizes cells to radiation via inhibition of homologous recombination

    International Nuclear Information System (INIS)

    Background and purpose: 5-Chloro-2,4-dihydroxypyridine (Gimeracil) is a component of an oral fluoropyrimidine derivative S-1. Gimeracil is originally added to S-1 to yield prolonged 5-FU concentrations in tumor tissues by inhibiting dihydropyrimidine dehydrogenase, which degrades 5-FU. We found that Gimeracil by itself had the radiosensitizing effect. Methods and materials: We used various cell lines deficient in non-homologous end-joining (NHEJ) or homologous recombination (HR) as well as DLD-1 and HeLa in clonogenic assay. ?-H2AX focus formation and SCneo assay was performed to examine the effects of Gimeracil on DNA double strand break (DSB) repair mechanisms. Results: Results of ?-H2AX focus assay indicated that Gimeracil inhibited DNA DSB repair. It did not sensitize cells deficient in HR but sensitized those deficient in NHEJ. In SCneo assay, Gimeracil reduced the frequency of neo-positive clones. Additionally, it sensitized the cells in S-phase more than in G0/G1. Conclusions: Gimeracil inhibits HR. Because HR plays key roles in the repair of DSBH caused by radiotherapy, Gimeracil may enhance the efficacy of radiotherapy through the suppression of HR-mediated DNA repair pathways.

  2. Short homologies efficiently generate detectable homologous recombination events.

    Science.gov (United States)

    Osahor, Andrew N; Tan, Chau-Yan; Sim, Edmund Ui-Hang; Lee, Choon-Weng; Narayanan, Kumaran

    2014-10-01

    When recombineering bacterial artificial chromosomes (BACs), it is common practice to design the ends of the donor molecule with 50 bp of homology specifying its insertion site. We demonstrate that desired recombinants can be produced using intermolecular homologies as short as 15 bp. Although the use of shorter donor end regions decreases total recombinants by several fold, the frequency of recombinants with correctly inserted donor molecules was high enough for easy detection by simple polymerase chain reaction (PCR) screening. This observation may have important implications for the design of oligonucleotides for recombineering, including significant cost savings, especially for high-throughput projects that use large quantities of primers. PMID:24929088

  3. Use of Helical Transport Channels for Bunch Recombination

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. Competitive repair by naturally dispersed repetitive DNA during non-allelic homologous recombination

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    Directory of Open Access Journals (Sweden)

    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.

  6. In utero recombinant adeno-associated virus gene transfer in mice, rats, and primates

    Directory of Open Access Journals (Sweden)

    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.

  7. FASEB Summer Research Conference. Genetic Recombination and Chromosome Rearrangements

    Energy Technology Data Exchange (ETDEWEB)

    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.

  8. Monitoring homologous recombination in rice (Oryza sativa L.)

    International Nuclear Information System (INIS)

    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 10-5 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.

  9. Production of recombinant proteins in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Wolfgang Schumann

    2004-01-01

    Full Text Available Attempts to obtain a recombinant protein using prokaryotic expression systems can go from a rewarding and rather fast procedure to a frustrating time-consuming experience. In most cases production of heterologous proteins in Escherichia coli K12 strains has remained an empirical exercise in which different systems are tested without a careful insight into the various factors affecting adequate expression of the encoded protein. The present review will deal with E. coli as protein factory and will cover some of the aspects related to transcriptional and translational expression signals, factors affecting protein stability and solubility and targeting of proteins to different cell compartments. Based on the knowledge accumulated over the last decade, we believe that the rate of success for those dedicated to expression of recombinant proteins based on the use E. coli strains can still be significantly improved.

  10. Recombination energy in double white dwarf formation

    CERN Document Server

    Nandez, Jose L A; Lombardi, James C

    2015-01-01

    In this Letter we investigate the role of recombination energy during a common envelope event. We confirm that taking this energy into account helps to avoid the formation of the circumbinary envelope commonly found in previous studies. For the first time, we can model a complete common envelope event, with a clean compact double white dwarf binary system formed at the end. The resulting binary orbit is almost perfectly circular. In addition to considering recombination energy, we also show that between 1/4 and 1/2 of the released orbital energy is taken away by the ejected material. We apply this new method to the case of the double-white dwarf system WD 1101+364, and we find that the progenitor system at the start of the common envelope event consisted of a $\\sim1.5M_\\odot$ red giant star in a $\\sim 30$ day orbit with a white dwarf companion.

  11. Recombination energy in double white dwarf formation

    Science.gov (United States)

    Nandez, J. L. A.; Ivanova, N.; Lombardi, J. C.

    2015-06-01

    In this Letter, we investigate the role of recombination energy during a common envelope event. We confirm that taking this energy into account helps to avoid the formation of the circumbinary envelope commonly found in previous studies. For the first time, we can model a complete common envelope event, with a clean compact double white dwarf binary system formed at the end. The resulting binary orbit is almost perfectly circular. In addition to considering recombination energy, we also show that between 1/4 and 1/2 of the released orbital energy is taken away by the ejected material. We apply this new method to the case of the double white dwarf system WD 1101+364, and we find that the progenitor system at the start of the common envelope event consisted of an ˜1.5 M? red giant star in an ˜30 d orbit with a white dwarf companion.

  12. Production of recombinant proteins in Escherichia coli

    Scientific Electronic Library Online (English)

    Wolfgang, Schumann; Luis Carlos S., Ferreira.

    Full Text Available Attempts to obtain a recombinant protein using prokaryotic expression systems can go from a rewarding and rather fast procedure to a frustrating time-consuming experience. In most cases production of heterologous proteins in Escherichia coli K12 strains has remained an empirical exercise in which di [...] fferent systems are tested without a careful insight into the various factors affecting adequate expression of the encoded protein. The present review will deal with E. coli as protein factory and will cover some of the aspects related to transcriptional and translational expression signals, factors affecting protein stability and solubility and targeting of proteins to different cell compartments. Based on the knowledge accumulated over the last decade, we believe that the rate of success for those dedicated to expression of recombinant proteins based on the use E. coli strains can still be significantly improved.

  13. Laser-induced recombination of D+

    International Nuclear Information System (INIS)

    Laser induced recombination into the L shell (n=2) of deuterium was investigated at the heavy-ion cooler storage ring CRYRING for two values of the electron density. The data shows the expected enhancement of radiative recombination, induced by the intense laser field applied, over the spontaneous case. Like in previous measurements, a strong intensity below the photoionization threshold was observed. Qualitatively, this gain is described by an average field induced threshold shift model. However, when reducing the electron density by roughly a factor of two, the effective field increased by an order of magnitude. This surprising result indicates that the precise origin of the observed fields is not simply related to the known field sources. (orig.)

  14. Studies on innovative hydrogen recombining devices

    International Nuclear Information System (INIS)

    In order to prevent the containment and other safety relevant components from incurring serious damage caused by a detonation of the hydrogen/air-mixture generated during a severe accident in Light Water Reactors (LWR) passive autocatalytic recombiners (PAR) are used for hydrogen removal in an increasing number of European plants. These devices make use of the fact that hydrogen and oxygen react exothermally on catalytic surfaces generating steam and heat. Experimental investigations at several research facilities indicate that existing plate-type PAR systems bear the risk of igniting the gaseous mixture due to an overheating of the catalyst elements caused by strong reaction heat generation. Innovative devices could overcome existing limitations making use of the knowledge deduced from experiments at the REKO facilities at FZJ. The paper analyses the mechanisms of the thermal behavior of catalytic plate-type recombiners and presents experimental results on existing and innovative concepts for hydrogen removal. (author)

  15. Mechanisms and factors that influence high frequency retroviral recombination

    DEFF Research Database (Denmark)

    Delviks-Frankenberry, Krista; Galli, Andrea

    2011-01-01

    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.

  16. Mechanisms and Factors that Influence High Frequency Retroviral Recombination

    Directory of Open Access Journals (Sweden)

    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.

  17. Induction of molecular and genetic recombination in eukaryotic cells

    International Nuclear Information System (INIS)

    The purpose of this review was to generally describe mitotic and meiotic recombination and to relate these processes to DNA repair. The results and mechanisms which have been described for genetic recombination in lower eukaryocytes are related to recent observations with mammalian cell systems in an attempt to bridge the gap between these categories of eukaryotes. The types of genetic recombinations which can be identified in lower eukaryotes, the effects of various mutagens and radiations, the similarities between spontaneous mitotic and meiotic recombination, the importance of recombination in DNA repair, and the role of various mechanisms in recombination are described. Molecular recombination is discussed. Gene conversion (informational transfer) is related to biochemical changes and is a very sensitive genetic tool for detecting DNA alterations

  18. Photoionization and recombination of Fe XIX

    OpenAIRE

    Zhang, Hong Lin; Anil K. Pradhan

    1999-01-01

    Photoionization cross sections and recombination rate coefficients are presented for the L-shell ground state fine structure levels $2s^22p^4 \\ ^3P_{2,0,1}$ of Fe~XIX. Several sets of calculations including relativistic effects are carried out: (i) Breit-Pauli R-matrix (BPRM), (ii) Relativistic Distorted Wave (RDW), and (iii) a semi-relativistic calculation. Non-relativistic LS coupling calculations are also done for comparison. The BPRM calculations employ a configuration i...

  19. Cultivating Insect Cells To Produce Recombinant Proteins

    Science.gov (United States)

    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).

  20. Telomerase RNA function in recombinant Tetrahymena telomerase

    OpenAIRE

    Licht, Jill D.; Collins, Kathleen

    1999-01-01

    Telomerase is a ribonucleoprotein reverse transcriptase specialized for use of a sequence within its integral RNA component as the template for DNA synthesis. Telomerase adds telomeric simple sequence repeats to single-stranded primers in vitro or chromosome ends in vivo. We have investigated the sequences and structures of recombinant Tetrahymena thermophila telomerase RNA necessary for physical association and activity with the catalytic protein subunit expressed in rabbit reticulocyte lysa...

  1. Designing recombinant Pseudomonas strains to enhance biodesulfurization.

    Science.gov (United States)

    Gallardo, M E; Ferrández, A; De Lorenzo, V; García, J L; Díaz, E

    1997-01-01

    The dsz biodesulfurization cluster from Rhodococcus erythropolis IGTS8 has been engineered under the control of heterologous broad-host-range regulatory signals to alleviate the mechanism of sulfur repression, and it was stably inserted into the chromosomes of different Pseudomonas strains. The recombinant bacteria were able to desulfurize dibenzothiophene more efficiently than the native host. Furthermore, these new biocatalysts combine relevant industrial and environmental traits, such as production of biosurfactants, with the enhanced biodesulfurization phenotype. PMID:9371464

  2. Designing recombinant Pseudomonas strains to enhance biodesulfurization.

    OpenAIRE

    Gallardo, M.E.; Ferrández, A.; de Lorenzo, V.; García, J. L.; Díaz, E.

    1997-01-01

    The dsz biodesulfurization cluster from Rhodococcus erythropolis IGTS8 has been engineered under the control of heterologous broad-host-range regulatory signals to alleviate the mechanism of sulfur repression, and it was stably inserted into the chromosomes of different Pseudomonas strains. The recombinant bacteria were able to desulfurize dibenzothiophene more efficiently than the native host. Furthermore, these new biocatalysts combine relevant industrial and environmental traits, such as p...

  3. Photoluminescence lifetime spectroscopy - Surface recombination analysis

    OpenAIRE

    Roth, T.; Rüdiger, M; Rosenits, P.; Diez, S; Trupke, T.; Bardos, R.A.; Glunz, S. W.

    2007-01-01

    The recently introduced quasi-steady-state photoluminescence technique (QSS-PL) for determining the injection-dependent carrier lifetime in silicon samples was shown to be a good complement of the well established quasi-steady-state photoconductance technique (QSS-PC) especially in the low injection regime, where the QSS-PC technique is prone to measurement artefacts due to trapping or depletion region modulation. The ability to measure the real recombination lifetime at low injection densiti...

  4. Optical recombination of biexcitons in semiconductors

    OpenAIRE

    Bauer, M.; Keeling, J; Parish, M. M.; Rios, P. Lopez; Littlewood, P B

    2011-01-01

    We calculate the photoluminescence spectrum and lifetime of a biexciton in a semiconductor using Fermi's golden rule. Our biexciton wavefunction is obtained using a Quantum Monte Carlo calculation. We consider a recombination process where one of the excitons within the biexciton annihilates. For hole masses greater than or equal to the electron mass, we find that the surviving exciton is most likely to populate the ground state. We also investigate how the confinement of ex...

  5. Rapid one-step recombinational cloning

    OpenAIRE

    Fu, Changlin; Wehr, Daniel R.; Edwards, Janice; Hauge, Brian

    2008-01-01

    As an increasing number of genes and open reading frames of unknown function are discovered, expression of the encoded proteins is critical toward establishing function. Accordingly, there is an increased need for highly efficient, high-fidelity methods for directional cloning. Among the available methods, site-specific recombination-based cloning techniques, which eliminate the use of restriction endonucleases and ligase, have been widely used for high-throughput (HTP) procedures. We have de...

  6. (Photoexcited charge pair escape and recombination)

    Energy Technology Data Exchange (ETDEWEB)

    Braun, C.L.

    1990-01-01

    Progress in four research areas on this project are summarized under the following topics: (1) Geminate charge pair recombination in hexane; (2) Fast current measurements resulting from excitation of charge transfer (CT) states; (3) Measurement of the dipole moment of excited states by DC conductivity; and (4) Charge separation at macroscopic interfaces between electron donor and acceptor solids. In a final section, personnel who have contributed to the project during the past budget period are described.

  7. Modelling of procecces in catalytic recombiners

    International Nuclear Information System (INIS)

    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.)

  8. Lasting recombination and cosmic background radiation spectrum

    International Nuclear Information System (INIS)

    The thermal decoupling during recombination and the endurance of this process can significantly deform the black-body background radiation spectrum even in the absence of heating sources. Observational data contradicts a flat standard universe but agrees with a low-density model and with a flat matter-antimatter symmetric universe. Evidence is presented against the presumption that matter-antimatter annihilation should necessarily produce large distortions. (Auth.)

  9. Recombinant spider silk with antimicrobial properties

    OpenAIRE

    Nilebäck, Linnea

    2013-01-01

    Immobilizing antimicrobial substances onto biocompatible materials is an important approach for the design of novel, functionalized medical devices. By choosing antimicrobial substances from innate immune systems, the risk for development of resistance in pathogenic microbes is lower than if conventional antibiotics are used. Combining natural antimicrobial peptides and bactericidal enzymes with strong and elastic spider silk through recombinant protein technology would enable large-scale pro...

  10. Assembly mechanism of recombinant spider silk proteins

    OpenAIRE

    Rammensee, S.; Slotta, U.; Scheibel, T.; Bausch, A.R.

    2008-01-01

    Spider silk threads are formed by the irreversible aggregation of silk proteins in a spinning duct with dimensions of only a few micrometers. Here, we present a microfluidic device in which engineered and recombinantly produced spider dragline silk proteins eADF3 (engineered Araneus diadematus fibroin) and eADF4 are assembled into fibers. Our approach allows the direct observation and identification of the essential parameters of dragline silk assembly. Changes in ionic conditions and pH resu...

  11. Jet Fragmentation via Recombination of Parton Showers

    CERN Document Server

    Han, Kyong Chol; Ko, Che Ming

    2012-01-01

    We study hadron production in jets by applying quark recombination to jet shower partons. With the jet showers obtained from PYTHIA and augmented by additional non-perturbative effects, we compute hadron spectra in e+ + e-collisions at sqrt(s)=200 GeV. Including contributions from resonance decays, we find that the resulting transverse momentum spectra for pions, kaons, and protons reproduce reasonably those from the string fragmentation as implemented in PYTHIA.

  12. Recombinant CBM-fusion technology : applications overview

    OpenAIRE

    Oliveira, Carla Cristina Marques de; Carvalho, Vera; Domingues, Lucília; Gama, F.M.

    2015-01-01

    Carbohydrate-binding modules (CBMs) are small components of several enzymes, which present an independent fold and function, and specific carbohydrate-binding activity. Their major function is to bind the enzyme to the substrate enhancing its catalytic activity, especially in the case of insoluble substrates. The immense diversity of CBMs, together with their unique properties, has long raised their attention for many biotechnological applications. Recombinant DNA technology has been used for...

  13. Recombinant Expression of Backbone-Cyclized Polypeptides

    OpenAIRE

    Borra, Radhika; Camarero, Julio A.

    2013-01-01

    Here we review the different biochemical approaches available for the expression of backbone-cyclized polypeptides, including peptides and proteins. These methods allow for the production of circular polypeptides either in vitro or in vivo using standard recombinant DNA expression techniques. Polypeptide circularization provides a valuable tool to study the effects of topology on protein stability and folding kinetics. Furthermore, having biosynthetic access to backbone-cyclized polypeptides ...

  14. Music, Radio, and Mediatization

    DEFF Research Database (Denmark)

    Krogh, Mads; Michelsen, Morten

    2015-01-01

    Mediatization has become a key concept for understanding the relations between media and other cultural and social fields. Contributing to the discussions related to the concept of mediatization this article discusses how practices of radio and music(al life) influence each other. We follow Deacon’s and Stanyer’s advice to supplement the concept of mediatization with ‘a series of additional concepts at lower levels of abstraction’ (2014: 1040), and suggest, in this respect, the notion of heterog...

  15. The Schizosaccharomyces pombe Mediator

    DEFF Research Database (Denmark)

    Venturi, Michela

    2013-01-01

    In the past several years great attention has been dedicated to the characterization of the Mediator complex in a different range of model organisms. Mediator is a conserved co-activator complex involved in transcriptional regulation and it conveys signals from regulatory transcription factors to the basal transcription machinery. Mediator was initially isolated from Saccharomyces cerevisiae based on its ability to render a RNA polymerase II in vitro transcription system responsive to activators...

  16. Dissociative recombination of small molecular ions

    International Nuclear Information System (INIS)

    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.)

  17. Novel HIV-1 long terminal repeat (LTR) sequences of subtype B and mosaic intersubtype B/C recombinants in North India.

    Science.gov (United States)

    Neogi, Ujjwal; Sood, Vikas; Goel, Nidhi; Wanchu, Ajay; Banerjea, Akhil C

    2008-01-01

    Although the HIV-1 epidemic in India is mainly due to subtype C, other subtypes have also been reported from different parts of India. HIV-1 LTR sequence analysis from six HIV-1 infected individuals from North India was carried out to determine the nature and extent of variations. Four out of six samples formed a unique phylogenetic cluster which was close to subtype B. The other two samples (A3 and S3) turned out to be novel mosaic recombinants showing resemblance to subtypes B, B/C-India and B/C-Myanmar gene segments. All four subtype B LTR samples and the two B/C recombinants showed conserved as well as unique polymorphisms in all of the putative transcription factor binding sites (TFBS). These changes may potentially alter basal as well as Tat-mediated HIV-1 LTR promoter activation. The two recombinants possessed three copies of the NF-kappaB TFBS as seen with the majority of subtype C and recombinant B/C isolates reported earlier, but the other four non-recombinant B-LTRs showed only two copies of the NF-kappaB site. This is the first study to show a dominance of unique subtype B-LTRs and strongly suggests that this region could also be a hot spot for the formation of highly complex inter subtype B/C recombinants. PMID:18818865

  18. Construction and immunogenicity of a recombinant pseudorabies virus co-expressing porcine circovirus type 2 capsid protein and interleukin 18.

    Science.gov (United States)

    Zheng, Lan-lan; Guo, Xiao-qing; Zhu, Qian-lei; Chao, An-jun; Fu, Peng-fei; Wei, Zhan-yong; Wang, Shu-juan; Chen, Hong-ying; Cui, Bao-an

    2015-04-01

    A novel recombinant pseudorabies virus (PRV) expressing porcine circovirus type 2 (PCV2) capsid protein and IL-18 was constructed. The PCV2 open reading frame 2 (ORF2) and porcine IL-18 genes were amplified by PCR and then inserted into the PRV transfer vector (pG) to produce a recombinant plasmid (pGO18). Plasmid pGO18 was transfected into porcine kidney cell (PK15) pre-infected with PRV HB98 vaccine strain to generate a recombinant virus. The recombinant virus PRV-ORF2-IL18 was purified by green fluorescent plaque purification and the inserts were confirmed by PCR, enzyme digestion, sequencing, and Western blot. The humoral and cellular responses induced by the recombinant virus were assessed in mice. Mice (n=10) were immunized with PRV-ORF2-IL18, PRV-ORF2, PRV HB98, or inactivated PCV2. PRV-ORF2-IL18 elicited high titers of ELISA and serum neutralizing antibodies and strong cell-mediated immune responses in mice as indicated by anti-PCV2 ELISA, PRV-neutralizing assay, PCV2 specific lymphocyte proliferation assay, CD3(+), CD4(+) and CD8(+) T lymphocytes analysis, respectively. And PRV-ORF2-IL18 immunization protected mice against a lethal challenge of a virulent PRV Fa strain and significantly reduced the amount of PCV2 viremia. These results suggest an adjuvant effect of IL-18 on cellular immune responses. The recombinant virus might be an attractive candidate vaccine for preventing PCV2 and PRV infections in pigs. PMID:25701744

  19. Plant mediator: Mediating the jasmonate response

    OpenAIRE

    Kidd, Brendan N; Aitken, Elizabeth A; Schenk, Peer M.; Manners, John M.; Kazan, Kemal

    2010-01-01

    Jasmonate (JA) signaling plays an important role in regulating both plant defense and development. We have recently reported that the PHYTOCHROME AND FLOWERING TIME1 (PFT1) gene, which encodes the MEDIATOR25 subunit of the plant Mediator complex, is a key regulator of JA regulated transcription. We showed that the pft1 mutant had attenuated expression of a wide range of JA responsive genes and altered resistance to fungal pathogens. Here we examine the position of PFT1/MED25 within the JA pat...

  20. Leaf proteome rebalancing in Nicotiana benthamiana for upstream enrichment of a transiently expressed recombinant protein.

    Science.gov (United States)

    Robert, Stéphanie; Goulet, Marie-Claire; D'Aoust, Marc-André; Sainsbury, Frank; Michaud, Dominique

    2015-10-01

    A key factor influencing the yield of biopharmaceuticals in plants is the ratio of recombinant to host proteins in crude extracts. Postextraction procedures have been devised to enrich recombinant proteins before purification. Here, we assessed the potential of methyl jasmonate (MeJA) as a generic trigger of recombinant protein enrichment in Nicotiana benthamiana leaves before harvesting. Previous studies have reported a significant rebalancing of the leaf proteome via the jasmonate signalling pathway, associated with ribulose 1,5-bisphosphate carboxylase oxygenase (RuBisCO) depletion and the up-regulation of stress-related proteins. As expected, leaf proteome alterations were observed 7 days post-MeJA treatment, associated with lowered RuBisCO pools and the induction of stress-inducible proteins such as protease inhibitors, thionins and chitinases. Leaf infiltration with the Agrobacterium tumefaciens bacterial vector 24 h post-MeJA treatment induced a strong accumulation of pathogenesis-related proteins after 6 days, along with a near-complete reversal of MeJA-mediated stress protein up-regulation. RuBisCO pools were partly restored upon infiltration, but most of the depletion effect observed in noninfiltrated plants was maintained over six more days, to give crude protein samples with 50% less RuBisCO than untreated tissue. These changes were associated with net levels reaching 425 ?g/g leaf tissue for the blood-typing monoclonal antibody C5-1 expressed in MeJA-treated leaves, compared to less than 200 ?g/g in untreated leaves. Our data confirm overall the ability of MeJA to trigger RuBisCO depletion and recombinant protein enrichment in N. benthamiana leaves, estimated here for C5-1 at more than 2-fold relative to host proteins. PMID:26286859

  1. Teaching Mediated Public Relations.

    Science.gov (United States)

    Kent, Michael L.

    2001-01-01

    Discusses approaches to teaching a mediated public relations course, emphasizing the World Wide Web. Outlines five course objectives, assignments and activities, evaluation, texts, and lecture topics. Argues that students mastering these course objectives will understand ethical issues relating to media use, using mediated technology in public…

  2. Mediation and Domestic Violence

    Directory of Open Access Journals (Sweden)

    Jacques Faget

    2005-07-01

    Full Text Available This paper analyzes the potential and limits of recourse to mediation for regulating domestic violence. On the basis of an empirical study of its implementation in France and of the existing academic literature, it shows the existence of two types of practices reflecting two conceptions of mediation and more generally, two conceptions of the articulation between social and penal regulation

  3. Mediator redefines itself

    OpenAIRE

    Sennett, Nicholas C; Taatjes, Dylan J.

    2014-01-01

    Mediator is a large and structurally dynamic protein complex that is globally required for eukaryotic transcription by RNA polymerase II. In a recent paper published in Cell Research, Wang et al. report for the first time the location of distinct subunits and redefine domains in the S. cerevisiae Mediator complex.

  4. Budding yeast Hed1 down-regulates the mitotic recombination machinery when meiotic recombination is impaired

    OpenAIRE

    Tsubouchi, Hideo; Roeder, G Shirleen

    2006-01-01

    In budding yeast, there are two RecA homologs: Rad51 and Dmc1. While Rad51 is involved in both mitotic and meiotic recombination, Dmc1 participates specifically in meiotic recombination. Here, we describe a meiosis-specific protein (Hed1) with a novel Rad51 regulatory function. Several observations indicate that Hed1 attenuates Rad51 activity when Dmc1 is absent. First, although double-strand breaks are normally poorly repaired in the dmc1 mutant, repair becomes efficient when Hed1 is absent,...

  5. An Efficient Procedure for Marker-Free Mutagenesis of S. coelicolor by Site-Specific Recombination for Secondary Metabolite Overproduction

    Science.gov (United States)

    Dai, Ruixue; Yu, Meiying; Zhao, Guoping; Ding, Xiaoming

    2013-01-01

    Streptomyces bacteria are known for producing important natural compounds by secondary metabolism, especially antibiotics with novel biological activities. Functional studies of antibiotic-biosynthesizing gene clusters are generally through homologous genomic recombination by gene-targeting vectors. Here, we present a rapid and efficient method for construction of gene-targeting vectors. This approach is based on Streptomyces phage ?BT1 integrase-mediated multisite in vitro site-specific recombination. Four ‘entry clones’ were assembled into a circular plasmid to generate the destination gene-targeting vector by a one-step reaction. The four ‘entry clones’ contained two clones of the upstream and downstream flanks of the target gene, a selectable marker and an E. coli-Streptomyces shuttle vector. After targeted modification of the genome, the selectable markers were removed by ?C31 integrase-mediated in vivo site-specific recombination between pre-placed attB and attP sites. Using this method, part of the calcium-dependent antibiotic (CDA) and actinorhodin (Act) biosynthetic gene clusters were deleted, and the rrdA encoding RrdA, a negative regulator of Red production, was also deleted. The final prodiginine production of the engineered strain was over five times that of the wild-type strain. This straightforward ?BT1 and ?C31 integrase-based strategy provides an alternative approach for rapid gene-targeting vector construction and marker removal in streptomycetes. PMID:23409083

  6. Intermolecular domain swapping induces intein-mediated protein alternative splicing.

    Science.gov (United States)

    Aranko, A Sesilja; Oeemig, Jesper S; Kajander, Tommi; Iwaï, Hideo

    2013-10-01

    Protein sequences are diversified on the DNA level by recombination and mutation and can be further increased on the RNA level by alternative RNA splicing, involving introns that have important roles in many biological processes. The protein version of introns (inteins), which catalyze protein splicing, were first reported in the 1990s. The biological roles of protein splicing still remain elusive because inteins neither provide any clear benefits nor have an essential role in their host organisms. We now report protein alternative splicing, in which new protein sequences can be produced by protein recombination by intermolecular domain swapping of inteins, as elucidated by NMR spectroscopy and crystal structures. We demonstrate that intein-mediated protein alternative splicing could be a new strategy to increase protein diversity (that is, functions) without any modification in genetic backgrounds. We also exploited it as a post-translational protein conformation-driven switch of protein functions (for example, as highly specific protein interference). PMID:23974115

  7. Rad51C deficiency destabilizes XRCC3, impairs recombination and radiosensitizes S/G2-phase cells

    Energy Technology Data Exchange (ETDEWEB)

    Lio, Yi-Ching; Schild, David; Brenneman, Mark A.; Redpath, J. Leslie; Chen, David J.

    2004-05-01

    The highly conserved Rad51 protein plays an essential role in repairing DNA damage through homologous recombination. In vertebrates, five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, XRCC3) are expressed in mitotically growing cells, and are thought to play mediating roles in homologous recombination, though their precise functions remain unclear. Here we report the use of RNA interference to deplete expression of Rad51C protein in human HT1080 and HeLa cells. In HT1080 cells, depletion of Rad51C by small interfering RNA caused a significant reduction of frequency in homologous recombination. The level of XRCC3 protein was also sharply reduced in Rad51C-depleted HeLa cells, suggesting that XRCC3 is dependent for its stability upon heterodimerization with Rad51C. In addition, Rad51C-depleted HeLa cells showed hypersensitivity to the DNA cross-linking agent mitomycin C, and moderately increased sensitivity to ionizing radiation. Importantly, the radiosensitivity of Rad51C-deficient HeLa cells was evident in S and G{sub 2}/M phases of the cell cycle but not in G{sub 1} phase. Together, these results provide direct cellular evidence for the importance of human Rad51C in homologous recombinational repair.

  8. Induction and assessment of class switch recombination in purified murine B cells.

    Science.gov (United States)

    Zaheen, Ahmad; Martin, Alberto

    2010-01-01

    Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse. PMID:20736917

  9. Eosinophil Cysteinyl Leukotriene Synthesis Mediated by Exogenous Secreted Phospholipase A2 Group X*

    OpenAIRE

    Lai, Ying; Oslund, Rob C.; Bollinger, James G.; Henderson, William R; Santana, Luis F.; Altemeier, William A; Gelb, Michael H.; Hallstrand, Teal S.

    2010-01-01

    Secreted phospholipase A2 group X (sPLA2-X) has recently been identified in the airways of patients with asthma and may participate in cysteinyl leukotriene (CysLT; C4, D4, and E4) synthesis. We examined CysLT synthesis and arachidonic acid (AA) and lysophospholipid release by eosinophils mediated by recombinant human sPLA2-X. We found that recombinant sPLA2-X caused marked AA release and a rapid onset of CysLT synthesis in human eosinophils that was blocked by a selective sPLA2-X inhibitor. ...

  10. Functions and structures of eukaryotic recombination proteins

    International Nuclear Information System (INIS)

    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.)

  11. Recombination-deficient mutants of Bacillus subtilis

    International Nuclear Information System (INIS)

    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

  12. Nonparadoxical evolutionary stability of the recombination initiation landscape in yeast.

    Science.gov (United States)

    Lam, Isabel; Keeney, Scott

    2015-11-20

    The nonrandom distribution of meiotic recombination shapes heredity and genetic diversification. Theoretically, hotspots--favored sites of recombination initiation--either evolve rapidly toward extinction or are conserved, especially if they are chromosomal features under selective constraint, such as promoters. We tested these theories by comparing genome-wide recombination initiation maps from widely divergent Saccharomyces species. We find that hotspots frequently overlap with promoters in the species tested, and consequently, hotspot positions are well conserved. Remarkably, the relative strength of individual hotspots is also highly conserved, as are larger-scale features of the distribution of recombination initiation. This stability, not predicted by prior models, suggests that the particular shape of the yeast recombination landscape is adaptive and helps in understanding evolutionary dynamics of recombination in other species. PMID:26586758

  13. Dielectronic recombination of Ar13+ at CRYRING

    Science.gov (United States)

    DeWitt, D. R.; Schuch, R.; Zong, W.; Asp, S.; Gao, H.; Biedermann, C.; Liljeby, L.; Beebe, E.; Pikin, A.

    1997-01-01

    We have investigated ?n = 0 dielectronic recombination resonances of Ar13+ at CRYRING. The Ar13+ ions were obtained from the electron beam ion source CRYSIS. The ultracold electron beam produced in the modified electron cooler was used as the electron target, resulting in resonance widths less than 30 meV FWHM. Determination of the observed resonance line energies, which requires transformation from laboratory to center of mass frame, includes acceleration of the ion beam due to the drag force encountered as the electron energy is scanned. With this transformation the uncertainty in absolute line positions has been reduced to less than 30 meV.

  14. Therapeutic use of recombinant methionyl human leptin.

    OpenAIRE

    Vatier, Camille; Gautier, Jean-François; Vigouroux, Corinne

    2012-01-01

    Recombinant methionyl human leptin (r-metHuLeptin) was first used as a replacement therapy in patients bearing inactivating mutations in the leptin gene. In this indication, it was shown since 1999 to be very efficient in inducing a dramatic weight loss in rare children and adults with severe obesity due to the lack of leptin. These first clinical trials clearly showed that r-metHuLeptin acted centrally to reduce food intake, inducing loss of fat mass, and to correct metabolic alterations, im...

  15. Hydrogen recombiner catalyst test supporting data

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. Hydrogen recombiner catalyst test supporting data

    International Nuclear Information System (INIS)

    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

  17. Patterns of recombination on human chromosome 22

    Energy Technology Data Exchange (ETDEWEB)

    Schlumpf, K.S.; Kim, D.; Haines, J.L. [Massachusetts General Hospital, Boston, MA (United States)

    1994-09-01

    Virtually all genetic linkage maps generated to date are gross averages across individuals, ages, and (often) sexes. In addition, although some level of positive interference has been assumed, until recently little evidence to support this in humans has been available. The major stumbling block has been the quality of the data available, since even a few genotypic errors can have drastic effects on both the map length and the number of apparent recombinants. In addition, variation in recombination by factors other than sex have pretty much been ignored. To explore recombination in more detail, we have generated a microsatellite marker map of human chromosome 22. This map includes 32 markers genotyped through 46 sibships of the Venezuelan Reference Pedigree (VRP). Extensive error checking and regenotyping was performed to remove as many genotypic errors as possible, but no genotypes were removed simply because they created unlikely events. The following 1000:1 odds map has been obtained: cen--F8VWFP1--11--S264--3-S311--4--S257--2--TOP1P2--3--S156--1--CRYB2--1--S258--2--S310--6--S193--1--S275--3--S268--1--S280--4--S304--3--S283--2--LiR1--3--IL2RB--3--S299--1--S302--1--S537--2--S270--4--PDGF--8--S274--qter. The female map (91 cM) is twice as long as the male map (46 cM) and the log-likelihood difference in the maps (22.3) is highly significant (P=0.001, df=22) and appears constant across the chromosome. Analysis of recombination with age showed no particular trends for either males or females when chromosomes were grouped into three categories (20, 20-30, 30+) by parental age at birth of child. Positive interference was found in maternally derived chromosomes ({chi}{sup 2}=30.5 (4), p<0.005), but not in paternally derived chromosomes ({chi}{sup 2}=6.24 (3), P=0.10). This contrasts to data from chromosomes 9 and 21 where positive interference was found for both sexes. More detailed analyses are in progress.

  18. Recombinational Landscape and Population Genomics of Caenorhabditis elegans

    OpenAIRE

    Rockman, Matthew V.; Kruglyak, Leonid

    2009-01-01

    Recombination rate and linkage disequilibrium, the latter a function of population genomic processes, are the critical parameters for mapping by linkage and association, and their patterns in Caenorhabditis elegans are poorly understood. We performed high-density SNP genotyping on a large panel of recombinant inbred advanced intercross lines (RIAILs) of C. elegans to characterize the landscape of recombination and, on a panel of wild strains, to characterize population genomic patterns. We co...

  19. Relative rates of homologous and nonhomologous recombination in transfected DNA.

    OpenAIRE

    Roth, D. B.; Wilson, J H

    1985-01-01

    Both homologous and nonhomologous recombination events occur at high efficiency in DNA molecules transfected into mammalian cells. Both types of recombination occur with similar overall efficiencies, as measured by an endpoint assay, but their relative rates are unknown. In this communication, we measure the relative rates of homologous and nonhomologous recombination in DNA transfected into monkey cells. This measurement is made by using a linear simian virus 40 genome that contains a 131-ba...

  20. Recombination in polymer-fullerene bulk heterojunction solar cells

    OpenAIRE

    Cowan, Sarah R.; Roy, Anshuman; Heeger, Alan J.

    2010-01-01

    Recombination of photogenerated charge carriers in polymer bulk heterojunction (BHJ) solar cells reduces the short circuit current (Jsc) and the fill factor (FF). Identifying the mechanism of recombination is, therefore, fundamentally important for increasing the power conversion efficiency. Light intensity and temperature dependent current-voltage measurements on polymer BHJ cells made from a variety of different semiconducting polymers and fullerenes show that the recombin...

  1. Intermediate bands versus levels in non-radiative recombination

    International Nuclear Information System (INIS)

    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

  2. Recombination Suppression by Heterozygous Robertsonian Chromosomes in the Mouse

    OpenAIRE

    Davisson, M T; Akeson, E. C.

    1993-01-01

    Robertsonian chromosomes are metacentric chromosomes formed by the joining of two telocentric chromosomes at their centromere ends. Many Robertsonian chromosomes of the mouse suppress genetic recombination near the centromere when heterozygous. We have analyzed genetic recombination and meiotic pairing in mice heterozygous for Robertsonian chromosomes and genetic markers to determine (1) the reason for this recombination suppression and (2) whether there are any consistent rules to predict wh...

  3. Low energy electron-ion recombination experiments at CRYRING

    International Nuclear Information System (INIS)

    Results from recent experiments at the heavy-ion synchrotron storage ring CRYRING are described. These experiments include radiative recombination of deuterons using several separate techniques to investigate specific n-level capture, and dielectronic recombination of He+ and Ar13+. New methods applied to the argon dielectronic recombination experiment provided an energy resolution better than 30meV FWHM and a determination of the peak positions to ± 30mev

  4. Meiotic recombination and chromosome segregation in Schizosaccharomyces pombe

    OpenAIRE

    Davis, Luther; Smith, Gerald R.

    2001-01-01

    In most organisms homologous recombination is vital for the proper segregation of chromosomes during meiosis, the formation of haploid sex cells from diploid precursors. This review compares meiotic recombination and chromosome segregation in the fission yeast Schizosaccharomyces pombe and the distantly related budding yeast Saccharomyces cerevisiae, two especially tractable microorganisms. Certain features, such as the occurrence of DNA breaks associated with recombination, appear similar, s...

  5. The Effect of Recombination on the Reconstruction of Ancestral Sequences

    OpenAIRE

    Arenas, Miguel; Posada, David

    2010-01-01

    While a variety of methods exist to reconstruct ancestral sequences, all of them assume that a single phylogeny underlies all the positions in the alignment and therefore that recombination has not taken place. Using computer simulations we show that recombination can severely bias ancestral sequence reconstruction (ASR), and quantify this effect. If recombination is ignored, the ancestral sequences recovered can be quite distinct from the grand most recent common ancestor (GMRCA) of the samp...

  6. Development of Recombinant Cationic Polymers for Gene Therapy Research

    OpenAIRE

    Canine, Brenda F.; Hatefi, Arash

    2010-01-01

    Cationic polymers created through recombinant DNA technology have the potential to fill a void in the area of gene delivery. The recombinant cationic polymers to be discussed here are amino acid based polymers synthesized in E.coli with the purpose to not only address the major barriers to efficient gene delivery but offer safety, biodegradability, targetability and cost-effectiveness. This review helps the readers to get a better understanding about the evolution of recombinant cationic poly...

  7. Exciton recombination in one-dimensional organic Mott insulators

    Science.gov (United States)

    Lenar?i?, Zala; Eckstein, Martin; Prelovšek, Peter

    2015-11-01

    We present a theory for the recombination of (charged) holons and doublons in one-dimensional organic Mott insulators, which is responsible for the decay of the photoexcited state. Due to the charge-spin separation, the dominant mechanism for recombination at low density of charges involves a multiphonon emission. We show that a reasonable coupling to phonons is sufficient to explain the fast recombination observed by pump-probe experiments in ET -F2TCNQ , whereby we can also account for the measured pressure dependence of the recombination rate.

  8. Density dependence of recombination in the electron cooler of CRYRING

    International Nuclear Information System (INIS)

    The electron-ion recombination rates of Ne10+ and D+ have been measured as a function of relative energy and electron density. We found a strong enhancement of e-Ne10+ recombination over expected radiative recombination rates below 1 meV relative energies, reaching a factor of 4 close to zero relative energy. Remarkably, the measured rate coefficients decrease as a function of electron density for both systems. Studies of recombination of D+ indicate that this density dependence may be due to temperature variations of the electron beam with the electron density. (orig.)

  9. General resonance mediation

    Energy Technology Data Exchange (ETDEWEB)

    McGarrie, Moritz

    2012-07-15

    We extend the framework of general gauge mediation to cases where the mediating fields have a nontrivial spectral function, as might arise from strong dynamics. We demonstrate through examples that this setup describes a broad class of possible models of gauge mediated supersymmetry breaking. A main emphasis is to give general formulas for cross sections for {sigma}(visible {yields} hidden) in these resonance models. We will also give formulas for soft masses, A-terms and demonstrate the framework with a holographic setup.

  10. Generation of hypoxanthine phosphoribosyltransferase gene knockout rabbits by homologous recombination and gene trapping through somatic cell nuclear transfer.

    Science.gov (United States)

    Yin, Mingru; Jiang, Weihua; Fang, Zhenfu; Kong, Pengcheng; Xing, Fengying; Li, Yao; Chen, Xuejin; Li, Shangang

    2015-01-01

    The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT. Gene trap strategies were employed to enhance the gene targeting rates. The male and female gene knockout fibroblast cell lines were derived by different strategies. When male HPRT knockout cells were used for SCNT, no live rabbits were obtained. However, when female HPRT(+/-) cells were used for SCNT, live, healthy rabbits were generated. The cloned HPRT(+/-) rabbits were fertile at maturity. We demonstrate a new technique to produce gene-targeted rabbits. This approach may also be used in the genetic manipulation of different genes or in other species. PMID:26522387

  11. Photoionization and recombination of Fe XIX

    CERN Document Server

    Zhang, H L; Zhang, Hong Lin; Pradhan, Anil K.

    1999-01-01

    Photoionization cross sections and recombination rate coefficients are presented for the L-shell ground state fine structure levels $2s^22p^4 \\ ^3P_{2,0,1}$ of Fe~XIX. Several sets of calculations including relativistic effects are carried out: (i) Breit-Pauli R-matrix (BPRM), (ii) Relativistic Distorted Wave (RDW), and (iii) a semi-relativistic calculation. Non-relativistic LS coupling calculations are also done for comparison. The BPRM calculations employ a configuration interaction target representation for Fe~XX consisting of 12 LS terms (23-fine structure levels), as in the recently reported BPRM calculations by Donnelly et al (MNRAS, 307, 595, 1999). The background cross sections in all three sets of present calculations agree with one another, but differ considerably from those of Donnelly et al. Owing to much more extensive resonance structures in the present BPRM calculations, the sum of the corresponding recombination rate coefficients for the $^3P_{2,0,1}$ levels are up to 50% higher than the LS ra...

  12. Dielectronic recombination of lithium-like gold

    International Nuclear Information System (INIS)

    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

  13. Radio Recombination Lines in Galactic HII Regions

    CERN Document Server

    Quireza, C; Bania, T M; Rood, R T; Balser, Dana S.; Quireza, Cintia; Rood, Robert T.

    2006-01-01

    We report radio recombination line (RRL) and continuum observations of a sample of 106 Galactic HII regions made with the NRAO 140 Foot radio telescope in Green Bank, WV. We believe this to be the most sensitive RRL survey ever made for a sample this large. Most of our source integration times range between 6 and 90 hours which yield typical r.m.s. noise levels of 1.0--3.5 milliKelvins. Our data result from two different experiments performed, calibrated, and analyzed in similar ways. A CII survey was made at 3.5 cm wavelength to obtain accurate measurements of carbon radio recombination lines. When combined with atomic (CI) and molecular (CO) data, these measurements will constrain the composition, structure, kinematics, and physical properties of the photodissociation regions that lie on the edges of HII regions. A second survey was made at 3.5 cm wavelength to determine the abundance of 3He in the interstellar medium of the Milky Way. Together with measurements of the 3He+ hyperfine line we get high precis...

  14. Numerical and experimental investigations on catalytic recombiners

    International Nuclear Information System (INIS)

    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)

  15. Sweetness characterization of recombinant human lysozyme.

    Science.gov (United States)

    Matano, Mami; Nakajima, Kana; Kashiwagi, Yutaka; Udaka, Shigezo; Maehashi, Kenji

    2015-10-01

    Lysozyme, a bacteriolytic enzyme, is widely distributed in nature and is a component of the innate immune system. It is established that chicken egg lysozyme elicits sweetness. However, the sweetness of human milk lysozyme, which is vital for combating microbial infections of the gastrointestinal tract of breast-fed infants, has not been characterized. This study aimed to assess the elicitation of sweetness using recombinant mammalian lysozymes expressed in Pichia pastoris. Recombinant human lysozyme (h-LZ) and other mammalian lysozymes of mouse, dog, cat and bovine milk elicited similar sweetness as determined using a sensory test, whereas bovine stomach lysozyme (bs-LZ) did not. Assays of cell cultures showed that h-LZ activated the human sweet taste receptor hT1R2/hT1R3, whereas bs-LZ did not. Point mutations confirmed that the sweetness of h-LZ was independent of enzyme activity and substrate-binding sites, although acidic amino acid residues of bs-LZ played a significant role in diminishing sweetness. Therefore, we conclude that elicitation of sweetness is a ubiquitous function among all lysozymes including mammalian lysozymes. These findings may provide novel insights into the biological implications of T1R2/T1R3-activation by mammalian lysozyme in the oral cavity and gastrointestinal tract. However, the function of lysozyme within species lacking the functional sweet taste receptor gene, such as cat, is currently unknown. PMID:26027787

  16. Recombinant protein scaffolds for tissue engineering

    International Nuclear Information System (INIS)

    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)

  17. Analysis of recombinants in female mouse meiosis.

    Science.gov (United States)

    de Boer, Esther; Jasin, Maria; Keeney, Scott

    2013-01-01

    During meiosis, homologous chromosomes (homologs) undergo recombinational interactions, resulting in the formation of crossovers (COs) or noncrossovers (NCOs). Both COs and NCOs are initiated by the same event: programmed double-strand DNA breaks (DSBs), which occur preferentially at hotspots throughout the genome. COs contribute to the genetic diversity of gametes and are needed to promote proper meiotic chromosome segregation. Accordingly, their formation is tightly controlled. In the mouse, the sites of preferred CO formation differ between male and female chromosomes, both on a regional level and on the level of individual hotspots. Sperm typing using (half-sided) allele-specific PCR has proven a powerful technique to characterize COs and all detectable NCOs at hotspots on male human and mouse chromosomes. In contrast, very little is known about the properties of hotspots in female meiosis. This chapter describes an adaptation of sperm typing to analyze recombinants in a hotspot, using DNA isolated from an ovary cell suspension enriched for oocytes. PMID:23138942

  18. Pre-dimensioning the catalytic recombiners

    International Nuclear Information System (INIS)

    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

  19. Rapid generation of markerless recombinant MVA vaccines by en passant recombineering of a self-excising bacterial artificial chromosome.

    OpenAIRE

    Cottingham, MG; Gilbert, SC

    2010-01-01

    The non-replicating poxviral vector modified vaccinia virus Ankara (MVA) is currently a leading candidate for development of novel recombinant vaccines against globally important diseases. The 1980s technology for making recombinant MVA (and other poxviruses) is powerful and robust, but relies on rare recombination events in poxviral-infected cells. In the 21st century, it has become possible to apply bacterial artificial chromosome (BAC) technology to poxviruses, as first demonstrated by B. ...

  20. Electron-mediating Cu(A) centers in proteins

    DEFF Research Database (Denmark)

    Epel, Boris; Slutter, Claire S; Neese, Frank; Kroneck, Peter M H; Zumft, Walter G; Pecht, Israel; Farver, Ole; Lu, Yi; Goldfarb, Daniella

    2002-01-01

    High field (W-band, 95 GHz) pulsed electron-nuclear double resonance (ENDOR) measurements were carried out on a number of proteins that contain the mixed-valence, binuclear electron-mediating Cu(A) center. These include nitrous oxide reductase (N(2)OR), the recombinant water-soluble fragment of subunit II of Thermus thermophilus cytochrome c oxidase (COX) ba(3) (M160T9), its M160QT0 mutant, where the weak axial methionine ligand has been replaced by a glutamine, and the engineered "purple" azuri...

  1. Comparison of in vivo effects of human recombinant IL 1 and human recombinant IL 6 in mice

    International Nuclear Information System (INIS)

    IL 1 and IL 6 share a number of biological activities, including induction of fever, neutrophilia and acute phase response, and IL 1 induces IL 6 production by fibroblasts and macrophages. Therefore, it was proposed that IL 6 mediates many of the activities of IL 1. To test this hypothesis in vivo, we assessed induction of IL 6 following IL 1 alpha administration to mice and tested IL 6 for radioprotection and induction of early (CSF) and late (fibrinogen and SAA) acute phase reactants. IL 1 alpha given to mice ip induced, in a dose dependent manner, detectable IL 6 in circulation, with maximal titers at 2-4 hrs. However, unlike IL 1 which is 10-1000 ng/mouse of human recombinant IL 6 did not result in increased survival of mice following lethal irradiation. In fact, such treatment given 20 hrs before LD50/30 doses of radiation resulted in reduced survival of mice. However, IL 6 augmented the radioprotective effect of IL 1. IL 1 in doses above 10 ng/mouse induced within 2 to 6 hrs a dose dependent increase in CSF in circulation, but IL 6 did not induce detectable levels of CSF at 2, 6 and 20 hrs after administration. Administration of IL 6 to mice produced a dose dependent increase in circulating fibrinogen and SAA. Similarly, administration of IL 1 resulted in much greater increases in levels of fibrinogen and SAA. Therefore, IL 1 is a more effective inducer of fibrinogen and SAA in mice than is IL 6. Although definitive conclusions concerning the relative roles for IL 1 and IL 6 in vivo will await availability of anti IL 1 and anti-IL 6 antibodies, our data do not support the suggestion that the above IL 1 effects can be attributed solely to IL 6

  2. Homologous recombination mediates S-phase-dependent radioresistance in cells deficient in DNA polymerase eta.

    OpenAIRE

    Nicolay, NH; Carter, R.; Hatch, SB; Schultz, N; Prevo, R; McKenna, WG; Helleday, T; Sharma, RA

    2012-01-01

    DNA polymerase eta (pol ?) is the only DNA polymerase causally linked to carcinogenesis in humans. Inherited deficiency of pol ? in the variant form of xeroderma pigmentosum (XPV) predisposes to UV-light-induced skin cancer. Pol ?-deficient cells demonstrate increased sensitivity to cisplatin and oxaliplatin chemotherapy. We have found that XP30R0 fibroblasts derived from a patient with XPV are more resistant to cell kill by ionising radiation (IR) than the same cells complemented with wild-t...

  3. The MMS22L-TONSL complex mediates recovery from replication stress and homologous recombination

    OpenAIRE

    Lara O'Donnell; Stephanie Panier; Jan Wildenhain; Tkach, Johnny M.; Abdallah Al-Hakim; Marie-Claude Landry; Cristina Escribano-Diaz; Szilard, Rachel K; Young, Jordan T. F.; Meagan Munro; Canny, Marella D.; Kolas, Nadine K.; , Wei Zhang; Harding, Shane M.; Jarkko Ylanko

    2010-01-01

    Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here, we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogen...

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

    OpenAIRE

    Safarnejad, Mohammad Reza

    2008-01-01

    In dieser Studie wird die Expression spezifischer scFv-Fragmente in pflanzlichen Zellen für die Unterdrückung der Krankheitssymptome durch TYLCV-Infektion genutzt. Die für Rep, CP und MP kodierenden Gene C1, V1 und V2 wurden mit spezifischen Primern mit dem kompletten klonierten TYLCV Genom als Template amplifiziert. Die PCR-Produkte wurden zunächst in den TOPO Vektor und später in die Expressionsvektoren pGEX-5x3-und pMALc2x kloniert. Die rekombinanten Proteine wurden in E. coli als C-termin...

  5. Homologous Recombination Mediates Functional Recovery of Dysferlin Deficiency following AAV5 Gene Transfer

    OpenAIRE

    Grose, William E.; Clark, K. Reed; Griffin, Danielle; Malik, Vinod; Shontz, Kimberly M.; Montgomery, Chrystal L.; Lewis, Sarah; Brown, Robert H.; Janssen, Paul M.L.; Mendell, Jerry R; RODINO-KLAPAC, LOUISE R.

    2012-01-01

    The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV) gene transfer is a particularly auspicious treatment strategy, however the size of the DYSF cDNA (6.5 kb) negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8, 9). Potential...

  6. Technology-Use Mediation

    DEFF Research Database (Denmark)

    Bansler, Jørgen P.; Havn, Erling C.

    2004-01-01

    Implementation of new computer-mediated communication (CMC) systems in organizations is a complex socio-technical endeavour, involving the mutual adaptation of technology and organization over time. Drawing on the analytic concept of sensemaking, this paper provides a theoretical perspective that deepens our understanding of how organizations appropriate new electronic communication media. The paper analyzes how a group of mediators in a large, multinational company adapted a new web-based CMC t...

  7. Neurally Mediated Syncope

    OpenAIRE

    Zaqqa, Munir; Massumi, Ali

    2000-01-01

    Neurally mediated syncope is a disorder of the autonomic regulation of postural tone, which results in hypotension, bradycardia, and loss of consciousness. A wide variety of stimuli can trigger this reflex, the most common stimulus being orthostatic stress. Typically, a patient with neurally mediated syncope experiences nausea, lightheadedness, a feeling of warmth, and pallor before abruptly losing consciousness. If the cause of syncope is unclear, a stepwise approach is necessary to arrive a...

  8. Hypercharged Anomaly Mediation

    CERN Document Server

    Dermisek, Radovan; Wang, Lian-Tao

    2007-01-01

    We show that, in string models with the MSSM residing on D-branes, the bino mass can be generated in a geometrically separated hidden sector. Hypercharge mediation thus naturally teams up with anomaly mediation. The mixed scenario predicts a distinctive yet viable superpartner spectrum, provided that the ratio \\alpha between the bino and gravitino mass lies in the range 0.05 35 TeV. We summarize some of the phenomenological features of this scenario.

  9. Hypercharged Anomaly Mediation

    OpenAIRE

    Dermisek, Radovan; Verlinde, Herman; Wang, Lian-Tao

    2007-01-01

    We show that, in string models with the MSSM residing on D-branes, the bino mass can be generated in a geometrically separated hidden sector. Hypercharge mediation thus naturally teams up with anomaly mediation. The mixed scenario predicts a distinctive yet viable superpartner spectrum, provided that the ratio \\alpha between the bino and gravitino mass lies in the range 0.05 35 TeV. We summarize some of the phenomenological features of this s...

  10. Recombinant mussel adhesive protein Mgfp-5 as cell adhesion biomaterial.

    Science.gov (United States)

    Hwang, Dong Soo; Gim, Youngsoo; Kang, Dong Gyun; Kim, Yeon Kyu; Cha, Hyung Joon

    2007-01-20

    Mytilus galloprovincialis foot protein type-5 (Mgfp-5) is one of the mussel adhesive proteins that participate in adhesion with the substratum. We previously reported the production of recombinant Mgfp-5 in Escherichia coli and showed that the recombinant protein had superior adhesion abilities versus those of Cell-Tak, a commercially available mussel adhesive protein mixture. In the present work, we investigated the feasibility of using recombinant Mgfp-5 as a cell adhesion agent. Purified and tyrosinase-modified recombinant Mgfp-5 was used to adhere living anchorage-independent cells such as insect Drosophila S2 cells and human MOLT-4 cells onto glass slides. Our results revealed that these cell lines efficiently attached to recombinant Mgfp-5-coated glass surfaces, and that surface-immobilized S2 cells were viable and able to undergo cell division for up to 1 week. Cytochemical studies with 4',6-diamidino-2-phenylindole (DAPI) staining of nuclei and immunofluorescence for secreted foreign human erythropoietin (hEPO) from recombinant S2 cells and quantitative comparative analyses of S2 cell binding ability with Cell-Tak and poly-L-lysine, the main cell adhesion agent, were performed to demonstrate successful usage of recombinant Mgfp-5 for cell biological applications. Collectively, these results indicate that recombinant Mgfp-5 may be a useful new cell adhesion biomaterial for anchorage-independent cells. PMID:16979252

  11. Procedures for monitoring recombinant erythropoietin and analogs in doping.

    Science.gov (United States)

    Lamon, Séverine; Robinson, Neil; Saugy, Martial

    2010-03-01

    Hemoglobin concentration is one of the principal factors of aerobic power and, consequently, of performance in many types of physical activities. The use of recombinant human erythropoietin is, therefore, particularly powerful for improving the physical performances of patients, and, more generally, improving their quality of life. This article discusses procedures for monitoring recombinant erythropoietin and its analogues in doping for athletic performance. PMID:20122455

  12. Heterogeneity in rates of recombination across the mouse genome

    Energy Technology Data Exchange (ETDEWEB)

    Nachman, M.W.; Churchill, G.A. [Cornell Univ., Ithaca, NY (United States)

    1996-02-01

    If loci are randomly distributed on a physical map, the density of markers on a genetic map will be inversely proportional to recombination rate. First proposed by Mary Lyon, we have used this idea to estimate recombination rates from the Drosophila melanogaster linkage map. These results were compared with results of two other studies that estimated regional recombination rates in D. melanogaster using both physical and genetic maps. The three methods were largely concordant in identifying large-scale genomic patterns of recombination. The marker density method was then applied to the Mus musculus microsatellite linkage map. The distribution of microsatellites provided evidence for heterogeneity in recombination rates. Centromeric regions for several mouse chromosomes had significantly greater numbers of markers than expected, suggesting that recombination rates were lower in these regions. In contrast, most telomeric regions contained significantly fewer markers than expected. This indicates that recombination rates are elevated at the telomeres of many mouse chromosomes and is consistent with a comparison of the genetic and cytogenetic maps in these regions. The density of markers on a genetic map may provide a generally useful way to estimate regional recombination rates in species for which genetic, but not physical, maps are available. 44 refs., 5 figs., 4 tabs.

  13. Recombination in Avian Gamma-Coronavirus Infectious Bronchitis Virus

    Directory of Open Access Journals (Sweden)

    Mark W. Jackwood

    2011-09-01

    Full Text Available Recombination in the family Coronaviridae has been well documented and is thought to be a contributing factor in the emergence and evolution of different coronaviral genotypes as well as different species of coronavirus. However, there are limited data available on the frequency and extent of recombination in coronaviruses in nature and particularly for the avian gamma-coronaviruses where only recently the emergence of a turkey coronavirus has been attributed solely to recombination. In this study, the full-length genomes of eight avian gamma-coronavirus infectious bronchitis virus (IBV isolates were sequenced and along with other full-length IBV genomes available from GenBank were analyzed for recombination. Evidence of recombination was found in every sequence analyzed and was distributed throughout the entire genome. Areas that have the highest occurrence of recombination are located in regions of the genome that code for nonstructural proteins 2, 3 and 16, and the structural spike glycoprotein. The extent of the recombination observed, suggests that this may be one of the principal mechanisms for generating genetic and antigenic diversity within IBV. These data indicate that reticulate evolutionary change due to recombination in IBV, likely plays a major role in the origin and adaptation of the virus leading to new genetic types and strains of the virus.

  14. Effects of humidity on recombination in ionization chambers

    International Nuclear Information System (INIS)

    The output current from an ionization chamber is reduced from saturation current by initial recombination, back-diffusion to electrodes and volume recombination. If the ions due to those phenomena are little, the expression for the collection efficiency is derived. The collection efficiency for an ionization chamber with parallel plate electrodes is determined. In order to measure the collection efficiency for initial recombination, it is necessary to irradiate an ionization chamber at the exposure rate as low as possible. A large ionization chamber is preferable to measure signal current precisely. A parallel plate ionization chamber with a collector was constructed, and it was irradiated with a 60Co ?-ray source. For measuring the collection efficiency for volume recombination, a similar ionization chamber was used. The humidity control system used for the experiment is shown. The experimental results of initial recombination and back diffusion loss, volume recombination and so on are reported. The ion loss due to initial recombination both in clusters and in columns was observed. It was found that the initial recombination in clusters increased with humidity, but that in columns did not depend on humidity. Back diffusion loss also did not depend on humidity. (K.I.)

  15. Momentum-space calculation of four-boson recombination

    OpenAIRE

    Deltuva, A.

    2012-01-01

    The system of four identical bosons with large two-boson scattering length is described using momentum-space integral equations for the four-particle transition operators. The creation of Efimov trimers via ultracold four-boson recombination is studied. The universal behavior of the recombination rate is demonstrated.

  16. Biochemical characterization of a recombinant TRIM5alpha protein that restricts human immunodeficiency virus type 1 replication.

    Science.gov (United States)

    Langelier, Charles R; Sandrin, Virginie; Eckert, Debra M; Christensen, Devin E; Chandrasekaran, Viswanathan; Alam, Steven L; Aiken, Christopher; Olsen, John C; Kar, Alak Kanti; Sodroski, Joseph G; Sundquist, Wesley I

    2008-12-01

    The rhesus monkey intrinsic immunity factor TRIM5alpha(rh) recognizes incoming capsids from a variety of retroviruses, including human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV), and inhibits the accumulation of viral reverse transcripts. However, direct interactions between restricting TRIM5alpha proteins and retroviral capsids have not previously been demonstrated using pure recombinant proteins. To facilitate structural and mechanistic studies of retroviral restriction, we have developed methods for expressing and purifying an active chimeric TRIM5alpha(rh) protein containing the RING domain from the related human TRIM21 protein. This recombinant TRIM5-21R protein was expressed in SF-21 insect cells and purified through three chromatographic steps. Two distinct TRIM5-21R species were purified and shown to correspond to monomers and dimers, as analyzed by analytical ultracentrifugation. Chemically cross-linked recombinant TRIM5-21R dimers and mammalian-expressed TRIM5-21R and TRIM5alpha proteins exhibited similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities, indicating that mammalian TRIM5alpha proteins are predominantly dimeric. Purified TRIM5-21R had ubiquitin ligase activity and could autoubquitylate with different E2 ubiquitin conjugating enzymes in vitro. TRIM5-21R bound directly to synthetic capsids composed of recombinant HIV-1 CA-NC proteins and to authentic EIAV core particles. HIV-1 CA-NC assemblies bound dimeric TRIM5-21R better than either monomeric TRIM5-21R or TRIM5-21R constructs that lacked the SPRY domain or its V1 loop. Thus, our studies indicate that TRIM5alpha proteins are dimeric ubiquitin E3 ligases that recognize retroviral capsids through direct interactions mediated by the SPRY domain and demonstrate that these activities can be recapitulated in vitro using pure recombinant proteins. PMID:18799573

  17. Recombinant Nogo-66 via soluble expression with SUMO fusion in Escherichia coli inhibits neurite outgrowth in vitro.

    Science.gov (United States)

    Dai, Xiaoyong; Sun, Zhongqing; Liang, Rui; Li, Yu; Luo, Huanmin; Huang, Yadong; Chen, Meiwan; Su, Zhijian; Xiao, Fei

    2015-07-01

    Nogo-66, a hydrophilic loop of 66 amino acids flank two hydrophobic domains of the Nogo-A C terminus, interacts with the Nogo-66 receptor (NgR) to exert numerous functions in the central nervous system (CNS). Nogo-66 has important roles in aspects of neuronal development, including cell migration, axon guidance, fasciculation, and dendritic branching, and in aspects of CNS plasticity, including oligodendrocyte differentiation and myelination. Here, the small ubiquitin-related modifier (SUMO) was fused to the target gene, Nogo-66, and the construct was expressed in Escherichia coli (E. coli). Under the optimal fermentation conditions, the soluble expression level of the fusion protein was 33 % of the total supernatant protein. After cleaving the fusion proteins with SUMO protease and purifying them by Ni-NTA affinity chromatography, the yield and purity of recombinant Nogo-66 obtained by 10-L scale fermentation were 23?±?1.5 mg/L and greater than 93 %, respectively. The authenticity of the recombinant Nogo-66 was confirmed by an electrospray ionization-mass spectrometry analysis. The functional analyses indicated that the recombinant Nogo-66 was capable of binding the NgR specifically. The immunofluorescence results showed that the recombinant Nogo-66 could significantly inhibit neurite outgrowth of rat pheochromocytoma (PC12) cells stimulated by nerve growth factor and cerebellar granule cells (CGCs). Furthermore, Nogo-66 inhibited neurite outgrowth by increasing the level of phosphorylated Rho-associated coiled-coil-containing protein kinase 2 (ROCK2), collapsin response mediator protein 2 (CRMP2), and myosin light chain (MLC). This study provided a feasible and convenient production method for generating sufficient recombinant Nogo-66 for experimental and clinical applications. PMID:25758955

  18. Influence of exogenous phytohormones, methyl jasmonate and suppressors of jasmonate biosynthesis on Agrobacterium-mediated transient expression in Nicotiana excelsior

    Directory of Open Access Journals (Sweden)

    Kuchuk M. V.

    2012-09-01

    Full Text Available 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 diethyldithiocarbamic acid added to the infiltration buffer did not influence either GFP transient expression nor total protein accumulation in plant tissue. Conclusions. The results suggest that the tested factors are not substantial for Agrobacterium-mediated transient expression.

  19. The mediation procedure in Romania

    OpenAIRE

    Alexandrina Zaharia; Monica Saleh-Ali

    2009-01-01

    The mediation activity as an alternative way of solving conflicts occupies an important place in modernsociety. Currently, the mediation reached its maturity worldwide being adopted without reservations.The future of solving conflicts is undoubtedly closely related to mediation. XXth century is the century of solvingconflicts amiably outside the court room. In Romania and the mediation profession were regulated by the Law no.192/2006, on the basis of the idea that mediation is one of the majo...

  20. Approximating stochastic volatility by recombinant trees

    CERN Document Server

    Akyildirim, Erdinc; Soner, H Mete

    2012-01-01

    A general method to construct recombinant tree approximations for stochastic volatility models is developed and applied to the Heston model for stock price dynamics. In this application, the resulting approximation is a four tuple Markov process. The ?first two components are related to the stock and volatility processes and take values in a two dimensional Binomial tree. The other two components of the Markov process are the increments of random walks with simple values in {-1; +1}. The resulting effi?cient option pricing equations are numerically implemented for general American and European options including the standard put and calls, barrier, lookback and Asian type pay-o?ffs. The weak and extended weak convergence are also proved.

  1. Dissociative recombination of LiH2+

    Science.gov (United States)

    Thomas, R. D.; Ehlerding, A.; Geppert, W. D.; Hellberg, F.; Zhaunerchyk, V.; Larsson, M.; Bahati, E.; Bannister, M. E.; Fogle, M. R.; Vane, C. R.

    2014-05-01

    In this paper, we report results regarding how LiH2+ fragments as a result of a low-energy collision with an electron (dissociative recombination), a reaction that contains only elements and particles created during the very first phase of the universe. The collision-energy-dependent reaction rate and cross sections show detailed structures, more so than predicted by theory, suggesting significant rovibrational coupling in the ion and a complex reaction surface. From the structure of the molecule, the reaction predominantly results in the formation of Li + H2. However, 23% of the reaction flux leads to more interesting products, with 17% producing Li + 2H and 6% producing LiH + H. These last two channels break the strongest molecular bond in the system and, in the case of the latter channel, form a significantly weaker ionic bond. Possible reasons behind this interesting behavior are discussed, together with the interaction between the available reaction channels.

  2. Recombinant production of spider silk proteins.

    Science.gov (United States)

    Heidebrecht, Aniela; Scheibel, Thomas

    2013-01-01

    Natural spider silk fibers combine extraordinary properties such as stability and flexibility which results in a toughness superseding that of all other fiber materials. As the spider's aggressive territorial behavior renders their farming not feasible, the biotechnological production of spider silk proteins (spidroins) is essential in order to investigate and employ them for applications. In order to accomplish this task, two approaches have been tested: firstly, the expression of partial cDNAs, and secondly, the expression of synthetic genes in several host organisms, including bacteria, yeast, plants, insect cells, mammalian cells, and transgenic animals. The experienced problems include genetic instability, limitations of the translational and transcriptional machinery, and low solubility of the produced proteins. Here, an overview of attempts to recombinantly produce spidroins will be given, and advantages and disadvantages of the different approaches and host organisms will be discussed. PMID:23415154

  3. Initiation of Meiotic Recombination in Mammals

    Directory of Open Access Journals (Sweden)

    Rajeev Kumar

    2010-12-01

    Full Text Available Meiotic recombination is initiated by the induction of programmed DNA double strand breaks (DSBs. DSB repair promotes homologous interactions and pairing and leads to the formation of crossovers (COs, which are required for the proper reductional segregation at the first meiotic division. In mammals, several hundred DSBs are generated at the beginning of meiotic prophase by the catalytic activity of SPO11. Currently it is not well understood how the frequency and timing of DSB formation and their localization are regulated. Several approaches in humans and mice have provided an extensive description of the localization of initiation events based on CO mapping, leading to the identification and characterization of preferred sites (hotspots of initiation. This review presents the current knowledge about the proteins known to be involved in this process, the sites where initiation takes place, and the factors that control hotspot localization.

  4. Resonant Scattering and Recombination in CAL 87

    CERN Document Server

    Greiner, J; Jiménez-Garate, M A; Burwitz, V; Schwarz, R; Di Stefano, R; Schulz, N

    2004-01-01

    The eclipsing supersoft X-ray binary CAL 87 has been observed with Chandra on August 13/14, 2001 for nearly 100 ksec, covering two full orbital cycles and three eclipses. The shape of the eclipse light curve derived from the zeroth-order photons indicates that the size of the X-ray emission region is about 1.5 solar radii. The ACIS/LETG spectrum is completely dominated by emission lines without any noticeable continuum. The brightest emission lines are significantly redshifted and double-peaked, suggestive of emanating in a 2000 km/s wind. We model the X-ray spectrum by a mixture of recombination and resonant scattering. This allows us to deduce the temperature and luminosity of the ionizing source to be kT = 50-100 eV and L_X = 5E37 erg/s.

  5. Recombinant bacterial lipoproteins as vaccine candidates.

    Science.gov (United States)

    Leng, Chih-Hsiang; Liu, Shih-Jen; Chen, Hsin-Wei; Chong, Pele

    2015-12-01

    Recombinant bacterial lipoproteins (RLP) with built-in immuno-stimulating properties for novel subunit vaccine development are reviewed. This platform technology offers the following advantages: easily converts antigens into highly immunogenic RLP using a fusion sequence containing lipobox; the lipid moiety of RLP is recognized as the danger signals in the immune system through the Toll-like receptor 2, so both innate and adaptive immune responses can be induced by RLP; serves as an efficient and cost-effective bioprocess for producing RLP in Escherichia coli and the feasibility and safety of this core platform technology has been successfully demonstrated in animal model studies including meningococcal group B subunit vaccine, dengue subunit vaccine, novel subunit vaccine against Clostridium difficile-associated diseases and HPV-based immunotherapeutic vaccines. PMID:26420467

  6. Recombinant expression of backbone-cyclized polypeptides.

    Science.gov (United States)

    Borra, Radhika; Camarero, Julio A

    2013-09-01

    Here we review the different biochemical approaches available for the expression of backbone-cyclized polypeptides, including peptides and proteins. These methods allow for the production of circular polypeptides either in vitro or in vivo using standard recombinant DNA expression techniques. Polypeptide circularization provides a valuable tool to study the effects of topology on protein stability and folding kinetics. Furthermore, having biosynthetic access to backbone-cyclized polypeptides makes the production of genetically encoded libraries of cyclic polypeptides possible. The production of such libraries, which was previously restricted to the domain of synthetic chemistry, now offers biologists access to highly diverse and stable molecular libraries that can be screened using high-throughput methods for the rapid selection of novel cyclic polypeptide sequences with new biological activities. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 502-509, 2013. PMID:23893781

  7. Collisional dissociative recombination in helium-hydrogen afterglow plasmas

    Science.gov (United States)

    Johnsen, Rainer

    2012-10-01

    The puzzling dependence of electron-ion recombination in helium-hydrogen afterglows on neutralfootnotetextGlos'ik et al., Phys. Rev.A 79, 052707 (2009) and electronfootnotetextGougousi et al., Int. J. Mass Spec. Ion Proc. 149-150, 131 (1995) densities is shown to be compatible with the ``Collisional Dissociative Recombination'' mechanism, originally proposed by Collins,footnotetextCollins, Phys. Rev.A 140, 1850 (1965) in which three-body capture of electrons into molecular high Rydberg states of H3^+ leads to predissociation of the molecular core. While both electrons and neutrals play a role in the three-body capture, their effects on recombination do not add in a simple manner, which makes it difficult to distinguish three-body and binary dissociative recombination. Collision-induced angular momentum mixing (l-mixing), invoked in earlier models, also occurs but does not provide the rate-limiting step that controls the overall recombination rate.

  8. Kinetic and inactivation studies of recombinant Drosophila choline acetyltransferase.

    Science.gov (United States)

    Carbini, L; Rodriguez, G; Hersh, L B

    1990-01-01

    A cDNA for Drosophila choline acetyltransferase (ChAT) was expressed in E. coli and the recombinant enzyme partially purified. Kinetic analysis yielded the following constants for the recombinant enzyme; KmAcCoA = 29 microM, KmCoA = 25 microM, Kmcholine = 330 microM, and Kmacetylcholine = 2 mM. The recombinant Drosophila enzyme, like the enzyme from other species, exhibited an increase in activity as a function of increased salt concentration. Chemical modification studies using dithio-bis-nitro-2-carboxylate, butanedione, and diethylpyrocarbonate showed that the recombinant enzyme contains active site cysteine, arginine, and histidine residues. These studies demonstrate that the recombinant Drosophila ChAT possesses the same catalytic properties as the enzyme from a variety of other sources. PMID:2310940

  9. Sex recombination, and reproductive fitness: an experimental study using Paramecium

    Energy Technology Data Exchange (ETDEWEB)

    Nyberg, D.

    1982-08-01

    The effect of sex and recombination on reproductive fitness are measured using five wild stocks of Paramecium primaurelia. Among the wild stocks there were highly significant differences in growth rates. No hybrid had as low a fitness as the least fit parental stock. Recombination produced genotypes of higher fitness than those of either parent only in the cross between the two stocks of lowest fitness. The increase in variance of fitness as a result of recombination was almost exclusively attributable to the generation lines with low fitness. The fitness consequences of sexuality and mate choice were stock specific; some individuals leaving the most descendants by inbreeding, others by outcrossing. For most crosses the short-term advantage of sex, if any, accrue from the fusion of different gametes (hybrid vigor) and not from recombination. Since the homozygous genotype with the highest fitnes left the most progeny by inbreeding (no recombination), the persistence of conjugation in P. primaurelia is paradoxical. (JMT)

  10. Integration Frequency and Intermolecular Recombination of rAAV Vectors in Non-human Primate Skeletal Muscle and Liver

    OpenAIRE

    Nowrouzi, Ali; Penaud-Budloo, Magalie; Kaeppel, Christine; Appelt, Uwe; Le Guiner, Caroline; Moullier, Philippe; Kalle, Christof von; Snyder, Richard O.; Schmidt, Manfred

    2012-01-01

    The comprehensive characterization of recombinant adeno-associated viral (rAAV) integration frequency and persistence for assessing rAAV vector biosafety in gene therapy is severely limited due to the predominance of episomal rAAV vector genomes maintained in vivo. Introducing rAAV insertional standards (rAIS), we show that linear amplification-mediated (LAM)-PCR and deep sequencing can be used for validated measurement of rAAV integration frequencies. Integration of rAAV2/1 or rAAV2/8, follo...

  11. Adsorption Properties of a Gold-Binding Peptide Assessed by its Attachment to a Recombinant Apoferritin Molecule

    Science.gov (United States)

    Ishikawa, Kazutaka; Yamada, Kiyohito; Kumagai, Shinya; Sano, Ken-Ichi; Shiba, Kiyotaka; Yamashita, Ichiro; Kobayashi, Mime

    2008-03-01

    The adsorption properties of a recombinant apoferritin protein fused to a gold-binding peptide were characterized. The results of quartz crystal microbalance measurements showed that the fusion protein preferentially adsorbs to gold surfaces. Scanning electron microscopy also revealed that the protein selectively adsorbed onto a nanometer-scale gold pattern on a SiO2 surface fabricated by electron-beam lithography. Our results indicate that nanodots and nanowires synthesized using a biotemplate can be selectively placed onto a gold surface by genetically modifying the outer surface of the biotemplate. This technique represents an important step toward biotemplate-mediated fabrication of a nanometer-scaled device that utilizes gold electrodes.

  12. Interferon gamma is involved in the recovery of athymic nude mice from recombinant vaccinia virus/interleukin 2 infection

    OpenAIRE

    1990-01-01

    Athymic nude mice recover from an infection with recombinant vaccinia virus (VV) encoding murine interleukin 2 (IL-2), but treatment with a mAb to IL-2 accentuated infection. Administration of a mAb against interferon gamma (IFN-gamma) to mice infected with the IL-2-encoding virus completely prevented the IL-2-induced mechanisms of recovery. Both asialo-GM1+ (NK) and asialo-GM1- (non-NK) cells were participants in the IFN-gamma-mediated recovery of nude mice from infection with the IL-2-encod...

  13. Green's function for multielectron ions and its application to radiative recombination involving dielectronic recombinations

    International Nuclear Information System (INIS)

    We propose a general method to calculate the full Green's function of multielectron atomic ions. The key point exists in the usage of L2 integrable functions as a complete basis set in a finite region together with an optical potential to guaranty the outgoing scattering boundary condition. In such a way, the cumbersome procedure of adjusting boundary conditions in solving the differential Schroedinger equation is avoided. To show the validity of the method, we studied the radiative recombination involving dielectronic recombinations of Be-like Hg (Z=80) ions. The radiative damping effect is taken into account naturally in the present method. The calculated results reproduce well the asymmetric line profile observed in the experiments.

  14. Protection of pigs against Taenia solium cysticercosis by immunization with novel recombinant antigens

    OpenAIRE

    Gauci, Charles G.; Jayashi, César M.; Gonzalez, Armando E; Lackenby, Julia; Lightowlers, Marshall W

    2012-01-01

    ? Experimental Taenia solium challenge of piglets vaccinated with recombinant antigens. ? Protection of piglets immunized with the TSOL16 recombinant antigen. ? Assessment of antibody responses in pigs vaccinated with recombinant antigens.

  15. 75 FR 42114 - Office of Biotechnology Activities; Recombinant DNA Research: Proposed Action Under the NIH...

    Science.gov (United States)

    2010-07-20

    ...Research Involving Recombinant DNA Molecules (NIH Guidelines...generation of transgenic rodents by recombinant DNA technology must be registered with the...the animal's genome using recombinant DNA technology or breeding one or more...

  16. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Science.gov (United States)

    2010-04-01

    ...surgical suture produced by recombinant DNA technology. 878.4494 Section...surgical suture produced by recombinant DNA technology. (a) Identification...Surgical Suture Produced by Recombinant DNA Technology.” For the...

  17. Sex and speciation: the paradox that non-recombining DNA promotes recombination

    OpenAIRE

    Idnurm, Alexander

    2011-01-01

    The benefits of sexual reproduction that outweigh its costs have long puzzled biologists. Increased genetic diversity generated by new allelic combinations, as enhanced by recombination during meiosis, is considered to be a primary benefit of sex. Sex-determining systems have evolved independently on numerous occasions. One of the most familiar is the use of sex chromosomes in vertebrates. Other eukaryotic groups also use sex chromosomes or smaller sex-determining regions within their chromos...

  18. Genome Sequence of Complex HIV-1 Unique Recombinant Forms Sharing a Common Recombination Breakpoint Identified in Malaysia

    Science.gov (United States)

    Cheong, Hui Ting; Ng, Kim Tien; Ong, Lai Yee; Takebe, Yutaka; Chan, Kok Gan; Koh, Clayton; Al-Darraji, Haider Abdulrazzaq Abed; Kamarulzaman, Adeeba

    2015-01-01

    Three strains of HIV-1 unique recombinant forms (URFs) descended from subtypes B, B?, and CRF01_AE were identified among people who inject drugs in Kuala Lumpur, Malaysia. These three URFs shared a common recombination breakpoint in the reverse transcriptase region, indicating frequent linkage within the drug-injecting networks in Malaysia. PMID:26543107

  19. Genome Sequence of Complex HIV-1 Unique Recombinant Forms Sharing a Common Recombination Breakpoint Identified in Malaysia.

    Science.gov (United States)

    Cheong, Hui Ting; Ng, Kim Tien; Ong, Lai Yee; Takebe, Yutaka; Chan, Kok Gan; Koh, Clayton; Al-Darraji, Haider Abdulrazzaq Abed; Kamarulzaman, Adeeba; Tee, Kok Keng

    2015-01-01

    Three strains of HIV-1 unique recombinant forms (URFs) descended from subtypes B, B', and CRF01_AE were identified among people who inject drugs in Kuala Lumpur, Malaysia. These three URFs shared a common recombination breakpoint in the reverse transcriptase region, indicating frequent linkage within the drug-injecting networks in Malaysia. PMID:26543107

  20. Cold Spring Harbor symposia on quantitative biology: Volume 49, Recombination at the DNA level

    International Nuclear Information System (INIS)

    This volume contains full papers prepared by the participants to the 1984 Cold Springs Harbor Symposia on Quantitative Biology. This year's theme is entitled Recombination at the DNA level. The volume consists of 93 articles grouped into subject areas entitled chromosome mechanics, yeast systems, mammalian homologous recombination, transposons, mu, plant transposons/T4 recombination, topoisomerase, resolvase and gyrase, Escherichia coli general recombination, RecA, repair, leukaryotic enzymes, integration and excision of bacteriophage, site-specific recombination, and recombination in vitro

  1. Metadata based mediator generation

    Energy Technology Data Exchange (ETDEWEB)

    Critchlow, T

    1998-03-01

    Mediators are a critical component of any data warehouse, particularly one utilizing partially materialized views; they transform data from its source format to the warehouse representation while resolving semantic and syntactic conflicts. The close relationship between mediators and databases, requires a mediator to be updated whenever an associated schema is modified. This maintenance may be a significant undertaking if a warehouse integrates several dynamic data sources. However, failure to quickly perform these updates significantly reduces the reliability of the warehouse because queries do not have access to the m current data. This may result in incorrect or misleading responses, and reduce user confidence in the warehouse. This paper describes a metadata framework, and associated software designed to automate a significant portion of the mediator generation task and thereby reduce the effort involved in adapting the schema changes. By allowing the DBA to concentrate on identifying the modifications at a high level, instead of reprogramming the mediator, turnaround time is reduced and warehouse reliability is improved.

  2. Suppression of Meiotic Recombination by CENP-B Homologs in Schizosaccharomyces pombe.

    Science.gov (United States)

    Johansen, Peter; Cam, Hugh P

    2015-11-01

    Meiotic homologous recombination (HR) is not uniform across eukaryotic genomes, creating regions of HR hot- and coldspots. Previous study reveals that the Spo11 homolog Rec12 responsible for initiation of meiotic double-strand breaks in the fission yeast Schizosaccharomyces pombe is not targeted to Tf2 retrotransposons. However, whether Tf2s are HR coldspots is not known. Here, we show that the rates of HR across Tf2s are similar to a genome average but substantially increase in mutants deficient for the CENP-B homologs. Abp1, which is the most prominent of the CENP-B family members and acts as the primary determinant of HR suppression at Tf2s, is required to prevent gene conversion and maintain proper recombination exchange of homologous alleles flanking Tf2s. In addition, Abp1-mediated suppression of HR at Tf2s requires all three of its domains with distinct functions in transcriptional repression and higher-order genome organization. We demonstrate that HR suppression of Tf2s can be robustly maintained despite disruption to chromatin factors essential for transcriptional repression and nuclear organization of Tf2s. Intriguingly, we uncover a surprising cooperation between the histone methyltransferase Set1 responsible for histone H3 lysine 4 methylation and the nonhomologous end joining pathway in ensuring the suppression of HR at Tf2s. Our study identifies a molecular pathway involving functional cooperation between a transcription factor with epigenetic regulators and a DNA repair pathway to regulate meiotic recombination at interspersed repeats. PMID:26354768

  3. Expression and Isolation of Recombinant Microneme 3 (MIC3 Protein of Toxoplasma gondii Local Isolate on Eschericia coli (BL21

    Directory of Open Access Journals (Sweden)

    D Indrasanti

    2011-05-01

    Full Text Available Toxoplasmosis is a disease that infects all warm-blooded animals, including livestocks and humans caused by Toxoplasma gondii parasites. There are major drugs used for the therapy, though they have some effects to the patients, such as allergy, toxic and teratogenic for fetus. In addition, toxoplasmosis treatment is only effective for tachyzoites T. gondii in acute infection, while tissue cysts cannot be eradicated in chronic toxoplasmosis Tissue cysts of T. gondii contained in meat that are consumed by humans and meat-derived products may be important sources of infection for humans. Microneme protein (MIC is one of proteins that belongs to excretory-secretory antigens (ESAs of Toxoplasma gondii. Microneme 3 protein (MIC3 is the protein that plays an important role in the invasion process during cell infection as a mediator attachment parasite to the host cell. Recombinant MIC3 protein has been already used for the detection of toxoplasmosis and it could induce humoral and cellular immune response in experimental animals. The aim of this research was to express MIC3 recombinant protein of T. gondii from local isolate that was cloned into expression vector and transformed to E. coli BL21. In the future, recombinant protein MIC3 can be used for vaccine candidate and diagnostic tools for toxoplasmosis in animals and humans. Gene of MIC3 T. gondii local isolate (1.2 Kbp was cloned into expression vector pET-32a(+ (5.9 Kbp and transformed to Escherichia coli BL21. Protein from plasmid recombinant (7.1 Kbp was expressed and performed by culturing recombinant bacteria into LB medium containing ampicillin and IPTG. Recombinant protein was isolated by sonication method and identified using SDS-PAGE. Finally, the recombinant protein was analyzed by immunoblotting using anti-ESAs polyclonal antibody. In conclusion, expression of the MIC3 gene with ~108 kDa has been successfully performed by cloning gene encoding for MIC3 protein of T. gondii local isolate that can be identified with polyclonal antibody anti-ESAs.Key Words: Toxoplasma gondii, expression, MIC3 protein

  4. Persistent Expression of Biologically Active Anti-HER2 Antibody by AAVrh.10-mediated Gene Transfer*

    OpenAIRE

    WANG Guoqing; Qiu, Jianping; Wang, Rui; Krause, Anja; Boyer, Julie L.; Hackett, Neil R.; Crystal, Ronald G.

    2010-01-01

    Trastuzumab (Herceptin) is a recombinant humanized monoclonal antibody directed against an extracellular region of the HER2 protein. We hypothesized that a single adeno-associated virus mediated genetic delivery of an anti-HER2 antibody should be effective in mediating long term production of anti-HER2 and in suppressing the growth of human tumors in a xenograft model in nude mice. The adeno-associated virus gene transfer vector AAVrh.10?HER2 was constructed based on non-human primate AAV ser...

  5. Recombinant glucose oxidase from Penicillium amagasakiense for efficient bioelectrochemical applications in physiological conditions.

    Science.gov (United States)

    Courjean, Olivier; Mano, Nicolas

    2011-01-10

    GOX is the most widely used enzyme for the development of electrochemical glucose biosensors and biofuel cell in physiological conditions. The present work describes the production of a recombinant glucose oxidase from Penicillium amagasakiense (yGOXpenag) displaying a more efficient glucose catalysis (k(cat)/K(M)(glucose)=93 ?M?¹ s?¹) than the native GOX from Aspergillus niger (nGOXaspng), which is the most industrially used (k(cat)/K(M)(glucose)=27 ?M?¹ s?¹). Expression in Pichia pastoris allowed easy production and purification of the recombinant active enzyme, without overglycosylation. Its biotechnological interest was further evaluated by measuring kinetics of ferrocinium-methanol (FM(ox)) reduction, which is commonly used for electron transfer to the electrode surface. Despite their homologies in sequence and structure, pH-dependent FM(ox) reduction was different between the two enzymes. At physiological pH and temperature, we observed that electron transfer to the redox mediator is also more efficient for yGOXpenag than for nGOXaspng(k(cat)/K(M)(FM(ox))=27 ?M?¹ s?¹ and 17 ?M?¹ s?¹ respectively). In our model system, the catalytic current observed in the presence of blood glucose concentration (5 mM) was two times higher with yGOXpenag than with nGOXaspng. All our results indicated that yGOXpenag is a better candidate for industrial development of efficient bioelectrochemical devices used in physiological conditions. PMID:21040747

  6. Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes

    Directory of Open Access Journals (Sweden)

    Jofre Juan

    2006-09-01

    Full Text Available Abstract Background The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products. The aim of this study was to induce mutations in the genome of some temperate Shiga toxin encoding bacteriophages. When phage genes are in the prophage state, they behave like chromosomal genes. This enables marker genes, such as antibiotic resistance genes, to be incorporated into the stx gene. Once the phages' lytic cycle is activated, recombinant Shiga toxin converting phages are produced. These phages can transfer the marker genes to the bacteria that they infect and convert. As the Red system's effectiveness decreased when used for our purposes, we had to introduce significant variations to the original method. These modifications included: confirming the stability of the target stx gene increasing the number of cells to be transformed and using a three-step PCR method to produce the amplimer containing the antibiotic resistance gene. Results Seven phages carrying two different antibiotic resistance genes were derived from phages that are directly involved in the pathogenesis of Shiga toxin-producing strains, using this modified protocol. Conclusion This approach facilitates exploration of the transduction processes and is a valuable tool for studying phage-mediated horizontal gene transfer.

  7. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand

    OpenAIRE

    Hsu, Hailing; Lacey, David L.; Dunstan, Colin R; Solovyev, Irina; Colombero, Anne; Timms, Emma; Tan, Hong-Lin; Elliott, Gary; Kelley, Michael J; Sarosi, Ildiko; Wang, Ling; Xia, Xing-Zhong; Elliott, Robin; Chiu, Laura; Black, Tabitha

    1999-01-01

    A receptor that mediates osteoprotegerin ligand (OPGL)-induced osteoclast differentiation and activation has been identified via genomic analysis of a primary osteoclast precursor cell cDNA library and is identical to the tumor necrosis factor receptor (TNFR) family member RANK. The RANK mRNA was highly expressed by isolated bone marrow-derived osteoclast progenitors and by mature osteoclasts in vivo. Recombinant OPGL binds specifically to RANK expressed by transfected cell lines and purified...

  8. Recombinant norovirus-specific scFv inhibit virus-like particle binding to cellular ligands

    Directory of Open Access Journals (Sweden)

    Hardy Michele E

    2008-01-01

    Full Text Available Abstract Background Noroviruses cause epidemic outbreaks of gastrointestinal illness in all age-groups. The rapid onset and ease of person-to-person transmission suggest that inhibitors of the initial steps of virus binding to susceptible cells have value in limiting spread and outbreak persistence. We previously generated a monoclonal antibody (mAb 54.6 that blocks binding of recombinant norovirus-like particles (VLP to Caco-2 intestinal cells and inhibits VLP-mediated hemagglutination. In this study, we engineered the antigen binding domains of mAb 54.6 into a single chain variable fragment (scFv and tested whether these scFv could function as cell binding inhibitors, similar to the parent mAb. Results The scFv54.6 construct was engineered to encode the light (VL and heavy (VH variable domains of mAb 54.6 separated by a flexible peptide linker, and this recombinant protein was expressed in Pichia pastoris. Purified scFv54.6 recognized native VLPs by immunoblot, inhibited VLP-mediated hemagglutination, and blocked VLP binding to H carbohydrate antigen expressed on the surface of a CHO cell line stably transfected to express ? 1,2-fucosyltransferase. Conclusion scFv54.6 retained the functional properties of the parent mAb with respect to inhibiting norovirus particle interactions with cells. With further engineering into a form deliverable to the gut mucosa, norovirus neutralizing antibodies represent a prophylactic strategy that would be valuable in outbreak settings.

  9. Images and mediation

    OpenAIRE

    Olivier, Bert

    2010-01-01

    This paper focuses on the question of mediation via images. Its point of departure is the work of Kant on the mediation of human reality by the faculty of reason, including imagination and the forms of space and time, as well as the categories of the understanding, all of which combine to render an intelligible, spatiotemporal world, as opposed to the inaccessible realm of ‘things-in-themselves’. This is followed by a scrutiny of Gombrich’s claim, that artistic schemata comprise a...

  10. Hypercharged Anomaly Mediation

    Science.gov (United States)

    Dermíšek, Radovan; Verlinde, Herman; Wang, Lian-Tao

    2008-04-01

    We show that, in string models with the minimal supersymmetric standard model residing on D-branes, the bino mass can be generated in a geometrically separated hidden sector. Hypercharge mediation thus naturally teams up with anomaly mediation. The mixed scenario predicts a distinctive yet viable superpartner spectrum, provided that the ratio ? between the bino and gravitino mass lies in the range 0.05?|?|?0.25 and m3/2?35TeV. We summarize some of the experimental signatures of this scenario.

  11. Numerical Study of Passive Catalytic Recombiner for Hydrogen Mitigation

    Directory of Open Access Journals (Sweden)

    Pavan K Sharma

    2010-10-01

    Full Text Available A significant amount of hydrogen is expected to be released within the containment of a water cooled power reactor after a severe accident. To reduce the risk of deflagration/detonation various means for hydrogen control have been adopted all over the world. Passive catalytic recombiner with vertical flat catalytic plate is one of such hydrogen mitigating device. Passive catalytic recombiners are designed for the removal of hydrogen generated in order to limit the impact of possible hydrogen combustion. Inside a passive catalytic recombiner, numerous thin steel sheets coated with catalyst material are vertically arranged at the bottom opening of a sheet metal housing forming parallel flow channels for the surrounding gas atmosphere. Already below conventional flammability limits, hydrogen and oxygen react exothermally on the catalytic surfaces forming harmless steam. Detailed numerical simulations and experiments are required for an in-depth knowledge of such plate type catalytic recombiners. Specific finite volume based in-house CFD code has been developed to model and analyse the working of these recombiner. The code has been used to simulate the recombiner device used in the Gx-test series of Battelle-Model Containment (B-MC experiments. The present paper briefly describes the working principle of such passive catalytic recombiner and salient feature of the CFD model developed at Bhabha Atomic Research Centre (BARC. Finally results of the calculations and comparison with existing data are discussed.

  12. Radiative recombination data for tungsten ions: III. W-W

    Science.gov (United States)

    Trzhaskovskaya, M. B.; Nikulin, V. K.

    2014-09-01

    This paper completes the cycle of our calculations of the radiative recombination and photoionization data for tungsten ions. Presented here are the photoionization and radiative recombination cross sections, radiative recombination rate coefficients, and radiated power loss rate coefficients for ten tungsten impurity ions from W14+ to W23+. These data are required in diagnostics and modeling fusion plasmas studied in such devices as ITER, ASDEX Upgrade, and EBIT. Partial photoionization cross sections have been fitted by an analytical expression with five fit parameters tabulated here. Total radiative recombination cross sections are presented in the electron energy range from 1 eV to ?80 keV. Radiative recombination rates and radiated power loss rates are given in the temperature range from 104 K to 109 K. Calculations have been performed on the basis of the fully relativistic treatment of photoionization and radiative recombination taking into account all significant multipoles of the radiative field. Electron wave functions have been obtained by the Dirac-Fock method with the proper consideration of the electron exchange. The relativistic Maxwell-Jüttner distribution of continuum electrons has been used in calculations of radiative recombination rates and radiated power loss rates. This decreases values of the rates noticeably at a high temperature as compared to the usual non-relativistic Maxwell-Boltzmann distribution.

  13. Plasmid-mediated resistance to protein biosynthesis inhibitors in staphylococci.

    Science.gov (United States)

    Schwarz, Stefan; Fessler, Andrea T; Hauschild, Tomasz; Kehrenberg, Corinna; Kadlec, Kristina

    2011-12-01

    Protein biosynthesis inhibitors (PBIs) represent powerful antimicrobial agents for the control of bacterial infections. In staphylococci, numerous resistance genes are known to be involved in resistance to PBIs, most of which mediate resistance to a specific class/subclass of PBIs, though a few genes do confer a multidrug resistance phenotype-up to five classes/subclasses of PBIs. Plasmids play a key role in the dissemination of PBI resistance among staphylococci, as they primarily carry plasmid-borne PBI resistance genes; however, plasmids also can be vectors for transposon-borne PBI resistance genes. Small plasmids that carry single PBI resistance genes are widespread among staphylococci of human and animal origin. Various mechanisms exist by which they can recombine, form cointegrates, or integrate into chromosomal DNA or larger plasmids. We provide an overview of the current knowledge of plasmid-mediated PBI resistance in staphylococci, with particular reference to the currently known PBI resistance genes, their association with mobile genetic elements, and the recombination/integration processes that control their mobility. PMID:22191528

  14. Analysis of intermolecular RNA-RNA recombination by rubella virus

    International Nuclear Information System (INIS)

    To investigate whether rubella virus (RUB) undergoes intermolecular RNA-RNA recombination, cells were cotransfected with pairs of in vitro transcripts from genomic cDNA plasmid vectors engineered to contain nonoverlapping deletions: the replicative transcript maintained the 5'-proximal nonstructural (NS) ORF (which contained the replicase, making it RNA replication competent), had a deletion in the 3'-proximal structural protein (SP) ORF, and maintained the 3' end of the genome, including the putative 3' cis-acting elements (CSE), while the nonreplicative transcript consisted of the 3' half of the genome including the SP-ORF and 3' CSE. Cotransfection yielded plaque-forming virus that synthesized the standard genomic and subgenomic RNAs and thus was generated by RNA-RNA recombination. Using transcripts tagged with a 3'-terminal deletion, it was found that recombinants contained the 3' end derived from the replicative strand, indicating a cis-preference for initiation of negative-strand synthesis. In cotransfections in which the replicative transcript lacked the 3' CSE, recombination occurred, albeit at lower efficiency, indicating that initiation in trans from the NS-ORF can occur. The 3' CSE was sufficient as a nonreplicative transcript, showing that it can serve as a promoter for negative-strand RNA synthesis. While deletion mutagenesis showed that the presence of the junction untranslated region (J-UTR) between the ORFs appeared to be necessary on both transcripts for recombination in this region of the genome, analysis with transcripts tagged with restriction sites showed that the J-UTR was not a hot spot for recombination compared to neighboring regions in both ORFs. Sequence analysis of recombinants revealed that both precise (homologous) and imprecise recombination (aberrant, homologous resulting in duplications) occurred; however, imprecise recombination only involved the J-UTR or the 3' end of the NS-ORF and the J-UTR (maintaining the NS-ORF), indicating selection pressure against duplications in other regions of the genome

  15. ACG: rapid inference of population history from recombining nucleotide sequences

    Directory of Open Access Journals (Sweden)

    O'Fallon Brendan D

    2013-02-01

    Full Text Available Abstract Background Reconstruction of population history from genetic data often requires Monte Carlo integration over the genealogy of the samples. Among tools that perform such computations, few are able to consider genetic histories including recombination events, precluding their use on most alignments of nuclear DNA. Explicit consideration of recombinations requires modeling the history of the sequences with an Ancestral Recombination Graph (ARG in place of a simple tree, which presents significant computational challenges. Results ACG is an extensible desktop application that uses a Bayesian Markov chain Monte Carlo procedure to estimate the posterior likelihood of an evolutionary model conditional on an alignment of genetic data. The ancestry of the sequences is represented by an ARG, which is estimated from the data with other model parameters. Importantly, ACG computes the full, Felsenstein likelihood of the ARG, not a pairwise or composite likelihood. Several strategies are used to speed computations, and ACG is roughly 100x faster than a similar, recombination-aware program. Conclusions Modeling the ancestry of the sequences with an ARG allows ACG to estimate the evolutionary history of recombining nucleotide sequences. ACG can accurately estimate the posterior distribution of population parameters such as the (scaled population size and recombination rate, as well as many aspects of the recombinant history, including the positions of recombination breakpoints, the distribution of time to most recent common ancestor along the sequence, and the non-recombining trees at individual sites. Multiple substitution models and population size models are provided. ACG also provides a richly informative graphical interface that allows users to view the evolution of model parameters and likelihoods in real time.

  16. Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

    International Nuclear Information System (INIS)

    We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by ?25-fold in WT MEFs, but only by ?4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency ?23-fold in WT MEFs, but only ?4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, ?59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only ?28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene therapy

  17. General theoretical description of N-body recombination.

    Science.gov (United States)

    Mehta, N P; Rittenhouse, Seth T; D'Incao, J P; von Stecher, J; Greene, Chris H

    2009-10-01

    Formulas for the cross section and event rate constant describing recombination of N particles are derived in terms of general S-matrix elements. Our result immediately yields the generalized Wigner threshold scaling for the recombination of N bosons. A semianalytical formula encapsulates the overall scaling with energy and scattering length, as well as resonant modifications by the presence of N-body states near the threshold collision energy in the entrance channel. We then apply our model to the case of four-boson recombination into an Efimov trimer and a free atom. PMID:19905635

  18. Sequence and recombination analyses of the geminivirus replication initiator protein

    Indian Academy of Sciences (India)

    T Vadivukarasi; K R Girish; R Usha

    2007-01-01

    The sequence motifs present in the replication initiator protein (Rep) of geminiviruses have been compared with those present in all known rolling circle replication initiators. The predicted secondary structures of Rep representing each group of organisms have been compared and found to be conserved. Regions of recombination in the Rep gene and the adjoining 5? intergenic region (IR) of representative species of Geminiviridae have been identified using Recombination Detection Programs. The possible implications of such recombinations on the increasing host range of geminivirus infections are discussed.

  19. Recombinative generalization of subword units using matching to sample.

    LENUS (Irish Health Repository)

    Mahon, Catherine

    2010-01-01

    The purpose of the current study was to develop and test a computerized matching-to-sample (MTS) protocol to facilitate recombinative generalization of subword units (onsets and rimes) and recognition of novel onset-rime and onset-rime-rime words. In addition, we sought to isolate the key training components necessary for recombinative generalization. Twenty-five literate adults participated. Conditional discrimination training emerged as a crucial training component. These findings support the effectiveness of MTS in facilitating recombinative generalization, particularly when conditional discrimination training with subword units is used.

  20. Intershell trielectronic recombination with K -shell excitation in Kr30+

    Science.gov (United States)

    Beilmann, C.; Postavaru, O.; L. H. Arntzen; Ginzel, R.; Keitel, C. H.; Mäckel, V.; Mokler, P. H.; Simon, M. C.; Tawara, H.; Tupitsyn, I. I.; Ullrich, J.; Crespo López-Urrutia, J. R.; Harman, Z.

    2009-11-01

    We report the observation of trielectronic recombination with simultaneous excitation of a K -shell and an L -shell electron, hence involving three active electrons. This process was identified in the x-ray emission spectrum of recombining highly charged Kr ions. An energy resolution three times higher than any reported for this collision energy range around 10 keV resulted in the separation of the associated lines from the stronger dielectronic resonances. For Kr30+ , intershell trielectronic recombination contributions of nearly 6% to the total resonant photorecombination rate were found.