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Sample records for recombinant full length

  1. Purification and Fibrillation of Full-Length Recombinant PrP

    OpenAIRE

    Makarava, Natallia; Baskakov, Ilia V.

    2012-01-01

    Misfolding and aggregation of prion protein (PrP) is related to several neurodegenerative diseases in humans such as Creutzfeldt–Jacob disease, fatal familial insomnia, and Gerstmann–Straussler–Sheinker disease. Certain applications in prion area require recombinant PrP of high purity and quality. Here, we report an experimental procedure for expression and purification of full-length mammalian PrP. This protocol has been proved to yield PrP of extremely high purity that lac...

  2. Purification and Fibrillation of Full-Length Recombinant PrP.

    Science.gov (United States)

    Makarava, Natallia; Savtchenko, Regina; Baskakov, Ilia V

    2017-01-01

    Misfolding and aggregation of prion protein are related to several neurodegenerative diseases in humans such as Creutzfeldt-Jakob disease, fatal familial insomnia, and Gerstmann-Straussler-Scheinker disease. A growing number of applications in the prion field including assays for detection of PrP Sc and methods for production of PrP Sc de novo require recombinant prion protein (PrP) of high purity and quality. Here, we report an experimental procedure for expression and purification of full-length mammalian prion protein. This protocol has been proved to yield PrP of extremely high purity that lacks PrP adducts, oxidative modifications, or truncation, which is typically generated as a result of spontaneous oxidation or degradation. We also describe methods for preparation of amyloid fibrils from recombinant PrP in vitro. Recombinant PrP fibrils can be used as a noninfectious synthetic surrogate of PrP Sc for development of prion diagnostics including generation of PrP Sc -specific antibody.

  3. Near Full-Length Identification of a Novel HIV-1 CRF01_AE/B/C Recombinant in Northern Myanmar.

    Science.gov (United States)

    Zhou, Yan-Heng; Chen, Xin; Liang, Yue-Bo; Pang, Wei; Qin, Wei-Hong; Zhang, Chiyu; Zheng, Yong-Tang

    2015-08-01

    The Myanmar-China border appears to be the "hot spot" region for the occurrence of HIV-1 recombination. The majority of the previous analyses of HIV-1 recombination were based on partial genomic sequences, which obviously cannot reflect the reality of the genetic diversity of HIV-1 in this area well. Here, we present a near full-length characterization of a novel HIV-1 CRF01_AE/B/C recombinant isolated from a long-distance truck driver in Northern Myanmar. It is the first description of a near full-length genomic sequence in Myanmar since 2003, and might be one of the most complicated HIV-1 chimeras ever detected in Myanmar, containing four CRF01_AE, six B segments, and five C segments separated by 14 breakpoints throughout its genome. The discovery and characterization of this new CRF01_AE/B/C recombinant indicate that intersubtype recombination is ongoing in Myanmar, continuously generating new forms of HIV-1. More work based on near full-length sequence analyses is urgently needed to better understand the genetic diversity of HIV-1 in these regions.

  4. Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

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    B.M. Ribeiro

    1998-06-01

    Full Text Available The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs. Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV. The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

  5. 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

    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...

  6. Cocrystallization studies of full-length recombinant butyrylcholinesterase (BChE) with cocaine

    Energy Technology Data Exchange (ETDEWEB)

    Asojo, Oluwatoyin Ajibola; Asojo, Oluyomi Adebola; Ngamelue, Michelle N.; Homma, Kohei; Lockridge, Oksana (Nebraska-Med)

    2011-09-16

    Human butyrylcholinesterase (BChE; EC 3.1.1.8) is a 340 kDa tetrameric glycoprotein that is present in human serum at about 5 mg l{sup -1} and has well documented therapeutic effects on cocaine toxicity. BChE holds promise as a therapeutic that reduces and finally eliminates the rewarding effects of cocaine, thus weaning an addict from the drug. There have been extensive computational studies of cocaine hydrolysis by BChE. Since there are no reported structures of BChE with cocaine or any of the hydrolysis products, full-length monomeric recombinant wild-type BChE was cocrystallized with cocaine. The refined 3 {angstrom} resolution structure appears to retain the hydrolysis product benzoic acid in sufficient proximity to form a hydrogen bond to the active-site Ser198.

  7. Potency of full-length MGF to induce maximal activation of the IGF-I R Is similar to recombinant human IGF-I at high equimolar concentrations

    NARCIS (Netherlands)

    J.A.M.J.L. Janssen (Joseph); L.J. Hofland (Leo); C.J. Strasburger; E.S.R.D. Van Dungen (Elisabeth S.R. Den); M. Thevis (Mario)

    2016-01-01

    textabstractAims To compare full-length mechano growth factor (full-length MGF) with human recombinant insulin-like growth factor-I (IGF-I) and human recombinant insulin (HI) in their ability to activate the human IGF-I receptor (IGF-IR), the human insulin receptor (IR-A) and the human insulin

  8. Copper Coordination in the Full-Length, Recombinant Prion Protein†

    Science.gov (United States)

    Burns, Colin S.; Aronoff-Spencer, Eliah; Legname, Giuseppe; Prusiner, Stanley B.; Antholine, William E.; Gerfen, Gary J.; Peisach, Jack; Millhauser, Glenn L.

    2010-01-01

    The prion protein (PrP) binds divalent copper at physiologically relevant conditions and is believed to participate in copper regulation or act as a copper-dependent enzyme. Ongoing studies aim at determining the molecular features of the copper binding sites. The emerging consensus is that most copper binds in the octarepeat domain, which is composed of four or more copies of the fundamental sequence PHGGGWGQ. Previous work from our laboratory using PrP-derived peptides, in conjunction with EPR and X-ray crystallography, demonstrated that the HGGGW segment constitutes the fundamental binding unit in the octarepeat domain [Burns et al. (2002) Biochemistry 41, 3991–4001; Aronoff-Spencer et al. (2000) Biochemistry 39, 13760–13771]. Copper coordination arises from the His imidazole and sequential deprotonated glycine amides. In this present work, recombinant, full-length Syrian hamster PrP is investigated using EPR methodologies. Four copper ions are taken up in the octarepeat domain, which supports previous findings. However, quantification studies reveal a fifth binding site in the flexible region between the octarepeats and the PrP globular C-terminal domain. A series of PrP peptide constructs show that this site involves His96 in the PrP(92–96) segment GGGTH. Further examination by X-band EPR, S-band EPR, and electron spin–echo envelope spectroscopy, demonstrates coordination by the His96 imidazole and the glycine preceding the threonine. The copper affinity for this type of binding site is highly pH dependent, and EPR studies here show that recombinant PrP loses its affinity for copper below pH 6.0. These studies seem to provide a complete profile of the copper binding sites in PrP and support the hypothesis that PrP function is related to its ability to bind copper in a pH-dependent fashion. PMID:12779334

  9. dsRNA binding characterization of full length recombinant wild type and mutants Zaire ebolavirus VP35.

    Science.gov (United States)

    Zinzula, Luca; Esposito, Francesca; Pala, Daniela; Tramontano, Enzo

    2012-03-01

    The Ebola viruses (EBOVs) VP35 protein is a multifunctional major virulence factor involved in EBOVs replication and evasion of the host immune system. EBOV VP35 is an essential component of the viral RNA polymerase, it is a key participant of the nucleocapsid assembly and it inhibits the innate immune response by antagonizing RIG-I like receptors through its dsRNA binding function and, hence, by suppressing the host type I interferon (IFN) production. Insights into the VP35 dsRNA recognition have been recently revealed by structural and functional analysis performed on its C-terminus protein. We report the biochemical characterization of the Zaire ebolavirus (ZEBOV) full-length recombinant VP35 (rVP35)-dsRNA binding function. We established a novel in vitro magnetic dsRNA binding pull down assay, determined the rVP35 optimal dsRNA binding parameters, measured the rVP35 equilibrium dissociation constant for heterologous in vitro transcribed dsRNA of different length and short synthetic dsRNA of 8bp, and validated the assay for compound screening by assessing the inhibitory ability of auryntricarboxylic acid (IC(50) value of 50μg/mL). Furthermore, we compared the dsRNA binding properties of full length wt rVP35 with those of R305A, K309A and R312A rVP35 mutants, which were previously reported to be defective in dsRNA binding-mediated IFN inhibition, showing that the latter have measurably increased K(d) values for dsRNA binding and modified migration patterns in mobility shift assays with respect to wt rVP35. Overall, these results provide the first characterization of the full-length wt and mutants VP35-dsRNA binding functions. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Assessment and optimization of theileria parva sporozoite full-length p67 antigen expression in mammalian cells

    Science.gov (United States)

    Delivery of various forms of recombinant Theileria parva sporozoite antigen (p67) has been shown to elicit antibody responses in cattle capable of providing protection against East Coast fever, the clinical disease caused by T. parva. Previous formulations of full-length and shorter recombinant vers...

  11. Production of enzymatically active recombinant full-length barley high pI alpha-glucosidase of glycoside family 31 by high cell-density fermentation of Pichia pastoris and affinity purification

    DEFF Research Database (Denmark)

    Næsted, Henrik; Kramhøft, Birte; Lok, F.

    2006-01-01

    Recombinant barley high pI alpha-glucosidase was produced by high cell-density fermentation of Pichia pastoris expressing the cloned full-length gene. The gene was amplified from a genomic clone and exons (coding regions) were assembled by overlap PCR. The resulting cDNA was expressed under contr...... nM x s(-1), and 85 s(-1) using maltose as substrate. This work presents the first production of fully active recombinant alpha-glucosidase of glycoside hydrolase family 31 from higher plants. (c) 2005 Elsevier Inc. All rights reserved....

  12. Evidence for a Complex Mosaic Genome Pattern in a Full-length Hepatitis C Virus Sequence

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    R.S. Ross

    2008-01-01

    Full Text Available The genome of the hepatitis C virus (HCV exhibits a high genetic variability. This remarkable heterogeneity is mainly attributed to the gradual accumulation of mutational changes, whereas the contribution of recombination events to the evolution of HCV remains controversial so far. While performing phylogenetic analyses including a large number of sequences deposited in the GenBank, we encountered a full-length HCV sequence (AY651061 that showed evidence for inter-subtype recombination and was, therefore, subjected to a detailed analysis of its molecular structure. The obtained results indicated that AY651061 does not represent a “simple” HCV 1c isolate, but a complex 1a/1c mosaic genome, showing five putative breakpoints in the core to NS3 regions. To our knowledge, this is the first report on a mosaic HCV full- length sequence with multiple breakpoints. The molecular structure of AY651061 is reminiscent of complex homologous recombinant variants occurring among other members of the flaviviridae family, e.g. GB virus C, dengue virus, and Japanese encephalitis virus. Our finding of a mosaic HCV sequence may have important implications for many fields of current HCV research which merit careful consideration.

  13. Use of Full-Length Recombinant Calflagin and Its C Fragment for Improvement of Diagnosis of Trypanosoma cruzi Infection†

    Science.gov (United States)

    Marcipar, Iván S.; Roodveldt, Cintia; Corradi, Gerardo; Cabeza, María L.; Brito, Maria Edileuza F.; Winter, Lucile M. Floeter; Marcipar, Alberto J.; Silber, Ariel M.

    2005-01-01

    Serological diagnosis of Trypanosoma cruzi infection is hampered by issues related to test specificity due to the cross-reactivity of most antigens with proteins of related parasites such as Leishmania spp. The recombinant calflagins are considered relevant antigens for the diagnosis of infection by Trypanosoma cruzi. In the present work, we describe two genes coding for putative calflagins in Leishmania major with the N-terminal moieties presenting high similarity with T. cruzi genes. This fact raised questions about their role in some cross-recognition of this antigen by sera from Leishmania spp.-infected individuals. The complete T. cruzi calflagin and two fragments of the protein, consisting of 146 amino acids of the N-terminal and 65 amino acids of the C-terminal regions, were expressed and evaluated against a panel of sera, which included well-characterized samples from T. cruzi, and Leishmania-infected patients. We were able to show that sera from Leishmania (Viannia) braziliensis-infected individuals recognized the recombinant full-length calflagin. Both the N-terminal and the complete protein presented the same high sensitivity (98.5% of sera from T. cruzi-infected patients was detected) but different specificities (94% and 98%, respectively, when evaluated against sera from people not infected by T. cruzi, including 15 sera from people infected with L. braziliensis). The C-terminal fragment presented low sensitivity (70%) but 100% specificity. We propose the use of these antigens in two sequential assays to optimize the serological diagnosis of T. cruzi infection in humans in geographic areas where Leishmania spp. infection is coendemic. PMID:16272476

  14. Genetic characterization of human herpesvirus type 1: Full-length genome sequence of strain obtained from an encephalitis case from India

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    Vijay P Bondre

    2016-01-01

    Interpretation & conclusions: Our results showed that the full-length genome sequence generated from an Indian HSV-1 isolate shared close genetic relationship with the American KOS and Chinese CR38 strains which belonged to the Asian genetic lineage. Recombination analysis of Indian isolate demonstrated multiple recombination crossover points throughout the genome. This full-length genome sequence amplified from the Indian isolate would be helpful to study HSV evolution, genetic basis of differential pathogenesis, host-virus interactions and viral factors contributing towards differential clinical outcome in human infections.

  15. Full-length characterization of A1/D intersubtype recombinant genomes from a therapy-induced HIV type 1 controller during acute infection and his noncontrolling partner

    DEFF Research Database (Denmark)

    Fomsgaard, A.; Vinner, L.; Therrien, D.

    2008-01-01

    To increase the understanding of mechanisms of HIV control we have genetically and immunologically characterized a full-length HIV-1 isolated from an acute infection in a rare case of undetectable viremia. The subject, a 43-year-old Danish white male (DK1), was diagnosed with acute HIV-1 infection...... and phylogenic trees were constructed and diversity and evolutionary distances were calculated. Intracellular IFN-gamma in CD8(+)CD3(+) T-lymphocyte reactions was investigated by intracellular flow cytometry (IC-FACS). Virus isolates from both patients were A1D intersubtype recombinants showing 98% sequence...

  16. A full-length Plasmodium falciparum recombinant circumsporozoite protein expressed by Pseudomonas fluorescens platform as a malaria vaccine candidate.

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    Amy R Noe

    Full Text Available The circumsporozoite protein (CSP of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite's surface. Preclinical research on CSP and clinical evaluation of a CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA and immunofluorescence assay (IFA, as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime

  17. Generation of a reliable full-length cDNA of infectiousTembusu virus using a PCR-based protocol.

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    Liang, Te; Liu, Xiaoxiao; Cui, Shulin; Qu, Shenghua; Wang, Dan; Liu, Ning; Wang, Fumin; Ning, Kang; Zhang, Bing; Zhang, Dabing

    2016-02-02

    Full-length cDNA of Tembusu virus (TMUV) cloned in a plasmid has been found instable in bacterial hosts. Using a PCR-based protocol, we generated a stable full-length cDNA of TMUV. Different cDNA fragments of TMUV were amplified by reverse transcription (RT)-PCR, and cloned into plasmids. Fragmented cDNAs were amplified and assembled by fusion PCR to produce a full-length cDNA using the recombinant plasmids as templates. Subsequently, a full-length RNA was transcribed from the full-length cDNA in vitro and transfected into BHK-21 cells; infectious viral particles were rescued successfully. Following several passages in BKH-21 cells, the rescued virus was compared with the parental virus by genetic marker checks, growth curve determinations and animal experiments. These assays clearly demonstrated the genetic and biological stabilities of the rescued virus. The present work will be useful for future investigations on the molecular mechanisms involved in replication and pathogenesis of TMUV. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Near-Full Genome Characterisation of Two Natural Intergenotypic 2k/1b Recombinant Hepatitis C Virus Isolates

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    Victoria L. Demetriou

    2011-01-01

    Full Text Available Few natural intergenotypic hepatitis C virus (HCV recombinants have been characterised, and only RF1_2k/1b has demonstrated widespread transmission. The near-full length genome sequences for two cases of 2k/1b recombinants (CYHCV037 and CYHCV093 sampled in Cyprus were obtained using strain-specific RT-PCR amplification and sequencing protocols. Sequence analysis confirmed their similarity with the original RF1_2k/1b strain from St. Petersburg, N687. These two isolates significantly contribute to the sequence data available on this recombinant and confirm its increasing spread among individuals from Eastern Europe, and its association with transmission through intravenous drug use. Phylogenetic analyses reveal clustering of the sequence 3′ to the recombination point, not seen in the topology of the 5′ sequences, implying a more complicated evolutionary history than that held to date. The increasing cases of HCV recombinant strains underline the requirement of their contribution to the standardised rules of HCV classification and nomenclature, molecular epidemiology, diagnosis, and treatment.

  19. Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system

    DEFF Research Database (Denmark)

    Li, Yi-Ping; Ramirez, Santseharay; Jensen, Sanne B

    2012-01-01

    Chronic infection with hepatitis C virus (HCV) is an important cause of end stage liver disease worldwide. In the United States, most HCV-related disease is associated with genotype 1 infection, which remains difficult to treat. Drug and vaccine development was hampered by inability to culture...... full-length TN infection dose-dependently. Given the unique importance of genotype 1 for pathogenesis, this infectious 1a culture system represents an important advance in HCV research. The approach used and the mutations identified might permit culture development for other HCV isolates, thus......) culture systems in Huh7.5 cells. Here, we developed a highly efficient genotype 1a (strain TN) full-length culture system. We initially found that the LSG substitutions conferred viability to an intergenotypic recombinant composed of TN 5' untranslated region (5'UTR)-NS5A and JFH1 NS5B-3'UTR; recovered...

  20. Virtually full-length subtype F and F/D recombinant HIV-1 from Africa and South America

    NARCIS (Netherlands)

    Laukkanen, T.; Carr, J. K.; Janssens, W.; Liitsola, K.; Gotte, D.; McCutchan, F. E.; Op de Coul, E.; Cornelissen, M.; Heyndrickx, L.; van der Groen, G.; Salminen, M. O.

    2000-01-01

    For reliable classification of HIV-1 strains appropriate reference sequences are needed. The HIV-1 genetic subtype F has a wide geographic spread, causing significant epidemics in South America, Africa, and some regions of Europe. Previously only two full-length sequences of each of the HIV-1

  1. Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells

    DEFF Research Database (Denmark)

    Rasch, Morten G; Lund, Ida K; Illemann, Martin

    2010-01-01

    MMP-9. Constructs encoding zymogens of full-length murine MMP-9 and a version lacking the O-glycosylated linker region and hemopexin domains were therefore generated and expressed in stably transfected Drosophila S2 insect cells. After 7 days of induction the expression levels of the full......-length and truncated versions were 5 mg/l and 2 mg/l, respectively. The products were >95% pure after gelatin Sepharose chromatography and possessed proteolytic activity when analyzed by gelatin zymography. Using the purified full-length murine MMP-9 we raised polyclonal antibodies by immunizations of rabbits......Matrix metalloproteinase-9 (MMP-9) is a 92-kDa soluble pro-enzyme implicated in pathological events including cancer invasion. It is therefore an attractive target for therapeutic intervention studies in mouse models. Development of inhibitors requires sufficient amounts of correctly folded murine...

  2. Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells

    DEFF Research Database (Denmark)

    Rasch, Morten G; Lund, Ida K.; Illemann, Martin

    2010-01-01

    -length and truncated versions were 5 mg/l and 2 mg/l, respectively. The products were >95% pure after gelatin Sepharose chromatography and possessed proteolytic activity when analyzed by gelatin zymography. Using the purified full-length murine MMP-9 we raised polyclonal antibodies by immunizations of rabbits...

  3. Scalable production in human cells and biochemical characterization of full-length normal and mutant huntingtin.

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    Bin Huang

    Full Text Available Huntingtin (Htt is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington's disease (HD is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q and mutant (46Q and 128Q Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on "gutless" adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.

  4. Generation and biological assessment of recombinant avian metapneumovirus subgroup C (aMPV-C) viruses containing different length of the G gene.

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    Yu, Qingzhong; Estevez, Carlos; Song, Minxun; Kapczynski, Darrell; Zsak, Laszlo

    2010-02-01

    Genetic variation in length of the G gene among different avian metapneumovirus subgroup C (aMPV-C) isolates has been reported. However, its biological significance in virus replication, pathogenicity and immunity is unknown. In this study, we developed a reverse genetics system for aMPV-C and generated two Colorado (CO) strain-based recombinant viruses containing either the full-length G gene derived from a Canadian goose isolate or a C-terminally truncated G gene of the CO strain. The truncated short G (sG) gene encoded 252 amino acids (aa), which is 333 aa shorter than the full-length G (585 aa). The biological properties of these two recombinant G variants were assessed in Vero cells and in specific-pathogen-free (SPF) turkeys. In Vero cells, the short G variant displayed a similar level of growth dynamics and virus titers as the parental aMPV-CO strain, whereas the full-length G variant replicated less efficiently than the sG variant during the first 72 h post-infection. Both of the G variants induced typical cytopathic effects (CPE) that were indistinguishable from those seen with the parental aMPV-CO infection. In SPF turkeys, both of the G variants were attenuated and caused little or no disease signs, but the full-length G variant appeared to grow more readily in tracheal tissue than the sG variant during the first 5 days post-infection. Both G variants were immunogenic and induced a slightly different level of antibody response. These results demonstrated that the large portion (333 aa) of the extracellular domain of the viral attachment protein is not essential for virus viability in vitro and in vivo, but may play a role in enhancing virus attachment specificity and immunity in a natural host. (c) 2009 Elsevier B.V. All rights reserved.

  5. Assessment of a robust model protocol with accelerated throughput for a human recombinant full length estrogen receptor-alpha binding assay: protocol optimization and intralaboratory assay performance as initial steps towards validation.

    Science.gov (United States)

    Freyberger, Alexius; Wilson, Vickie; Weimer, Marc; Tan, Shirlee; Tran, Hoai-Son; Ahr, Hans-Jürgen

    2010-08-01

    Despite about two decades of research in the field of endocrine active compounds, still no validated human recombinant (hr) estrogen receptor-alpha (ERalpha) binding assay is available, although hr-ERalpha is available from several sources. In a joint effort, US EPA and Bayer Schering Pharma with funding from the EU-sponsored 6th framework project, ReProTect, developed a model protocol for such a binding assay. Important features of this assay are the use of a full length hr-ERalpha and performance in a 96-well plate format. A full length hr-ERalpha was chosen, as it was considered to provide the most accurate and human-relevant results, whereas truncated receptors could perform differently. Besides three reference compounds [17beta-estradiol, norethynodrel, dibutylphthalate] nine test compounds with different affinities for the ERalpha [diethylstilbestrol (DES), ethynylestradiol, meso-hexestrol, equol, genistein, o,p'-DDT, nonylphenol, n-butylparaben, and corticosterone] were used to explore the performance of the assay. Three independent experiments per compound were performed on different days, and dilutions of test compounds from deep-frozen stocks, solutions of radiolabeled ligand and receptor preparation were freshly prepared for each experiment. The ERalpha binding properties of reference and test compounds were well detected. As expected dibutylphthalate and corticosterone were non-binders in this assay. In terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using a human recombinant ERalpha ligand binding domain. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.5. Our data demonstrate that the assay was robust and reliably ranked compounds with strong, weak, and no affinity for the ERalpha with high accuracy. It avoids the manipulation and use of animals, i.e., the preparation of uterine cytosol as

  6. Construction of a Full-Length Enriched cDNA Library and Preliminary Analysis of Expressed Sequence Tags from Bengal Tiger Panthera tigris tigris

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    Changqing Liu

    2013-05-01

    Full Text Available In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers.

  7. Effect of the electrostatic surface potential on the oligomerization of full-length human recombinant prion protein at single-molecule level

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Bin; Xu, Bingqian, E-mail: bxu@engr.uga.edu [Single Molecule Study Laboratory, College of Engineering and Nanoscale Science, and Engineering Center, University of Georgia, Athens, Georgia 30605 (United States); Lou, Zhichao [Single Molecule Study Laboratory, College of Engineering and Nanoscale Science, and Engineering Center, University of Georgia, Athens, Georgia 30605 (United States); College of Materials Science and Technology, Nanjing University of Aeronautics and Astronautics, Nanjing 210016 (China); Zhang, Haiqian [College of Materials Science and Technology, Nanjing University of Aeronautics and Astronautics, Nanjing 210016 (China)

    2016-03-21

    The electrostatic surface potential (ESP) of prion oligomers has critical influences on the aggregating processes of the prion molecules. The atomic force microscopy (AFM) and structural simulation were combined to investigate the molecular basis of the full-length human recombinant prion oligomerization on mica surfaces. The high resolution non-intrusive AFM images showed that the prion oligomers formed different patterns on mica surfaces at different buffer pH values. The basic binding units for the large oligomers were determined to be prion momoners (Ms), dimers (Ds), and trimers (Ts). The forming of the D and T units happened through the binding of hydrophobic β-sheets of the M units. In contrast, the α-helices of these M, D, and T units were the binding areas for the formation of large oligomers. At pH 4.5, the binding units M, D, and T showed clear polarized ESP distributions on the surface domains, while at pH 7.0, they showed more evenly distributed ESPs. Based on the conformations of oligomers observed from AFM images, the D and T units were more abundantly on mica surface at pH 4.5 because the ESP re-distribution of M units helped to stabilize these larger oligomers. The amino acid side chains involved in the binding interfaces were stabilized by hydrogen bonds and electrostatic interactions. The detailed analysis of the charged side chains at pH 4.5 indicated that the polarized ESPs induced the aggregations among M, D, and T to form larger oligomers. Therefore, the hydrogen bonds and electrostatic interactions worked together to form the stabilized prion oligomers.

  8. Effect of the electrostatic surface potential on the oligomerization of full-length human recombinant prion protein at single-molecule level

    International Nuclear Information System (INIS)

    Wang, Bin; Xu, Bingqian; Lou, Zhichao; Zhang, Haiqian

    2016-01-01

    The electrostatic surface potential (ESP) of prion oligomers has critical influences on the aggregating processes of the prion molecules. The atomic force microscopy (AFM) and structural simulation were combined to investigate the molecular basis of the full-length human recombinant prion oligomerization on mica surfaces. The high resolution non-intrusive AFM images showed that the prion oligomers formed different patterns on mica surfaces at different buffer pH values. The basic binding units for the large oligomers were determined to be prion momoners (Ms), dimers (Ds), and trimers (Ts). The forming of the D and T units happened through the binding of hydrophobic β-sheets of the M units. In contrast, the α-helices of these M, D, and T units were the binding areas for the formation of large oligomers. At pH 4.5, the binding units M, D, and T showed clear polarized ESP distributions on the surface domains, while at pH 7.0, they showed more evenly distributed ESPs. Based on the conformations of oligomers observed from AFM images, the D and T units were more abundantly on mica surface at pH 4.5 because the ESP re-distribution of M units helped to stabilize these larger oligomers. The amino acid side chains involved in the binding interfaces were stabilized by hydrogen bonds and electrostatic interactions. The detailed analysis of the charged side chains at pH 4.5 indicated that the polarized ESPs induced the aggregations among M, D, and T to form larger oligomers. Therefore, the hydrogen bonds and electrostatic interactions worked together to form the stabilized prion oligomers.

  9. Effect of the electrostatic surface potential on the oligomerization of full-length human recombinant prion protein at single-molecule level

    Science.gov (United States)

    Wang, Bin; Lou, Zhichao; Zhang, Haiqian; Xu, Bingqian

    2016-03-01

    The electrostatic surface potential (ESP) of prion oligomers has critical influences on the aggregating processes of the prion molecules. The atomic force microscopy (AFM) and structural simulation were combined to investigate the molecular basis of the full-length human recombinant prion oligomerization on mica surfaces. The high resolution non-intrusive AFM images showed that the prion oligomers formed different patterns on mica surfaces at different buffer pH values. The basic binding units for the large oligomers were determined to be prion momoners (Ms), dimers (Ds), and trimers (Ts). The forming of the D and T units happened through the binding of hydrophobic β-sheets of the M units. In contrast, the α-helices of these M, D, and T units were the binding areas for the formation of large oligomers. At pH 4.5, the binding units M, D, and T showed clear polarized ESP distributions on the surface domains, while at pH 7.0, they showed more evenly distributed ESPs. Based on the conformations of oligomers observed from AFM images, the D and T units were more abundantly on mica surface at pH 4.5 because the ESP re-distribution of M units helped to stabilize these larger oligomers. The amino acid side chains involved in the binding interfaces were stabilized by hydrogen bonds and electrostatic interactions. The detailed analysis of the charged side chains at pH 4.5 indicated that the polarized ESPs induced the aggregations among M, D, and T to form larger oligomers. Therefore, the hydrogen bonds and electrostatic interactions worked together to form the stabilized prion oligomers.

  10. Full-length enriched multistage cDNA library construction covering ...

    African Journals Online (AJOL)

    DR TONUKARI NYEROVWO

    2012-04-10

    Apr 10, 2012 ... Full Length Research Paper. Full-length enriched ... complementary DNA; pfu, plaque-forming unit. ... Chinese-native tree species in Populus section Leuce ... the infected bacteria, 2 ml melted top agar was added, and the.

  11. 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; Moore, Michael D

    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...

  12. 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.

  13. Safety of PEGylated recombinant human full-length coagulation factor VIII (BAX 855) in the overall context of PEG and PEG conjugates.

    Science.gov (United States)

    Stidl, R; Fuchs, S; Bossard, M; Siekmann, J; Turecek, P L; Putz, M

    2016-01-01

    BAX 855 is a PEGylated human full-length recombinant factor VIII (rFVIII) based on licensed rFVIII (ADVATE). The applied PEGylation technology has been optimized to retain functionality of the FVIII molecule, improve its pharmacokinetic properties and allow less frequent injections while maintaining efficacy. The aim of this study was to confirm that the excellent safety profile of ADVATE remains unchanged after PEGylation. Non-clinical safety studies with BAX 855 and its respective unbound polyethylene glycol (PEG) were conducted in several species. The distribution of a single dose of radiolabelled BAX 855 was further investigated in rats. Publically available safety data on PEG alone and PEGylated biomolecules were summarized and reviewed for specific safety findings attributable to PEG or PEGylated biopharmaceuticals. Safety pharmacology studies in rabbits and macaques and repeated dose toxicity studies in rats and macaques identified no safety issues. Results of a distribution study in rats administered radiolabelled BAX 855 showed that radioactivity was completely excreted; urine was the major elimination route. A 28-day study in rats dosed with the unbound PEG constituent (PEG2ru20KCOOH) of BAX 855 showed no adverse or non-adverse effects. Safety data for PEG and PEG-protein conjugates indicate no safety concerns associated with PEG at clinically relevant dose levels. Although vacuolation of certain cell types has been reported in mammals, no such vacuolation was observed with BAX 855 or with the unbound PEG constituent. Non-clinical safety evaluation of PEG and BAX 855 identified no safety signals; the compound is now in clinical development for the treatment of patients with haemophilia A. © 2015 Baxalta Innovations GmbH. Haemophilia Published by John Wiley & Sons Ltd.

  14. Construction of a Full-Length Enriched cDNA Library and Preliminary Analysis of Expressed Sequence Tags from Bengal Tiger Panthera tigris tigris

    Science.gov (United States)

    Liu, Changqing; Liu, Dan; Guo, Yu; Lu, Taofeng; Li, Xiangchen; Zhang, Minghai; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun

    2013-01-01

    In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers. PMID:23708105

  15. Conformational change in full-length mouse prion: A site-directed spin-labeling study

    International Nuclear Information System (INIS)

    Inanami, Osamu; Hashida, Shukichi; Iizuka, Daisuke; Horiuchi, Motohiro; Hiraoka, Wakako; Shimoyama, Yuhei; Nakamura, Hideo; Inagaki, Fuyuhiko; Kuwabara, Mikinori

    2005-01-01

    The structure of the mouse prion (moPrP) was studied using site-directed spin-labeling electron spin resonance (SDSL-ESR). Since a previous NMR study by Hornemanna et al., [Hornemanna, Korthb, Oeschb, Rieka, Widera, Wuethricha, Glockshubera, Recombinant full-length murine prion protein, mPrP (23-231): purification and spectroscopic characterization, FEBS Lett. 413 (1997) 277-281] has indicated that N96, D143, and T189 in moPrP are localized in a Cu 2+ binding region, Helix1 and Helix2, respectively, three recombinant moPrP mutations (N96C, D143C, and T189C) were expressed in an Escherichia coli system, and then refolded by dialysis under low pH and purified by reverse-phase HPLC. By using the preparation, we succeeded in preserving a target cystein residue without alteration of the α-helix structure of moPrP and were able to apply SDSL-ESR with a methane thiosulfonate spin label to the full-length prion protein. The rotational correlation times (τ) of 1.1, 3.3, and 4.8 ns were evaluated from the X-band ESR spectra at pH 7.4 and 20 deg C for N96R1, D143R1, and T189R1, respectively. τ reflects the fact that the Cu 2+ binding region is more flexible than Helix1 or Helix2. ESR spectra recorded at various temperatures revealed two phases together with a transition point at around 20 deg C in D143R1 and T189R1, but not in N96R1. With the variation of pH from 4.0 to 7.8, ESR spectra of T189R1 at 20 deg C showed a gradual increase of τ from 2.9 to 4.8 ns. On the other hand, the pH-dependent conformational changes in N96R1 and D143R1 were negligible. These results indicated that T189 located in Helix2 possessed a structure sensitive to physiological pH changes; simultaneously, N96 in the Cu 2+ binding region and D143 in Helix1 were conserved

  16. Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures.

    Directory of Open Access Journals (Sweden)

    Asma Rehman

    Full Text Available Vesicle fusion is an indispensable cellular process required for eukaryotic cargo delivery. The Sec/Munc18 protein Munc18c is essential for insulin-regulated trafficking of glucose transporter4 (GLUT4 vesicles to the cell surface in muscle and adipose tissue. Previously, our biophysical and structural studies have used Munc18c expressed in SF9 insect cells. However to maximize efficiency, minimize cost and negate any possible effects of post-translational modifications of Munc18c, we investigated the use of Escherichia coli as an expression host for Munc18c. We were encouraged by previous reports describing Munc18c production in E. coli cultures for use in in vitro fusion assay, pulldown assays and immunoprecipitations. Our approach differs from the previously reported method in that it uses a codon-optimized gene, lower temperature expression and autoinduction media. Three N-terminal His-tagged constructs were engineered, two with a tobacco etch virus (TEV or thrombin protease cleavage site to enable removal of the fusion tag. The optimized protocol generated 1-2 mg of purified Munc18c per L of culture at much reduced cost compared to Munc18c generated using insect cell culture. The purified recombinant Munc18c protein expressed in bacteria was monodisperse, monomeric, and functional. In summary, we developed methods that decrease the cost and time required to generate functional Munc18c compared with previous insect cell protocols, and which generates sufficient purified protein for structural and biophysical studies.

  17. Cloning and sequencing of full-length cDNAs of RNA1 and RNA2 of a Tomato black ring virus isolate from Poland.

    Science.gov (United States)

    Jończyk, M; Le Gall, O; Pałucha, A; Borodynko, N; Pospieszny, H

    2004-04-01

    Full-length cDNA clones corresponding to the RNA1 and RNA2 of the Polish isolate MJ of Tomato black ring virus (TBRV, genus Nepovirus) were obtained using a direct recombination strategy in yeast, and their complete nucleotide sequences were established. RNA1 is 7358 nucleotides and RNA2 is 4633 nucleotides in length, excluding the poly(A) tails. Both RNAs contain a single open reading frame encoding polyproteins of 254 kDa and 149 kDa for RNA1 and RNA2 respectively. Putative cleavage sites were identified, and the relationships between TBRV and related nepoviruses were studied by sequence comparison.

  18. Full length channel Pressure Tube sagging under completely voided full length pressure tube of an Indian PHWR

    Energy Technology Data Exchange (ETDEWEB)

    Negi, Sujay, E-mail: negi.sujay@gmail.com [Indian Institute of Technology, Roorkee 247667 (India); Kumar, Ravi, E-mail: ravikfme@gmail.com [Indian Institute of Technology, Roorkee 247667 (India); Majumdar, P., E-mail: pmajum@barc.gov.in [Bhabha Atomic Research Centre, Mumbai 400085 (India); Mukopadhyay, D., E-mail: dmukho@barc.gov.in [Bhabha Atomic Research Centre, Mumbai 400085 (India)

    2017-03-15

    Highlights: • At 16 kW/m input, thermal stability was attained at 595 °C, without PT-CT contact. • At 20 kW/m step input, PT-CT contact occurred at 637 °C near bottom-center of the tube. • PT integrity was maintained throughout the experiment. - Abstract: An experimental investigation was conducted to simulate the sagging behavior of a full length Pressure Tube of a channel of 220 MWe Indian PHWR. The investigation aimed to recreate a condition resembling Loss of Coolant Accident (LOCA) with Emergency Core Cooling System (ECCS) failure in a nuclear power plant. A full length channel assembly immersed in moderator was subjected to electrical resistance heating of Pressure Tube (PT) to simulate the residual heat after shutting down of reactor. The temperature of PT started rising and the contact between PT and CT was established at the center of the tube where average bottom temperature was 637 °C. The integrity of PT was maintained throughout the experiment and the PT heat up was arrested on contact with the CT due to transfer of heat to the moderator.

  19. Human uroporphyrinogen III synthase: Molecular cloning, nucleotide sequence, and expression of a full-length cDNA

    International Nuclear Information System (INIS)

    Tsai, Shihfeng; Bishop, D.F.; Desnick, R.J.

    1988-01-01

    Uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 x 10 6 recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria

  20. Full-length fuel rod behavior under severe accident conditions

    International Nuclear Information System (INIS)

    Lombardo, N.J.; Lanning, D.D.; Panisko, F.E.

    1992-12-01

    This document presents an assessment of the severe accident phenomena observed from four Full-Length High-Temperature (FLHT) tests that were performed by the Pacific Northwest Laboratory (PNL) in the National Research Universal (NRU) reactor at Chalk River, Ontario, Canada. These tests were conducted for the US Nuclear Regulatory Commission (NRC) as part of the Severe Accident Research Program. The objectives of the test were to simulate conditions and provide information on the behavior of full-length fuel rods during hypothetical, small-break, loss-of-coolant severe accidents, in commercial light water reactors

  1. Full mitochondrial genome sequences of two endemic Philippine hornbill species (Aves: Bucerotidae provide evidence for pervasive mitochondrial DNA recombination

    Directory of Open Access Journals (Sweden)

    Bleidorn Christoph

    2011-01-01

    Full Text Available Abstract Background Although nowaday it is broadly accepted that mitochondrial DNA (mtDNA may undergo recombination, the frequency of such recombination remains controversial. Its estimation is not straightforward, as recombination under homoplasmy (i.e., among identical mt genomes is likely to be overlooked. In species with tandem duplications of large mtDNA fragments the detection of recombination can be facilitated, as it can lead to gene conversion among duplicates. Although the mechanisms for concerted evolution in mtDNA are not fully understood yet, recombination rates have been estimated from "one per speciation event" down to 850 years or even "during every replication cycle". Results Here we present the first complete mt genome of the avian family Bucerotidae, i.e., that of two Philippine hornbills, Aceros waldeni and Penelopides panini. The mt genomes are characterized by a tandemly duplicated region encompassing part of cytochrome b, 3 tRNAs, NADH6, and the control region. The duplicated fragments are identical to each other except for a short section in domain I and for the length of repeat motifs in domain III of the control region. Due to the heteroplasmy with regard to the number of these repeat motifs, there is some size variation in both genomes; with around 21,657 bp (A. waldeni and 22,737 bp (P. panini, they significantly exceed the hitherto longest known avian mt genomes, that of the albatrosses. We discovered concerted evolution between the duplicated fragments within individuals. The existence of differences between individuals in coding genes as well as in the control region, which are maintained between duplicates, indicates that recombination apparently occurs frequently, i.e., in every generation. Conclusions The homogenised duplicates are interspersed by a short fragment which shows no sign of recombination. We hypothesize that this region corresponds to the so-called Replication Fork Barrier (RFB, which has been

  2. A Rapid and Improved Method to Generate Recombinant Dengue Virus Vaccine Candidates.

    Science.gov (United States)

    Govindarajan, Dhanasekaran; Guan, Liming; Meschino, Steven; Fridman, Arthur; Bagchi, Ansu; Pak, Irene; ter Meulen, Jan; Casimiro, Danilo R; Bett, Andrew J

    2016-01-01

    Dengue is one of the most important mosquito-borne infections accounting for severe morbidity and mortality worldwide. Recently, the tetravalent chimeric live attenuated Dengue vaccine Dengvaxia® was approved for use in several dengue endemic countries. In general, live attenuated vaccines (LAV) are very efficacious and offer long-lasting immunity against virus-induced disease. Rationally designed LAVs can be generated through reverse genetics technology, a method of generating infectious recombinant viruses from full length cDNA contained in bacterial plasmids. In vitro transcribed (IVT) viral RNA from these infectious clones is transfected into susceptible cells to generate recombinant virus. However, the generation of full-length dengue virus cDNA clones can be difficult due to the genetic instability of viral sequences in bacterial plasmids. To circumvent the need for a single plasmid containing a full length cDNA, in vitro ligation of two or three cDNA fragments contained in separate plasmids can be used to generate a full-length dengue viral cDNA template. However, in vitro ligation of multiple fragments often yields low quality template for IVT reactions, resulting in inconsistent low yield RNA. These technical difficulties make recombinant virus recovery less efficient. In this study, we describe a simple, rapid and efficient method of using LONG-PCR to recover recombinant chimeric Yellow fever dengue (CYD) viruses as potential dengue vaccine candidates. Using this method, we were able to efficiently generate several viable recombinant viruses without introducing any artificial mutations into the viral genomes. We believe that the techniques reported here will enable rapid and efficient recovery of recombinant flaviviruses for evaluation as vaccine candidates and, be applicable to the recovery of other RNA viruses.

  3. Direct recovery of infectious Pestivirus from a full-length RT-PCR amplicon

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, Ilona; Hoffmann, Bernd

    2008-01-01

    This study describes the use of a novel and rapid long reverse transcription (RT)-PCR for the generation of infectious full-length cDNA of pestiviruses. To produce rescued viruses, full-length RT-PCR amplicons of 12.3 kb, including a T7-promotor, were transcribed directly in vitro, and the result......This study describes the use of a novel and rapid long reverse transcription (RT)-PCR for the generation of infectious full-length cDNA of pestiviruses. To produce rescued viruses, full-length RT-PCR amplicons of 12.3 kb, including a T7-promotor, were transcribed directly in vitro......, and the resulting RNA transcripts were electroporated into ovine cells. Infectious virus was obtained after one cell culture passage. The rescued viruses had a phenotype similar to the parental Border Disease virus strain. Therefore, direct generation of infectious pestiviruses from full-length RT-PCR cDNA products...

  4. A Rapid and Improved Method to Generate Recombinant Dengue Virus Vaccine Candidates.

    Directory of Open Access Journals (Sweden)

    Dhanasekaran Govindarajan

    Full Text Available Dengue is one of the most important mosquito-borne infections accounting for severe morbidity and mortality worldwide. Recently, the tetravalent chimeric live attenuated Dengue vaccine Dengvaxia® was approved for use in several dengue endemic countries. In general, live attenuated vaccines (LAV are very efficacious and offer long-lasting immunity against virus-induced disease. Rationally designed LAVs can be generated through reverse genetics technology, a method of generating infectious recombinant viruses from full length cDNA contained in bacterial plasmids. In vitro transcribed (IVT viral RNA from these infectious clones is transfected into susceptible cells to generate recombinant virus. However, the generation of full-length dengue virus cDNA clones can be difficult due to the genetic instability of viral sequences in bacterial plasmids. To circumvent the need for a single plasmid containing a full length cDNA, in vitro ligation of two or three cDNA fragments contained in separate plasmids can be used to generate a full-length dengue viral cDNA template. However, in vitro ligation of multiple fragments often yields low quality template for IVT reactions, resulting in inconsistent low yield RNA. These technical difficulties make recombinant virus recovery less efficient. In this study, we describe a simple, rapid and efficient method of using LONG-PCR to recover recombinant chimeric Yellow fever dengue (CYD viruses as potential dengue vaccine candidates. Using this method, we were able to efficiently generate several viable recombinant viruses without introducing any artificial mutations into the viral genomes. We believe that the techniques reported here will enable rapid and efficient recovery of recombinant flaviviruses for evaluation as vaccine candidates and, be applicable to the recovery of other RNA viruses.

  5. Effects of Organic Cation Length on Exciton Recombination in Two-Dimensional Layered Lead Iodide Hybrid Perovskite Crystals.

    Science.gov (United States)

    Gan, Lu; Li, Jing; Fang, Zhishan; He, Haiping; Ye, Zhizhen

    2017-10-19

    In recent years, 2D layered organic-inorganic lead halide perovskites have attracted considerable attention due to the distinctive quantum confinement effects as well as prominent excitonic luminescence. Herein, we show that the recombination dynamics and photoluminescence (PL) of the 2D layered perovskites can be tuned by the organic cation length. 2D lead iodide perovskite crystals with increased length of the organic chains reveal blue-shifted PL as well as enhanced relative internal quantum efficiency. Furthermore, we provide experimental evidence that the formation of face-sharing [PbI 6 ] 4- octahedron in perovskites with long alkyls induces additional confinement for the excitons, leading to 1D-like recombination. As a result, the PL spectra show enhanced inhomogeneous broadening at low temperature. Our work provides physical understanding of the role of organic cation in the optical properties of 2D layered perovskites, and would benefit the improvement of luminescence efficiency of such materials.

  6. Low risk of inhibitor formation in haemophilia A patients following en masse switch in treatment to a third generation full length plasma and albumin-free recombinant factor VIII product (ADVATE®).

    LENUS (Irish Health Repository)

    Bacon, C L

    2011-05-01

    Previous studies have suggested that development of inhibitors in previously treated patients (PTPs) may be attributable to a switch in factor VIII (FVIII) therapeutic product. Consequently, it is widely recognized that inhibitor development must be assessed in PTPs following the introduction of any new FVIII product. Following a national tender process in 2006, all patients with haemophilia A in Ireland changed their FVIII treatment product en masse to a plasma and albumin-free recombinant full-length FVIII product (ADVATE(®)). In this study, we retrospectively reviewed the case records of Irish PTPs to evaluate risk of inhibitor formation following this treatment switch. One hundred and thirteen patients participated in the study. Most patients (89%) had severe haemophilia. Only one of 96 patients with no inhibitor history developed an inhibitor. Prior to the switch in his recombinant FVIII (rFVIII) treatment of choice, this child had only experienced three exposure days (EDs). Consequently, in total he had only received 6 EDs when his inhibitor was first diagnosed. In keeping with this lack of de novo inhibitor development, we observed no evidence of any recurrent inhibitor formation in any of 16 patients with previously documented inhibitors. Similarly, following a previous en masse switch, we have previously reported that changing from a Chinese hamster ovary cell-produced to a baby hamster kidney cell-produced rFVIII was also associated with a low risk of inhibitor formation in PTPs. Our cumulative findings from these two studies clearly emphasizes that the risk of inhibitor development for PTPs following changes in commercial rFVIII product is low, at least in the Irish population.

  7. Technology development for gene discovery and full-length sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Marcelo Bento Soares

    2004-07-19

    In previous years, with support from the U.S. Department of Energy, we developed methods for construction of normalized and subtracted cDNA libraries, and constructed hundreds of high-quality libraries for production of Expressed Sequence Tags (ESTs). Our clones were made widely available to the scientific community through the IMAGE Consortium, and millions of ESTs were produced from our libraries either by collaborators or by our own sequencing laboratory at the University of Iowa. During this grant period, we focused on (1) the development of a method for preferential cloning of tissue-specific and/or rare transcripts, (2) its utilization to expedite EST-based gene discovery for the NIH Mouse Brain Molecular Anatomy Project, (3) further development and optimization of a method for construction of full-length-enriched cDNA libraries, and (4) modification of a plasmid vector to maximize efficiency of full-length cDNA sequencing by the transposon-mediated approach. It is noteworthy that the technology developed for preferential cloning of rare mRNAs enabled identification of over 2,000 mouse transcripts differentially expressed in the hippocampus. In addition, the method that we optimized for construction of full-length-enriched cDNA libraries was successfully utilized for the production of approximately fifty libraries from the developing mouse nervous system, from which over 2,500 full-ORF-containing cDNAs have been identified and accurately sequenced in their entirety either by our group or by the NIH-Mammalian Gene Collection Program Sequencing Team.

  8. Full-length Ebola glycoprotein accumulates in the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Bhattacharyya Suchita

    2011-01-01

    Full Text Available Abstract The Filoviridae family comprises of Ebola and Marburg viruses, which are known to cause lethal hemorrhagic fever. However, there is no effective anti-viral therapy or licensed vaccines currently available for these human pathogens. The envelope glycoprotein (GP of Ebola virus, which mediates entry into target cells, is cytotoxic and this effect maps to a highly glycosylated mucin-like region in the surface subunit of GP (GP1. However, the mechanism underlying this cytotoxic property of GP is unknown. To gain insight into the basis of this GP-induced cytotoxicity, HEK293T cells were transiently transfected with full-length and mucin-deleted (Δmucin Ebola GP plasmids and GP localization was examined relative to the nucleus, endoplasmic reticulum (ER, Golgi, early and late endosomes using deconvolution fluorescent microscopy. Full-length Ebola GP was observed to accumulate in the ER. In contrast, GPΔmucin was uniformly expressed throughout the cell and did not localize in the ER. The Ebola major matrix protein VP40 was also co-expressed with GP to investigate its influence on GP localization. GP and VP40 co-expression did not alter GP localization to the ER. Also, when VP40 was co-expressed with the nucleoprotein (NP, it localized to the plasma membrane while NP accumulated in distinct cytoplasmic structures lined with vimentin. These latter structures are consistent with aggresomes and may serve as assembly sites for filoviral nucleocapsids. Collectively, these data suggest that full-length GP, but not GPΔmucin, accumulates in the ER in close proximity to the nuclear membrane, which may underscore its cytotoxic property.

  9. Construction of a full-length infectious bacterial artificial chromosome clone of duck enteritis virus vaccine strain

    Science.gov (United States)

    2013-01-01

    Background Duck enteritis virus (DEV) is the causative agent of duck viral enteritis, which causes an acute, contagious and lethal disease of many species of waterfowl within the order Anseriformes. In recent years, two laboratories have reported on the successful construction of DEV infectious clones in viral vectors to express exogenous genes. The clones obtained were either created with deletion of viral genes and based on highly virulent strains or were constructed using a traditional overlapping fosmid DNA system. Here, we report the construction of a full-length infectious clone of DEV vaccine strain that was cloned into a bacterial artificial chromosome (BAC). Methods A mini-F vector as a BAC that allows the maintenance of large circular DNA in E. coli was introduced into the intergenic region between UL15B and UL18 of a DEV vaccine strain by homologous recombination in chicken embryoblasts (CEFs). Then, the full-length DEV clone pDEV-vac was obtained by electroporating circular viral replication intermediates containing the mini-F sequence into E. coli DH10B and identified by enzyme digestion and sequencing. The infectivity of the pDEV-vac was validated by DEV reconstitution from CEFs transfected with pDEV-vac. The reconstructed virus without mini-F vector sequence was also rescued by co-transfecting the Cre recombinase expression plasmid pCAGGS-NLS/Cre and pDEV-vac into CEF cultures. Finally, the in vitro growth properties and immunoprotection capacity in ducks of the reconstructed viruses were also determined and compared with the parental virus. Results The full genome of the DEV vaccine strain was successfully cloned into the BAC, and this BAC clone was infectious. The in vitro growth properties of these reconstructions were very similar to parental DEV, and ducks immunized with these viruses acquired protection against virulent DEV challenge. Conclusions DEV vaccine virus was cloned as an infectious bacterial artificial chromosome maintaining full-length

  10. Construction of a normalized full-length cDNA library of cephalopod Amphioctopus fangsiao and development of microsatellite markers

    Science.gov (United States)

    Feng, Yanwei; Liu, Wenfen; Xu, Xin; Yang, Jianmin; Wang, Weijun; Wei, Xiumei; Liu, Xiangquan; Sun, Guohua

    2017-10-01

    Amphioctopus fangsiao is one of the most economically important species and has been considered to be a candidate for aquaculture. In order to facilitate its fine-scale genetic analyses, we constructed a normalized full-length library successfully and developed a set of microsatellite markers in this study. The normalized full-length library had a storage capacity of 6.9×105 independent clones. The recombination efficiency was 95% and the average size of inserted fragments was longer than 1000 bp. A total of 3440 high quality ESTs were obtained, which were assembled into 1803 unigenes. Of these unigenes, 450 (25%) were assigned into 33 Gene Ontology terms, 576 (31.9%) into 153 Kyoto Encyclopedia of Genes and Genomes pathways, and 275 (15.3%) into 22 Clusters of Orthologous Groups. Seventy-six polymorphic microsatellite markers were identified. The number of alleles per locus ranged from 4 to 17, and the observed and expected heterozygosities varied between 0.167 and 0.967 and between 0.326 and 0.944, respectively. Twelve loci were significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction and no linkage disequilibrium was found between different loci. This study provided not only a useful resource for the isolation of the functional genes, but also a set of informative microsatellites for the assessment of population structure and conservation genetics of A. fangsiao.

  11. A comparative phylogenetic analysis of full-length mariner elements

    Indian Academy of Sciences (India)

    Mariner like elements (MLEs) are widely distributed type II transposons with an open reading frame (ORF) for transposase. We studied comparative phylogenetic evolution and inverted terminal repeat (ITR) conservation of MLEs from Indian saturniid silkmoth, Antheraea mylitta with other full length MLEs submitted in the ...

  12. The Length of N-Glycans of Recombinant H5N1 Hemagglutinin Influences the Oligomerization and Immunogenicity of Vaccine Antigen

    Directory of Open Access Journals (Sweden)

    Edyta Kopera

    2017-04-01

    Full Text Available Hemagglutinin glycoprotein (HA is a principle influenza vaccine antigen. Recombinant HA-based vaccines become a potential alternative for traditional approach. Complexity and variation of HA N-glycosylation are considered as the important factors for the vaccine design. The number and location of glycan moieties in the HA molecule are also crucial. Therefore, we decided to study the effect of N-glycosylation pattern on the H5 antigen structure and its ability to induce immunological response. We also decided to change neither the number nor the position of the HA glycosylation sites but only the glycan length. Two variants of the H5 antigen with high mannose glycosylation (H5hm and with low-mannose glycosylation (H5Man5 were prepared utilizing different Pichia strains. Our structural studies demonstrated that only the highly glycosylated H5 antigen formed high molecular weight oligomers similar to viral particles. Further, the H5hm was much more immunogenic for mice than H5Man5. In summary, our results suggest that high mannose glycosylation of vaccine antigen is superior to the low glycosylation pattern. Our findings have strong implications for the recombinant HA-based influenza vaccine design.

  13. Amplification and pyrosequencing of near-full-length hepatitis C virus for typing and monitoring antiviral resistant strains.

    Science.gov (United States)

    Trémeaux, P; Caporossi, A; Ramière, C; Santoni, E; Tarbouriech, N; Thélu, M-A; Fusillier, K; Geneletti, L; François, O; Leroy, V; Burmeister, W P; André, P; Morand, P; Larrat, S

    2016-05-01

    Directly acting antiviral drugs have contributed considerable progress to hepatitis C virus (HCV) treatment, but they show variable activity depending on virus genotypes and subtypes. Therefore, accurate genotyping including recombinant form detection is still of major importance, as is the detection of resistance-associated mutations in case of therapeutic failure. To meet these goals, an approach to amplify the HCV near-complete genome with a single long-range PCR and sequence it with Roche GS Junior was developed. After optimization, the overall amplification success rate was 73% for usual genotypes (i.e. HCV 1a, 1b, 3a and 4a, 16/22) and 45% for recombinant forms RF_2k/1b (5/11). After pyrosequencing and subsequent de novo assembly, a near-full-length genomic consensus sequence was obtained for 19 of 21 samples. The genotype and subtype were confirmed by phylogenetic analysis for every sample, including the suspected recombinant forms. Resistance-associated mutations were detected in seven of 13 samples at baseline, in the NS3 (n = 3) or NS5A (n = 4) region. Of these samples, the treatment of one patient included daclatasvir, and that patient experienced a relapse. Virus sequences from pre- and posttreatment samples of four patients who experienced relapse after sofosbuvir-based therapy were compared: the selected variants seem too far from the NS5B catalytic site to be held responsible. Although tested on a limited set of samples and with technical improvements still necessary, this assay has proven to be successful for both genotyping and resistance-associated variant detection on several HCV types. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  14. Recombiner

    International Nuclear Information System (INIS)

    Kikuchi, Nobuo.

    1983-01-01

    Purpose: To shorten the pre-heating time for a recombiner and obtain a uniform temperature distribution for the charged catalyst layer in a BWR type reactor. Constitution: A pre-heating heater is disposed to the outer periphery of a vessel for a recombiner packed with catalysts for recombining hydrogen and oxygen in gases flowing through a radioactive gaseous wastes processing system. Heat pipes for transmitting the heat applied to said container to the catalyst are disposed vertically and horizontally within the container. Different length of the heat pipes are combined. In this way, pre-heating time for the recombiner before the operation start and before the system switching can be shortened and the uniform pre-heating for the inside of the recombiner is also made possible. Further, heater control in the pre-heating can be carried out effectively and with ease. (Moriyama, K.)

  15. Single-Molecule Tethered Particle Motion: Stepwise Analyses of Site-Specific DNA Recombination

    Directory of Open Access Journals (Sweden)

    Hsiu-Fang Fan

    2018-05-01

    Full Text Available Tethered particle motion/microscopy (TPM is a biophysical tool used to analyze changes in the effective length of a polymer, tethered at one end, under changing conditions. The tether length is measured indirectly by recording the Brownian motion amplitude of a bead attached to the other end. In the biological realm, DNA, whose interactions with proteins are often accompanied by apparent or real changes in length, has almost exclusively been the subject of TPM studies. TPM has been employed to study DNA bending, looping and wrapping, DNA compaction, high-order DNA–protein assembly, and protein translocation along DNA. Our TPM analyses have focused on tyrosine and serine site-specific recombinases. Their pre-chemical interactions with DNA cause reversible changes in DNA length, detectable by TPM. The chemical steps of recombination, depending on the substrate and the type of recombinase, may result in a permanent length change. Single molecule TPM time traces provide thermodynamic and kinetic information on each step of the recombination pathway. They reveal how mechanistically related recombinases may differ in their early commitment to recombination, reversibility of individual steps, and in the rate-limiting step of the reaction. They shed light on the pre-chemical roles of catalytic residues, and on the mechanisms by which accessory proteins regulate recombination directionality.

  16. Irradiation performance of full-length metallic IFR fuels

    International Nuclear Information System (INIS)

    Tsai, H.; Neimark, L.A.

    1992-07-01

    An assembly irradiation of 169 full-length U-Pu-Zr metallic fuel pins was successfully completed in FFTF to a goal burnup of 10 at.%. All test fuel pins maintained their cladding integrity during the irradiation. Postirradiation examination showed minimal fuel/cladding mechanical interaction and excellent stability of the fuel column. Fission-gas release was normal and consistent with the existing data base from irradiation testing of shorter metallic fuel pins in EBR-II

  17. The function analysis of full-length cDNA sequence from IRM-2 mouse cDNA library

    International Nuclear Information System (INIS)

    Wang Qin; Liu Xiaoqiu; Xu Chang; Du Liqing; Sun Zhijuan; Wang Yan; Liu Qiang; Song Li; Li Jin; Fan Feiyue

    2013-01-01

    Objective: To identify the function of full-length cDNA sequence from IRM-2 mouse cDNA library. Methods: Full-length cDNA products were amplified by PCR from IRM-2 mouse cDNA library according to twenty-one pieces of expressed sequence tag. The expression of full-length cDNAs were detected after mouse embryonic fibroblasts were exposed to 6.5 Gy γ-ray radiation. And the effect on the growth of radiosensitivity cells AT5B1VA transfected with full-length cDNAs was investigated. Results: The expression of No.4, 5 and 2 full-length cDNAs from IRM-2 mouse were higher than that of parental ICR and 615 mouse after mouse embryonic fibroblasts irradiated with γ-ray radiation. And the survival rate of AT5B1VA cells transfected with No.4, 5 and 2 full-length cDNAs was high. Conclusion: No.4, 5 and 2 full-length cDNAs of IRM-2 mouse are of high radioresistance. (authors)

  18. Assessment of adaptive evolution between wheat and rice as deduced from full-length common wheat cDNA sequence data and expression patterns

    Directory of Open Access Journals (Sweden)

    Hayashizaki Yoshihide

    2009-06-01

    Full Text Available Abstract Background Wheat is an allopolyploid plant that harbors a huge, complex genome. Therefore, accumulation of expressed sequence tags (ESTs for wheat is becoming particularly important for functional genomics and molecular breeding. We prepared a comprehensive collection of ESTs from the various tissues that develop during the wheat life cycle and from tissues subjected to stress. We also examined their expression profiles in silico. As full-length cDNAs are indispensable to certify the collected ESTs and annotate the genes in the wheat genome, we performed a systematic survey and sequencing of the full-length cDNA clones. This sequence information is a valuable genetic resource for functional genomics and will enable carrying out comparative genomics in cereals. Results As part of the functional genomics and development of genomic wheat resources, we have generated a collection of full-length cDNAs from common wheat. By grouping the ESTs of recombinant clones randomly selected from the full-length cDNA library, we were able to sequence 6,162 independent clones with high accuracy. About 10% of the clones were wheat-unique genes, without any counterparts within the DNA database. Wheat clones that showed high homology to those of rice were selected in order to investigate their expression patterns in various tissues throughout the wheat life cycle and in response to abiotic-stress treatments. To assess the variability of genes that have evolved differently in wheat and rice, we calculated the substitution rate (Ka/Ks of the counterparts in wheat and rice. Genes that were preferentially expressed in certain tissues or treatments had higher Ka/Ks values than those in other tissues and treatments, which suggests that the genes with the higher variability expressed in these tissues is under adaptive selection. Conclusion We have generated a high-quality full-length cDNA resource for common wheat, which is essential for continuation of the

  19. Generation of recombinant pestiviruses using a full genome amplification strategy

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, Ilona; Uttenthal, Åse

    Aim Complete genome amplification of viral RNA provides a new tool for generation of modified pestiviruses. We have recently reported a full genome amplification strategy for direct recovery of infectious pestivirus (Rasmussen et al., 2008). This comprised rescue of BDV strain “Gifhorn” from a full......-length RT-PCR amplicon demonstrating that long RT-PCR can be used for direct generation of an infectious pestivirus. The strategy is not limited to amplification of BDV “Gifhorn”, but can be further utilized for amplification of a diverse selection of pestivirus strains and for the generation of modified...... was reverse transcribed to cDNA at 50C for 90 minutes using SuperScript III reverse transcriptase (Invitrogen). Full-length PCR amplification was performed using primers specific for the extreme 5’- and 3’-ends of the viral genomes. A T7 promoter was incorporated in the 5’-primers for direct in vitro...

  20. Seismic inference of 57 stars using full-length Kepler data sets

    Directory of Open Access Journals (Sweden)

    Creevey Orlagh

    2017-01-01

    Full Text Available We present stellar properties of 57 stars from a seismic inference using full-length data sets from Kepler (mass, age, radius, distances. These stars comprise active stars, planet-hosts, solar-analogs, and binary systems. We validate the distances derived from the astrometric Gaia-Tycho solution. Ensemble analysis of the stellar properties reveals a trend of mixing-length parameter with the surface gravity and effective temperature. We derive a linear relationship with the seismic quantity ‹r02› to estimate the stellar age. Finally, we define the stellar regimes where the Kjeldsen et al (2008 empirical surface correction for 1D model frequencies is valid.

  1. Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes

    Science.gov (United States)

    Throop, Andrea L.; LaBaer, Joshua

    2015-01-01

    The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full-length cDNAs in species-specific expression vectors and subsequent functional analysis of the expressed protein. Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence of interest, which differentiates this method from the classical restriction enzyme-based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression-ready clones using the most popular recombinational cloning technologies, including the commercially available Gateway® (Life Technologies) and In-Fusion® (Clontech) cloning technologies. PMID:25827088

  2. Full length prototype SSC dipole test results

    International Nuclear Information System (INIS)

    Strait, J.; Brown, B.C.; Carson, J.

    1987-01-01

    Results are presented from tests of the first full length prototype SSC dipole magnet. The cryogenic behavior of the magnet during a slow cooldown to 4.5K and a slow warmup to room temperature has been measured. Magnetic field quality was measured at currents up to 2000 A. Averaged over the body field all harmonics with the exception of b 2 and b 8 are at or within the tolerances specified by the SSC Central Design Group. (The values of b 2 and b 8 result from known design and construction defects which will be be corrected in later magnets.) Using an NMR probe the average body field strength is measured to be 10.283 G/A with point to point variations on the order of one part in 1000. Data are presented on quench behavior of the magnet up to 3500 A (approximately 55% of full field) including longitudinal and transverse velocities for the first 250 msec of the quench

  3. Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

    International Nuclear Information System (INIS)

    Han, J.H.; Stratowa, C.; Rutter, W.J.

    1987-01-01

    The authors have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy using [ 32 P]-labelled nucleotides. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase

  4. 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 (mt......DNA 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...

  5. First identification of a recombinant form of hepatitis C virus in Austrian patients by full-genome next generation sequencing.

    Directory of Open Access Journals (Sweden)

    Evelyn Stelzl

    Full Text Available Hepatitis C virus (HCV intergenotypic recombinant forms have been reported for various HCV genotypes/subtypes in several countries worldwide. In a recent study, four patients living in Austria had been identified to be possibly infected with a recombinant HCV strain. To clarify results and determine the point of recombination, full-genome next-generation sequencing using the Illumina MiSeq v2 300 cycle kit (Illumina, San Diego, CA, USA was performed in the present study. Samples of all of the patients contained the recombinant HCV strain 2k/1b. The point of recombination was found to be within the HCV NS2 gene between nucleotide positions 3189-3200 based on H77 numbering. While three of four patients were male and had migration background from Chechnya (n = 2 and Azerbaijan (n = 1, the forth patient was a female born in Austria. Three of the four patients including the female had intravenous drug abuse as a risk factor for HCV transmission. While sequencing techniques are limited to a few specialized laboratories, a genotyping assay that uses both ends of the HCV genome should be employed to identify patients infected with a recombinant HCV strain. The correct identification of recombinant strains also has an impact considering the tailored choice of anti-HCV treatment.

  6. Development and characterization of recombinant ovine coagulation factor VIII.

    Directory of Open Access Journals (Sweden)

    Philip M Zakas

    Full Text Available Animal models of the bleeding disorder, hemophilia A, have been an integral component of the biopharmaceutical development process and have facilitated the development of recombinant coagulation factor VIII (fVIII products capable of restoring median survival of persons with hemophilia A to that of the general population. However, there remain several limitations to recombinant fVIII as a biotherapeutic, including invasiveness of intravenous infusion, short half-life, immunogenicity, and lack of availability to the majority of the world's population. The recently described ovine model of hemophilia A is the largest and most accurate phenocopy. Affected sheep die prematurely due to bleeding-related pathogenesis and display robust adaptive humoral immunity to non-ovine fVIII. Herein, we describe the development and characterization of recombinant ovine fVIII (ofVIII to support further the utility of the ovine hemophilia A model. Full-length and B-domain deleted (BDD ofVIII cDNAs were generated and demonstrated to facilitate greater biosynthetic rates than their human fVIII counterparts while both BDD constructs showed greater expression rates than the same-species full-length versions. A top recombinant BDD ofVIII producing baby hamster kidney clone was identified and used to biosynthesize raw material for purification and biochemical characterization. Highly purified recombinant BDD ofVIII preparations possess a specific activity nearly 2-fold higher than recombinant BDD human fVIII and display a differential glycosylation pattern. However, binding to the carrier protein, von Willebrand factor, which is critical for stability of fVIII in circulation, is indistinguishable. Decay of thrombin-activated ofVIIIa is 2-fold slower than human fVIII indicating greater intrinsic stability. Furthermore, intravenous administration of ofVIII effectively reverses the bleeding phenotype in the murine model of hemophilia A. Recombinant ofVIII should facilitate

  7. Prophylaxis vs. on-demand treatment with BAY 81-8973, a full-length plasma protein-free recombinant factor VIII product: results from a randomized trial (LEOPOLD II).

    Science.gov (United States)

    Kavakli, K; Yang, R; Rusen, L; Beckmann, H; Tseneklidou-Stoeter, D; Maas Enriquez, M

    2015-03-01

    BAY 81-8973 is a new full-length human recombinant factor VIII product manufactured with technologies to improve consistency in glycosylation and expression to optimize clinical performance. To demonstrate superiority of prophylaxis vs. on demand therapy with BAY 81-8973 in patients with severe hemophilia A. In this multinational,randomized, open-label crossover study (LEOPOLD II;ClinicalTrials.gov identifier: NCT01233258), males aged 12–65 years with severe hemophilia A were randomized to twice-weekly prophylaxis (20-30 IU kg(-1)), 3-times-weekly prophylaxis (30-40 IU kg(-1)), or on-demand treatment with BAY 81-8973. Potency labeling for BAY 81-8973 was based on the chromogenic substrate assay or adjusted to the one-stage assay. Primary efficacy endpoint was annualized number of all bleeds (ABR). Adverse events (AEs)and immunogenicity were also assessed. Eighty patients (on demand, n = 21; twice-weekly prophylaxis, n = 28; 3-times-weekly prophylaxis, n = 31) were treated and analyzed. Mean ± SD ABR was significantly lower with prophylaxis (twice-weekly, 5.7 ± 7.2; 3-times-weekly, 4.3 ± 6.5; combined, 4.9 ± 6.8) vs. on-demand treatment (57.7 ± 24.6; P demand treatment (60.0). Median ABR was higher with twice-weekly vs. 3-times-weekly prophylaxis during the first 6-month treatment period (4.1 vs. 2.0) but was comparable in the second 6-month period (1.1 vs. 2.0). Few patients reported treatment-related AEs (4%); no treatment-related serious AEs or inhibitors were reported. Twice weekly or 3-times-weekly prophylaxis with BAY 81-8973 reduced median ABR by 97% compared with on-demand therapy, confirming the superiority of prophylaxis. Treatment with BAY 81-8973 was well tolerated.

  8. Differential sensitivity of 5'UTR-NS5A recombinants of hepatitis C virus genotypes 1-6 to protease and NS5A inhibitors

    DEFF Research Database (Denmark)

    Li, Yi-Ping; Ramirez, Santseharay; Humes, Daryl

    2014-01-01

    BACKGROUND & AIMS: Hepatitis C virus (HCV) therapy will benefit from the preclinical evaluation of direct-acting antiviral (DAA) agents in infectious culture systems that test the effects on different virus genotypes. We developed HCV recombinants comprising the 5' untranslated region-NS5A (5-5A...... daclatasvir. The 1a(TN) 5-5A and JFH1-independent full-length viruses had similar levels of sensitivity to the DAA agents, validating the 5-5A recombinants as surrogates for full-length viruses in DAA testing. Compared with the 1a(TN) full-length virus, the 3a(S52) 5-5A recombinant was highly resistant to all...... protease inhibitors, and the 4a(ED43) recombinant was highly resistant to telaprevir and boceprevir, but most sensitive to other protease inhibitors. Compared with other protease inhibitors, MK-5172 had exceptional potency against all HCV genotypes. The NS5A inhibitor daclatasvir had the highest potency...

  9. PRODUCTION OF RECOMBINANT HIGH pI-BARLEY α-GLUCOSIDASE

    DEFF Research Database (Denmark)

    Næsted, Henrik; Svensson, Birte

    plantlet [1]. Recently, expression and characterization of the recombinant full length, fully functional barley high pI α-glucosidase in Pichia pastoris has been achieved. To enable production of recombinant protein in mg amounts, a transformant harbouring a clone encoding the N-terminally hexa histidine...... tagged recombinant form of the enzyme was propagated using a high cell-density fermentation procedure. This system resulted in successful expression under the highly sensitive methanol utilization phase conducting the fermentation process using a BiostatB 5 L reactor. The recombinant high pI α...... glycosylation of the recombinant α-glucosidase. The enzyme activity was highly stable during the 5 day long fermentation. Characterisation of the enzymatic properties confirmed the specific activity actually to be superior to that of the native enzyme purified from malt [2]. The kinetic parameters Km, Vmax...

  10. Increased circulating full-length betatrophin levels in drug-naïve metabolic syndrome.

    Science.gov (United States)

    Liu, Dan; Li, Sheyu; He, He; Yu, Chuan; Li, Xiaodan; Liang, Libo; Chen, Yi; Li, Jianwei; Li, Jianshu; Sun, Xin; Tian, Haoming; An, Zhenmei

    2017-03-14

    Betatrophin is a newly identified circulating adipokine playing a role in the regulation of glucose homeostasis and lipid metabolism. But its role in metabolic syndrome (MetS) remains unknown. Therefore, we aimed to compare the circulating betatrophin concentrations between patients with MetS and healthy controls. We recruited 47 patients with MetS and 47 age and sex matched healthy controls. Anthropometric and biochemical measurements were performed, and serum betatrophin levels were detected by ELISA. Full-length betatrophin levels in patients with MetS were significantly higher than those in controls (694.84 ± 365.51 pg/ml versus 356.64 ± 287.92 pg/ml; P <0.001). While no significant difference of total betatrophin levels was found between the two groups (1.20 ± 0.79 ng/ml versus 1.31 ± 1.08 ng/ml; P = 0.524). Full-length betatrophin level was positively correlated with fasting plasma glucose (FPG) (r = 0.357, P = 0.014) and 2-hour plasma glucose (2hPG) (r = 0.38, P <0.01). Binary logistic regression models indicated that subjects in the tertile of the highest full-length betatrophin level experienced higher odds of having MetS (OR, 8.6; 95% CI 2.8-26.8; P <0.001). Our study showed that full-length betatrophin concentrations were increased in drug-naïve MetS patients.

  11. Production of Recombinant Peanut Allergen Ara h 2 using Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    Frøkiær Hanne

    2007-08-01

    Full Text Available Abstract Background Natural allergen sources can supply large quantities of authentic allergen mixtures for use as immunotherapeutics. However, such extracts are complex, difficult to define, vary from batch to batch, which may lead to unpredictable efficacy and/or unacceptable levels of side effects. The use of recombinant expression systems for allergen production can alleviate some of these issues. Several allergens have been tested in high-level expression systems and in most cases show immunereactivity comparable to their natural counterparts. The gram positive lactic acid bacterium Lactococcus lactis is an attractive microorganism for use in the production of protein therapeutics. L. lactis is considered food grade, free of endotoxins, and is able to secrete the heterologous product together with few other native proteins. Hypersensitivity to peanut represents a serious allergic problem. Some of the major allergens in peanut have been described. However, for therapeutic usage more information about the individual allergenic components is needed. In this paper we report recombinant production of the Ara h 2 peanut allergen using L. lactis. Results A synthetic ara h 2 gene was cloned into an L. lactis expression plasmid containing the P170 promoter and the SP310mut2 signal sequence. Flask cultures grown overnight showed secretion of the 17 kDa Ara h 2 protein. A batch fermentation resulted in 40 mg/L recombinant Ara h 2. Purification of Ara h 2 from the culture supernatant was done by hydrophobic exclusion and size separation. Mass spectrometry and N-terminal analysis showed a recombinant Ara h 2 of full length and correctly processed by the signal peptidase. The immunological activity of recombinant Ara h 2 was analysed by ELISA using antibodies specific for native Ara h 2. The recombinant Ara h 2 showed comparable immunereactivity to that of native Ara h 2. Conclusion Recombinant production of Ara h 2 using L. lactis can offer high yields

  12. Recombination events and variability among full-length genomes of co-circulating molluscum contagiosum virus subtypes 1 and 2.

    Science.gov (United States)

    López-Bueno, Alberto; Parras-Moltó, Marcos; López-Barrantes, Olivia; Belda, Sylvia; Alejo, Alí

    2017-05-01

    Molluscum contagiosum virus (MCV) is the sole member of the Molluscipoxvirus genus and causes a highly prevalent human disease of the skin characterized by the formation of a variable number of lesions that can persist for prolonged periods of time. Two major genotypes, subtype 1 and subtype 2, are recognized, although currently only a single complete genomic sequence corresponding to MCV subtype 1 is available. Using next-generation sequencing techniques, we report the complete genomic sequence of four new MCV isolates, including the first one derived from a subtype 2. Comparisons suggest a relatively distant evolutionary split between both MCV subtypes. Further, our data illustrate concurrent circulation of distinct viruses within a population and reveal the existence of recombination events among them. These results help identify a set of MCV genes with potentially relevant roles in molluscum contagiosum epidemiology and pathogenesis.

  13. Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells.

    Science.gov (United States)

    Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani; Streicher, Werner; Wikström, Mats; Cazzamali, Giuseppe

    2015-04-01

    Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme

    Science.gov (United States)

    Bezem, Maria T.; Baumann, Anne; Skjærven, Lars; Meyer, Romain; Kursula, Petri; Martinez, Aurora; Flydal, Marte I.

    2016-01-01

    Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners. PMID:27462005

  15. Full mitochondrial genome sequences of two endemic Philippine hornbill species (Aves: Bucerotidae) provide evidence for pervasive mitochondrial DNA recombination.

    Science.gov (United States)

    Sammler, Svenja; Bleidorn, Christoph; Tiedemann, Ralph

    2011-01-14

    Although nowaday it is broadly accepted that mitochondrial DNA (mtDNA) may undergo recombination, the frequency of such recombination remains controversial. Its estimation is not straightforward, as recombination under homoplasmy (i.e., among identical mt genomes) is likely to be overlooked. In species with tandem duplications of large mtDNA fragments the detection of recombination can be facilitated, as it can lead to gene conversion among duplicates. Although the mechanisms for concerted evolution in mtDNA are not fully understood yet, recombination rates have been estimated from "one per speciation event" down to 850 years or even "during every replication cycle". Here we present the first complete mt genome of the avian family Bucerotidae, i.e., that of two Philippine hornbills, Aceros waldeni and Penelopides panini. The mt genomes are characterized by a tandemly duplicated region encompassing part of cytochrome b, 3 tRNAs, NADH6, and the control region. The duplicated fragments are identical to each other except for a short section in domain I and for the length of repeat motifs in domain III of the control region. Due to the heteroplasmy with regard to the number of these repeat motifs, there is some size variation in both genomes; with around 21,657 bp (A. waldeni) and 22,737 bp (P. panini), they significantly exceed the hitherto longest known avian mt genomes, that of the albatrosses. We discovered concerted evolution between the duplicated fragments within individuals. The existence of differences between individuals in coding genes as well as in the control region, which are maintained between duplicates, indicates that recombination apparently occurs frequently, i.e., in every generation. The homogenised duplicates are interspersed by a short fragment which shows no sign of recombination. We hypothesize that this region corresponds to the so-called Replication Fork Barrier (RFB), which has been described from the chicken mitochondrial genome. As this RFB

  16. Recent and historical recombination in the admixed Norwegian Red cattle breed

    Directory of Open Access Journals (Sweden)

    Grove Harald

    2011-01-01

    Full Text Available Abstract Background Comparison of recent patterns of recombination derived from linkage maps to historical patterns of recombination from linkage disequilibrium (LD could help identify genomic regions affected by strong artificial selection, appearing as reduced recent recombination. Norwegian Red cattle (NRF make an interesting case study for investigating these patterns as it is an admixed breed with an extensively recorded pedigree. NRF have been under strong artificial selection for traits such as milk and meat production, fertility and health. While measures of LD is also crucial for determining the number of markers required for association mapping studies, estimates of recombination rate can be used to assess quality of genomic assemblies. Results A dataset containing more than 17,000 genome-wide distributed SNPs and 2600 animals was used to assess recombination rates and LD in NRF. Although low LD measured by r2 was observed in NRF relative to some of the breeds from which this breed originates, reports from breeds other than those assessed in this study have described more rapid decline in r2 at short distances than what was found in NRF. Rate of decline in r2 for NRF suggested that to obtain an expected r2 between markers and a causal polymorphism of at least 0.5 for genome-wide association studies, approximately one SNP every 15 kb or a total of 200,000 SNPs would be required. For well known quantitative trait loci (QTLs for milk production traits on Bos Taurus chromosomes 1, 6 and 20, map length based on historic recombination was greater than map length based on recent recombination in NRF. Further, positions for 130 previously unpositioned contigs from assembly of the bovine genome sequence (Btau_4.0 found using comparative sequence analysis were validated by linkage analysis, and 28% of these positions corresponded to extreme values of population recombination rate. Conclusion While LD is reduced in NRF compared to some of the

  17. Identification of a recombinant Muscovy Duck parvovirus (MDPV) in Shanghai, China.

    Science.gov (United States)

    Zhu, Yumin; Zhou, Zongqing; Huang, Yu; Yu, Ruisong; Dong, Shijuan; Li, Zhen; Zhang, Yuanshu

    2014-12-05

    The full-length genome of strain SAAS-SHNH, a MDPV isolated from Muscovy Duck in Shanghai, has been sequenced and shown to share 93.7% nucleotide identity with MDPV strain FM (NC_006147). Two putative genetic recombination events were identified as occurring within the 419-610 nt and 3113-4241 nt regions of the SAAS-SHNH genome which, for the first time, provide evidence of recombination between MDPVs and GPVs. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Evidence of recombination in natural populations of hepatitis A virus

    International Nuclear Information System (INIS)

    Costa-Mattioli, Mauro; Ferre, Virginie; Casane, Didier; Perez-Bercoff, Raoul; Coste-Burel, Marianne; Imbert-Marcille, Berthe-Marie; Andre, Elisabeth Claude Monique; Bressollette-Bodin, Celine; Billaudel, Sylviane; Cristina, Juan

    2003-01-01

    Genetic analysis of selected genome regions of hepatitis A virus (HAV) suggested that distinct genotypes of HAV could be found in different geographical regions. At least seven HAV genotypes have been identified all over the world, including four human genotypes (I, II, III, and VII) and three simian strains (IV, V, and VI). Phylogenetic analysis using full-length VP1 sequences revealed that human strain 9F94 has a close genetic relation with strain SLF-88 (sub-genotype VII). Nevertheless, the same analysis using full-length VP2 or VP3 sequences revealed that strain 9F94 has a close genetic relation with strain MBB (sub-genotype IB). To test the possibility of genetic recombination, phylogenetic studies were carried out, revealing that a crossing over had taken place in the VP1 capsid protein. These findings indicate that capsid-recombination can play a significant role in shaping the genetic diversity of HAV and, as such, can have important implications for its evolution, biology, and control

  19. Oral Salmonella: malaria circumsporozoite recombinants induce specific CD8+ cytotoxic T cells

    OpenAIRE

    1990-01-01

    Oral immunization with an attenuated Salmonella typhimurium recombinant containing the full-length Plasmodium berghei circumsporozoite (CS) gene induces protective immunity against P. berghei sporozoite challenge in the absence of antibody. We found that this immunity was mediated through the induction of specific CD8+ T cells since in vivo elimination of CD8+ cells abrogated protection. In vitro studies revealed that this Salmonella-P. berghei CS recombinant induced class I- restricted CD8+ ...

  20. Generation of recombinant pestiviruses using a full-genome amplification strategy

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, I.; Uttenthal, Åse

    2010-01-01

    -Gifhorn genome was generated by long RTPCR and then RNA transcripts derived from this amplicon were used to rescue infectious virus. Here, we have now used this full-genome amplification strategy for efficient and robust amplification of three additional pestivirus strains: the vaccine strain C and the virulent...... Paderborn strain of Classical swine fever virus plus the CP7 strain of Bovine viral diarrhoea virus. The amplicons were cloned directly into a stable single-copy bacterial artificial chromosome generating full-length pestivirus DNAs from which infectious RNA transcripts could be also derived....

  1. Effect of alkyl chain length of imidazolium cations on the electron transport and recombination kinetics in ionic gel electrolytes based quasi-solid-state dye-sensitized solar cells

    International Nuclear Information System (INIS)

    Huo, Zhipeng; Tao, Li; Wang, Lu; Zhu, Jun; Chen, Shuanghong; Zhang, Changneng; Dai, Songyuan; Zhang, Bing

    2015-01-01

    Highlights: •A series of novel IGEs based on 12-hydroxystearicacid as LMOG were prepared. •The QS-DSSCs exhibit excellent stability during the accelerated aging tests. •The influence of Im + alkyl chain length on the electron kinetic process is investigated. -- Abstract: A series of stable quasi-solid-state dye-sensitized solar cells (QS-DSSCs) are prepared by the 12-hydroxystearicacid as low molecular mass organogelator (LMOG) to gelate the ionic liquid with different alkyl chain lengths (3, 4, and 7). The influence of alkyl chain length of imidazolium cations (Im + ) on the kinetic processes of electron transport and recombination are investigated by Electrochemical impedance spectroscopy (EIS) and intensity-modulated photocurrent spectroscopy/intensity-modulated photovoltage spectroscopy (IMPS/IMVS). It is found that the ionic gel electrolytes (IGEs) with different alkyl chain lengths of Im + can influence the competitive adsorption effects of imidazolium cations (Im + ) and Li + , and further affect the charge diffusion, the electron recombination/transport processes, the shift of TiO 2 conduction band edge and surface states distribution. The IGE with longer alkyl chain length of Im + can prolong the electron recombination lifetime, promote the incidental photon-to-electron conversion efficiency (IPCE) and the short circuit photocurrent density (J sc ). An excellent QS-DSSC based on the IGE with the longer alkyl chain of Im + gives the highest photoelectric conversion efficiency. Moreover, all the QS-DSSCs based on IGEs exhibit excellent durability without losing their photovoltaic performances during the accelerated thermal and light–soaking test. These results are very important to the researches on the electrochemical mechanism and application of QS-DSSCs based on IGEs

  2. Simulations of The Dalles Dam Proposed Full Length Spillwall

    Energy Technology Data Exchange (ETDEWEB)

    Rakowski, Cynthia L.; Perkins, William A.; Richmond, Marshall C.; Serkowski, John A.

    2008-02-25

    This report presents results of a computational fluid dynamics (CFD) modeling study to evaluatethe impacts of a full-length spillwall at The Dalles Dam. The full-length spillwall is being designed and evaluated as a structural means to improve tailrace egress and thus survival of juvenile fish passing through the spillway. During the course of this study, a full-length spillwall at Bays 6/7 and 8/9 were considered. The U.S. Army Corps of Engineers (USACE) has proposed extending the spillwall constructed in the stilling basin between spillway Bays 6 and 7 about 590 ft farther downstream. It is believed that the extension of the spillwall will improve egress conditions for downstream juvenile salmonids by moving them more rapidly into the thalweg of the river hence reducing their exposure to predators. A numerical model was created, validated, and applied the The Dalles Dam tailrace. The models were designed to assess impacts to flow, tailrace egress, navigation, and adult salmon passage of a proposed spill wall extension. The more extensive model validation undertaken in this study greatly improved our confidence in the numerical model to represent the flow conditions in The Dalles tailrace. This study used these validated CFD models to simulate the potential impacts of a spillwall extension for The Dalles Dam tailrace for two locations. We determined the following: (1)The construction of an extended wall (between Bays 6/7) will not adversely impact entering or exiting the navigation lock. Impact should be less if a wall were constructed between Bays 8/9. (2)The construction of a wall between Bays 6/7 will increase the water surface elevation between the wall and the Washington shore. Although the increased water surface elevation would be beneficial to adult upstream migrants in that it decreases velocities on the approach to the adult ladder, the increased flow depth would enhance dissolved gas production, impacting potential operations of the project because of

  3. Recombination properties of dislocations in GaN

    Science.gov (United States)

    Yakimov, Eugene B.; Polyakov, Alexander Y.; Lee, In-Hwan; Pearton, Stephen J.

    2018-04-01

    The recombination activity of threading dislocations in n-GaN with different dislocation densities and different doping levels was studied using electron beam induced current (EBIC). The recombination velocity on a dislocation, also known as the dislocation recombination strength, was calculated. The results suggest that dislocations in n-GaN giving contrast in EBIC are charged and surrounded by a space charge region, as evidenced by the observed dependence of dislocation recombination strength on dopant concentration. For moderate (below ˜108 cm-2) dislocation densities, these defects do not primarily determine the average diffusion length of nonequilibrium charge carriers, although locally, dislocations are efficient recombination sites. In general, it is observed that the effect of the growth method [standard metalorganic chemical vapor deposition (MOCVD), epitaxial lateral overgrowth versions of MOCVD, and hydride vapor phase epitaxy] on the recombination activity of dislocations is not very pronounced, although the average diffusion lengths can widely differ for various samples. The glide of basal plane dislocations at room temperature promoted by low energy electron irradiation does not significantly change the recombination properties of dislocations.

  4. Dissection of Recombination Attributes for Multiple Maize Populations Using a Common SNP Assay

    Directory of Open Access Journals (Sweden)

    Haiying Guan

    2017-11-01

    Full Text Available Recombination is a vital characteristic for quantitative trait loci mapping and breeding to enhance the yield potential of maize. However, recombination characteristics in globally used segregating populations have never been evaluated at similar genetic marker densities. This study aimed to divulge the characteristics of recombination events, recombinant chromosomal segments, and recombination frequency for four dissimilar populations. These populations were doubled haploid (DH, recombination inbred line (RIL, intermated B73xMo17 (IBM, and multi-parent advanced generation inter-cross (MAGIC, using the Illumina MaizeSNP50 BeadChip to provide markers. Our results revealed that the average number of recombination events was 16, 41, 72, and 86 per line in DH, RIL, IBM, and MAGIC populations, respectively. Accordingly, the average length of recombinant chromosomal segments was 84.8, 47.3, 29.2, and 20.4 Mb in DH, RIL, IBM, and MAGIC populations, respectively. Furtherly, the recombination frequency varied in different genomic regions and population types [DH (0–12.7 cM/Mb, RIL (0–15.5 cM/Mb, IBM (0–24.1 cM/Mb, MAGIC (0–42.3 cM/Mb]. Utilizing different sub-sets of lines, the recombination bin number and size were analyzed in each population. Additionally, different sub-sets of markers and lines were employed to estimate the recombination bin number and size via formulas for relationship in these populations. The relationship between recombination events and recombination bin length was also examined. Our results contribute to determining the most suitable number of genetic markers, lines in each population, and population type for successful mapping and breeding.

  5. Construction and characterisation of a full-length infectious molecular clone from a fast replicating, X4-tropic HIV-1 CRF02.AG primary isolate

    International Nuclear Information System (INIS)

    Tebit, Denis M.; Zekeng, Leopold; Kaptue, Lazare; Kraeusslich, Hans-Georg; Herchenroeder, Ottmar

    2003-01-01

    Based on our previous analysis of HIV-1 isolates from Cameroon, we constructed a full-length infectious molecular clone from a primary isolate belonging to the CRF02.AG group of recombinant viruses which dominate the HIV-epidemic in West and Central Africa. The virus derived by transfection of the proviral clone pBD6-15 replicated with similar efficiency compared to its parental isolate and used CXCR4 as coreceptor as well. Furthermore, HIV-1 BD6-15 exhibited similar replication properties and virus yield as the reference B-type HIV-1 strain NL4-3. Sequence analysis revealed open reading frames for all structural and accessory genes apart from vpr. Phylogenetic and bootscanning analyses confirmed that BD6-15 clusters with CRF02.AG recombinant strains from West and Central Africa with similar cross-over points as described for the CRF02.AG prototype strain lbNG. Thus, pBD6-15 represents the first non-subtype B infectious molecular clone of a fast replicating, high producer, X4-tropic primary HIV-1 isolate, which had only been briefly passaged in primary cells

  6. Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5.

    Science.gov (United States)

    Keniya, Mikhail V; Holmes, Ann R; Niimi, Masakazu; Lamping, Erwin; Gillet, Jean-Pierre; Gottesman, Michael M; Cannon, Richard D

    2014-10-06

    ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-β mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance.

  7. Analysis of expressed sequence tags generated from full-length enriched cDNA libraries of melon

    Directory of Open Access Journals (Sweden)

    Bendahmane Abdelhafid

    2011-05-01

    Full Text Available Abstract Background Melon (Cucumis melo, an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited. Result We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs and 3,073 single nucleotide polymorphisms (SNPs in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but

  8. A simple strategy for the purification of native recombinant full-length human RPL10 protein from inclusion bodies.

    Science.gov (United States)

    Pereira, Larissa M; Silva, Luana R; Alves, Joseane F; Marin, Nélida; Silva, Flavio Sousa; Morganti, Ligia; Silva, Ismael D C G; Affonso, Regina

    2014-09-01

    The L10 ribosomal protein (RPL10) plays a role in the binding of the 60 S and 40 S ribosomal subunits and in mRNA translation. The evidence indicates that RPL10 also has multiple extra-ribosomal functions, including tumor suppression. Recently, the presence of RPL10 in prostate and ovarian cancers was evaluated, and it was demonstrated to be associated with autistic disorders and premature ovarian failure. In the present work, we successfully cloned and expressed full-length human RPL10 (hRPL10) protein and isolated inclusion bodies containing this protein that had formed under mild growth conditions. The culture produced 376mg of hRPL10 protein per liter of induced bacterial culture, of which 102.4mg was present in the soluble fraction, and 25.6mg was recovered at approximately 94% purity. These results were obtained using a two-step process of non-denaturing protein extraction from pelleted inclusion bodies. We studied the characteristics of this protein using circular dichroism spectroscopy and by monitoring the changes induced by the presence or absence of zinc ions using fluorescence spectrometry. The results demonstrated that the protein obtained using these non-conventional methods retained its secondary and tertiary structure. The conformational changes induced by the incorporation of zinc suggested that this protein could interact with Jun or the SH3 domain of c-yes. The results suggested that the strategy used to obtain hRPL10 is simple and could be applied to obtaining other proteins that are susceptible to degradation. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Production of full length and splicing form of chymosin using pETexpression system in E-coli and investigation its enzyme activity and preplasmic secretion

    Directory of Open Access Journals (Sweden)

    M. Ahmadi Zeydabadi

    2008-05-01

    Full Text Available Introduction: Chymosin (Rennin EC 3.4.23.4 is an aspartyl proteinas (the major proteolyticenzyme in the fourth stomach of the unweaned calf that is formed by proteolytic activation fromzymogene prochymosin. The aim of his study was to produce the full length and splicing form ofchymosin using pETexpression system in E-coli and to assay the activity of expressed enzyme andpreplasmic secretion.Materials and Methods: The sense primer F-prochy(+ (5´-ggggccatgGCTGAGATCACCAGGAincluding NCOI restriction site. The anti sense R-prochy(- (5´-gggcggccgcGATGGCTTTGGCCAGC -3´ hybridizing to the C-terminal end of calf preprocymosincDNA and contains an additional NotI restriction site at its 5´-end . The cells were disrupted bysonication and proteins were purified by using Ni-NTA beads from Qiagen under native conditional.The preprochymosin and AS6 preprochymosin were activated at pH 4.7. The enzyme solutions werediluted 20-fold with 50 mM phosphate buffer .Results: Sequencing data analysis showed that the exon six has been spliced out and, therefore thegene product is 114 bp shorter in length, both chymosin forms were expressed together in E.coli.Under the same expression conditions, at least AS6 preprochymosin was produced 7-fold highexpression in comparison to a full-length recombinant chymosin. Following acid activation andneutralization, the purified fractions were tested in a qualitative milk clotting assay. The clottingactivity of preprochymosin and exon6-less preprochymosin were comparable.Conclusion: High expression of this alternatively expressed transcript in bacteria, and properfolding of the AS6 chymosin protein molecule in the absence of exon six are the two most importantaspects distinguished in this research.

  10. Characterization of DNA binding, transcriptional activation, and regulated nuclear association of recombinant human NFATp

    Directory of Open Access Journals (Sweden)

    Seto Anita G

    2000-11-01

    Full Text Available Abstract Background NFATp is one member of a family of transcriptional activators whose nuclear accumulation and hence transcriptional activity is regulated in mammalian cells. Human NFATp exists as a phosphoprotein in the cytoplasm of naive T cells. Upon antigen stimulation, NFATp is dephosphorylated, accumulates in nuclei, and functions to regulate transcription of genes including those encoding cytokines. While the properties of the DNA binding domain of NFATp have been investigated in detail, biochemical studies of the transcriptional activation and regulated association with nuclei have remained unexplored because of a lack of full length, purified recombinant NFATp. Results We developed methods for expressing and purifying full length recombinant human NFATp that has all of the properties known to be associated with native NFATp. The recombinant NFATp binds DNA on its own and cooperatively with AP-1 proteins, activates transcription in vitro, is phosphorylated, can be dephosphorylated by calcineurin, and exhibits regulated association with nuclei in vitro. Importantly, activation by recombinant NFATp in a reconstituted transcription system required regions of the protein outside of the central DNA binding domain. Conclusions We conclude that NFATp is a bona fide transcriptional activator. Moreover, the reagents and methods that we developed will facilitate future studies on the mechanisms of transcriptional activation and nuclear accumulation by NFATp, a member of an important family of transcriptional regulatory proteins.

  11. Expression of full-length and splice forms of FoxP3 in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Ryder, L R; Woetmann, A; Madsen, H O

    2010-01-01

    OBJECTIVE: The aim of our study was to compare the presence of full-length and alternative splice forms of FoxP3 mRNA in CD4 cells from rheumatoid arthritis (RA) patients and healthy controls. METHODS: A quantitative real-time polymerase chain reaction (QRT-PCR) method was used to measure...... the amount of FoxP3 mRNA full-length and splice forms. CD4-positive T cells were isolated from peripheral blood from 50 RA patients by immunomagnetic separation, and the FoxP3 mRNA expression was compared with the results from 10 healthy controls. RESULTS: We observed an increased expression of full......-length FoxP3 mRNA in RA patients when compared to healthy controls, as well as an increase in CD25 mRNA expression, but no corresponding increase in CTLA-4 mRNA expression. The presence of an alternative splice form of FoxP3 lacking exon 2 was confirmed in both RA patients and healthy controls...

  12. Recombination pattern reanalysis of some HIV-1 circulating recombination forms suggest the necessity and difficulty of revision.

    Directory of Open Access Journals (Sweden)

    Lei Jia

    Full Text Available Recombination is one of the major mechanisms underlying the generation of HIV-1 variability. Currently 61 circulating recombinant forms of HIV-1 have been identified. With the development of recombination detection techniques and accumulation of HIV-1 reference stains, more accurate mosaic structures of circulating recombinant forms (CRFs, like CRF04 and CRF06, have undergone repeated analysis and upgrades. Such revisions may also be necessary for other CRFs. Unlike previous studies, whose results are based primarily on a single recombination detection program, the current study was based on multiple recombination analysis, which may have produced more impartial results.Representative references of 3 categories of intersubtype recombinants were selected, including BC recombinants (CRF07 and CRF08, BG recombinants (CRF23 and CRF24, and BF recombinants (CRF38 and CRF44. They were reanalyzed in detail using both the jumping profile hidden Markov model and RDP3.The results indicate that revisions and upgrades are very necessary and the entire re-analysis suggested 2 types of revision: (i length of inserted fragments; and (ii number of inserted fragments. The reanalysis also indicated that determination of small regions of about 200 bases or fewer should be performed with more caution.Results indicated that the involvement of multiple recombination detection programs is very necessary. Additionally, results suggested two major challenges, one involving the difficulty of accurately determining the locations of breakpoints and the second involving identification of small regions of about 200 bases or fewer with greater caution. Both indicate the complexity of HIV-1 recombination. The resolution would depend critically on development of a recombination analysis algorithm, accumulation of HIV-1 stains, and a higher sequencing quality. With the changes in recombination pattern, phylogenetic relationships of some CRFs may also change. All these results may

  13. High-level secretion of native recombinant human calreticulin in yeast

    DEFF Research Database (Denmark)

    Čiplys, Evaldas; Žitkus, Eimantas; Gold, Leslie I.

    2015-01-01

    , Saccharomyces cerevisiae and Pichia pastoris. RESULTS: Expression of a full-length human CRT precursor including its native signal sequence resulted in high-level secretion of mature recombinant protein into the culture medium by both S. cerevisiae and P. pastoris. To ensure the structural and functional...... by non-denaturing PAGE. Moreover, limited trypsin digestion yielded identical fragment patterns of calcium-binding recombinant and native CRT suggesting that the yeast-derived CRT was correctly folded. Furthermore, both native and recombinant CRT induced cellular proliferation (MTS assay) and migration...... recombinant CRT protein with yields reaching 75 % of total secreted protein and with production levels of 60 and 200 mg/l from S. cerevisiae and P. pastoris, respectively. Finally, cultivation of P. pastoris in a bioreactor yielded CRT secretion titer to exceed 1.5 g/l of culture medium. CONCLUSIONS: Yeasts...

  14. First identification of a recombinant form of hepatitis C virus in Austrian patients by full-genome next generation sequencing.

    Science.gov (United States)

    Stelzl, Evelyn; Haas, Bernhard; Bauer, Bernd; Zhang, Sherry; Fiss, Ellen H; Hillman, Grantland; Hamilton, Aaron T; Mehta, Rochak; Heil, Marintha L; Marins, Ed G; Santner, Brigitte I; Kessler, Harald H

    2017-01-01

    Hepatitis C virus (HCV) intergenotypic recombinant forms have been reported for various HCV genotypes/subtypes in several countries worldwide. In a recent study, four patients living in Austria had been identified to be possibly infected with a recombinant HCV strain. To clarify results and determine the point of recombination, full-genome next-generation sequencing using the Illumina MiSeq v2 300 cycle kit (Illumina, San Diego, CA, USA) was performed in the present study. Samples of all of the patients contained the recombinant HCV strain 2k/1b. The point of recombination was found to be within the HCV NS2 gene between nucleotide positions 3189-3200 based on H77 numbering. While three of four patients were male and had migration background from Chechnya (n = 2) and Azerbaijan (n = 1), the forth patient was a female born in Austria. Three of the four patients including the female had intravenous drug abuse as a risk factor for HCV transmission. While sequencing techniques are limited to a few specialized laboratories, a genotyping assay that uses both ends of the HCV genome should be employed to identify patients infected with a recombinant HCV strain. The correct identification of recombinant strains also has an impact considering the tailored choice of anti-HCV treatment.

  15. Construction experience with Fermilab-built full length 50mm SSC dipoles

    International Nuclear Information System (INIS)

    Blessing, M.J.; Hoffman, D.E.; Packer, M.D.; Gordon, M.; Higinbotham, W.; Sims, R.

    1992-03-01

    Fourteen full length SSC dipole magnets are being built and tested at Fermilab. Their purpose is to verify the magnet design as well as transfer the construction technology to industry. Magnet design is summarized. Construction problems and their solutions are discussed. Topics include coil winding, curing and measuring, collaring, instrumentation, end clamp installation, yoking and electrical and mechanical interconnection

  16. 3G vector-primer plasmid for constructing full-length-enriched cDNA libraries.

    Science.gov (United States)

    Zheng, Dong; Zhou, Yanna; Zhang, Zidong; Li, Zaiyu; Liu, Xuedong

    2008-09-01

    We designed a 3G vector-primer plasmid for the generation of full-length-enriched complementary DNA (cDNA) libraries. By employing the terminal transferase activity of reverse transcriptase and the modified strand replacement method, this plasmid (assembled with a polydT end and a deoxyguanosine [dG] end) combines priming full-length cDNA strand synthesis and directional cDNA cloning. As a result, the number of steps involved in cDNA library preparation is decreased while simplifying downstream gene manipulation, sequencing, and subcloning. The 3G vector-primer plasmid method yields fully represented plasmid primed libraries that are equivalent to those made by the SMART (switching mechanism at 5' end of RNA transcript) approach.

  17. Full-length high-temperature severe fuel damage test No. 2

    International Nuclear Information System (INIS)

    Hesson, G.M.; Lombardo, N.J.; Pilger, J.P.; Rausch, W.N.; King, L.L.; Hurley, D.E.; Parchen, L.J.; Panisko, F.E.

    1993-09-01

    Hazardous conditions associated with performing the Full-Length High- Temperature (FLHT). Severe Fuel Damage Test No. 2 experiment have been analyzed. Major hazards that could cause harm or damage are (1) radioactive fission products, (2) radiation fields, (3) reactivity changes, (4) hydrogen generation, (5) materials at high temperature, (6) steam explosion, and (7) steam pressure pulse. As a result of this analysis, it is concluded that with proper precautions the FLHT- 2 test can be safely conducted

  18. A new strategy for full-length Ebola virus glycoprotein expression in E.coli.

    Science.gov (United States)

    Zai, Junjie; Yi, Yinhua; Xia, Han; Zhang, Bo; Yuan, Zhiming

    2016-12-01

    Ebola virus (EBOV) causes severe hemorrhagic fever in humans and non-human primates with high rates of fatality. Glycoprotein (GP) is the only envelope protein of EBOV, which may play a critical role in virus attachment and entry as well as stimulating host protective immune responses. However, the lack of expression of full-length GP in Escherichia coli hinders the further study of its function in viral pathogenesis. In this study, the vp40 gene was fused to the full-length gp gene and cloned into a prokaryotic expression vector. We showed that the VP40-GP and GP-VP40 fusion proteins could be expressed in E.coli at 16 °C. In addition, it was shown that the position of vp40 in the fusion proteins affected the yields of the fusion proteins, with a higher level of production of the fusion protein when vp40 was upstream of gp compared to when it was downstream. The results provide a strategy for the expression of a large quantity of EBOV full-length GP, which is of importance for further analyzing the relationship between the structure and function of GP and developing an antibody for the treatment of EBOV infection.

  19. The correlation between the length of repetitive domain and mechanical properties of the recombinant flagelliform spidroin

    Directory of Open Access Journals (Sweden)

    Xue Li

    2017-03-01

    Full Text Available Spider silk is an attractive biopolymer with numerous potential applications due to its remarkable characteristics. Among the six categories of spider silks, flagelliform (Flag spider silk possesses longer and more repetitive core domains than others, therefore performing the highest extensibility. To investigate the correlation between the recombinant spidroin size and the synthetic fiber properties, four recombinant proteins with different sizes [N-Scn-C (n=1-4] were constructed and expressed using IMPACT system. Subsequently, different recombinant spidroins were spun into fibers through wet-spinning via a custom-made continuous post-drawing device. Mechanical tests of the synthetic fibers with four parameters (maximum stress, maximum extension, Young's modulus and toughness demonstrated that the extensibility of the fibers showed a positive correlation with spidroin size, consequently resulting in the extensibility of N-Sc4-C fiber ranked the highest (58.76% among four fibers. Raman data revealed the relationship between secondary structure content and mechanical properties. The data here provide a deeper insight into the relationship between the function and structure of Flag silk for future design of artificial fibers.

  20. Llama immunization with full-length VAR2CSA generates cross-reactive and inhibitory single-domain antibodies against the DBL1X domain.

    Science.gov (United States)

    Nunes-Silva, Sofia; Gangnard, Stéphane; Vidal, Marta; Vuchelen, Anneleen; Dechavanne, Sebastien; Chan, Sherwin; Pardon, Els; Steyaert, Jan; Ramboarina, Stephanie; Chêne, Arnaud; Gamain, Benoît

    2014-12-09

    VAR2CSA stands today as the leading vaccine candidate aiming to protect future pregnant women living in malaria endemic areas against the severe clinical outcomes of pregnancy associated malaria (PAM). The rational design of an efficient VAR2CSA-based vaccine relies on a profound understanding of the molecular interactions associated with P. falciparum infected erythrocyte sequestration in the placenta. Following immunization of a llama with the full-length VAR2CSA recombinant protein, we have expressed and characterized a panel of 19 nanobodies able to recognize the recombinant VAR2CSA as well as the surface of erythrocytes infected with parasites originating from different parts of the world. Domain mapping revealed that a large majority of nanobodies targeted DBL1X whereas a few of them were directed towards DBL4ε, DBL5ε and DBL6ε. One nanobody targeting the DBL1X was able to recognize the native VAR2CSA protein of the three parasite lines tested. Furthermore, four nanobodies targeting DBL1X reproducibly inhibited CSA adhesion of erythrocytes infected with the homologous NF54-CSA parasite strain, providing evidences that DBL1X domain is part or close to the CSA binding site. These nanobodies could serve as useful tools to identify conserved epitopes shared between different variants and to characterize the interactions between VAR2CSA and CSA.

  1. First report of an HIV-1 triple recombinant of subtypes B, C and F in Buenos Aires, Argentina

    Directory of Open Access Journals (Sweden)

    Weissenbacher Mercedes

    2006-09-01

    Full Text Available Abstract We describe the genetic diversity of currently transmitted strains of HIV-1 in men who have sex with men (MSM in Buenos Aires, Argentina between 2000 and 2004. Nearly full-length sequence analysis of 10 samples showed that 6 were subtype B, 3 were BF recombinant and 1 was a triple recombinant of subtypes B, C and F. The 3 BF recombinants were 3 different unique recombinant forms. Full genome analysis of one strain that was subtype F when sequenced in pol was found to be a triple recombinant. Gag and pol were predominantly subtype F, while gp120 was subtype B; there were regions of subtype C interspersed throughout. The young man infected with this strain reported multiple sexual partners and sero-converted between May and November of 2004. This study reported for the first time the full genome analysis of a triple recombinant between subtypes B, C and F, that combines in one virus the three most common subtypes in South America.

  2. Development of three full-length infectious cDNA clones of distinct brassica yellows virus genotypes for agrobacterium-mediated inoculation.

    Science.gov (United States)

    Zhang, Xiao-Yan; Dong, Shu-Wei; Xiang, Hai-Ying; Chen, Xiang-Ru; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

    2015-02-02

    Brassica yellows virus is a newly identified species in the genus of Polerovirus within the family Luteoviridae. Brassica yellows virus (BrYV) is prevalently distributed throughout Mainland China and South Korea, is an important virus infecting cruciferous crops. Based on six BrYV genomic sequences of isolates from oilseed rape, rutabaga, radish, and cabbage, three genotypes, BrYV-A, BrYV-B, and BrYV-C, exist, which mainly differ in the 5' terminal half of the genome. BrYV is an aphid-transmitted and phloem-limited virus. The use of infectious cDNA clones is an alternative means of infecting plants that allows reverse genetic studies to be performed. In this study, full-length cDNA clones of BrYV-A, recombinant BrYV5B3A, and BrYV-C were constructed under control of the cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system of Nicotiana benthamiana was developed using these cDNA clones. Three days after infiltration with full-length BrYV cDNA clones, necrotic symptoms were observed in the inoculated leaves of N. benthamiana; however, no obvious symptoms appeared in the upper leaves. Reverse transcription-PCR (RT-PCR) and western blot detection of samples from the upper leaves showed that the maximum infection efficiency of BrYVs could reach 100%. The infectivity of the BrYV-A, BrYV-5B3A, and BrYV-C cDNA clones was further confirmed by northern hybridization. The system developed here will be useful for further studies of BrYV, such as host range, pathogenicity, viral gene functions, and plant-virus-vector interactions, and especially for discerning the differences among the three genotypes. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. VP1u phospholipase activity is critical for infectivity of full-length parvovirus B19 genomic clones.

    Science.gov (United States)

    Filippone, Claudia; Zhi, Ning; Wong, Susan; Lu, Jun; Kajigaya, Sachiko; Gallinella, Giorgio; Kakkola, Laura; Söderlund-Venermo, Maria; Young, Neal S; Brown, Kevin E

    2008-05-10

    Three full-length genomic clones (pB19-M20, pB19-FL and pB19-HG1) of parvovirus B19 were produced in different laboratories. pB19-M20 was shown to produce infectious virus. To determine the differences in infectivity, all three plasmids were tested by transfection and infection assays. All three clones were similar in viral DNA replication, RNA transcription, and viral capsid protein production. However, only pB19-M20 and pB19-HG1 produced infectious virus. Comparison of viral sequences showed no significant differences in ITR or NS regions. In the capsid region, there was a nucleotide sequence difference conferring an amino acid substitution (E176K) in the phospholipase A2-like motif of the VP1-unique (VP1u) region. The recombinant VP1u with the E176K mutation had no catalytic activity as compared with the wild-type. When this mutation was introduced into pB19-M20, infectivity was significantly attenuated, confirming the critical role of this motif. Investigation of the original serum from which pB19-FL was cloned confirmed that the phospholipase mutation was present in the native B19 virus.

  4. VP1u phospholipase activity is critical for infectivity of full-length parvovirus B19 genomic clones✰

    Science.gov (United States)

    Filippone, Claudia; Zhi, Ning; Wong, Susan; Lu, Jun; Kajigaya, Sachiko; Gallinella, Giorgio; Kakkola, Laura; Venermo, Maria S Söderlund; Young, Neal S.; Brown, Kevin E.

    2008-01-01

    Three full-length genomic clones (pB19-M20, pB19-FL and pB19-HG1) of parvovirus B19 were produced in different laboratories. pB19-M20 was shown to produce infectious virus. To determine the differences in infectivity, all three plasmids were tested by transfection and infection assays. All three clones were similar in viral DNA replication, RNA transcription, and viral capsid protein production. However, only pB19-M20 and pB19-HG1 produced infectious virus. Comparison of viral sequences showed no significant differences in ITR or NS regions. In the capsid region, there was a nucleotide sequence difference conferring an amino acid substitution (E176K) in the phospholipase A2-like motif of the VP1-unique (VP1u) region. The recombinant VP1u with the E176K mutation had no catalytic activity as compared with the wild-type. When this mutation was introduced into pB19-M20, infectivity was significantly attenuated, confirming the critical role of this motif. Investigation of the original serum from which pB19-FL was cloned confirmed that the phospholipase mutation was present in the native B19 virus. PMID:18252260

  5. Characterization of recombinant high pI Barley α-Glucosidase

    DEFF Research Database (Denmark)

    Næsted, Henrik; Svensson, Birte

    (MacGregor A.W.). Here we present the recent results of the expression and characterization of the recombinant full length barley high pI α-glucosidase in Pichia Pastoris. In order to facilitate in the range of mg protein yield, a clone representing an N-terminal hexa histidine tagged recombinant form...... of the enzyme was grown under high cell-density fermentation conditions. This approach enabled a successful protein expression profile under the highly sensitive methanol utilization phase of the fermentation procedure. The enzyme was purified using a four step purification strategy. Interestingly, the purified...... enzyme exhibits a higher molecular mass than expected from its primary sequence when applied on SDS-PAGE, indicating a possible post translational modification of the recombinant α-glucosidase. Preliminary enzyme kinetic analysis has demonstrated that the purified α-glucosidase is “fully” active when...

  6. Full-length cDNA sequences from Rhesus monkey placenta tissue: analysis and utility for comparative mapping

    Directory of Open Access Journals (Sweden)

    Lee Sang-Rae

    2010-07-01

    Full Text Available Abstract Background Rhesus monkeys (Macaca mulatta are widely-used as experimental animals in biomedical research and are closely related to other laboratory macaques, such as cynomolgus monkeys (Macaca fascicularis, and to humans, sharing a last common ancestor from about 25 million years ago. Although rhesus monkeys have been studied extensively under field and laboratory conditions, research has been limited by the lack of genetic resources. The present study generated placenta full-length cDNA libraries, characterized the resulting expressed sequence tags, and described their utility for comparative mapping with human RefSeq mRNA transcripts. Results From rhesus monkey placenta full-length cDNA libraries, 2000 full-length cDNA sequences were determined and 1835 rhesus placenta cDNA sequences longer than 100 bp were collected. These sequences were annotated based on homology to human genes. Homology search against human RefSeq mRNAs revealed that our collection included the sequences of 1462 putative rhesus monkey genes. Moreover, we identified 207 genes containing exon alterations in the coding region and the untranslated region of rhesus monkey transcripts, despite the highly conserved structure of the coding regions. Approximately 10% (187 of all full-length cDNA sequences did not represent any public human RefSeq mRNAs. Intriguingly, two rhesus monkey specific exons derived from the transposable elements of AluYRa2 (SINE family and MER11B (LTR family were also identified. Conclusion The 1835 rhesus monkey placenta full-length cDNA sequences described here could expand genomic resources and information of rhesus monkeys. This increased genomic information will greatly contribute to the development of evolutionary biology and biomedical research.

  7. Full-length high-temperature severe fuel damage test No. 5

    International Nuclear Information System (INIS)

    Lanning, D.D.; Lombardo, N.J.; Hensley, W.K.; Fitzsimmons, D.E.; Panisko, F.E.; Hartwell, J.K.

    1993-09-01

    This report describes and presents data from a severe fuel damage test that was conducted in the National Research Universal (NRU) reactor at Chalk River Nuclear Laboratories (CRNL), Ontario, Canada. The test, designated FLHT-5, was the fourth in a series of full-length high-temperature (FLHT) tests on light-water reactor fuel. The tests were designed and performed by staff from the US Department of Energy's Pacific Northwest Laboratory (PNL), operated by Battelle Memorial Institute. The test operation and test results are described in this report. The fuel bundle in the FLHT-5 experiment included 10 unirradiated full-length pressurized-water reactor (PWR) rods, 1 irradiated PWR rod and 1 dummy gamma thermometer. The fuel rods were subjected to a very low coolant flow while operating at low fission power. This caused coolant boilaway, rod dryout and overheating to temperatures above 2600 K, severe fuel rod damage, hydrogen generation, and fission product release. The test assembly and its effluent path were extensively instrumented to record temperatures, pressures, flow rates, hydrogen evolution, and fission product release during the boilaway/heatup transient. Post-test gamma scanning of the upper plenum indicated significant iodine and cesium release and deposition. Both stack gas activity and on-line gamma spectrometer data indicated significant (∼50%) release of noble fission gases. Post-test visual examination of one side of the fuel bundle revealed no massive relocation and flow blockage; however, rundown of molten cladding was evident

  8. Meiotic recombination analyses of individual chromosomes in male domestic pigs (Sus scrofa domestica.

    Directory of Open Access Journals (Sweden)

    Nicolas Mary

    Full Text Available For the first time in the domestic pig, meiotic recombination along the 18 porcine autosomes was directly studied by immunolocalization of MLH1 protein. In total, 7,848 synaptonemal complexes from 436 spermatocytes were analyzed, and 13,969 recombination sites were mapped. Individual chromosomes for 113 of the 436 cells (representing 2,034 synaptonemal complexes were identified by immunostaining and fluorescence in situ hybridization (FISH. The average total length of autosomal synaptonemal complexes per cell was 190.3 µm, with 32.0 recombination sites (crossovers, on average, per cell. The number of crossovers and the lengths of the autosomal synaptonemal complexes showed significant intra- (i.e. between cells and inter-individual variations. The distributions of recombination sites within each chromosomal category were similar: crossovers in metacentric and submetacentric chromosomes were concentrated in the telomeric regions of the p- and q-arms, whereas two hotspots were located near the centromere and in the telomeric region of acrocentrics. Lack of MLH1 foci was mainly observed in the smaller chromosomes, particularly chromosome 18 (SSC18 and the sex chromosomes. All autosomes displayed positive interference, with a large variability between the chromosomes.

  9. Four Closely Related HIV-1 CRF01_AE/CRF07_BC Recombinant Forms Identified in East China.

    Science.gov (United States)

    Li, Fan; Li, Yuxueyun; Feng, Yi; Hu, Jing; Ruan, Yuhua; Xing, Hui; Shao, Yiming

    2017-07-01

    Five near full-length genomes of novel second-generation HIV-1 recombinant virus (JS150021, JS150029, JS150129, JS150132, and AH150183) were identified from five HIV-positive people in Jiangsu and Anhui province, east China. Phylogenic analyses showed that these five sequences are all composed of two well-established circulating recombinant forms (CRFs) CRF07_BC and CRF01_AE, grouped into four new discovered recombinant forms, which show several very similar but not identical recombinant breakpoints. The four recombinant forms are also identified to be a sort of family or related viruses, seems to be the results of different recombination events. The emergence of a serious new closely related CRF07_BC/CRF01_AE recombinant strain indicates the increasing complexity of sexual transmission of the HIV-1 epidemic in China.

  10. Pulp regeneration in a full-length human tooth root using a hierarchical nanofibrous microsphere system.

    Science.gov (United States)

    Li, Xiangwei; Ma, Chi; Xie, Xiaohua; Sun, Hongchen; Liu, Xiaohua

    2016-04-15

    While pulp regeneration using tissue engineering strategy has been explored for over a decade, successful regeneration of pulp tissues in a full-length human root with a one-end seal that truly simulates clinical endodontic treatment has not been achieved. To address this challenge, we designed and synthesized a unique hierarchical growth factor-loaded nanofibrous microsphere scaffolding system. In this system, vascular endothelial growth factor (VEGF) binds with heparin and is encapsulated in heparin-conjugated gelatin nanospheres, which are further immobilized in the nanofibers of an injectable poly(l-lactic acid) (PLLA) microsphere. This hierarchical microsphere system not only protects the VEGF from denaturation and degradation, but also provides excellent control of its sustained release. In addition, the nanofibrous PLLA microsphere integrates the extracellular matrix-mimicking architecture with a highly porous injectable form, efficiently accommodating dental pulp stem cells (DPSCs) and supporting their proliferation and pulp tissue formation. Our in vivo study showed the successful regeneration of pulp-like tissues that fulfilled the entire apical and middle thirds and reached the coronal third of the full-length root canal. In addition, a large number of blood vessels were regenerated throughout the canal. For the first time, our work demonstrates the success of pulp tissue regeneration in a full-length root canal, making it a significant step toward regenerative endodontics. The regeneration of pulp tissues in a full-length tooth root canal has been one of the greatest challenges in the field of regenerative endodontics, and one of the biggest barriers for its clinical application. In this study, we developed a unique approach to tackle this challenge, and for the first time, we successfully regenerated living pulp tissues in a full-length root canal, making it a significant step toward regenerative endodontics. This study will make positive scientific

  11. Full-length genomic characterization and molecular evolution of canine parvovirus in China.

    Science.gov (United States)

    Zhou, Ling; Tang, Qinghai; Shi, Lijun; Kong, Miaomiao; Liang, Lin; Mao, Qianqian; Bu, Bin; Yao, Lunguang; Zhao, Kai; Cui, Shangjin; Leal, Élcio

    2016-06-01

    Canine parvovirus type 2 (CPV-2) can cause acute haemorrhagic enteritis in dogs and myocarditis in puppies. This disease has become one of the most serious infectious diseases of dogs. During 2014 in China, there were many cases of acute infectious diarrhoea in dogs. Some faecal samples were negative for the CPV-2 antigen based on a colloidal gold test strip but were positive based on PCR, and a viral strain was isolated from one such sample. The cytopathic effect on susceptible cells and the results of the immunoperoxidase monolayer assay, PCR, and sequencing indicated that the pathogen was CPV-2. The strain was named CPV-NY-14, and the full-length genome was sequenced and analysed. A maximum likelihood tree was constructed using the full-length genome and all available CPV-2 genomes. New strains have replaced the original strain in Taiwan and Italy, although the CPV-2a strain is still predominant there. However, CPV-2a still causes many cases of acute infectious diarrhoea in dogs in China.

  12. Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1

    DEFF Research Database (Denmark)

    End, Caroline; Lyer, Stefan; Renner, Marcus

    2005-01-01

    of a vector system that facilitates cloning of DMBT1 variants. We demonstrate applicability of the vector system by expression of the largest DMBT1 variant in a tetracycline-inducible mammalian expression system using the Chinese hamster ovary cell line. Yields up to 30 mg rDMBT1 per litre of cell culture......Deleted in malignant brain tumours 1 (DMBT1) codes for a approximately 340kDa glycoprotein with highly repetitive scavenger receptor cysteine-rich (SRCR) domains. DMBT1 was implicated in cancer, defence against viral and bacterial infections, and differentiation of epithelial cells. Recombinant...... yields, and protein preparations which may substantially vary due to differential processing and genetic polymorphism, all of which impedes functional research on DMBT1. Cloning of DMBT1 cDNAs is hampered because of the size and the 13 highly homologous SRCR exons. In this study, we report on the setup...

  13. Fast Dissemination of New HIV-1 CRF02/A1 Recombinants in Pakistan.

    Directory of Open Access Journals (Sweden)

    Yue Chen

    Full Text Available A number of HIV-1 subtypes are identified in Pakistan by characterization of partial viral gene sequences. Little is known whether new recombinants are generated and how they disseminate since whole genome sequences for these viruses have not been characterized. Near full-length genome (NFLG sequences were obtained by amplifying two overlapping half genomes or next generation sequencing from 34 HIV-1-infected individuals in Pakistan. Phylogenetic tree analysis showed that the newly characterized sequences were 16 subtype As, one subtype C, and 17 A/G recombinants. Further analysis showed that all 16 subtype A1 sequences (47%, together with the vast majority of sequences from Pakistan from other studies, formed a tight subcluster (A1a within the subtype A1 clade, suggesting that they were derived from a single introduction. More in-depth analysis of 17 A/G NFLG sequences showed that five shared similar recombination breakpoints as in CRF02 (15% but were phylogenetically distinct from the prototype CRF02 by forming a tight subcluster (CRF02a while 12 (38% were new recombinants between CRF02a and A1a or a divergent A1b viruses. Unique recombination patterns among the majority of the newly characterized recombinants indicated ongoing recombination. Interestingly, recombination breakpoints in these CRF02/A1 recombinants were similar to those in prototype CRF02 viruses, indicating that recombination at these sites more likely generate variable recombinant viruses. The dominance and fast dissemination of new CRF02a/A1 recombinants over prototype CRF02 suggest that these recombinant have more adapted and may become major epidemic strains in Pakistan.

  14. Revised genomic consensus for the hypermethylated CpG island region of the human L1 transposon and integration sites of full length L1 elements from recombinant clones made using methylation-tolerant host strains

    DEFF Research Database (Denmark)

    Crowther, P J; Doherty, J P; Linsenmeyer, M E

    1991-01-01

    preferentially from L1 members which have accumulated mutations that have removed sites of methylation. We present a revised consensus from the 5' presumptive control region of these elements. This revised consensus contains a consensus RNA polymerase III promoter which would permit the synthesis of transcripts......Efficient recovery of clones from the 5' end of the human L1 dispersed repetitive elements necessitates the use of deletion mcr- host strains since this region contains a CpG island which is hypermethylated in vivo. Clones recovered with conventional mcr+ hosts seem to have been derived...... from the 5' end of full length L1 elements. Such potential transcripts are likely to exhibit a high degree of secondary structure. In addition, we have determined the flanking sequences for 6 full length L1 elements. The majority of full length L1 clones show no convincing evidence for target site...

  15. Purification and characterization of recombinant high pI Barley α-Glucosidase

    DEFF Research Database (Denmark)

    Næsted, Henrik; Bojsen, Kirsten; Svensson, Birte

    (MACGREGOR & sissons). recently expression and characterization of the recombinant full length and fully functional barley high pi α-glucosidase in pichia pastoris has been achieved. in order to facilitate protein yield in the mg range, a clone representing an n-terminal hexa histidine tagged recombinant...... form of the enzyme was grown under high cell-density fermentation conditions. this approach enabled a successful protein expression profile under the highly sensitive methanol utilization phase using a biotatb 5 l reactor for the fermentation procedure. the enzyme was purified from 3.5 liter α...... of the native enzyme indicates a possible post-translational glycosylation of the recombinant α-glucosidase. preliminary enzyme kinetic analysis has demonstrated that the purified α-glucosidase displayed high stability during the 5 day long fermenentation and exhibited a specific activity in the range...

  16. Evaluation of full-length, cleaved and nitrosylated serum surfactant protein D as biomarkers for COPD

    DEFF Research Database (Denmark)

    Duvoix, Annelyse; Miranda, Elena; Perez, Juan

    2011-01-01

    . Serum levels of SP-D are raised in individuals with COPD but there is no correlation between the serum level of SP-D and the severity of airflow obstruction. Serum SP-D is present in different forms that may have more utility as a biomarker for COPD. We report here the development of new monoclonal...... antibodies to full length and cleaved SP-D. We have assessed these and existing antibodies in 98 individuals with COPD recruited to the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) cohort. Our data show that neither monoclonal antibodies to full length nor cleaved SP...

  17. Insertion of Introns: A Strategy to Facilitate Assembly of Infectious Full Length Clones

    DEFF Research Database (Denmark)

    Johansen, Ida Elisabeth; Lund, Ole Søgaard

    2008-01-01

    Some DNA fragments are difficult to clone in Escherichia coli by standard methods. It has been speculated that unintended transcription and translation result in expression of proteins that are toxic to the bacteria. This problem is frequently observed during assembly of infectious full-length vi...

  18. Impaired heat shock response in cells expressing full-length polyglutamine-expanded huntingtin.

    Directory of Open Access Journals (Sweden)

    Sidhartha M Chafekar

    Full Text Available The molecular mechanisms by which polyglutamine (polyQ-expanded huntingtin (Htt causes neurodegeneration in Huntington's disease (HD remain unclear. The malfunction of cellular proteostasis has been suggested as central in HD pathogenesis and also as a target of therapeutic interventions for the treatment of HD. We present results that offer a previously unexplored perspective regarding impaired proteostasis in HD. We find that, under non-stress conditions, the proteostatic capacity of cells expressing full length polyQ-expanded Htt is adequate. Yet, under stress conditions, the presence of polyQ-expanded Htt impairs the heat shock response, a key component of cellular proteostasis. This impaired heat shock response results in a reduced capacity to withstand the damage caused by cellular stress. We demonstrate that in cells expressing polyQ-expanded Htt the levels of heat shock transcription factor 1 (HSF1 are reduced, and, as a consequence, these cells have an impaired a heat shock response. Also, we found reduced HSF1 and HSP70 levels in the striata of HD knock-in mice when compared to wild-type mice. Our results suggests that full length, non-aggregated polyQ-expanded Htt blocks the effective induction of the heat shock response under stress conditions and may thus trigger the accumulation of cellular damage during the course of HD pathogenesis.

  19. Performance of initial full-length RHIC [Relativistic Heavy Ion Collider] dipoles

    International Nuclear Information System (INIS)

    Dahl, P.; Cottingham, J.; Garber, M.

    1987-01-01

    The first four full-length (9.7 m) R and D dipoles for the proposed Relativistic Heavy Ion Collider (RHIC) have been successfully tested. The magnets reached a quench plateau of approximately 4.5 T with very reasonable training - a field level comfortably above the design field of 3.45 T required for operation with beams of 100 GeV/amu gold nuclei. Measured field multipoles are considered to be quite acceptable for this series of R and D magnets

  20. Bicarbonate-dependent secretion and proteolytic processing of recombinant myocilin.

    Directory of Open Access Journals (Sweden)

    José-Daniel Aroca-Aguilar

    Full Text Available Myocilin is an extracellular glycoprotein of poorly understood function. Mutations of this protein are involved in glaucoma, an optic neuropathy characterized by a progressive and irreversible visual loss and frequently associated with elevated intraocular pressure. We previously showed that recombinant myocilin undergoes an intracellular proteolytic processing by calpain II which cleaves the central region of the protein, releasing one N- and one C-terminal fragment. Myocilin cleavage is reduced by glaucoma mutations and it has been proposed to participate in intraocular pressure modulation. To identify possible factors regulating the proteolytic processing of recombinant myocilin, we used a cellular model in which we analyzed how different culture medium parameters (i.e., culture time, cell density, pH, bicarbonate concentration, etc. affect the presence of the extracellular C-terminal fragment. Extracellular bicarbonate depletion associated with culture medium acidification produced a reversible intracellular accumulation of full-length recombinant myocilin and incremented its intracellular proteolytic processing, raising the extracellular C-terminal fragment percentage. It was also determined that myocilin intracellular accumulation depends on its N-terminal region. These data suggest that aqueous humor bicarbonate variations could also modulate the secretion and cleavage of myocilin present in ocular tissues.

  1. Construction of recombinant DNA clone for bovine viral diarrhea virus

    International Nuclear Information System (INIS)

    Yeo, S.G.; Cho, H.J.; Masri, S.A.

    1992-01-01

    Molecular cloning was carried out on the Danish strain of bovine viral diarrhea virus (BVDV) to construct strategy for the diagnostic tools and effective vaccine of BVD afterwards. A recombinant DNA clone (No. 29) was established successfully from cDNA for viral RNA tailed with adenine homopolymer at 3 -end. 32 P-labeled DNA probes of 300~1, 800bp fragments, originating from the clone 29, directed specific DNA-RNA hybridization results with BVDV RNA. Recombinant DNA of the clone 29 was about 5,200bp representing 41.6% of the full length of Danish strain's RNA, and restriction sites were recognized for EooR I, Sst I, Hind III and Pst I restriction enzymes in the DNA fragment

  2. Evolutionary advantage via common action of recombination and neutrality

    Science.gov (United States)

    Saakian, David B.; Hu, Chin-Kun

    2013-11-01

    We investigate evolution models with recombination and neutrality. We consider the Crow-Kimura (parallel) mutation-selection model with the neutral fitness landscape, in which there is a central peak with high fitness A, and some of 1-point mutants have the same high fitness A, while the fitness of other sequences is 0. We find that the effect of recombination and neutrality depends on the concrete version of both neutrality and recombination. We consider three versions of neutrality: (a) all the nearest neighbor sequences of the peak sequence have the same high fitness A; (b) all the l-point mutations in a piece of genome of length l≥1 are neutral; (c) the neutral sequences are randomly distributed among the nearest neighbors of the peak sequences. We also consider three versions of recombination: (I) the simple horizontal gene transfer (HGT) of one nucleotide; (II) the exchange of a piece of genome of length l, HGT-l; (III) two-point crossover recombination (2CR). For the case of (a), the 2CR gives a rather strong contribution to the mean fitness, much stronger than that of HGT for a large genome length L. For the random distribution of neutral sequences there is a critical degree of neutrality νc, and for μmutations affect the mean-field-like and fluctuation-like factors similarly. Consequently, recombination can accelerate the non-mean-field (fluctuation) type dynamics without considerably affecting the mean-field-like factors.

  3. Bacterial phylogenetic reconstruction from whole genomes is robust to recombination but demographic inference is not.

    Science.gov (United States)

    Hedge, Jessica; Wilson, Daniel J

    2014-11-25

    Phylogenetic inference in bacterial genomics is fundamental to understanding problems such as population history, antimicrobial resistance, and transmission dynamics. The field has been plagued by an apparent state of contradiction since the distorting effects of recombination on phylogeny were discovered more than a decade ago. Researchers persist with detailed phylogenetic analyses while simultaneously acknowledging that recombination seriously misleads inference of population dynamics and selection. Here we resolve this paradox by showing that phylogenetic tree topologies based on whole genomes robustly reconstruct the clonal frame topology but that branch lengths are badly skewed. Surprisingly, removing recombining sites can exacerbate branch length distortion caused by recombination. Phylogenetic tree reconstruction is a popular approach for understanding the relatedness of bacteria in a population from differences in their genome sequences. However, bacteria frequently exchange regions of their genomes by a process called homologous recombination, which violates a fundamental assumption of phylogenetic methods. Since many researchers continue to use phylogenetics for recombining bacteria, it is important to understand how recombination affects the conclusions drawn from these analyses. We find that whole-genome sequences afford great accuracy in reconstructing evolutionary relationships despite concerns surrounding the presence of recombination, but the branch lengths of the phylogenetic tree are indeed badly distorted. Surprisingly, methods to reduce the impact of recombination on branch lengths can exacerbate the problem. Copyright © 2014 Hedge and Wilson.

  4. Three-body recombination of cold fermionic atoms

    International Nuclear Information System (INIS)

    Suno, H; Esry, B D; Greene, Chris H

    2003-01-01

    Recombination of identical, spin-polarized fermions in cold three-body collisions is investigated. We parametrize the mechanisms for recombination in terms of the 'scattering volume' V p and another length scale r 0 . Model two-body interactions were used within the framework of the adiabatic hyperspherical representation. We examine the recombination rate K 3 as a function of the collision energy E for various values of V p . Not only do we consider the dominant J Π = 1 + case, but also the next-leading order contributions from J Π = 1 - and 3 - . We discuss the behaviour near a two-body resonance and the expected universality of fermionic recombination. Comparisons with boson recombination are considered in detail

  5. XenDB: Full length cDNA prediction and cross species mapping in Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Giegerich Robert

    2005-09-01

    Full Text Available Abstract Background Research using the model system Xenopus laevis has provided critical insights into the mechanisms of early vertebrate development and cell biology. Large scale sequencing efforts have provided an increasingly important resource for researchers. To provide full advantage of the available sequence, we have analyzed 350,468 Xenopus laevis Expressed Sequence Tags (ESTs both to identify full length protein encoding sequences and to develop a unique database system to support comparative approaches between X. laevis and other model systems. Description Using a suffix array based clustering approach, we have identified 25,971 clusters and 40,877 singleton sequences. Generation of a consensus sequence for each cluster resulted in 31,353 tentative contig and 4,801 singleton sequences. Using both BLASTX and FASTY comparison to five model organisms and the NR protein database, more than 15,000 sequences are predicted to encode full length proteins and these have been matched to publicly available IMAGE clones when available. Each sequence has been compared to the KOG database and ~67% of the sequences have been assigned a putative functional category. Based on sequence homology to mouse and human, putative GO annotations have been determined. Conclusion The results of the analysis have been stored in a publicly available database XenDB http://bibiserv.techfak.uni-bielefeld.de/xendb/. A unique capability of the database is the ability to batch upload cross species queries to identify potential Xenopus homologues and their associated full length clones. Examples are provided including mapping of microarray results and application of 'in silico' analysis. The ability to quickly translate the results of various species into 'Xenopus-centric' information should greatly enhance comparative embryological approaches. Supplementary material can be found at http://bibiserv.techfak.uni-bielefeld.de/xendb/.

  6. Genome-wide recombination rate variation in a recombination map of cotton.

    Science.gov (United States)

    Shen, Chao; Li, Ximei; Zhang, Ruiting; Lin, Zhongxu

    2017-01-01

    Recombination is crucial for genetic evolution, which not only provides new allele combinations but also influences the biological evolution and efficacy of natural selection. However, recombination variation is not well understood outside of the complex species' genomes, and it is particularly unclear in Gossypium. Cotton is the most important natural fibre crop and the second largest oil-seed crop. Here, we found that the genetic and physical maps distances did not have a simple linear relationship. Recombination rates were unevenly distributed throughout the cotton genome, which showed marked changes along the chromosome lengths and recombination was completely suppressed in the centromeric regions. Recombination rates significantly varied between A-subgenome (At) (range = 1.60 to 3.26 centimorgan/megabase [cM/Mb]) and D-subgenome (Dt) (range = 2.17 to 4.97 cM/Mb), which explained why the genetic maps of At and Dt are similar but the physical map of Dt is only half that of At. The translocation regions between A02 and A03 and between A04 and A05, and the inversion regions on A10, D10, A07 and D07 indicated relatively high recombination rates in the distal regions of the chromosomes. Recombination rates were positively correlated with the densities of genes, markers and the distance from the centromere, and negatively correlated with transposable elements (TEs). The gene ontology (GO) categories showed that genes in high recombination regions may tend to response to environmental stimuli, and genes in low recombination regions are related to mitosis and meiosis, which suggested that they may provide the primary driving force in adaptive evolution and assure the stability of basic cell cycle in a rapidly changing environment. Global knowledge of recombination rates will facilitate genetics and breeding in cotton.

  7. Expression of recombinant Clostridium difficile toxin A and B in Bacillus megaterium

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    Nie Weijia

    2008-11-01

    Full Text Available Abstract Background Major Clostridium difficile virulence factors are the exotoxins TcdA and TcdB. Due to the large size and poor stability of the proteins, the active recombinant TcdA and TcdB have been difficult to produce. Results The toxin genes tcdA and tcdB were amplified by PCR using chromosomal DNA from a toxigenic strain as a template, and cloned into a shuttle vector pHis1522. The sequences of both tcdA and tcdB genes in the vector have been verified by DNA sequencing. The constructs were transformed into B. megaterium protoplasts and the protein expression was controlled under a xylose promoter. The recombinant toxins (rTcdA and rTcdB were purified from bacterial crude extracts. Approximately 5 – 10 mg of highly purified recombinant toxins were obtained from one liter of bacterial culture. The resulting rTcdA and rTcdB had similar molecular masses to the native toxins, and their biological activities were found to be similar to their native counterparts after an extensive examination. Conclusion We have generated the full length and active recombinant TcdA and TcdB in Bacillus megaterium.

  8. Expression of Recombinant Human Coagulation Factor VII by the Lizard Leishmania Expression System

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    Sina Mirzaahmadi

    2011-01-01

    Full Text Available The variety of recombinant protein expression systems have been developed as a resource of FVII gene expression. In the current study, the authors used a novel protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host for expression of FVII. Plasmid containing cDNA encoding full-length human FVII was introduced into Lizard Leishmania and positive transfectants were analyzed by SDS-PAGE and Western blot analysis. Furthermore, biological activity of purified protein was detected by PT assay. The recombinant strain harboring a construct was analyzed for expression of FVII at the mRNA and protein level. Purified rFVII was obtained and in order to confirm the purified compound was in fact rFVII. Western blot analysis was carried out. Clotting time in PT assay was reduced about 30 seconds with the purified rFVII. In Conclusion, this study has demonstrated, for the first time, that Leishmania cells can be used as an expression system for producing recombinant FVII.

  9. Homologous recombination is a force in the evolution of canine distemper virus.

    Science.gov (United States)

    Yuan, Chaowen; Liu, Wenxin; Wang, Yingbo; Hou, Jinlong; Zhang, Liguo; Wang, Guoqing

    2017-01-01

    Canine distemper virus (CDV) is the causative agent of canine distemper (CD) that is a highly contagious, lethal, multisystemic viral disease of receptive carnivores. The prevalence of CDV is a major concern in susceptible animals. Presently, it is unclear whether intragenic recombination can contribute to gene mutations and segment reassortment in the virus. In this study, 25 full-length CDV genome sequences were subjected to phylogenetic and recombinational analyses. The results of phylogenetic analysis, intragenic recombination, and nucleotide selection pressure indicated that mutation and recombination occurred in the six individual genes segment (H, F, P, N, L, M) of the CDV genome. The analysis also revealed pronounced genetic diversity in the CDV genome according to the geographically distinct lineages (genotypes), namely Asia-1, Asia-2, Asia-3, Europe, America-1, and America-2. The six recombination events were detected using SimPlot and RDP programs. The analysis of selection pressure demonstrated that a majority of the nucleotides in the CDV individual gene were under negative selection. Collectively, these data suggested that homologous recombination acts as a key force driving the genetic diversity and evolution of canine distemper virus.

  10. Full-length Dysferlin Transfer by the Hyperactive Sleeping Beauty Transposase Restores Dysferlin-deficient Muscle

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    Helena Escobar

    2016-01-01

    Full Text Available Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target genome. We constructed an SB transposon-based vector to deliver full-length human DYSF cDNA into dysferlin-deficient H2K A/J myoblasts. We demonstrate proper dysferlin expression as well as highly efficient engraftment (>1,100 donor-derived fibers of the engineered myoblasts in the skeletal muscle of dysferlin- and immunodeficient B6. Cg-Dysfprmd Prkdcscid/J (Scid/BLA/J mice. Nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle are important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy.

  11. Regulation of homologous recombination at telomeres in budding yeast

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine; Lisby, Michael

    2010-01-01

    Homologous recombination is suppressed at normal length telomere sequences. In contrast, telomere recombination is allowed when telomeres erode in the absence of telomerase activity or as a consequence of nucleolytic degradation or incomplete replication. Here, we review the mechanisms that contr...... that contribute to regulating mitotic homologous recombination at telomeres and the role of these mechanisms in signalling short telomeres in the budding yeast Saccharomyces cerevisiae....

  12. Generation of Recombinant Ebola Viruses Using Reverse Genetics.

    Science.gov (United States)

    Groseth, Allison

    2017-01-01

    Reverse genetics systems encompass a wide array of tools aimed at recapitulating some or all of the virus life cycle. In their most complete form, full-length clone systems allow us to use plasmid-encoded versions of the ribonucleoprotein (RNP) components to initiate the transcription and replication of a plasmid-encoded version of the complete viral genome, thereby initiating the complete virus life cycle and resulting in infectious virus. As such this approach is ideal for the generation of tailor-made recombinant filoviruses, which can be used to study virus biology. In addition, the generation of tagged and particularly fluorescent or luminescent viruses can be applied as tools for both diagnostic applications and for screening to identify novel countermeasures. Here we describe the generation and basic characterization of recombinant Ebola viruses rescued from cloned cDNA using a T7-driven system.

  13. Species-Specific Expression of Full-Length and Alternatively Spliced Variant Forms of CDK5RAP2.

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    John S Y Park

    Full Text Available CDK5RAP2 is one of the primary microcephaly genes that are associated with reduced brain size and mental retardation. We have previously shown that human CDK5RAP2 exists as a full-length form (hCDK5RAP2 or an alternatively spliced variant form (hCDK5RAP2-V1 that is lacking exon 32. The equivalent of hCDK5RAP2-V1 has been reported in rat and mouse but the presence of full-length equivalent hCDK5RAP2 in rat and mouse has not been examined. Here, we demonstrate that rat expresses both a full length and an alternatively spliced variant form of CDK5RAP2 that are equivalent to our previously reported hCDK5RAP2 and hCDK5RAP2-V1, repectively. However, mouse expresses only one form of CDK5RAP2 that is equivalent to the human and rat alternatively spliced variant forms. Knowledge of this expression of different forms of CDK5RAP2 in human, rat and mouse is essential in selecting the appropriate model for studies of CDK5RAP2 and primary microcephaly but our findings further indicate the evolutionary divergence of mouse from the human and rat species.

  14. Homologous recombination mediates functional recovery of dysferlin deficiency following AAV5 gene transfer.

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    William E Grose

    Full Text Available 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 advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique plasticity with regards to packaging capacity and recombination of virions containing homologous regions of cDNA inserts has been implicated in the generation of full-length transcripts. Herein we show for the first time in vivo that homologous recombination following AAV5.DYSF gene transfer leads to the production of full length transcript and protein. Moreover, gene transfer of full-length dysferlin protein in dysferlin deficient mice resulted in expression levels sufficient to correct functional deficits in the diaphragm and importantly in skeletal muscle membrane repair. Intravascular regional gene transfer through the femoral artery produced high levels of transduction and enabled targeting of specific muscle groups affected by the dysferlinopathies setting the stage for potential translation to clinical trials. We provide proof of principle that AAV5 mediated delivery of dysferlin is a highly promising strategy for treatment of dysferlinopathies and has far-reaching implications for the therapeutic delivery of other large genes.

  15. Full-length mRNA sequencing uncovers a widespread coupling between transcription initiation and mRNA processing.

    Science.gov (United States)

    Anvar, Seyed Yahya; Allard, Guy; Tseng, Elizabeth; Sheynkman, Gloria M; de Klerk, Eleonora; Vermaat, Martijn; Yin, Raymund H; Johansson, Hans E; Ariyurek, Yavuz; den Dunnen, Johan T; Turner, Stephen W; 't Hoen, Peter A C

    2018-03-29

    The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription initiation, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing. In MCF-7 breast cancer cells, we find 2700 genes with interdependent alternative transcription initiation, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules, including examples of coupling between transcription start sites and polyadenylation sites. The analysis of three human primary tissues (brain, heart and liver) reveals similar patterns of interdependency between transcription initiation and mRNA processing events. We predict thousands of novel open reading frames from full-length mRNA sequences and obtained evidence for their translation by shotgun proteomics. The mapping database rescues 358 previously unassigned peptides and improves the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improves proteogenomics analysis of MCF-7 cells. Our findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription initiation and mRNA processing.

  16. A molecular recombination map of Antirrhinum majus

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    Hudson Andrew

    2010-12-01

    Full Text Available Abstract Background Genetic recombination maps provide important frameworks for comparative genomics, identifying gene functions, assembling genome sequences and for breeding. The molecular recombination map currently available for the model eudicot Antirrhinum majus is the result of a cross with Antirrhinum molle, limiting its usefulness within A. majus. Results We created a molecular linkage map of A. majus based on segregation of markers in the F2 population of two inbred lab strains of A. majus. The resulting map consisted of over 300 markers in eight linkage groups, which could be aligned with a classical recombination map and the A. majus karyotype. The distribution of recombination frequencies and distorted transmission of parental alleles differed from those of a previous inter-species hybrid. The differences varied in magnitude and direction between chromosomes, suggesting that they had multiple causes. The map, which covered an estimated of 95% of the genome with an average interval of 2 cM, was used to analyze the distribution of a newly discovered family of MITE transposons and tested for its utility in positioning seven mutations that affect aspects of plant size. Conclusions The current map has an estimated interval of 1.28 Mb between markers. It shows a lower level of transmission ratio distortion and a longer length than the previous inter-species map, making it potentially more useful. The molecular recombination map further indicates that the IDLE MITE transposons are distributed throughout the genome and are relatively stable. The map proved effective in mapping classical morphological mutations of A. majus.

  17. Production of biologically active recombinant human factor H in Physcomitrella.

    Science.gov (United States)

    Büttner-Mainik, Annette; Parsons, Juliana; Jérôme, Hanna; Hartmann, Andrea; Lamer, Stephanie; Schaaf, Andreas; Schlosser, Andreas; Zipfel, Peter F; Reski, Ralf; Decker, Eva L

    2011-04-01

    The human complement regulatory serum protein factor H (FH) is a promising future biopharmaceutical. Defects in the gene encoding FH are associated with human diseases like severe kidney and retinal disorders in the form of atypical haemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis II (MPGN II) or age-related macular degeneration (AMD). There is a current need to apply intact full-length FH for the therapy of patients with congenital or acquired defects of this protein. Application of purified or recombinant FH (rFH) to these patients is an important and promising approach for the treatment of these diseases. However, neither protein purified from plasma of healthy individuals nor recombinant protein is currently available on the market. Here, we report the first stable expression of the full-length human FH cDNA and the subsequent production of this glycoprotein in a plant system. The moss Physcomitrella patens perfectly suits the requirements for the production of complex biopharmaceuticals as this eukaryotic system not only offers an outstanding genetical accessibility, but moreover, proteins can be produced safely in scalable photobioreactors without the need for animal-derived medium compounds. Transgenic moss lines were created, which express the human FH cDNA and target the recombinant protein to the culture supernatant via a moss-derived secretion signal. Correct processing of the signal peptide and integrity of the moss-produced rFH were verified via peptide mapping by mass spectrometry. Ultimately, we show that the rFH displays complement regulatory activity comparable to FH purified from plasma. © 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  18. Characterization of partial and near full-length genomes of HIV-1 strains sampled from recently infected individuals in São Paulo, Brazil.

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    Sabri Saeed Sanabani

    Full Text Available BACKGROUND: Genetic variability is a major feature of human immunodeficiency virus type 1 (HIV-1 and is considered the key factor frustrating efforts to halt the HIV epidemic. A proper understanding of HIV-1 genomic diversity is a fundamental prerequisite for proper epidemiology, genetic diagnosis, and successful drugs and vaccines design. Here, we report on the partial and near full-length genomic (NFLG variability of HIV-1 isolates from a well-characterized cohort of recently infected patients in São Paul, Brazil. METHODOLOGY: HIV-1 proviral DNA was extracted from the peripheral blood mononuclear cells of 113 participants. The NFLG and partial fragments were determined by overlapping nested PCR and direct sequencing. The data were phylogenetically analyzed. RESULTS: Of the 113 samples (90.3% male; median age 31 years; 79.6% homosexual men studied, 77 (68.1% NFLGs and 32 (29.3% partial fragments were successfully subtyped. Of the successfully subtyped sequences, 88 (80.7% were subtype B sequences, 12 (11% BF1 recombinants, 3 (2.8% subtype C sequences, 2 (1.8% BC recombinants and subclade F1 each, 1 (0.9% CRF02 AG, and 1 (0.9% CRF31 BC. Primary drug resistance mutations were observed in 14/101 (13.9% of samples, with 5.9% being resistant to protease inhibitors and nucleoside reverse transcriptase inhibitors (NRTI and 4.9% resistant to non-NRTIs. Predictions of viral tropism were determined for 86 individuals. X4 or X4 dual or mixed-tropic viruses (X4/DM were seen in 26 (30.2% of subjects. The proportion of X4 viruses in homosexuals was detected in 19/69 (27.5%. CONCLUSIONS: Our results confirm the existence of various HIV-1 subtypes circulating in São Paulo, and indicate that subtype B account for the majority of infections. Antiretroviral (ARV drug resistance is relatively common among recently infected patients. The proportion of X4 viruses in homosexuals was significantly higher than the proportion seen in other study populations.

  19. Novel HIV-1 recombinants spreading across multiple risk groups in the United Kingdom: the identification and phylogeography of Circulating Recombinant Form (CRF 50_A1D.

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    Geraldine M Foster

    Full Text Available An increase in non-B HIV-1 infections among men who have sex with men (MSM in the United Kingdom (UK has created opportunities for novel recombinants to arise and become established. We used molecular mapping to characterize the importance of such recombinants to the UK HIV epidemic, in order to gain insights into transmission dynamics that can inform control strategies.A total of 55,556 pol (reverse transcriptase and protease sequences in the UK HIV Drug Resistance Database were analyzed using Subtype Classification Using Evolutionary Algorithms (SCUEAL. Overall 72 patients shared the same A1/D recombination breakpoint in pol, comprising predominantly MSM but also heterosexuals and injecting drug users (IDUs. In six MSM, full-length single genome amplification of plasma HIV-1 RNA was performed in order to characterize the A1/D recombinant. Subtypes and recombination breakpoints were identified using sliding window and jumping profile hidden markov model approaches. Global maximum likelihood trees of gag, pol and env genes were drawn using FastTree version 2.1. Five of the six strains showed the same novel A1/D recombinant (8 breakpoints, which has been classified as CRF50_A1D. The sixth strain showed a complex CRF50_A1D/B/U structure. Divergence dates and phylogeographic inferences were determined using Bayesian Evolutionary Analysis using Sampling Trees (BEAST. This estimated that CRF50_A1D emerged in the UK around 1992 in MSM, with subsequent transmissions to heterosexuals and IDUs. Analysis of CRF50_A1D/B/U demonstrated that around the year 2000 CRF50_A1D underwent recombination with a subtype B strain.We report the identification of CRF50_A1D, a novel circulating recombinant that emerged in UK MSM around 1992, with subsequent onward transmission to heterosexuals and IDUs, and more recent recombination with subtype B. These findings highlight the changing dynamics of HIV transmission in the UK and the converging of the two previously

  20. Evidence of recombination and positive selection in cetacean papillomaviruses

    International Nuclear Information System (INIS)

    Robles-Sikisaka, Refugio; Rivera, Rebecca; Nollens, Hendrik H.; St Leger, Judy; Durden, Wendy N.; Stolen, Megan; Burchell, Jennifer; Wellehan, James F.X.

    2012-01-01

    Papillomaviruses (PVs) are small DNA viruses that have been associated with increased epithelial proliferation. Over one hundred PV types have been identified in humans; however, only three have been identified in bottlenose dolphins (Tursiops truncatus) to date. Using rolling circle amplification and degenerate PCR, we identified four novel PV genomes of bottlenose dolphins. TtPV4, TtPV5 and TtPV6 were identified in genital lesions while TtPV7 was identified in normal genital mucosa. Bayesian analysis of the full-length L1 genes found that TtPV4 and TtPV7 group within the Upsilonpapillomavirus genus while TtPV5 and TtPV6 group with Omikronpapillomavirus. However, analysis of the E1 gene did not distinguish these genera, implying that these genes may not share a common history, consistent with recombination. Recombination analyses identified several probable events. Signals of positive selection were found mostly in the E1 and E2 genes. Recombination and diversifying selection pressures constitute important driving forces of cetacean PV evolution.

  1. Evidence of recombination and positive selection in cetacean papillomaviruses

    Energy Technology Data Exchange (ETDEWEB)

    Robles-Sikisaka, Refugio, E-mail: refugio.robles1@gmail.com [Hubbs-SeaWorld Research Institute, Center for Marine Veterinary Virology, 2595 Ingraham Street, San Diego, CA 92109 (United States); Rivera, Rebecca, E-mail: RRivera@hswri.org [Hubbs-SeaWorld Research Institute, Center for Marine Veterinary Virology, 2595 Ingraham Street, San Diego, CA 92109 (United States); Nollens, Hendrik H., E-mail: Hendrik.Nollens@SeaWorld.com [Hubbs-SeaWorld Research Institute, Center for Marine Veterinary Virology, 2595 Ingraham Street, San Diego, CA 92109 (United States); College of Veterinary Medicine, University of Florida, PO Box 110885, Gainesville, FL 32611 (United States); SeaWorld San Diego, 500 SeaWorld Drive, San Diego, CA 92109 (United States); St Leger, Judy, E-mail: Judy.St.Leger@SeaWorld.com [SeaWorld San Diego, 500 SeaWorld Drive, San Diego, CA 92109 (United States); Durden, Wendy N., E-mail: WNoke@hswri.org [Hubbs-SeaWorld Research Institute, 3830 South Highway A1A 4-181, Melbourne Beach, FL 32951 (United States); Stolen, Megan, E-mail: MStolen@hswri.org [Hubbs-SeaWorld Research Institute, 3830 South Highway A1A 4-181, Melbourne Beach, FL 32951 (United States); Burchell, Jennifer, E-mail: JBurchell@hswri.org [Hubbs-SeaWorld Research Institute, Center for Marine Veterinary Virology, 2595 Ingraham Street, San Diego, CA 92109 (United States); Wellehan, James F.X., E-mail: WellehanJ@ufl.edu [College of Veterinary Medicine, University of Florida, PO Box 110885, Gainesville, FL 32611 (United States)

    2012-06-05

    Papillomaviruses (PVs) are small DNA viruses that have been associated with increased epithelial proliferation. Over one hundred PV types have been identified in humans; however, only three have been identified in bottlenose dolphins (Tursiops truncatus) to date. Using rolling circle amplification and degenerate PCR, we identified four novel PV genomes of bottlenose dolphins. TtPV4, TtPV5 and TtPV6 were identified in genital lesions while TtPV7 was identified in normal genital mucosa. Bayesian analysis of the full-length L1 genes found that TtPV4 and TtPV7 group within the Upsilonpapillomavirus genus while TtPV5 and TtPV6 group with Omikronpapillomavirus. However, analysis of the E1 gene did not distinguish these genera, implying that these genes may not share a common history, consistent with recombination. Recombination analyses identified several probable events. Signals of positive selection were found mostly in the E1 and E2 genes. Recombination and diversifying selection pressures constitute important driving forces of cetacean PV evolution.

  2. SequenceLDhot: detecting recombination hotspots.

    Science.gov (United States)

    Fearnhead, Paul

    2006-12-15

    There is much local variation in recombination rates across the human genome--with the majority of recombination occurring in recombination hotspots--short regions of around approximately 2 kb in length that have much higher recombination rates than neighbouring regions. Knowledge of this local variation is important, e.g. in the design and analysis of association studies for disease genes. Population genetic data, such as that generated by the HapMap project, can be used to infer the location of these hotspots. We present a new, efficient and powerful method for detecting recombination hotspots from population data. We compare our method with four current methods for detecting hotspots. It is orders of magnitude quicker, and has greater power, than two related approaches. It appears to be more powerful than HotspotFisher, though less accurate at inferring the precise positions of the hotspot. It was also more powerful than LDhot in some situations: particularly for weaker hotspots (10-40 times the background rate) when SNP density is lower (maths.lancs.ac.uk/~fearnhea/Hotspot.

  3. Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

    Directory of Open Access Journals (Sweden)

    Sandra Arenhart

    Full Text Available Abstract The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3. The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.

  4. ION-EXCHANGE IMMUNOAFFINITY PURIFICATION OF A RECOMBINANT BACULOVIRUS PLASMODIUM-FALCIPARUM APICAL MEMBRANE ANTIGEN, PF83/AMA-1

    NARCIS (Netherlands)

    NARUM, DL; WELLING, GW; THOMAS, AW

    1993-01-01

    A two-step purification regime has been developed for a quantitatively minor, putatively transmembrane, M(r) 83 000, apical membrane blood stage vaccine candidate antigen of Plasmodium falciparum (PF83/AMA-1), that has been expressed as a full-length baculovirus recombinant protein, PF83-FG8-1. The

  5. Non-destructive testing of full-length bonded rock bolts based on HHT signal analysis

    Science.gov (United States)

    Shi, Z. M.; Liu, L.; Peng, M.; Liu, C. C.; Tao, F. J.; Liu, C. S.

    2018-04-01

    Full-length bonded rock bolts are commonly used in mining, tunneling and slope engineering because of their simple design and resistance to corrosion. However, the length of a rock bolt and grouting quality do not often meet the required design standards in practice because of the concealment and complexity of bolt construction. Non-destructive testing is preferred when testing a rock bolt's quality because of the convenience, low cost and wide detection range. In this paper, a signal analysis method for the non-destructive sound wave testing of full-length bonded rock bolts is presented, which is based on the Hilbert-Huang transform (HHT). First, we introduce the HHT analysis method to calculate the bolt length and identify defect locations based on sound wave reflection test signals, which includes decomposing the test signal via empirical mode decomposition (EMD), selecting the intrinsic mode functions (IMF) using the Pearson Correlation Index (PCI) and calculating the instantaneous phase and frequency via the Hilbert transform (HT). Second, six model tests are conducted using different grouting defects and bolt protruding lengths to verify the effectiveness of the HHT analysis method. Lastly, the influence of the bolt protruding length on the test signal, identification of multiple reflections from defects, bolt end and protruding end, and mode mixing from EMD are discussed. The HHT analysis method can identify the bolt length and grouting defect locations from signals that contain noise at multiple reflected interfaces. The reflection from the long protruding end creates an irregular test signal with many frequency peaks on the spectrum. The reflections from defects barely change the original signal because they are low energy, which cannot be adequately resolved using existing methods. The HHT analysis method can identify reflections from the long protruding end of the bolt and multiple reflections from grouting defects based on mutations in the instantaneous

  6. Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase

    Directory of Open Access Journals (Sweden)

    Kim Chan-Mi

    2007-07-01

    Full Text Available Abstract Background Japanese encephalitis virus (JEV NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust in vitro RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated. Results To characterize the biochemical properties of JEV RdRp, we expressed in Escherichia coli and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U81, at the two nucleotides upstream of the 3'-end of the template. Conclusion As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the in vitro reconstitution of viral RNA replicase complex, we for the first time established an in vitro JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the

  7. Cost-effective sequencing of full-length cDNA clones powered by a de novo-reference hybrid assembly.

    Science.gov (United States)

    Kuroshu, Reginaldo M; Watanabe, Junichi; Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka; Kasahara, Masahiro

    2010-05-07

    Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence approximately 800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only approximately US$3 per clone, demonstrating a significant advantage over previous approaches.

  8. Sequencing, mapping, and analysis of 27,455 maize full-length cDNAs.

    Directory of Open Access Journals (Sweden)

    Carol Soderlund

    2009-11-01

    Full Text Available Full-length cDNA (FLcDNA sequencing establishes the precise primary structure of individual gene transcripts. From two libraries representing 27 B73 tissues and abiotic stress treatments, 27,455 high-quality FLcDNAs were sequenced. The average transcript length was 1.44 kb including 218 bases and 321 bases of 5' and 3' UTR, respectively, with 8.6% of the FLcDNAs encoding predicted proteins of fewer than 100 amino acids. Approximately 94% of the FLcDNAs were stringently mapped to the maize genome. Although nearly two-thirds of this genome is composed of transposable elements (TEs, only 5.6% of the FLcDNAs contained TE sequences in coding or UTR regions. Approximately 7.2% of the FLcDNAs are putative transcription factors, suggesting that rare transcripts are well-enriched in our FLcDNA set. Protein similarity searching identified 1,737 maize transcripts not present in rice, sorghum, Arabidopsis, or poplar annotated genes. A strict FLcDNA assembly generated 24,467 non-redundant sequences, of which 88% have non-maize protein matches. The FLcDNAs were also assembled with 41,759 FLcDNAs in GenBank from other projects, where semi-strict parameters were used to identify 13,368 potentially unique non-redundant sequences from this project. The libraries, ESTs, and FLcDNA sequences produced from this project are publicly available. The annotated EST and FLcDNA assemblies are available through the maize FLcDNA web resource (www.maizecdna.org.

  9. Identification of a natural intergenotypic recombinant hepatitis delta virus genotype 1 and 2 in Vietnamese HBsAg-positive patients.

    Science.gov (United States)

    Sy, B T; Nguyen, H M; Toan, N L; Song, L H; Tong, H V; Wolboldt, C; Binh, V Q; Kremsner, P G; Velavan, T P; Bock, C-T

    2015-01-01

    Hepatitis D virus (HDV) infection is acquired as a co- /superinfection of Hepatitis B virus (HBV) and can modulate the pathophysiology of chronic hepatitis B and related liver diseases including hepatocellular carcinoma. Among the eight distinct HDV genotypes reported, relatively few studies have attempted to investigate the prevalence of HDV mixed genotypes and RNA recombination of HDV. With a recorded prevalence of 10-20% HBV infection in Vietnam, this study investigated the HDV variability, HDV genotypes and HDV recombination among twenty-one HDV isolates in Vietnamese HBsAg-positive patients. HDV subgenomic and full-length genome sequences were obtained using newly established HDV-specific RT-PCR techniques. The nucleotide homology was observed from 74.6% to 99.4% among the investigated full-length genome of the HDV isolates. We observed HDV genotype 1 and HDV genotype 2 in the investigated Vietnamese patients. Although no HDV genotype mixtures were observed, we report here a newly identified recombinant of HDV genotypes (HDV 1 and HDV 2). The identified recombinant HDV isolate C03 revealed sequence homology to both HDV genotype 1 (nt1 to nt907) and HDV genotype 2 (nt908 to nt1675; HDAg coding region) with a breakpoint at nt908. Our findings demonstrate the prevalence of intergenotypic recombination between HDV genotypes 1 and 2 in a Vietnamese HBsAg-positive patient. Extended investigation on the distribution and prevalence of HDV, HDV mixed genotypes and recombinant HDV genotypes in a larger Vietnamese population offers vital insights into understanding of the micro-epidemiology of HDV and subsequent pathophysiology in chronic HBV- /HDV-related liver diseases. © 2014 John Wiley & Sons Ltd.

  10. Genetic Characterization of a Novel HIV-1 Circulating Recombinant Form (CRF74_01B Identified among Intravenous Drug Users in Malaysia: Recombination History and Phylogenetic Linkage with Previously Defined Recombinant Lineages.

    Directory of Open Access Journals (Sweden)

    Hui Ting Cheong

    Full Text Available In many parts of Southeast Asia, the HIV-1 epidemic has been driven by the sharing of needles and equipment among intravenous drug users (IDUs. Over the last few decades, many studies have proven time and again that the diversity of HIV-1 epidemics can often be linked to the route of infection transmission. That said, the diversity and complexity of HIV-1 molecular epidemics in the region have been increasing at an alarming rate, due in part to the high tendency of the viral RNA to recombine. This scenario was exemplified by the discovery of numerous circulating recombinant forms (CRFs, especially in Thailand and Malaysia. In this study, we characterized a novel CRF designated CRF74_01B, which was identified in six epidemiologically unlinked IDUs in Kuala Lumpur, Malaysia. The near-full length genomes were composed of CRF01_AE and subtype B', with eight breakpoints dispersed in the gag-pol and nef regions. Remarkably, this CRF shared four and two recombination hotspots with the previously described CRF33_01B and the less prevalent CRF53_01B, respectively. Genealogy-based Bayesian phylogenetic analysis of CRF74_01B genomic regions showed that it is closely related to both CRF33_01B and CRF53_01B. This observation suggests that CRF74_01B was probably a direct descendent from specific lineages of CRF33_01B, CRF53_01B and subtype B' that could have emerged in the mid-1990s. Additionally, it illustrated the active recombination processes between prevalent HIV-1 subtypes and recombinants in Malaysia. In summary, we report a novel HIV-1 genotype designated CRF74_01B among IDUs in Kuala Lumpur, Malaysia. The characterization of the novel CRF74_01B is of considerable significance towards the understanding of the genetic diversity and population dynamics of HIV-1 circulating in the region.

  11. The ability to form full-length intron RNA circles is a general property of nuclear group I introns

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Fiskaa, Tonje; Birgisdottir, Asa Birna

    2003-01-01

    at the expense of the host. The circularization pathway has distinct structural requirements that differ from those of splicing and appears to be specifically suppressed in vivo. The ability to form full-length circles is found in all types of nuclear group I introns, including those from the Tetrahymena...... ribosomal DNA. The biological function of the full-length circles is not known, but the fact that the circles contain the entire genetic information of the intron suggests a role in intron mobility....

  12. Sequence requirement of the ade6-4095 meiotic recombination hotspot in Schizosaccharomyces pombe.

    Science.gov (United States)

    Foulis, Steven J; Fowler, Kyle R; Steiner, Walter W

    2018-02-01

    Homologous recombination occurs at a greatly elevated frequency in meiosis compared to mitosis and is initiated by programmed double-strand DNA breaks (DSBs). DSBs do not occur at uniform frequency throughout the genome in most organisms, but occur preferentially at a limited number of sites referred to as hotspots. The location of hotspots have been determined at nucleotide-level resolution in both the budding and fission yeasts, and while several patterns have emerged regarding preferred locations for DSB hotspots, it remains unclear why particular sites experience DSBs at much higher frequency than other sites with seemingly similar properties. Short sequence motifs, which are often sites for binding of transcription factors, are known to be responsible for a number of hotspots. In this study we identified the minimum sequence required for activity of one of such motif identified in a screen of random sequences capable of producing recombination hotspots. The experimentally determined sequence, GGTCTRGACC, closely matches the previously inferred sequence. Full hotspot activity requires an effective sequence length of 9.5 bp, whereas moderate activity requires an effective sequence length of approximately 8.2 bp and shows significant association with DSB hotspots. In combination with our previous work, this result is consistent with a large number of different sequence motifs capable of producing recombination hotspots, and supports a model in which hotspots can be rapidly regenerated by mutation as they are lost through recombination.

  13. Fast Dissemination of New HIV-1 CRF02/A1 Recombinants in Pakistan

    Science.gov (United States)

    Chen, Yue; Hora, Bhavna; DeMarco, Todd; Shah, Sharaf Ali; Ahmed, Manzoor; Sanchez, Ana M.; Su, Chang; Carter, Meredith; Stone, Mars; Hasan, Rumina; Hasan, Zahra; Busch, Michael P.; Denny, Thomas N.; Gao, Feng

    2016-01-01

    A number of HIV-1 subtypes are identified in Pakistan by characterization of partial viral gene sequences. Little is known whether new recombinants are generated and how they disseminate since whole genome sequences for these viruses have not been characterized. Near full-length genome (NFLG) sequences were obtained by amplifying two overlapping half genomes or next generation sequencing from 34 HIV-1-infected individuals in Pakistan. Phylogenetic tree analysis showed that the newly characterized sequences were 16 subtype As, one subtype C, and 17 A/G recombinants. Further analysis showed that all 16 subtype A1 sequences (47%), together with the vast majority of sequences from Pakistan from other studies, formed a tight subcluster (A1a) within the subtype A1 clade, suggesting that they were derived from a single introduction. More in-depth analysis of 17 A/G NFLG sequences showed that five shared similar recombination breakpoints as in CRF02 (15%) but were phylogenetically distinct from the prototype CRF02 by forming a tight subcluster (CRF02a) while 12 (38%) were new recombinants between CRF02a and A1a or a divergent A1b viruses. Unique recombination patterns among the majority of the newly characterized recombinants indicated ongoing recombination. Interestingly, recombination breakpoints in these CRF02/A1 recombinants were similar to those in prototype CRF02 viruses, indicating that recombination at these sites more likely generate variable recombinant viruses. The dominance and fast dissemination of new CRF02a/A1 recombinants over prototype CRF02 suggest that these recombinant have more adapted and may become major epidemic strains in Pakistan. PMID:27973597

  14. Novel full-length major histocompatibility complex class I allele discovery and haplotype definition in pig-tailed macaques.

    Science.gov (United States)

    Semler, Matthew R; Wiseman, Roger W; Karl, Julie A; Graham, Michael E; Gieger, Samantha M; O'Connor, David H

    2017-11-13

    Pig-tailed macaques (Macaca nemestrina, Mane) are important models for human immunodeficiency virus (HIV) studies. Their infectability with minimally modified HIV makes them a uniquely valuable animal model to mimic human infection with HIV and progression to acquired immunodeficiency syndrome (AIDS). However, variation in the pig-tailed macaque major histocompatibility complex (MHC) and the impact of individual transcripts on the pathogenesis of HIV and other infectious diseases is understudied compared to that of rhesus and cynomolgus macaques. In this study, we used Pacific Biosciences single-molecule real-time circular consensus sequencing to describe full-length MHC class I (MHC-I) transcripts for 194 pig-tailed macaques from three breeding centers. We then used the full-length sequences to infer Mane-A and Mane-B haplotypes containing groups of MHC-I transcripts that co-segregate due to physical linkage. In total, we characterized full-length open reading frames (ORFs) for 313 Mane-A, Mane-B, and Mane-I sequences that defined 86 Mane-A and 106 Mane-B MHC-I haplotypes. Pacific Biosciences technology allows us to resolve these Mane-A and Mane-B haplotypes to the level of synonymous allelic variants. The newly defined haplotypes and transcript sequences containing full-length ORFs provide an important resource for infectious disease researchers as certain MHC haplotypes have been shown to provide exceptional control of simian immunodeficiency virus (SIV) replication and prevention of AIDS-like disease in nonhuman primates. The increased allelic resolution provided by Pacific Biosciences sequencing also benefits transplant research by allowing researchers to more specifically match haplotypes between donors and recipients to the level of nonsynonymous allelic variation, thus reducing the risk of graft-versus-host disease.

  15. High yield purification of full-length functional hERG K+ channels produced in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Molbaek, Karen; Scharff-Poulsen, Peter; Hélix-Nielsen, Claus

    2015-01-01

    knowledge this is the first reported high-yield production and purification of full length, tetrameric and functional hERG. This significant breakthrough will be paramount in obtaining hERG crystal structures, and in establishment of new high-throughput hERG drug safety screening assays....

  16. Development of a novel recombinant encapsidated RNA particle: evaluation as an internal control for diagnostic RT-PCR

    DEFF Research Database (Denmark)

    King, Donald P.; Montague, Nick; Ebert, Katja

    2007-01-01

    -and-mouth disease virus (FMDV) and a set for swine vesicular disease virus (SVDV) was engineered into a full-length cDNA clone containing the RNA-2 segment of Cowpea Mosaic Virus (CPMV). After co-inoculation with a plasmid that expressed CPMV RNA-1, recombinant virus particles were rescued from cowpea plants (Vigna...

  17. Meiotic gene-conversion rate and tract length variation in the human genome.

    Science.gov (United States)

    Padhukasahasram, Badri; Rannala, Bruce

    2013-02-27

    Meiotic recombination occurs in the form of two different mechanisms called crossing-over and gene-conversion and both processes have an important role in shaping genetic variation in populations. Although variation in crossing-over rates has been studied extensively using sperm-typing experiments, pedigree studies and population genetic approaches, our knowledge of variation in gene-conversion parameters (ie, rates and mean tract lengths) remains far from complete. To explore variability in population gene-conversion rates and its relationship to crossing-over rate variation patterns, we have developed and validated using coalescent simulations a comprehensive Bayesian full-likelihood method that can jointly infer crossing-over and gene-conversion rates as well as tract lengths from population genomic data under general variable rate models with recombination hotspots. Here, we apply this new method to SNP data from multiple human populations and attempt to characterize for the first time the fine-scale variation in gene-conversion parameters along the human genome. We find that the estimated ratio of gene-conversion to crossing-over rates varies considerably across genomic regions as well as between populations. However, there is a great degree of uncertainty associated with such estimates. We also find substantial evidence for variation in the mean conversion tract length. The estimated tract lengths did not show any negative relationship with the local heterozygosity levels in our analysis.European Journal of Human Genetics advance online publication, 27 February 2013; doi:10.1038/ejhg.2013.30.

  18. Quench start localization in full-length SSC R ampersand D dipoles

    International Nuclear Information System (INIS)

    Devred, A.; Chapman, M.; Cortella, J.; Desportes, A.; Kaugerts, J.; Kirk, T.; Mirk, K.; Schermer, R.; Tompkins, J.C.; Turner, J.; Bleadon, M.; Brown, B.C.; Hanft, R.; Kuchnir, M.; Lamm, M.; Mantsch, P.; Mazur, P.O.; Orris, D.; Peoples, J.; Strait, J.; Tool, G.; Caspi, S.; Gilbert, W.; Meuser, R.; Peters, C.; Rechen, J.; Royet, J.; Scanlan, R.; Taylor, C.; Zbasnik, J.

    1989-04-01

    Full-length SSC R ampersand D dipole magnets instrumented with four voltage taps on each turn of the inner quarter coils have been tested. These voltage taps enable accurate location of the point at which the quenches start and detailed studies of quench development in the coil. Attention here is focused on localizing the quench source. After recalling the basic mechanism of a quench (why it occurs and how it propagates), the method of quench origin analysis is described: the quench propagation velocity on the turn where the quench occurs is calculated, and the quench location is then verified by reiterating the analysis on the adjacent turns. Last, the velocity value, which appears to be higher than previously measured, is discussed

  19. Characterization of full-length sequenced cDNA inserts (FLIcs from Atlantic salmon (Salmo salar

    Directory of Open Access Journals (Sweden)

    Lunner Sigbjørn

    2009-10-01

    Full Text Available Abstract Background Sequencing of the Atlantic salmon genome is now being planned by an international research consortium. Full-length sequenced inserts from cDNAs (FLIcs are an important tool for correct annotation and clustering of the genomic sequence in any species. The large amount of highly similar duplicate sequences caused by the relatively recent genome duplication in the salmonid ancestor represents a particular challenge for the genome project. FLIcs will therefore be an extremely useful resource for the Atlantic salmon sequencing project. In addition to be helpful in order to distinguish between duplicate genome regions and in determining correct gene structures, FLIcs are an important resource for functional genomic studies and for investigation of regulatory elements controlling gene expression. In contrast to the large number of ESTs available, including the ESTs from 23 developmental and tissue specific cDNA libraries contributed by the Salmon Genome Project (SGP, the number of sequences where the full-length of the cDNA insert has been determined has been small. Results High quality full-length insert sequences from 560 pre-smolt white muscle tissue specific cDNAs were generated, accession numbers [GenBank: BT043497 - BT044056]. Five hundred and ten (91% of the transcripts were annotated using Gene Ontology (GO terms and 440 of the FLIcs are likely to contain a complete coding sequence (cCDS. The sequence information was used to identify putative paralogs, characterize salmon Kozak motifs, polyadenylation signal variation and to identify motifs likely to be involved in the regulation of particular genes. Finally, conserved 7-mers in the 3'UTRs were identified, of which some were identical to miRNA target sequences. Conclusion This paper describes the first Atlantic salmon FLIcs from a tissue and developmental stage specific cDNA library. We have demonstrated that many FLIcs contained a complete coding sequence (cCDS. This

  20. The full-length form of the Drosophila amyloid precursor protein is involved in memory formation.

    Science.gov (United States)

    Bourdet, Isabelle; Preat, Thomas; Goguel, Valérie

    2015-01-21

    The APP plays a central role in AD, a pathology that first manifests as a memory decline. Understanding the role of APP in normal cognition is fundamental in understanding the progression of AD, and mammalian studies have pointed to a role of secreted APPα in memory. In Drosophila, we recently showed that APPL, the fly APP ortholog, is required for associative memory. In the present study, we aimed to characterize which form of APPL is involved in this process. We show that expression of a secreted-APPL form in the mushroom bodies, the center for olfactory memory, is able to rescue the memory deficit caused by APPL partial loss of function. We next assessed the impact on memory of the Drosophila α-secretase kuzbanian (KUZ), the enzyme initiating the nonamyloidogenic pathway that produces secreted APPLα. Strikingly, KUZ overexpression not only failed to rescue the memory deficit caused by APPL loss of function, it exacerbated this deficit. We further show that in addition to an increase in secreted-APPL forms, KUZ overexpression caused a decrease of membrane-bound full-length species that could explain the memory deficit. Indeed, we observed that transient expression of a constitutive membrane-bound mutant APPL form is sufficient to rescue the memory deficit caused by APPL reduction, revealing for the first time a role of full-length APPL in memory formation. Our data demonstrate that, in addition to secreted APPL, the noncleaved form is involved in memory, raising the possibility that secreted and full-length APPL act together in memory processes. Copyright © 2015 the authors 0270-6474/15/351043-09$15.00/0.

  1. Pharmacological efficacy of anti-IL-1β scFv, Fab and full-length antibodies in treatment of rheumatoid arthritis.

    Science.gov (United States)

    Qi, Jianying; Ye, Xianlong; Ren, Guiping; Kan, Fangming; Zhang, Yu; Guo, Mo; Zhang, Zhiyi; Li, Deshan

    2014-02-01

    Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that mainly causes the synovial joint inflammation and cartilage destruction. Interleukin-1β (IL-1β) is an important proinflammatory cytokine involved in the pathogenesis of RA. In this study, we constructed and expressed anti-IL-1β-full-length antibody in CHO-K1-SV, anti-IL-1β-Fab and anti-IL-1β-scFv in Rosetta. We compared the therapeutic efficacy of three anti-IL-1β antibodies for CIA mice. Mice with CIA were subcutaneously injected with humanized anti-IL-1β-scFv, anti-IL-1β-Fab or anti-IL-1β-full-length antibody. The effects of treatment were determined by arthritis severity score, autoreactive humoral, cellular immune responses, histological lesion and cytokines production. Compared with anti-IL-1β-scFv treatments, anti-IL-1β-Fab and anti-IL-1β-full-length antibody therapy resulted in more significant effect in alleviating the severity of arthritis by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and down-regulating the expression of IL-1β, IL-6, IL-2, IFN-γ, TNF-α and MMP-3 in inflammatory tissue. The therapeutic effects of anti-IL-1β-Fab and anti-IL-1β-full-length antibodies on CIA mice had no significant difference. However, production of anti-IL-1β-full-length antibody in eukaryotic system is, in general, time-consuming and more expensive than that of anti-IL-1β-Fab in prokaryotic systems. In conclusion, as a small molecule antibody, anti-IL-1β-Fab is an ideal candidate for RA therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Analysis of ORF5 and Full-Length Genome Sequences of Porcine Reproductive and Respiratory Syndrome Virus Isolates of Genotypes 1 and 2 Retrieved Worldwide Provides Evidence that Recombination Is a Common Phenomenon and May Produce Mosaic Isolates

    DEFF Research Database (Denmark)

    Martín-Valls, G. E.; Kvisgaard, Lise Kirstine; Tello, M.

    2014-01-01

    Recombination is currently recognized as a factor for high genetic diversity, but the frequency of such recombination events and the genome segments involved are not well known. In the present study, we initially focused on the detection of recombinant porcine reproductive and respiratory syndrom...

  3. Three-body recombination of two-component cold atomic gases into deep dimers in an optical model

    International Nuclear Information System (INIS)

    Mikkelsen, M; Jensen, A S; Fedorov, D V; Zinner, N T

    2015-01-01

    We consider three-body recombination into deep dimers in a mass-imbalanced two-component atomic gas. We use an optical model where a phenomenological imaginary potential is added to the lowest adiabatic hyper-spherical potential. The consequent imaginary part of the energy eigenvalue corresponds to the decay rate or recombination probability of the three-body system. The method is formulated in details and the relevant qualitative features are discussed as functions of scattering lengths and masses. We use zero-range model in analyses of recent recombination data. The dominating scattering length is usually related to the non-equal two-body systems. We account for temperature smearing which tends to wipe out the higher-lying Efimov peaks. The range and the strength of the imaginary potential determine positions and shapes of the Efimov peaks as well as the absolute value of the recombination rate. The Efimov scaling between recombination peaks is calculated and shown to depend on both scattering lengths. Recombination is predicted to be largest for heavy–heavy–light systems. Universal properties of the optical parameters are indicated. We compare to available experiments and find in general very satisfactory agreement. (paper)

  4. Identification and genetic characterization of unique HIV-1 A1/C recombinant strain in South Africa.

    Science.gov (United States)

    Musyoki, Andrew M; Rakgole, Johnny N; Selabe, Gloria; Mphahlele, Jeffrey

    2015-03-01

    HIV isolates from South Africa are predominantly subtype C. Sporadic isolation of non-C strains has been reported mainly in cosmopolitan cities. HIV isolate j51 was recovered from a rural South African heterosexual female aged 51 years. Near full length amplification of the genome was attempted using PCR with primers targeting overlapping segments of the HIV genome. Analysis of 5593 bp (gag to vpu) at a bootstrap value greater than 70% found that all but the vpu gene was HIV-1 subtype A1. The vpu gene was assigned HIV-1 subtype C. The recombination breaking point was estimated at position 6035+/- 15 bp with reference to the beginning of the HXB2 reference strain. Isolate j51 revealed a unique genome constellation to previously reported recombinant strains with parental A/C backbones from South Africa though a common recombination with subtype C within the vpu gene. Identification of recombinant strains supports continued surveillance of HIV genetic diversity.

  5. Failure Mode and Effects Analysis (FMEA) of the solid state full length rod control system

    International Nuclear Information System (INIS)

    Shopsky, W.E.

    1977-01-01

    The Full Length Rod Control System (FLRCS) controls the power to the rod drive mechanisms for rod movement in response to signals received from the Reactor Control System or from signals generated through Reactor Operator action. Rod movement is used to control reactivity of the reactor during plant operation. The Full Length Rod Control System is designed to perform its reactivity control function in conjunction with the Reactor Control and Protection System, to maintain the reactor core within design safety limits. By the use of a Failure Mode and Effects Analysis, it is shown that the FLRCS will perform its reactivity control functions considering the loss of single active components. That is, sufficient fault limiting control circuits are provided which blocks control rod movement and/or indicates presence of a fault condition at the Control Board. Reactor operator action or automatic reactor trip will thus mitigate the consequences of potential failure of the FLRCS. The analysis also qualitatively demonstrates the reliability of the FLRCS to perform its intended function

  6. A Novel Strategy to Engineer Pre-Vascularized Full-Length Dental Pulp-like Tissue Constructs.

    Science.gov (United States)

    Athirasala, Avathamsa; Lins, Fernanda; Tahayeri, Anthony; Hinds, Monica; Smith, Anthony J; Sedgley, Christine; Ferracane, Jack; Bertassoni, Luiz E

    2017-06-12

    The requirement for immediate vascularization of engineered dental pulp poses a major hurdle towards successful implementation of pulp regeneration as an effective therapeutic strategy for root canal therapy, especially in adult teeth. Here, we demonstrate a novel strategy to engineer pre-vascularized, cell-laden hydrogel pulp-like tissue constructs in full-length root canals for dental pulp regeneration. We utilized gelatin methacryloyl (GelMA) hydrogels with tunable physical and mechanical properties to determine the microenvironmental conditions (microstructure, degradation, swelling and elastic modulus) that enhanced viability, spreading and proliferation of encapsulated odontoblast-like cells (OD21), and the formation of endothelial monolayers by endothelial colony forming cells (ECFCs). GelMA hydrogels with higher polymer concentration (15% w/v) and stiffness enhanced OD21 cell viability, spreading and proliferation, as well as endothelial cell spreading and monolayer formation. We then fabricated pre-vascularized, full-length, dental pulp-like tissue constructs by dispensing OD21 cell-laden GelMA hydrogel prepolymer in root canals of extracted teeth and fabricating 500 µm channels throughout the root canals. ECFCs seeded into the microchannels successfully formed monolayers and underwent angiogenic sprouting within 7 days in culture. In summary, the proposed approach is a simple and effective strategy for engineering of pre-vascularized dental pulp constructs offering potentially beneficial translational outcomes.

  7. Efimov trimers in a harmonic potential and universality in three-body recombination

    NARCIS (Netherlands)

    Kokkelmans, S.J.J.M.F.; Portegies, J.W.; Gross, N.; Shotan, Z.; Khaykovich, L.

    2009-01-01

    We report on experimental evidence of universality in ultracold 7Li atoms’three-body recombination loss in the vicinity of a Feshbach resonance [1]. We observe a recombination minimum and an Efimov resonance in regions of positive and negative scattering lengths. Both observed features lie deeply

  8. Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

    Directory of Open Access Journals (Sweden)

    Tadashi Imanishi

    2004-06-01

    Full Text Available The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/. It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs, identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA

  9. Initial recombination of ions in gases (Air). Anfangsrekombination von Ionen in gasen (Luft)

    Energy Technology Data Exchange (ETDEWEB)

    Booz, J.; Ebert, H. G.

    1962-08-15

    The dependence of the initial recombination coefficients on the ion density, the ionization length, and the recombination time was theoretically and experimentally investigated for the case of ionization of air with x rays. The experiments were carried out at initial ion densities between 5 x 10/sup 4/ and 2 x 10/sup 6/ ion pairs/cm/sup 3/ and at ionization lengths between 100 mu sec and 3 msec. There was satisfactory agreement with the theoretically derived formula. (tr-auth)

  10. Research Update: Relativistic origin of slow electron-hole recombination in hybrid halide perovskite solar cells

    Directory of Open Access Journals (Sweden)

    Pooya Azarhoosh

    2016-09-01

    Full Text Available The hybrid perovskite CH3NH3PbI3 (MAPI exhibits long minority-carrier lifetimes and diffusion lengths. We show that slow recombination originates from a spin-split indirect-gap. Large internal electric fields act on spin-orbit-coupled band extrema, shifting band-edges to inequivalent wavevectors, making the fundamental gap indirect. From a description of photoluminescence within the quasiparticle self-consistent GW approximation for MAPI, CdTe, and GaAs, we predict carrier lifetime as a function of light intensity and temperature. At operating conditions we find radiative recombination in MAPI is reduced by a factor of more than 350 compared to direct gap behavior. The indirect gap is retained with dynamic disorder.

  11. A comparative phylogenetic analysis of full-length mariner elements ...

    Indian Academy of Sciences (India)

    Unknown

    recent study showing non-occurance of inter-subfamily excisions because of .... length shown in our figure is greater because of the gaps introduced to maintain an ... to test the feasibility of transforming silkmoths with a foreign gene of ...

  12. A network approach to analyzing highly recombinant malaria parasite genes.

    Directory of Open Access Journals (Sweden)

    Daniel B Larremore

    Full Text Available The var genes of the human malaria parasite Plasmodium falciparum present a challenge to population geneticists due to their extreme diversity, which is generated by high rates of recombination. These genes encode a primary antigen protein called PfEMP1, which is expressed on the surface of infected red blood cells and elicits protective immune responses. Var gene sequences are characterized by pronounced mosaicism, precluding the use of traditional phylogenetic tools that require bifurcating tree-like evolutionary relationships. We present a new method that identifies highly variable regions (HVRs, and then maps each HVR to a complex network in which each sequence is a node and two nodes are linked if they share an exact match of significant length. Here, networks of var genes that recombine freely are expected to have a uniformly random structure, but constraints on recombination will produce network communities that we identify using a stochastic block model. We validate this method on synthetic data, showing that it correctly recovers populations of constrained recombination, before applying it to the Duffy Binding Like-α (DBLα domain of var genes. We find nine HVRs whose network communities map in distinctive ways to known DBLα classifications and clinical phenotypes. We show that the recombinational constraints of some HVRs are correlated, while others are independent. These findings suggest that this micromodular structuring facilitates independent evolutionary trajectories of neighboring mosaic regions, allowing the parasite to retain protein function while generating enormous sequence diversity. Our approach therefore offers a rigorous method for analyzing evolutionary constraints in var genes, and is also flexible enough to be easily applied more generally to any highly recombinant sequences.

  13. The effect of alcohol on recombinant proteins derived from mammalian adenylyl cyclase

    Directory of Open Access Journals (Sweden)

    Emily Qualls-Creekmore

    2017-07-01

    Full Text Available The cyclic AMP (cAMP signaling pathway is implicated in the development of alcohol use disorder. Previous studies have demonstrated that ethanol enhances the activity of adenylyl cyclase (AC in an isoform specific manner; AC7 is most enhanced by ethanol, and regions responsible for enhancement by ethanol are located in the cytoplasmic domains of the AC7 protein. We hypothesize that ethanol modulates AC activity by directly interacting with the protein and that ethanol effects on AC can be studied using recombinant AC in vitro. AC recombinant proteins containing only the C1a or C2 domains of AC7 and AC9 individually were expressed in bacteria, and purified. The purified recombinant AC proteins retained enzymatic activity and isoform specific alcohol responsiveness. The combination of the C1a or C2 domains of AC7 maintained the same alcohol cutoff point as full-length AC7. We also find that the recombinant AC7 responds to alcohol differently in the presence of different combinations of activators including MnCl2, forskolin, and Gsα. Through a series of concentration-response experiments and curve fitting, the values for maximum activities, Hill coefficients, and EC50 were determined in the absence and presence of butanol as a surrogate of ethanol. The results suggest that alcohol modulates AC activity by directly interacting with the AC protein and that the alcohol interaction with the AC protein occurs at multiple sites with positive cooperativity. This study indicates that the recombinant AC proteins expressed in bacteria can provide a useful model system to investigate the mechanism of alcohol action on their activity.

  14. Salmo salar and Esox lucius full-length cDNA sequences reveal changes in evolutionary pressures on a post-tetraploidization genome

    Directory of Open Access Journals (Sweden)

    Holt Robert A

    2010-04-01

    Full Text Available Abstract Background Salmonids are one of the most intensely studied fish, in part due to their economic and environmental importance, and in part due to a recent whole genome duplication in the common ancestor of salmonids. This duplication greatly impacts species diversification, functional specialization, and adaptation. Extensive new genomic resources have recently become available for Atlantic salmon (Salmo salar, but documentation of allelic versus duplicate reference genes remains a major uncertainty in the complete characterization of its genome and its evolution. Results From existing expressed sequence tag (EST resources and three new full-length cDNA libraries, 9,057 reference quality full-length gene insert clones were identified for Atlantic salmon. A further 1,365 reference full-length clones were annotated from 29,221 northern pike (Esox lucius ESTs. Pairwise dN/dS comparisons within each of 408 sets of duplicated salmon genes using northern pike as a diploid out-group show asymmetric relaxation of selection on salmon duplicates. Conclusions 9,057 full-length reference genes were characterized in S. salar and can be used to identify alleles and gene family members. Comparisons of duplicated genes show that while purifying selection is the predominant force acting on both duplicates, consistent with retention of functionality in both copies, some relaxation of pressure on gene duplicates can be identified. In addition, there is evidence that evolution has acted asymmetrically on paralogs, allowing one of the pair to diverge at a faster rate.

  15. Characterization of full-length sequenced cDNA inserts (FLIcs) from Atlantic salmon (Salmo salar)

    Science.gov (United States)

    Andreassen, Rune; Lunner, Sigbjørn; Høyheim, Bjørn

    2009-01-01

    Background Sequencing of the Atlantic salmon genome is now being planned by an international research consortium. Full-length sequenced inserts from cDNAs (FLIcs) are an important tool for correct annotation and clustering of the genomic sequence in any species. The large amount of highly similar duplicate sequences caused by the relatively recent genome duplication in the salmonid ancestor represents a particular challenge for the genome project. FLIcs will therefore be an extremely useful resource for the Atlantic salmon sequencing project. In addition to be helpful in order to distinguish between duplicate genome regions and in determining correct gene structures, FLIcs are an important resource for functional genomic studies and for investigation of regulatory elements controlling gene expression. In contrast to the large number of ESTs available, including the ESTs from 23 developmental and tissue specific cDNA libraries contributed by the Salmon Genome Project (SGP), the number of sequences where the full-length of the cDNA insert has been determined has been small. Results High quality full-length insert sequences from 560 pre-smolt white muscle tissue specific cDNAs were generated, accession numbers [GenBank: BT043497 - BT044056]. Five hundred and ten (91%) of the transcripts were annotated using Gene Ontology (GO) terms and 440 of the FLIcs are likely to contain a complete coding sequence (cCDS). The sequence information was used to identify putative paralogs, characterize salmon Kozak motifs, polyadenylation signal variation and to identify motifs likely to be involved in the regulation of particular genes. Finally, conserved 7-mers in the 3'UTRs were identified, of which some were identical to miRNA target sequences. Conclusion This paper describes the first Atlantic salmon FLIcs from a tissue and developmental stage specific cDNA library. We have demonstrated that many FLIcs contained a complete coding sequence (cCDS). This suggests that the remaining c

  16. Fiscal 2000 report on result of the full-length cDNA structure analysis; 2000 nendo kanzen cho cDNA kozo kaiseki seika hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

    This paper explains the results of research on full-length cDNA structure analysis for the period from April, 2000 to March, 2001. The outline of human genome sequence was published in June, 2000. In Japan, human gene analysis was such that, as the basic technology of the bio industry, a millennium project was decided in the budget of fiscal 2000. The full-length cDNA structure analysis is the core of the project. The libraries of cDNA were prepared using full-length and more than 4-5kbp-long cDNAs by oligo-capping method. It began from determining partial sequence data at end cDNA, and then, with new clones selected therefrom, full-length human cDNA sequence data were determined. The partial sequence data determined by fiscal 2000 were 1,035,000 clones while the full-length sequence data were 12,144 clones. The sequence data obtained were analyzed by homology search and translated into amino acid coding sequences, with predictions conducted on protein functions. A clustering method was examined that selects new clones from partial sequences. Database was constructed on gene expression profiles and disease-related gene sequence data. (NEDO)

  17. Diversity, distribution and dynamics of full-length Copia and Gypsy LTR retroelements in Solanum lycopersicum.

    Science.gov (United States)

    Paz, Rosalía Cristina; Kozaczek, Melisa Eliana; Rosli, Hernán Guillermo; Andino, Natalia Pilar; Sanchez-Puerta, Maria Virginia

    2017-10-01

    Transposable elements are the most abundant components of plant genomes and can dramatically induce genetic changes and impact genome evolution. In the recently sequenced genome of tomato (Solanum lycopersicum), the estimated fraction of elements corresponding to retrotransposons is nearly 62%. Given that tomato is one of the most important vegetable crop cultivated and consumed worldwide, understanding retrotransposon dynamics can provide insight into its evolution and domestication processes. In this study, we performed a genome-wide in silico search of full-length LTR retroelements in the tomato nuclear genome and annotated 736 full-length Gypsy and Copia retroelements. The dispersion level across the 12 chromosomes, the diversity and tissue-specific expression of those elements were estimated. Phylogenetic analysis based on the retrotranscriptase region revealed the presence of 12 major lineages of LTR retroelements in the tomato genome. We identified 97 families, of which 77 and 20 belong to the superfamilies Copia and Gypsy, respectively. Each retroelement family was characterized according to their element size, relative frequencies and insertion time. These analyses represent a valuable resource for comparative genomics within the Solanaceae, transposon-tagging and for the design of cultivar-specific molecular markers in tomato.

  18. Full-Length High-Temperature Severe Fuel Damage Test No. 5: Final safety analysis

    International Nuclear Information System (INIS)

    Lanning, D.D.; Lombardo, N.J.; Panisko, F.E.

    1993-09-01

    This report presents the final safety analysis for the preparation, conduct, and post-test discharge operation for the Full-Length High Temperature Experiment-5 (FLHT-5) to be conducted in the L-24 position of the National Research Universal (NRU) Reactor at Chalk River Nuclear Laboratories (CRNL), Ontario, Canada. The test is sponsored by an international group organized by the US Nuclear Regulatory Commission. The test is designed and conducted by staff from Pacific Northwest Laboratory with CRNL staff support. The test will study the consequences of loss-of-coolant and the progression of severe fuel damage

  19. Functional characterization of a full length pregnane X receptor, expression in vivo, and identification of PXR alleles, in Zebrafish (Danio rerio)

    Energy Technology Data Exchange (ETDEWEB)

    Bainy, Afonso C.D. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Departamento de Bioquímica, CCB, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900 (Brazil); Kubota, Akira; Goldstone, Jared V. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Lille-Langøy, Roger [Department of Biology, University of Bergen, N-5020 Bergen (Norway); Karchner, Sibel I. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Celander, Malin C. [Department of Biological and Environmental Sciences, University of Gothenburg, SE 405 30 Göteborg (Sweden); Hahn, Mark E. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Goksøyr, Anders [Department of Biology, University of Bergen, N-5020 Bergen (Norway); Stegeman, John J., E-mail: jstegeman@whoi.edu [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States)

    2013-10-15

    Highlights: •Full-length pxr has been cloned from zebrafish. •Alleles of pxr were identified in zebrafish. •Full length Pxr was activated less strongly than ligand binding domain in cell-based reporter assays. •High levels of pxr expression were found in eye and brain as well as in liver. •TCPOBOP and PB did not significantly alter expression of pxr in liver. -- Abstract: The pregnane X receptor (PXR) (nuclear receptor NR1I2) is a ligand activated transcription factor, mediating responses to diverse xenobiotic and endogenous chemicals. The properties of PXR in fish are not fully understood. Here we report on cloning and characterization of full-length PXR of zebrafish, Danio rerio, and pxr expression in vivo. Initial efforts gave a cDNA encoding a 430 amino acid protein identified as zebrafish pxr by phylogenetic and synteny analysis. The sequence of the cloned Pxr DNA binding domain (DBD) was highly conserved, with 74% identity to human PXR-DBD, while the ligand-binding domain (LBD) of the cloned sequence was only 44% identical to human PXR-LBD. Sequence variation among clones in the initial effort prompted sequencing of multiple clones from a single fish. There were two prominent variants, one sequence with S183, Y218 and H383 and the other with I183, C218 and N383, which we designate as alleles pxr*1 (nr1i2*1) and pxr*2 (nr1i2*2), respectively. In COS-7 cells co-transfected with a PXR-responsive reporter gene, the full-length Pxr*1 (the more common variant) was activated by known PXR agonists clotrimazole and pregnenolone 16α-carbonitrile but to a lesser extent than the full-length human PXR. Activation of full-length Pxr*1 was only 10% of that with the Pxr*1 LBD. Quantitative real time PCR analysis showed prominent expression of pxr in liver and eye, as well as brain and intestine of adult zebrafish. The pxr was expressed in heart and kidney at levels similar to that in intestine. The expression of pxr in liver was weakly induced by ligands for

  20. Analysis of HIV-1 intersubtype recombination breakpoints suggests region with high pairing probability may be a more fundamental factor than sequence similarity affecting HIV-1 recombination.

    Science.gov (United States)

    Jia, Lei; Li, Lin; Gui, Tao; Liu, Siyang; Li, Hanping; Han, Jingwan; Guo, Wei; Liu, Yongjian; Li, Jingyun

    2016-09-21

    With increasing data on HIV-1, a more relevant molecular model describing mechanism details of HIV-1 genetic recombination usually requires upgrades. Currently an incomplete structural understanding of the copy choice mechanism along with several other issues in the field that lack elucidation led us to perform an analysis of the correlation between breakpoint distributions and (1) the probability of base pairing, and (2) intersubtype genetic similarity to further explore structural mechanisms. Near full length sequences of URFs from Asia, Europe, and Africa (one sequence/patient), and representative sequences of worldwide CRFs were retrieved from the Los Alamos HIV database. Their recombination patterns were analyzed by jpHMM in detail. Then the relationships between breakpoint distributions and (1) the probability of base pairing, and (2) intersubtype genetic similarities were investigated. Pearson correlation test showed that all URF groups and the CRF group exhibit the same breakpoint distribution pattern. Additionally, the Wilcoxon two-sample test indicated a significant and inexplicable limitation of recombination in regions with high pairing probability. These regions have been found to be strongly conserved across distinct biological states (i.e., strong intersubtype similarity), and genetic similarity has been determined to be a very important factor promoting recombination. Thus, the results revealed an unexpected disagreement between intersubtype similarity and breakpoint distribution, which were further confirmed by genetic similarity analysis. Our analysis reveals a critical conflict between results from natural HIV-1 isolates and those from HIV-1-based assay vectors in which genetic similarity has been shown to be a very critical factor promoting recombination. These results indicate the region with high-pairing probabilities may be a more fundamental factor affecting HIV-1 recombination than sequence similarity in natural HIV-1 infections. Our

  1. Thousands of primer-free, high-quality, full-length SSU rRNA sequences from all domains of life

    DEFF Research Database (Denmark)

    Karst, Soeren M; Dueholm, Morten S; McIlroy, Simon J

    2016-01-01

    Ribosomal RNA (rRNA) genes are the consensus marker for determination of microbial diversity on the planet, invaluable in studies of evolution and, for the past decade, high-throughput sequencing of variable regions of ribosomal RNA genes has become the backbone of most microbial ecology studies...... (SSU) rRNA genes and synthetic long read sequencing by molecular tagging, to generate primer-free, full-length SSU rRNA gene sequences from all domains of life, with a median raw error rate of 0.17%. We generated thousands of full-length SSU rRNA sequences from five well-studied ecosystems (soil, human...... gut, fresh water, anaerobic digestion, and activated sludge) and obtained sequences covering all domains of life and the majority of all described phyla. Interestingly, 30% of all bacterial operational taxonomic units were novel, compared to the SILVA database (less than 97% similarity...

  2. Secretory production of tetrameric native full-length streptavidin with thermostability using Streptomyces lividans as a host.

    Science.gov (United States)

    Noda, Shuhei; Matsumoto, Takuya; Tanaka, Tsutomu; Kondo, Akihiko

    2015-01-13

    Streptavidin is a tetrameric protein derived from Streptomyces avidinii, and has tight and specific biotin binding affinity. Applications of the streptavidin-biotin system have been widely studied. Streptavidin is generally produced using protein expression in Escherichia coli. In the present study, the secretory production of streptavidin was carried out using Streptomyces lividans as a host. In this study, we used the gene encoding native full-length streptavidin, whereas the core region is generally used for streptavidin production in E. coli. Tetrameric streptavidin composed of native full-length streptavidin monomers was successfully secreted in the culture supernatant of S. lividans transformants, and had specific biotin binding affinity as strong as streptavidin produced by E. coli. The amount of Sav using S. lividans was about 9 times higher than using E. coli. Surprisingly, streptavidin produced by S. lividans exhibited affinity to biotin after boiling, despite the fact that tetrameric streptavidin is known to lose its biotin binding ability after brief boiling. We successfully produced a large amount of tetrameric streptavidin as a secretory-form protein with unique thermotolerance.

  3. PCR-based isolation and identification of full-length low-molecular-weight glutenin subunit genes in bread wheat (Triticum aestivum L.).

    Science.gov (United States)

    Zhang, Xiaofei; Liu, Dongcheng; Jiang, Wei; Guo, Xiaoli; Yang, Wenlong; Sun, Jiazhu; Ling, Hongqing; Zhang, Aimin

    2011-12-01

    Low-molecular-weight glutenin subunits (LMW-GSs) are encoded by a multi-gene family and are essential for determining the quality of wheat flour products, such as bread and noodles. However, the exact role or contribution of individual LMW-GS genes to wheat quality remains unclear. This is, at least in part, due to the difficulty in characterizing complete sequences of all LMW-GS gene family members in bread wheat. To identify full-length LMW-GS genes, a polymerase chain reaction (PCR)-based method was established, consisting of newly designed conserved primers and the previously developed LMW-GS gene molecular marker system. Using the PCR-based method, 17 LMW-GS genes were identified and characterized in Xiaoyan 54, of which 12 contained full-length sequences. Sequence alignments showed that 13 LMW-GS genes were identical to those found in Xiaoyan 54 using the genomic DNA library screening, and the other four full-length LMW-GS genes were first isolated from Xiaoyan 54. In Chinese Spring, 16 unique LMW-GS genes were isolated, and 13 of them contained full-length coding sequences. Additionally, 16 and 17 LMW-GS genes in Dongnong 101 and Lvhan 328 (chosen from the micro-core collections of Chinese germplasm), respectively, were also identified. Sequence alignments revealed that at least 15 LMW-GS genes were common in the four wheat varieties, and allelic variants of each gene shared high sequence identities (>95%) but exhibited length polymorphism in repetitive regions. This study provides a PCR-based method for efficiently identifying LMW-GS genes in bread wheat, which will improve the characterization of complex members of the LMW-GS gene family and facilitate the understanding of their contributions to wheat quality.

  4. Hibiscus latent Fort Pierce virus in Brazil and synthesis of its biologically active full-length cDNA clone.

    Science.gov (United States)

    Gao, Ruimin; Niu, Shengniao; Dai, Weifang; Kitajima, Elliot; Wong, Sek-Man

    2016-10-01

    A Brazilian isolate of Hibiscus latent Fort Pierce virus (HLFPV-BR) was firstly found in a hibiscus plant in Limeira, SP, Brazil. RACE PCR was carried out to obtain the full-length sequences of HLFPV-BR which is 6453 nucleotides and has more than 99.15 % of complete genomic RNA nucleotide sequence identity with that of HLFPV Japanese isolate. The genomic structure of HLFPV-BR is similar to other tobamoviruses. It includes a 5' untranslated region (UTR), followed by open reading frames encoding for a 128-kDa protein and a 188-kDa readthrough protein, a 38-kDa movement protein, 18-kDa coat protein, and a 3' UTR. Interestingly, the unique feature of poly(A) tract is also found within its 3'-UTR. Furthermore, from the total RNA extracted from the local lesions of HLFPV-BR-infected Chenopodium quinoa leaves, a biologically active, full-length cDNA clone encompassing the genome of HLFPV-BR was amplified and placed adjacent to a T7 RNA polymerase promoter. The capped in vitro transcripts from the cloned cDNA were infectious when mechanically inoculated into C. quinoa and Nicotiana benthamiana plants. This is the first report of the presence of an isolate of HLFPV in Brazil and the successful synthesis of a biologically active HLFPV-BR full-length cDNA clone.

  5. RT-PCR and sequence analysis of the full-length fusion protein of Canine Distemper Virus from domestic dogs.

    Science.gov (United States)

    Romanutti, Carina; Gallo Calderón, Marina; Keller, Leticia; Mattion, Nora; La Torre, José

    2016-02-01

    During 2007-2014, 84 out of 236 (35.6%) samples from domestic dogs submitted to our laboratory for diagnostic purposes were positive for Canine Distemper Virus (CDV), as analyzed by RT-PCR amplification of a fragment of the nucleoprotein gene. Fifty-nine of them (70.2%) were from dogs that had been vaccinated against CDV. The full-length gene encoding the Fusion (F) protein of fifteen isolates was sequenced and compared with that of those of other CDVs, including wild-type and vaccine strains. Phylogenetic analysis using the F gene full-length sequences grouped all the Argentinean CDV strains in the SA2 clade. Sequence identity with the Onderstepoort vaccine strain was 89.0-90.6%, and the highest divergence was found in the 135 amino acids corresponding to the F protein signal-peptide, Fsp (64.4-66.7% identity). In contrast, this region was highly conserved among the local strains (94.1-100% identity). One extra putative N-glycosylation site was identified in the F gene of CDV Argentinean strains with respect to the vaccine strain. The present report is the first to analyze full-length F protein sequences of CDV strains circulating in Argentina, and contributes to the knowledge of molecular epidemiology of CDV, which may help in understanding future disease outbreaks. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Human microcephaly protein RTTN interacts with STIL and is required to build full-length centrioles.

    Science.gov (United States)

    Chen, Hsin-Yi; Wu, Chien-Ting; Tang, Chieh-Ju C; Lin, Yi-Nan; Wang, Won-Jing; Tang, Tang K

    2017-08-15

    Mutations in many centriolar protein-encoding genes cause primary microcephaly. Using super-resolution and electron microscopy, we find that the human microcephaly protein, RTTN, is recruited to the proximal end of the procentriole at early S phase, and is located at the inner luminal walls of centrioles. Further studies demonstrate that RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly. CRISPR/Cas9-mediated RTTN gene knockout in p53-deficient cells induce amplification of primitive procentriole bodies that lack the distal-half centriolar proteins, POC5 and POC1B. Additional analyses show that RTTN serves as an upstream effector of CEP295, which mediates the loading of POC1B and POC5 to the distal-half centrioles. Interestingly, the naturally occurring microcephaly-associated mutant, RTTN (A578P), shows a low affinity for STIL binding and blocks centriole assembly. These findings reveal that RTTN contributes to building full-length centrioles and illuminate the molecular mechanism through which the RTTN (A578P) mutation causes primary microcephaly.Mutations in many centriolar protein-encoding genes cause primary microcephaly. Here the authors show that human microcephaly protein RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly, contributing to building full-length centrioles.

  7. Recombinant human prion protein inhibits prion propagation in vitro.

    Science.gov (United States)

    Yuan, Jue; Zhan, Yi-An; Abskharon, Romany; Xiao, Xiangzhu; Martinez, Manuel Camacho; Zhou, Xiaochen; Kneale, Geoff; Mikol, Jacqueline; Lehmann, Sylvain; Surewicz, Witold K; Castilla, Joaquín; Steyaert, Jan; Zhang, Shulin; Kong, Qingzhong; Petersen, Robert B; Wohlkonig, Alexandre; Zou, Wen-Quan

    2013-10-09

    Prion diseases are associated with the conformational conversion of the cellular prion protein (PrP(C)) into the pathological scrapie isoform (PrP(Sc)) in the brain. Both the in vivo and in vitro conversion of PrP(C) into PrP(Sc) is significantly inhibited by differences in amino acid sequence between the two molecules. Using protein misfolding cyclic amplification (PMCA), we now report that the recombinant full-length human PrP (rHuPrP23-231) (that is unglycosylated and lacks the glycophosphatidylinositol anchor) is a strong inhibitor of human prion propagation. Furthermore, rHuPrP23-231 also inhibits mouse prion propagation in a scrapie-infected mouse cell line. Notably, it binds to PrP(Sc), but not PrP(C), suggesting that the inhibitory effect of recombinant PrP results from blocking the interaction of brain PrP(C) with PrP(Sc). Our findings suggest a new avenue for treating prion diseases, in which a patient's own unglycosylated and anchorless PrP is used to inhibit PrP(Sc) propagation without inducing immune response side effects.

  8. Broad-scale recombination patterns underlying proper disjunction in humans.

    Directory of Open Access Journals (Sweden)

    Adi Fledel-Alon

    2009-09-01

    Full Text Available Although recombination is essential to the successful completion of human meiosis, it remains unclear how tightly the process is regulated and over what scale. To assess the nature and stringency of constraints on human recombination, we examined crossover patterns in transmissions to viable, non-trisomic offspring, using dense genotyping data collected in a large set of pedigrees. Our analysis supports a requirement for one chiasma per chromosome rather than per arm to ensure proper disjunction, with additional chiasmata occurring in proportion to physical length. The requirement is not absolute, however, as chromosome 21 seems to be frequently transmitted properly in the absence of a chiasma in females, a finding that raises the possibility of a back-up mechanism aiding in its correct segregation. We also found a set of double crossovers in surprisingly close proximity, as expected from a second pathway that is not subject to crossover interference. These findings point to multiple mechanisms that shape the distribution of crossovers, influencing proper disjunction in humans.

  9. Genetic Characterization of a Novel HIV-1 Circulating Recombinant Form (CRF74_01B) Identified among Intravenous Drug Users in Malaysia: Recombination History and Phylogenetic Linkage with Previously Defined Recombinant Lineages.

    Science.gov (United States)

    Cheong, Hui Ting; Chow, Wei Zhen; Takebe, Yutaka; Chook, Jack Bee; Chan, Kok Gan; Al-Darraji, Haider Abdulrazzaq Abed; Koh, Clayton; Kamarulzaman, Adeeba; Tee, Kok Keng

    2015-01-01

    In many parts of Southeast Asia, the HIV-1 epidemic has been driven by the sharing of needles and equipment among intravenous drug users (IDUs). Over the last few decades, many studies have proven time and again that the diversity of HIV-1 epidemics can often be linked to the route of infection transmission. That said, the diversity and complexity of HIV-1 molecular epidemics in the region have been increasing at an alarming rate, due in part to the high tendency of the viral RNA to recombine. This scenario was exemplified by the discovery of numerous circulating recombinant forms (CRFs), especially in Thailand and Malaysia. In this study, we characterized a novel CRF designated CRF74_01B, which was identified in six epidemiologically unlinked IDUs in Kuala Lumpur, Malaysia. The near-full length genomes were composed of CRF01_AE and subtype B', with eight breakpoints dispersed in the gag-pol and nef regions. Remarkably, this CRF shared four and two recombination hotspots with the previously described CRF33_01B and the less prevalent CRF53_01B, respectively. Genealogy-based Bayesian phylogenetic analysis of CRF74_01B genomic regions showed that it is closely related to both CRF33_01B and CRF53_01B. This observation suggests that CRF74_01B was probably a direct descendent from specific lineages of CRF33_01B, CRF53_01B and subtype B' that could have emerged in the mid-1990s. Additionally, it illustrated the active recombination processes between prevalent HIV-1 subtypes and recombinants in Malaysia. In summary, we report a novel HIV-1 genotype designated CRF74_01B among IDUs in Kuala Lumpur, Malaysia. The characterization of the novel CRF74_01B is of considerable significance towards the understanding of the genetic diversity and population dynamics of HIV-1 circulating in the region.

  10. Rare HIV-1 Subtype J Genomes and a New H/U/CRF02_AG Recombinant Genome Suggests an Ancient Origin of HIV-1 in Angola.

    Science.gov (United States)

    Bártolo, Inês; Calado, Rita; Borrego, Pedro; Leitner, Thomas; Taveira, Nuno

    2016-08-01

    Angola has an extremely diverse HIV-1 epidemic fueled in part by the frequent interchange of people with the Democratic Republic of Congo (DRC) and Republic of Congo (RC). Characterization of HIV-1 strains circulating in Angola should help to better understand the origin of HIV-1 subtypes and recombinant forms and their transmission dynamics. In this study we characterize the first near full-length HIV-1 genomic sequences from HIV-1 infected individuals from Angola. Samples were obtained in 1993 from three HIV-1 infected patients living in Cabinda, Angola. Near full-length genomic sequences were obtained from virus isolates. Maximum likelihood phylogenetic tree inference and analyses of potential recombination patterns were performed to evaluate the sequence classifications and origins. Phylogenetic and recombination analyses revealed that one virus was a pure subtype J, another mostly subtype J with a small uncertain region, and the final virus was classified as a H/U/CRF02_AG recombinant. Consistent with their epidemiological data, the subtype J sequences were more closely related to each other than to other J sequences previously published. Based on the env gene, taxa from Angola occur throughout the global subtype J phylogeny. HIV-1 subtypes J and H are present in Angola at low levels since at least 1993. Low transmission efficiency and/or high recombination potential may explain their limited epidemic success in Angola and worldwide. The high diversity of rare subtypes in Angola suggests that Angola was part of the early establishment of the HIV-1 pandemic.

  11. Molecular cloning and expression of full-length DNA copies of the genomic RNAs of cowpea mosaic virus

    NARCIS (Netherlands)

    Vos, P.

    1987-01-01

    The experiments described in this thesis were designed to unravel various aspects of the mechanism of gene expression of cowpea mosaic virus (CPMV). For this purpose full-length DNA copies of both genomic RNAs of CPMV were constructed. Using powerful invitro

  12. TypeLoader: A fast and efficient automated workflow for the annotation and submission of novel full-length HLA alleles.

    Science.gov (United States)

    Surendranath, V; Albrecht, V; Hayhurst, J D; Schöne, B; Robinson, J; Marsh, S G E; Schmidt, A H; Lange, V

    2017-07-01

    Recent years have seen a rapid increase in the discovery of novel allelic variants of the human leukocyte antigen (HLA) genes. Commonly, only the exons encoding the peptide binding domains of novel HLA alleles are submitted. As a result, the IPD-IMGT/HLA Database lacks sequence information outside those regions for the majority of known alleles. This has implications for the application of the new sequencing technologies, which deliver sequence data often covering the complete gene. As these technologies simplify the characterization of the complete gene regions, it is desirable for novel alleles to be submitted as full-length sequences to the database. However, the manual annotation of full-length alleles and the generation of specific formats required by the sequence repositories is prone to error and time consuming. We have developed TypeLoader to address both these facets. With only the full-length sequence as a starting point, Typeloader performs automatic sequence annotation and subsequently handles all steps involved in preparing the specific formats for submission with very little manual intervention. TypeLoader is routinely used at the DKMS Life Science Lab and has aided in the successful submission of more than 900 novel HLA alleles as full-length sequences to the European Nucleotide Archive repository and the IPD-IMGT/HLA Database with a 95% reduction in the time spent on annotation and submission when compared with handling these processes manually. TypeLoader is implemented as a web application and can be easily installed and used on a standalone Linux desktop system or within a Linux client/server architecture. TypeLoader is downloadable from http://www.github.com/DKMS-LSL/typeloader. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.

    Directory of Open Access Journals (Sweden)

    Nan Zhong

    Full Text Available We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP protocols.

  14. Looking for the optimal rate of recombination for evolutionary dynamics

    Science.gov (United States)

    Saakian, David B.

    2018-01-01

    We consider many-site mutation-recombination models of evolution with selection. We are looking for situations where the recombination increases the mean fitness of the population, and there is an optimal recombination rate. We found two fitness landscapes supporting such nonmonotonic behavior of the mean fitness versus the recombination rate. The first case is related to the evolution near the error threshold on a neutral-network-like fitness landscape, for moderate genome lengths and large population. The more realistic case is the second one, in which we consider the evolutionary dynamics of a finite population on a rugged fitness landscape (the smooth fitness landscape plus some random contributions to the fitness). We also give the solution to the horizontal gene transfer model in the case of asymmetric mutations. To obtain nonmonotonic behavior for both mutation and recombination, we need a specially designed (ideal) fitness landscape.

  15. Yeast expressed recombinant Hemagglutinin protein of Novel H1N1 elicits neutralising antibodies in rabbits and mice

    Directory of Open Access Journals (Sweden)

    Athmaram TN

    2011-11-01

    Full Text Available Abstract Currently available vaccines for the pandemic Influenza A (H1N1 2009 produced in chicken eggs have serious impediments viz limited availability, risk of allergic reactions and the possible selection of sub-populations differing from the naturally occurring virus, whereas the cell culture derived vaccines are time consuming and may not meet the demands of rapid global vaccination required to combat the present/future pandemic. Hemagglutinin (HA based subunit vaccine for H1N1 requires the HA protein in glycosylated form, which is impossible with the commonly used bacterial expression platform. Additionally, bacterial derived protein requires extensive purification and refolding steps for vaccine applications. For these reasons an alternative heterologous system for rapid, easy and economical production of Hemagglutinin protein in its glycosylated form is required. The HA gene of novel H1N1 A/California/04/2009 was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA- synthetic gene having α-secretory tag was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having multiple copy integrants of the transgene expressed full length HA protein in the culture supernatant. The Recombinant yeast derived H1N1 HA protein elicited neutralising antibodies both in mice and rabbits. The sera from immunised animals also exhibited Hemagglutination Inhibition (HI activity. Considering the safety, reliability and also economic potential of Pichia expression platform, our preliminary data indicates the feasibility of using this system as an alternative for large-scale production of recombinant influenza HA protein in the face of influenza pandemic threat.

  16. Recombination Catalysts for Hypersonic Fuels

    Science.gov (United States)

    Chinitz, W.

    1998-01-01

    The goal of commercially-viable access to space will require technologies that reduce propulsion system weight and complexity, while extracting maximum energy from the products of combustion. This work is directed toward developing effective nozzle recombination catalysts for the supersonic and hypersonic aeropropulsion engines used to provide such access to space. Effective nozzle recombination will significantly reduce rk=le length (hence, propulsion system weight) and reduce fuel requirements, further decreasing the vehicle's gross lift-off weight. Two such catalysts have been identified in this work, barium and antimony compounds, by developing chemical kinetic reaction mechanisms for these materials and determining the engine performance enhancement for a typical flight trajectory. Significant performance improvements are indicated, using only 2% (mole or mass) of these compounds in the combustor product gas.

  17. Three-body recombination of two-component cold atomic gases into deep dimers in an optical model

    DEFF Research Database (Denmark)

    Mikkelsen, Mathias; Jensen, A. S.; Fedorov, D. V.

    2015-01-01

    to the decay rate or recombination probability of the three-body system. The method is formulated in details and the relevant qualitative features are discussed as functions of scattering lengths and masses. We use zero-range model in analyses of recent recombination data. The dominating scattering length......We consider three-body recombination into deep dimers in a mass-imbalanced two-component atomic gas. We use an optical model where a phenomenological imaginary potential is added to the lowest adiabatic hyper-spherical potential. The consequent imaginary part of the energy eigenvalue corresponds...... is usually related to the non-equal two-body systems. We account for temperature smearing which tends to wipe out the higher-lying Efimov peaks. The range and the strength of the imaginary potential determine positions and shapes of the Efimov peaks as well as the absolute value of the recombination rate...

  18. Construction and characterization of a full-length infectious cDNA clone of foot-and-mouth disease virus strain O/JPN/2010 isolated in Japan in 2010.

    Science.gov (United States)

    Nishi, Tatsuya; Onozato, Hiroyuki; Ohashi, Seiichi; Fukai, Katsuhiko; Yamada, Manabu; Morioka, Kazuki; Kanno, Toru

    2016-06-01

    A full-length infectious cDNA clone of the genome of a foot-and-mouth disease virus isolated from the 2010 epidemic in Japan was constructed and designated pSVL-f02. Transfection of Cos-7 or IBRS-2 cells with this clone allowed the recovery of infectious virus. The recovered virus had the same in vitro characterization as the parental virus with regard to antigenicity in neutralization and indirect immunofluorescence tests, plaque size and one-step growth. Pigs were experimentally infected with the parental virus or the recombinant virus recovered from pSVL-f02 transfected cells. There were no significant differences in clinical signs or antibody responses between the two groups, and virus isolation and viral RNA detection from clinical samples were similar. Virus recovered from transfected cells therefore retained the in vitro characteristics and the in vivo pathogenicity of their parental strain. This cDNA clone should be a valuable tool to analyze determinants of pathogenicity and mechanisms of virus replication, and to develop genetically engineered vaccines against foot-and-mouth disease virus. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Generation and Analysis of Full-length cDNA Sequences from Elephant Shark (Callorhinchus milii)

    KAUST Repository

    Kodzius, Rimantas

    2009-03-17

    Cartilaginous fishes are the oldest living group of jawed vertebrates and therefore is an important group for understanding the evolution of vertebrate genomes including the human genome. Our laboratory has proposed elephant shark (C. milii) as a model cartilaginous fish genome because of its relatively small genome size (910 Mb). The whole genome of C. milii is being sequenced (first cartilaginous fish genome to be sequenced completely). To characterize the transcriptome of C. milii and to assist in annotating exon-intron boundaries, transcriptional start sites and alternatively spliced transcripts, we are generating full-length cDNA sequences from C. milii.

  20. Transformation of Cowpea Vigna unguiculata with a Full-Length DNA Copy of Cowpea Mosaic Virus M-RNA

    NARCIS (Netherlands)

    Hille, Jacques; Goldbach, Rob

    1987-01-01

    A full-length DNA copy of the M-RNA of cowpea mosaic virus (CPMV), supplied with either the 35S promoter from cauliflower mosaic virus (CaMV) or the nopaline synthase promoter from Agrobacterium tumefaciens, was introduced into the T-DNA region of a Ti-plasmid-derived gene vector and transferred to

  1. An analysis of expressed sequence tags of developing castor endosperm using a full-length cDNA library

    Directory of Open Access Journals (Sweden)

    Wallis James G

    2007-07-01

    Full Text Available Abstract Background Castor seeds are a major source for ricinoleate, an important industrial raw material. Genomics studies of castor plant will provide critical information for understanding seed metabolism, for effectively engineering ricinoleate production in transgenic oilseeds, or for genetically improving castor plants by eliminating toxic and allergic proteins in seeds. Results Full-length cDNAs are useful resources in annotating genes and in providing functional analysis of genes and their products. We constructed a full-length cDNA library from developing castor endosperm, and obtained 4,720 ESTs from 5'-ends of the cDNA clones representing 1,908 unique sequences. The most abundant transcripts are genes encoding storage proteins, ricin, agglutinin and oleosins. Several other sequences are also very numerous, including two acidic triacylglycerol lipases, and the oleate hydroxylase (FAH12 gene that is responsible for ricinoleate biosynthesis. The role(s of the lipases in developing castor seeds are not clear, and co-expressing of a lipase and the FAH12 did not result in significant changes in hydroxy fatty acid accumulation in transgenic Arabidopsis seeds. Only one oleate desaturase (FAD2 gene was identified in our cDNA sequences. Sequence and functional analyses of the castor FAD2 were carried out since it had not been characterized previously. Overexpression of castor FAD2 in a FAH12-expressing Arabidopsis line resulted in decreased accumulation of hydroxy fatty acids in transgenic seeds. Conclusion Our results suggest that transcriptional regulation of FAD2 and FAH12 genes maybe one of the mechanisms that contribute to a high level of ricinoleate accumulation in castor endosperm. The full-length cDNA library will be used to search for additional genes that affect ricinoleate accumulation in seed oils. Our EST sequences will also be useful to annotate the castor genome, which whole sequence is being generated by shotgun sequencing at

  2. A trans-Complementing Recombination Trap Demonstrates a Low Propensity of Flaviviruses for Intermolecular Recombination▿

    Science.gov (United States)

    Taucher, Christian; Berger, Angelika; Mandl, Christian W.

    2010-01-01

    Intermolecular recombination between the genomes of closely related RNA viruses can result in the emergence of novel strains with altered pathogenic potential and antigenicity. Although recombination between flavivirus genomes has never been demonstrated experimentally, the potential risk of generating undesirable recombinants has nevertheless been a matter of concern and controversy with respect to the development of live flavivirus vaccines. As an experimental system for investigating the ability of flavivirus genomes to recombine, we developed a “recombination trap,” which was designed to allow the products of rare recombination events to be selected and amplified. To do this, we established reciprocal packaging systems consisting of pairs of self-replicating subgenomic RNAs (replicons) derived from tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) that could complement each other in trans and thus be propagated together in cell culture over multiple passages. Any infectious viruses with intact, full-length genomes that were generated by recombination of the two replicons would be selected and enriched by end point dilution passage, as was demonstrated in a spiking experiment in which a small amount of wild-type virus was mixed with the packaged replicons. Using the recombination trap and the JEV system, we detected two aberrant recombination events, both of which yielded unnatural genomes containing duplications. Infectious clones of both of these genomes yielded viruses with impaired growth properties. Despite the fact that the replicon pairs shared approximately 600 nucleotides of identical sequence where a precise homologous crossover event would have yielded a wild-type genome, this was not observed in any of these systems, and the TBEV and WNV systems did not yield any viable recombinant genomes at all. Our results show that intergenomic recombination can occur in the structural region of flaviviruses

  3. Global identification of the full-length transcripts and alternative splicing related to phenolic acid biosynthetic genes in Salvia miltiorrhiza

    Directory of Open Access Journals (Sweden)

    Zhichao eXu

    2016-02-01

    Full Text Available Salvianolic acids are among the main bioactive components in Salvia miltiorrhiza, and their biosynthesis has attracted widespread interest. However, previous studies on the biosynthesis of phenolic acids using next-generation sequencing platforms are limited with regard to the assembly of full-length transcripts. Based on hybrid-seq (next-generation and single molecular real-time sequencing of the S. miltiorrhiza root transcriptome, we experimentally identified 15 full-length transcripts and 4 alternative splicing events of enzyme-coding genes involved in the biosynthesis of rosmarinic acid. Moreover, we herein demonstrate that lithospermic acid B accumulates in the phloem and xylem of roots, in agreement with the expression patterns of the identified key genes related to rosmarinic acid biosynthesis. According to co-expression patterns, we predicted that 6 candidate cytochrome P450s and 5 candidate laccases participate in the salvianolic acid pathway. Our results provide a valuable resource for further investigation into the synthetic biology of phenolic acids in S. miltiorrhiza.

  4. The effect of two different renal denervation strategies on blood pressure in resistant hypertension: Comparison of full-length versus proximal renal artery ablation.

    Science.gov (United States)

    Chen, Weijie; Ling, Zhiyu; Du, Huaan; Song, Wenxin; Xu, Yanping; Liu, Zengzhang; Su, Li; Xiao, Peilin; Yuan, Yuelong; Lu, Jiayi; Zhang, Jianhong; Li, Zhifeng; Shao, Jiang; Zhong, Bin; Zhou, Bei; Woo, Kamsang; Yin, Yuehui

    2016-11-01

    Renal denervation (RDN) is used to manage blood pressure (BP) in patients with resistant hypertension (rHT), but effectiveness is still a concern, and key arterial portion for successful RDN is not clear. The aim of this study was to investigate the efficacy and safety of proximal versus full-length renal artery ablation in patients with resistant hypertension (rHT). Forty-seven patients with rHT were randomly assigned to receive full-length ablation (n = 23) or proximal ablation (n = 24) of the renal arteries. All lesions were treated with radiofrequency energy via a saline-irrigated catheter. Office BP was measured during 12 months of follow-up and ambulatory BP at baseline and 6 months (n = 15 in each group). Compared with full-length ablation, proximal ablation reduced the number of ablation points in both the right (6.1 ± 0.7 vs. 3.3 ± 0.6, P renal arteries (6.2 ± 0.7 vs. 3.3 ± 0.8, P  0.5). Similar office BPs was reduced by -39.4 ± 11.5/-20.9 ± 7.1 mm Hg at 6 months and -38.2 ± 10.3/-21.5 ± 5.8 mm Hg at 12 months in the full-length group (P efficacy and safety profile compared with full-length RDN, and propose the proximal artery as the key portion for RDN. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  5. Bioconversion of Agricultural Waste to Ethanol by SSF Using Recombinant Cellulase from Clostridium thermocellum

    Directory of Open Access Journals (Sweden)

    Ruchi Mutreja

    2011-01-01

    Full Text Available The effect of different pretreatment methods, temperature, and enzyme concentration on ethanol production from 8 lignocellulosic agrowaste by simultaneous saccharification and fermentation (SSF using recombinant cellulase and Saccharomyces cerevisiae were studied. Recombinant cellulase was isolated from E. coli BL21 cells transformed with CtLic26A-Cel5-CBM11 full-length gene from Clostridium thermocellum and produced in both batch and fed-batch processes. The maximum cell OD and specific activity in batch mode were 1.6 and 1.91 U/mg, respectively, whereas in the fed-batch mode, maximum cell OD and specific activity were 3.8 and 3.5 U/mg, respectively, displaying a 2-fold increase. Eight substrates, Syzygium cumini (jamun, Azadirachta indica (neem, Saracens indica (asoka, bambusa dendrocalmus (bamboo, Populas nigra (poplar, Achnatherum hymenoides (wild grass, Eucalyptus marginata (eucalyptus, and Mangifera indica (mango, were subjected to SSF. Of three pretreatments, acid, alkali, and steam explosion, acid pretreatment Syzygium cumini (Jamun at 30°C gave maximum ethanol yield of 1.42 g/L.

  6. Genetic deletion of muscle RANK or selective inhibition of RANKL is not as effective as full-length OPG-fc in mitigating muscular dystrophy.

    Science.gov (United States)

    Dufresne, Sébastien S; Boulanger-Piette, Antoine; Bossé, Sabrina; Argaw, Anteneh; Hamoudi, Dounia; Marcadet, Laetitia; Gamu, Daniel; Fajardo, Val A; Yagita, Hideo; Penninger, Josef M; Russell Tupling, A; Frenette, Jérôme

    2018-04-24

    Although there is a strong association between osteoporosis and skeletal muscle atrophy/dysfunction, the functional relevance of a particular biological pathway that regulates synchronously bone and skeletal muscle physiopathology is still elusive. Receptor-activator of nuclear factor κB (RANK), its ligand RANKL and the soluble decoy receptor osteoprotegerin (OPG) are the key regulators of osteoclast differentiation and bone remodelling. We thus hypothesized that RANK/RANKL/OPG, which is a key pathway for bone regulation, is involved in Duchenne muscular dystrophy (DMD) physiopathology. Our results show that muscle-specific RANK deletion (mdx-RANK mko ) in dystrophin deficient mdx mice improves significantly specific force [54% gain in force] of EDL muscles with no protective effect against eccentric contraction-induced muscle dysfunction. In contrast, full-length OPG-Fc injections restore the force of dystrophic EDL muscles [162% gain in force], protect against eccentric contraction-induced muscle dysfunction ex vivo and significantly improve functional performance on downhill treadmill and post-exercise physical activity. Since OPG serves a soluble receptor for RANKL and as a decoy receptor for TRAIL, mdx mice were injected with anti-RANKL and anti-TRAIL antibodies to decipher the dual function of OPG. Injections of anti-RANKL and/or anti-TRAIL increase significantly the force of dystrophic EDL muscle [45% and 17% gains in force, respectively]. In agreement, truncated OPG-Fc that contains only RANKL domains produces similar gains, in terms of force production, than anti-RANKL treatments. To corroborate that full-length OPG-Fc also acts independently of RANK/RANKL pathway, dystrophin/RANK double-deficient mice were treated with full-length OPG-Fc for 10 days. Dystrophic EDL muscles exhibited a significant gain in force relative to untreated dystrophin/RANK double-deficient mice, indicating that the effect of full-length OPG-Fc is in part independent of the RANKL

  7. Contrasted patterns of crossover and non-crossover at Arabidopsis thaliana meiotic recombination hotspots.

    Directory of Open Access Journals (Sweden)

    Jan Drouaud

    2013-11-01

    Full Text Available The vast majority of meiotic recombination events (crossovers (COs and non-crossovers (NCOs cluster in narrow hotspots surrounded by large regions devoid of recombinational activity. Here, using a new molecular approach in plants, called "pollen-typing", we detected and characterized hundreds of CO and NCO molecules in two different hotspot regions in Arabidopsis thaliana. This analysis revealed that COs are concentrated in regions of a few kilobases where their rates reach up to 50 times the genome average. The hotspots themselves tend to cluster in regions less than 8 kilobases in size with overlapping CO distribution. Non-crossover (NCO events also occurred in the two hotspots but at very different levels (local CO/NCO ratios of 1/1 and 30/1 and their track lengths were quite small (a few hundred base pairs. We also showed that the ZMM protein MSH4 plays a role in CO formation and somewhat unexpectedly we also found that it is involved in the generation of NCOs but with a different level of effect. Finally, factors acting in cis and in trans appear to shape the rate and distribution of COs at meiotic recombination hotspots.

  8. 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.

  9. Recombination every day: abundant recombination in a virus during a single multi-cellular host infection.

    Directory of Open Access Journals (Sweden)

    Remy Froissart

    2005-03-01

    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.

  10. Full splitting of the first zero-field steps in the I-V curve of Josephson junctions of intermediate length

    International Nuclear Information System (INIS)

    Hansen, J.B.; Divin, Y.Y.; Mygind, J.

    1986-01-01

    We report on the observation of full splitting of the first zero-field steps in the I-V curves of Josephson transmission lines of intermediate length Lroughly-equal(3--5)lambda/sub J/, where lambda/sub J/ is the Josephson penetration length. We study in detail how this splitting of the step into two branches depends on the temperature of the junction and on a weak applied magnetic field. We relate the splitting to excitations in the junctions whose behavior is described by the perturbed Sine-Gordon equation

  11. Parameters of mitotic recombination in minute mutants of Drosophila melanogaster

    International Nuclear Information System (INIS)

    Ferrus, A.

    1975-01-01

    A sample of 16 Minutes, representing 12 loci distributed over all the chromosome arms and including 3 pairs of alleles and 4 deficiencies, has been studied with respect to several developmental and recombinational parameters. Cell marker mutants located in most of the chromosome arms were used to assess (1) spontaneous and x-ray-induced mitotic recombination frequencies of each Minute, and (2) clone sizes of the different cell marker clones. These parameters were analyzed both in the wing disc and in the abdominal histoblasts. Whereas spontaneous frequencies are not affected by the presence of the Minutes studied, the different Minutes characteristically increase the frequency of recombination clones arising after x irradiation. The recombinant clones which are M + /M + are significantly larger than clones in the same fly which retain the M + /M condition. This is particularly striking in clones in the wing disc, slightly so in clones in the tergites. The occurrence of mitotic recombination in the fourth chromosome is reported for the first time. Chaeta length and developmental delay correlates with the recombinational parameters in different ways. Possible causal interrelationships of the different traits of the Minute syndrome are discussed. (U.S.)

  12. Beam test of a full-length prototype of the BESIII drift chamber with the readout electronics

    International Nuclear Information System (INIS)

    Qin, Z.H.; Chen, Y.B.; Sheng, H.Y.; Wu, L.H.; Liu, J.B.; Zhuang, B.A.; Jiang, X.S.; Zhao, Y.B.; Zhu, K.J.; Yan, Z.K.; Chen, C.; Xu, M.H.; Wang, L.; Ma, X.Y.; Tang, X.; Liu, R.G.; Jin, Y.; Zhu, Q.M.; Zhang, G.F.; Wu, Z.; Li, R.Y.; Zhao, P.P.; Dai, H.L.; Li, X.P.; Li, J.

    2007-01-01

    A full-length prototype of the BESIII drift chamber together with its readout electronics was built and a beam test was performed. Two different methods, namely 'single-threshold method' and 'double-threshold method' for timing measurement, were studied. Test results show that the BESIII drift chamber and its readout electronics can reach their design specifications. The 'double-threshold method' results in a better timing accuracy and noise suppression capabilities as compared with the 'single-threshold method'

  13. Cytological studies of human meiosis: sex-specific differences in recombination originate at, or prior to, establishment of double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Jennifer R Gruhn

    Full Text Available Meiotic recombination is sexually dimorphic in most mammalian species, including humans, but the basis for the male:female differences remains unclear. In the present study, we used cytological methodology to directly compare recombination levels between human males and females, and to examine possible sex-specific differences in upstream events of double-strand break (DSB formation and synaptic initiation. Specifically, we utilized the DNA mismatch repair protein MLH1 as a marker of recombination events, the RecA homologue RAD51 as a surrogate for DSBs, and the synaptonemal complex proteins SYCP3 and/or SYCP1 to examine synapsis between homologs. Consistent with linkage studies, genome-wide recombination levels were higher in females than in males, and the placement of exchanges varied between the sexes. Subsequent analyses of DSBs and synaptic initiation sites indicated similar male:female differences, providing strong evidence that sex-specific differences in recombination rates are established at or before the formation of meiotic DSBs. We then asked whether these differences might be linked to variation in the organization of the meiotic axis and/or axis-associated DNA and, indeed, we observed striking male:female differences in synaptonemal complex (SC length and DNA loop size. Taken together, our observations suggest that sex specific differences in recombination in humans may derive from chromatin differences established prior to the onset of the recombination pathway.

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

    Directory of Open Access Journals (Sweden)

    Matthew G Cottingham

    2008-02-01

    Full Text Available The production, manipulation and rescue of a bacterial artificial chromosome clone of Vaccinia virus (VAC-BAC in order to expedite construction of expression vectors and mutagenesis of the genome has been described (Domi & Moss, 2002, PNAS99 12415-20. The genomic BAC clone was 'rescued' back to infectious virus using a Fowlpox virus helper to supply transcriptional machinery. We apply here a similar approach to the attenuated strain Modified Vaccinia virus Ankara (MVA, now widely used as a safe non-replicating recombinant vaccine vector in mammals, including humans. Four apparently full-length, rescuable clones were obtained, which had indistinguishable immunogenicity in mice. One clone was shotgun sequenced and found to be identical to the parent. We employed GalK recombination-mediated genetic engineering (recombineering of MVA-BAC to delete five selected viral genes. Deletion of C12L, A44L, A46R or B7R did not significantly affect CD8(+ T cell immunogenicity in BALB/c mice, but deletion of B15R enhanced specific CD8(+ T cell responses to one of two endogenous viral epitopes (from the E2 and F2 proteins, in accordance with published work (Staib et al., 2005, J. Gen. Virol.86, 1997-2006. In addition, we found a higher frequency of triple-positive IFN-gamma, TNF-alpha and IL-2 secreting E3-specific CD8+ T-cells 8 weeks after vaccination with MVA lacking B15R. Furthermore, a recombinant vaccine capable of inducing CD8(+ T cells against an epitope from Plasmodium berghei was created using GalK counterselection to insert an antigen expression cassette lacking a tandem marker gene into the traditional thymidine kinase locus of MVA-BAC. MVA continues to feature prominently in clinical trials of recombinant vaccines against diseases such as HIV-AIDS, malaria and tuberculosis. Here we demonstrate in proof-of-concept experiments that MVA-BAC recombineering is a viable route to more rapid and efficient generation of new candidate mutant and recombinant

  15. On the Use of Hydrogen Recombination Energy during Common Envelope Events

    Science.gov (United States)

    Ivanova, Natalia

    2018-05-01

    In this Letter we discuss what happens to hydrogen recombination energy that is released during regular common envelope (CE) events as opposed to self-regulated CE events. We show that the amount of recombination energy that can be transferred away by either convection or radiation from the regions where recombination takes place is negligible. Instead, recombination energy is destined to be used either to help CE expansion, as a work term, or to accelerate local fluid elements. The exceptions are donors that initially have very high entropy material, S/(k B N A) > 37 mol g‑1. The analysis and conclusions are independent of specific stellar models or evolutionary codes, and rely on fundamental properties of stellar matter such as the equation of state, Saha equation, and opacities, as well as on stellar structure equations and the mixing length theory of convection.

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

    DEFF Research Database (Denmark)

    Cottingham, Matthew G; Andersen, Rikke F; Spencer, Alexandra J

    2008-01-01

    -length, rescuable clones were obtained, which had indistinguishable immunogenicity in mice. One clone was shotgun sequenced and found to be identical to the parent. We employed GalK recombination-mediated genetic engineering (recombineering) of MVA-BAC to delete five selected viral genes. Deletion of C12L, A44L, A...

  17. Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

    Science.gov (United States)

    Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

    2015-05-01

    Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  18. Conventional and real time RT-PCR assays for the detection and differentiation of variant rabbit hemorrhagic disease virus (RHDVb) and its recombinants.

    Science.gov (United States)

    Dalton, K P; Arnal, J L; Benito, A A; Chacón, G; Martín Alonso, J M; Parra, F

    2018-01-01

    Since its emergence, variant RHDV (RHDVb/RHDV2) has spread throughout the Iberian Peninsula aided by the apparent lack of cross protection provided by classic (genogroup 1; G1) strain derived vaccines. In addition to RHDVb, full-length genome sequencing of RHDV strains has recently revealed the circulation of recombinant viruses on the Iberian Peninsula. These recombinant viruses contain the RHDVb structural protein encoding sequences and the non-structural coding regions of either pathogenic RHDV-G1 strains or non-pathogenic (np) rabbit caliciviruses. The aim of the work was twofold: firstly to validate a diagnostic real time RT-PCR developed in 2012 for the detection of RHDVb strains and secondly, to design a conventional RT-PCR for the differentiation of RHDVb strains from RHDVb recombinants by subsequent sequencing of the amplicon. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. BIOTECHNOLOGY OF RECOMBINANT HORMONES IN DOPING

    Directory of Open Access Journals (Sweden)

    Biljana Vitošević

    2011-09-01

    Full Text Available Recombinant DNA technology has allowed rapid progress in creating biosynthetic gene products for the treatment of many diseases. In this way it can produce large amounts of hormone, which is intended for the treatment of many pathological conditions. Recombinant hormones that are commonly used are insulin, growth hormone and erythropoietin. Precisely because of the availability of these recombinant hormones, it started their abuse by athletes. Experiments in animal models confirmed the potential effects of some of these hormones in increasing physical abilities, which attracted the attention of athletes who push the limits of their competitive capability by such manipulation. The risks of the use of recombinant hormones in doping include serious consequences for the health of athletes. Methods of detection of endogenous hormones from recombined based on the use of a monoclonal antibodies, capillary zone electrophoresis and protein biomarkers

  20. Recombination analysis based on the complete genome of bocavirus

    Directory of Open Access Journals (Sweden)

    Chen Shengxia

    2011-04-01

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

  1. Caspase 3 inactivates biologically active full length interleukin-33 as a classical cytokine but does not prohibit nuclear translocation

    International Nuclear Information System (INIS)

    Ali, Shafaqat; Nguyen, Dang Quan; Falk, Werner; Martin, Michael Uwe

    2010-01-01

    IL-33 is a member of the IL-1 family of cytokines with dual function which either activates cells via the IL-33 receptor in a paracrine fashion or translocates to the nucleus to regulate gene transcription in an intracrine manner. We show that full length murine IL-33 is active as a cytokine and that it is not processed by caspase 1 to mature IL-33 but instead cleaved by caspase 3 at aa175 to yield two products which are both unable to bind to the IL-33 receptor. Full length IL-33 and its N-terminal caspase 3 breakdown product, however, translocate to the nucleus. Finally, bioactive IL-33 is not released by cells constitutively or after activation. This suggests that IL-33 is not a classical cytokine but exerts its function in the nucleus of intact cells and only activates others cells via its receptor as an alarm mediator after destruction of the producing cell.

  2. Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications.

    Science.gov (United States)

    Gupta, Sanjeev K; Shukla, Pratyoosh

    2016-12-01

    Prokaryotic expression systems are superior in producing valuable recombinant proteins, enzymes and therapeutic products. Conventional microbial technology is evolving gradually and amalgamated with advanced technologies in order to give rise to improved processes for the production of metabolites, recombinant biopharmaceuticals and industrial enzymes. Recently, several novel approaches have been employed in a bacterial expression platform to improve recombinant protein expression. These approaches involve metabolic engineering, use of strong promoters, novel vector elements such as inducers and enhancers, protein tags, secretion signals, high-throughput devices for cloning and process screening as well as fermentation technologies. Advancement of the novel technologies in E. coli systems led to the production of "difficult to express" complex products including small peptides, antibody fragments, few proteins and full-length aglycosylated monoclonal antibodies in considerable large quantity. Wacker's secretion technologies, Pfenex system, inducers, cell-free systems, strain engineering for post-translational modification, such as disulfide bridging and bacterial N-glycosylation, are still under evaluation for the production of complex proteins and peptides in E. coli in an efficient manner. This appraisal provides an impression of expression technologies developed in recent times for enhanced production of heterologous proteins in E. coli which are of foremost importance for diverse applications in microbiology and biopharmaceutical production.

  3. {sup 68}Ga-labelled recombinant antibody variants for immuno-PET imaging of solid tumours

    Energy Technology Data Exchange (ETDEWEB)

    Eder, Matthias; Eisenhut, Michael [German Cancer Research Center, Radiopharmaceutical Chemistry, Heidelberg (Germany); Knackmuss, Stefan; Gall, Fabrice Le; Reusch, Uwe; Little, Melvyn [Affimed Therapeutics AG, Heidelberg (Germany); Rybin, Vladimir [European Molecular Biology Laboratory, Heidelberg (Germany); Haberkorn, Uwe; Mier, Walter [University of Heidelberg, Department of Nuclear Medicine, Heidelberg (Germany)

    2010-07-15

    Recombinant antibodies isolated from human antibody libraries have excellent affinities and high target specificity. As full-length IgGs are cleared inadequately slowly from the circulation, the aim of this work was to figure out which kind of recombinant antibody fragment proves to be appropriate for imaging epithelial cell adhesion molecule (EpCAM)-expressing tumours with the short-living radioisotope {sup 68}Ga. In order to combine the promising tumour targeting properties of antibodies with {sup 68}Ga, four antibody variants with the same specificity and origin only differing in molecular weight were constructed for comparison. Therefore, the binding domains of a single-chain fragment variable (scFv) isolated from a human naive antibody library were modified genetically to construct the respective full-length IgG, the tria- and diabody variants. These molecules were conjugated with the bifunctional chelating agent N,N{sup '}-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N{sup '}-diacetic acid (HBED-CC) to enable {sup 68}Ga labelling at ambient temperature and compared in biodistribution and immuno-PET imaging experiments. The antibody variants with identical specificity proved to have the correct molecular weight, high binding affinity and specificity to their antigen, EpCAM. Radiometal complexation was efficiently performed at room temperature leading to {sup 68}Ga-labelled antibodies with unchanged binding properties compared to the original antibody variants. The best targeting properties were obtained with the scFv and especially with the diabody. The triabody showed higher absolute tumour uptake but only moderate clearance from circulation. The antibody variants differed considerably in normal organ uptake, clearance from circulation and tumour accumulation. The data demonstrate the feasibility of imaging solid tumours with the {sup 68}Ga-labelled diabody format. This type of recombinant protein might be a promising carrier even for the

  4. Construction and characterization of a full-length cDNA library for the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici

    Directory of Open Access Journals (Sweden)

    Chen Xianming

    2007-06-01

    Full Text Available Abstract Background Puccinia striiformis is a plant pathogenic fungus causing stripe rust, one of the most important diseases on cereal crops and grasses worldwide. However, little is know about its genome and genes involved in the biology and pathogenicity of the pathogen. We initiated the functional genomic research of the fungus by constructing a full-length cDNA and determined functions of the first group of genes by sequence comparison of cDNA clones to genes reported in other fungi. Results A full-length cDNA library, consisting of 42,240 clones with an average cDNA insert of 1.9 kb, was constructed using urediniospores of race PST-78 of P. striiformis f. sp. tritici. From 196 sequenced cDNA clones, we determined functions of 73 clones (37.2%. In addition, 36 clones (18.4% had significant homology to hypothetical proteins, 37 clones (18.9% had some homology to genes in other fungi, and the remaining 50 clones (25.5% did not produce any hits. From the 73 clones with functions, we identified 51 different genes encoding protein products that are involved in amino acid metabolism, cell defense, cell cycle, cell signaling, cell structure and growth, energy cycle, lipid and nucleotide metabolism, protein modification, ribosomal protein complex, sugar metabolism, transcription factor, transport metabolism, and virulence/infection. Conclusion The full-length cDNA library is useful in identifying functional genes of P. striiformis.

  5. Construction and expression of eukaryotic expression vectors of full-length, amino-terminus and carboxyl-terminus Raf gene

    Directory of Open Access Journals (Sweden)

    Zhuomin WANG

    2008-06-01

    Full Text Available Background and objective Raf is a key molecule in the Ras-Raf-MEK-ERK signal transduction pathway and is highly activated in different human carcinomas. However, its biological functions and regulation mechanisms are still unclear. The aims of this study were to construct eukaryotic expression vectors with Raf full encoding region, truncated amino-terminus and carboxyl-terminus, respectively. Methods Eukaryotic expression vectors of pCMV-Tag2b-Raf-1, pCMV-Tag2b-N-Raf and pCMV-Tag2b-C-Raf were constructed by gene recombination technique and confirmed by restriction enzyme analysis and DNA sequencing. Furthermore, the expression of these fusion proteins was detected by western blot in transient transfected 293T cells. Results The sequences and open reading frames of these three vectors were completely consistent with experimental design. All target proteins can be detected in 293T cells. Conclusion Eukaryotic expression vectors of pCMV-Tag2b-Raf-1, pCMV-Tag2b-N-Raf and pCMV-Tag2b-C-Raf were successfully constructed and can be expressed in 293T cells.

  6. Full-Length Fibronectin Drives Fibroblast Accumulation at the Surface of Collagen Microtissues during Cell-Induced Tissue Morphogenesis.

    Directory of Open Access Journals (Sweden)

    Jasper Foolen

    Full Text Available Generating and maintaining gradients of cell density and extracellular matrix (ECM components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is vital. In a 3D model system of tissue morphogenesis, a fibronectin-FRET sensor recently revealed the existence of two separate fibronectin populations with different conformations in microtissues, i.e. 'compact and adsorbed to collagen' versus 'extended and fibrillar' fibronectin that does not colocalize with the collagen scaffold. Here we asked how the presence of fibronectin might drive this cell-induced tissue morphogenesis, more specifically the formation of gradients in cell density and ECM composition. Microtissues were engineered in a high-throughput model system containing rectangular microarrays of 12 posts, which constrained fibroblast-populated collagen gels, remodeled by the contractile cells into trampoline-shaped microtissues. Fibronectin's contribution during the tissue maturation process was assessed using fibronectin-knockout mouse embryonic fibroblasts (Fn-/- MEFs and floxed equivalents (Fnf/f MEFs, in fibronectin-depleted growth medium with and without exogenously added plasma fibronectin (full-length, or various fragments. In the absence of full-length fibronectin, Fn-/- MEFs remained homogenously distributed throughout the cell-contracted collagen gels. In contrast, in the presence of full-length fibronectin, both cell types produced shell-like tissues with a predominantly cell-free compacted collagen core and a peripheral surface layer rich in cells. Single cell assays then revealed that Fn-/- MEFs applied lower total strain energy on nanopillar arrays coated with either fibronectin or vitronectin when compared to Fnf/f MEFs, but that the presence of exogenously added plasma fibronectin rescued their contractility. While collagen

  7. First full length sequences of the S gene of European isolates reveal further diversity among turkey coronaviruses.

    OpenAIRE

    2011-01-01

    Abstract An increasing incidence of enteric disorders clinically evocative of the poult enteritis complex has been observed in turkeys in France since 2003. Using a newly designed real-time RT-PCR assay specific for the nucleocapsid (N) gene of infectious bronchitis virus (IBV) and turkey coronaviruses (TCoV), coronaviruses were identified in 37 % of the intestinal samples collected from diseased turkey flocks. The full length Spike (S) gene of these viruses was amplified, cloned a...

  8. A high HIV-1 strain variability in London, UK, revealed by full-genome analysis: Results from the ICONIC project

    Science.gov (United States)

    Frampton, Dan; Gallo Cassarino, Tiziano; Raffle, Jade; Hubb, Jonathan; Ferns, R. Bridget; Waters, Laura; Tong, C. Y. William; Kozlakidis, Zisis; Hayward, Andrew; Kellam, Paul; Pillay, Deenan; Clark, Duncan; Nastouli, Eleni; Leigh Brown, Andrew J.

    2018-01-01

    Background & methods The ICONIC project has developed an automated high-throughput pipeline to generate HIV nearly full-length genomes (NFLG, i.e. from gag to nef) from next-generation sequencing (NGS) data. The pipeline was applied to 420 HIV samples collected at University College London Hospitals NHS Trust and Barts Health NHS Trust (London) and sequenced using an Illumina MiSeq at the Wellcome Trust Sanger Institute (Cambridge). Consensus genomes were generated and subtyped using COMET, and unique recombinants were studied with jpHMM and SimPlot. Maximum-likelihood phylogenetic trees were constructed using RAxML to identify transmission networks using the Cluster Picker. Results The pipeline generated sequences of at least 1Kb of length (median = 7.46Kb, IQR = 4.01Kb) for 375 out of the 420 samples (89%), with 174 (46.4%) being NFLG. A total of 365 sequences (169 of them NFLG) corresponded to unique subjects and were included in the down-stream analyses. The most frequent HIV subtypes were B (n = 149, 40.8%) and C (n = 77, 21.1%) and the circulating recombinant form CRF02_AG (n = 32, 8.8%). We found 14 different CRFs (n = 66, 18.1%) and multiple URFs (n = 32, 8.8%) that involved recombination between 12 different subtypes/CRFs. The most frequent URFs were B/CRF01_AE (4 cases) and A1/D, B/C, and B/CRF02_AG (3 cases each). Most URFs (19/26, 73%) lacked breakpoints in the PR+RT pol region, rendering them undetectable if only that was sequenced. Twelve (37.5%) of the URFs could have emerged within the UK, whereas the rest were probably imported from sub-Saharan Africa, South East Asia and South America. For 2 URFs we found highly similar pol sequences circulating in the UK. We detected 31 phylogenetic clusters using the full dataset: 25 pairs (mostly subtypes B and C), 4 triplets and 2 quadruplets. Some of these were not consistent across different genes due to inter- and intra-subtype recombination. Clusters involved 70 sequences, 19.2% of the dataset. Conclusions

  9. Measurement of Debye length in laser-produced plasma.

    Science.gov (United States)

    Ehler, W.

    1973-01-01

    The Debye length of an expanded plasma created by placing an evacuated chamber with an entrance slit in the path of a freely expanding laser produced plasma was measured, using the slab geometry. An independent measurement of electron density together with the observed value for the Debye length also provided a means for evaluating the plasma electron temperature. This temperature has applications in ascertaining plasma conductivity and magnetic field necessary for confinement of the laser produced plasma. Also, the temperature obtained would be useful in analyzing electron-ion recombination rates in the expanded plasma and the dynamics of the cooling process of the plasma expansion.

  10. Evidence that meiotic pairing starts at the telomeres: Molecular analysis of recombination in a family with a pericentric X chromosome inversion

    Energy Technology Data Exchange (ETDEWEB)

    Shashi, V.; Allinson, P.S.; Golden, W.L.; Kelly, T.E. [Univ. of Virginia, Charlottesville, VA (United States)

    1994-09-01

    Recent studies in yeast have shown that telomeres rather than centromeres lead in chromosome movement just prior to meiosis and may have a role in recombination. Cytological studies of meiosis in Drosophila and mice have shown that in pericentric inversion heterozygotes there is lack of loop formation, with recobmination seen only outside the inversion. In a family with Duchenne muscular dystrophy (DMD) we recognized that only affected males and carrier females had a pericentric X chromosome inversion (inv X(p11.4;q26)). Since the short arm inversion breakpoint was proximal to the DMD locus, it could not be implicated in the mutational event causing DMD. There was no history of infertility, recurrent miscarriages or liveborn unbalanced females to suggest there was recombination within the inversion. We studied 22 members over three generations to understand the pattern of meiotic recombination between the normal and the inverted X chromosome. In total, 17 meioses involving the inverted X chromosome in females were studied by cytogenetic analysis and 16 CA repeat polymorphisms along the length of the X chromosome. Results: (a) There was complete concordance between the segregation of the DMD mutation and the inverted X chromosome. (b) On DNA analysis, there was complete absence of recombination within the inverted segment. We also found no recombination at the DMD locus. Recombination was seen only at Xp22 and Xq27-28. (c) Recombination was seen in the same individual at both Xp22 and Xq27-28 without recombination otherwise. Conclusions: (1) Pericentric X inversions reduce the genetic map length of the chromosome, with the physical map length being normal. (2) Meiotic X chromosome pairing in this family is initiated at the telomeres. (3) Following telomeric pairing in pericentric X chromosome inversions, there is inhibition of recombination within the inversion and adjacent regions.

  11. Toxoplasma gondii: biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6.

    Science.gov (United States)

    Bittame, Amina; Effantin, Grégory; Pètre, Graciane; Ruffiot, Pauline; Travier, Laetitia; Schoehn, Guy; Weissenhorn, Winfried; Cesbron-Delauw, Marie-France; Gagnon, Jean; Mercier, Corinne

    2015-03-27

    The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6-8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8-15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Photoconductivity, pH Sensitivity, Noise, and Channel Length Effects in Si Nanowire FET Sensors

    Science.gov (United States)

    Gasparyan, Ferdinand; Zadorozhnyi, Ihor; Khondkaryan, Hrant; Arakelyan, Armen; Vitusevich, Svetlana

    2018-03-01

    Silicon nanowire (NW) field-effect transistor (FET) sensors of various lengths were fabricated. Transport properties of Si NW FET sensors were investigated involving noise spectroscopy and current-voltage (I-V) characterization. The static I-V dependencies demonstrate the high quality of fabricated silicon FETs without leakage current. Transport and noise properties of NW FET structures were investigated under different light illumination conditions, as well as in sensor configuration in an aqueous solution with different pH values. Furthermore, we studied channel length effects on the photoconductivity, noise, and pH sensitivity. The magnitude of the channel current is approximately inversely proportional to the length of the current channel, and the pH sensitivity increases with the increase of channel length approaching the Nernst limit value of 59.5 mV/pH. We demonstrate that dominant 1/f-noise can be screened by the generation-recombination plateau at certain pH of the solution or external optical excitation. The characteristic frequency of the generation-recombination noise component decreases with increasing of illumination power. Moreover, it is shown that the measured value of the slope of 1/f-noise spectral density dependence on the current channel length is 2.7 which is close to the theoretically predicted value of 3.

  13. Protein misfolding cyclic amplification induces the conversion of recombinant prion protein to PrP oligomers causing neuronal apoptosis.

    Science.gov (United States)

    Yuan, Zhen; Yang, Lifeng; Chen, Baian; Zhu, Ting; Hassan, Mohammad Farooque; Yin, Xiaomin; Zhou, Xiangmei; Zhao, Deming

    2015-06-01

    The formation of neurotoxic prion protein (PrP) oligomers is thought to be a key step in the development of prion diseases. Recently, it was determined that the sonication and shaking of recombinant PrP can convert PrP monomers into β-state oligomers. Herein, we demonstrate that β-state oligomeric PrP can be generated through protein misfolding cyclic amplification from recombinant full-length hamster, human, rabbit, and mutated rabbit PrP, and that these oligomers can be used for subsequent research into the mechanisms of PrP-induced neurotoxicity. We have characterized protein misfolding cyclic amplification-induced monomer-to-oligomer conversion of PrP from three species using western blotting, circular dichroism, size-exclusion chromatography, and resistance to proteinase K (PK) digestion. We have further shown that all of the resulting β-oligomers are toxic to primary mouse cortical neurons independent of the presence of PrP(C) in the neurons, whereas the corresponding monomeric PrP were not toxic. In addition, we found that this toxicity is the result of oligomer-induced apoptosis via regulation of Bcl-2, Bax, and caspase-3 in both wild-type and PrP(-/-) cortical neurons. It is our hope that these results may contribute to our understanding of prion transformation within the brain. We found that β-state oligomeric PrPs can be generated through protein misfolding cyclic amplification (PMCA) from recombinant full-length hamster, human, rabbit, and mutated rabbit PrP. β-oligomers are toxic to primary mouse cortical neurons independent of the presence of PrP(C) in the neurons, while the corresponding monomeric PrPs were not toxic. This toxicity is the result of oligomers-induced apoptosis via regulation of Bcl-2, Bax, and caspase-3. These results may contribute to our understanding of prion transformation within the brain. © 2015 International Society for Neurochemistry.

  14. The Meiotic Recombination Activator PRDM9 Trimethylates Both H3K36 and H3K4 at Recombination Hotspots In Vivo.

    Science.gov (United States)

    Powers, Natalie R; Parvanov, Emil D; Baker, Christopher L; Walker, Michael; Petkov, Petko M; Paigen, Kenneth

    2016-06-01

    In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we

  15. Late replicating domains are highly recombining in females but have low male recombination rates: implications for isochore evolution.

    Directory of Open Access Journals (Sweden)

    Catherine J Pink

    Full Text Available In mammals sequences that are either late replicating or highly recombining have high rates of evolution at putatively neutral sites. As early replicating domains and highly recombining domains both tend to be GC rich we a priori expect these two variables to covary. If so, the relative contribution of either of these variables to the local neutral substitution rate might have been wrongly estimated owing to covariance with the other. Against our expectations, we find that sex-averaged recombination rates show little or no correlation with replication timing, suggesting that they are independent determinants of substitution rates. However, this result masks significant sex-specific complexity: late replicating domains tend to have high recombination rates in females but low recombination rates in males. That these trends are antagonistic explains why sex-averaged recombination is not correlated with replication timing. This unexpected result has several important implications. First, although both male and female recombination rates covary significantly with intronic substitution rates, the magnitude of this correlation is moderately underestimated for male recombination and slightly overestimated for female recombination, owing to covariance with replicating timing. Second, the result could explain why male recombination is strongly correlated with GC content but female recombination is not. If to explain the correlation between GC content and replication timing we suppose that late replication forces reduced GC content, then GC promotion by biased gene conversion during female recombination is partly countered by the antagonistic effect of later replicating sequence tending increase AT content. Indeed, the strength of the correlation between female recombination rate and local GC content is more than doubled by control for replication timing. Our results underpin the need to consider sex-specific recombination rates and potential covariates in

  16. Sequencing and analysis of full-length cDNAs, 5'-ESTs and 3'-ESTs from a cartilaginous fish, the elephant shark (Callorhinchus milii).

    KAUST Repository

    Brenner, Sydney

    2012-10-08

    Cartilaginous fishes are the most ancient group of living jawed vertebrates (gnathostomes) and are, therefore, an important reference group for understanding the evolution of vertebrates. The elephant shark (Callorhinchus milii), a holocephalan cartilaginous fish, has been identified as a model cartilaginous fish genome because of its compact genome (∼910 Mb) and a genome project has been initiated to obtain its whole genome sequence. In this study, we have generated and sequenced full-length enriched cDNA libraries of the elephant shark using the \\'oligo-capping\\' method and Sanger sequencing. A total of 6,778 full-length protein-coding cDNA and 10,701 full-length noncoding cDNA were sequenced from six tissues (gills, intestine, kidney, liver, spleen, and testis) of the elephant shark. Analysis of their polyadenylation signals showed that polyadenylation usage in elephant shark is similar to that in mammals. Furthermore, both coding and noncoding transcripts of the elephant shark use the same proportion of canonical polyadenylation sites. Besides BLASTX searches, protein-coding transcripts were annotated by Gene Ontology, InterPro domain, and KEGG pathway analyses. By comparing elephant shark genes to bony vertebrate genes, we identified several ancient genes present in elephant shark but differentially lost in tetrapods or teleosts. Only ∼6% of elephant shark noncoding cDNA showed similarity to known noncoding RNAs (ncRNAs). The rest are either highly divergent ncRNAs or novel ncRNAs. In addition to full-length transcripts, 30,375 5\\'-ESTs and 41,317 3\\'-ESTs were sequenced and annotated. The clones and transcripts generated in this study are valuable resources for annotating transcription start sites, exon-intron boundaries, and UTRs of genes in the elephant shark genome, and for the functional characterization of protein sequences. These resources will also be useful for annotating genes in other cartilaginous fishes whose genomes have been targeted for

  17. Sequencing and analysis of full-length cDNAs, 5'-ESTs and 3'-ESTs from a cartilaginous fish, the elephant shark (Callorhinchus milii).

    KAUST Repository

    Brenner, Sydney; Kodzius, Rimantas; Tan, Yue Ying; Tay, Alice; Tay, Boon-Hui; Venkatesh, Byrappa

    2012-01-01

    Cartilaginous fishes are the most ancient group of living jawed vertebrates (gnathostomes) and are, therefore, an important reference group for understanding the evolution of vertebrates. The elephant shark (Callorhinchus milii), a holocephalan cartilaginous fish, has been identified as a model cartilaginous fish genome because of its compact genome (∼910 Mb) and a genome project has been initiated to obtain its whole genome sequence. In this study, we have generated and sequenced full-length enriched cDNA libraries of the elephant shark using the 'oligo-capping' method and Sanger sequencing. A total of 6,778 full-length protein-coding cDNA and 10,701 full-length noncoding cDNA were sequenced from six tissues (gills, intestine, kidney, liver, spleen, and testis) of the elephant shark. Analysis of their polyadenylation signals showed that polyadenylation usage in elephant shark is similar to that in mammals. Furthermore, both coding and noncoding transcripts of the elephant shark use the same proportion of canonical polyadenylation sites. Besides BLASTX searches, protein-coding transcripts were annotated by Gene Ontology, InterPro domain, and KEGG pathway analyses. By comparing elephant shark genes to bony vertebrate genes, we identified several ancient genes present in elephant shark but differentially lost in tetrapods or teleosts. Only ∼6% of elephant shark noncoding cDNA showed similarity to known noncoding RNAs (ncRNAs). The rest are either highly divergent ncRNAs or novel ncRNAs. In addition to full-length transcripts, 30,375 5'-ESTs and 41,317 3'-ESTs were sequenced and annotated. The clones and transcripts generated in this study are valuable resources for annotating transcription start sites, exon-intron boundaries, and UTRs of genes in the elephant shark genome, and for the functional characterization of protein sequences. These resources will also be useful for annotating genes in other cartilaginous fishes whose genomes have been targeted for whole

  18. Full length articles published in BJOMS during 2010-11--an analysis by sub-specialty and study type.

    Science.gov (United States)

    Arakeri, Gururaj; Colbert, Serryth; Rosenbaum, Gavin; Brennan, Peter A

    2012-12-01

    Full length articles such as prospective and retrospective studies, case series, laboratory-based research and reviews form the majority of papers published in the British Journal of Oral and Maxillofacial Surgery (BJOMS). We were interested to evaluate the breakdown of these types of articles both by sub-specialty and the type of study as well as the proportion that are written by UK colleagues compared to overseas authors over a 2 year period (2010-11). A total of 191 full length articles across all sub-specialties of our discipline were published, with 107 papers (56%) coming from UK authors. There were proportionately more oncology papers arising from the UK than overseas (60 and 30% of total respectively) while the opposite was found for cleft/deformity studies (10% and 22%). There was only one laboratory-based study published from the UK compared with 27 papers from overseas. The number of quality papers being submitted to the Journal continues to increase, and the type of article being published between UK and overseas probably reflects different practices and case-loads amongst colleagues. The relatively few UK laboratory based studies published in BJOMS compared to overseas authors are most likely due to authors seeking the most prestigious journals possible for their work. Copyright © 2012 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

  19. Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning

    Energy Technology Data Exchange (ETDEWEB)

    Deymier, Martin J., E-mail: mdeymie@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Claiborne, Daniel T., E-mail: dclaibo@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Ende, Zachary, E-mail: zende@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Ratner, Hannah K., E-mail: hannah.ratner@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Kilembe, William, E-mail: wkilembe@rzhrg-mail.org [Zambia-Emory HIV Research Project (ZEHRP), B22/737 Mwembelelo, Emmasdale Post Net 412, P/BagE891, Lusaka (Zambia); Allen, Susan, E-mail: sallen5@emory.edu [Zambia-Emory HIV Research Project (ZEHRP), B22/737 Mwembelelo, Emmasdale Post Net 412, P/BagE891, Lusaka (Zambia); Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA (United States); Hunter, Eric, E-mail: eric.hunter2@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA (United States)

    2014-11-15

    The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual's diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens. - Highlights: • Our novel methodology demonstrates accurate amplification and cloning of full-length HIV-1 genomes. • A majority of plasma derived HIV variants from a chronically infected individual are infectious. • The transmitted/founder was more infectious than the majority of the variants from the chronically infected donor.

  20. Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning

    International Nuclear Information System (INIS)

    Deymier, Martin J.; Claiborne, Daniel T.; Ende, Zachary; Ratner, Hannah K.; Kilembe, William; Allen, Susan; Hunter, Eric

    2014-01-01

    The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual's diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens. - Highlights: • Our novel methodology demonstrates accurate amplification and cloning of full-length HIV-1 genomes. • A majority of plasma derived HIV variants from a chronically infected individual are infectious. • The transmitted/founder was more infectious than the majority of the variants from the chronically infected donor

  1. The effect of gamma radiation on recombination frequency in Caenorhabditis elegans

    International Nuclear Information System (INIS)

    Kim, J.S.; Rose, A.M.

    1987-01-01

    We have studied the effect of gamma radiation on recombination frequency for intervals across the cluster of linkage group I in Caenorhabditis elegans. Recombination frequency increased approximately twofold across the dpy-5-unc-13 interval after treatment with 2000 rads (1 rad = 10 mGy) of cobalt 60 gamma radiation. Several factors affecting the magnitude of the increase have been characterized. Recombination frequency increased more with higher doses of radiation. However, the increase in recombination frequency with increasing dose was accompanied by a reduced average number of progeny from radiation-treated individuals. The amount of the increase was affected by meiotic stage, age at the time of treatment (premeiotic), and radiation dose. The increase in recombination was detectable in the B brood and remained elevated for the remainder of egg production. X-chromosome nondisjunction was also increased by radiation treatment. A high frequency of the recombinant progeny produced with radiation treatment were sterile unlike their nonrecombinant siblings. When parameters affecting recombination frequency are held constant during treatment, chromosomal regions of high gene density on the meiotic map increased more (fourfold) than an adjacent region of low gene density (no increase). The greatest was across the dpy-14-unc-13 interval near the center of the gene cluster. These results may suggest that the physical length of DNA per map unit is greater within the cluster than outside

  2. Inconsistencies of genome annotations in apicomplexan parasites revealed by 5'-end-one-pass and full-length sequences of oligo-capped cDNAs

    Directory of Open Access Journals (Sweden)

    Sugano Sumio

    2009-07-01

    Full Text Available Abstract Background Apicomplexan parasites are causative agents of various diseases including malaria and have been targets of extensive genomic sequencing. We generated 5'-EST collections for six apicomplexa parasites using our full-length oligo-capping cDNA library method. To improve upon the current genome annotations, as well as to validate the importance for physical cDNA clone resources, we generated a large-scale collection of full-length cDNAs for several apicomplexa parasites. Results In this study, we used a total of 61,056 5'-end-single-pass cDNA sequences from Plasmodium falciparum, P. vivax, P. yoelii, P. berghei, Cryptosporidium parvum, and Toxoplasma gondii. We compared these partially sequenced cDNA sequences with the currently annotated gene models and observed significant inconsistencies between the two datasets. In particular, we found that on average 14% of the exons in the current gene models were not supported by any cDNA evidence, and that 16% of the current gene models may contain at least one mis-annotation and should be re-evaluated. We also identified a large number of transcripts that had been previously unidentified. For 732 cDNAs in T. gondii, the entire sequences were determined in order to evaluate the annotated gene models at the complete full-length transcript level. We found that 41% of the T. gondii gene models contained at least one inconsistency. We also identified and confirmed by RT-PCR 140 previously unidentified transcripts found in the intergenic regions of the current gene annotations. We show that the majority of these discrepancies are due to questionable predictions of one or two extra exons in the upstream or downstream regions of the genes. Conclusion Our data indicates that the current gene models are likely to still be incomplete and have much room for improvement. Our unique full-length cDNA information is especially useful for further refinement of the annotations for the genomes of

  3. Meiotic recombination in human oocytes.

    Directory of Open Access Journals (Sweden)

    Edith Y Cheng

    2009-09-01

    Full Text Available Studies of human trisomies indicate a remarkable relationship between abnormal meiotic recombination and subsequent nondisjunction at maternal meiosis I or II. Specifically, failure to recombine or recombination events located either too near to or too far from the centromere have been linked to the origin of human trisomies. It should be possible to identify these abnormal crossover configurations by using immunofluorescence methodology to directly examine the meiotic recombination process in the human female. Accordingly, we initiated studies of crossover-associated proteins (e.g., MLH1 in human fetal oocytes to analyze their number and distribution on nondisjunction-prone human chromosomes and, more generally, to characterize genome-wide levels of recombination in the human female. Our analyses indicate that the number of MLH1 foci is lower than predicted from genetic linkage analysis, but its localization pattern conforms to that expected for a crossover-associated protein. In studies of individual chromosomes, our observations provide evidence for the presence of "vulnerable" crossover configurations in the fetal oocyte, consistent with the idea that these are subsequently translated into nondisjunctional events in the adult oocyte.

  4. A highly efficient targeted recombination system for engineering linear chromosomes of industrial bacteria Streptomyces.

    Science.gov (United States)

    Pan, Hung-Yin; Chen, Carton W; Huang, Chih-Hung

    2018-04-17

    Soil bacteria Streptomyces are the most important producers of secondary metabolites, including most known antibiotics. These bacteria and their close relatives are unique in possessing linear chromosomes, which typically harbor 20 to 30 biosynthetic gene clusters of tens to hundreds of kb in length. Many Streptomyces chromosomes are accompanied by linear plasmids with sizes ranging from several to several hundred kb. The large linear plasmids also often contain biosynthetic gene clusters. We have developed a targeted recombination procedure for arm exchanges between a linear plasmid and a linear chromosome. A chromosomal segment inserted in an artificially constructed plasmid allows homologous recombination between the two replicons at the homology. Depending on the design, the recombination may result in two recombinant replicons or a single recombinant chromosome with the loss of the recombinant plasmid that lacks a replication origin. The efficiency of such targeted recombination ranges from 9 to 83% depending on the locations of the homology (and thus the size of the chromosomal arm exchanged), essentially eliminating the necessity of selection. The targeted recombination is useful for the efficient engineering of the Streptomyces genome for large-scale deletion, addition, and shuffling.

  5. Patient-Specific Instruments Based on Knee Joint Computed Tomography and Full-Length Lower Extremity Radiography in Total Knee Replacement

    Directory of Open Access Journals (Sweden)

    Hua Tian

    2018-01-01

    Conclusions: The use of PSIs based on knee joint CT and standing full-length lower extremity radiography in TKR resulted in acceptable alignment compared with the use of conventional instruments, although the marginal advantage was not statistically different. Surgical time and clinical results were also similar between the two groups. However, the PSI group had less postoperative drainage.

  6. Molecular assembly of recombinant chicken type II collagen in the yeast Pichia pastoris.

    Science.gov (United States)

    Xi, Caixia; Liu, Nan; Liang, Fei; Zhao, Xiao; Long, Juan; Yuan, Fang; Yun, Song; Sun, Yuying; Xi, Yongzhi

    2018-01-09

    Effective treatment of rheumatoid arthritis can be mediated by native chicken type II collagen (nCCII), recombinant peptide containing nCCII tolerogenic epitopes (CTEs), or a therapeutic DNA vaccine encoding the full-length CCOL2A1 cDNA. As recombinant CCII (rCCII) might avoid potential pathogenic virus contamination during nCCII preparation or chromosomal integration and oncogene activation associated with DNA vaccines, here we evaluated the importance of propeptide and telopeptide domains on rCCII triple helix molecular assembly. We constructed pC- and pN-procollagen (without N- or Cpropeptides, respectively) as well as CTEs located in the triple helical domain lacking both propeptides and telopeptides, and expressed these in yeast Pichia pastoris host strain GS115 (his4, Mut + ) simultaneously with recombinant chicken prolyl-4-hydroxylase α and β subunits. Both pC- and pN-procollagen monomers accumulated inside P. pastoris cells, whereas CTE was assembled into homotrimers with stable conformation and secreted into the supernatants, suggesting that the large molecular weight pC-or pN-procollagens were retained within the endoplasmic reticulum whereas the smaller CTEs proceeded through the secretory pathway. Furthermore, resulting recombinant chicken type II collagen pCα1(II) can induced collagen-induced arthritis (CIA) rat model, which seems to be as effective as the current standard nCCII. Notably, protease digestion assays showed that rCCII could assemble in the absence of C- and N-propeptides or telopeptides. These findings provide new insights into the minimal structural requirements for rCCII expression and folding.

  7. Recombinant Programming

    OpenAIRE

    Pawlak , Renaud; Cuesta , Carlos; Younessi , Houman

    2004-01-01

    This research report presents a promising new approach to computation called Recombinant Programming. The novelty of our approach is that it separates the program into two layers of computation: the recombination and the interpretation layer. The recombination layer takes sequences as inputs and allows the programmer to recombine these sequences through the definition of cohesive code units called extensions. The output of such recombination is a mesh that can be used by the interpretation la...

  8. An RNA-Seq strategy to detect the complete coding and non-coding transcriptome including full-length imprinted macro ncRNAs.

    Directory of Open Access Journals (Sweden)

    Ru Huang

    Full Text Available Imprinted macro non-protein-coding (nc RNAs are cis-repressor transcripts that silence multiple genes in at least three imprinted gene clusters in the mouse genome. Similar macro or long ncRNAs are abundant in the mammalian genome. Here we present the full coding and non-coding transcriptome of two mouse tissues: differentiated ES cells and fetal head using an optimized RNA-Seq strategy. The data produced is highly reproducible in different sequencing locations and is able to detect the full length of imprinted macro ncRNAs such as Airn and Kcnq1ot1, whose length ranges between 80-118 kb. Transcripts show a more uniform read coverage when RNA is fragmented with RNA hydrolysis compared with cDNA fragmentation by shearing. Irrespective of the fragmentation method, all coding and non-coding transcripts longer than 8 kb show a gradual loss of sequencing tags towards the 3' end. Comparisons to published RNA-Seq datasets show that the strategy presented here is more efficient in detecting known functional imprinted macro ncRNAs and also indicate that standardization of RNA preparation protocols would increase the comparability of the transcriptome between different RNA-Seq datasets.

  9. An integrated PCR colony hybridization approach to screen cDNA libraries for full-length coding sequences.

    Science.gov (United States)

    Pollier, Jacob; González-Guzmán, Miguel; Ardiles-Diaz, Wilson; Geelen, Danny; Goossens, Alain

    2011-01-01

    cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) is a commonly used technique for genome-wide expression analysis that does not require prior sequence knowledge. Typically, quantitative expression data and sequence information are obtained for a large number of differentially expressed gene tags. However, most of the gene tags do not correspond to full-length (FL) coding sequences, which is a prerequisite for subsequent functional analysis. A medium-throughput screening strategy, based on integration of polymerase chain reaction (PCR) and colony hybridization, was developed that allows in parallel screening of a cDNA library for FL clones corresponding to incomplete cDNAs. The method was applied to screen for the FL open reading frames of a selection of 163 cDNA-AFLP tags from three different medicinal plants, leading to the identification of 109 (67%) FL clones. Furthermore, the protocol allows for the use of multiple probes in a single hybridization event, thus significantly increasing the throughput when screening for rare transcripts. The presented strategy offers an efficient method for the conversion of incomplete expressed sequence tags (ESTs), such as cDNA-AFLP tags, to FL-coding sequences.

  10. Caenorhabditis briggsae recombinant inbred line genotypes reveal inter-strain incompatibility and the evolution of recombination.

    Directory of Open Access Journals (Sweden)

    Joseph A Ross

    2011-07-01

    Full Text Available The nematode Caenorhabditis briggsae is an emerging model organism that allows evolutionary comparisons with C. elegans and exploration of its own unique biological attributes. To produce a high-resolution C. briggsae recombination map, recombinant inbred lines were generated from reciprocal crosses between two strains and genotyped at over 1,000 loci. A second set of recombinant inbred lines involving a third strain was also genotyped at lower resolution. The resulting recombination maps exhibit discrete domains of high and low recombination, as in C. elegans, indicating these are a general feature of Caenorhabditis species. The proportion of a chromosome's physical size occupied by the central, low-recombination domain is highly correlated between species. However, the C. briggsae intra-species comparison reveals striking variation in the distribution of recombination between domains. Hybrid lines made with the more divergent pair of strains also exhibit pervasive marker transmission ratio distortion, evidence of selection acting on hybrid genotypes. The strongest effect, on chromosome III, is explained by a developmental delay phenotype exhibited by some hybrid F2 animals. In addition, on chromosomes IV and V, cross direction-specific biases towards one parental genotype suggest the existence of cytonuclear epistatic interactions. These interactions are discussed in relation to surprising mitochondrial genome polymorphism in C. briggsae, evidence that the two strains diverged in allopatry, the potential for local adaptation, and the evolution of Dobzhansky-Muller incompatibilities. The genetic and genomic resources resulting from this work will support future efforts to understand inter-strain divergence as well as facilitate studies of gene function, natural variation, and the evolution of recombination in Caenorhabditis nematodes.

  11. The extent and importance of intragenic recombination

    Directory of Open Access Journals (Sweden)

    de Silva Eric

    2004-11-01

    Full Text Available Abstract We have studied the recombination rate behaviour of a set of 140 genes which were investigated for their potential importance in inflammatory disease. Each gene was extensively sequenced in 24 individuals of African descent and 23 individuals of European descent, and the recombination process was studied separately in the two population samples. The results obtained from the two populations were highly correlated, suggesting that demographic bias does not affect our population genetic estimation procedure. We found evidence that levels of recombination correlate with levels of nucleotide diversity. High marker density allowed us to study recombination rate variation on a very fine spatial scale. We found that about 40 per cent of genes showed evidence of uniform recombination, while approximately 12 per cent of genes carried distinct signatures of recombination hotspots. On studying the locations of these hotspots, we found that they are not always confined to introns but can also stretch across exons. An investigation of the protein products of these genes suggested that recombination hotspots can sometimes separate exons belonging to different protein domains; however, this occurs much less frequently than might be expected based on evolutionary studies into the origins of recombination. This suggests that evolutionary analysis of the recombination process is greatly aided by considering nucleotide sequences and protein products jointly.

  12. Termini of human chromosomes display elevated rates of mitotic recombination.

    Science.gov (United States)

    Cornforth, M N; Eberle, R L

    2001-01-01

    The strand-specific in situ hybridization technique of CO-FISH was used to probe telomeres of human mitotic cells in order to determine the spontaneous frequency of crossover. This approach allowed the detection of recombinational crossovers occurring anywhere along the length of individual chromosomes, including reciprocal events taking place between sister chromatids. Although the process of sister chromatid exchange (SCE) is the most prominent type of recombination in somatic mammalian cells, our results show that SCEs accounted for less than a third of the recombinational events revealed by CO-FISH. It is concluded that chromosomal regions near the termini of chromosome arms undergo extraordinarily high rates of spontaneous recombination, producing terminal crossovers whose small size precludes detection by standard cytogenetic methods. That similar results were observed for transformed epithelial cells, as well as primary fibroblasts, suggests that the phenomenon is a common characteristic of human cells. These findings are noteworthy because, although telomeric and subtelomeric DNA is known to be preferentially involved in certain types of recombination, the tips of somatic mammalian chromosomes have not previously been identified as preferred sites for crossover. Implications of these results are discussed in terms of limitations imposed on CO-FISH for its proposed use in directional hybridization mapping.

  13. Pre-Steady-State Kinetic Analysis of Truncated and Full-Length Saccharomyces cerevisiae DNA Polymerase Eta

    Directory of Open Access Journals (Sweden)

    Jessica A. Brown

    2010-01-01

    Full Text Available Understanding polymerase fidelity is an important objective towards ascertaining the overall stability of an organism's genome. Saccharomyces cerevisiae DNA polymerase η (yPolη, a Y-family DNA polymerase, is known to efficiently bypass DNA lesions (e.g., pyrimidine dimers in vivo. Using pre-steady-state kinetic methods, we examined both full-length and a truncated version of yPolη which contains only the polymerase domain. In the absence of yPolη's C-terminal residues 514–632, the DNA binding affinity was weakened by 2-fold and the base substitution fidelity dropped by 3-fold. Thus, the C-terminus of yPolη may interact with DNA and slightly alter the conformation of the polymerase domain during catalysis. In general, yPolη discriminated between a correct and incorrect nucleotide more during the incorporation step (50-fold on average than the ground-state binding step (18-fold on average. Blunt-end additions of dATP or pyrene nucleotide 5′-triphosphate revealed the importance of base stacking during the binding of incorrect incoming nucleotides.

  14. A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

    Directory of Open Access Journals (Sweden)

    Alamar Santiago

    2009-09-01

    Full Text Available Abstract Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information and biological knowledge. Full-length cDNA clones facilitate functional analysis of the corresponding genes enabling manipulation of their expression in heterologous systems and the generation of a variety of tagged versions of the native protein. In addition, the development of full-length cDNA sequences has the power to improve the quality of genome annotation. Results We developed an integrated method to generate a new normalized EST collection enriched in full-length and rare transcripts of different citrus species from multiple tissues and developmental stages. We constructed a total of 15 cDNA libraries, from which we isolated 10,898 high-quality ESTs representing 6142 different genes. Percentages of redundancy and proportion of full-length clones range from 8 to 33, and 67 to 85, respectively, indicating good efficiency of the approach employed. The new EST collection adds 2113 new citrus ESTs, representing 1831 unigenes, to the collection of citrus genes available in the public databases. To facilitate functional analysis, cDNAs were introduced in a Gateway-based cloning vector for high-throughput functional analysis of genes in planta. Herein, we describe the technical methods used in the library construction, sequence analysis of clones and the overexpression of CitrSEP, a citrus homolog to the Arabidopsis SEP3 gene, in Arabidopsis as an example of a practical application of the engineered Gateway vector for functional analysis. Conclusion The new

  15. Hydrogen recombiner development at AECL

    International Nuclear Information System (INIS)

    Dewit, W.A.; Koroll, G.W.; Loesel Sitar, J.; Graham, W.R.C.

    1997-01-01

    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

  16. Characterization of near full-length genomes of HIV type 1 strains in Denmark: Basis for a universal therapeutic vaccine

    DEFF Research Database (Denmark)

    Andresen, Betina S.; Vinner, Lasse; Tang, Sheila Tuyet

    2007-01-01

    We report here the near full-length sequence characterization of 17 Danish clinical HIV-1 strains isolated from HLA-A02 patients not in need of ART, with relatively low viral loads and normal CD4 cell counts. Sequencing was performed directly on DNA extracted from short-term cocultures of PBMCs...... of a universal immunotherapeutic vaccine construct based on these epitopes....

  17. Development of a recombinant toxin fragment vaccine for Clostridium difficile infection.

    Science.gov (United States)

    Karczewski, Jerzy; Zorman, Julie; Wang, Su; Miezeiewski, Matthew; Xie, Jinfu; Soring, Keri; Petrescu, Ioan; Rogers, Irene; Thiriot, David S; Cook, James C; Chamberlin, Mihaela; Xoconostle, Rachel F; Nahas, Debbie D; Joyce, Joseph G; Bodmer, Jean-Luc; Heinrichs, Jon H; Secore, Susan

    2014-05-19

    Clostridium difficile infection (CDI) is the major cause of antibiotic-associated diarrhea and pseudomembranous colitis, a disease associated with significant morbidity and mortality. The disease is mostly of nosocomial origin, with elderly patients undergoing anti-microbial therapy being particularly at risk. C. difficile produces two large toxins: Toxin A (TcdA) and Toxin B (TcdB). The two toxins act synergistically to damage and impair the colonic epithelium, and are primarily responsible for the pathogenesis associated with CDI. The feasibility of toxin-based vaccination against C. difficile is being vigorously investigated. A vaccine based on formaldehyde-inactivated Toxin A and Toxin B (toxoids) was reported to be safe and immunogenic in healthy volunteers and is now undergoing evaluation in clinical efficacy trials. In order to eliminate cytotoxic effects, a chemical inactivation step must be included in the manufacturing process of this toxin-based vaccine. In addition, the large-scale production of highly toxic antigens could be a challenging and costly process. Vaccines based on non-toxic fragments of genetically engineered versions of the toxins alleviate most of these limitations. We have evaluated a vaccine assembled from two recombinant fragments of TcdB and explored their potential as components of a novel experimental vaccine against CDI. Golden Syrian hamsters vaccinated with recombinant fragments of TcdB combined with full length TcdA (Toxoid A) developed high titer IgG responses and potent neutralizing antibody titers. We also show here that the recombinant vaccine protected animals against lethal challenge with C. difficile spores, with efficacy equivalent to the toxoid vaccine. The development of a two-segment recombinant vaccine could provide several advantages over toxoid TcdA/TcdB such as improvements in manufacturability. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Integration of vectors by homologous recombination in the plant pathogen Glomerella cingulata.

    Science.gov (United States)

    Rikkerink, E H; Solon, S L; Crowhurst, R N; Templeton, M D

    1994-03-01

    An homologous transformation system has been developed for the plant pathogenic fungus Glomerella cingulata (Colletotrichum gloeosporioides). A transformation vector containing the G. cingulata gpdA promoter fused to the hygromycin phosphotransferase gene was constructed. Southern analyses indicated that this vector integrated at single sites in most transformants. A novel method of PCR amplification across the recombination junction point indicated that the integration event occurred by homologous recombination in more than 95% of the transformants. Deletion studies demonstrated that 505 bp (the minimum length of homologous promoter DNA analysed which was still capable of promoter function) was sufficient to target integration events. Homologous integration of the vector resulted in duplication of the gdpA promoter region. When transformants were grown without selective pressure, a high incidence of vector excision by recombination between the duplicated regions was evident. The significance of these recombination characteristics is discussed with reference to the feasibility of performing gene disruption experiments.

  19. Experimental evidence that RNA recombination occurs in the Japanese encephalitis virus

    International Nuclear Information System (INIS)

    Chuang, C.-K.; Chen, W.-J.

    2009-01-01

    Due to the lack of a proofreading function and error-repairing ability of genomic RNA, accumulated mutations are known to be a force driving viral evolution in the genus Flavivirus, including the Japanese encephalitis (JE) virus. Based on sequencing data, RNA recombination was recently postulated to be another factor associated with genomic variations in these viruses. We herein provide experimental evidence to demonstrate the occurrence of RNA recombination in the JE virus using two local pure clones (T1P1-S1 and CJN-S1) respectively derived from the local strains, T1P1 and CJN. Based on results from a restriction fragment length polymorphism (RFLP) assay on the C/preM junction comprising a fragment of 868 nucleotides (nt 10-877), the recombinant progeny virus was primarily formed in BHK-21 cells that had been co-infected with the two clones used in this study. Nine of 20 recombinant forms of the JE virus had a crossover in the nt 123-323 region. Sequencing data derived from these recombinants revealed that no nucleotide deletion or insertion occurred in this region favoring crossovers, indicating that precisely, not aberrantly, homologous recombination was involved. With site-directed mutagenesis, three stem-loop secondary structures were destabilized and re-stabilized in sequence, leading to changes in the frequency of recombination. This suggests that the conformation, not the free energy, of the secondary structure is important in modulating RNA recombination of the virus. It was concluded that because RNA recombination generates genetic diversity in the JE virus, this must be considered particularly in studies of viral evolution, epidemiology, and possible vaccine safety.

  20. Binding site analysis of full-length α1a adrenergic receptor using homology modeling and molecular docking

    International Nuclear Information System (INIS)

    Pedretti, Alessandro; Elena Silva, Maria; Villa, Luigi; Vistoli, Giulio

    2004-01-01

    The recent availability of crystal structure of bovine rhodopsin offers new opportunities in order to approach the construction of G protein coupled receptors. This study focuses the attention on the modeling of full-length α 1a adrenergic receptor (α 1a -AR) due to its biological role and significant implications in pharmacological treatment of benign prostate hyperplasia. This work could be considered made up by two main steps: (a) the construction of full structure of α 1a -AR, through homology modeling methods; (b) the automated docking of an endogenous agonist, norepinephrine, and of an antagonist, WB-4101, using BioDock program. The obtained results highlight the key residues involved in binding sites of both agonists and antagonists, confirming the mutagenesis data and giving new suggestions for the rational design of selective ligands

  1. The Impact of Recombination Hotspots on Genome Evolution of a Fungal Plant Pathogen.

    Science.gov (United States)

    Croll, Daniel; Lendenmann, Mark H; Stewart, Ethan; McDonald, Bruce A

    2015-11-01

    Recombination has an impact on genome evolution by maintaining chromosomal integrity, affecting the efficacy of selection, and increasing genetic variability in populations. Recombination rates are a key determinant of the coevolutionary dynamics between hosts and their pathogens. Historic recombination events created devastating new pathogens, but the impact of ongoing recombination in sexual pathogens is poorly understood. Many fungal pathogens of plants undergo regular sexual cycles, and sex is considered to be a major factor contributing to virulence. We generated a recombination map at kilobase-scale resolution for the haploid plant pathogenic fungus Zymoseptoria tritici. To account for intraspecific variation in recombination rates, we constructed genetic maps from two independent crosses. We localized a total of 10,287 crossover events in 441 progeny and found that recombination rates were highly heterogeneous within and among chromosomes. Recombination rates on large chromosomes were inversely correlated with chromosome length. Short accessory chromosomes often lacked evidence for crossovers between parental chromosomes. Recombination was concentrated in narrow hotspots that were preferentially located close to telomeres. Hotspots were only partially conserved between the two crosses, suggesting that hotspots are short-lived and may vary according to genomic background. Genes located in hotspot regions were enriched in genes encoding secreted proteins. Population resequencing showed that chromosomal regions with high recombination rates were strongly correlated with regions of low linkage disequilibrium. Hence, genes in pathogen recombination hotspots are likely to evolve faster in natural populations and may represent a greater threat to the host. Copyright © 2015 by the Genetics Society of America.

  2. Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction.

    Directory of Open Access Journals (Sweden)

    Bhagyashree S Birla

    Full Text Available Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple double-stranded DNA fragments. Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor. In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number dsDNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the Gibson Assembly protocol, we have assembled 3 different genes from up to 45 dsDNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to most gene sequences, and can improve both the efficiency and cost of gene assembly.

  3. VP1u phospholipase activity is critical for infectivity of full-length parvovirus B19 genomic clones

    OpenAIRE

    Filippone, Claudia; Zhi, Ning; Wong, Susan; Lu, Jun; Kajigaya, Sachiko; Gallinella, Giorgio; Kakkola, Laura; Söderlund-Venermo, Maria; Young, Neal S.; Brown, Kevin E.

    2008-01-01

    Three full-length genomic clones (pB19-M20, pB19-FL and pB19-HG1) of parvovirus B19 were produced in different laboratories. pB19-M20 was shown to produce infectious virus. To determine the differences in infectivity, all three plasmids were tested by transfection and infection assays. All three clones were similar in viral DNA replication, RNA transcription, and viral capsid protein production. However, only pB19-M20 and pB19-HG1 produced infectious virus. Comparison of viral sequences showe...

  4. Hybrid Perovskites for Photovoltaics: Charge-Carrier Recombination, Diffusion, and Radiative Efficiencies.

    Science.gov (United States)

    Johnston, Michael B; Herz, Laura M

    2016-01-19

    values extracted from OPTP measurements and their dependence on perovskite composition and morphology. The significance of the reviewed charge-carrier recombination and mobility parameters is subsequently evaluated in terms of the charge-carrier diffusion lengths and radiative efficiencies that may be obtained for such hybrid perovskites. We particularly focus on calculating such quantities in the limit of ultra-low trap-related recombination, which has not yet been demonstrated but could be reached through further advances in material processing. We find that for thin films of hybrid lead iodide perovskites with typical charge-carrier mobilities of ∼30cm(2)/(V s), charge-carrier diffusion lengths at solar (AM1.5) irradiation are unlikely to exceed ∼10 μm even if all trap-related recombination is eliminated. We further examine the radiative efficiency for hybrid lead halide perovskite films and show that if high efficiencies are to be obtained for intermediate charge-carrier densities (n ≈ 10(14) cm(-3)) trap-related recombination lifetimes will have to be enhanced well into the microsecond range.

  5. Bioconversion of Agricultural Waste to Ethanol by SSF Using Recombinant Cellulase from Clostridium thermocellum.

    Science.gov (United States)

    Mutreja, Ruchi; Das, Debasish; Goyal, Dinesh; Goyal, Arun

    2011-01-01

    The effect of different pretreatment methods, temperature, and enzyme concentration on ethanol production from 8 lignocellulosic agrowaste by simultaneous saccharification and fermentation (SSF) using recombinant cellulase and Saccharomyces cerevisiae were studied. Recombinant cellulase was isolated from E. coli BL21 cells transformed with CtLic26A-Cel5-CBM11 full-length gene from Clostridium thermocellum and produced in both batch and fed-batch processes. The maximum cell OD and specific activity in batch mode were 1.6 and 1.91 U/mg, respectively, whereas in the fed-batch mode, maximum cell OD and specific activity were 3.8 and 3.5 U/mg, respectively, displaying a 2-fold increase. Eight substrates, Syzygium cumini (jamun), Azadirachta indica (neem), Saracens indica (asoka), bambusa dendrocalmus (bamboo), Populas nigra (poplar), Achnatherum hymenoides (wild grass), Eucalyptus marginata (eucalyptus), and Mangifera indica (mango), were subjected to SSF. Of three pretreatments, acid, alkali, and steam explosion, acid pretreatment Syzygium cumini (Jamun) at 30°C gave maximum ethanol yield of 1.42 g/L.

  6. Temporally-controlled site-specific recombination in zebrafish.

    Directory of Open Access Journals (Sweden)

    Stefan Hans

    Full Text Available Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreER(T2. Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM or its active metabolite, 4-hydroxy-tamoxifen (4-OHT. Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms.

  7. Differing Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models

    Directory of Open Access Journals (Sweden)

    Tania Rivera-Hernandez

    2016-06-01

    Full Text Available Group A Streptococcus (GAS is an important human pathogen responsible for both superficial infections and invasive diseases. Autoimmune sequelae may occur upon repeated infection. For this reason, development of a vaccine against GAS represents a major challenge, since certain GAS components may trigger autoimmunity. We formulated three combination vaccines containing the following: (i streptolysin O (SLO, interleukin 8 (IL-8 protease (Streptococcus pyogenes cell envelope proteinase [SpyCEP], group A streptococcal C5a peptidase (SCPA, arginine deiminase (ADI, and trigger factor (TF; (ii the conserved M-protein-derived J8 peptide conjugated to ADI; and (iii group A carbohydrate lacking the N-acetylglucosamine side chain conjugated to ADI. We compared these combination vaccines to a “gold standard” for immunogenicity, full-length M1 protein. Vaccines were adjuvanted with alum, and mice were immunized on days 0, 21, and 28. On day 42, mice were challenged via cutaneous or subcutaneous routes. High-titer antigen-specific antibody responses with bactericidal activity were detected in mouse serum samples for all vaccine candidates. In comparison with sham-immunized mice, all vaccines afforded protection against cutaneous challenge. However, only full-length M1 protein provided protection in the subcutaneous invasive disease model.

  8. Improved antifungal activity of barley derived chitinase I gene that overexpress a 32 kDa recombinant chitinase in Escherichia coli host

    Directory of Open Access Journals (Sweden)

    Nida Toufiq

    Full Text Available Abstract Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L. cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935 bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35 kDa exhibited highest expression at 0.5 mM concentration of IPTG. Expressed recombinant protein of 35 kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35 kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80 µg and 200 µg.

  9. Genomic organization, sequence divergence, and recombination of feline immunodeficiency virus from lions in the wild

    Directory of Open Access Journals (Sweden)

    Sondgeroth Kerry

    2008-02-01

    Full Text Available Abstract Background Feline immunodeficiency virus (FIV naturally infects multiple species of cat and is related to human immunodeficiency virus in humans. FIV infection causes AIDS-like disease and mortality in the domestic cat (Felis catus and serves as a natural model for HIV infection in humans. In African lions (Panthera leo and other exotic felid species, disease etiology introduced by FIV infection are less clear, but recent studies indicate that FIV causes moderate to severe CD4 depletion. Results In this study, comparative genomic methods are used to evaluate the full proviral genome of two geographically distinct FIV subtypes isolated from free-ranging lions. Genome organization of FIVPle subtype B (9891 bp from lions in the Serengeti National Park in Tanzania and FIVPle subtype E (9899 bp isolated from lions in the Okavango Delta in Botswana, both resemble FIV genome sequence from puma, Pallas cat and domestic cat across 5' LTR, gag, pol, vif, orfA, env, rev and 3'LTR regions. Comparative analyses of available full-length FIV consisting of subtypes A, B and C from FIVFca, Pallas cat FIVOma and two puma FIVPco subtypes A and B recapitulate the species-specific monophyly of FIV marked by high levels of genetic diversity both within and between species. Across all FIVPle gene regions except env, lion subtypes B and E are monophyletic, and marginally more similar to Pallas cat FIVOma than to other FIV. Sequence analyses indicate the SU and TM regions of env vary substantially between subtypes, with FIVPle subtype E more related to domestic cat FIVFca than to FIVPle subtype B and FIVOma likely reflecting recombination between strains in the wild. Conclusion This study demonstrates the necessity of whole-genome analysis to complement population/gene-based studies, which are of limited utility in uncovering complex events such as recombination that may lead to functional differences in virulence and pathogenicity. These full-length lion

  10. A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

    Science.gov (United States)

    Marques, M Carmen; Alonso-Cantabrana, Hugo; Forment, Javier; Arribas, Raquel; Alamar, Santiago; Conejero, Vicente; Perez-Amador, Miguel A

    2009-01-01

    Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information and biological knowledge. Full-length cDNA clones facilitate functional analysis of the corresponding genes enabling manipulation of their expression in heterologous systems and the generation of a variety of tagged versions of the native protein. In addition, the development of full-length cDNA sequences has the power to improve the quality of genome annotation. Results We developed an integrated method to generate a new normalized EST collection enriched in full-length and rare transcripts of different citrus species from multiple tissues and developmental stages. We constructed a total of 15 cDNA libraries, from which we isolated 10,898 high-quality ESTs representing 6142 different genes. Percentages of redundancy and proportion of full-length clones range from 8 to 33, and 67 to 85, respectively, indicating good efficiency of the approach employed. The new EST collection adds 2113 new citrus ESTs, representing 1831 unigenes, to the collection of citrus genes available in the public databases. To facilitate functional analysis, cDNAs were introduced in a Gateway-based cloning vector for high-throughput functional analysis of genes in planta. Herein, we describe the technical methods used in the library construction, sequence analysis of clones and the overexpression of CitrSEP, a citrus homolog to the Arabidopsis SEP3 gene, in Arabidopsis as an example of a practical application of the engineered Gateway vector for functional analysis. Conclusion The new EST collection denotes an

  11. EXPRESSION AND CHARACTERIZATION OF FULL-LENGTH HUMAN HEME OXYGENASE-1: PRESENCE OF INTACT MEMBRANE-BINDING REGION LEADS TO INCREASED BINDING AFFINITY FOR NADPH-CYTOCHROME P450 REDUCTASE

    Science.gov (United States)

    Huber, Warren J.; Backes, Wayne L.

    2009-01-01

    Heme oxygenase (HO) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, this NADPH and cytochrome P450 reductase (CPR)-dependent oxidation of heme also releases free iron and carbon monoxide. Much of the recent research involving heme oxygenase is done using a 30-kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a GST-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30-kDa degradation product that could not be eliminated. Therefore, we attempted to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces lysine with arginine. This mutation allowed the expression and purification of a full length hHO-1 protein. Unlike wild-type HO-1, the K254R mutant could be purified to a single 32-kDa protein capable of degrading heme at the same rate as the wild-type enzyme. The K254R full-length form had a specific activity of ~200–225 nmol bilirubin hr−1nmol−1 HO-1 as compared to ~140–150 nmol bilirubin hr−1nmol−1 for the WT form, which contains the 30-kDa contaminant. This is a 2–3-fold increase from the previously reported soluble 30-kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other ER-resident enzymes. PMID:17915953

  12. Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

    Directory of Open Access Journals (Sweden)

    Cashman Kathleen A

    2008-06-01

    Full Text Available Abstract Background There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP, glycoprotein 1 (GP1, and glycoprotein 2 (GP2. Results Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP fusions in the Rosetta strains of Escherichia coli (E. coli using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC. Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA. Conclusion These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.

  13. A Novel mouse model of enhanced proteostasis: Full-length human heat shock factor 1 transgenic mice

    International Nuclear Information System (INIS)

    Pierce, Anson; Wei, Rochelle; Halade, Dipti; Yoo, Si-Eun; Ran, Qitao; Richardson, Arlan

    2010-01-01

    Research highlights: → Development of mouse overexpressing native human HSF1 in all tissues including CNS. → HSF1 overexpression enhances heat shock response at whole-animal and cellular level. → HSF1 overexpression protects from polyglutamine toxicity and favors aggresomes. → HSF1 overexpression enhances proteostasis at the whole-animal and cellular level. -- Abstract: The heat shock response (HSR) is controlled by the master transcriptional regulator heat shock factor 1 (HSF1). HSF1 maintains proteostasis and resistance to stress through production of heat shock proteins (HSPs). No transgenic model exists that overexpresses HSF1 in tissues of the central nervous system (CNS). We generated a transgenic mouse overexpressing full-length non-mutant HSF1 and observed a 2-4-fold increase in HSF1 mRNA and protein expression in all tissues studied of HSF1 transgenic (HSF1 +/0 ) mice compared to wild type (WT) littermates, including several regions of the CNS. Basal expression of HSP70 and 90 showed only mild tissue-specific changes; however, in response to forced exercise, the skeletal muscle HSR was more elevated in HSF1 +/0 mice compared to WT littermates and in fibroblasts following heat shock, as indicated by levels of inducible HSP70 mRNA and protein. HSF1 +/0 cells elicited a significantly more robust HSR in response to expression of the 82 repeat polyglutamine-YFP fusion construct (Q82YFP) and maintained proteasome-dependent processing of Q82YFP compared to WT fibroblasts. Overexpression of HSF1 was associated with fewer, but larger Q82YFP aggregates resembling aggresomes in HSF1 +/0 cells, and increased viability. Therefore, our data demonstrate that tissues and cells from mice overexpressing full-length non-mutant HSF1 exhibit enhanced proteostasis.

  14. The effect of recombination under short-circuit conditions on the determination of charge transport properties in nanostructured photoelectrodes.

    Science.gov (United States)

    Villanueva-Cab, J; Anta, J A; Oskam, G

    2016-01-28

    We report on the commonly unaccounted for process of recombination under short-circuit conditions in nanostructured photoelectrodes with special attention to the charge collection efficiency. It is observed that when recombination under short circuit conditions is significant, small perturbation methods overestimate the charge-collection efficiency, which is related to the inaccurate determination of the electron diffusion coefficient and diffusion length.

  15. Computational Insight Into the Structural Organization of Full-Length Toll-Like Receptor 4 Dimer in a Model Phospholipid Bilayer

    Directory of Open Access Journals (Sweden)

    Mahesh Chandra Patra

    2018-03-01

    Full Text Available Toll-like receptors (TLRs are a unique category of pattern recognition receptors that recognize distinct pathogenic components, often utilizing the same set of downstream adaptors. Specific molecular features of extracellular, transmembrane (TM, and cytoplasmic domains of TLRs are crucial for coordinating the complex, innate immune signaling pathway. Here, we constructed a full-length structural model of TLR4—a widely studied member of the interleukin-1 receptor/TLR superfamily—using homology modeling, protein–protein docking, and molecular dynamics simulations to understand the differential domain organization of TLR4 in a membrane-aqueous environment. Results showed that each functional domain of the membrane-bound TLR4 displayed several structural transitions that are biophysically essential for plasma membrane integration. Specifically, the extracellular and cytoplasmic domains were partially immersed in the upper and lower leaflets of the membrane bilayer. Meanwhile, TM domains tilted considerably to overcome the hydrophobic mismatch with the bilayer core. Our analysis indicates an alternate dimerization or a potential oligomerization interface of TLR4-TM. Moreover, the helical properties of an isolated TM dimer partly agree with that of the full-length receptor. Furthermore, membrane-absorbed or solvent-exposed surfaces of the toll/interleukin-1 receptor domain are consistent with previous X-ray crystallography and biochemical studies. Collectively, we provided a complete structural model of membrane-bound TLR4 that strengthens our current understanding of the complex mechanism of receptor activation and adaptor recruitment in the innate immune signaling pathway.

  16. Terahertz Conductivity within Colloidal CsPbBr3 Perovskite Nanocrystals: Remarkably High Carrier Mobilities and Large Diffusion Lengths.

    Science.gov (United States)

    Yettapu, Gurivi Reddy; Talukdar, Debnath; Sarkar, Sohini; Swarnkar, Abhishek; Nag, Angshuman; Ghosh, Prasenjit; Mandal, Pankaj

    2016-08-10

    Colloidal CsPbBr3 perovskite nanocrystals (NCs) have emerged as an excellent light emitting material in last one year. Using time domain and time-resolved THz spectroscopy and density functional theory based calculations, we establish 3-fold free carrier recombination mechanism, namely, nonradiative Auger, bimolecular electron-hole recombination, and inefficient trap-assisted recombination in 11 nm sized colloidal CsPbBr3 NCs. Our results confirm a negligible influence of surface defects in trapping charge carriers, which in turn results into desirable intrinsic transport properties, from the perspective of device applications, such as remarkably high carrier mobility (∼4500 cm(2) V(-1) s(-1)), large diffusion length (>9.2 μm), and high luminescence quantum yield (80%). Despite being solution processed and possessing a large surface to volume ratio, this combination of high carrier mobility and diffusion length, along with nearly ideal photoluminescence quantum yield, is unique compared to any other colloidal quantum dot system.

  17. Effect of Conjugation Length on Photoinduced Charge-Transfer in π-Conjugated Oligomer-Acceptor Dyads

    KAUST Repository

    Jiang, Junlin

    2017-05-25

    A series of -conjugated oligomer-acceptor dyads were synthesized that feature oligo(phenylene ethynylene) (OPE) conjugated backbones end-capped with a naphthalene diimide (NDI) acceptor. The OPE segments vary in length from 4 to 8 phenylene ethynene units (PEn-NDI, where n = 4, 6 and 8). Fluorescence and transient absorption spectroscopy reveals that intramolecular OPE NDI charge transfer dominates the deactivation of excited states of the PEn-NDI oligomers. Both charge separation (CS) and charge recombination (CR) are strongly exothermic (G0CS ~ -1.1 and G0CR ~ -2.0 eV), and the driving forces do not vary much across the series because the oxidation and reduction potentials and singlet energies of the OPEs do not vary much with their length. Bimolecular photoinduced charge transfer between model OPEs that do not contain the NDI acceptors with methyl viologen was studied, and the results reveal that the absorption of the cation radical state (OPE+•) remains approximately constant ( ~ 575 nm) regardless of oligomer length. This finding suggests that the cation radical (polaron) of the OPE is relatively localized, effectively occupying a confined segment of n 4 repeat units in the longer oligomers. Photoinduced intramolecular electron transfer dynamics in the PEn-NDI series was investigated by UV-visible femtosecond transient absorption spectroscopy with visible and mid-infrared probes. Charge separation occurs on the 1 – 10 ps timescale, with the rates decreasing slightly with increased oligomer length (βCS ~ 0.15 Å-1). The rate for charge-recombination decreases in the sequence PE4-NDI > PE6-NDI ~ PE8-NDI. The discontinuous distance dependence in the rate for charge recombination may be related to the spatial localization of the positive polaron state in the longer oligomers.

  18. Effect of Conjugation Length on Photoinduced Charge-Transfer in π-Conjugated Oligomer-Acceptor Dyads

    KAUST Repository

    Jiang, Junlin; Alsam, Amani Abdu; Wang, Shanshan; Aly, Shawkat Mohammede; Pan, Zhenxing; Mohammed, Omar F.; Schanze, Kirk S.

    2017-01-01

    A series of -conjugated oligomer-acceptor dyads were synthesized that feature oligo(phenylene ethynylene) (OPE) conjugated backbones end-capped with a naphthalene diimide (NDI) acceptor. The OPE segments vary in length from 4 to 8 phenylene ethynene units (PEn-NDI, where n = 4, 6 and 8). Fluorescence and transient absorption spectroscopy reveals that intramolecular OPE NDI charge transfer dominates the deactivation of excited states of the PEn-NDI oligomers. Both charge separation (CS) and charge recombination (CR) are strongly exothermic (G0CS ~ -1.1 and G0CR ~ -2.0 eV), and the driving forces do not vary much across the series because the oxidation and reduction potentials and singlet energies of the OPEs do not vary much with their length. Bimolecular photoinduced charge transfer between model OPEs that do not contain the NDI acceptors with methyl viologen was studied, and the results reveal that the absorption of the cation radical state (OPE+•) remains approximately constant ( ~ 575 nm) regardless of oligomer length. This finding suggests that the cation radical (polaron) of the OPE is relatively localized, effectively occupying a confined segment of n 4 repeat units in the longer oligomers. Photoinduced intramolecular electron transfer dynamics in the PEn-NDI series was investigated by UV-visible femtosecond transient absorption spectroscopy with visible and mid-infrared probes. Charge separation occurs on the 1 – 10 ps timescale, with the rates decreasing slightly with increased oligomer length (βCS ~ 0.15 Å-1). The rate for charge-recombination decreases in the sequence PE4-NDI > PE6-NDI ~ PE8-NDI. The discontinuous distance dependence in the rate for charge recombination may be related to the spatial localization of the positive polaron state in the longer oligomers.

  19. Facile method for the site-specific, covalent attachment of full-length IgG onto nanoparticles.

    Science.gov (United States)

    Hui, James Zhe; Al Zaki, Ajlan; Cheng, Zhiliang; Popik, Vladimir; Zhang, Hongtao; Luning Prak, Eline T; Tsourkas, Andrew

    2014-08-27

    Antibodies, most commonly IgGs, have been widely used as targeting ligands in research and therapeutic applications due to their wide array of targets, high specificity and proven efficacy. Many of these applications require antibodies to be conjugated onto surfaces (e.g. nanoparticles and microplates); however, most conventional bioconjugation techniques exhibit low crosslinking efficiencies, reduced functionality due to non-site-specific labeling and random surface orientation, and/or require protein engineering (e.g. cysteine handles), which can be technically challenging. To overcome these limitations, we have recombinantly expressed Protein Z, which binds the Fc region of IgG, with an UV active non-natural amino acid benzoylphenyalanine (BPA) within its binding domain. Upon exposure to long wavelength UV light, the BPA is activated and forms a covalent link between the Protein Z and the bound Fc region of IgG. This technology was combined with expressed protein ligation (EPL), which allowed for the introduction of a fluorophore and click chemistry-compatible azide group onto the C-terminus of Protein Z during the recombinant protein purification step. This enabled the crosslinked-Protein Z-IgG complexes to be efficiently and site-specifically attached to aza-dibenzocyclooctyne-modified nanoparticles, via copper-free click chemistry. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Photoionization and Recombination

    Science.gov (United States)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  1. A new set of BXD recombinant inbred lines from advanced intercross populations in mice

    Directory of Open Access Journals (Sweden)

    Gu Jing

    2004-04-01

    Full Text Available Abstract Background Recombinant inbred (RI strains are an important resource for mapping complex traits in many species. While large RI panels are available for Arabidopsis, maize, C. elegans, and Drosophila, mouse RI panels typically consist of fewer than 30 lines. This is a severe constraint on the power and precision of mapping efforts and greatly hampers analysis of epistatic interactions. Results In order to address these limitations and to provide the community with a more effective collaborative RI mapping panel we generated new BXD RI strains from two independent advanced intercrosses (AI between C57BL/6J (B6 and DBA/2J (D2 progenitor strains. Progeny were intercrossed for 9 to 14 generations before initiating inbreeding, which is still ongoing for some strains. Since this AI base population is highly recombinant, the 46 advanced recombinant inbred (ARI strains incorporate approximately twice as many recombinations as standard RI strains, a fraction of which are inevitably shared by descent. When combined with the existing BXD RI strains, the merged BXD strain set triples the number of previously available unique recombinations and quadruples the total number of recombinations in the BXD background. Conclusion The combined BXD strain set is the largest mouse RI mapping panel. It is a powerful tool for collaborative analysis of quantitative traits and gene function that will be especially useful to study variation in transcriptome and proteome data sets under multiple environments. Additional strains also extend the value of the extensive phenotypic characterization of the previously available strains. A final advantage of expanding the BXD strain set is that both progenitors have been sequenced, and approximately 1.8 million SNPs have been characterized. This provides unprecedented power in screening candidate genes and can reduce the effective length of QTL intervals. It also makes it possible to reverse standard mapping strategies and

  2. Genetic analysis of variation in human meiotic recombination.

    Directory of Open Access Journals (Sweden)

    Reshmi Chowdhury

    2009-09-01

    Full Text Available The number of recombination events per meiosis varies extensively among individuals. This recombination phenotype differs between female and male, and also among individuals of each gender. In this study, we used high-density SNP genotypes of over 2,300 individuals and their offspring in two datasets to characterize recombination landscape and to map the genetic variants that contribute to variation in recombination phenotypes. We found six genetic loci that are associated with recombination phenotypes. Two of these (RNF212 and an inversion on chromosome 17q21.31 were previously reported in the Icelandic population, and this is the first replication in any other population. Of the four newly identified loci (KIAA1462, PDZK1, UGCG, NUB1, results from expression studies provide support for their roles in meiosis. Each of the variants that we identified explains only a small fraction of the individual variation in recombination. Notably, we found different sequence variants associated with female and male recombination phenotypes, suggesting that they are regulated by different genes. Characterization of genetic variants that influence natural variation in meiotic recombination will lead to a better understanding of normal meiotic events as well as of non-disjunction, the primary cause of pregnancy loss.

  3. Hydroxylation of recombinant human collagen type I alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylase

    Directory of Open Access Journals (Sweden)

    Pappu Kameshwari M

    2011-06-01

    Full Text Available Abstract Background Collagens require the hydroxylation of proline (Pro residues in their triple-helical domain repeating sequence Xaa-Pro-Gly to function properly as a main structural component of the extracellular matrix in animals at physiologically relevant conditions. The regioselective proline hydroxylation is catalyzed by a specific prolyl 4-hydroxylase (P4H as a posttranslational processing step. Results A recombinant human collagen type I α-1 (rCIα1 with high percentage of hydroxylated prolines (Hyp was produced in transgenic maize seeds when co-expressed with both the α- and β- subunits of a recombinant human P4H (rP4H. Germ-specific expression of rCIα1 using maize globulin-1 gene promoter resulted in an average yield of 12 mg/kg seed for the full-length rCIα1 in seeds without co-expression of rP4H and 4 mg/kg seed for the rCIα1 (rCIα1-OH in seeds with co-expression of rP4H. High-resolution mass spectrometry (HRMS analysis revealed that nearly half of the collagenous repeating triplets in rCIα1 isolated from rP4H co-expressing maize line had the Pro residues changed to Hyp residues. The HRMS analysis determined the Hyp content of maize-derived rCIα1-OH as 18.11%, which is comparable to the Hyp level of yeast-derived rCIα1-OH (17.47% and the native human CIa1 (14.59%, respectively. The increased Hyp percentage was correlated with a markedly enhanced thermal stability of maize-derived rCIα1-OH when compared to the non-hydroxylated rCIα1. Conclusions This work shows that maize has potential to produce adequately modified exogenous proteins with mammalian-like post-translational modifications that may be require for their use as pharmaceutical and industrial products.

  4. A newly emerging HIV-1 recombinant lineage (CRF58_01B disseminating among people who inject drugs in Malaysia.

    Directory of Open Access Journals (Sweden)

    Wei Zhen Chow

    Full Text Available The HIV epidemic is primarily characterised by the circulation of HIV-1 group M (main comprising of 11 subtypes and sub-subtypes (A1, A2, B-D, F1, F2, G, H, J, and K and to date 55 circulating recombinant forms (CRFs. In Southeast Asia, active inter-subtype recombination involving three main circulating genotypes--subtype B (including subtype B', the Thai variant of subtype B, CRF01_AE, and CRF33_01B--have contributed to the emergence of novel unique recombinant forms. In the present study, we conducted the molecular epidemiological surveillance of HIV-1 gag-RT genes among 258 people who inject drugs (PWIDs in Kuala Lumpur, Malaysia, between 2009 and 2011 whereby a novel CRF candidate was recently identified. The near full-length genome sequences obtained from six epidemiologically unlinked individuals showed identical mosaic structures consisting of subtype B' and CRF01_AE, with six unique recombination breakpoints in the gag-RT, pol, and env regions. Among the high-risk population of PWIDs in Malaysia, which was predominantly infected by CRF33_01B (>70%, CRF58_01B circulated at a low but significant prevalence (2.3%, 6/258. Interestingly, the CRF58_01B shared two unique recombination breakpoints with other established CRFs in the region: CRF33_01B, CRF48_01B, and CRF53_01B in the gag gene, and CRF15_01B (from Thailand in the env gene. Extended Bayesian Markov chain Monte Carlo sampling analysis showed that CRF58_01B and other recently discovered CRFs were most likely to have originated in Malaysia, and that the recent spread of recombinant lineages in the country had little influence from neighbouring countries. The isolation, genetic characterization, and evolutionary features of CRF58_01B among PWIDs in Malaysia signify the increasingly complex HIV-1 diversity in Southeast Asia that may hold an implication on disease treatment, control, and prevention.

  5. Algae-based oral recombinant vaccines

    Directory of Open Access Journals (Sweden)

    Elizabeth A Specht

    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.

  6. High avidity antibodies to full-length VAR2CSA correlate with absence of placental malaria

    DEFF Research Database (Denmark)

    Tutterrow, Yeung Lo; Salanti, Ali; Avril, Marion

    2012-01-01

    VAR2CSA mediates sequestration of Plasmodium falciparum-infected erythrocytes in the placenta, increasing the risk of poor pregnancy outcomes. Naturally acquired antibodies (Ab) to placental parasites at delivery have been associated with improved pregnancy outcomes, but Ab levels and how early...... in pregnancy Ab must be present in order to eliminate placental parasites before delivery remains unknown. Antibodies to individual Duffy-binding like domains of VAR2CSA have been studied, but the domains lack many of the conformational epitopes present in full-length VAR2CSA (FV2). Thus, the purpose...... of this study was to describe the acquisition of Ab to FV2 in women residing in high and low transmission areas and determine how Ab levels during pregnancy correlate with clearance of placental parasites. Plasma samples collected monthly throughout pregnancy from pregnant women living in high and low...

  7. Correlation of Meiotic DSB Formation and Transcription Initiation Around Fission Yeast Recombination Hotspots.

    Science.gov (United States)

    Yamada, Shintaro; Okamura, Mika; Oda, Arisa; Murakami, Hiroshi; Ohta, Kunihiro; Yamada, Takatomi

    2017-06-01

    Meiotic homologous recombination, a critical event for ensuring faithful chromosome segregation and creating genetic diversity, is initiated by programmed DNA double-strand breaks (DSBs) formed at recombination hotspots. Meiotic DSB formation is likely to be influenced by other DNA-templated processes including transcription, but how DSB formation and transcription interact with each other has not been understood well. In this study, we used fission yeast to investigate a possible interplay of these two events. A group of hotspots in fission yeast are associated with sequences similar to the cyclic AMP response element and activated by the ATF/CREB family transcription factor dimer Atf1-Pcr1. We first focused on one of those hotspots, ade6-3049 , and Atf1. Our results showed that multiple transcripts, shorter than the ade6 full-length messenger RNA, emanate from a region surrounding the ade6-3049 hotspot. Interestingly, we found that the previously known recombination-activation region of Atf1 is also a transactivation domain, whose deletion affected DSB formation and short transcript production at ade6-3049 These results point to a possibility that the two events may be related to each other at ade6-3049 In fact, comparison of published maps of meiotic transcripts and hotspots suggested that hotspots are very often located close to meiotically transcribed regions. These observations therefore propose that meiotic DSB formation in fission yeast may be connected to transcription of surrounding regions. Copyright © 2017 by the Genetics Society of America.

  8. Vaccination with recombinant heat shock protein 60 from Histoplasma capsulatum protects mice against pulmonary histoplasmosis.

    Science.gov (United States)

    Gomez, F J; Allendoerfer, R; Deepe, G S

    1995-07-01

    HIS-62 is a glycoprotein that has been isolated from the cell wall and cell membrane fraction of the pathogenic fungus Histoplasma capsulatum. It is a target of the cellular immune response to this fungus, and it protects mice against a lethal intravenous inoculum of H. capsulatum yeast cells. In this study, we cloned the gene encoding this antigen to reveal its biological nature and studied the immunological activity of recombinant antigen. The amino acid sequences of the NH2 terminus and internal peptides were obtained by Edman degradation. Degenerate oligonucleotides were used to isolate a gene fragment of HIS-62 by PCR. One 680-bp segment that corresponded to the known peptide sequence was amplified from H. capsulatum DNA. This DNA was used to screen a genomic library, and the full-length gene was isolated and sequenced. The deduced amino acid sequence of the gene demonstrated approximately 70 and approximately 50% identity to heat shock protein 60 (hsp 60) from Saccharomyces cerevisiae and hsp 60 from Escherichia coli, respectively. A cDNA was synthesized by reverse transcription PCR and was expressed in E. coli. Recombinant protein reacted with a monospecific polyclonal rabbit antiserum raised against native HIS-62, with monoclonal HIS-62-reactive T cells, and with splenocytes from mice immunized with viable yeast cells. Moreover, vaccination with the recombinant protein conferred protection in mice against a lethal intranasal inoculation with yeast cells. Thus, HIS-62 is a member of the hsp 60 family, and the recombinant hsp 60 is protective against pulmonary histoplasmosis in mice.

  9. Retrieval of a million high-quality, full-length microbial 16S and 18S rRNA gene sequences without primer bias

    DEFF Research Database (Denmark)

    Karst, Søren Michael; Dueholm, Morten Simonsen; McIlroy, Simon Jon

    2018-01-01

    Small subunit ribosomal RNA (SSU rRNA) genes, 16S in bacteria and 18S in eukaryotes, have been the standard phylogenetic markers used to characterize microbial diversity and evolution for decades. However, the reference databases of full-length SSU rRNA gene sequences are skewed to well-studied e...

  10. Construction of C35 gene bait recombinants and T47D cell cDNA library.

    Science.gov (United States)

    Yin, Kun; Xu, Chao; Zhao, Gui-Hua; Liu, Ye; Xiao, Ting; Zhu, Song; Yan, Ge

    2017-11-20

    C35 is a novel tumor biomarker associated with metastasis progression. To investigate the interaction factors of C35 in its high expressed breast cancer cell lines, we constructed bait recombinant plasmids of C35 gene and T47D cell cDNA library for yeast two-hybrid screening. Full length C35 sequences were subcloned using RT-PCR from cDNA template extracted from T47D cells. Based on functional domain analysis, the full-length C35 1-348bp was also truncated into two fragments C351-153bp and C35154-348bp to avoid auto-activation. The three kinds of C35 genes were successfully amplified and inserted into pGBKT7 to construct bait recombinant plasmids pGBKT7-C351-348bp, pGBKT7-C351-153bp and pGBKT7-C35154-348bp, then transformed into Y187 yeast cells by the lithium acetate method. Auto-activation and toxicity of C35 baits were detected using nutritional deficient medium and X-α-Gal assays. The T47D cell ds cDNA was generated by SMART TM technology and the library was constructed using in vivo recombination-mediated cloning in the AH109 yeast strain using a pGADT7-Rec plasmid. The transformed Y187/pGBKT7-C351-348bp line was intensively inhibited while the truncated Y187/pGBKT7-C35 lines had no auto-activation and toxicity in yeast cells. The titer of established cDNA library was 2 × 10 7 pfu/mL with high transformation efficiency of 1.4 × 10 6 , and the insert size of ds cDNA was distributed homogeneously between 0.5-2.0 kb. Our research generated a T47D cell cDNA library with high titer, and the constructed two C35 "baits" contained a respective functional immunoreceptor tyrosine based activation motif (ITAM) and the conserved last four amino acids Cys-Ile-Leu-Val (CILV) motif, and therefore laid a foundation for screening the C35 interaction factors in a BC cell line.

  11. Molecular comparisons of full length metapneumovirus (MPV genomes, including newly determined French AMPV-C and -D isolates, further supports possible subclassification within the MPV Genus.

    Directory of Open Access Journals (Sweden)

    Paul A Brown

    Full Text Available Four avian metapneumovirus (AMPV subgroups (A-D have been reported previously based on genetic and antigenic differences. However, until now full length sequences of the only known isolates of European subgroup C and subgroup D viruses (duck and turkey origin, respectively have been unavailable. These full length sequences were determined and compared with other full length AMPV and human metapneumoviruses (HMPV sequences reported previously, using phylogenetics, comparisons of nucleic and amino acid sequences and study of codon usage bias. Results confirmed that subgroup C viruses were more closely related to HMPV than they were to the other AMPV subgroups in the study. This was consistent with previous findings using partial genome sequences. Closer relationships between AMPV-A, B and D were also evident throughout the majority of results. Three metapneumovirus "clusters" HMPV, AMPV-C and AMPV-A, B and D were further supported by codon bias and phylogenetics. The data presented here together with those of previous studies describing antigenic relationships also between AMPV-A, B and D and between AMPV-C and HMPV may call for a subclassification of metapneumoviruses similar to that used for avian paramyxoviruses, grouping AMPV-A, B and D as type I metapneumoviruses and AMPV-C and HMPV as type II.

  12. MVA recombinants expressing the fusion and hemagglutinin genes of PPRV protects goats against virulent challenge.

    Science.gov (United States)

    Chandran, Dev; Reddy, Kolli Bhaktavatsala; Vijayan, Shahana Pallichera; Sugumar, Parthasarthy; Rani, Gudavalli Sudha; Kumar, Ponsekaran Santha; Rajendra, Lingala; Srinivasan, Villuppanoor Alwar

    2010-09-01

    Peste des Petits Ruminants (PPR) is a highly contagious animal disease caused by the Peste des Petits Ruminants virus (PPRV) belonging to the genus morbillivirus and family Paramyxoviridae. The disease results in high morbidity and mortality in goats, sheep and in some small wild ruminants. The presence of large number of small ruminants reared in endemic areas makes PPR a notorious disease threatening the livelihood of poor farmers. Conventional vaccination using a live, attenuated vaccine gives adequate protection but cannot be used in case of eradication of the disease due to difficulty in differentiation of infected animals from the vaccinated ones.In the present study, we constructed two recombinant viruses using attenuated Modified Vaccinia virus Ankara virus (MVA) namely MVA-F and MVA-H expressing the full length PPRV fusion (F) and hemagglutinin (H) glycoproteins, respectively. Goats were vaccinated intramuscularly with 105 plaque forming units (PFU) each of the recombinant viruses and a live attenuated vaccine (RAKSHA PPR) and challenged 4 months later with PPRV challenge virus (10(3) goat LD(50)). All goats were completely protected from the clinical disease. This study gave an indication that mass vaccination of small ruminants with either of the above or both recombinant inexpensive virus vaccines could help in possible eradication of PPRV from endemic countries like India and subsequent seromonitoring of the disease for differentiation of infected animals from vaccinated ones.

  13. Retroviral Vectors for Analysis of Viral Mutagenesis and Recombination

    Directory of Open Access Journals (Sweden)

    Jonathan M.O. Rawson

    2014-09-01

    Full Text Available Retrovirus population diversity within infected hosts is commonly high due in part to elevated rates of replication, mutation, and recombination. This high genetic diversity often complicates the development of effective diagnostics, vaccines, and antiviral drugs. This review highlights the diverse vectors and approaches that have been used to examine mutation and recombination in retroviruses. Retroviral vectors for these purposes can broadly be divided into two categories: those that utilize reporter genes as mutation or recombination targets and those that utilize viral genes as targets of mutation or recombination. Reporter gene vectors greatly facilitate the detection, quantification, and characterization of mutants and/or recombinants, but may not fully recapitulate the patterns of mutagenesis or recombination observed in native viral gene sequences. In contrast, the detection of mutations or recombination events directly in viral genes is more biologically relevant but also typically more challenging and inefficient. We will highlight the advantages and disadvantages of the various vectors and approaches used as well as propose ways in which they could be improved.

  14. Use of Dried Blood Spots to Elucidate Full-Length Transmitted/Founder HIV-1 Genomes

    Directory of Open Access Journals (Sweden)

    Jesus F. Salazar-Gonzalez

    2016-07-01

    Full Text Available Background: Identification of HIV-1 genomes responsible for establishing clinical infection in newly infected individuals is fundamental to prevention and pathogenesis research. Processing, storage, and transportation of the clinical samples required to perform these virologic assays in resource-limited settings requires challenging venipuncture and cold chain logistics. Here, we validate the use of dried-blood spots (DBS as a simple and convenient alternative to collecting and storing frozen plasma. Methods: We performed parallel nucleic acid extraction, single genome amplification (SGA, next generation sequencing (NGS, and phylogenetic analyses on plasma and DBS. Results: We demonstrated the capacity to extract viral RNA from DBS and perform SGA to infer the complete nucleotide sequence of the transmitted/founder (TF HIV-1 envelope gene and full-length genome in two acutely infected individuals. Using both SGA and NGS methodologies, we showed that sequences generated from DBS and plasma display comparable phylogenetic patterns in both acute and chronic infection. SGA was successful on samples with a range of plasma viremia, including samples as low as 1,700 copies/ml and an estimated ~50 viral copies per blood spot. Further, we demonstrated reproducible efficiency in gp160 env sequencing in DBS stored at ambient temperature for up to three weeks or at -20ºC for up to five months. Conclusions: These findings support the use of DBS as a practical and cost-effective alternative to frozen plasma for clinical trials and translational research conducted in resource-limited settings.

  15. Single-molecule study of full-length NaChBac by planar lipid bilayer recording.

    Directory of Open Access Journals (Sweden)

    Andrew Jo

    Full Text Available Planar lipid bilayer device, alternatively known as BLM, is a powerful tool to study functional properties of conducting membrane proteins such as ion channels and porins. In this work, we used BLM to study the prokaryotic voltage-gated sodium channel (Nav NaChBac in a well-defined membrane environment. Navs are an essential component for the generation and propagation of electric signals in excitable cells. The successes in the biochemical, biophysical and crystallographic studies on prokaryotic Navs in recent years has greatly promoted the understanding of the molecular mechanism that underlies these proteins and their eukaryotic counterparts. In this work, we investigated the single-molecule conductance and ionic selectivity behavior of NaChBac. Purified NaChBac protein was first reconstituted into lipid vesicles, which is subsequently incorporated into planar lipid bilayer by fusion. At single-molecule level, we were able to observe three distinct long-lived conductance sub-states of NaChBac. Change in the membrane potential switches on the channel mainly by increasing its opening probability. In addition, we found that individual NaChBac has similar permeability for Na+, K+, and Ca2+. The single-molecule behavior of the full-length protein is essentially highly stochastic. Our results show that planar lipid bilayer device can be used to study purified ion channels at single-molecule level in an artificial environment, and such studies can reveal new protein properties that are otherwise not observable in in vivo ensemble studies.

  16. Calculation of evolutionary correlation between individual genes and full-length genome: a method useful for choosing phylogenetic markers for molecular epidemiology.

    Directory of Open Access Journals (Sweden)

    Shuai Wang

    Full Text Available Individual genes or regions are still commonly used to estimate the phylogenetic relationships among viral isolates. The genomic regions that can faithfully provide assessments consistent with those predicted with full-length genome sequences would be preferable to serve as good candidates of the phylogenetic markers for molecular epidemiological studies of many viruses. Here we employed a statistical method to evaluate the evolutionary relationships between individual viral genes and full-length genomes without tree construction as a way to determine which gene can match the genome well in phylogenetic analyses. This method was performed by calculation of linear correlations between the genetic distance matrices of aligned individual gene sequences and aligned genome sequences. We applied this method to the phylogenetic analyses of porcine circovirus 2 (PCV2, measles virus (MV, hepatitis E virus (HEV and Japanese encephalitis virus (JEV. Phylogenetic trees were constructed for comparisons and the possible factors affecting the method accuracy were also discussed in the calculations. The results revealed that this method could produce results consistent with those of previous studies about the proper consensus sequences that could be successfully used as phylogenetic markers. And our results also suggested that these evolutionary correlations could provide useful information for identifying genes that could be used effectively to infer the genetic relationships.

  17. [Genetic evidence for recombination and mutation in the emergence of human enterovirus 71].

    Science.gov (United States)

    Liu, Ai-Ping; Tan, Hui; Xie, Qun; Chen, Bai-Tang; Liu, Xiao-Feng; Zhang, Yong

    2014-09-01

    We wished to understand the genetic recombination and phylogenetic characteristics of human en- terovirus A71 (EV-A71) and to explore its potential virulence-related sites. Full-length genomes of three EV-A71 strains isolated from patients in Chenzhou City (China) were sequenced and analyzed. Possible re- combination events and crossover sites were analyzed with Recombination Detection Program v4. 1. 6 by comparison with the complete genome sequences of 231 strains of EV-A71. Similarly, plot and bootscanning analyses were undertaken with SimPlot v3. 5. 1. Phylogenetic trees based on the sequences of VP1 regions were constructed with MEGA v5. 2 using the Kimura two-parameter model and neighbor-joining method. Results suggested that recombination events were detected among the three EV-A71 isolates from Chenzhou City. The common main parent sequence was from JF799986 isolated from samples in Guang- zhou City (China) in 2009, and the minor parent sequence was TW/70516/08. Intertypic recombination e- vents were found in the C4b strain (strain SHZH98 isolated in 1998) and C4a strain (Fuyang strain isola- ted in 2008) with the prototype strains of CVA4 and CVA14 in the 3D region. The chi-square test was used to screen-out potential virulence-related sites with nucleotide substitutions of different types of hand, foot, and mouth disease (HFMD) cases using SPSS v19.0. Results suggested that there were no significant nucleotide substitutions between death cases and severe-HFMD cases. Eighteen significant nucleotide substitutions were found between death/severe-HFMD cases and mild-HFMD cases, and all these 18 substitutions were distributed only in P2 and P3 regions. Intertypic recombination among the predominant circulating EV-A71 strains in the Chinese mainland and other EV-A strains probably dates before 1998, and intratypic recombination might have occurred frequently in the HFMD outbreak from 2008 to 2012. Substitutions in the non-capsid region may be correlated with the

  18. Increased understanding of the biochemistry and biosynthesis of MUC2 and other gel-forming mucins through the recombinant expression of their protein domains.

    Science.gov (United States)

    Bäckström, Malin; Ambort, Daniel; Thomsson, Elisabeth; Johansson, Malin E V; Hansson, Gunnar C

    2013-06-01

    The gel-forming mucins are large and heavily O-glycosylated proteins which build up mucus gels. The recombinant production of full-length gel-forming mucins has not been possible to date. In order to study mucin biosynthesis and biochemistry, we and others have taken the alternative approach of constructing different recombinant proteins consisting of one or several domains of these large proteins and expressing them separately in different cell lines. Using this approach, we have determined that MUC2, the intestinal gel-forming mucin, dimerizes via its C-terminal cysteine-knot domain and also trimerizes via one of the N-terminal von Willebrand D domains. Both of these interactions are disulfide bond mediated. Via this assembly, a molecular network is built by which the mucus gel is formed. Here we discuss not only the functional understanding obtained from studies of the recombinant proteins, but also highlight the difficulties encountered when these proteins were produced recombinantly. We often found an accumulation of the proteins in the ER and consequently no secretion. This was especially apparent when the cysteine-rich domains of the N- and C-terminal parts of the mucins were expressed. Other proteins that we constructed were either not secreted or not expressed at all. Despite these problems, the knowledge of mucin biosynthesis and assembly has advanced considerably through the studies of these recombinant proteins.

  19. Genomic organization, sequence divergence, and recombination of feline immunodeficiency virus from lions in the wild

    Science.gov (United States)

    Pecon-Slattery, Jill; McCracken, Carrie L; Troyer, Jennifer L; VandeWoude, Sue; Roelke, Melody; Sondgeroth, Kerry; Winterbach, Christiaan; Winterbach, Hanlie; O'Brien, Stephen J

    2008-01-01

    Background Feline immunodeficiency virus (FIV) naturally infects multiple species of cat and is related to human immunodeficiency virus in humans. FIV infection causes AIDS-like disease and mortality in the domestic cat (Felis catus) and serves as a natural model for HIV infection in humans. In African lions (Panthera leo) and other exotic felid species, disease etiology introduced by FIV infection are less clear, but recent studies indicate that FIV causes moderate to severe CD4 depletion. Results In this study, comparative genomic methods are used to evaluate the full proviral genome of two geographically distinct FIV subtypes isolated from free-ranging lions. Genome organization of FIVPle subtype B (9891 bp) from lions in the Serengeti National Park in Tanzania and FIVPle subtype E (9899 bp) isolated from lions in the Okavango Delta in Botswana, both resemble FIV genome sequence from puma, Pallas cat and domestic cat across 5' LTR, gag, pol, vif, orfA, env, rev and 3'LTR regions. Comparative analyses of available full-length FIV consisting of subtypes A, B and C from FIVFca, Pallas cat FIVOma and two puma FIVPco subtypes A and B recapitulate the species-specific monophyly of FIV marked by high levels of genetic diversity both within and between species. Across all FIVPle gene regions except env, lion subtypes B and E are monophyletic, and marginally more similar to Pallas cat FIVOma than to other FIV. Sequence analyses indicate the SU and TM regions of env vary substantially between subtypes, with FIVPle subtype E more related to domestic cat FIVFca than to FIVPle subtype B and FIVOma likely reflecting recombination between strains in the wild. Conclusion This study demonstrates the necessity of whole-genome analysis to complement population/gene-based studies, which are of limited utility in uncovering complex events such as recombination that may lead to functional differences in virulence and pathogenicity. These full-length lion lentiviruses are integral to

  20. Conversion of recombinant hirudin to the natural form by in vitro tyrosine sulfation. Differential substrate specificities of leech and bovine tyrosylprotein sulfotransferases.

    Science.gov (United States)

    Niehrs, C; Huttner, W B; Carvallo, D; Degryse, E

    1990-06-05

    Hirudin, a tyrosine-sulfated protein secreted by the leech Hirudo medicinalis, is one of the most potent anticoagulants known. The hirudin cDNA has previously been cloned and has been expressed in yeast, but the resulting recombinant protein was found to be produced in the unsulfated form, which is known to have an at least 10 times lower affinity for thrombin than the naturally occurring tyrosine-sulfated hirudin. Here we describe the in vitro tyrosine sulfation of recombinant hirudin by leech and bovine tyrosylprotein sulfotransferase (TPST). With both enzymes, in vitro sulfation of recombinant hirudin occurred at the physiological site (Tyr-63) and rendered the protein biochemically and biologically indistinguishable from natural hirudin. However, leech TPST had an over 20-fold lower apparent Km value for recombinant hirudin than bovine TPST. Further differences in the catalytic properties of leech and bovine TPSTs were observed when synthetic peptides were tested as substrates. Moreover, a synthetic peptide corresponding to the 9 carboxyl-terminal residues of hirudin (which include Tyr-63) was sulfated by leech TPST with a similar apparent Km value as full length hirudin, indicating that structural determinants residing in the immediate vicinity of Tyr-63 are sufficient for sulfation to occur.

  1. Cold Spring Harbor symposia on quantitative biology: Volume 49, Recombination at the DNA level

    International Nuclear Information System (INIS)

    1984-01-01

    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

  2. Recombiner

    International Nuclear Information System (INIS)

    Osumi, Morimichi.

    1979-01-01

    Purpose: To provide a recombiner which is capable of converting hydrogen gas into water by use of high-frequency heating at comparatively low temperatures and is safe and cheap in cost. Constitution: Hydrogen gas is introduced from an outer pipeline to the main structure of a recombiner, and when it passes through the vicinity of the central part of the recombiner, it is reacted with copper oxide (CuO 2 ) heated to a temperature more than 300 0 C by a high-frequency heater, and converted gently into water by reduction operation (2H 2 + CuO 2 → Cu + 2H 2 O). The thus prepared water is exhausted through the outer pipeline to a suppression pool. A part of hydrogen gas which has not been converted completely into water by the reaction and is remaining as hydrogen is recovered through exhaust nozzles and again introduced into the main structure of the recombiner. (Yoshino, Y.)

  3. A Network Approach to Analyzing Highly Recombinant Malaria Parasite Genes

    Science.gov (United States)

    Larremore, Daniel B.; Clauset, Aaron; Buckee, Caroline O.

    2013-01-01

    The var genes of the human malaria parasite Plasmodium falciparum present a challenge to population geneticists due to their extreme diversity, which is generated by high rates of recombination. These genes encode a primary antigen protein called PfEMP1, which is expressed on the surface of infected red blood cells and elicits protective immune responses. Var gene sequences are characterized by pronounced mosaicism, precluding the use of traditional phylogenetic tools that require bifurcating tree-like evolutionary relationships. We present a new method that identifies highly variable regions (HVRs), and then maps each HVR to a complex network in which each sequence is a node and two nodes are linked if they share an exact match of significant length. Here, networks of var genes that recombine freely are expected to have a uniformly random structure, but constraints on recombination will produce network communities that we identify using a stochastic block model. We validate this method on synthetic data, showing that it correctly recovers populations of constrained recombination, before applying it to the Duffy Binding Like-α (DBLα) domain of var genes. We find nine HVRs whose network communities map in distinctive ways to known DBLα classifications and clinical phenotypes. We show that the recombinational constraints of some HVRs are correlated, while others are independent. These findings suggest that this micromodular structuring facilitates independent evolutionary trajectories of neighboring mosaic regions, allowing the parasite to retain protein function while generating enormous sequence diversity. Our approach therefore offers a rigorous method for analyzing evolutionary constraints in var genes, and is also flexible enough to be easily applied more generally to any highly recombinant sequences. PMID:24130474

  4. A network approach to analyzing highly recombinant malaria parasite genes.

    Science.gov (United States)

    Larremore, Daniel B; Clauset, Aaron; Buckee, Caroline O

    2013-01-01

    The var genes of the human malaria parasite Plasmodium falciparum present a challenge to population geneticists due to their extreme diversity, which is generated by high rates of recombination. These genes encode a primary antigen protein called PfEMP1, which is expressed on the surface of infected red blood cells and elicits protective immune responses. Var gene sequences are characterized by pronounced mosaicism, precluding the use of traditional phylogenetic tools that require bifurcating tree-like evolutionary relationships. We present a new method that identifies highly variable regions (HVRs), and then maps each HVR to a complex network in which each sequence is a node and two nodes are linked if they share an exact match of significant length. Here, networks of var genes that recombine freely are expected to have a uniformly random structure, but constraints on recombination will produce network communities that we identify using a stochastic block model. We validate this method on synthetic data, showing that it correctly recovers populations of constrained recombination, before applying it to the Duffy Binding Like-α (DBLα) domain of var genes. We find nine HVRs whose network communities map in distinctive ways to known DBLα classifications and clinical phenotypes. We show that the recombinational constraints of some HVRs are correlated, while others are independent. These findings suggest that this micromodular structuring facilitates independent evolutionary trajectories of neighboring mosaic regions, allowing the parasite to retain protein function while generating enormous sequence diversity. Our approach therefore offers a rigorous method for analyzing evolutionary constraints in var genes, and is also flexible enough to be easily applied more generally to any highly recombinant sequences.

  5. Meiotic Recombination Analyses in Pigs Carrying Different Balanced Structural Chromosomal Rearrangements.

    Directory of Open Access Journals (Sweden)

    Nicolas Mary

    Full Text Available Correct pairing, synapsis and recombination between homologous chromosomes are essential for normal meiosis. All these events are strongly regulated, and our knowledge of the mechanisms involved in this regulation is increasing rapidly. Chromosomal rearrangements are known to disturb these processes. In the present paper, synapsis and recombination (number and distribution of MLH1 foci were studied in three boars (Sus scrofa domestica carrying different chromosomal rearrangements. One (T34he was heterozygote for the t(3;4(p1.3;q1.5 reciprocal translocation, one (T34ho was homozygote for that translocation, while the third (T34Inv was heterozygote for both the translocation and a pericentric inversion inv(4(p1.4;q2.3. All three boars were normal for synapsis and sperm production. This particular situation allowed us to rigorously study the impact of rearrangements on recombination. Overall, the rearrangements induced only minor modifications of the number of MLH1 foci (per spermatocyte or per chromosome and of the length of synaptonemal complexes for chromosomes 3 and 4. The distribution of MLH1 foci in T34he was comparable to that of the controls. Conversely, the distributions of MLH1 foci on chromosome 4 were strongly modified in boar T34Inv (lack of crossover in the heterosynaptic region of the quadrivalent, and crossover displaced to the chromosome extremities, and also in boar T34ho (two recombination peaks on the q-arms compared with one of higher magnitude in the controls. Analyses of boars T34he and T34Inv showed that the interference was propagated through the breakpoints. A different result was obtained for boar T34ho, in which the breakpoints (transition between SSC3 and SSC4 chromatin on the bivalents seemed to alter the transmission of the interference signal. Our results suggest that the number of crossovers and crossover interference could be regulated by partially different mechanisms.

  6. Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction.

    Science.gov (United States)

    Birla, Bhagyashree S; Chou, Hui-Hsien

    2015-01-01

    Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple double-stranded DNA fragments. Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor. In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number dsDNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the Gibson Assembly protocol, we have assembled 3 different genes from up to 45 dsDNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to most gene sequences, and can improve both the efficiency and cost of gene assembly.

  7. Intragenotypic JFH1 based recombinant hepatitis C virus produces high levels of infectious particles but causes increased cell death

    DEFF Research Database (Denmark)

    Mateu, Guaniri; Donis, Ruben O; Wakita, Takaji

    2008-01-01

    The full-length hepatitis C virus (HCV) JFH1 genome (genotype 2a) produces moderate titers of infectious particles in cell culture but the optimal determinants required for virion production are unclear. It has been shown that intragenotypic recombinants encoding core to NS2 from J6CF...... in the context of JFH1 are more robust in the release of viral particles. To understand the contributions of structural and nonstructural genes to HCV replication potential and infectivity, we have characterized intragenotypic recombinant genotype 2a viruses with different portions of the J6 isolate engineered...... into the JFH1 infectious clone. All genomes produced high levels of intracellular HCV RNA and NS3 protein in Huh-7.5 transfected cells. However, JFH1 genomes containing J6 sequences from C to E2 (CE2) or C to p7 (Cp7) secreted up to 100-fold more infectious HCV particles than the parental JFH1 clone...

  8. Genetic characterization of somatic recombination in Trichoderma pseudokoningii

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    Barcellos Fernando Gomes

    2003-01-01

    Full Text Available Crossing experiments via hyphal anastomosis between two strains contrasting for auxotrophic markers of Trichoderma pseudokoningii were conducted to characterize the somatic recombination process in this specie. Four crossings were made and a total of 1052 colonies obtained from conidial suspensions of the heterokaryotic colonies were analyzed. Sixty-eight recombinant colonies, from four growing generations, were analyzed for the auxotrophic markers. Of the 68 colonies analyzed, 58 were stable after four generations and the remainders were unstable, reverting to one of the parentals. Most of the recombinant colonies were unstable through subculture and after four growing generations they showed the leu ino met markers (auxotrophic for leucin, inositol and metionin respectively. The unstable recombinant colonies showed irregular growing borders, sparse sporulation and frequent sector formation. The results suggest the occurrence of recombination mechanisms in the heterokaryon (somatic recombination, different from those described for the parasexual cycle or parameiosis. Therefore, we proposed the ocurrence of nuclei degradation from one parental (non prevalent parental in the heterokaryon and that the resulting chromosomal fragments may be incorporated into whole nuclei of the another parental (prevalent parental. However the parameiosis as originally described cannot be excluded.

  9. INTERACTION OF RECOMBINANT DIPHTHERIA TOXOIDS WITH CELLULAR RECEPTORS in vitro

    Directory of Open Access Journals (Sweden)

    K. Yu. Manoilov

    2016-06-01

    Full Text Available The aim of the research was to compare in vitro characteristics of reception of the natural diphtheria toxin — DT and its nontoxic recombinant analogs — toxoids. For assessing ligand-receptor interaction the method of immunoenzyme analysis and ELISA was used, where the bonding layer recombinant analogues of diphtheria toxin cell receptor HB-EGF from sensitive and resistant to the toxin of the organisms were served. According to the results of ELISA the natural diphtheria toxin, in contrast to recombinant toxoids — CRM197, and B subunit, interacted with mouse HB-EGF with a very low affinity. While human HB-EGF with an equally high affinity connected as toxoids as native diphtheria toxin. Therefore, the analyzed recombinant analogs of toxin obtained in E. coli cells did not reproduce in full measure the receptor specificity of the natural toxin, which should be considered in the case of using these proteins as biotech products.

  10. The length-weight and length-length relationships of bluefish, Pomatomus saltatrix (Linnaeus, 1766 from Samsun, middle Black Sea region

    Directory of Open Access Journals (Sweden)

    Melek Özpiçak

    2017-10-01

    Full Text Available In this study, length-weight relationship (LWR and length-length relationship (LLR of bluefish, Pomatomus saltatrix were determined. A total of 125 specimens were sampled from Samsun, the middle Black Sea in 2014 fishing season. Bluefish specimens were monthly collected from commercial fishing boats from October to December 2014. All captured individuals (N=125 were measured to the nearest 0.1 cm for total, fork and standard lengths. The weight of each fish (W was recorded to the nearest 0.01 g. According to results of analyses, there were no statistically significant differences between sexes in term of length and weight (P˃0.05. The minimum and maximum total, fork and standard lengths of bluefish ranged between 13.5-23.6 cm, 12.50-21.80 cm and 10.60-20.10 cm, respectively. The equation of length-weight relationship were calculated as W=0.008TL3.12 (r2>0.962. Positive allometric growth was observed for bluefish (b>3. Length-length relationship was also highly significant (P<0.001 with coefficient of determination (r2 ranging from 0.916 to 0.988.

  11. Frequent intra-subtype recombination among HIV-1 circulating in Tanzania.

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    Ireen E Kiwelu

    Full Text Available The study estimated the prevalence of HIV-1 intra-subtype recombinant variants among female bar and hotel workers in Tanzania. While intra-subtype recombination occurs in HIV-1, it is generally underestimated. HIV-1 env gp120 V1-C5 quasispecies from 45 subjects were generated by single-genome amplification and sequencing (median (IQR of 38 (28-50 sequences per subject. Recombination analysis was performed using seven methods implemented within the recombination detection program version 3, RDP3. HIV-1 sequences were considered recombinant if recombination signals were detected by at least three methods with p-values of ≤0.05 after Bonferroni correction for multiple comparisons. HIV-1 in 38 (84% subjects showed evidence for intra-subtype recombination including 22 with HIV-1 subtype A1, 13 with HIV-1 subtype C, and 3 with HIV-1 subtype D. The distribution of intra-patient recombination breakpoints suggested ongoing recombination and showed selective enrichment of recombinant variants in 23 (60% subjects. The number of subjects with evidence of intra-subtype recombination increased from 29 (69% to 36 (82% over one year of follow-up, although the increase did not reach statistical significance. Adjustment for intra-subtype recombination is important for the analysis of multiplicity of HIV infection. This is the first report of high prevalence of intra-subtype recombination in the HIV/AIDS epidemic in Tanzania, a region where multiple HIV-1 subtypes co-circulate. HIV-1 intra-subtype recombination increases viral diversity and presents additional challenges for HIV-1 vaccine design.

  12. Full-length genomic and molecular characterization of Canine parvovirus in dogs from North of Brazil.

    Science.gov (United States)

    Silva, S P; Silva, L N P P; Rodrigues, E D L; Cardoso, J F; Tavares, F N; Souza, W M; Santos, C M P; Martins, F M S; Jesus, I S; Brito, T C; Moura, T P C; Nunes, M R T; Casseb, L M N; Silva Filho, E; Casseb, A R

    2017-09-21

    With the objective of characterizing Canine parvovirus (CPV) from some suspected fecal samples of dogs collected from the Veterinarian Hospital in Belém city, five positive samples were found by PCR assay and an update molecular characterization was provided of the CPV-2 circulation in Belém. Through sequencing of the complete DNA sequences (NS1, NS2, VP1, and VP2 genes), the CPV-2 strain was identified as CPV-2b (Asn426Asp) circulating in Belém. The CPV-2b strain with a different change at the position Tyr324Leu was detected in all samples assessed and thus reported for the first time for the scientific community. Phylogenetic analysis indicated that Belém CPV-2b and CPV-2a strains would be related to a cluster with samples after the 1990s, suggesting that CPV-2b in Belém originated from CPV-2a circulating in Brazil after the 1990s. Potential recombination events were analyzed using RDP4 and SplitsTree4; therefore, results suggest that CPV-2 sequences here described were not potentially recombination events. Continuous monitoring and molecular characterization of CPV-2 samples are needed not only to identify possible genetic and antigenic changes that may interfere with the effectiveness of vaccines but also to bring a better understanding of the mechanisms that drive the evolution of CPV-2 in Brazil.

  13. A Glance at Recombination Hotspots in the Domestic Cat.

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    Hasan Alhaddad

    Full Text Available Recombination has essential roles in increasing genetic variability within a population and in ensuring successful meiotic events. The objective of this study is to (i infer the population-scaled recombination rate (ρ, and (ii identify and characterize regions of increased recombination rate for the domestic cat, Felis silvestris catus. SNPs (n = 701 were genotyped in twenty-two East Asian feral cats (random bred. The SNPs covered ten different chromosomal regions (A1, A2, B3, C2, D1, D2, D4, E2, F2, X with an average region size of 850 Kb and an average SNP density of 70 SNPs/region. The Bayesian method in the program inferRho was used to infer regional population recombination rates and hotspots localities. The regions exhibited variable population recombination rates and four decisive recombination hotspots were identified on cat chromosome A2, D1, and E2 regions. As a description of the identified hotspots, no correlation was detected between the GC content and the locality of recombination spots, and the hotspots enclosed L2 LINE elements and MIR and tRNA-Lys SINE elements.

  14. Frequency and genetic characterization of V(DD)J recombinants in the human peripheral blood antibody repertoire.

    Science.gov (United States)

    Briney, Bryan S; Willis, Jordan R; Hicar, Mark D; Thomas, James W; Crowe, James E

    2012-09-01

    Antibody heavy-chain recombination that results in the incorporation of multiple diversity (D) genes, although uncommon, contributes substantially to the diversity of the human antibody repertoire. Such recombination allows the generation of heavy chain complementarity determining region 3 (HCDR3) regions of extreme length and enables junctional regions that, because of the nucleotide bias of N-addition regions, are difficult to produce through normal V(D)J recombination. Although this non-classical recombination process has been observed infrequently, comprehensive analysis of the frequency and genetic characteristics of such events in the human peripheral blood antibody repertoire has not been possible because of the rarity of such recombinants and the limitations of traditional sequencing technologies. Here, through the use of high-throughput sequencing of the normal human peripheral blood antibody repertoire, we analysed the frequency and genetic characteristics of V(DD)J recombinants. We found that these recombinations were present in approximately 1 in 800 circulating B cells, and that the frequency was severely reduced in memory cell subsets. We also found that V(DD)J recombination can occur across the spectrum of diversity genes, indicating that virtually all recombination signal sequences that flank diversity genes are amenable to V(DD)J recombination. Finally, we observed a repertoire bias in the diversity gene repertoire at the upstream (5') position, and discovered that this bias was primarily attributable to the order of diversity genes in the genomic locus. © 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.

  15. Expression and characterization of full-length human heme oxygenase-1: the presence of intact membrane-binding region leads to increased binding affinity for NADPH cytochrome P450 reductase.

    Science.gov (United States)

    Huber, Warren J; Backes, Wayne L

    2007-10-30

    Heme oxygenase-1 (HO-1) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, HO-1 receives the electrons necessary for catalysis from the flavoprotein NADPH cytochrome P450 reductase (CPR), releasing free iron and carbon monoxide. Much of the recent research involving heme oxygenase has been done using a 30 kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a glutathione-s-transferase (GST)-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30 kDa degradation product that could not be eliminated. Therefore, attempts were made to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces arginine with lysine. This mutation allowed the expression and purification of a full-length hHO-1 protein. Unlike wild type (WT) HO-1, the R254K mutant could be purified to a single 32 kDa protein capable of degrading heme at the same rate as the WT enzyme. The R254K full-length form had a specific activity of approximately 200-225 nmol of bilirubin h-1 nmol-1 HO-1 as compared to approximately 140-150 nmol of bilirubin h-1 nmol-1 for the WT form, which contains the 30 kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30 kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane-spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other endoplasmic reticulum resident enzymes.

  16. Strong and long: effects of word length on phonological binding in verbal short-term memory.

    Science.gov (United States)

    Jefferies, Elizabeth; Frankish, Clive; Noble, Katie

    2011-02-01

    This study examined the effects of item length on the contribution of linguistic knowledge to immediate serial recall (ISR). Long words are typically recalled more poorly than short words, reflecting the greater demands that they place on phonological encoding, rehearsal, and production. However, reverse word length effects--that is, better recall of long than short words--can also occur in situations in which phonological maintenance is difficult, suggesting that long words derive greater support from long-term lexical knowledge. In this study, long and short words and nonwords (containing one vs. three syllables) were presented for immediate serial recall in (a) pure lists and (b) unpredictable mixed lists of words and nonwords. The mixed-list paradigm is known to disrupt the phonological stability of words, encouraging their phonemes to recombine with the elements of other list items. In this situation, standard length effects were seen for nonwords, while length effects for words were absent or reversed. A detailed error analysis revealed that long words were more robust to the mixed-list manipulation than short words: Their phonemes were less likely to be omitted and to recombine with phonemes from other list items. These findings support an interactive view of short-term memory, in which long words derive greater benefits from lexical knowledge than short words-especially when their phonological integrity is challenged by the inclusion of nonwords in mixed lists.

  17. Significance of urinary full-length megalin in patients with IgA nephropathy.

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    Takuto Seki

    Full Text Available BACKGROUND AND OBJECTIVES: Megalin is highly expressed at the apical membranes of proximal tubular epithelial cells. A urinary full-length megalin (C-megalin assay is linked to the severity of diabetic nephropathy in type 2 diabetes. This study examined the relationship between levels of urinary C-megalin and histological findings in adult patients with IgA nephropathy (IgAN. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Urine samples voided in the morning on the day of renal biopsy were obtained from 73 patients with IgAN (29 men and 44 women; mean age, 33 years and 5 patients with membranous nephropathy (MN. Renal pathologic variables were analyzed using the Oxford classification of IgAN, the Shigematsu classification and the Clinical Guidelines of IgAN in Japan. The levels of urinary C-megalin were measured by sandwich ELISA. RESULTS: Histological analysis based on the Oxford classification revealed that the levels of urinary C-megalin were correlated with mesangial hypercellularity in IgAN patients (OR = 1.76, 95% CI: 1.04-3.27, P<0.05. There was a significant correlation between the levels of urinary C-megalin and the severity of chronic extracapillary abnormalities according to the Shigematsu classification in IgAN patients (β = 0.33, P = 0.008. The levels of urinary C-megalin were significantly higher in all risk levels of IgAN patients requiring dialysis using the Clinical Guidelines of IgAN in Japan than in the control group. The levels of urinary C-megalin were significantly higher in the high risk and very high risk grades than in the low risk grade (P<0.05. The levels of urinary C-megalin were significantly higher in MN patients compared to the control group. CONCLUSIONS: The levels of urinary C-megalin are associated with histological abnormalities in adult IgAN patients. There is a possibility that urinary C-megalin is an independent predictor of disease progression of IgAN. In addition, our results suggest that

  18. The α-helical C-terminal domain of full-length recombinant PrP converts to an in-register parallel β-sheet structure in PrP fibrils: evidence from solid state nuclear magnetic resonance.

    Science.gov (United States)

    Tycko, Robert; Savtchenko, Regina; Ostapchenko, Valeriy G; Makarava, Natallia; Baskakov, Ilia V

    2010-11-09

    We report the results of solid state nuclear magnetic resonance (NMR) measurements on amyloid fibrils formed by the full-length prion protein PrP (residues 23−231, Syrian hamster sequence). Measurements of intermolecular 13C−13C dipole−dipole couplings in selectively carbonyl-labeled samples indicate that β-sheets in these fibrils have an in-register parallel structure, as previously observed in amyloid fibrils associated with Alzheimer’s disease and type 2 diabetes and in yeast prion fibrils. Two-dimensional 13C−13C and 15N−13C solid state NMR spectra of a uniformly 15N- and 13C-labeled sample indicate that a relatively small fraction of the full sequence, localized to the C-terminal end, forms the structurally ordered, immobilized core. Although unique site-specific assignments of the solid state NMR signals cannot be obtained from these spectra, analysis with a Monte Carlo/simulated annealing algorithm suggests that the core is comprised primarily of residues in the 173−224 range. These results are consistent with earlier electron paramagnetic resonance studies of fibrils formed by residues 90−231 of the human PrP sequence, formed under somewhat different conditions [Cobb, N. J., Sonnichsen, F. D., McHaourab, H., and Surewicz, W. K. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 18946−18951], suggesting that an in-register parallel β-sheet structure formed by the C-terminal end may be a general feature of PrP fibrils prepared in vitro.

  19. Genetic recombination within the human T-cell receptor α-chain gene complex

    International Nuclear Information System (INIS)

    Robinson, M.A.; Kindt, T.J.

    1987-01-01

    Genetic analyses of the human T-cell receptor (TCR) α-chain genes indicate that recombination events may occur frequently within this gene complex. Examination of the inheritance of restriction fragment length polymorphisms (RFLP) detected by using probes for constant or variable region gene segments made it possible to assign TCRα haplotypes to the 16 parents and 43 offspring of eight families studied. A total of six RFLP, three for the constant region and three for variable region segments, were examined in the present studies. Most enzyme and probe combinations tested revealed no polymorphism and those finally selected for the study showed limited polymorphism in that only two or, in one case, three allelic forms of the gene were seen. In spite of limited variability at this level, extensive heterogeneity was observed for the combinations of markers present in haplotypes, suggesting that frequent recombination events have occurred. Most strikingly, multiple combinations of RFLP occurring in close proximity of the TCRα constant region gene were observed in this study. A high recombination frequency for the TCRα gene complex is further supported by the observation that two children, one in each of two families, inherited recombinant TCRα haplotypes

  20. The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis.

    Science.gov (United States)

    Aoubala, M; Holt, J; Clegg, R A; Rowlands, D J; Harris, M

    2001-07-01

    Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system--under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.

  1. Effect of Full-Length Carbon Fiber Insoles on Lower Limb Kinetics in Patients With Midfoot Osteoarthritis: A Pilot Study.

    Science.gov (United States)

    Yi, Taeim; Kim, Jung Hyun; Oh-Park, Mooyeon; Hwang, Ji Hye

    2018-03-01

    We investigated the effects of full-length carbon fiber (FCF) insoles on gait, muscle activity, kinetics, and pain in patients with midfoot osteoarthritis (OA). We enrolled 13 patients with unilateral midfoot OA (mild: Visual Analog Scale [VAS] range, 1-3; moderate, VAS range, 4-7) and healthy controls. All participants were asked to walk under two conditions: with and without FCF insole. The outcome measures were ground reaction force, quantitative gait parameters, electromyography activities and pain severity (VAS). In the patients with moderate midfoot OA, significantly longer gait cycle and higher muscle activity of lower limb during loading-response phase were observed while walking without FCF insoles. In the mild midfoot OA group, there was no significant difference in VAS score (without, 2.0 ± 1.0 vs. with, 2.0 ± 0.5) with FCF insole use. However, significantly reduced VAS score (without, 5.5 ± 1.4 vs. with, 2.0 ± 0.5) and muscle activity of the tibialis anterior and increased muscle activity of gastrocnemius were observed in the moderate midfoot OA group by using an FCF insole (P < 0.05). Full-length carbon fiber insoles can improve pain in individuals with moderate midfoot OA, which might be associated with changes in the kinetics and muscle activities of the lower limb. Taken together, the results of the present study suggest that FCF insoles may be used as a helpful option for midfoot OA.

  2. Potential role of a new PEGylated recombinant factor VIII for hemophilia A

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    Wynn TT

    2016-06-01

    Full Text Available Tung Thanh Wynn,1 Burak Gumuscu,2,3 1Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of Florida, Gainesville, FL, 2Pediatric Hematology-Oncology, Bon Secours Health System, St. Mary’s Hospital, Richmond, VA, 3Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of Virginia, Charlottesville, VA, USA Abstract: Hemophilia A, a deficiency in the activity of coagulation factor (F VIII, is an X-linked bleeding disorder with an approximate incidence of one in 5,000 male infants. Bleeding-related complications often result in greater severity of disease, poor quality of life, surgical interventions for severe joint destruction, and shortened life span. With the availability of plasma-derived and recombinant FVIII products, the benefits of primary prophylaxis were demonstrated and is now the standard of care for patients with severe factor deficiencies. Current hemophilia research is focusing on the creation of new factor replacement therapies with longer half-lives; accessing alternative mechanisms to achieve desired hemostasis and enhance bypassing ­activity; and limiting the immunogenicity of the protein. PEGylation involves the covalent attachment of polyethylene glycol (PEG to a protein, peptide, or a small molecule drug. PEG effectively increases the molecular weight and size of the protein by creating a hydrophilic cloud around the molecule. This molecular change may reduce susceptibility of the molecule to proteolytic activity and degradation. It is also believed that PEGylation changes the surface charge of the protein that ultimately interferes with some receptor-mediated clearance processes. The half-life of PEGylated factor is more prolonged when compared to non-PEGylated full-length recombinant FVIII. The dawn of a new era in the care of hemophilia patients is upon us with the release of recombinant FVIII products with extended half-lives, and products with even more extended half

  3. Reduced genetic distance and high replication levels increase the RNA recombination rate of hepatitis delta virus.

    Science.gov (United States)

    Lin, Chia-Chi; Yang, Zhi-Wei; Iang, Shan-Bei; Chao, Mei

    2015-01-02

    Hepatitis delta virus (HDV) replication is carried out by host RNA polymerases. Since homologous inter-genotypic RNA recombination is known to occur in HDV, possibly via a replication-dependent process, we hypothesized that the degree of sequence homology and the replication level should be related to the recombination frequency in cells co-expressing two HDV sequences. To confirm this, we separately co-transfected cells with three different pairs of HDV genomic RNAs and analyzed the obtained recombinants by RT-PCR followed by restriction fragment length polymorphism and sequencing analyses. The sequence divergence between the clones ranged from 24% to less than 0.1%, and the difference in replication levels was as high as 100-fold. As expected, significant differences were observed in the recombination frequencies, which ranged from 0.5% to 47.5%. Furthermore, varying the relative amounts of parental RNA altered the dominant recombinant species produced, suggesting that template switching occurs frequently during the synthesis of genomic HDV RNA. Taken together, these data suggest that during the host RNA polymerase-driven RNA recombination of HDV, both inter- and intra-genotypic recombination events are important in shaping the genetic diversity of HDV. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Fine Mapping of QUICK ROOTING 1 and 2, Quantitative Trait Loci Increasing Root Length in Rice

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    Yuka Kitomi

    2018-02-01

    Full Text Available The volume that the root system can occupy is associated with the efficiency of water and nutrient uptake from soil. Genetic improvement of root length, which is a limiting factor for root distribution, is necessary for increasing crop production. In this report, we describe identification of two quantitative trait loci (QTLs for maximal root length, QUICK ROOTING 1 (QRO1 on chromosome 2 and QRO2 on chromosome 6, in cultivated rice (Oryza sativa L.. We measured the maximal root length in 26 lines carrying chromosome segments from the long-rooted upland rice cultivar Kinandang Patong in the genetic background of the short-rooted lowland cultivar IR64. Five lines had longer roots than IR64. By rough mapping of the target regions in BC4F2 populations, we detected putative QTLs for maximal root length on chromosomes 2, 6, and 8. To fine-map these QTLs, we used BC4F3 recombinant homozygous lines. QRO1 was mapped between markers RM5651 and RM6107, which delimit a 1.7-Mb interval on chromosome 2, and QRO2 was mapped between markers RM20495 and RM3430-1, which delimit an 884-kb interval on chromosome 6. Both QTLs may be promising gene resources for improving root system architecture in rice.

  5. Identification of Novel Recombinant Forms of Hepatitis B Virus Generated from Genotypes Ae and G in HIV-1-Positive Japanese Men Who Have Sex with Men.

    Science.gov (United States)

    Kojima, Yoko; Kawahata, Takuya; Mori, Haruyo; Furubayashi, Keiichi; Taniguchi, Yasushi; Itoda, Ichiro; Komano, Jun

    2015-07-01

    The rare hepatitis B virus (HBV) genotype G (HBV/G) coinfects HIV-1-positive individuals along with HBV/A and generates recombinants. However, the circulation of HBV A/G recombinants remains poorly understood. This molecular epidemiologic study examined HBV A/G recombinants in Japanese HIV-1-positive men who have sex with men (MSM). Initially, blood specimens submitted for confirmatory tests of HIV infection in Osaka and Tokyo, Japan, from 2006 to 2013 were examined for HIV-1, and HIV-1-positive specimens were screened for HBV. Among 817 specimens from HIV-1-positive individuals, HBsAg was detected in 59 specimens; of these, HBV/Ae (alternatively A2), a subgenotype of HBV/A prevalent in Europe and North America, was identified in 70.2%, HBV/C in 17.5%, and HBV/G in 10.5%, and HBV/E in 1.8% according to the core gene sequence. The full-length genome analysis of HBV was performed on HBV/G-positive specimens because some HBV A/G recombinants were historically overlooked by genotyping based on a partial genome analysis. It revealed that five of the specimens contained novel Ae/G recombinants, the core gene of which had a high sequence similarity to HBV/G. Detailed analyses showed that novel recombinants were coinfected with HBV/Ae in a recombinant-dominant fashion. No major drug-resistant mutations were found in the newly identified HBV Ae/G recombinants. Some of the individuals asymptomatically coinfected with HIV/HBV suffered mild liver injury. This study demonstrated that novel Ae/G HBV recombinants were identified in Japanese HIV-1-positive MSM. The pathogenicity of novel HBV Ae/G recombinants should be examined in a future longitudinal study. Surveillance of such viruses in HIV-1-positive individuals should be emphasized.

  6. Expression and purification of recombinant hemoglobin in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Chandrasekhar Natarajan

    Full Text Available Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species.As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus, a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications.Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis.

  7. The Effect of Casting Ring Liner Length and Prewetting on the Marginal Adaptation and Dimensional Accuracy of Full Crown Castings.

    Science.gov (United States)

    Haralur, Satheesh B; Hamdi, Osama A; Al-Shahrani, Abdulaziz A; Alhasaniah, Sultan

    2017-01-01

    To evaluate the effect of varying cellulose casting ring liner length and its prewetting on the marginal adaptation and dimensional accuracy of full veneer metal castings. The master die was milled in stainless steel to fabricate the wax pattern. Sixty wax patterns were fabricated with a uniform thickness of 1.5 mm at an occlusal surface and 1 mm axial surface, cervical width at 13.5 mm, and 10 mm cuspal height. The samples were divided into six groups ( n = 10). Groups I and II samples had the full-length cellulose prewet and dry ring liner, respectively. The groups III and IV had 2 mm short prewet and dry cellulose ring liner, respectively, whereas groups V and VI were invested in 6 mm short ring liner. The wax patterns were immediately invested in phosphate bonded investment, and casting procedure was completed with nickel-chrome alloy. The castings were cleaned and mean score of measurements at four reference points for marginal adaption, casting height, and cervical width was calculated. The marginal adaption was calculated with Imaje J software, whereas the casting height and cervical width was determined using a digital scale. The data was subjected to one-way analysis of varaince and Tukey post hoc statistical analysis with Statistical Package for the Social Sciences version 20 software. The group II had the best marginal adaption with a gap of 63.786 μm followed by group I (65.185 μm), group IV (87.740 μm), and group III (101.455 μm). A large marginal gap was observed in group V at 188.871 μm. Cuspal height was more accurate with group V (10.428 mm), group VI (10.421 mm), and group II (10.488 mm). The cervical width was approximately similar in group I, group III, and group V. Statistically significant difference was observed in Tukey post hoc analysis between group V and group VI with all the other groups with regards to marginal adaptation. The dry cellulose ring liners provided better marginal adaptation in comparison to prewet cellulose ring

  8. Tula hantavirus isolate with the full-length ORF for nonstructural protein NSs survives for more consequent passages in interferon-competent cells than the isolate having truncated NSs ORF.

    Science.gov (United States)

    Jääskeläinen, Kirsi M; Plyusnina, Angelina; Lundkvist, Ake; Vaheri, Antti; Plyusnin, Alexander

    2008-01-11

    The competitiveness of two Tula hantavirus (TULV) isolates, TULV/Lodz and TULV/Moravia, was evaluated in interferon (IFN) -competent and IFN-deficient cells. The two isolates differ in the length of the open reading frame (ORF) encoding the nonstructural protein NSs, which has previously been shown to inhibit IFN response in infected cells. In IFN-deficient Vero E6 cells both TULV isolates survived equally well. In contrast, in IFN-competent MRC5 cells TULV/Lodz isolate, that possesses the NSs ORF for the full-length protein of 90 aa, survived for more consequent passages than TULV/Moravia isolate, which contains the ORF for truncated NSs protein (66-67 aa). Our data show that expression of a full-length NSs protein is beneficial for the virus survival and competitiveness in IFN-competent cells and not essential in IFN-deficient cells. These results suggest that the N-terminal aa residues are important for the full activity of the NSs protein.

  9. Full-Length Characterization of Hepatitis C Virus Subtype 3a Reveals Novel Hypervariable Regions under Positive Selection during Acute Infection▿

    OpenAIRE

    Humphreys, Isla; Fleming, Vicki; Fabris, Paolo; Parker, Joe; Schulenberg, Bodo; Brown, Anthony; Demetriou, Charis; Gaudieri, Silvana; Pfafferott, Katja; Lucas, Michaela; Collier, Jane; Huang, Kuan-Hsiang Gary; Pybus, Oliver G.; Klenerman, Paul; Barnes, Eleanor

    2009-01-01

    Hepatitis C virus subtype 3a is a highly prevalent and globally distributed strain that is often associated with infection via injection drug use. This subtype exhibits particular phenotypic characteristics. In spite of this, detailed genetic analysis of this subtype has rarely been performed. We performed full-length viral sequence analysis in 18 patients with chronic HCV subtype 3a infection and assessed genomic viral variability in comparison to other HCV subtypes. Two novel regions of int...

  10. Construction and evaluation of normalized cDNA libraries enriched with full-length sequences for rapid discovery of new genes from Sisal (Agave sisalana Perr.) different developmental stages.

    Science.gov (United States)

    Zhou, Wen-Zhao; Zhang, Yan-Mei; Lu, Jun-Ying; Li, Jun-Feng

    2012-10-12

    To provide a resource of sisal-specific expressed sequence data and facilitate this powerful approach in new gene research, the preparation of normalized cDNA libraries enriched with full-length sequences is necessary. Four libraries were produced with RNA pooled from Agave sisalana multiple tissues to increase efficiency of normalization and maximize the number of independent genes by SMART™ method and the duplex-specific nuclease (DSN). This procedure kept the proportion of full-length cDNAs in the subtracted/normalized libraries and dramatically enhanced the discovery of new genes. Sequencing of 3875 cDNA clones of libraries revealed 3320 unigenes with an average insert length about 1.2 kb, indicating that the non-redundancy of libraries was about 85.7%. These unigene functions were predicted by comparing their sequences to functional domain databases and extensively annotated with Gene Ontology (GO) terms. Comparative analysis of sisal unigenes and other plant genomes revealed that four putative MADS-box genes and knotted-like homeobox (knox) gene were obtained from a total of 1162 full-length transcripts. Furthermore, real-time PCR showed that the characteristics of their transcripts mainly depended on the tight expression regulation of a number of genes during the leaf and flower development. Analysis of individual library sequence data indicated that the pooled-tissue approach was highly effective in discovering new genes and preparing libraries for efficient deep sequencing.

  11. The full-length E1-circumflexE4 protein of human papillomavirus type 18 modulates differentiation-dependent viral DNA amplification and late gene expression

    International Nuclear Information System (INIS)

    Wilson, Regina; Ryan, Gordon B.; Knight, Gillian L.; Laimins, Laimonis A.; Roberts, Sally

    2007-01-01

    Activation of the productive phase of the human papillomavirus (HPV) life cycle in differentiated keratinocytes is coincident with high-level expression of E1-circumflexE4 protein. To determine the role of E1-circumflexE4 in the HPV replication cycle, we constructed HPV18 mutant genomes in which expression of the full-length E1-circumflexE4 protein was abrogated. Undifferentiated keratinocytes containing mutant genomes showed enhanced proliferation when compared to cells containing wildtype genomes, but there were no differences in maintenance of viral episomes. Following differentiation, cells with mutant genomes exhibited reduced levels of viral DNA amplification and late gene expression, compared to wildtype genome-containing cells. This indicates that HPV18 E1-circumflexE4 plays an important role in regulating HPV late functions, and it may also function in the early phase of the replication cycle. Our finding that full-length HPV18 E1-circumflexE4 protein plays a significant role in promoting viral genome amplification concurs with a similar report with HPV31, but is in contrast to an HPV11 study where viral DNA amplification was not dependent on full-length E1-circumflexE4 expression, and to HPV16 where only C-terminal truncations in E1-circumflexE4 abrogated vegetative genome replication. This suggests that type-specific differences exist between various E1-circumflexE4 proteins

  12. The evolutionary value of recombination is constrained by genome modularity.

    Directory of Open Access Journals (Sweden)

    Darren P Martin

    2005-10-01

    Full Text Available Genetic recombination is a fundamental evolutionary mechanism promoting biological adaptation. Using engineered recombinants of the small single-stranded DNA plant virus, Maize streak virus (MSV, we experimentally demonstrate that fragments of genetic material only function optimally if they reside within genomes similar to those in which they evolved. The degree of similarity necessary for optimal functionality is correlated with the complexity of intragenomic interaction networks within which genome fragments must function. There is a striking correlation between our experimental results and the types of MSV recombinants that are detectable in nature, indicating that obligatory maintenance of intragenome interaction networks strongly constrains the evolutionary value of recombination for this virus and probably for genomes in general.

  13. Production of a full-length infectious GFP-tagged cDNA clone of Beet mild yellowing virus for the study of plant-polerovirus interactions.

    Science.gov (United States)

    Stevens, Mark; Viganó, Felicita

    2007-04-01

    The full-length cDNA of Beet mild yellowing virus (Broom's Barn isolate) was sequenced and cloned into the vector pLitmus 29 (pBMYV-BBfl). The sequence of BMYV-BBfl (5721 bases) shared 96% and 98% nucleotide identity with the other complete sequences of BMYV (BMYV-2ITB, France and BMYV-IPP, Germany respectively). Full-length capped RNA transcripts of pBMYV-BBfl were synthesised and found to be biologically active in Arabidopsis thaliana protoplasts following electroporation or PEG inoculation when the protoplasts were subsequently analysed using serological and molecular methods. The BMYV sequence was modified by inserting DNA that encoded the jellyfish green fluorescent protein (GFP) into the P5 gene close to its 3' end. A. thaliana protoplasts electroporated with these RNA transcripts were biologically active and up to 2% of transfected protoplasts showed GFP-specific fluorescence. The exploitation of these cDNA clones for the study of the biology of beet poleroviruses is discussed.

  14. Phylogenetic and recombination analysis of tomato spotted wilt virus.

    Directory of Open Access Journals (Sweden)

    Sen Lian

    Full Text Available Tomato spotted wilt virus (TSWV severely damages and reduces the yield of many economically important plants worldwide. In this study, we determined the whole-genome sequences of 10 TSWV isolates recently identified from various regions and hosts in Korea. Phylogenetic analysis of these 10 isolates as well as the three previously sequenced isolates indicated that the 13 Korean TSWV isolates could be divided into two groups reflecting either two different origins or divergences of Korean TSWV isolates. In addition, the complete nucleotide sequences for the 13 Korean TSWV isolates along with previously sequenced TSWV RNA segments from Korea and other countries were subjected to phylogenetic and recombination analysis. The phylogenetic analysis indicated that both the RNA L and RNA M segments of most Korean isolates might have originated in Western Europe and North America but that the RNA S segments for all Korean isolates might have originated in China and Japan. Recombination analysis identified a total of 12 recombination events among all isolates and segments and five recombination events among the 13 Korea isolates; among the five recombinants from Korea, three contained the whole RNA L segment, suggesting reassortment rather than recombination. Our analyses provide evidence that both recombination and reassortment have contributed to the molecular diversity of TSWV.

  15. Homologous Recombination as a Replication Fork Escort: Fork-Protection and Recovery

    Directory of Open Access Journals (Sweden)

    Audrey Costes

    2012-12-01

    Full Text Available Homologous recombination is a universal mechanism that allows DNA repair and ensures the efficiency of DNA replication. The substrate initiating the process of homologous recombination is a single-stranded DNA that promotes a strand exchange reaction resulting in a genetic exchange that promotes genetic diversity and DNA repair. The molecular mechanisms by which homologous recombination repairs a double-strand break have been extensively studied and are now well characterized. However, the mechanisms by which homologous recombination contribute to DNA replication in eukaryotes remains poorly understood. Studies in bacteria have identified multiple roles for the machinery of homologous recombination at replication forks. Here, we review our understanding of the molecular pathways involving the homologous recombination machinery to support the robustness of DNA replication. In addition to its role in fork-recovery and in rebuilding a functional replication fork apparatus, homologous recombination may also act as a fork-protection mechanism. We discuss that some of the fork-escort functions of homologous recombination might be achieved by loading of the recombination machinery at inactivated forks without a need for a strand exchange step; as well as the consequence of such a model for the stability of eukaryotic genomes.

  16. Fine-Scale Recombination Maps of Fungal Plant Pathogens Reveal Dynamic Recombination Landscapes and Intragenic Hotspots.

    Science.gov (United States)

    Stukenbrock, Eva H; Dutheil, Julien Y

    2018-03-01

    Meiotic recombination is an important driver of evolution. Variability in the intensity of recombination across chromosomes can affect sequence composition, nucleotide variation, and rates of adaptation. In many organisms, recombination events are concentrated within short segments termed recombination hotspots. The variation in recombination rate and positions of recombination hotspot can be studied using population genomics data and statistical methods. In this study, we conducted population genomics analyses to address the evolution of recombination in two closely related fungal plant pathogens: the prominent wheat pathogen Zymoseptoria tritici and a sister species infecting wild grasses Z. ardabiliae We specifically addressed whether recombination landscapes, including hotspot positions, are conserved in the two recently diverged species and if recombination contributes to rapid evolution of pathogenicity traits. We conducted a detailed simulation analysis to assess the performance of methods of recombination rate estimation based on patterns of linkage disequilibrium, in particular in the context of high nucleotide diversity. Our analyses reveal overall high recombination rates, a lack of suppressed recombination in centromeres, and significantly lower recombination rates on chromosomes that are known to be accessory. The comparison of the recombination landscapes of the two species reveals a strong correlation of recombination rate at the megabase scale, but little correlation at smaller scales. The recombination landscapes in both pathogen species are dominated by frequent recombination hotspots across the genome including coding regions, suggesting a strong impact of recombination on gene evolution. A significant but small fraction of these hotspots colocalize between the two species, suggesting that hotspot dynamics contribute to the overall pattern of fast evolving recombination in these species. Copyright © 2018 Stukenbrock and Dutheil.

  17. Assessing the genetic diversity of Cu resistance in mine tailings through high-throughput recovery of full-length copA genes

    Science.gov (United States)

    Li, Xiaofang; Zhu, Yong-Guan; Shaban, Babak; Bruxner, Timothy J. C.; Bond, Philip L.; Huang, Longbin

    2015-01-01

    Characterizing the genetic diversity of microbial copper (Cu) resistance at the community level remains challenging, mainly due to the polymorphism of the core functional gene copA. In this study, a local BLASTN method using a copA database built in this study was developed to recover full-length putative copA sequences from an assembled tailings metagenome; these sequences were then screened for potentially functioning CopA using conserved metal-binding motifs, inferred by evolutionary trace analysis of CopA sequences from known Cu resistant microorganisms. In total, 99 putative copA sequences were recovered from the tailings metagenome, out of which 70 were found with high potential to be functioning in Cu resistance. Phylogenetic analysis of selected copA sequences detected in the tailings metagenome showed that topology of the copA phylogeny is largely congruent with that of the 16S-based phylogeny of the tailings microbial community obtained in our previous study, indicating that the development of copA diversity in the tailings might be mainly through vertical descent with few lateral gene transfer events. The method established here can be used to explore copA (and potentially other metal resistance genes) diversity in any metagenome and has the potential to exhaust the full-length gene sequences for downstream analyses. PMID:26286020

  18. Natural type 3/type 2 intertypic vaccine-related poliovirus recombinants with the first crossover sites within the VP1 capsid coding region.

    Directory of Open Access Journals (Sweden)

    Yong Zhang

    Full Text Available BACKGROUND: Ten uncommon natural type 3/type 2 intertypic poliovirus recombinants were isolated from stool specimens from nine acute flaccid paralysis case patients and one healthy vaccinee in China from 2001 to 2008. PRINCIPAL FINDINGS: Complete genomic sequences revealed their vaccine-related genomic features and showed that their first crossover sites were randomly distributed in the 3' end of the VP1 coding region. The length of donor Sabin 2 sequences ranged from 55 to 136 nucleotides, which is the longest donor sequence reported in the literature for this type of poliovirus recombination. The recombination resulted in the introduction of Sabin 2 neutralizing antigenic site 3a (NAg3a into a Sabin 3 genomic background in the VP1 coding region, which may have been altered by some of the type 3-specific antigenic properties, but had not acquired any type 2-specific characterizations. NAg3a of the Sabin 3 strain seems atypical; other wild-type poliovirus isolates that have circulated in recent years have sequences of NAg3a more like the Sabin 2 strain. CONCLUSIONS: 10 natural type 3/type 2 intertypic VP1 capsid-recombinant polioviruses, in which the first crossover sites were found to be in the VP1 coding region, were isolated and characterized. In spite of the complete replacement of NAg3a by type 2-specific amino acids, the serotypes of the recombinants were not altered, and they were totally neutralized by polyclonal type 3 antisera but not at all by type 2 antisera. It is possible that recent type 3 wild poliovirus isolates may be a recombinant having NAg3a sequences derived from another strain during between 1967 and 1980, and the type 3/type 2 recombination events in the 3' end of the VP1 coding region may result in a higher fitness.

  19. The influence of recombination on human genetic diversity.

    Directory of Open Access Journals (Sweden)

    Chris C A Spencer

    2006-09-01

    Full Text Available In humans, the rate of recombination, as measured on the megabase scale, is positively associated with the level of genetic variation, as measured at the genic scale. Despite considerable debate, it is not clear whether these factors are causally linked or, if they are, whether this is driven by the repeated action of adaptive evolution or molecular processes such as double-strand break formation and mismatch repair. We introduce three innovations to the analysis of recombination and diversity: fine-scale genetic maps estimated from genotype experiments that identify recombination hotspots at the kilobase scale, analysis of an entire human chromosome, and the use of wavelet techniques to identify correlations acting at different scales. We show that recombination influences genetic diversity only at the level of recombination hotspots. Hotspots are also associated with local increases in GC content and the relative frequency of GC-increasing mutations but have no effect on substitution rates. Broad-scale association between recombination and diversity is explained through covariance of both factors with base composition. To our knowledge, these results are the first evidence of a direct and local influence of recombination hotspots on genetic variation and the fate of individual mutations. However, that hotspots have no influence on substitution rates suggests that they are too ephemeral on an evolutionary time scale to have a strong influence on broader scale patterns of base composition and long-term molecular evolution.

  20. Virulence of a chimeric recombinant infectious haematopoietic necrosis virus expressing the spring viraemia of carp virus glycoprotein in salmonid and cyprinid fish

    Science.gov (United States)

    Emmenegger, Eveline; Biacchesi, Stéphane; Mérour, Emilie; Glenn, Jolene. A; Palmer, Alexander D.; Brémont, Michel; Kurath, Gael

    2018-01-01

    Infectious haematopoietic necrosis virus (IHNV) and spring viraemia of carp virus (SVCV) are both rhabdoviruses of fish, listed as notifiable disease agents by the World Organization for Animal Health. Recombinant rhabdoviruses with heterologous gene substitutions have been engineered to study genetic determinants and assess the potential of these recombinant viruses for vaccine development. A recombinant IHNV (rIHNV), containing the full-length genome of a European IHNV strain, was modified by deleting the glycoprotein (G) gene and replacing it with a European SVCV G-gene to make the rIHNV-Gsvcv. The chimeric rIHNV-Gsvcv level of virulence in rainbow trout, common carp and koi was assessed, and its ability to induce a protective immune response in surviving koi against wild-type SVCV infection was tested. The rIHNV-Gsvcv infection of trout led to high mortality, ranging from 78% to 92.5%, after immersion. In contrast, no deaths occurred in juvenile common carp after infection with rIHNV-Gsvcv by either immersion or intraperitoneal (IP) injection. Similarly, koi infected with rIHNV-Gsvcv via IP injection had little to no mortality (≤9%). Koi that survived initial infection with a high dose of recombinant virus rIHNV-Gsvcv were protected against a virulent SVCV challenge resulting in a high relative per cent survival of 82.5%.

  1. Theoretical simulation of soft x-rays for recombining pump

    International Nuclear Information System (INIS)

    Peng Huimin; Zhang Guoping; Sheng Jiatian

    1990-05-01

    The theoretical study and computational simulation of soft X-ray laser produced by the recombination of highly ionized plasma are given. An one-dimensional non LTE radiative hydrodynamic code JB-19 is used for simulating the process of soft X-ray laser produced by the recombination. The incident laser light is focused linearly onto the thin carbon fibre. In the duration of incident laser pulse a highly ionized plasma is generated. After the incident laser has been ended the plasma adiabatically expands and rapidly cools down. During the time of three-body recombination and cascading transition, the population inversion between n = 3 and n = 2 is produced and transition gain is obtained. The analysis and evolution is presented, and factors effected on the gain are also discussed. The calculated results have been compared with the experimental data of RAL. It is found that some were in good agreement with them but some are not. Under the limitation of laser energy, the gain is inversely proportional to the wave-length and pulse width of incident laser. For obtaining high gain it is necessary to have double frequency and to shorten the pulse width of Nd-glass laser. Finally the preliminary results about H-like F ion are also given

  2. Effect of metal ions on de novo aggregation of full-length prion protein

    International Nuclear Information System (INIS)

    Giese, Armin; Levin, Johannes; Bertsch, Uwe; Kretzschmar, Hans

    2004-01-01

    It is well established that the prion protein (PrP) contains metal ion binding sites with specificity for copper. Changes in copper levels have been suggested to influence incubation time in experimental prion disease. Therefore, we studied the effect of heavy metal ions (Cu 2+ , Mn 2+ , Ni 2+ , Co 2+ , and Zn 2+ ) in vitro in a model system that utilizes changes in the concentration of SDS to induce structural conversion and aggregation of recombinant PrP. To quantify and characterize PrP aggregates, we used fluorescently labelled PrP and cross-correlation analysis as well as scanning for intensely fluorescent targets in a confocal single molecule detection system. We found a specific strong pro-aggregatory effect of Mn 2+ at low micromolar concentrations that could be blocked by nanomolar concentration of Cu 2+ . These findings suggest that metal ions such as copper and manganese may also affect PrP conversion in vivo

  3. Pre-Steady-State Kinetic Analysis of Truncated and Full-Length Saccharomyces cerevisiae DNA Polymerase Eta

    Science.gov (United States)

    Brown, Jessica A.; Zhang, Likui; Sherrer, Shanen M.; Taylor, John-Stephen; Burgers, Peter M. J.; Suo, Zucai

    2010-01-01

    Understanding polymerase fidelity is an important objective towards ascertaining the overall stability of an organism's genome. Saccharomyces cerevisiae DNA polymerase η (yPolη), a Y-family DNA polymerase, is known to efficiently bypass DNA lesions (e.g., pyrimidine dimers) in vivo. Using pre-steady-state kinetic methods, we examined both full-length and a truncated version of yPolη which contains only the polymerase domain. In the absence of yPolη's C-terminal residues 514–632, the DNA binding affinity was weakened by 2-fold and the base substitution fidelity dropped by 3-fold. Thus, the C-terminus of yPolη may interact with DNA and slightly alter the conformation of the polymerase domain during catalysis. In general, yPolη discriminated between a correct and incorrect nucleotide more during the incorporation step (50-fold on average) than the ground-state binding step (18-fold on average). Blunt-end additions of dATP or pyrene nucleotide 5′-triphosphate revealed the importance of base stacking during the binding of incorrect incoming nucleotides. PMID:20798853

  4. Recombination facilitates neofunctionalization of duplicate genes via originalization

    Directory of Open Access Journals (Sweden)

    Huang Ren

    2010-06-01

    Full Text Available Abstract Background Recently originalization was proposed to be an effective way of duplicate-gene preservation, in which recombination provokes the high frequency of original (or wild-type allele on both duplicated loci. Because the high frequency of wild-type allele might drive the arising and accumulating of advantageous mutation, it is hypothesized that recombination might enlarge the probability of neofunctionalization (Pneo of duplicate genes. In this article this hypothesis has been tested theoretically. Results Results show that through originalization recombination might not only shorten mean time to neofunctionalizaiton, but also enlarge Pneo. Conclusions Therefore, recombination might facilitate neofunctionalization via originalization. Several extensive applications of these results on genomic evolution have been discussed: 1. Time to nonfunctionalization can be much longer than a few million generations expected before; 2. Homogenization on duplicated loci results from not only gene conversion, but also originalization; 3. Although the rate of advantageous mutation is much small compared with that of degenerative mutation, Pneo cannot be expected to be small.

  5. The telomere length dynamic and methods of its assessment.

    Science.gov (United States)

    Lin, Kah-Wai; Yan, Ju

    2005-01-01

    Human telomeres are composed of long repeating sequences of TTAGGG, associated with a variety of telomere-binding proteins. Its function as an end-protector of chromosomes prevents the chromosome from end-to-end fusion, recombination and degradation. Telomerase acts as reverse transcriptase in the elongation of telomeres, which prevent the loss of telomeres due to the end replication problems. However, telomerase activity is detected at low level in somatic cells and high level in embryonic stem cells and tumor cells. It confers immortality to embryonic stem cells and tumor cells. In most tumor cells, telomeres are extremely short and stable. Telomere length is an important indicator of the telomerase activity in tumor cells and it may be used in the prognosis of malignancy. Thus, the assessment of telomeres length is of great experimental and clinical significance. This review describes the role of telomere and telomerase in cancer pathogenesis and the dynamics of the telomeres length in different cell types. The various methods of measurement of telomeres length, i.e. southern blot, hybridization protection assay, fluorescence in situ hybridization, primed in situ, quantitative PCR and single telomere length analysis are discussed. The principle and comparative evaluation of these methods are reviewed. The detection of G-strand overhang by telomeric-oligonucleotide ligation assay, primer extension/nick translation assay and electron microscopy are briefly discussed.

  6. Full protection in mink against mink enteritis virus with new generation canine parvovirus vaccines based on synthetic peptide or recombinant protein

    DEFF Research Database (Denmark)

    Langeveld, J. P.; Kamstrup, Søren; Uttenthal, Åse

    1995-01-01

    Two recently developed vaccines—one based on synthetic peptide and one based on recombinant capsid protein—fully protected dogs against heavy experimental canine parvovirus (CPV) infection. The high sequence homology (>98%) and antigenic similarity between CPV and mink enteritis virus (MEV), feline...... on inactivated virus. Surprisingly, this protection was obtained after only a single injection. Furthermore, the vaccinal dose of 150 μg of conjugated peptide or 3 μg of recombinant VP2 particles per animal, are sufficiently low to be cost-effective and applicable on a large scale....

  7. Expression of the amino-terminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein

    International Nuclear Information System (INIS)

    Funk, W.D.; MacGillivray, R.T.A.; Mason, A.B.; Brown, S.A.; Woodworth, R.C.

    1990-01-01

    A human liver cDNA library was screened with a synthetic oligonucleotide, complementary to the 5' region of human transferrin mRNA, as a hybridization probe. The full-length human cDNA clone isolated from this screen contained part of the 5' untranslated region, the complete coding region for the signal peptide and the two lobes of transferrin, the 3' untranslated region, and a poly(A) tail. By use of oligonucleotide-directed mutagenesis in vitro, two translational stop codons and a HindIII site were introduced after the codon for Asp-337. This fragment was inserted into two different expression vectors that were then introduced into Escherichia coli. As judged by NaDodSO 4 -polyacrylamide gel electrophoresis and Western blot analysis, however, recombinant hTF/2N was undetectable in bacteria transformed by these plasmids. Concurrently, the authors developed a plasmid vector for the expression of recombinant hTF/2N in eukaryotic cells. The recombinant hTF/2N appeared to behave identically with the proteolytically derived half-molecule, but to show a higher degree of monodispersity than the latter protein. Addition of m-fluorotyrosine to the culture medium resulted in random incorporation of this amino acid into cellular protein in lieu of tyrosine. Purified recombinant 19 F-Tyr hTF/2N gave four well-resolved 19 F NMR resonances of 20-40 Hz line width, two with suggestions of shoulders

  8. Construction of phosphorylation interaction networks by text mining of full-length articles using the eFIP system.

    Science.gov (United States)

    Tudor, Catalina O; Ross, Karen E; Li, Gang; Vijay-Shanker, K; Wu, Cathy H; Arighi, Cecilia N

    2015-01-01

    Protein phosphorylation is a reversible post-translational modification where a protein kinase adds a phosphate group to a protein, potentially regulating its function, localization and/or activity. Phosphorylation can affect protein-protein interactions (PPIs), abolishing interaction with previous binding partners or enabling new interactions. Extracting phosphorylation information coupled with PPI information from the scientific literature will facilitate the creation of phosphorylation interaction networks of kinases, substrates and interacting partners, toward knowledge discovery of functional outcomes of protein phosphorylation. Increasingly, PPI databases are interested in capturing the phosphorylation state of interacting partners. We have previously developed the eFIP (Extracting Functional Impact of Phosphorylation) text mining system, which identifies phosphorylated proteins and phosphorylation-dependent PPIs. In this work, we present several enhancements for the eFIP system: (i) text mining for full-length articles from the PubMed Central open-access collection; (ii) the integration of the RLIMS-P 2.0 system for the extraction of phosphorylation events with kinase, substrate and site information; (iii) the extension of the PPI module with new trigger words/phrases describing interactions and (iv) the addition of the iSimp tool for sentence simplification to aid in the matching of syntactic patterns. We enhance the website functionality to: (i) support searches based on protein roles (kinases, substrates, interacting partners) or using keywords; (ii) link protein entities to their corresponding UniProt identifiers if mapped and (iii) support visual exploration of phosphorylation interaction networks using Cytoscape. The evaluation of eFIP on full-length articles achieved 92.4% precision, 76.5% recall and 83.7% F-measure on 100 article sections. To demonstrate eFIP for knowledge extraction and discovery, we constructed phosphorylation-dependent interaction

  9. Recombinant human erythropoietin in sports: a review

    Directory of Open Access Journals (Sweden)

    Rafael Maia de Almeida Bento

    2003-06-01

    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.

  10. Production and recombination of gluons

    International Nuclear Information System (INIS)

    Temiraliev, A.T.

    2006-01-01

    Full text: Nonlinear Markov process of parton production has been considered. The Kolmogorov equation is applied for the evolution equation based on the approximation of independent gluons production in every decay act. We introduced a 'crossing' parameter and used the combination relations to obtain nonlinear recombination equation for the evolution of gluon structure function. (author)

  11. Molecular Comparisons of Full Length Metapneumovirus (MPV) Genomes, Including Newly Determined French AMPV-C and –D Isolates, Further Supports Possible Subclassification within the MPV Genus

    Science.gov (United States)

    Brown, Paul A.; Lemaitre, Evelyne; Briand, François-Xavier; Courtillon, Céline; Guionie, Olivier; Allée, Chantal; Toquin, Didier; Bayon-Auboyer, Marie-Hélène; Jestin, Véronique; Eterradossi, Nicolas

    2014-01-01

    Four avian metapneumovirus (AMPV) subgroups (A–D) have been reported previously based on genetic and antigenic differences. However, until now full length sequences of the only known isolates of European subgroup C and subgroup D viruses (duck and turkey origin, respectively) have been unavailable. These full length sequences were determined and compared with other full length AMPV and human metapneumoviruses (HMPV) sequences reported previously, using phylogenetics, comparisons of nucleic and amino acid sequences and study of codon usage bias. Results confirmed that subgroup C viruses were more closely related to HMPV than they were to the other AMPV subgroups in the study. This was consistent with previous findings using partial genome sequences. Closer relationships between AMPV-A, B and D were also evident throughout the majority of results. Three metapneumovirus “clusters” HMPV, AMPV-C and AMPV-A, B and D were further supported by codon bias and phylogenetics. The data presented here together with those of previous studies describing antigenic relationships also between AMPV-A, B and D and between AMPV-C and HMPV may call for a subclassification of metapneumoviruses similar to that used for avian paramyxoviruses, grouping AMPV-A, B and D as type I metapneumoviruses and AMPV-C and HMPV as type II. PMID:25036224

  12. Scattering Length Scaling Laws for Ultracold Three-Body Collisions

    International Nuclear Information System (INIS)

    D'Incao, J.P.; Esry, B.D.

    2005-01-01

    We present a simple and unifying picture that provides the energy and scattering length dependence for all inelastic three-body collision rates in the ultracold regime for three-body systems with short-range two-body interactions. Here, we present the scaling laws for vibrational relaxation, three-body recombination, and collision-induced dissociation for systems that support s-wave two-body collisions. These systems include three identical bosons, two identical bosons, and two identical fermions. Our approach reproduces all previous results, predicts several others, and gives the general form of the scaling laws in all cases

  13. Genetic Recombination

    Science.gov (United States)

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  14. Recombinant expression and purification of the RNA-binding LARP6 proteins from fish genetic model organisms.

    Science.gov (United States)

    Castro, José M; Horn, Daniel A; Pu, Xinzhu; Lewis, Karen A

    2017-06-01

    The RNA-binding proteins that comprise the La-related protein (LARP) superfamily have been implicated in a wide range of cellular functions, from tRNA maturation to regulation of protein synthesis. To more expansively characterize the biological function of the LARP6 subfamily, we have recombinantly expressed the full-length LARP6 proteins from two teleost fish, platyfish (Xiphophorus maculatus) and zebrafish (Danio rerio). The yields of the recombinant proteins were enhanced to >2 mg/L using a tandem approach of an N-terminal His 6 -SUMO tag and an iterative solubility screening assay to identify structurally stabilizing buffer components. The domain topologies of the purified fish proteins were probed with limited proteolysis. The fish proteins contain an internal, protease-resistant 40 kDa domain, which is considerably more stable than the comparable domain from the human LARP6 protein. The fish proteins are therefore a lucrative model system in which to study both the evolutionary divergence of this family of La-related proteins and the structure and conformational dynamics of the domains that comprise the LARP6 protein. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Full-length genome sequences of five hepatitis C virus isolates representing subtypes 3g, 3h, 3i and 3k, and a unique genotype 3 variant.

    Science.gov (United States)

    Lu, Ling; Li, Chunhua; Yuan, Jie; Lu, Teng; Okamoto, Hiroaki; Murphy, Donald G

    2013-03-01

    We characterized the full-length genomes of five distinct hepatitis C virus (HCV)-3 isolates. These represent the first complete genomes for subtypes 3g and 3h, the second such genomes for 3k and 3i, and of one novel variant presently not assigned to a subtype. Each genome was determined from 18-25 overlapping fragments. They had lengths of 9579-9660 nt and each contained a single ORF encoding 3020-3025 aa. They were isolated from five patients residing in Canada; four were of Asian origin and one was of Somali origin. Phylogenetic analysis using 64 partial NS5B sequences differentiated 10 assigned subtypes, 3a-3i and 3k, and two additional lineages within genotype 3. From the data of this study, HCV-3 full-length sequences are now available for six of the assigned subtypes and one unassigned. Our findings should add insights to HCV evolutionary studies and clinical applications.

  16. Correlated evolution of sternal keel length and ilium length in birds

    Directory of Open Access Journals (Sweden)

    Tao Zhao

    2017-07-01

    Full Text Available The interplay between the pectoral module (the pectoral girdle and limbs and the pelvic module (the pelvic girdle and limbs plays a key role in shaping avian evolution, but prior empirical studies on trait covariation between the two modules are limited. Here we empirically test whether (size-corrected sternal keel length and ilium length are correlated during avian evolution using phylogenetic comparative methods. Our analyses on extant birds and Mesozoic birds both recover a significantly positive correlation. The results provide new evidence regarding the integration between the pelvic and pectoral modules. The correlated evolution of sternal keel length and ilium length may serve as a mechanism to cope with the effect on performance caused by a tradeoff in muscle mass between the pectoral and pelvic modules, via changing moment arms of muscles that function in flight and in terrestrial locomotion.

  17. Full-length model of the human galectin-4 and insights into dynamics of inter-domain communication

    Science.gov (United States)

    Rustiguel, Joane K.; Soares, Ricardo O. S.; Meisburger, Steve P.; Davis, Katherine M.; Malzbender, Kristina L.; Ando, Nozomi; Dias-Baruffi, Marcelo; Nonato, Maria Cristina

    2016-09-01

    Galectins are proteins involved in diverse cellular contexts due to their capacity to decipher and respond to the information encoded by β-galactoside sugars. In particular, human galectin-4, normally expressed in the healthy gastrointestinal tract, displays differential expression in cancerous tissues and is considered a potential drug target for liver and lung cancer. Galectin-4 is a tandem-repeat galectin characterized by two carbohydrate recognition domains connected by a linker-peptide. Despite their relevance to cell function and pathogenesis, structural characterization of full-length tandem-repeat galectins has remained elusive. Here, we investigate galectin-4 using X-ray crystallography, small- and wide-angle X-ray scattering, molecular modelling, molecular dynamics simulations, and differential scanning fluorimetry assays and describe for the first time a structural model for human galectin-4. Our results provide insight into the structural role of the linker-peptide and shed light on the dynamic characteristics of the mechanism of carbohydrate recognition among tandem-repeat galectins.

  18. Identification of three new isolates of Tomato spotted wilt virus from different hosts in China: molecular diversity, phylogenetic and recombination analyses.

    Science.gov (United States)

    Zhang, Zhenjia; Wang, Deya; Yu, Chengming; Wang, Zenghui; Dong, Jiahong; Shi, Kerong; Yuan, Xuefeng

    2016-01-14

    Destructive diseases caused by Tomato spotted wilt virus (TSWV) have been reported associated with many important plants worldwide. Recently, TSWV was reported to infect different hosts in China. It is of value to clone TSWV isolates from different hosts and examine diversity and evolution among different TSWV isolates in China as well as worldwide. RT-PCR was used to clone the full-length genome (L, M and S segments) of three new isolates of TSWV that infected different hosts (tobacco, red pepper and green pepper) in China. Identity of nucleotide and amino acid sequences among TSWV isolates were analyzed by DNAMAN. MEGA 5.0 was used to construct phylogenetic trees. RDP4 was used to detect recombination events during evolution of these isolates. Whole-genome sequences of three new TSWV isolates in China were determined. Together with other available isolates, 29 RNA L, 62 RNA M and 66 RNA S of TSWV isolates were analyzed for molecular diversity, phylogenetic and recombination events. This analysis revealed that the entire TSWV genome, especially the M and S RNAs, had major variations in genomic size that mainly involve the A-U rich intergenic region (IGR). Phylogenetic analyses on TSWV isolates worldwide revealed evidence for frequent reassortments in the evolution of tripartite negative-sense RNA genome. Significant numbers of recombination events with apparent 5' regional preference were detected among TSWV isolates worldwide. Moreover, TSWV isolates with similar recombination events usually had closer relationships in phylogenetic trees. All five Chinese TSWV isolates including three TSWV isolates of this study and previously reported two isolates can be divided into two groups with different origins based on molecular diversity and phylogenetic analysis. During their evolution, both reassortment and recombination played roles. These results suggest that recombination could be an important mechanism in the evolution of multipartite RNA viruses, even negative

  19. Inference of historical changes in migration rate from the lengths of migrant tracts

    DEFF Research Database (Denmark)

    Pool, John E; Nielsen, Rasmus

    2009-01-01

    After migrant chromosomes enter a population, they are progressively sliced into smaller pieces by recombination. Therefore, the length distribution of "migrant tracts" (chromosome segments with recent migrant ancestry) contains information about historical patterns of migration. Here we introduce...... a theoretical framework describing the migrant tract length distribution and propose a likelihood inference method to test demographic hypotheses and estimate parameters related to a historical change in migration rate. Applying this method to data from the hybridizing subspecies Mus musculus domesticus and M....... m. musculus, we find evidence for an increase in the rate of hybridization. Our findings could indicate an evolutionary trajectory toward fusion rather than speciation in these taxa....

  20. First full-length genome sequence of the polerovirus luffa aphid-borne yellows virus (LABYV) reveals the presence of at least two consensus sequences in an isolate from Thailand.

    Science.gov (United States)

    Knierim, Dennis; Maiss, Edgar; Kenyon, Lawrence; Winter, Stephan; Menzel, Wulf

    2015-10-01

    Luffa aphid-borne yellows virus (LABYV) was proposed as the name for a previously undescribed polerovirus based on partial genome sequences obtained from samples of cucurbit plants collected in Thailand between 2008 and 2013. In this study, we determined the first full-length genome sequence of LABYV. Based on phylogenetic analysis and genome properties, it is clear that this virus represents a distinct species in the genus Polerovirus. Analysis of sequences from sample TH24, which was collected in 2010 from a luffa plant in Thailand, reveals the presence of two different full-length genome consensus sequences.

  1. Diffusion length measurements in bulk and epitaxially grown 3-5 semiconductors using charge collection microscopy

    Science.gov (United States)

    Leon, R. P.

    1987-01-01

    Diffusion lengths and surface recombination velocities were measured in GaAs diodes and InP finished solar cells. The basic techniques used was charge collection microscopy also known as electron beam induced current (EBIC). The normalized currents and distances from the pn junction were read directly from the calibrated curves obtained while using the line scan mode in an SEM. These values were then equated to integral and infinite series expressions resulting from the solution of the diffusion equation with both extended generation and point generation functions. This expands previous work by examining both thin and thick samples. The surface recombination velocity was either treated as an unknown in a system of two equations, or measured directly using low e(-) beam accelerating voltages. These techniques give accurate results by accounting for the effects of surface recombination and the finite size of the generation volume.

  2. Gene discovery from Jatropha curcas by sequencing of ESTs from normalized and full-length enriched cDNA library from developing seeds

    Directory of Open Access Journals (Sweden)

    Sugantham Priyanka Annabel

    2010-10-01

    Full Text Available Abstract Background Jatropha curcas L. is promoted as an important non-edible biodiesel crop worldwide. Jatropha oil, which is a triacylglycerol, can be directly blended with petro-diesel or transesterified with methanol and used as biodiesel. Genetic improvement in jatropha is needed to increase the seed yield, oil content, drought and pest resistance, and to modify oil composition so that it becomes a technically and economically preferred source for biodiesel production. However, genetic improvement efforts in jatropha could not take advantage of genetic engineering methods due to lack of cloned genes from this species. To overcome this hurdle, the current gene discovery project was initiated with an objective of isolating as many functional genes as possible from J. curcas by large scale sequencing of expressed sequence tags (ESTs. Results A normalized and full-length enriched cDNA library was constructed from developing seeds of J. curcas. The cDNA library contained about 1 × 106 clones and average insert size of the clones was 2.1 kb. Totally 12,084 ESTs were sequenced to average high quality read length of 576 bp. Contig analysis revealed 2258 contigs and 4751 singletons. Contig size ranged from 2-23 and there were 7333 ESTs in the contigs. This resulted in 7009 unigenes which were annotated by BLASTX. It showed 3982 unigenes with significant similarity to known genes and 2836 unigenes with significant similarity to genes of unknown, hypothetical and putative proteins. The remaining 191 unigenes which did not show similarity with any genes in the public database may encode for unique genes. Functional classification revealed unigenes related to broad range of cellular, molecular and biological functions. Among the 7009 unigenes, 6233 unigenes were identified to be potential full-length genes. Conclusions The high quality normalized cDNA library was constructed from developing seeds of J. curcas for the first time and 7009 unigenes coding

  3. γ-rays from pair recombination in a travelling wave structure

    International Nuclear Information System (INIS)

    Bertolotti, M.; Sibilla, C.

    1978-01-01

    To obtain stimulated annihilation of pairs, two photon beams arriving from opposite directions are made to impinge with a region where e - - e + pairs are interacting, thus stimulating their recombination. At the exit of the interaction region photons are amplified by a factor β. The change in photon density is studied as a function of the length of the interaction region. The pair density needed to have a given gain is calculated and results to be proportional to log β 2 /(1+β)

  4. Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline

    Directory of Open Access Journals (Sweden)

    Christelle Folio

    2017-11-01

    Full Text Available Feline immunodeficiency virus (FIV is a member of the Retroviridae family. It is the causative agent of an acquired immunodeficiency syndrome (AIDS in cats and wild felines. Its capsid protein (CA drives the assembly of the viral particle, which is a critical step in the viral replication cycle. Here, the first atomic structure of full-length FIV CA to 1.67 Å resolution is determined. The crystallized protein exhibits an original tetrameric assembly, composed of dimers which are stabilized by an intermolecular disulfide bridge induced by the crystallogenesis conditions. The FIV CA displays a standard α-helical CA topology with two domains, separated by a linker shorter than other retroviral CAs. The β-hairpin motif at its amino terminal end, which interacts with nucleotides in HIV-1, is unusually long in FIV CA. Interestingly, this functional β-motif is formed in this construct in the absence of the conserved N-terminal proline. The FIV CA exhibits a cis Arg–Pro bond in the CypA-binding loop, which is absent in known structures of lentiviral CAs. This structure represents the first tri-dimensional structure of a functional, full-length FIV CA.

  5. Combination treatment with hepatitis C virus protease and NS5A inhibitors is effective against recombinant genotype 1a, 2a, and 3a viruses

    DEFF Research Database (Denmark)

    Gottwein, Judith M; Jensen, Sanne B; Li, Yi-Ping

    2013-01-01

    With the development of directly acting antivirals, hepatitis C virus (HCV) therapy entered a new era. However, rapid selection of resistance mutations necessitates combination therapy. To study combination therapy in infectious culture systems, we aimed at developing HCV semi-full-length (semi...... to single-drug treatment, combination treatment with relatively low concentrations of asunaprevir and daclatasvir suppressed infection with all five recombinants. Escaped viruses primarily had substitutions at amino acids in the NS3 protease and NS5A domain I reported to be genotype 1 resistance mutations...

  6. Isolation and characterization of a replication-competent molecular clone of an HIV-1 circulating recombinant form (CRF33_01B.

    Directory of Open Access Journals (Sweden)

    Kok Keng Tee

    Full Text Available A growing number of emerging HIV-1 recombinants classified as circulating recombinant forms (CRFs have been identified in Southeast Asia in recent years, establishing a molecular diversity of increasing complexity in the region. Here, we constructed a replication-competent HIV-1 clone for CRF33_01B (designated p05MYKL045.1, a newly identified recombinant comprised of CRF01_AE and subtype B. p05MYKL045.1 was reconstituted by cloning of the near full-length HIV-1 sequence from a newly-diagnosed individual presumably infected heterosexually in Kuala Lumpur, Malaysia. The chimeric clone, which contains the 5' LTR (long terminal repeat region of p93JP-NH1 (a previously isolated CRF01_AE infectious clone, showed robust viral replication in the human peripheral blood mononuclear cells. This clone demonstrated robust viral propagation and profound syncytium formation in CD4+, CXCR4-expressing human glioma NP-2 cells, indicating that p05MYKL045.1 is a CXCR4-using virus. Viral propagation, however, was not detected in various human T cell lines including MT-2, M8166, Sup-T1, H9, Jurkat, Molt-4 and PM1. p05MYKL045.1 appears to proliferate only in restricted host range, suggesting that unknown viral and/or cellular host factors may play a role in viral infectivity and replication in human T cell lines. Availability of a CRF33_01B molecular clone will be useful in facilitating the development of vaccine candidates that match the HIV-1 strains circulating in Southeast Asia.

  7. Analysis of the full-length VP2 protein of canine parvoviruses circulating in Hungary.

    Science.gov (United States)

    Cságola, Attila; Varga, Szilvia; Lőrincz, Márta; Tuboly, Tamás

    2014-09-01

    In recent years, the number of cases of disease caused by canine parvovirus 2 (CPV-2) in vaccinated dogs has increased. The aim of the present study was to identify CPV-2 strains present in Hungary. Forty-two out of 50 faecal specimens examined were positive, and 25 VP2 sequences were determined and analysed. Based on the current classification, the Hungarian viruses belong to New CPV-2a type, except two viruses that are recombinants of vaccine viruses and CPV-2a strains. The Tyr324Ile alteration was detected for the first time in Europe, and a "Hungarian-specific" substitution (Ala516Thr) was also identified in this study. The immunologically important parts of the currently spreading canine parvoviruses were examined and found to differ greatly from the vaccine strains that are widely used in Hungary.

  8. Polarization mechanism for Bremsstrahlung and radiative recombination in a plasma with heavy ions

    International Nuclear Information System (INIS)

    Astapenko, V.A.; Bureeva, L.A.; Lisitsa, V.S.

    2002-01-01

    Contribution of polarization channel into radiation and recombination of electrons in plasma with heavy ions is investigated. Cases of hot plasma with temperature T e = 0.5 keV and Fe, Mo, W, U ions and relatively cold plasma with temperature 0.1-10 eV are considered. Calculations of spectral characteristics, full cross sections and recombination rates in plasma are made, bearing in mind its real ionization equilibrium. The calculations are made on the basis of quasiclassical approximation for electron scattering and statistical model of ions. It is shown that contribution of polarization channel is essential both for effective radiation and full rate of radiative recombination [ru

  9. Rapid customised operon assembly by yeast recombinational cloning.

    Science.gov (United States)

    Liu, Michael A; Kenyon, Johanna J; Lee, Jason; Reeves, Peter R

    2017-06-01

    We have developed a system called the Operon Assembly Protocol (OAP), which takes advantage of the homologous recombination DNA repair pathway in Saccharomyces cerevisiae to assemble full-length operons from a series of overlapping PCR products into a specially engineered yeast-Escherichia coli shuttle vector. This flexible, streamlined system can be used to assemble several operon clones simultaneously, and each clone can be expressed in the same E. coli tester strain to facilitate direct functional comparisons. We demonstrated the utility of the OAP by assembling and expressing a series of E. coli O1A O-antigen gene cluster clones containing various gene deletions or replacements. We then used these constructs to assess the substrate preferences of several Wzx flippases, which are responsible for translocation of oligosaccharide repeat units (O units) across the inner membrane during O-antigen biosynthesis. We were able to identify several O unit structural features that appear to be important determinants of Wzx substrate preference. The OAP system should be broadly applicable for the genetic manipulation of any bacterial operon and can be modified for use in other host species. It could also have potential uses in fields such as glycoengineering.

  10. 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.

  11. Assembly and dynamics of the bacteriophage T4 homologous recombination machinery

    Directory of Open Access Journals (Sweden)

    Morrical Scott W

    2010-12-01

    Full Text Available Abstract Homologous recombination (HR, a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining the genetic diversity and genome stability of species. Bacteriophage T4 is one of the classic systems for studies of homologous recombination. T4 uses HR for high-frequency genetic exchanges, for homology-directed DNA repair (HDR processes including DNA double-strand break repair, and for the initiation of DNA replication (RDR. T4 recombination proteins are expressed at high levels during T4 infection in E. coli, and share strong sequence, structural, and/or functional conservation with their counterparts in cellular organisms. Biochemical studies of T4 recombination have provided key insights on DNA strand exchange mechanisms, on the structure and function of recombination proteins, and on the coordination of recombination and DNA synthesis activities during RDR and HDR. Recent years have seen the development of detailed biochemical models for the assembly and dynamics of presynaptic filaments in the T4 recombination system, for the atomic structure of T4 UvsX recombinase, and for the roles of DNA helicases in T4 recombination. The goal of this chapter is to review these recent advances and their implications for HR and HDR mechanisms in all organisms.

  12. Report of recombinant norovirus GII.g/GII.12 in Beijing, China.

    Directory of Open Access Journals (Sweden)

    Shaowei Sang

    Full Text Available BACKGROUND: Norovirus (NoV has been recognized as the most important cause of nonbacterial acute gastroenteritis affecting all age group people in the world. Genetic recombination is a common occurance in RNA viruses and many recombinant NoV strains have been described since it was first reported in 1997. However, the knowledge of recombinant NoV in China is extremely limited. METHODS: A total of 685 stool specimens were tested for NoV infection from the acute gastroenteritis patients who visited one general hospital in Beijing from April 2009 to November 2011. The virus recombination was identified by constructing phylogenetic trees of two genes, further SimPlot and the maximum chi-square analysis. RESULTS: The overall positive rate was 9.6% (66/685. GII.4 New Orleans 2009 and GII.4 2006b variants were the dominant genotype. Four GII.g/GII.12 and one GII.12/GII.3 recombinant strains were confirmed, and all derived from adult outpatients. The predictive recombination point occurred at the open reading frame (ORF1/ORF2 overlap. CONCLUSIONS: The GII.g ORF1/GII.12ORF2 recombinant has been reported in several countries and it was the first report of this recombinant in China.

  13. The repetitive portion of the Xenopus IgH Mu switch region mediates orientation-dependent class switch recombination.

    Science.gov (United States)

    Zhang, Zheng Z; Pannunzio, Nicholas R; Lu, Zhengfei; Hsu, Ellen; Yu, Kefei; Lieber, Michael R

    2015-10-01

    Vertebrates developed immunoglobulin heavy chain (IgH) class switch recombination (CSR) to express different IgH constant regions. Most double-strand breaks for Ig CSR occur within the repetitive portion of the switch regions located upstream of each set of constant domain exons for the Igγ, Igα or Igϵ heavy chain. Unlike mammalian switch regions, Xenopus switch regions do not have a high G-density on the non-template DNA strand. In previous studies, when Xenopus Sμ DNA was moved to the genome of mice, it is able to support substantial CSR when it is used to replace the murine Sγ1 region. Here, we tested both the 2kb repetitive portion and the 4.6 kb full-length portions of the Xenopus Sμ in both their natural (forward) orientation relative to the constant domain exons, as well as the opposite (reverse) orientation. Consistent with previous work, we find that the 4.6 kb full-length Sμ mediates similar levels of CSR in both the forward and reverse orientations. Whereas, the forward orientation of the 2kb portion can restore the majority of the CSR level of the 4.6 kb full-length Sμ, the reverse orientation poorly supports R-looping and no CSR. The forward orientation of the 2kb repetitive portion has more GG dinucleotides on the non-template strand than the reverse orientation. The correlation of R-loop formation with CSR efficiency, as demonstrated in the 2kb repetitive fragment of the Xenopus switch region, confirms a role played by R-looping in CSR that appears to be conserved through evolution. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Preparation of a differentially expressed, full-length cDNA expression library by RecA-mediated triple-strand formation with subtractively enriched cDNA fragments

    NARCIS (Netherlands)

    Hakvoort, T. B.; Spijkers, J. A.; Vermeulen, J. L.; Lamers, W. H.

    1996-01-01

    We have developed a fast and general method to obtain an enriched, full-length cDNA expression library with subtractively enriched cDNA fragments. The procedure relies on RecA-mediated triple-helix formation of single-stranded cDNA fragments with a double-stranded cDNA plasmid library. The complexes

  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. A newly emerging HIV-1 recombinant lineage (CRF58_01B) disseminating among people who inject drugs in Malaysia.

    Science.gov (United States)

    Chow, Wei Zhen; Takebe, Yutaka; Syafina, Nur Ezreen; Prakasa, Malarvelli Soorya; Chan, Kok Gan; Al-Darraji, Haider Abdulrazzaq Abed; Koh, Clayton; Kamarulzaman, Adeeba; Tee, Kok Keng

    2014-01-01

    The HIV epidemic is primarily characterised by the circulation of HIV-1 group M (main) comprising of 11 subtypes and sub-subtypes (A1, A2, B-D, F1, F2, G, H, J, and K) and to date 55 circulating recombinant forms (CRFs). In Southeast Asia, active inter-subtype recombination involving three main circulating genotypes--subtype B (including subtype B', the Thai variant of subtype B), CRF01_AE, and CRF33_01B--have contributed to the emergence of novel unique recombinant forms. In the present study, we conducted the molecular epidemiological surveillance of HIV-1 gag-RT genes among 258 people who inject drugs (PWIDs) in Kuala Lumpur, Malaysia, between 2009 and 2011 whereby a novel CRF candidate was recently identified. The near full-length genome sequences obtained from six epidemiologically unlinked individuals showed identical mosaic structures consisting of subtype B' and CRF01_AE, with six unique recombination breakpoints in the gag-RT, pol, and env regions. Among the high-risk population of PWIDs in Malaysia, which was predominantly infected by CRF33_01B (>70%), CRF58_01B circulated at a low but significant prevalence (2.3%, 6/258). Interestingly, the CRF58_01B shared two unique recombination breakpoints with other established CRFs in the region: CRF33_01B, CRF48_01B, and CRF53_01B in the gag gene, and CRF15_01B (from Thailand) in the env gene. Extended Bayesian Markov chain Monte Carlo sampling analysis showed that CRF58_01B and other recently discovered CRFs were most likely to have originated in Malaysia, and that the recent spread of recombinant lineages in the country had little influence from neighbouring countries. The isolation, genetic characterization, and evolutionary features of CRF58_01B among PWIDs in Malaysia signify the increasingly complex HIV-1 diversity in Southeast Asia that may hold an implication on disease treatment, control, and prevention.

  17. Identification of a heterologous cellulase and its N-terminus that can guide recombinant proteins out of Escherichia coli.

    Science.gov (United States)

    Gao, Dongfang; Wang, Shengjun; Li, Haoran; Yu, Huili; Qi, Qingsheng

    2015-04-10

    The Gram-negative bacterium Escherichia coli has been widely used as a cell factory for the production of proteins and specialty chemicals because it is the best characterized host with many available expression and regulation systems. However, recombinant proteins produced in Escherichia coli are generally intracellular and often found in the form of inclusion bodies. Extracellular production of proteins is advantageous compared with intracellular production because extracellular proteins can be purified more easily and can avoid protease attack, which results in higher product quality. In this study, we found a catalytic domain of a cellulase (Cel-CD) and its N-terminus can be employed as carriers for extracellular production of recombinant proteins. In this report, we identified the catalytic domain of a cellulase (Cel-CD) from Bacillus sp. that can be secreted into the medium from recombinant E. coli BL21 (DE3) in large quantities without its native signal peptide. By subcellular location analysis, we proved that the secretion was a two-step process and the N-terminal sequence of the full length Cel-CD played a crucial function in secretion. Both the Cel-CD and its N-terminal sequence can serve as carriers for efficient extracellular production of select target proteins. Fusion of heterologous proteins with N20 from Cel-CD can carry the target proteins out of the cells with a concentration from 101 to 691 mg/L in flask cultivation. The extracellular recombinant proteins with a relative high purity. The results suggested that this system has a potential application in plant biomass conversion and industrial production of enzymes and therapeutic proteins.

  18. Toxoplasma gondii: Biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6

    International Nuclear Information System (INIS)

    Bittame, Amina; Effantin, Grégory; Pètre, Graciane; Ruffiot, Pauline; Travier, Laetitia; Schoehn, Guy; Weissenhorn, Winfried; Cesbron-Delauw, Marie-France; Gagnon, Jean; Mercier, Corinne

    2015-01-01

    The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6–8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8–15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed. - Highlights: • Toxoplasma gondii: soluble GRA2 forms 2 populations of particles. • T. gondii: the dense granule protein GRA2 folds intrinsically as an alpha-helix. • T. gondii: monomeric soluble GRA6 forms particles of 6–8 nm in diameter. • T. gondii: monomeric soluble GRA6 is random coiled. • Unusual biophysical properties of the dense granule protein GRA2 from T. gondii

  19. Toxoplasma gondii: Biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6

    Energy Technology Data Exchange (ETDEWEB)

    Bittame, Amina [CNRS, UMR 5163, 38042 Grenoble (France); Université Grenoble Alpes, 38042 Grenoble (France); Effantin, Grégory [Université Grenoble Alpes, Institut de Biologie Structurale (IBS), 38044 Grenoble (France); CNRS, IBS, 38044 Grenoble (France); CEA, IBS, 38044 Grenoble (France); Unit for Virus Host-Cell Interactions (UVHCI), UMI 3265 (UJF-EMBL-CNRS), 38027 Grenoble (France); Pètre, Graciane; Ruffiot, Pauline; Travier, Laetitia [CNRS, UMR 5163, 38042 Grenoble (France); Université Grenoble Alpes, 38042 Grenoble (France); Schoehn, Guy; Weissenhorn, Winfried [Université Grenoble Alpes, Institut de Biologie Structurale (IBS), 38044 Grenoble (France); CNRS, IBS, 38044 Grenoble (France); CEA, IBS, 38044 Grenoble (France); Unit for Virus Host-Cell Interactions (UVHCI), UMI 3265 (UJF-EMBL-CNRS), 38027 Grenoble (France); Cesbron-Delauw, Marie-France; Gagnon, Jean [CNRS, UMR 5163, 38042 Grenoble (France); Université Grenoble Alpes, 38042 Grenoble (France); Mercier, Corinne, E-mail: corinne.mercier@ujf-grenoble.fr [CNRS, UMR 5163, 38042 Grenoble (France); Université Grenoble Alpes, 38042 Grenoble (France)

    2015-03-27

    The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6–8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8–15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed. - Highlights: • Toxoplasma gondii: soluble GRA2 forms 2 populations of particles. • T. gondii: the dense granule protein GRA2 folds intrinsically as an alpha-helix. • T. gondii: monomeric soluble GRA6 forms particles of 6–8 nm in diameter. • T. gondii: monomeric soluble GRA6 is random coiled. • Unusual biophysical properties of the dense granule protein GRA2 from T. gondii.

  20. Full Length Research Article

    African Journals Online (AJOL)

    Dr Ahmed

    other areas of business administration. ... mathematics, real estate, insurance, actuarial science and business administration (McCutcheon & Scott, 1989). Most textbooks written in these ..... Mathematics of Finance; Heinemann; Oxford. Murray ...

  1. Full length Research Article

    African Journals Online (AJOL)

    Dr Ahmed

    ABSTRACT. This paper considered contamination of aquifer resulting from petroleum spillage, which is a common phenomenal in the. Niger Delta area of Nigeria. We used the model given by. Bestman (1987) and assumed that some endogenous variables are built into the system. To achieve a level of desirable state, we ...

  2. Full Length Research Article

    African Journals Online (AJOL)

    Dr Ahmed

    Mbah & Amao (SWJ):11-14. Natural Foods and Feeding Habits Of The African Honey bee ... Keywords: natural food, nectar, pollen, african honeybee, Apis mellifera adansonii ..... Crailsheim, K. (1990). The Protein balance of the honeybee.

  3. Full Length Research Article

    African Journals Online (AJOL)

    Dr Ahmed

    ... Solarin (SWJ):1-6. On Existence Of Control For A Class Of Uncertain Dynamical Systems ... .m We apply the Conjugate Gradient Method. (C.G.M) in ...... Numerical Technique .... Some of its Applications; Journal of Mathematical Analysis and.

  4. Full Length Research Article

    African Journals Online (AJOL)

    Dr Ahmed

    providing energy storage and metabolic fuel, acting as functional and structural ... zoonotic and non-zoonotic diseases are been imported and exported in and out ..... rabbits infected with T. brucie and Gow et al., (2007) in which dog naturally ...

  5. Full Length Research Article

    African Journals Online (AJOL)

    Dr Ahmed

    In recent years, a new technique was introduced that combines both hydrolysis and ... Cassava Starch Preparation: The cassava starch used for this investigation was prepared ... One normal HCl and one normal NaOH were used to adjust the ...

  6. Full length Research Article

    African Journals Online (AJOL)

    Administrator

    chemical oxidation and reduction, electro-chemical treatment and ion-pair extraction were extensively used ... The present study is an effort to develop a fungal-based treatment system for the cleaning of dye industrial ..... Biochemical and Biophysical Research Communications 178:1056-. 1063. Raju, N. S., Venkataramana ...

  7. Full Length Research Article

    African Journals Online (AJOL)

    Dr Ahmed

    SPECKLED PIGEON (Columba guinea HARTLAUB AND. FINSCH ... area of the Nigerian northern guinea savanna (Fry, 1965). The habit of the .... defined by Margolis et al., (1982). Chi-square ..... Southern Africa, Academic Press, London.. 4 ...

  8. Full Length Research paper

    African Journals Online (AJOL)

    Marcos

    2012-08-21

    Aug 21, 2012 ... Cut flowers have a very limited life after they have been cut off from the mother plant, as survival on their own reserves is ... One of the techniques used for the removal ... and vegetables (Pellegrini and Belle, 2008). There are ...

  9. Full Length Research Article

    African Journals Online (AJOL)

    Administrator

    inorganic, synthetic organic polymer and naturally occurring coagulants .... Flash fast mixing was done for 2 mins at a speed of 100rpm, followed by slow mixing for ..... Moringa leaves from germplasm to plant, to food, to health. Symposium on ...

  10. Full Length Research Article

    African Journals Online (AJOL)

    Dr Ahmed

    The isotope techniques used in hydrology may be classified into three groups: environmental ..... In that case a solution may be found in measurements of 14C content of groundwaters. ... Applied Isotope Hydrogeology. Elsevier, New. York.

  11. Full Length Research Article

    African Journals Online (AJOL)

    Dr Ahmed

    some rural communities of Zaria, Nigeria for microbial index of water quality in relation to ... These factors, together with the inadequate waste treatment facilities and ..... The Need for an Integrated Approach to Water Supply and. Sanitation in ...

  12. Full Length Research paper

    African Journals Online (AJOL)

    Marcos

    2012-08-21

    Aug 21, 2012 ... 1Universidade Estadual Paulista, Faculdade de Ciências Agronômicas, CEP: 18601-060, Botucatu, SP, Brasil. 2Universidade Federal de Alagoas, Centro de Ciências Agrárias-CECA, Maceio, AL, Brasil. 3Universidade Federal Rural do Semi-Árido, Departamento de Ciências Vegetais, Mossoró, RN, Brasil ...

  13. Full Length Research Article

    African Journals Online (AJOL)

    MEMUNA

    markovian process could be reduced to a markovian chain with the homogenous .... x and y respectively. n= the value of the strategy i.e. number of dummies/virtual ..... Groove, CA. Feinberg, E. A. & Shwartz, A. (2002), Handbook of Markov.

  14. Molecular Cloning and Characterization of Full-Length cDNA of Calmodulin Gene from Pacific Oyster Crassostrea gigas

    Directory of Open Access Journals (Sweden)

    Xing-Xia Li

    2016-01-01

    Full Text Available The shell of the pearl oyster (Pinctada fucata mainly comprises aragonite whereas that of the Pacific oyster (Crassostrea gigas is mainly calcite, thereby suggesting the different mechanisms of shell formation between above two mollusks. Calmodulin (CaM is an important gene for regulating the uptake, transport, and secretion of calcium during the process of shell formation in pearl oyster. It is interesting to characterize the CaM in oysters, which could facilitate the understanding of the different shell formation mechanisms among mollusks. We cloned the full-length cDNA of Pacific oyster CaM (cgCaM and found that the cgCaM ORF encoded a peptide of 113 amino acids containing three EF-hand calcium-binding domains, its expression level was highest in the mantle, hinting that the cgCaM gene is probably involved in shell formation of Pacific oyster, and the common ancestor of Gastropoda and Bivalvia may possess at least three CaM genes. We also found that the numbers of some EF hand family members in highly calcified species were higher than those in lowly calcified species and the numbers of these motifs in oyster genome were the highest among the mollusk species with whole genome sequence, further hinting the correlation between CaM and biomineralization.

  15. Characterization of a Full-Length Endogenous Beta-Retrovirus, EqERV-Beta1, in the Genome of the Horse (Equus caballus

    Directory of Open Access Journals (Sweden)

    Antoinette C. van der Kuyl

    2011-06-01

    Full Text Available Information on endogenous retroviruses fixed in the horse (Equus caballus genome is scarce. The recent availability of a draft sequence of the horse genome enables the detection of such integrated viruses by similarity search. Using translated nucleotide fragments from gamma-, beta-, and delta-retroviral genera for initial searches, a full-length beta-retrovirus genome was retrieved from a horse chromosome 5 contig. The provirus, tentatively named EqERV-beta1 (for the first equine endogenous beta-retrovirus, was 10434 nucleotide (nt in length with the usual retroviral genome structure of 5’LTR-gag-pro-pol-env-3’LTR. The LTRs were 1361 nt long, and differed approximately 1% from each other, suggestive of a relatively recent integration. Coding sequences for gag, pro and pol were present in three different reading-frames, as common for beta-retroviruses, and the reading frames were completely open, except that the env gene was interrupted by a single stopcodon. No reading frame was apparent downstream of the env gene, suggesting that EqERV-beta1 does not encode a superantigen like mouse mammary tumor virus (MMTV. A second proviral genome of EqERV-beta1, with no stopcodon in env, is additionally integrated on chromosome 5 downstream of the first virus. Single EqERV-beta1 LTRs were abundantly present on all chromosomes except chromosome 24. Phylogenetically, EqERV-beta1 most closely resembles an unclassified retroviral sequence from cattle (Bos taurus, and the murine beta-retrovirus MMTV.

  16. Study of canine parvovirus evolution: comparative analysis of full-length VP2 gene sequences from Argentina and international field strains.

    Science.gov (United States)

    Gallo Calderón, Marina; Wilda, Maximiliano; Boado, Lorena; Keller, Leticia; Malirat, Viviana; Iglesias, Marcela; Mattion, Nora; La Torre, Jose

    2012-02-01

    The continuous emergence of new strains of canine parvovirus (CPV), poorly protected by current vaccination, is a concern among breeders, veterinarians, and dog owners around the world. Therefore, the understanding of the genetic variation in emerging CPV strains is crucial for the design of disease control strategies, including vaccines. In this paper, we obtained the sequences of the full-length gene encoding for the main capsid protein (VP2) of 11 canine parvovirus type 2 (CPV-2) Argentine representative field strains, selected from a total of 75 positive samples studied in our laboratory in the last 9 years. A comparative sequence analysis was performed on 9 CPV-2c, one CPV-2a, and one CPV-2b Argentine strains with respect to international strains reported in the GenBank database. In agreement with previous reports, a high degree of identity was found among CPV-2c Argentine strains (99.6-100% and 99.7-100% at nucleotide and amino acid levels, respectively). However, the appearance of a new substitution in the 440 position (T440A) in four CPV-2c Argentine strains obtained after the year 2009 gives support to the variability observed for this position located within the VP2, three-fold spike. This is the first report on the genetic characterization of the full-length VP2 gene of emerging CPV strains in South America and shows that all the Argentine CPV-2c isolates cluster together with European and North American CPV-2c strains.

  17. High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

    Directory of Open Access Journals (Sweden)

    You Bang-Jau

    2011-07-01

    Full Text Available Abstract Background Chicken anemia virus (CAV, the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention. Results Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay. Conclusions Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.

  18. Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila

    Science.gov (United States)

    Smukowski Heil, Caiti S.; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A.F.

    2015-01-01

    Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human–chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. PMID:26430062

  19. Characterization of novel human papillomavirus types 157, 158 and 205 from healthy skin and recombination analysis in genus γ-Papillomavirus.

    Science.gov (United States)

    Bolatti, Elisa M; Chouhy, Diego; Casal, Pablo E; Pérez, Germán R; Stella, Emma J; Sanchez, Adriana; Gorosito, Mario; Bussy, Ramón Fernandez; Giri, Adriana A

    2016-08-01

    Gammapapillomavirus (γ-PV) is a diverse and rapidly expanding genus, currently consisting of 79 fully characterized human PV (HPV) types. In this study, three novel types, HPV157, HPV158 and HPV205, obtained from healthy sun-exposed skin of two immunocompetent individuals, were amplified by the "Hanging droplet" long PCR technique, cloned, sequenced and characterized. HPV157, HPV158 and HPV205 genomes comprise 7154-bp, 7192-bp and 7298-bp, respectively, and contain four early (E1, E2, E6 and E7) and two late genes (L1 and L2). Phylogenetic analysis of the L1 ORF placed all novel types within the γ-PV genus: HPV157 was classified as a new member of species γ-12 while HPV158 and HPV205 belong to species γ-1. We then explored potential recombination events in genus γ-PV with the RDP4 program in a dataset of 74 viruses (71 HPV types with available full-length genomes and the 3 novel types). Two events, both located in the E1 ORF, met the inclusion criterion (p-values methods) and persisted in different ORF combinations: an inter-species recombination in species γ-8 (major and minor parents: species γ-24 and γ-11, respectively), and an intra-species recombination in species γ-7 (recombinant strain: HPV170; major and minor parents: HPV-109 and HPV-149, respectively). These findings were confirmed by phylogenetic tree incongruence analysis. An additional incongruence was found in members of species γ-9 but it was not detected by the RDP4. This report expands our knowledge of the family Papillomaviridae and provides for the first time in silico evidence of recombination in genus γ-PV. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Large-scale analysis of full-length cDNAs from the tomato (Solanum lycopersicum cultivar Micro-Tom, a reference system for the Solanaceae genomics

    Directory of Open Access Journals (Sweden)

    Kikuchi Mari

    2010-03-01

    Full Text Available Abstract Background The Solanaceae family includes several economically important vegetable crops. The tomato (Solanum lycopersicum is regarded as a model plant of the Solanaceae family. Recently, a number of tomato resources have been developed in parallel with the ongoing tomato genome sequencing project. In particular, a miniature cultivar, Micro-Tom, is regarded as a model system in tomato genomics, and a number of genomics resources in the Micro-Tom-background, such as ESTs and mutagenized lines, have been established by an international alliance. Results To accelerate the progress in tomato genomics, we developed a collection of fully-sequenced 13,227 Micro-Tom full-length cDNAs. By checking redundant sequences, coding sequences, and chimeric sequences, a set of 11,502 non-redundant full-length cDNAs (nrFLcDNAs was generated. Analysis of untranslated regions demonstrated that tomato has longer 5'- and 3'-untranslated regions than most other plants but rice. Classification of functions of proteins predicted from the coding sequences demonstrated that nrFLcDNAs covered a broad range of functions. A comparison of nrFLcDNAs with genes of sixteen plants facilitated the identification of tomato genes that are not found in other plants, most of which did not have known protein domains. Mapping of the nrFLcDNAs onto currently available tomato genome sequences facilitated prediction of exon-intron structure. Introns of tomato genes were longer than those of Arabidopsis and rice. According to a comparison of exon sequences between the nrFLcDNAs and the tomato genome sequences, the frequency of nucleotide mismatch in exons between Micro-Tom and the genome-sequencing cultivar (Heinz 1706 was estimated to be 0.061%. Conclusion The collection of Micro-Tom nrFLcDNAs generated in this study will serve as a valuable genomic tool for plant biologists to bridge the gap between basic and applied studies. The nrFLcDNA sequences will help annotation of the

  1. Laser-induced electron--ion recombination used to study enhanced spontaneous recombination during electron cooling

    International Nuclear Information System (INIS)

    Schramm, U.; Wolf, A.; Schuess ler, T.; Habs, D.; Schwalm, D.; Uwira, O.; Linkemann, J.; Mueller, A.

    1997-01-01

    Spontaneous recombination of highly charged ions with free electrons in merged velocity matched electron and ion beams has been observed in earlier experiments to occur at rates significantly higher than predicted by theoretical estimates. To study this enhanced spontaneous recombination, laser induced recombination spectra were measured both in velocity matched beams and in beams with well defined relative velocities, corresponding to relative electron-ion detuning energies ranging from 1 meV up to 6.5 meV where the spontaneous recombination enhancement was found to be strongly reduced. Based on a comparison with simplified calculations, the development of the recombination spectra for decreasing detuning energies indicates additional contributions at matched velocities which could be related to the energy distribution of electrons causing the spontaneous recombination rate enhancement

  2. Polyclonal antibodies against the recombinantly expressed coat protein of the Citrus psorosis virus

    Directory of Open Access Journals (Sweden)

    Reda Salem

    2018-05-01

    Full Text Available Psorosis is a damaging disease of citrus that is widespread in many parts of the world. Citrus psorosis virus (CPsV, the type species of the genus Ophiovirus, is the putative causal agent of psorosis. Detection of CPsV by laboratory methods, serology in particular is a primary requirement for large-scale surveys but their production has been impaired by the difficulty of obtaining sufficient clean antigen for immunization. Specific PAbs against coat protein were produced in E. coli using recombinant DNA approach. The full length CP gene fragment was amplified by RT-PCR using total RNA extracted from CPsV infected citrus leaves and CP specific primers. The obtained product (1320bp was cloned, sequenced and sub-cloned into pET-30(+ expression vector. Expression was induced and screened in different bacterial clones by the presence of the expressed protein (48kDa and optimized in one clone. Expressed CP was purified using batch chromatography under denaturing conditions. Specificity of expressed protein was demonstrated by ELISA before used as antigen for raising PAbs in mice. Specificity of the raised PAbs to CPsV was verified by ELISA and western blotting. The raised PAbs were showed highly effectiveness in screening by ELISA comparing with the commercial antibodies purchased from Agritest, Valanzano, Italy.The expression of CPsV CP gene in E. coli, production of PAbs using recombinant protein as an antigen, the suitability of these antibodies for use in immunodiagnostics against the CPsV Egyptian isolate have been accomplished in this work. Keywords: CPsV, CP, PAbs, RT-PCR, ELISA, Western blotting

  3. Asthma and Therapeutics: Recombinant Therapies in Asthma

    Directory of Open Access Journals (Sweden)

    Cockcroft Donald W

    2005-03-01

    Full Text Available Abstract Numerous recombinant therapies are being investigated for the treatment of asthma. This report reviews the current status of several of these novel agents. Anti-immunoglobulin (IgE (omalizumab, Xolair markedly inhibits all aspects of the allergen challenge in subjects who have reduction of free serum IgE to undetectable levels. Several clinical studies in atopic asthma have demonstrated benefit by improved symptoms and lung function and a reduction in corticosteroid requirements. Early use in atopic asthmatics may be even more effective. Several approaches target interleukin (IL-4. Soluble IL-4 receptor has been shown to effectively replace inhaled corticosteroid; further studies are under way. Recombinant anti-IL-5 and recombinant IL-12 inhibit blood and sputum eosinophils and allergen-induced eosinophilia without any effect on airway responsiveness, allergen-induced airway responses, or allergen-induced airway hyperresponsiveness. Efalizumab, a recombinant antibody that inhibits lymphocyte trafficking, is effective in psoriasis. A bronchoprovocation study showed a reduction in allergen-induced late asthmatic response and allergen-induced eosinophilia, which suggests that it should be effective in clinical asthma. These exciting novel therapies provide not only promise of new therapies for asthma but also valuable tools for investigation of asthma mechanisms.

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

    International Nuclear Information System (INIS)

    Fraenkel, A.H.M.

    1974-01-01

    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.)

  5. The RAG1 and RAG2 proteins establish the 12/23 rule in V(D)J recombination

    NARCIS (Netherlands)

    D.C. van Gent (Dik)

    1996-01-01

    textabstractV(D)J recombination requires a pair of signal sequences with spacer lengths of 12 and 23 base pairs. Cleavage by the RAG1 AND RAG2 proteins was previously shown to demand only a single signal sequence. Here, we established conditions where 12- and 23-spacer signal

  6. A VNTR element associated with steroid sulfatase gene deletions stimulates recombination in cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Gong, Y.; Li, X.M.; Shapiro, L.J. [UCSF School of Medicine, San Francisco, CA (United States)] [and others

    1994-09-01

    Steroid sulfatase deficiency is a common genetic disorder, with a prevalence of approximately one in every 3500 males world wide. About 90% of these patients have complete gene deletions, which appear to result from recombination between members of a low-copy repeat family (CRI-232 is the prototype) that flank the gene. RU1 and RU2 are two VNTR elements found within each of these family members. RU1 consists of 30 bp repeating units and its length shows minimal variation among individuals. The RU2 element consists of repeating sequences which are highly asymmetric, with about 90% purines and no C`s on one strand, and range from 0.6 kb to over 23 kb among different individuals. We conducted a study to determine if the RU1 or RU2 elements can promote recombination in an in vivo test system. We inserted these elements adjacent to the neo gene in each of two pSV2neo derivatives, one of which has a deletion in the 5{prime} portion of the neo gene and the other having a deletion in the 3{prime} portion. These plasmids were combined and used to transfect EJ cells. Survival of cells in G418 indicates restoration of a functional neo gene by recombination between two deletion constructs. Thus counting G418 resistant colonies gives a quantitative measure of the enhancement of recombination by the inserted VNTR elements. The results showed no effect on recombination by the inserted RU1 element (compared to the insertion of a nonspecific sequence), while the RU2 element stimulated recombination by 3.5-fold (P<0.01). A separate set of constructs placed RU1 or RU2 within the intron of an exon trapping vector. Following tranfection of cells, recombination events were monitored by a PCR assay that detected the approximation of primer binding sites (as a result of recombination). These studies showed that, as in the first set of experiments, the highly variable RU2 element is capable of stimulating somatic recombination in mammalian cells.

  7. Recombinant Chromosome 4 from a Familial Pericentric Inversion: Prenatal and Adulthood Wolf-Hirschhorn Phenotypes

    Directory of Open Access Journals (Sweden)

    Francesca Malvestiti

    2013-01-01

    Full Text Available Pericentric inversion of chromosome 4 can give rise to recombinant chromosomes by duplication or deletion of 4p. We report on a familial case of Wolf-Hirschhorn Syndrome characterized by GTG-banding karyotypes, FISH, and array CGH analysis, caused by a recombinant chromosome 4 with terminal 4p16.3 deletion and terminal 4q35.2 duplication. This is an aneusomy due to a recombination which occurred during the meiosis of heterozygote carrier of cryptic pericentric inversion. We also describe the adulthood and prenatal phenotypes associated with the recombinant chromosome 4.

  8. Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila.

    Science.gov (United States)

    Smukowski Heil, Caiti S; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A F

    2015-10-01

    Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human-chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  9. Evidence for natural recombination between mink enteritis virus and canine parvovirus

    Directory of Open Access Journals (Sweden)

    Wang Jianke

    2012-10-01

    Full Text Available Abstract A virus was isolated from mink showing clinical and pathological signs of enteritis in China. This virus, designated MEV/LN-10, was identified as mink enteritis virus (MEV based on its cytopathic effect in the feline F81 cell line, the hemagglutination (HA and hemagglutination inhibition (HI assay, electron microscopy (EM and animal infection experiments. The complete viral genome was cloned and sequenced. Phylogenetic and recombination analyses on the complete MEV/LN-10 genome showed evidence of recombination between MEV and canine parvovirus (CPV. The genome was composed of the NS1 gene originating from CPV while the VP1 gene was of MEV origin. This is the first demonstration of recombination between a CPV and MEV in nature. Our findings not only provide valuable evidence indicating that recombination is an important genetic mechanism contributing to the variation and evolution of MEV, but also that heterogeneous recombination can occur in the feline parvovirus subspecies.

  10. Isolation of Specific Clones from Nonarrayed BAC Libraries through Homologous Recombination

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    Mikhail Nefedov

    2011-01-01

    Full Text Available We have developed a new approach to screen bacterial artificial chromosome (BAC libraries by recombination selection. To test this method, we constructed an orangutan BAC library using an E. coli strain (DY380 with temperature inducible homologous recombination (HR capability. We amplified one library segment, induced HR at 42∘C to make it recombination proficient, and prepared electrocompetent cells for transformation with a kanamycin cassette to target sequences in the orangutan genome through terminal recombineering homologies. Kanamycin-resistant colonies were tested for the presence of BACs containing the targeted genes by the use of a PCR-assay to confirm the presence of the kanamycin insertion. The results indicate that this is an effective approach for screening clones. The advantage of recombination screening is that it avoids the high costs associated with the preparation, screening, and archival storage of arrayed BAC libraries. In addition, the screening can be conceivably combined with genetic engineering to create knockout and reporter constructs for functional studies.

  11. 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…

  12. Recombining overlapping BACs into a single larger BAC

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    Huxley Clare

    2004-01-01

    Full Text Available Abstract Background BAC clones containing entire mammalian genes including all the transcribed region and long range controlling elements are very useful for functional analysis. Sequenced BACs are available for most of the human and mouse genomes and in many cases these contain intact genes. However, large genes often span more than one BAC, and single BACs covering the entire region of interest are not available. Here we describe a system for linking two or more overlapping BACs into a single clone by homologous recombination. Results The method was used to link a 61-kb insert carrying the final 5 exons of the human CFTR gene onto a 160-kb BAC carrying the first 22 exons. Two rounds of homologous recombination were carried out in the EL350 strain of bacteria which can be induced for the Red genes. In the first round, the inserts of the two overlapping BACs were subcloned into modified BAC vectors using homologous recombination. In the second round, the BAC to be added was linearised with the very rare-cutting enzyme I-PpoI and electroporated into recombination efficient EL350 bacteria carrying the other BAC. Recombined BACs were identified by antibiotic selection and PCR screening and 10% of clones contained the correctly recombined 220-kb BAC. Conclusion The system can be used to link the inserts from any overlapping BAC or PAC clones. The original orientation of the inserts is not important and desired regions of the inserts can be selected. The size limit for the fragments recombined may be larger than the 61 kb used here and multiple BACs in a contig could be combined by alternating use of the two pBACLink vectors. This system should be of use to many investigators wishing to carry out functional analysis on large mammalian genes which are not available in single BAC clones.

  13. Generation of Length Distribution, Length Diagram, Fibrogram, and Statistical Characteristics by Weight of Cotton Blends

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    B. Azzouz

    2007-01-01

    Full Text Available The textile fibre mixture as a multicomponent blend of variable fibres imposes regarding the proper method to predict the characteristics of the final blend. The length diagram and the fibrogram of cotton are generated. Then the length distribution, the length diagram, and the fibrogram of a blend of different categories of cotton are determined. The length distributions by weight of five different categories of cotton (Egyptian, USA (Pima, Brazilian, USA (Upland, and Uzbekistani are measured by AFIS. From these distributions, the length distribution, the length diagram, and the fibrogram by weight of four binary blends are expressed. The length parameters of these cotton blends are calculated and their variations are plotted against the mass fraction x of one component in the blend .These calculated parameters are compared to those of real blends. Finally, the selection of the optimal blends using the linear programming method, based on the hypothesis that the cotton blend parameters vary linearly in function of the components rations, is proved insufficient.

  14. Research Update: Relativistic origin of slow electron-hole recombination in hybrid halide perovskite solar cells

    Science.gov (United States)

    Azarhoosh, Pooya; McKechnie, Scott; Frost, Jarvist M.; Walsh, Aron; van Schilfgaarde, Mark

    2016-09-01

    The hybrid perovskite CH3NH3PbI3 (MAPI) exhibits long minority-carrier lifetimes and diffusion lengths. We show that slow recombination originates from a spin-split indirect-gap. Large internal electric fields act on spin-orbit-coupled band extrema, shifting band-edges to inequivalent wavevectors, making the fundamental gap indirect. From a description of photoluminescence within the quasiparticle self-consistent GW approximation for MAPI, CdTe, and GaAs, we predict carrier lifetime as a function of light intensity and temperature. At operating conditions we find radiative recombination in MAPI is reduced by a factor of more than 350 compared to direct gap behavior. The indirect gap is retained with dynamic disorder.

  15. Recombinant expression, purification, and characterization of an acyl-CoA binding protein from Aspergillus oryzae.

    Science.gov (United States)

    Hao, Qing; Liu, Xiaoguang; Zhao, Guozhong; Jiang, Lu; Li, Ming; Zeng, Bin

    2016-03-01

    To characterize biochemically the lipid metabolism-regulating acyl-CoA binding protein (ACBP) from the industrially-important fungus Aspergillus oryzae. A full-length cDNA encoding a candidate ACBP from A. oryzae (AoACBP) was cloned and expressed in Escherichia coli as a maltose-binding protein (MBP) fusion protein. The MBP-AoACBP protein was purified by an amylose resin chromatography column. SDS-PAGE showed that MBP-AoACBP has an estimated molecular weight of 82 kDa. Microscale thermophoresis binding assay showed that the recombinant AoACBP displayed much greater affinity for palmitoyl-CoA (K d = 80 nM) than for myristoyl-CoA (K d = 510 nM), thus demonstrating the preference of AoACBP for long-chain acyl-CoA. The data support the identification of AoACBP as a long-chain ACBP in A. oryzae.

  16. Data for the co-expression and purification of human recombinant CaMKK2 in complex with calmodulin in Escherichia coli

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    Lisa Gerner

    2016-09-01

    Full Text Available Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2 has been implicated in a range of conditions and pathologies from prostate to hepatic cancer. Here, we describe the expression in Escherichia coli and the purification protocol for the following constructs: full-length CaMKK2 in complex with CaM, CaMKK2 ‘apo’, CaMKK2 (165-501 in complex with CaM, and the CaMKK2 F267G mutant. The protocols described have been optimized for maximum yield and purity with minimal purification steps required and the proteins subsequently used to develop a fluorescence-based assay for drug binding to the kinase, “Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2” [1]. Keywords: CaMKK2, Calmodulin, Fermentation

  17. Construction of a full-length prototype of the BESIII drift chamber and on-detector test for the BESIII drift chamber electronics

    International Nuclear Information System (INIS)

    Qin Zhonghua; Wu Linghui; Liu Jianbei; Chinese Academy of Sciences, Beijing; Yan Zhikang; Hunan Univ., Changsha; Chen Yuanbo; Chen Chang; Xu Meihang; Wang Lan; Ma Xiaoyan; Jin Yan; Liu Rongguang; Tang Xiao; Zhang Guifang; Zhu Qiming; Sheng Huayi; Zhu Kejun

    2007-01-01

    A full-length prototype of the BESIII drift chamber was built. The experience gained on gas sealing, high voltage supply and front-end electronics installation should be greatly beneficial to the successful construction of the BESIII drift chamber. An on-detector test of the BESIII drift chamber electronics was carried out with the constructed prototype chamber. The noise performance, drift time and charge measurements, and electronics gains were examined specifically. The final test results indicate that the electronics have a good performance and can satisfy their design requirements. (authors)

  18. GROWTH RESPONSE OF CLOWN LOACH (Chromobotia macracanthus Bleeker 1852 JUVENILES IMMERSED IN WATER CONTAINING RECOMBINANT GROWTH HORMONE

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    Asep Permana

    2015-12-01

    Full Text Available The main problem in the culture of clown loach (Chromobotia macracanthus is the slow growth rate, which takes about six months to reach its market size (two inches total body length. Slow growth eventually cause a long production time and increase the production costs. An alternative solution can be proposed in order to enhance the growth is by using recombinant growth hormone. The aim of this study was to determine the immersion dose of recombinant Epinephelus lanceolatus growth hormone (rElGH which can generate the highest growth in clown loach. Larvae at seven day after hatching were hyperosmotic treated with NaCl 2.0% for one minute, then immersed for one hour in water containing 0.3% NaCl, 0.01% bovine serum albumin (BSA, and different doses of rElGH, namely: 0.12 (treatment A, 1.2 (B, 12 (C, and 120 mg/L (D. As control, fish were immersed in water without rElGH and NaCl (control-1, water containing 0.3% NaCl and 0.01% BSA (control-2, and 0.3% NaCl water (control-3. Each treatment was replicated three times. The results showed that clown loach juveniles in treatment B, C, and D had longer total body length (P0.05. In addition, the percentage of large size juveniles increased approximately 5% in treatment B, almost the same as in the medium size, while the small size were decrease compared to the control-1. Thus, the best immersion dose of rElGH was 1.2 mg/L water.

  19. Prediction of recombinant protein overexpression in Escherichia coli using a machine learning based model (RPOLP).

    Science.gov (United States)

    Habibi, Narjeskhatoon; Norouzi, Alireza; Mohd Hashim, Siti Z; Shamsir, Mohd Shahir; Samian, Razip

    2015-11-01

    Recombinant protein overexpression, an important biotechnological process, is ruled by complex biological rules which are mostly unknown, is in need of an intelligent algorithm so as to avoid resource-intensive lab-based trial and error experiments in order to determine the expression level of the recombinant protein. The purpose of this study is to propose a predictive model to estimate the level of recombinant protein overexpression for the first time in the literature using a machine learning approach based on the sequence, expression vector, and expression host. The expression host was confined to Escherichia coli which is the most popular bacterial host to overexpress recombinant proteins. To provide a handle to the problem, the overexpression level was categorized as low, medium and high. A set of features which were likely to affect the overexpression level was generated based on the known facts (e.g. gene length) and knowledge gathered from related literature. Then, a representative sub-set of features generated in the previous objective was determined using feature selection techniques. Finally a predictive model was developed using random forest classifier which was able to adequately classify the multi-class imbalanced small dataset constructed. The result showed that the predictive model provided a promising accuracy of 80% on average, in estimating the overexpression level of a recombinant protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Cosmic microwave background bispectrum from recombination.

    Science.gov (United States)

    Huang, Zhiqi; Vernizzi, Filippo

    2013-03-08

    We compute the cosmic microwave background temperature bispectrum generated by nonlinearities at recombination on all scales. We use CosmoLib2nd, a numerical Boltzmann code at second order to compute cosmic microwave background bispectra on the full sky. We consistently include all effects except gravitational lensing, which can be added to our result using standard methods. The bispectrum is peaked on squeezed triangles and agrees with the analytic approximation in the squeezed limit at the few percent level for all the scales where this is applicable. On smaller scales, we recover previous results on perturbed recombination. For cosmic-variance limited data to l(max)=2000, its signal-to-noise ratio is S/N=0.47, corresponding to f(NL)(eff)=-2.79, and will bias a local signal by f(NL)(loc) ~/= 0.82.

  1. Recombinant PrPSc shares structural features with brain-derived PrPSc: Insights from limited proteolysis.

    Science.gov (United States)

    Sevillano, Alejandro M; Fernández-Borges, Natalia; Younas, Neelam; Wang, Fei; R Elezgarai, Saioa; Bravo, Susana; Vázquez-Fernández, Ester; Rosa, Isaac; Eraña, Hasier; Gil, David; Veiga, Sonia; Vidal, Enric; Erickson-Beltran, Melissa L; Guitián, Esteban; Silva, Christopher J; Nonno, Romolo; Ma, Jiyan; Castilla, Joaquín; R Requena, Jesús

    2018-01-01

    Very solid evidence suggests that the core of full length PrPSc is a 4-rung β-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the β-strands and connecting loops that make up the PrPSc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions ~116/118, 133-134, 141, 152-153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrPSc. Such sites likely define loops and/or borders of β-strands, helping us to predict the threading of the β-solenoid. We have now extended this approach to recombinant PrPSc (recPrPSc). The term recPrPSc refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrPSc species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrPSc, albeit with varying relative yields. Along with these fragments, doubly N- and C-terminally truncated fragments, in particular ~89/97-152, were detected in some recPrPSc preparations; similar fragments are characteristic of atypical strains of brain-derived PrPSc. Our results suggest a shared architecture of recPrPSc and brain PrPSc prions. The observed differences, in particular the distinct yields of specific PK-resistant fragments, are likely due to differences in threading which result in the specific biochemical characteristics of recPrPSc. Furthermore, recombinant PrPSc offers exciting opportunities for structural studies unachievable with brain-derived PrPSc.

  2. The Genetic Architecture of Natural Variation in Recombination Rate in Drosophila melanogaster.

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    Chad M Hunter

    2016-04-01

    Full Text Available Meiotic recombination ensures proper chromosome segregation in many sexually reproducing organisms. Despite this crucial function, rates of recombination are highly variable within and between taxa, and the genetic basis of this variation remains poorly understood. Here, we exploit natural variation in the inbred, sequenced lines of the Drosophila melanogaster Genetic Reference Panel (DGRP to map genetic variants affecting recombination rate. We used a two-step crossing scheme and visible markers to measure rates of recombination in a 33 cM interval on the X chromosome and in a 20.4 cM interval on chromosome 3R for 205 DGRP lines. Though we cannot exclude that some biases exist due to viability effects associated with the visible markers used in this study, we find ~2-fold variation in recombination rate among lines. Interestingly, we further find that recombination rates are uncorrelated between the two chromosomal intervals. We performed a genome-wide association study to identify genetic variants associated with recombination rate in each of the two intervals surveyed. We refined our list of candidate variants and genes associated with recombination rate variation and selected twenty genes for functional assessment. We present strong evidence that five genes are likely to contribute to natural variation in recombination rate in D. melanogaster; these genes lie outside the canonical meiotic recombination pathway. We also find a weak effect of Wolbachia infection on recombination rate and we confirm the interchromosomal effect. Our results highlight the magnitude of population variation in recombination rate present in D. melanogaster and implicate new genetic factors mediating natural variation in this quantitative trait.

  3. Recombination-ready Sindbis replicon expression vectors for transgene expression

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    Olson Ken E

    2007-10-01

    Full Text Available Abstract Background Sindbis viruses have been widely used as tools to study gene function in cells. Despite the utility of these systems, the construction and production of alphavirus replicons is time consuming and inefficient due to potential additional restriction sites within the insert region and lack of directionality for insert ligation. In this report, we present a system useful for producing recombinant Sindbis replicons that uses lambda phage recombination technology to rapidly and specifically construct replicon expression plasmids that contain insert regions in the desired orientation. Results Recombination of the gene of interest with the replicon plasmid resulted in nearly 100% recombinants, each of which contained a correctly orientated insert. Replicons were easily produced in cell culture and packaged into pseudo-infectious viral particles. Insect and mammalian cells infected with pseudo-infectious viral particles expressed various transgenes at high levels. Finally, inserts from persistently replicating replicon RNA were easily isolated and recombined back into entry plasmids for sequencing and subsequent analysis. Conclusion Replication-ready replicon expression plasmids make the use of alphavirus replicons fast and easy as compared to traditional replicon production methods. This system represents a significant step forward in the utility and ease of use of alphavirus replicons in the study of gene function.

  4. No Evidence that Infection Alters Global Recombination Rate in House Mice.

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    Beth L Dumont

    Full Text Available Recombination rate is a complex trait, with genetic and environmental factors shaping observed patterns of variation. Although recent studies have begun to unravel the genetic basis of recombination rate differences between organisms, less attention has focused on the environmental determinants of crossover rates. Here, we test the effect of one ubiquitous environmental pressure-bacterial infection-on global recombination frequency in mammals. We applied MLH1 mapping to assay global crossover rates in male mice infected with the pathogenic bacterium Borrelia burgdorferi, the causative agent of Lyme Disease, and uninfected control animals. Despite ample statistical power to identify biologically relevant differences between infected and uninfected animals, we find no evidence for a global recombination rate response to bacterial infection. Moreover, broad-scale patterns of crossover distribution, including the number of achiasmate bivalents, are not affected by infection status. Although pathogen exposure can plastically increase recombination in some species, our findings suggest that recombination rates in house mice may be resilient to at least some forms of infection stress. This negative result motivates future experiments with alternative house mouse pathogens to evaluate the generality of this conclusion.

  5. Expression of recombinant Antibodies

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    André eFrenzel

    2013-07-01

    Full Text Available Recombinant antibodies are highly specific detection probes in research, diagnostics and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines and transgenic plants are promising to obtain antibodies with human-like post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  6. Detection and frequency of recombination in tomato-infecting begomoviruses of South and Southeast Asia

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    Rai Mathura

    2007-10-01

    Full Text Available Abstract Background Tomato-infecting begomoviruses are widely distributed across the world and cause diseases of high economic impact on wide range of agriculturally important crops. Though recombination plays a pivotal role in diversification and evolution of these viruses, it is currently unknown whether there are differences in the number and quality of recombination events amongst different tomato-infecting begomovirus species. To examine this we sought to characterize the recombination events, estimate the frequency of recombination, and map recombination hotspots in tomato-infecting begomoviruses of South and Southeast Asia. Results Different methods used for recombination breakpoint analysis provided strong evidence for presence of recombination events in majority of the sequences analyzed. However, there was a clear evidence for absence or low Recombination events in viruses reported from North India. In addition, we provide evidence for non-random distribution of recombination events with the highest frequency of recombination being mapped in the portion of the N-terminal portion of Rep. Conclusion The variable recombination observed in these viruses signified that all begomoviruses are not equally prone to recombination. Distribution of recombination hotspots was found to be reliant on the relatedness of the genomic region involved in the exchange. Overall the frequency of phylogenetic violations and number of recombination events decreased with increasing parental sequence diversity. These findings provide valuable new information for understanding the diversity and evolution of tomato-infecting begomoviruses in Asia.

  7. Optimal recombination in genetic algorithms for combinatorial optimization problems: Part I

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    Eremeev Anton V.

    2014-01-01

    Full Text Available This paper surveys results on complexity of the optimal recombination problem (ORP, which consists in finding the best possible offspring as a result of a recombination operator in a genetic algorithm, given two parent solutions. We consider efficient reductions of the ORPs, allowing to establish polynomial solvability or NP-hardness of the ORPs, as well as direct proofs of hardness results. Part I presents the basic principles of optimal recombination with a survey of results on Boolean Linear Programming Problems. Part II (to appear in a subsequent issue is devoted to the ORPs for problems which are naturally formulated in terms of search for an optimal permutation.

  8. The small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma and full-length FOXP1 exert similar oncogenic and transcriptional activity in human B cells.

    Science.gov (United States)

    van Keimpema, Martine; Grüneberg, Leonie J; Schilder-Tol, Esther J M; Oud, Monique E C M; Beuling, Esther A; Hensbergen, Paul J; de Jong, Johann; Pals, Steven T; Spaargaren, Marcel

    2017-03-01

    The forkhead transcription factor FOXP1 is generally regarded as an oncogene in activated B cell-like diffuse large B-cell lymphoma. Previous studies have suggested that a small isoform of FOXP1 rather than full-length FOXP1, may possess this oncogenic activity. Corroborating those studies, we herein show that activated B cell-like diffuse large B-cell lymphoma cell lines and primary activated B cell-like diffuse large B-cell lymphoma cells predominantly express a small FOXP1 isoform, and that the 5'-end of the Foxp1 gene is a common insertion site in murine lymphomas in leukemia virus- and transposon-mediated insertional mutagenesis screens. By combined mass spectrometry, (quantative) reverse transcription polymerase chain reaction/sequencing, and small interfering ribonucleic acid-mediated gene silencing, we determined that the small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma lacks the N-terminal 100 amino acids of full-length FOXP1. Aberrant overexpression of this FOXP1 isoform (ΔN100) in primary human B cells revealed its oncogenic capacity; it repressed apoptosis and plasma cell differentiation. However, no difference in potency was found between this small FOXP1 isoform and full-length FOXP1. Furthermore, overexpression of full-length FOXP1 or this small FOXP1 isoform in primary B cells and diffuse large B-cell lymphoma cell lines resulted in similar gene regulation. Taken together, our data indicate that this small FOXP1 isoform and full-length FOXP1 have comparable oncogenic and transcriptional activity in human B cells, suggesting that aberrant expression or overexpression of FOXP1, irrespective of the specific isoform, contributes to lymphomagenesis. These novel insights further enhance the value of FOXP1 for the diagnostics, prognostics, and treatment of diffuse large B-cell lymphoma patients. Copyright© Ferrata Storti Foundation.

  9. Identifying recombinants in human and primate immunodeficiency virus sequence alignments using quartet scanning

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    Martin Darren P

    2009-04-01

    Full Text Available Abstract Background Recombination has a profound impact on the evolution of viruses, but characterizing recombination patterns in molecular sequences remains a challenging endeavor. Despite its importance in molecular evolutionary studies, identifying the sequences that exhibit such patterns has received comparatively less attention in the recombination detection framework. Here, we extend a quartet-mapping based recombination detection method to enable identification of recombinant sequences without prior specifications of either query and reference sequences. Through simulations we evaluate different recombinant identification statistics and significance tests. We compare the quartet approach with triplet-based methods that employ additional heuristic tests to identify parental and recombinant sequences. Results Analysis of phylogenetic simulations reveal that identifying the descendents of relatively old recombination events is a challenging task for all methods available, and that quartet scanning performs relatively well compared to the triplet based methods. The use of quartet scanning is further demonstrated by analyzing both well-established and putative HIV-1 recombinant strains. In agreement with recent findings, we provide evidence that the presumed circulating recombinant CRF02_AG is a 'pure' lineage, whereas the presumed parental lineage subtype G has a recombinant origin. We also demonstrate HIV-1 intrasubtype recombination, confirm the hybrid origin of SIV in chimpanzees and further disentangle the recombinant history of SIV lineages in a primate immunodeficiency virus data set. Conclusion Quartet scanning makes a valuable addition to triplet-based methods for identifying recombinant sequences without prior specifications of either query and reference sequences. The new method is available in the VisRD v.3.0 package http://www.cmp.uea.ac.uk/~vlm/visrd.

  10. A new theraphosid spider toxin causes early insect cell death by necrosis when expressed in vitro during recombinant baculovirus infection.

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    Daniel Mendes Pereira Ardisson-Araújo

    Full Text Available Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3 has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV expression. Five different forms of Ba3 were assessed; (1 the full-length sequence, (2 the pro-peptide and mature region, (3 only the mature region, and the mature region fused to an (4 insect or a (5 virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect

  11. Bacteriophage T5 encodes a homolog of the eukaryotic transcription coactivator PC4 implicated in recombination-dependent DNA replication.

    Science.gov (United States)

    Steigemann, Birthe; Schulz, Annina; Werten, Sebastiaan

    2013-11-15

    The RNA polymerase II cofactor PC4 globally regulates transcription of protein-encoding genes through interactions with unwinding DNA, the basal transcription machinery and transcription activators. Here, we report the surprising identification of PC4 homologs in all sequenced representatives of the T5 family of bacteriophages, as well as in an archaeon and seven phyla of eubacteria. We have solved the crystal structure of the full-length T5 protein at 1.9Å, revealing a striking resemblance to the characteristic single-stranded DNA (ssDNA)-binding core domain of PC4. Intriguing novel structural features include a potential regulatory region at the N-terminus and a C-terminal extension of the homodimerisation interface. The genome organisation of T5-related bacteriophages points at involvement of the PC4 homolog in recombination-dependent DNA replication, strongly suggesting that the protein corresponds to the hitherto elusive replicative ssDNA-binding protein of the T5 family. Our findings imply that PC4-like factors intervene in multiple unwinding-related processes by acting as versatile modifiers of nucleic acid conformation and raise the possibility that the eukaryotic transcription coactivator derives from ancestral DNA replication, recombination and repair factors. © 2013.

  12. Study of Yield and Effective Traits in Bread Wheat Recombinant Inbred Lines (Triticum aestivum L. under Water Deficit Condition

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    S. Mohammad zadeh

    2013-11-01

    Full Text Available The effects some traits on seed yield of recombinant inbred lines of wheat under water deficit stress was studied. This research was done at the Agricultural Research Stations, Islamic Azad University, Tabriz Branch in 2010- 2011. 28 recombinant inbred lines of wheat bread with two parents (Norstar and Zagros in split plot experiment based on a randomized complete block design with three replications at two irrigation levels (70 and 140 mm evaporation from pan class A were studied. Analysis of variance indicated a significant genetic differences in all traits under study among the lines. Lines No. 32, 163 and 182 produced highest yield under both irrigation levels. Number of spikes, grains per spike and harvest index had the highest positive correlation with grain yield. Path analysis based on stepwise regression showed that under the normal irrigation conditions, number spike (0.556, number of grains per spike (0.278, weight of 1000 grain (0.259 and the drought stress number spike (0.430, straw yield (0.276 and peduncle length (0.323 had the most direct and positive effect on yield respectively.

  13. A large outbreak of acute gastroenteritis in Shippensburg, Pennsylvania, 1972 revisited: evidence for common source exposure to a recombinant GII.Pg/GII.3 norovirus.

    Science.gov (United States)

    Johnson, J A; Parra, G I; Levenson, E A; Green, K Y

    2017-06-01

    Historical outbreaks can be an important source of information in the understanding of norovirus evolution and epidemiology. Here, we revisit an outbreak of undiagnosed gastroenteritis that occurred in Shippensburg, Pennsylvania in 1972. Nearly 5000 people fell ill over the course of 10 days. Symptoms included diarrhea, vomiting, stomach cramps, and fever, lasting for a median of 24 h. Using current techniques, including next-generation sequencing of full-length viral genomic amplicons, we identified an unusual norovirus recombinant (GII.Pg/GII.3) in nine of 15 available stool samples from the outbreak. This particular recombinant virus has not been reported in recent decades, although GII.3 and GII.Pg genotypes have been detected individually in current epidemic strains. The consensus nucleotide sequences were nearly identical among the four viral genomes analysed, although each strain had three to seven positions in the genome with heterogenous non-synonymous nucleotide subpopulations. Two of these resulting amino acid polymorphisms were conserved in frequency among all four cases, consistent with common source exposure and successful transmission of a mixed viral population. Continued investigation of variant nucleotide populations and recombination events among ancestral norovirus strains such as the Shippensburg virus may provide unique insight into the origin of contemporary strains.

  14. Study of a photovoltaic cell to silicon tri grain under illumination in static mode: determination of the parameters of recombination

    International Nuclear Information System (INIS)

    ZERBO Issa

    2000-01-01

    A study of the photovoltaic cell to silicon tri grain under illumination functioning at a static normal rate is presented. The determination of the parameters of recombination relies on the analysis of the photo-answer of the photovoltaic cell. The length of diffusion L, the speeds of recombination of minority carriers and respectively on the surface of the junction and with the back face of the base of the photovoltaic cell are extracted thanks to the measurement from the from short-circuit electricity and the tension from open circuit [fr

  15. 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

  16. Full-length genome sequences of porcine epidemic diarrhoea virus strain CV777; Use of NGS to analyse genomic and sub-genomic RNAs

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Boniotti, Maria Beatrice; Papetti, Alice

    2018-01-01

    Porcine epidemic diarrhoea virus, strain CV777, was initially characterized in 1978 as the causative agent of a disease first identified in the UK in 1971. This coronavirus has been widely distributed among laboratories and has been passaged both within pigs and in cell culture. To determine...... the variability between different stocks of the PEDV strain CV777, sequencing of the full-length genome (ca. 28kb) has been performed in 6 different laboratories, using different protocols. Not surprisingly, each of the different full genome sequences were distinct from each other and from the reference sequence...... the analysis of sub-genomic mRNAs from infected cells. It is clearly important to know the features of the specific sample of CV777 being used for experimental studies....

  17. Immunoglobulin class-switch recombination deficiencies.

    Science.gov (United States)

    Durandy, Anne; Kracker, Sven

    2012-07-30

    Immunoglobulin class-switch recombination deficiencies (Ig-CSR-Ds) are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect in question, the Ig-CSR-D may be combined with an impairment in somatic hypermutation (SHM). Some of the mechanisms underlying Ig-CSR and SHM have been described by studying natural mutants in humans. This approach has revealed that T cell-B cell interaction (resulting in CD40-mediated signaling), intrinsic B-cell mechanisms (activation-induced cytidine deaminase-induced DNA damage), and complex DNA repair machineries (including uracil-N-glycosylase and mismatch repair pathways) are all involved in class-switch recombination and SHM. However, several of the mechanisms required for full antibody maturation have yet to be defined. Elucidation of the molecular defects underlying the diverse set of Ig-CSR-Ds is essential for understanding Ig diversification and has prompted better definition of the clinical spectrum of diseases and the development of increasingly accurate diagnostic and therapeutic approaches.

  18. Recombination in feline immunodeficiency virus from feral and companion domestic cats

    Directory of Open Access Journals (Sweden)

    Rodrigo Allen G

    2008-06-01

    Full Text Available Abstract Background Recombination is a relatively common phenomenon in retroviruses. We investigated recombination in Feline Immunodeficiency Virus from naturally-infected New Zealand domestic cats (Felis catus by sequencing regions of the gag, pol and env genes. Results The occurrence of intragenic recombination was highest in env, with evidence of recombination in 6.4% (n = 156 of all cats. A further recombinant was identified in each of the gag (n = 48 and pol (n = 91 genes. Comparisons of phylogenetic trees across genes identified cases of incongruence, indicating intergenic recombination. Three (7.7%, n = 39 of these incongruencies were found to be significantly different using the Shimodaira-Hasegawa test. Surprisingly, our phylogenies from the gag and pol genes showed that no New Zealand sequences group with reference subtype C sequences within intrasubtype pairwise distances. Indeed, we find one and two distinct unknown subtype groups in gag and pol, respectively. These observations cause us to speculate that these New Zealand FIV strains have undergone several recombination events between subtype A parent strains and undefined unknown subtype strains, similar to the evolutionary history hypothesised for HIV-1 "subtype E". Endpoint dilution sequencing was used to confirm the consensus sequences of the putative recombinants and unknown subtype groups, providing evidence for the authenticity of these sequences. Endpoint dilution sequencing also resulted in the identification of a dual infection event in the env gene. In addition, an intrahost recombination event between variants of the same subtype in the pol gene was established. This is the first known example of naturally-occurring recombination in a cat with infection of the parent strains. Conclusion Evidence of intragenic recombination in the gag, pol and env regions, and complex intergenic recombination, of FIV from naturally-infected domestic cats in New Zealand was found. Strains

  19. A Novel Cold-Active Lipase from Candida albicans: Cloning, Expression and Characterization of the Recombinant Enzyme

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    Dong-Ming Lan

    2011-06-01

    Full Text Available A novel lipase gene lip5 from the yeast Candida albicans was cloned and sequenced. Alignment of amino acid sequences revealed that 86–34% identity exists with lipases from other Candida species. The lipase and its mutants were expressed in the yeast Pichia pastoris, where alternative codon usage caused the mistranslation of 154-Ser and 293-Ser as leucine. 154-Ser to leucine resulted in loss of expression of Lip5, and 293-Ser to leucine caused a marked reduction in the lipase activity. Lip5-DM, which has double mutations that revert 154 and 293 to serine residues, showed good lipase activity, and was overexpressed and purified by (NH42SO4 precipitation and ion-exchange chromatography. The pure Lip5-DM was stable at low temperatures ranging from 15–35 °C and pH 5–9, with the optimal conditions being 15–25 °C and pH 5–6. The activation energy of recombinant lipase was 8.5 Kcal/mol between 5 and 25 °C, suggesting that Lip5-DM was a cold–active lipase. Its activity was found to increase in the presence of Zn2+, but it was strongly inhibited by Fe2+, Fe3+, Hg2+ and some surfactants. In addition, the Lip5-DM could not tolerate water-miscible organic solvents. Lip5-DM exhibited a preference for the short- and medium-chain length p-nitrophenyl (C4 and C8 acyl group esters rather than the long chain length p-nitrophenyl esters (C12, C16 and C18 acyl group with highest activity observed with the C8 derivatives. The recombinant enzyme displayed activity toward triacylglycerols, such as olive oil and safflower oil.

  20. Induction of mutation and recombination following UV irradiation during meiosis in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Kelly, S.L.; Parry, J.M. (University Coll. of Swansea (UK). Dept. of Genetics)

    1983-03-01

    Irradiation of yeast cultures with ultraviolet light at discrete stages during meiosis produces cyclic variations in sensitivity, i.e. cells are more sensitive to the lethal effects of UV light prior to entry into the meiotic DNA synthesis, and this corresponds to a peak of induction of point mutation. Cells become more resistant to both induced point mutation and lethality as they enter meiotic DNA synthesis, but become more sensitive again during spore formation. The induced level of intragenic recombination rises during the period of commitment ot recombination to a level indistinguishable from the full meiotic level of spontaneous intragenic recombination. Induced reciprocal recombination remains above the spontaneous level up to the point of commitment to sporulation.

  1. Insights into the Functions of a Prophage Recombination Directionality Factor

    Directory of Open Access Journals (Sweden)

    Mireille Ansaldi

    2012-10-01

    Full Text Available Recombination directionality factors (RDFs, or excisionases, are essential players of prophage excisive recombination. Despite the essentially catalytic role of the integrase in both integrative and excisive recombination, RDFs are required to direct the reaction towards excision and to prevent re-integration of the prophage genome when entering a lytic cycle. KplE1, HK620 and numerous (prophages that integrate at the same site in enterobacteria genomes (such as the argW tRNA gene all share a highly conserved recombination module. This module comprises the attL and attR recombination sites and the RDF and integrase genes. The KplE1 RDF was named TorI after its initial identification as a negative regulator of the tor operon. However, it was characterized as an essential factor of excisive recombination. In this study, we designed an extensive random mutagenesis protocol of the torI gene and identified key residues involved in both functions of the TorI protein. We show that, in addition to TorI-TorR protein-protein interaction, TorI interacts in solution with the IntS integrase. Moreover, in vitro, TorR and IntS appear to compete for TorI binding. Finally, our mutagenesis results suggest that the C-terminal part of the TorI protein is dedicated to protein-protein interactions with both proteins TorR and IntS.

  2. Lineage specific recombination rates and microevolution in Listeria monocytogenes

    Directory of Open Access Journals (Sweden)

    Nightingale Kendra K

    2008-10-01

    Full Text Available Abstract Background The bacterium Listeria monocytogenes is a saprotroph as well as an opportunistic human foodborne pathogen, which has previously been shown to consist of at least two widespread lineages (termed lineages I and II and an uncommon lineage (lineage III. While some L. monocytogenes strains show evidence for considerable diversification by homologous recombination, our understanding of the contribution of recombination to L. monocytogenes evolution is still limited. We therefore used STRUCTURE and ClonalFrame, two programs that model the effect of recombination, to make inferences about the population structure and different aspects of the recombination process in L. monocytogenes. Analyses were performed using sequences for seven loci (including the house-keeping genes gap, prs, purM and ribC, the stress response gene sigB, and the virulence genes actA and inlA for 195 L. monocytogenes isolates. Results Sequence analyses with ClonalFrame and the Sawyer's test showed that recombination is more prevalent in lineage II than lineage I and is most frequent in two house-keeping genes (ribC and purM and the two virulence genes (actA and inlA. The relative occurrence of recombination versus point mutation is about six times higher in lineage II than in lineage I, which causes a higher genetic variability in lineage II. Unlike lineage I, lineage II represents a genetically heterogeneous population with a relatively high proportion (30% average of genetic material imported from external sources. Phylograms, constructed with correcting for recombination, as well as Tajima's D data suggest that both lineages I and II have suffered a population bottleneck. Conclusion Our study shows that evolutionary lineages within a single bacterial species can differ considerably in the relative contributions of recombination to genetic diversification. Accounting for recombination in phylogenetic studies is critical, and new evolutionary models that

  3. Effects of Dissociation/Recombination on the Day–Night Temperature Contrasts of Ultra-hot Jupiters

    Science.gov (United States)

    Komacek, Thaddeus D.; Tan, Xianyu

    2018-05-01

    Secondary eclipse observations of ultra-hot Jupiters have found evidence that hydrogen is dissociated on their daysides. Additionally, full-phase light curve observations of ultra-hot Jupiters show a smaller day-night emitted flux contrast than that expected from previous theory. Recently, it was proposed by Bell & Cowan (2018) that the heat intake to dissociate hydrogen and heat release due to recombination of dissociated hydrogen can affect the atmospheric circulation of ultra-hot Jupiters. In this work, we add cooling/heating due to dissociation/recombination into the analytic theory of Komacek & Showman (2016) and Zhang & Showman (2017) for the dayside-nightside temperature contrasts of hot Jupiters. We find that at high values of incident stellar flux, the day-night temperature contrast of ultra-hot Jupiters may decrease with increasing incident stellar flux due to dissociation/recombination, the opposite of that expected without including the effects of dissociation/recombination. We propose that a combination of a greater number of full-phase light curve observations of ultra-hot Jupiters and future General Circulation Models that include the effects of dissociation/recombination could determine in detail how the atmospheric circulation of ultra-hot Jupiters differs from that of cooler planets.

  4. PCR artifact in testing for homologous recombination in genomic editing in zebrafish.

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    Minho Won

    Full Text Available We report a PCR-induced artifact in testing for homologous recombination in zebrafish. We attempted to replace the lnx2a gene with a donor cassette, mediated by a TALEN induced double stranded cut. The donor construct was flanked with homology arms of about 1 kb at the 5' and 3' ends. Injected embryos (G0 were raised and outcrossed to wild type fish. A fraction of the progeny appeared to have undergone the desired homologous recombination, as tested by PCR using primer pairs extending from genomic DNA outside the homology region to a site within the donor cassette. However, Southern blots revealed that no recombination had taken place. We conclude that recombination happened during PCR in vitro between the donor integrated elsewhere in the genome and the lnx2a locus. We conclude that PCR alone may be insufficient to verify homologous recombination in genome editing experiments in zebrafish.

  5. Recombination of cluster ions

    Science.gov (United States)

    Johnsen, Rainer

    1993-01-01

    Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

  6. Bacterial Artificial Chromosome Mutagenesis Using Recombineering

    Directory of Open Access Journals (Sweden)

    Kumaran Narayanan

    2011-01-01

    Full Text Available Gene expression from bacterial artificial chromosome (BAC clones has been demonstrated to facilitate physiologically relevant levels compared to viral and nonviral cDNA vectors. BACs are large enough to transfer intact genes in their native chromosomal setting together with flanking regulatory elements to provide all the signals for correct spatiotemporal gene expression. Until recently, the use of BACs for functional studies has been limited because their large size has inherently presented a major obstacle for introducing modifications using conventional genetic engineering strategies. The development of in vivo homologous recombination strategies based on recombineering in E. coli has helped resolve this problem by enabling facile engineering of high molecular weight BAC DNA without dependence on suitably placed restriction enzymes or cloning steps. These techniques have considerably expanded the possibilities for studying functional genetics using BACs in vitro and in vivo.

  7. In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors.

    Science.gov (United States)

    Guijarro-Pardo, Eva; Gómez-Sebastián, Silvia; Escribano, José M

    2017-12-01

    Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a

  8. Large-scale analysis of full-length cDNAs from the tomato (Solanum lycopersicum) cultivar Micro-Tom, a reference system for the Solanaceae genomics.

    Science.gov (United States)

    Aoki, Koh; Yano, Kentaro; Suzuki, Ayako; Kawamura, Shingo; Sakurai, Nozomu; Suda, Kunihiro; Kurabayashi, Atsushi; Suzuki, Tatsuya; Tsugane, Taneaki; Watanabe, Manabu; Ooga, Kazuhide; Torii, Maiko; Narita, Takanori; Shin-I, Tadasu; Kohara, Yuji; Yamamoto, Naoki; Takahashi, Hideki; Watanabe, Yuichiro; Egusa, Mayumi; Kodama, Motoichiro; Ichinose, Yuki; Kikuchi, Mari; Fukushima, Sumire; Okabe, Akiko; Arie, Tsutomu; Sato, Yuko; Yazawa, Katsumi; Satoh, Shinobu; Omura, Toshikazu; Ezura, Hiroshi; Shibata, Daisuke

    2010-03-30

    The Solanaceae family includes several economically important vegetable crops. The tomato (Solanum lycopersicum) is regarded as a model plant of the Solanaceae family. Recently, a number of tomato resources have been developed in parallel with the ongoing tomato genome sequencing project. In particular, a miniature cultivar, Micro-Tom, is regarded as a model system in tomato genomics, and a number of genomics resources in the Micro-Tom-background, such as ESTs and mutagenized lines, have been established by an international alliance. To accelerate the progress in tomato genomics, we developed a collection of fully-sequenced 13,227 Micro-Tom full-length cDNAs. By checking redundant sequences, coding sequences, and chimeric sequences, a set of 11,502 non-redundant full-length cDNAs (nrFLcDNAs) was generated. Analysis of untranslated regions demonstrated that tomato has longer 5'- and 3'-untranslated regions than most other plants but rice. Classification of functions of proteins predicted from the coding sequences demonstrated that nrFLcDNAs covered a broad range of functions. A comparison of nrFLcDNAs with genes of sixteen plants facilitated the identification of tomato genes that are not found in other plants, most of which did not have known protein domains. Mapping of the nrFLcDNAs onto currently available tomato genome sequences facilitated prediction of exon-intron structure. Introns of tomato genes were longer than those of Arabidopsis and rice. According to a comparison of exon sequences between the nrFLcDNAs and the tomato genome sequences, the frequency of nucleotide mismatch in exons between Micro-Tom and the genome-sequencing cultivar (Heinz 1706) was estimated to be 0.061%. The collection of Micro-Tom nrFLcDNAs generated in this study will serve as a valuable genomic tool for plant biologists to bridge the gap between basic and applied studies. The nrFLcDNA sequences will help annotation of the tomato whole-genome sequence and aid in tomato functional

  9. Electron-ion recombination in merged beams

    International Nuclear Information System (INIS)

    Wolf, A.; Habs, D.; Lampert, A.; Neumann, R.; Schramm, U.; Schuessler, T.; Schwalm, D.

    1993-01-01

    Detailed studies of recombination processes between electrons and highly charged ions have become possible by recent improvements of merged-beams experiments. We discuss in particular measurements with stored cooled ion beams at the Test Storage Ring (TSR) in Heidelberg. The cross section of dielectronic recombination was measured with high energy resolution for few-electron systems up to the nuclear charge of Cu at a relative energy up to 2.6 keV. At low energy (∼0.1 eV) total recombination rates of several ions were measured and compared with calculated radiative recombination rates. Laser-stimulated recombination of protons and of C 6+ ions was investigated as a function of the photon energy using visible radiation. Both the total recombination rates and the stimulated recombination spectra indicate that in spite of the short interaction time in merged beams, also collisional capture of electrons into weakly bound levels (related to three-body recombination) could be important

  10. Novel HBV recombinants between genotypes B and C in 3'-terminal reverse transcriptase (RT) sequences are associated with enhanced viral DNA load, higher RT point mutation rates and place of birth among Chinese patients.

    Science.gov (United States)

    Liu, Baoming; Yang, Jing-Xian; Yan, Ling; Zhuang, Hui; Li, Tong

    2018-01-01

    As one of the major global public health concerns, hepatitis B virus (HBV) can be divided into at least eight genotypes, which may be related to disease severity and treatment response. We previously demonstrated that genotypes B and C HBV, with distinct geographical distribution in China, had divergent genotype-dependent amino acid polymorphisms and variations in reverse transcriptase (RT) gene region, a target of antiviral therapy using nucleos(t)ide analogues. Recently recombination between HBV genotypes B and C was reported to occur in the RT region. However, their frequency and clinical significance is poorly understood. Here full-length HBV RT sequences from 201 Chinese chronic hepatitis B (CHB) patients were amplified and sequenced, among which 31.34% (63/201) were genotype B whereas 68.66% (138/201) genotype C. Although no intergenotypic recombination was detected among C-genotype HBV, 38.10% (24/63) of B-genotype HBV had recombination with genotype C in the 3'-terminal RT sequences. The patients with B/C intergenotypic recombinants had significantly (Pdistribution feature in China. Our findings provide novel insight into the virological, clinical and epidemiological features of new HBV B/C intergenotypic recombinants at the 3' end of RT sequences among Chinese CHB patients. The highly complex genetic background of the novel recombinant HBV carrying new mutations affecting RT protein may contribute to an enhanced heterogeneity in treatment response or prognosis among CHB patients. Published by Elsevier B.V.

  11. Rate constant of free electrons and holes recombination in thin films CdSe

    International Nuclear Information System (INIS)

    Radychev, N.A.; Novikov, G.F.

    2006-01-01

    Destruction kinetics of electrons generated in thin films CdSe by laser impulse (wave length is 337 nm, period of impulse - 8 nc) is studied by the method of microwave photoconductivity (36 GHz) at 295 K. Model of the process was suggested using the analysis of kinetics of photo-responses decay, and it allowed determination of rate constant of recombination of free electrons and holes in cadmium selenide - (4-6)x10 -11 cm 3 s -1 [ru

  12. Frequent sgRNA-barcode recombination in single-cell perturbation assays.

    Directory of Open Access Journals (Sweden)

    Shiqi Xie

    Full Text Available Simultaneously detecting CRISPR-based perturbations and induced transcriptional changes in the same cell is a powerful approach to unraveling genome function. Several lentiviral approaches have been developed, some of which rely on the detection of distally located genetic barcodes as an indirect proxy of sgRNA identity. Since barcodes are often several kilobases from their corresponding sgRNAs, viral recombination-mediated swapping of barcodes and sgRNAs is feasible. Using a self-circularization-based sgRNA-barcode library preparation protocol, we estimate the recombination rate to be ~50% and we trace this phenomenon to the pooled viral packaging step. Recombination is random, and decreases the signal-to-noise ratio of the assay. Our results suggest that alternative approaches can increase the throughput and sensitivity of single-cell perturbation assays.

  13. Oxygen-hydrogen recombination system

    International Nuclear Information System (INIS)

    Sato, Shuichiro; Takejima, Masaki.

    1981-01-01

    Purpose: To avoid reduction in the performance of catalyst used for an oxygen-hydrogen recombiner in the off gas processing system of a nuclear reactor. Constitution: A thermometer is provided for the detection of temperature in an oxygen-hydrogen recombiner. A cooling pipe is provided in the recombiner and cooling medium is introduced externally. The cooling medium may be water or air. In accordance with the detection value from the thermometer, ON-OFF control is carried out for a valve to control the flow rate of the cooling medium thereby rendering the temperature in the recombiner to a predetermined value. This can prevent the catalyst from being exposed to high temperature and avoid the reduction in the performance of the catalyst. (Ikeda, J.)

  14. Evidence of recombination in Hepatitis C Virus populations infecting a hemophiliac patient

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    Cristina Juan

    2009-11-01

    Full Text Available Abstract Background/Aim Hepatitis C virus (HCV infection is an important cause of morbidity and mortality in patients affected by hereditary bleeding disorders. HCV, as others RNA virus, exploit all possible mechanisms of genetic variation to ensure their survival, such as recombination and mutation. In order to gain insight into the genetic variability of HCV virus strains circulating in hemophiliac patients, we have performed a phylogenetic analysis of HCV strains isolated from 10 patients with this kind of pathology. Methods Putative recombinant sequence was identified with the use of GARD program. Statistical support for the presence of a recombination event was done by the use of LARD program. Results A new intragenotypic recombinant strain (1b/1a was detected in 1 out of the 10 hemophiliac patient studied. The recombination event was located at position 387 of the HCV genome (relative to strain AF009606, sub-type 1a corresponding to the core gene region. Conclusion Although recombination may not appear to be common among natural populations of HCV it should be considered as a possible mechanism for generating genetic diversity in hemophiliacs patients.

  15. Functional Requirements for DjlA- and RraA-Mediated Enhancement of Recombinant Membrane Protein Production in the Engineered Escherichia coli Strains SuptoxD and SuptoxR.

    Science.gov (United States)

    Gialama, Dimitra; Delivoria, Dafni Chrysanthi; Michou, Myrsini; Giannakopoulou, Artemis; Skretas, Georgios

    2017-06-16

    In previous work, we have generated the engineered Escherichia coli strains SuptoxD and SuptoxR, which upon co-expression of the effector genes djlA or rraA, respectively, are capable of suppressing the cytotoxicity caused by membrane protein (MP) overexpression and of producing dramatically enhanced yields for a variety of recombinant MPs of both prokaryotic and eukaryotic origin. Here, we investigated the functional requirements for DnaJ-like protein A (DjlA)- and regulator of ribonuclease activity A (RraA)-mediated enhancement of recombinant MP production in these strains and show that: (i) DjlA and RraA act independently, that is, the beneficial effects of each protein on recombinant MP production occur through a mechanism that does not involve the other, and in a non-additive manner; (ii) full-length and membrane-bound DjlA is required for exerting its beneficial effects on recombinant MP production in E. coli SuptoxD; (iii) the MP production-promoting properties of DjlA in SuptoxD involve the action of the molecular chaperone DnaK but do not rely on the activation of the regulation of capsular synthesis response, a well-established consequence of djlA overexpression; (iv) the observed RraA-mediated effects in E. coli SuptoxR involve the ribonucleolytic activity of RNase E, but not that of its paralogous ribonuclease RNase G; and (v) DjlA and RraA are unique among similar E. coli proteins in their ability to promote bacterial recombinant MP production. These observations provide important clues about the molecular requirements for suppressed toxicity and enhanced MP accumulation in SuptoxD/SuptoxR and will guide future studies aiming to decipher the exact mechanism of DjlA- and RraA-mediated enhancement of recombinant MP production in these strains. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Recombination difference between sexes: a role for haploid selection.

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    Thomas Lenormand

    2005-03-01

    Full Text Available Why the autosomal recombination rate differs between female and male meiosis in most species has been a genetic enigma since the early study of meiosis. Some hypotheses have been put forward to explain this widespread phenomenon and, up to now, only one fact has emerged clearly: In species in which meiosis is achiasmate in one sex, it is the heterogametic one. This pattern, known as the Haldane-Huxley rule, is thought to be a side effect, on autosomes, of the suppression of recombination between the sex chromosomes. However, this rule does not hold for heterochiasmate species (i.e., species in which recombination is present in both sexes but varies quantitatively between sexes and does not apply to species lacking sex chromosomes, such as hermaphroditic plants. In this paper, we show that in plants, heterochiasmy is due to a male-female difference in gametic selection and is not influenced by the presence of heteromorphic sex chromosomes. This finding provides strong empirical support in favour of a population genetic explanation for the evolution of heterochiasmy and, more broadly, for the evolution of sex and recombination.

  17. Perspectives of medium chain length poly(hydroxyalkanoates), a versatile set of bacterial bioplastics

    Science.gov (United States)

    Witholt; Kessler

    1999-06-01

    Medium chain length (mcl) poly(hydroxyalkanoic acids) (PHAs) are polyesters accumulated by fluorescent Pseudomonads and other bacteria. Work on the genetics of mcl-PHA formation has led to polymer synthesis in recombinant bacteria and plants. Several high and medium cost applications are now emerging. With optimized bacterial mcl-PHA synthesis on inexpensive agro-substrates and the development of plant-based mcl-PHAs in the next decade, the production economics of these bioplastics will ultimately permit their sustainable production for bulk applications.

  18. Dither Cavity Length Controller with Iodine Locking

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    Lawson Marty

    2016-01-01

    Full Text Available A cavity length controller for a seeded Q-switched frequency doubled Nd:YAG laser is constructed. The cavity length controller uses a piezo-mirror dither voltage to find the optimum length for the seeded cavity. The piezo-mirror dither also dithers the optical frequency of the output pulse. [1]. This dither in optical frequency is then used to lock to an Iodine absorption line.

  19. High avidity antibodies to full-length VAR2CSA correlate with absence of placental malaria.

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    Yeung Lo Tutterrow

    Full Text Available VAR2CSA mediates sequestration of Plasmodium falciparum-infected erythrocytes in the placenta, increasing the risk of poor pregnancy outcomes. Naturally acquired antibodies (Ab to placental parasites at delivery have been associated with improved pregnancy outcomes, but Ab levels and how early in pregnancy Ab must be present in order to eliminate placental parasites before delivery remains unknown. Antibodies to individual Duffy-binding like domains of VAR2CSA have been studied, but the domains lack many of the conformational epitopes present in full-length VAR2CSA (FV2. Thus, the purpose of this study was to describe the acquisition of Ab to FV2 in women residing in high and low transmission areas and determine how Ab levels during pregnancy correlate with clearance of placental parasites. Plasma samples collected monthly throughout pregnancy from pregnant women living in high and low transmission areas in Cameroon were evaluated for Ab to FV2 and the proportion of high avidity Ab (i.e., Ab that remain bound in the presence of 3M NH(4SCN was assessed. Ab levels and proportion of high avidity Ab were compared between women with placental malaria (PM(+ and those without (PM(- at delivery. Results showed that PM(- women had significantly higher Ab levels (p = 0.0047 and proportion of high avidity Ab (p = 0.0009 than PM(+ women throughout pregnancy. Specifically, women with moderate to high Ab levels (>5,000 MFI and those with ≥ 35% high avidity Ab at 5-6 months were found to have 2.3 (95% CI, 1.0-4.9 and 7.6-fold (p = 0.0013, 95% CI: 1.2-50.0 reduced risk of placental malaria, respectively. These data show that high levels of Ab to FV2, particularly those with high avidity for FV2, produced by mid-pregnancy are important in clearing parasites from the placenta. Both high Ab levels and proportion of high avidity Ab to FV2 may serve as correlates of protection for assessing immunity against placental malaria.

  20. The role of recombination in the emergence of a complex and dynamic HIV epidemic

    Directory of Open Access Journals (Sweden)

    Morgenstern Burkhard

    2010-03-01

    Full Text Available Abstract Background Inter-subtype recombinants dominate the HIV epidemics in three geographical regions. To better understand the role of HIV recombinants in shaping the current HIV epidemic, we here present the results of a large-scale subtyping analysis of 9435 HIV-1 sequences that involve subtypes A, B, C, G, F and the epidemiologically important recombinants derived from three continents. Results The circulating recombinant form CRF02_AG, common in West Central Africa, appears to result from recombination events that occurred early in the divergence between subtypes A and G, followed by additional recent recombination events that contribute to the breakpoint pattern defining the current recombinant lineage. This finding also corrects a recent claim that G is a recombinant and a descendant of CRF02, which was suggested to be a pure subtype. The BC and BF recombinants in China and South America, respectively, are derived from recent recombination between contemporary parental lineages. Shared breakpoints in South America BF recombinants indicate that the HIV-1 epidemics in Argentina and Brazil are not independent. Therefore, the contemporary HIV-1 epidemic has recombinant lineages of both ancient and more recent origins. Conclusions Taken together, we show that these recombinant lineages, which are highly prevalent in the current HIV epidemic, are a mixture of ancient and recent recombination. The HIV pandemic is moving towards having increasing complexity and higher prevalence of recombinant forms, sometimes existing as "families" of related forms. We find that the classification of some CRF designations need to be revised as a consequence of (1 an estimated > 5% error in the original subtype assignments deposited in the Los Alamos sequence database; (2 an increasing number of CRFs are defined while they do not readily fit into groupings for molecular epidemiology and vaccine design; and (3 a dynamic HIV epidemic context.

  1. A recombination hotspot in a schizophrenia-associated region of GABRB2.

    Directory of Open Access Journals (Sweden)

    Siu-Kin Ng

    Full Text Available BACKGROUND: Schizophrenia is a major disorder with complex genetic mechanisms. Earlier, population genetic studies revealed the occurrence of strong positive selection in the GABRB2 gene encoding the beta(2 subunit of GABA(A receptors, within a segment of 3,551 bp harboring twenty-nine single nucleotide polymorphisms (SNPs and containing schizophrenia-associated SNPs and haplotypes. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, the possible occurrence of recombination in this 'S1-S29' segment was assessed. The occurrence of hotspot recombination was indicated by high resolution recombination rate estimation, haplotype diversity, abundance of rare haplotypes, recurrent mutations and torsos in haplotype networks, and experimental haplotyping of somatic and sperm DNA. The sub-segment distribution of relative recombination strength, measured by the ratio of haplotype diversity (H(d over mutation rate (theta, was indicative of a human specific Alu-Yi6 insertion serving as a central recombining sequence facilitating homologous recombination. Local anomalous DNA conformation attributable to the Alu-Yi6 element, as suggested by enhanced DNase I sensitivity and obstruction to DNA sequencing, could be a contributing factor of the increased sequence diversity. Linkage disequilibrium (LD analysis yielded prominent low LD points that supported ongoing recombination. LD contrast revealed significant dissimilarity between control and schizophrenic cohorts. Among the large array of inferred haplotypes, H26 and H73 were identified to be protective, and H19 and H81 risk-conferring, toward the development of schizophrenia. CONCLUSIONS/SIGNIFICANCE: The co-occurrence of hotspot recombination and positive selection in the S1-S29 segment of GABRB2 has provided a plausible contribution to the molecular genetics mechanisms for schizophrenia. The present findings therefore suggest that genome regions characterized by the co-occurrence of positive selection and

  2. On the relict recombination lines

    International Nuclear Information System (INIS)

    Bershtejn, I.N.; Bernshtejn, D.N.; Dubrovich, V.K.

    1977-01-01

    Accurate numerical calculation of intensities and profiles of hydrogen recombination lines of cosmological origin is made. Relie radiation distortions stipulated by recombination quantum release at the irrevocable recombination are investigated. Mean number calculation is given for guantums educing for one irrevocably-lost electron. The account is taken of the educed quantums interraction with matter. The main quantum-matter interrraction mechanisms are considered: electronic blow broadening; free-free, free-bound, bound-bound absorptions Recombination dynamics is investigated depending on hydrogen density and total density of all the matter kinds in the Universe

  3. Conserved Genetic Architecture Underlying Individual Recombination Rate Variation in a Wild Population of Soay Sheep (Ovis aries).

    Science.gov (United States)

    Johnston, Susan E; Bérénos, Camillo; Slate, Jon; Pemberton, Josephine M

    2016-05-01

    Meiotic recombination breaks down linkage disequilibrium (LD) and forms new haplotypes, meaning that it is an important driver of diversity in eukaryotic genomes. Understanding the causes of variation in recombination rate is important in interpreting and predicting evolutionary phenomena and in understanding the potential of a population to respond to selection. However, despite attention in model systems, there remains little data on how recombination rate varies at the individual level in natural populations. Here we used extensive pedigree and high-density SNP information in a wild population of Soay sheep (Ovis aries) to investigate the genetic architecture of individual autosomal recombination rates. Individual rates were high relative to other mammal systems and were higher in males than in females (autosomal map lengths of 3748 and 2860 cM, respectively). The heritability of autosomal recombination rate was low but significant in both sexes (h(2) = 0.16 and 0.12 in females and males, respectively). In females, 46.7% of the heritable variation was explained by a subtelomeric region on chromosome 6; a genome-wide association study showed the strongest associations at locus RNF212, with further associations observed at a nearby ∼374-kb region of complete LD containing three additional candidate loci, CPLX1, GAK, and PCGF3 A second region on chromosome 7 containing REC8 and RNF212B explained 26.2% of the heritable variation in recombination rate in both sexes. Comparative analyses with 40 other sheep breeds showed that haplotypes associated with recombination rates are both old and globally distributed. Both regions have been implicated in rate variation in mice, cattle, and humans, suggesting a common genetic architecture of recombination rate variation in mammals. Copyright © 2016 by the Genetics Society of America.

  4. CFD Analysis of Passive Autocatalytic Recombiner

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    B. Gera

    2011-01-01

    Full Text Available In water-cooled nuclear power reactors, significant quantities of hydrogen could be produced following a postulated loss-of-coolant accident (LOCA along with nonavailability of emergency core cooling system (ECCS. Passive autocatalytic recombiners (PAR are implemented in the containment of water-cooled power reactors to mitigate the risk of hydrogen combustion. In the presence of hydrogen with available oxygen, a catalytic reaction occurs spontaneously at the catalyst surfaces below conventional ignition concentration limits and temperature and even in presence of steam. Heat of reaction produces natural convection flow through the enclosure and promotes mixing in the containment. For the assessment of the PAR performance in terms of maximum temperature of catalyst surface and outlet hydrogen concentration an in-house 3D CFD model has been developed. The code has been used to study the mechanism of catalytic recombination and has been tested for two literature-quoted experiments.

  5. Full-length VP2 gene analysis of canine parvovirus reveals emergence of newer variants in India.

    Science.gov (United States)

    Nookala, Mangadevi; Mukhopadhyay, Hirak Kumar; Sivaprakasam, Amsaveni; Balasubramanian, Brindhalakshmi; Antony, Prabhakar Xavier; Thanislass, Jacob; Srinivas, Mouttou Vivek; Pillai, Raghavan Madhusoodanan

    2016-12-01

    The canine parvovirus (CPV) infection is a highly contagious and serious enteric disease of dogs with high fatality rate. The present study was taken up to characterize the full-length viral polypeptide 2 (VP2) gene of CPV of Indian origin along with the commercially available vaccines. The faecal samples from parvovirus suspected dogs were collected from various states of India for screening by PCR assay and 66.29% of samples were found positive. Six CPV-2a, three CPV-2b, and one CPV-2c types were identified by sequence analysis. Several unique and existing mutations have been noticed in CPV types analyzed indicating emergence of newer variants of CPV in India. The phylogenetic analysis revealed that all the field CPV types were grouped in different subclades within two main clades, but away from the commercial vaccine strains. CPV-2b and CPV-2c types with unique mutations were found to be establishing in India apart from the prevailing CPV-2a type. Mutations and the positive selection of the mutants were found to be the major mechanism of emergence and evolution of parvovirus. Therefore, the incorporation of local strain in the vaccine formulation may be considered for effective control of CPV infections in India.

  6. Bi facial silicon solar cell study in modelling in frequency dynamic regime under multispectral illumination: Recombination parameters determination methods

    International Nuclear Information System (INIS)

    ZERBO Issa

    2010-01-01

    A bibliographic study on the techniques of characterization of silicon solar cell, diodes, massifs and silicon wafer are presented. The influence of the modulation frequency and recombination in volume and in surface phenomena of on the profiles of carriers' densities, photocurrent and photovoltage has been put in evidence. The study of surface recombination velocities permitted to show that the bi facial silicon solar cell of Back Surface Field type behaves like an ohmic contacts solar cell for modulation frequencies above 40 khz. pplicability in frequency dynamic regime in the frequency range [0 - 40 khz] of three techniques of steady state recombination parameters determination is shown. A technique of diffusion length determination, in the range of (200 Hz - 40 khz] is proposed. It rests on the measurement of the short circuit current phase that is compared with the theoretical curve of short circuit current phase. The intersection of the experimental short circuit current phase and the theoretical curve of short circuit current phase permits to get the minority carriers effective diffusion length. An equivalent electric model of a solar cell in frequency dynamic regime is proposed. A study in modelling of the bi facial solar cell shunt resistance and space charge zone capacity is led from a determination method of these parameters proposed in steady state. (Author [fr

  7. The induction of mutation and recombination following UV irradiation during meiosis in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Kelly, S.L.; Parry, J.M.

    1983-01-01

    Irradiation of yeast cultures with ultraviolet light at discrete stages during meiosis produces cyclic variations in sensitivity, i.e. cells are more sensitive to the lethal effects of UV light prior to entry into the meiotic DNA synthesis, and this corresponds to a peak of induction of point mutation. Cells become more resistant to both induced point mutation and lethality as they enter meiotic DNA synthesis, but become more sensitive again during spore formation. The induced level of intragenic recombination rises during the period of commitment ot recombination to a level indistinguishable from the full meiotic level of spontaneous intragenic recombination. Induced reciprocal recombination remains above the spontaneous level up to the point of commitment to sporulation. (orig.)

  8. The induction of mutation and recombination following UV irradiation during meiosis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kelly, S L; Parry, J M

    1983-03-01

    Irradiation of yeast cultures with ultraviolet light at discrete stages during meiosis produces cyclic variations in sensitivity, i.e. cells are more sensitive to the lethal effects of UV light prior to entry into the meiotic DNA synthesis, and this corresponds to a peak of induction of point mutation. Cells become more resistant to both induced point mutation and lethality as they enter meiotic DNA synthesis, but become more sensitive again during spore formation. The induced level of intragenic recombination rises during the period of commitment to recombination to a level indistinguishable from the full meiotic level of spontaneous intragenic recombination. Induced reciprocal recombination remains above the spontaneous level up to the point of commitment to sporulation.

  9. Pressure for Pattern-Specific Intertypic Recombination between Sabin Polioviruses: Evolutionary Implications

    Directory of Open Access Journals (Sweden)

    Ekaterina Korotkova

    2017-11-01

    Full Text Available Complete genomic sequences of a non-redundant set of 70 recombinants between three serotypes of attenuated Sabin polioviruses as well as location (based on partial sequencing of crossover sites of 28 additional recombinants were determined and compared with the previously published data. It is demonstrated that the genomes of Sabin viruses contain distinct strain-specific segments that are eliminated by recombination. The presumed low fitness of these segments could be linked to mutations acquired upon derivation of the vaccine strains and/or may have been present in wild-type parents of Sabin viruses. These “weak” segments contribute to the propensity of these viruses to recombine with each other and with other enteroviruses as well as determine the choice of crossover sites. The knowledge of location of such segments opens additional possibilities for the design of more genetically stable and/or more attenuated variants, i.e., candidates for new oral polio vaccines. The results also suggest that the genome of wild polioviruses, and, by generalization, of other RNA viruses, may harbor hidden low-fitness segments that can be readily eliminated only by recombination.

  10. Within-host dynamics of the emergence of Tomato yellow leaf curl virus recombinants.

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    Cica Urbino

    Full Text Available Tomato yellow leaf curl virus (TYLCV is a highly damaging begomovirus native to the Middle East. TYLCV has recently spread worldwide, recombining with other begomoviruses. Recent analysis of mixed infections between TYLCV and Tomato leaf curl Comoros begomovirus (ToLCKMV has shown that, although natural selection preserves certain co-evolved intra-genomic interactions, numerous and diverse recombinants are produced at 120 days post-inoculation (dpi, and recombinant populations from different tomato plants are very divergent. Here, we investigate the population dynamics that lead to such patterns in tomato plants co-infected with TYLCV and ToLCKMV either by agro-inoculation or using the natural whitefly vector Bemisia tabaci. We monitored the frequency of parental and recombinant genotypes independently in 35 plants between 18 and 330 dpi and identified 177 recombinants isolated at different times. Recombinants were detected from 18 dpi and their frequency increased over time to reach about 50% at 150 dpi regardless of the inoculation method. The distribution of breakpoints detected on 96 fully sequenced recombinants was consistent with a continuous generation of new recombinants as well as random and deterministic effects in their maintenance. A severe population bottleneck of around 10 genomes was estimated during early systemic infection-a phenomenon that could account partially for the heterogeneity in recombinant patterns observed among plants. The detection of the same recombinant genome in six of the thirteen plants analysed beyond 30 dpi supported the influence of selection on observed recombination patterns. Moreover, a highly virulent recombinant genotype dominating virus populations within one plant has, apparently, the potential to be maintained in the natural population according to its infectivity, within-host accumulation, and transmission efficiency - all of which were similar or intermediate to those of the parent genotypes. Our

  11. In Vivo Toxicity and Immunological Characterization of Detoxified Recombinant Botulinum Neurotoxin Type A.

    Science.gov (United States)

    Ravichandran, Easwaran; Janardhanan, Pavithra; Patel, Kruti; Riding, Stephen; Cai, Shuowei; Singh, Bal Ram

    2016-03-01

    A double-mutant E224A/E262A full-length botulinum neurotoxin (BoNT) Type A with structural similarity to native BoNT/A but lacking the endopeptidase activity provides an ideal surrogate for testing pharmacokinetics and immunochemical characteristics of BoNT. We determined lethality (LD50) of deactivated recombinant botulinum neurotoxin (drBoNT/A) to be 24.0 μg by intraperitoneal route (i.p). The polypeptide drBoNT/A labeled with near infra-red dye 800 (NIR 800) was used to examine its distribution to different organs using whole body imaging when administered to mice via intravenous (i.v) or i.p route. Also, drBoNT/A was used to evaluate its immunogenicity in Balb/C mice model. drBoNT/A was found to be highly immunogenic when tested under various in vivo conditions in Balb/C mice model. For the first time we have demonstrated that a full length 150 kDa drBoNT/A, by administering via inhalation route in mice model, has evoked both circulating immunoglobulin levels of IgG and secretory IgA at the mucosal surface. The immunoglobulin levels were sufficient enough to protect against the challenge dose of native BoNT toxin in mice model. Tissue distribution of drBoNT/A seems to be similar to that of native toxin. Based on the characteristics described in this report this nontoxic holotoxin protein will assist us to explore the window of opportunity available for therapeutic treatment in case of unnatural poisoning, and also it can be an effective vaccine candidate.

  12. Structure and function of the first full-length murein peptide ligase (Mpl cell wall recycling protein.

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    Debanu Das

    2011-03-01

    Full Text Available Bacterial cell walls contain peptidoglycan, an essential polymer made by enzymes in the Mur pathway. These proteins are specific to bacteria, which make them targets for drug discovery. MurC, MurD, MurE and MurF catalyze the synthesis of the peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine by the sequential addition of amino acids onto UDP-N-acetylmuramic acid (UDP-MurNAc. MurC-F enzymes have been extensively studied by biochemistry and X-ray crystallography. In gram-negative bacteria, ∼30-60% of the bacterial cell wall is recycled during each generation. Part of this recycling process involves the murein peptide ligase (Mpl, which attaches the breakdown product, the tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate, to UDP-MurNAc. We present the crystal structure at 1.65 Å resolution of a full-length Mpl from the permafrost bacterium Psychrobacter arcticus 273-4 (PaMpl. Although the Mpl structure has similarities to Mur enzymes, it has unique sequence and structure features that are likely related to its role in cell wall recycling, a function that differentiates it from the MurC-F enzymes. We have analyzed the sequence-structure relationships that are unique to Mpl proteins and compared them to MurC-F ligases. We have also characterized the biochemical properties of this enzyme (optimal temperature, pH and magnesium binding profiles and kinetic parameters. Although the structure does not contain any bound substrates, we have identified ∼30 residues that are likely to be important for recognition of the tripeptide and UDP-MurNAc substrates, as well as features that are unique to Psychrobacter Mpl proteins. These results provide the basis for future mutational studies for more extensive function characterization of the Mpl sequence-structure relationships.

  13. Efficient Production of an Engineered Apoptin from Chicken Anemia Virus in a Recombinant E. coli for Tumor Therapeutic Applications

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    Lee Meng-Shiou

    2012-06-01

    Full Text Available Abstract Background Apoptin, a nonstructural protein encoded by the VP3 gene of chicken anemia virus (CAV, has been shown to not only induce apoptosis when introduced into the precursors of chicken thymocytes, but has been found to specifically kill human cancer cells, tumor cell and transformed cells without affecting the proliferation of normal cells. This tumor-specific apoptotic characteristic of the protein potentially may allow the development of a protein drug that has applications in tumor therapy. However, several major problems, which include poor expression and poor protein solubility, have hampered the production of apoptin in bacteria. Results Significantly increased expression of recombinant full-length apoptin that originated from chicken anemia virus was demonstrated using an E. coli expression system. The CAV VP3 gene was fused with a synthetic sequence containing a trans-acting activator of transcription (TAT protein transduction domain (PTD. The resulting construct was cloned into various different expression vectors and these were then expressed in various E. coli strains. The expression of the TAT-Apoptin in E. coli was significantly increased when TAT-Apoptin was fused with GST-tag rather than a His-tag. When the various rare amino acid codons of apoptin were optimized, the expression level of the GST-TAT-Apoptinopt in E. coli BL21(DE3 was significantly further increased. The highest protein expression level obtained was 8.33 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 4 h at 25 °C. Moreover, approximately 90% of the expressed GST-TAT-Apoptinopt under these conditions was soluble. After purification by GST affinity chromatography, the purified recombinant TAT-Apoptinopt protein was used to evaluate the recombinant protein’s apoptotic activity on tumor cells. The results demonstrated that the E. coli-expressed GST-TAT-apoptinopt showed apoptotic activity and was able to induce human

  14. Sex-specific aspects of endogenous retroviral insertion and deletion.

    Science.gov (United States)

    Gemmell, Patrick; Hein, Jotun; Katzourakis, Aris

    2013-11-07

    We wish to understand how sex and recombination affect endogenous retroviral insertion and deletion. While theory suggests that the risk of ectopic recombination will limit the accumulation of repetitive DNA in areas of high meiotic recombination, the experimental evidence so far has been inconsistent. Under the assumption of neutrality, we examine the genomes of eighteen species of animal in order to compute the ratio of solo-LTRs that derive from insertions occurring down the male germ line as opposed to the female one (male bias). We also extend the simple idea of comparing autosome to allosome in order to predict the ratio of full-length proviruses we would expect to see under conditions of recombination linked deletion or otherwise. Using our model, we predict the ratio of allosomal to autosomal full-length proviruses to lie between32 and 23 under increasing male bias in mammals and between 1 and 2 under increasing male bias in birds. In contrast to our expectations, we find that a pattern of male bias is not universal across species and that there is a frequent overabundance of full-length proviruses on the allosome beyond the ratios predicted by our model. We use our data as a whole to argue that full-length proviruses should be treated as deleterious mutations or as effectively neutral mutations whose persistence in a full-length state is linked to the rate of meiotic recombination and whose origin is not universally male biased. These conclusions suggest that retroviral insertions on the allosome may be more prolific and that it might be possible to identify mechanisms of replication that are enhanced in the female sex.

  15. Diagnostic value of newborn foot length to predict gestational age

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    Mutia Farah Fawziah

    2017-08-01

    Full Text Available Background  Identification of gestational age, especially within 48 hours of birth, is crucial for newborns, as the earlier preterm status is detected, the earlier the child can receive optimal management. Newborn foot length is an anthropometric measurement which is easy to perform, inexpensive, and potentially efficient for predicting gestational age. Objective  To analyze the diagnostic value of newborn foot length in predicting gestational age. Methods  This diagnostic study was performed between October 2016 and February 2017 in the High Care Unit of Neonates at Dr. Moewardi General Hospital, Surakarta. A total of 152 newborns were consecutively selected and underwent right foot length measurements before 96 hours of age. The correlation between newborn foot length to classify as full term and gestational age was analyzed with Spearman’s correlation test because of non-normal data distribution. The cut-off point of newborn foot length was calculated by receiver operating characteristic (ROC curve and diagnostic values of newborn foot length were analyzed by 2 x 2 table with SPSS 21.0 software. Results There were no significant differences between male and female newborns in terms of gestational age, birth weight, choronological age, and newborn foot length (P>0.05. Newborn foot length and gestational age had a significant correlation (r=0.53; P=0.000. The optimal cut-off newborn foot length to predict full term status was 7.1 cm. Newborn foot length below 7.1 cm had sensitivity 75%, specificity 98%, positive predictive value 94.3%, negative predictive value 90.6%, positive likelihood ratio 40.5, negative likelihood ratio 0.25, and post-test probability 94.29%, to predict preterm status in newborns. Conclusion  Newborn foot length can be used to predict gestational age, especially for the purpose of differentiating between preterm and full term newborns.

  16. Auger recombination in sodium iodide

    Science.gov (United States)

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

    2014-03-01

    Scintillators are an important tool used to detect high energy radiation - both in the interest of national security and in medicine. However, scintillator detectors currently suffer from lower energy resolutions than expected from basic counting statistics. This has been attributed to non-proportional light yield compared to incoming radiation, but the specific mechanism for this non-proportionality has not been identified. Auger recombination is a non-radiative process that could be contributing to the non-proportionality of scintillating materials. Auger recombination comes in two types - direct and phonon-assisted. We have used first-principles calculations to study Auger recombination in sodium iodide, a well characterized scintillating material. Our findings indicate that phonon-assisted Auger recombination is stronger in sodium iodide than direct Auger recombination. Computational resources provided by LLNL and NERSC. Funding provided by NA-22.

  17. Characterization and frequency of a newly identified HIV-1 BF1 intersubtype circulating recombinant form in São Paulo, Brazil

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    Neto Walter

    2010-04-01

    Full Text Available Abstract Background HIV circulating recombinant forms (CRFs play an important role in the global and regional HIV epidemics, particularly in regions where multiple subtypes are circulating. To date, several (>40 CRFs are recognized worldwide with five currently circulating in Brazil. Here, we report the characterization of near full-length genome sequences (NFLG of six phylogenetically related HIV-1 BF1 intersubtype recombinants (five from this study and one from other published sequences representing CRF46_BF1. Methods Initially, we selected 36 samples from 888 adult patients residing in São Paulo who had previously been diagnosed as being infected with subclade F1 based on pol subgenomic fragment sequencing. Proviral DNA integrated in peripheral blood mononuclear cells (PBMC was amplified from the purified genomic DNA of all 36-blood samples by five overlapping PCR fragments followed by direct sequencing. Sequence data were obtained from the five fragments that showed identical genomic structure and phylogenetic trees were constructed and compared with previously published sequences. Genuine subclade F1 sequences and any other sequences that exhibited unique mosaic structures were omitted from further analysis Results Of the 36 samples analyzed, only six sequences, inferred from the pol region as subclade F1, displayed BF1 identical mosaic genomes with a single intersubtype breakpoint identified at the nef-U3 overlap (HXB2 position 9347-9365; LTR region. Five of these isolates formed a rigid cluster in phylogentic trees from different subclade F1 fragment regions, which we can now designate as CRF46_BF1. According to our estimate, the new CRF accounts for 0.56% of the HIV-1 circulating strains in São Paulo. Comparison with previously published sequences revealed an additional five isolates that share an identical mosaic structure with those reported in our study. Despite sharing a similar recombinant structure, only one sequence appeared to

  18. Cutting Whole Length or Partial Length of Internal Anal Sphincter in Managementof Fissure in Ano

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    Furat Shani Aoda

    2017-12-01

    Full Text Available A chronic anal fissure is a common painful perianal condition.The main operative procedure to treat this painful condition is a lateral internal sphincteretomy (LIS.The aim of study is to compare the outcome and complications of closed LIS up to the dentate line (whole length of internal sphincter or up to the fissure apex (partial length of internal sphincter in the treatment of anal fissure.It is a prospective comparativestudy including 100 patients with chronic fissure in ano. All patients assigned to undergo closed LIS. Those patients were randomly divided into two groups: 50 patients underwent LIS to the level of dentate line (whole length and other 50 patients underwent LIS to the level of fissure apex (partial length. Patients were followed up weekly in the 1st month, twice monthly in the second month then monthly   for next 2 months and finally after 1 year. There was satisfactory relief of pain in all patients in both groups & complete healing of the fissure occurred. Regarding post operative incontinence no major degree of incontinence occur in both group but minor degree of incontinence persists In 7 patients after whole length LIS after one year. In conclusion, both whole length & partial length LIS associated with improvement of pain, good chance of healing but whole length LIS associated with more chance of long term  flatus incontinence. Hence,we recommend partial length LIS as treatment forchronic anal fissure.

  19. Regulation of homologous recombination in eukaryotes

    OpenAIRE

    Heyer, Wolf-Dietrich; Ehmsen, Kirk T.; Liu, Jie

    2010-01-01

    Homologous recombination is required for accurate chromosome segregation during the first meiotic division and constitutes a key repair and tolerance pathway for complex DNA damage including DNA double-stranded breaks, interstrand crosslinks, and DNA gaps. In addition, recombination and replication are inextricably linked, as recombination recovers stalled and broken replication forks enabling the evolution of larger genomes/replicons. Defects in recombination lead to genomic instability and ...

  20. FULL LENGTH RESEARCH ARTICLE Yoriyo et al.(2008) SWJ:35 ...

    African Journals Online (AJOL)

    Dr. Ahmed

    poultry industry to its full capacity. These factors include poor management systems and diseases. Poultry diseases are the major cause of financial loss in poultry production (Oluyemi & Rober, 1979). Intestinal parasitism is a common problem in poultry especially those reared under extensive systems. Ajayi & Ajayi (1983) ...

  1. 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.

  2. Manipulating or superseding host recombination functions: a dilemma that shapes phage evolvability.

    Directory of Open Access Journals (Sweden)

    Louis-Marie Bobay

    Full Text Available Phages, like many parasites, tend to have small genomes and may encode autonomous functions or manipulate those of their hosts'. Recombination functions are essential for phage replication and diversification. They are also nearly ubiquitous in bacteria. The E. coli genome encodes many copies of an octamer (Chi motif that upon recognition by RecBCD favors repair of double strand breaks by homologous recombination. This might allow self from non-self discrimination because RecBCD degrades DNA lacking Chi. Bacteriophage Lambda, an E. coli parasite, lacks Chi motifs, but escapes degradation by inhibiting RecBCD and encoding its own autonomous recombination machinery. We found that only half of 275 lambdoid genomes encode recombinases, the remaining relying on the host's machinery. Unexpectedly, we found that some lambdoid phages contain extremely high numbers of Chi motifs concentrated between the phage origin of replication and the packaging site. This suggests a tight association between replication, packaging and RecBCD-mediated recombination in these phages. Indeed, phages lacking recombinases strongly over-represent Chi motifs. Conversely, phages encoding recombinases and inhibiting host recombination machinery select for the absence of Chi motifs. Host and phage recombinases use different mechanisms and the latter are more tolerant to sequence divergence. Accordingly, we show that phages encoding their own recombination machinery have more mosaic genomes resulting from recent recombination events and have more diverse gene repertoires, i.e. larger pan genomes. We discuss the costs and benefits of superseding or manipulating host recombination functions and how this decision shapes phage genome structure and evolvability.

  3. Recombination coefficients in extrinsic n-InSb

    International Nuclear Information System (INIS)

    Schneider, W.; Groh, H.; Huebner, K.

    1976-01-01

    The bulk recombination coefficients for linear recombination via recombination centers as well as for direct recombination have been determined measuring the conductivity decay after two-photon absorption with a CO 2 laser. The Suhl effect was applied to measure the surface recombination velocity. The corresponding literature is discussed and compared with our results. We conclude that two different kinds of recombination centers are possible in n-InSb, with energy levels (0.1-0.12)eV above the valence band, or (0.14-0.2)eV respectively. (orig.) [de

  4. Identification of the full-length β-actin sequence and expression profiles in the tree shrew (Tupaia belangeri).

    Science.gov (United States)

    Zheng, Yu; Yun, Chenxia; Wang, Qihui; Smith, Wanli W; Leng, Jing

    2015-02-01

    The tree shrew (Tupaia belangeri) diverges from the primate order (Primates) and is classified as a separate taxonomic group of mammals - Scandentia. It has been suggested that the tree shrew can be used as an animal model for studying human diseases; however, the genomic sequence of the tree shrew is largely unidentified. In the present study, we reported the full-length cDNA sequence of the housekeeping gene, β-actin, in the tree shrew. The amino acid sequence of β-actin in the tree shrew was compared to that of humans and other species; a simple phylogenetic relationship was discovered. Quantitative polymerase chain reaction (qPCR) and western blot analysis further demonstrated that the expression profiles of β-actin, as a general conservative housekeeping gene, in the tree shrew were similar to those in humans, although the expression levels varied among different types of tissue in the tree shrew. Our data provide evidence that the tree shrew has a close phylogenetic association with humans. These findings further enhance the potential that the tree shrew, as a species, may be used as an animal model for studying human disorders.

  5. The new albumin-free recombinant factor VIII concentrates for treatment of hemophilia: do they represent an actual incremental improvement?

    Science.gov (United States)

    Josephson, Cassandra D; Abshire, Thomas

    2004-07-01

    The goal of eliminating the low levels of infectious disease risk from hemophilia treatment has resulted in the development of multiple generations of recombinant factor VIII (rFVIII) products. The ideal product should be devoid of human and animal proteins, which may transmit infectious agents. These products should also maintain molecular integrity, hemostatic efficacy, similar immunogenicity, and acceptable side effect profiles as compared to plasma-derived factor VIII. Currently available first-, second-, and third-generation rFVIII products include Recombinate; Kogenate FS/Helixate FS and ReFacto; and Advate, respectively. During the evolution of rFVIII products, either full-length or B-domain-deleted factor VIII were transfected into immortalized cell lines. The B-domain-deleted product, ReFacto, has resulted in an additional method to monitor factor VIII levels. The third-generation products offer the theoretical advantage of being produced without human and/or animal proteins. Upon initial introduction into the marketplace, the newer products have a higher cost. However, when analyzing historical trends, the prices of these products are almost equivalent to first-generation products within 3 years of licensure. Thus, the initial cost of the product may be a minimal issue in the medical decision process when selecting rFVIII replacement therapy.

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

    International Nuclear Information System (INIS)

    Pajek, M.

    1994-01-01

    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

  7. Gene Delivery into Plant Cells for Recombinant Protein Production

    Directory of Open Access Journals (Sweden)

    Qiang Chen

    2015-01-01

    Full Text Available Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications.

  8. Variation in human recombination rates and its genetic determinants.

    Directory of Open Access Journals (Sweden)

    Adi Fledel-Alon

    Full Text Available Despite the fundamental role of crossing-over in the pairing and segregation of chromosomes during human meiosis, the rates and placements of events vary markedly among individuals. Characterizing this variation and identifying its determinants are essential steps in our understanding of the human recombination process and its evolution.Using three large sets of European-American pedigrees, we examined variation in five recombination phenotypes that capture distinct aspects of crossing-over patterns. We found that the mean recombination rate in males and females and the historical hotspot usage are significantly heritable and are uncorrelated with one another. We then conducted a genome-wide association study in order to identify loci that influence them. We replicated associations of RNF212 with the mean rate in males and in females as well as the association of Inversion 17q21.31 with the female mean rate. We also replicated the association of PRDM9 with historical hotspot usage, finding that it explains most of the genetic variance in this phenotype. In addition, we identified a set of new candidate regions for further validation.These findings suggest that variation at broad and fine scales is largely separable and that, beyond three known loci, there is no evidence for common variation with large effects on recombination phenotypes.

  9. Electron-ion recombination at low energy

    International Nuclear Information System (INIS)

    Andersen, L.H.

    1993-01-01

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

  10. Recombinant Innovation and Endogenous Transitions

    OpenAIRE

    Koen Frenken; Luis R. Izquierdo; Paolo Zeppini

    2012-01-01

    We propose a model of technological transitions based on two different types of innovations. Branching innovations refer to technological improvements along a particular path, while recombinant innovations represent fusions of multiple paths. Recombinant innovations create “short-cuts” which reduce switching costs allowing agents to escape a technological lock-in. As a result, recombinant innovations speed up technological progress allowing transitions that are impossible with only branching ...

  11. Interface recombination influence on carrier transport

    International Nuclear Information System (INIS)

    Konin, A

    2013-01-01

    A theory of interface recombination in the semiconductor–semiconductor junction is developed. The interface recombination rate dependence on the nonequilibrium carrier densities is derived on the basis of a model in which the interface recombination occurs through the mechanism of trapping. The general relation between the interface recombination parameters at small carrier density deviation from the equilibrium ones is obtained. The validity of this relation is proved considering the generation of the Hall electric field in the extrinsic semiconductor sample. The anomalous Hall electromotive force in a weak magnetic field was investigated and interpreted by means of a new interface recombination model. The experimental data corroborate the developed theory. (paper)

  12. A Novel Recombinant Enterovirus Type EV-A89 with Low Epidemic Strength in Xinjiang, China.

    Science.gov (United States)

    Fan, Qin; Zhang, Yong; Hu, Lan; Sun, Qiang; Cui, Hui; Yan, Dongmei; Sikandaner, Huerxidan; Tang, Haishu; Wang, Dongyan; Zhu, Zhen; Zhu, Shuangli; Xu, Wenbo

    2015-12-21

    Enterovirus A89 (EV-A89) is a novel member of the EV-A species. To date, only one full-length genome sequence (the prototype strain) has been published. Here, we report the molecular identification and genomic characterization of a Chinese EV-A89 strain, KSYPH-TRMH22F/XJ/CHN/2011, isolated in 2011 from a contact of an acute flaccid paralysis (AFP) patient during AFP case surveillance in Xinjiang China. This was the first report of EV-A89 in China. The VP1 coding sequence of this strain demonstrated 93.2% nucleotide and 99.3% amino acid identity with the EV-A89 prototype strain. In the P2 and P3 regions, the Chinese EV-A89 strain demonstrated markedly higher identity than the prototype strains of EV-A76, EV-A90, and EV-A91, indicating that one or more recombination events between EV-A89 and these EV-A types might have occurred. Long-term evolution of these EV types originated from the same ancestor provides the spatial and temporal circumstances for recombination to occur. An antibody sero-prevalence survey against EV-A89 in two Xinjiang prefectures demonstrated low positive rates and low titres of EV-A89 neutralization antibody, suggesting limited range of transmission and exposure to the population. This study provides a solid foundation for further studies on the biological and pathogenic properties of EV-A89.

  13. Hydrogen Recombination Rates of Plate-type Passive Auto-catalytic Recombiner

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jongtae; Hong, Seong-Wan [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Kim, Gun Hong [Kyungwon E-C Co., Seongnam (Korea, Republic of)

    2014-10-15

    The hydrogen mitigation system may include igniters, passive autocatalytic recombiner (PAR), and venting or dilution system. Recently PAR is commonly used as a main component of HMS in a NPP containment because of its passive nature. PARs are categorized by the shape and material of catalytic surface. Catalytic surface coated by platinum is mostly used for the hydrogen recombiners. The shapes of the catalytic surface can be grouped into plate type, honeycomb type and porous media type. Among them, the plate-type PAR is well tested by many experiments. PAR performance analysis can be approached by a multi-scale method which is composed of micro, meso and macro scales. The criterion of the scaling is the ratio of thickness of boundary layer developed on a catalytic surface to representative length of a computational domain. Mass diffusion in the boundary layer must be resolved in the micro scale analysis. In a lumped parameter (LP) analysis using a system code such as MAAP or MELCOR, the chamber of the PAR is much smaller than a computational node. The hydrogen depletion by a PAR is modeled as a source of mass and energy conservation equations. Te catalytic surface reaction of hydrogen must be modeled by a volume-averaged correlation. In this study, a micro scale analysis method is developed using libraries in OpenFOAM to evaluate a hydrogen depletion rate depending on parameters such as size and number of plates and plate arrangement. The analysis code is validated by simulating REKO-3 experiment. And hydrogen depletion analysis is conducted by changing the plate arrangement as a trial of the performance enhancement of a PAR. In this study, a numerical code for an analysis of a PAR performance in a micro scale has been developed by using OpenFOAM libraries. The physical and numerical models were validated by simulating the REKO-3 experiment. As a try to enhance the performance of the plate-type PAR, it was proposed to apply a staggered two-layer arrangement of the

  14. Hydrogen Recombination Rates of Plate-type Passive Auto-catalytic Recombiner

    International Nuclear Information System (INIS)

    Kim, Jongtae; Hong, Seong-Wan; Kim, Gun Hong

    2014-01-01

    The hydrogen mitigation system may include igniters, passive autocatalytic recombiner (PAR), and venting or dilution system. Recently PAR is commonly used as a main component of HMS in a NPP containment because of its passive nature. PARs are categorized by the shape and material of catalytic surface. Catalytic surface coated by platinum is mostly used for the hydrogen recombiners. The shapes of the catalytic surface can be grouped into plate type, honeycomb type and porous media type. Among them, the plate-type PAR is well tested by many experiments. PAR performance analysis can be approached by a multi-scale method which is composed of micro, meso and macro scales. The criterion of the scaling is the ratio of thickness of boundary layer developed on a catalytic surface to representative length of a computational domain. Mass diffusion in the boundary layer must be resolved in the micro scale analysis. In a lumped parameter (LP) analysis using a system code such as MAAP or MELCOR, the chamber of the PAR is much smaller than a computational node. The hydrogen depletion by a PAR is modeled as a source of mass and energy conservation equations. Te catalytic surface reaction of hydrogen must be modeled by a volume-averaged correlation. In this study, a micro scale analysis method is developed using libraries in OpenFOAM to evaluate a hydrogen depletion rate depending on parameters such as size and number of plates and plate arrangement. The analysis code is validated by simulating REKO-3 experiment. And hydrogen depletion analysis is conducted by changing the plate arrangement as a trial of the performance enhancement of a PAR. In this study, a numerical code for an analysis of a PAR performance in a micro scale has been developed by using OpenFOAM libraries. The physical and numerical models were validated by simulating the REKO-3 experiment. As a try to enhance the performance of the plate-type PAR, it was proposed to apply a staggered two-layer arrangement of the

  15. Visualized and precise design of artificial small RNAs for regulating T7 RNA polymerase and enhancing recombinant protein folding in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Yujia Zhao

    2016-12-01

    Full Text Available Small non-coding RNAs (sRNAs have received much attention in recent years due to their unique biological properties, which can efficiently and specifically tune target gene expressions in bacteria. Inspired by natural sRNAs, recent works have proposed the use of artificial sRNAs (asRNAs as genetic tools to regulate desired gene that has been applied in several fields, such as metabolic engineering and bacterial physiology studies. However, the rational design of asRNAs is still a challenge. In this study, we proposed structure and length as two criteria to implement rational visualized and precise design of asRNAs. T7 expression system was one of the most useful recombinant protein expression systems. However, it was deeply limited by the formation of inclusion body. To settle this problem, we designed a series of asRNAs to inhibit the T7 RNA polymerase (Gene1 expression to balance the rate between transcription and folding of recombinant protein. Based on the heterologous expression of Aspergillus oryzae Li-3 glucuronidase in E. coli, the asRNA-antigene1-17bp can effectively decrease the inclusion body and increase the enzyme activity by 169.9%.

  16. Impedance of finite length resistive cylinder

    Directory of Open Access Journals (Sweden)

    S. Krinsky

    2004-11-01

    Full Text Available We determine the impedance of a cylindrical metal tube (resistor of radius a, length g, and conductivity σ attached at each end to perfect conductors of semi-infinite length. Our main interest is in the asymptotic behavior of the impedance at high frequency (k≫1/a. In the equilibrium regime, ka^{2}≪g, the impedance per unit length is accurately described by the well-known result for an infinite length tube with conductivity σ. In the transient regime, ka^{2}≫g, where the contribution of transition radiation arising from the discontinuity in conductivity is important, we derive an analytic expression for the impedance and compute the short-range wakefield. The analytic results are shown to agree with numerical evaluation of the impedance.

  17. Investigation of the extraction of short diffusion lengths from simulated electron-beam induced current

    Energy Technology Data Exchange (ETDEWEB)

    Wee, D.; Parish, G.; Nener, B. [Microelectronics Research Group, The University of Western Australia, 35 Stirling Highway, 6009 Crawley (Perth) (Australia)

    2010-10-15

    This paper reports on the investigations via 2-D simulation into the accuracy of diffusion length extraction from scanning electron-beam induced current measurements when the diffusion length, L is very short. L is extracted by using the direct method proposed by Chan et al.[1] and later refined by Kurniawan and Ong[2] to take finite junction depth into account. The 2-D simulations were undertaken using Synopsys {sup registered} Sentaurus TCAD and a realistic electron-hole pair generation volume was created using CASINO v2.42[3], a Monte Carlo Scanning Electron Microscope interaction simulation software, and imported into Sentaurus. The voltage and diameter of the electron beam and diffusion length and surface recombination velocity of the semiconductor materials were varied in the simulations to determine the errors in the diffusion length extracted from the EBIC signals as a function of these parameters. The results of the simulation show that the accuracy of the method proposed in[1] is reasonably accurate and that the beam voltage and spot size do not have significant effects on the accuracy (copyright 2010 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  18. Investigation of the extraction of short diffusion lengths from simulated electron-beam induced current

    International Nuclear Information System (INIS)

    Wee, D.; Parish, G.; Nener, B.

    2010-01-01

    This paper reports on the investigations via 2-D simulation into the accuracy of diffusion length extraction from scanning electron-beam induced current measurements when the diffusion length, L is very short. L is extracted by using the direct method proposed by Chan et al.[1] and later refined by Kurniawan and Ong[2] to take finite junction depth into account. The 2-D simulations were undertaken using Synopsys registered Sentaurus TCAD and a realistic electron-hole pair generation volume was created using CASINO v2.42[3], a Monte Carlo Scanning Electron Microscope interaction simulation software, and imported into Sentaurus. The voltage and diameter of the electron beam and diffusion length and surface recombination velocity of the semiconductor materials were varied in the simulations to determine the errors in the diffusion length extracted from the EBIC signals as a function of these parameters. The results of the simulation show that the accuracy of the method proposed in[1] is reasonably accurate and that the beam voltage and spot size do not have significant effects on the accuracy (copyright 2010 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  19. Molecular requirements for radiation-activated recombination

    International Nuclear Information System (INIS)

    Stevens, Craig W.; Zeng Ming; Stamato, Thomas; Cerniglia, George

    1997-01-01

    Purpose/Objective: The major stumbling block to successful gene therapy today is poor gene transfer. We hypothesized that ionizing radiation might activate cellular recombination, and so improve stable gene transfer. We further hypothesized that known DNA-damage-repair proteins might also be important in radiation-activated recombination. Materials and Methods: The effect of irradiation on stable gene transfer efficiency was determined in human (A549 and 39F) and rodent (NIH/3T3) cell lines. Continuous low dose rate and multiple radiation fractions were also tested. Nuclear extracts were made and the effect of irradiation on inter-plasmid recombination/ligation determined. Multiple DNA damage-repair deficient cell lines were tested for radiation-activated recombination. Results: A significant radiation dose-dependent improvement in stable plasmid transfection (by as much as 1300 fold) is demonstrated in neoplastic and primary cells. An improvement in transient plasmid transfection is also seen, with as much as 85% of cells transiently expressing b-galactosidase (20-50 fold improvement). Stable transfection is only improved for linearized or nicked plasmids. Cells have improved gene transfer for at least 96 hours after irradiation. Both fractionated and continuous low dose rate irradiation are effective at improving stable gene transfer in mammalian cells, thus making relatively high radiation dose delivery clinically feasible. Inter-plasmid recombination is radiation dose dependent in nuclear extract assays, and the type of overhang (3', 5' or blunt end) significantly affects recombination efficiency and the type of product. The most common end-joining activity involves filling-in of the overhang followed by blunt end ligation. Adenovirus is a linear, double stranded DNA virus. We demonstrate that adenoviral infection efficiency is increased by irradiation. The duration of transgene expression is lengthened because the virus integrates with high efficiency (∼10

  20. Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

    Science.gov (United States)

    Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.

    2001-04-03

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.