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Sample records for monooxygenase gene recombinant

  1. Enhanced production of ε-caprolactone by coexpression of bacterial hemoglobin gene in recombinant Escherichia coli expressing cyclohexanone monooxygenase gene.

    Science.gov (United States)

    Lee, Won-Heong; Park, Eun-Hee; Kim, Myoung-Dong

    2014-12-28

    Baeyer-Villiger (BV) oxidation of cyclohexanone to epsilon-caprolactone in a microbial system expressing cyclohexanone monooxygenase (CHMO) can be influenced by not only the efficient regeneration of NADPH but also a sufficient supply of oxygen. In this study, the bacterial hemoglobin gene from Vitreoscilla stercoraria (vhb) was introduced into the recombinant Escherichia coli expressing CHMO to investigate the effects of an oxygen-carrying protein on microbial BV oxidation of cyclohexanone. Coexpression of Vhb allowed the recombinant E. coli strain to produce a maximum epsilon-caprolactone concentration of 15.7 g/l in a fed-batch BV oxidation of cyclohexanone, which corresponded to a 43% improvement compared with the control strain expressing CHMO only under the same conditions.

  2. Enhanced production of epsilon-caprolactone by overexpression of NADPH-regenerating glucose 6-phosphate dehydrogenase in recombinant Escherichia coli harboring cyclohexanone monooxygenase gene.

    Science.gov (United States)

    Lee, Won-Heong; Park, Jin-Byung; Park, Kyungmoon; Kim, Myoung-Dong; Seo, Jin-Ho

    2007-08-01

    Whole-cell conversion of cyclohexanone to epsilon-caprolactone was attempted by recombinant Escherichia coli BL21(DE3) expressing cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871. High concentrations of cyclohexanone and epsilon-caprolactone reduced CHMO-mediated bioconversion of cyclohexanone to epsilon-caprolactone in the resting recombinant E. coli cells. Metabolically active cells were employed by adopting a fed-batch culture to improve the production of epsilon-caprolactone from cyclohexanone. A glucose-limited fed-batch Baeyer-Villiger oxidation where a cyclohexanone level was maintained less than 6 g/l resulted in a maximum epsilon-caprolactone concentration of 11.0 g/l. The maximum epsilon-caprolactone concentration was improved further to 15.3 g/l by coexpression of glucose-6-phosphate dehydrogenase, an NADPH-generating enzyme encoded by the zwf gene which corresponded to a 39% enhancement in epsilon-caprolactone concentration compared with the control experiment performed under the same conditions.

  3. Bioconversion of Mono- and Sesquiterpenoids by Recombinant Human Cytochrome P450 Monooxygenases

    NARCIS (Netherlands)

    Julsing, Mattijs K.; Fichera, Mario A.; Malz, Frank; Ebbelaar, Monique; Bos, Rein; Woerdenbag, Herman J.; Quax, Wim J.; Kayser, Oliver

    2008-01-01

    Cytochrome P450 monooxygenases play an important role in the biosynthesis and metabolism of terpenoids. We explored the potential of recombinant human liver cytochrome P450 monooxygenases CYP1A2, CYP2C9, and CYP3A4, heterologously expressed in Escherichia coli, to convert mono- and sesquiterpenoids

  4. Expression and purification of the recombinant subunits of toluene/o-xylene monooxygenase and reconstitution of the active complex.

    Science.gov (United States)

    Cafaro, Valeria; Scognamiglio, Roberta; Viggiani, Ambra; Izzo, Viviana; Passaro, Irene; Notomista, Eugenio; Piaz, Fabrizio Dal; Amoresano, Angela; Casbarra, Annarita; Pucci, Piero; Di Donato, Alberto

    2002-11-01

    This paper describes the cloning of the genes coding for each component of the complex of toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1, their expression, purification and characterization. Moreover, the reconstitution of the active complex from the recombinant subunits has been obtained, and the functional role of each component in the electron transfer from the electron donor to molecular oxygen has been determined. The coexpression of subunits B, E and A leads to the formation of a subcomplex, named H, with a quaternary structure (BEA)2, endowed with hydroxylase activity. Tomo F component is an NADH oxidoreductase. The purified enzyme contains about 1 mol of FAD, 2 mol of iron, and 2 mol of acid labile sulfide per mol of protein, as expected for the presence of one [2Fe-2S] cluster, and exhibits a typical flavodoxin absorption spectrum. Interestingly, the sequence of the protein does not correspond to that previously predicted on the basis of DNA sequence. We have shown that this depends on minor errors in the gene sequence that we have corrected. C component is a Rieske-type ferredoxin, whose iron and acid labile sulfide content is in agreement with the presence of one [2Fe-2S] cluster. The cluster is very sensitive to oxygen damage. Mixtures of the subcomplex H and of the subunits F, C and D are able to oxidize p-cresol into 4-methylcathecol, thus demonstrating the full functionality of the recombinant subunits as purified. Finally, experimental evidence is reported which strongly support a model for the electron transfer. Subunit F is the first member of an electron transport chain which transfers electrons from NADH to C, which tunnels them to H subcomplex, and eventually to molecular oxygen.

  5. Continuous testing system for Baeyer-Villiger biooxidation using recombinant Escherichia coli expressing cyclohexanone monooxygenase encapsulated in polyelectrolyte complex capsules.

    Science.gov (United States)

    Bučko, Marek; Schenkmayerová, Andrea; Gemeiner, Peter; Vikartovská, Alica; Mihovilovič, Marko D; Lacík, Igor

    2011-08-10

    An original strategy for universal laboratory testing of Baeyer-Villiger monooxygenases based on continuous packed-bed minireactor connected with flow calorimeter and integrated with bubble-free oxygenation is reported. Model enantioselective Baeyer-Villiger biooxidations of rac-bicyclo[3.2.0]hept-2-en-6-one to corresponding lactones (1R,5S)-3-oxabicyclo-[3.3.0]oct-6-en-3-one and (1S,5R)-2-oxabicyclo-[3.3.0]oct-6-en-3-one as important chiral synthons for the synthesis of bioactive compounds were performed in the minireactor equipped with a column packed with encapsulated recombinant cells Escherichia coli overexpressing cyclohexanone monooxygenase. The cells were encapsulated in polyelectrolyte complex capsules formed by reaction of oppositely charged polymers utilizing highly reproducible and controlled encapsulation process. Encapsulated cells tested in minireactor exhibited high operational stability with 4 complete substrate conversions to products and 6 conversions above 80% within 14 repeated consecutive biooxidation tests. Moreover, encapsulated cells showed high enzyme stability during 91 days of storage with substrate conversions above 80% up to 60 days of storage. Furthermore, usable thermometric signal of Baeyer-Villiger biooxidation obtained by flow calorimetry using encapsulated cells was utilized for preparatory kinetic study in order to guarantee sub-inhibitory initial substrate concentration for biooxidation tests.

  6. Simultaneous biocatalyst production and Baeyer-Villiger oxidation for bioconversion of cyclohexanone by recombinant Escherichia coli expressing cyclohexanone monooxygenase.

    Science.gov (United States)

    Lee, Won-Heong; Park, Yong-Cheol; Lee, Dae-Hee; Park, Kyungmoon; Seo, Jin-Ho

    2005-01-01

    Cyclohexanone monooxygenase (CHMO) catalyzing Baeyer-Villiger oxidation converts cyclic ketones into optically pure lactones, which have been used as building blocks in organic synthesis. A recombinant Escherichia coli BL21(DE3)/pMM4 expressing CHMO originated from Acinetobacter sp. NCIB 9871 was used to produce epsilon-caprolactone through a simultaneous biocatalyst production and Baeyer-Villiger oxidation (SPO) process. A fed-batch process was designed to obtain high cell density for improving production of epsilon-caprolactone. The fed-batch SPO process gave the best results, 10.2 g/L of epsilon-caprolactone and 0.34 g/(L.h) of productivity, corresponding to a 10.5- and 3.4-fold enhancement compared with those of the batch SPO, respectively.

  7. Gene cloning and functional analysis of triple alkane monooxygenases from Geobacillus thermoleovorans B23

    OpenAIRE

    2014-01-01

    An extremely thermophilic bacterium, Geobacillus thermoleovorans B23 which was isolated from a deep subterranean oil reservoir at Niigata, Japan, is capable of degrading broad range alkanes (C11-C32) at 70℃ by terminal oxidation pathway, followed by β-oxidation pathway. Whole genome sequence analysis revealed that B23 did not have alkB-type alkane monooxygenases genes like most alkane degrading bacteria but it carried three gene homologs namely ladAαB23, ladAβB23 and ladBB23 on its chromosome...

  8. PCR-DGGE method to assess the diversity of BTEX mono-oxygenase genes at contaminated sites

    NARCIS (Netherlands)

    Hendrickx, B; Dejonghe, W; Faber, F; Boenne, W; Bastiaens, L; Verstraete, W; Top, EM; Springael, D

    2006-01-01

    tmoA and related genes encode the alpha-subunit of the hydroxylase component of the major group (subgroup 1 of subfamily 2) of bacterial multicomponent mono-oxygenase enzyme complexes involved in aerobic benzene, toluene, ethylbenzene and xylene (BTEX) degradation. A PCR-denaturing gradient gel elec

  9. Genome-wide identification and characterization of cytochrome P450 monooxygenase genes in the ciliate Tetrahymena thermophila

    Directory of Open Access Journals (Sweden)

    Xiong Jie

    2009-05-01

    Full Text Available Abstract Background Cytochrome P450 monooxygenases play key roles in the metabolism of a wide variety of substrates and they are closely associated with endocellular physiological processes or detoxification metabolism under environmental exposure. To date, however, none has been systematically characterized in the phylum Ciliophora. T. thermophila possess many advantages as a eukaryotic model organism and it exhibits rapid and sensitive responses to xenobiotics, making it an ideal model system to study the evolutionary and functional diversity of the P450 monooxygenase gene family. Results A total of 44 putative functional cytochrome P450 genes were identified and could be classified into 13 families and 21 sub-families according to standard nomenclature. The characteristics of both the conserved intron-exon organization and scaffold localization of tandem repeats within each P450 family clade suggested that the enlargement of T. thermophila P450 families probably resulted from recent separate small duplication events. Gene expression patterns of all T. thermophila P450s during three important cell physiological stages (vegetative growth, starvation and conjugation were analyzed based on EST and microarray data, and three main categories of expression patterns were postulated. Evolutionary analysis including codon usage preference, site-specific selection and gene-expression evolution patterns were investigated and the results indicated remarkable divergences among the T. thermophila P450 genes. Conclusion The characterization, expression and evolutionary analysis of T. thermophila P450 monooxygenase genes in the current study provides useful information for understanding the characteristics and diversities of the P450 genes in the Ciliophora, and provides the baseline for functional analyses of individual P450 isoforms in this model ciliate species.

  10. Pseudomonas aeruginosa LysR PA4203 regulator NmoR acts as a repressor of the PA4202 nmoA> gene, encoding a nitronate monooxygenase

    DEFF Research Database (Denmark)

    Vercammen, Ken; Wei, Qing; Charlier, Daniel;

    2015-01-01

    The PA4203 gene encodes a LysR regulator and lies between the ppgL gene (PA4204), which encodes a periplasmic gluconolactonase, and, in the opposite orientation, the PA4202 (nmoA) gene, coding for a nitronate monooxygenase, and ddlA (PA4201), encoding a d-alanine alanine ligase. The intergenic re...

  11. Tuning the specificity of the recombinant multicomponent toluene o-xylene monooxygenase from Pseudomonas sp. strain OX1 for the biosynthesis of tyrosol from 2-phenylethanol.

    Science.gov (United States)

    Notomista, Eugenio; Scognamiglio, Roberta; Troncone, Luca; Donadio, Giuliana; Pezzella, Alessandro; Di Donato, Alberto; Izzo, Viviana

    2011-08-01

    Biocatalysis is today a standard technology for the industrial production of several chemicals, and the number of biotransformation processes running on a commercial scale is constantly increasing. Among biocatalysts, bacterial multicomponent monooxygenases (BMMs), a diverse group of nonheme diiron enzymes that activate dioxygen, are of primary interest due to their ability to catalyze a variety of complex oxidations, including reactions of mono- and dihydroxylation of phenolic compounds. In recent years, both directed evolution and rational design have been successfully used to identify the molecular determinants responsible for BMM regioselectivity and to improve their activity toward natural and nonnatural substrates. Toluene o-xylene monooxygenase (ToMO) is a BMM isolated from Pseudomonas sp. strain OX1 which hydroxylates a wide spectrum of aromatic compounds. In this work we investigate the use of recombinant ToMO for the biosynthesis in recombinant cells of Escherichia coli strain JM109 of 4-hydroxyphenylethanol (tyrosol), an antioxidant present in olive oil, from 2-phenylethanol, a cheap and commercially available substrate. We initially found that wild-type ToMO is unable to convert 2-phenylethanol to tyrosol. This was explained by using a computational model which analyzed the interactions between ToMO active-site residues and the substrate. We found that residue F176 is the major steric hindrance for the correct positioning of the reaction intermediate leading to tyrosol production into the active site of the enzyme. Several mutants were designed and prepared, and we found that the combination of different mutations at position F176 with mutation E103G allows ToMO to convert up to 50% of 2-phenylethanol into tyrosol in 2 h.

  12. Development of a plant viral-vector-based gene expression assay for the screening of yeast cytochrome p450 monooxygenases.

    Science.gov (United States)

    Hanley, Kathleen; Nguyen, Long V; Khan, Faizah; Pogue, Gregory P; Vojdani, Fakhrieh; Panda, Sanjay; Pinot, Franck; Oriedo, Vincent B; Rasochova, Lada; Subramanian, Mani; Miller, Barbara; White, Earl L

    2003-02-01

    Development of a gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult. The activity assays are labor-intensive and not amenable to parallel screening. Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity. Plant virus gene expression systems have been utilized for a diverse group of organisms. In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid omega-hydroxylase activity. Yarrowia lipolytica CYP52 gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant virus gene expression vector, and used as a model system for determining heterologous expression. Plants infected with virus vectors expressing the yeast CYP52 genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves. No phenotype was detected on negative control constructs. YlALK3-, YlALK5-, and YlALK7-inoculated plants all catalyzed the terminal hydroxylation of lauric acid as confirmed using thin-layer and gas chromatography/mass spectrometry methods. The plant-based cytochrome P450 phenotypic screen was tested on an n-alkane-induced Yarrowia lipolytica plant virus expression library. A subset of 1,025 random library clones, including YlALK1-YlALK7 constructs, were tested on plants. All YlALK gene constructs scored positive in the randomized screen. Following nucleotide sequencing of the clones that scored positive using a phenotypic screen, approximately 5% were deemed appropriate for further biochemical analysis. This report illustrates the utility of a plant-based system for expression of heterologous cytochrome P450 monooxygenases and for the assignment of gene function.

  13. Oxidation of chlorinated olefins by Escherichia coli transformed with dimethyl sulfide monooxygenase genes or cumene dioxygenase genes.

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    Takami, Wako; Yoshida, Takako; Nojiri, Hideaki; Yamane, Hisakazu; Omori, Toshio

    1999-04-01

    In the present work, it was shown that the dimethyl sulfide (DMS) monooxygenase and the cumene dioxygenase catalyzed oxidation of various chlorinated ethenes, propenes, and butenes. The specific activities of these oxygenases were determined for C(2) to C(4) chlorinated olefins, and the oxidation rates ranged from 0.19 to 4.18 nmol.min(-1).mg(-1) of dry cells by the DMS monooxygenase and from 0.19 to 1.29 nmol.min(-1).mg(-1) of dry cells by the cumene dioxygenase. The oxidation products were identified by gas chromatography-mass spectrometry. Most chlorinated olefins were monooxygenated by the DMS monooxygenase to yield chlorinated epoxides. In the case of the cumene dioxygenase, the substrates lacking any chlorine atom on double-bond carbon atoms were dioxygenated, and those with chlorine atoms attaching to double-bond carbon atoms were monooxygenated to yield allyl alcohols.

  14. Rescue of white egg 1 mutant by introduction of the wild-type Bombyx kynurenine 3-monooxygenase gene

    Institute of Scientific and Technical Information of China (English)

    GUO-XING QUAN; ISAO KOBAYASHI; KATSURA KOJIMA; KEIRO UCHINO; TOSHIO KANDA; HIDEKI SEZUTSU; TORU SHIMADA; TOSHIKI TAMURA

    2007-01-01

    In silkworms, the white egg 1 (w-1) mutant, which is characterized by white eyes and white eggs, is deficient in Bombyx kynurenine 3-monooxygenase (KMO) activity. To investigate whether the w-1 mutant phenotype is rescued by introducing the wild-type KMO gene, we constructed transgenic silkworms with the wild-type Bombyx KMO gene under the control of either the cytoplasmic actin gene promoter (A3KMO) or the native KMO gene promoter (KKMO). We created two transgenic lines with A3KMO and one line with KKMO constructs. The eyes of adults in these lines were brown, and the eggs laid by the transgenic females were also brown. Reverse transcription-polymerase chain reaction(RT-PCR) analysis showed that the A3KMO silkworm lines expressed the transcript in the mid-gut, fat bodies, and Malpighian tubules. The KKMO line expressed the transcript only in the fat bodies and Malpighian tubules. The intensity of eye and egg color in the transgenic lines was proportional to the KMO expression level. Interestingly, transgenic larvae with the A3KMO construct had a light brown larval cuticle, but the KKMO line did not. These results indicate that the wild-type KMO gene can be used as a marker gene for visually screening transgenic silkworms.

  15. The detection and phylogenetic analysis of the alkane 1-monooxygenase gene of members of the genus Rhodococcus.

    Science.gov (United States)

    Táncsics, András; Benedek, Tibor; Szoboszlay, Sándor; Veres, Péter G; Farkas, Milán; Máthé, István; Márialigeti, Károly; Kukolya, József; Lányi, Szabolcs; Kriszt, Balázs

    2015-02-01

    Naturally occurring and anthropogenic petroleum hydrocarbons are potential carbon sources for many bacteria. The AlkB-related alkane hydroxylases, which are integral membrane non-heme iron enzymes, play a key role in the microbial degradation of many of these hydrocarbons. Several members of the genus Rhodococcus are well-known alkane degraders and are known to harbor multiple alkB genes encoding for different alkane 1-monooxygenases. In the present study, 48 Rhodococcus strains, representing 35 species of the genus, were investigated to find out whether there was a dominant type of alkB gene widespread among species of the genus that could be used as a phylogenetic marker. Phylogenetic analysis of rhodococcal alkB gene sequences indicated that a certain type of alkB gene was present in almost every member of the genus Rhodococcus. These alkB genes were common in a unique nucleotide sequence stretch absent from other types of rhodococcal alkB genes that encoded a conserved amino acid motif: WLG(I/V/L)D(G/D)GL. The sequence identity of the targeted alkB gene in Rhodococcus ranged from 78.5 to 99.2% and showed higher nucleotide sequence variation at the inter-species level compared to the 16S rRNA gene (93.9-99.8%). The results indicated that the alkB gene type investigated might be applicable for: (i) differentiating closely related Rhodococcus species, (ii) properly assigning environmental isolates to existing Rhodococcus species, and finally (iii) assessing whether a new Rhodococcus isolate represents a novel species of the genus. Copyright © 2014 Elsevier GmbH. All rights reserved.

  16. Somatic recombination, gene amplification and cancer.

    Science.gov (United States)

    Ramel, C; Cederberg, H; Magnusson, J; Vogel, E; Natarajan, A T; Mullender, L H; Nivard, J M; Parry, J M; Leyson, A; Comendador, M A; Sierra, L M; Ferreiro, J A; Consuegra, S

    1996-06-12

    The principle objective of this research programme, to analyse chemical induction of somatic recombination and related endpoints, i.e., mobilization of transposing elements and gene amplification, has been approached by means of several assay systems. These have included Drosophila, Saccharomyces and mammalian cell cultures. 6.1. Screening assays for mitotic recombination. A large number of chemicals have been investigated in the three Drosophila assay systems employed--the multiple wing hair/flare wing spot system developed by Graf et al., 1984, the white-ivory system developed by Green et al., 1986 and the white/white+ eye spot assay developed by Vogel (Vogel and Nivard, 1993). Particularly the screening of 181 chemicals, covering a wide array of chemical classes, by the last mentioned assay has shown that measurement of somatic recombination in Drosophila constitutes a sensitive and efficient short-term test which shows a remarkably good correlation with the agent score of 83 short-term tests analysed by ICPEMC (Mendelsohn et al., 1992; Table 2) as well as the assay performance in international collaborative programmes measuring carcinogen/non-carcinogens (de Serres and Ashby, 1981; Ashby et al., 1985, 1988). Also the wing spot assay has gained wide international recognition as a similarly sensitive test. These two assay systems in Drosophila measure both intrachromosomal events and interchromosomal recombination. The white-ivory system on the other hand is based on the loss of a tandem duplication in the white locus, the mechanism of which is less known, but probably involves intrachromosomal recombination. The difference in the mechanism between this assay and the former two was indicated by the lack of response to methotrexate in the white-ivory assay, while this compound was strongly recombinogenic in both the wing spot and white/white+ assays. The use of different strains of Drosophila with the white/white+ assay demonstrated the importance of the

  17. Cloning and expression of three ladA-type alkane monooxygenase genes from an extremely thermophilic alkane-degrading bacterium Geobacillus thermoleovorans B23.

    Science.gov (United States)

    Boonmak, Chanita; Takahashi, Yasunori; Morikawa, Masaaki

    2014-05-01

    An extremely thermophilic bacterium, Geobacillus thermoleovorans B23, is capable of degrading a broad range of alkanes (with carbon chain lengths ranging between C11 and C32) at 70 °C. Whole-genome sequence analysis revealed that unlike most alkane-degrading bacteria, strain B23 does not possess an alkB-type alkane monooxygenase gene. Instead, it possesses a cluster of three ladA-type genes, ladAαB23, ladAβB23, and ladB B23, on its chromosome, whose protein products share significant amino acid sequence identities, 49.8, 34.4, and 22.7 %, respectively, with that of ladA alkane monooxygenase gene found on a plasmid of Geobacillus thermodetrificans NG 80-2. Each of the three genes, ladAαB23, ladAβB23, and ladB B23, was heterologously expressed individually in an alkB1 deletion mutant strain, Pseudomonas fluorescens KOB2Δ1. It was found that all three genes were functional in P. fluorescens KOB2Δ1, and partially restored alkane degradation activity. In this study, we suggest that G. thermoleovorans B23 utilizes multiple LadA-type alkane monooxygenases for the degradation of a broad range of alkanes.

  18. The multifunctional peptidylglycine alpha-amidating monooxygenase gene: exon/intron organization of catalytic, processing, and routing domains.

    Science.gov (United States)

    Ouafik, L H; Stoffers, D A; Campbell, T A; Johnson, R C; Bloomquist, B T; Mains, R E; Eipper, B A

    1992-10-01

    Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction and is dependent on copper, ascorbate, and molecular oxygen. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyzes the second step of the reaction. Previous studies demonstrated that alternative splicing results in the production of bifunctional PAM proteins that are integral membrane or soluble proteins as well as soluble monofunctional PHM proteins. Rat PAM is encoded by a complex single copy gene that consists of 27 exons and encompasses more than 160 kilobases (kb) of genomic DNA. The 12 exons comprising PHM are distributed over at least 76 kb genomic DNA and range in size from 49-185 base pairs; four of the introns within the PHM domain are over 10 kb in length. Alternative splicing in the PHM region can result in a truncated, inactive PHM protein (rPAM-5), or a soluble, monofunctional PHM protein (rPAM-4) instead of a bifunctional protein. The eight exons comprising PAL are distributed over at least 19 kb genomic DNA. The exons encoding PAL range in size from 54-209 base pairs and have not been found to undergo alternative splicing. The PHM and PAL domains are separated by a single alternatively spliced exon surrounded by lengthy introns; inclusion of this exon results in the production of a form of PAM (rPAM-1) in which endoproteolytic cleavage at a paired basic site can separate the two catalytic domains. The exon following the PAL domain encodes the trans-membrane domain of PAM; alternative splicing at this site produces integral membrane or soluble PAM proteins. The COOH-terminal domain of PAM is comprised of a short exon subject to alternative splicing and a long exon encoding the final 68 amino acids present in all bifunctional PAM proteins along

  19. Cooperation between MEF2 and PPARγ in human intestinal β,β-carotene 15,15'-monooxygenase gene expression

    Directory of Open Access Journals (Sweden)

    Yan Bingfang

    2006-02-01

    Full Text Available Abstract Background Vitamin A and its derivatives, the retinoids, are essential for normal embryonic development and maintenance of cell differentiation. β, β-carotene 15,15'-monooxygenase 1 (BCMO1 catalyzes the central cleavage of β-carotene to all-trans retinal and is the key enzyme in the intestinal metabolism of carotenes to vitamin A. However, human and various rodent species show markedly different efficiencies in intestinal BCMO1-mediated carotene to retinoid conversion. The aim of this study is to identify potentially human-specific regulatory control mechanisms of BCMO1 gene expression. Results We identified and functionally characterized the human BCMO1 promoter sequence and determined the transcriptional regulation of the BCMO1 gene in a BCMO1 expressing human intestinal cell line, TC-7. Several functional transcription factor-binding sites were identified in the human promoter that are absent in the mouse BCMO1 promoter. We demonstrate that the proximal promoter sequence, nt -190 to +35, confers basal transcriptional activity of the human BCMO1 gene. Site-directed mutagenesis of the myocyte enhancer factor 2 (MEF2 and peroxisome proliferator-activated receptor (PPAR binding elements resulted in decreased basal promoter activity. Mutation of both promoter elements abrogated the expression of intestinal cell BCMO1. Electrophoretic mobility shift and supershift assays and transcription factor co-expression in TC-7 cells showed MEF2C and PPARγ bind to their respective DNA elements and synergistically transactivate BCMO1 expression. Conclusion We demonstrate that human intestinal cell BCMO1 expression is dependent on the functional cooperation between PPARγ and MEF2 isoforms. The findings suggest that the interaction between MEF2 and PPAR factors may provide a molecular basis for interspecies differences in the transcriptional regulation of the BCMO1 gene.

  20. Cloning and expressing DBT (dibenzothiophene) monooxygenase gene(dszC) from Rhodococcus sp.DS-3 in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    MA Ting; LI Shanshan; LI Guoqiang; WANG Renjing; LIANG Fenglai; LIU Rulin

    2006-01-01

    Dibenzothiophene (DBT) monooxygenase (DszC)catalysis,the first and also the key step in the microbial DBT desulfurization,is the conversion of DBT to DBT sulfone (DBTO2).In this study,dszC of a DBT-desulfiaizing bacterium Rhodococcus sp.DS-3 was cloned by PCR.The sequence cloned was 99% homologous to Rhodococcus erythropolis IGTS8 that was reported in the Genebank.The gene dszC could be overexpressed effectively after being inserted into plasmid pET28a and transformed into E.coli BL21 strain.The expression amount of DszC was about 20% of total supernatant at low temperature.The soluble DszC in the supematant was purified by Ni2+ chelating His-Tag resin column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to electronics purity.Only one band was detected by Western-blotting,which is for the antibody released in mouse against purified DszC in the expression product of BL21 (DE3,paC5) and Rhodococcus sp.DS-3.The activity of purified DszC was 0.36 U.DszC can utilize the organic compound such as DBT and methyl-DBT,hut not DBT derivates such as DBF,which has no sulfur or inorganic sulfur.

  1. Recombinant fungal entomopathogen RNAi target insect gene.

    Science.gov (United States)

    Hu, Qiongbo; Wu, Wei

    2016-11-01

    RNA interference (RNAi) technology is considered as an alternative for control of pests. However, RNAi has not been used in field conditions yet, since delivering exogenous ds/siRNA to target pests is very difficult. The laboratory methods of introducing the ds/siRNA into insects through feeding, micro feeding / dripping and injecting cannot be used in fields. Transgenic crop is perhaps the most effective application of RNAi for pest control, but it needs long-time basic researches in order to reduce the cost and evaluate the safety. Therefore, transgenic microbe is maybe a better choice. Entomopathogenic fungi generally invade the host insects through cuticle like chemical insecticides contact insect to control sucking sap pests. Isaria fumosorosea is a common fungal entomopathogen in whitefly, Bemisia tabaci. We constructed a recombinant strain of I. fumosorosea expressing specific dsRNA of whitefly's TLR7 gene. It could silence the TLR7 gene and improve the virulence against whitefly. Transgenic fungal entomopathogen has shown great potential to attain the application of RNAi technology for pests control in fields. In the future, the research interests should be focused on the selection of susceptible target pests and their vital genes, and optimizing the methods for screening genes and recombinants as well.

  2. Diversity of flavin-binding monooxygenase genes (almA) in marine bacteria capable of degradation long-chain alkanes.

    Science.gov (United States)

    Wang, Wanpeng; Shao, Zongze

    2012-06-01

    Many bacteria have been reported as degraders of long-chain (LC) n-alkanes, but the mechanism is poorly understood. Flavin-binding monooxygenase (AlmA) was recently found to be involved in LC-alkane degradation in bacteria of the Acinetobacter and Alcanivorax genera. However, the diversity of this gene and the role it plays in other bacteria remains unclear. In this study, we surveyed the diversity of almA in marine bacteria and in bacteria found in oil-enrichment communities. To identify the presence of this gene, a pair of degenerate PCR primers were was designed based on conserved motifs of the almA gene sequences in public databases. Using this approach, we identified diverse almA genes in the hydrocarbon-degrading bacteria and in bacterial communities from the surface seawater of the Xiamen coastal area, the South China Sea, the Indian Ocean, and the Atlantic Ocean. As a result, almA was positively detected in 35 isolates belonging to four genera, and a total of 39 different almA sequences were obtained. Five isolates were confirmed to harbor two to three almA genes. From the Xiamen coastal area and the Atlantic Ocean oil-enrichment communities, a total of 60 different almA sequences were obtained. These sequences mainly formed two clusters in the phylogenetic tree, named Class I and Class II, and these shared 45-56% identity at the amino acid level. Class I contained 11 sequences from bacteria represented by the Salinisphaera and Parvibaculum genera. Class II was larger and more diverse, and it was composed of 88 sequences from Proteobacteria, Gram-negative bacteria, and the enriched bacterial communities. These communities were represented by the Alcanivorax and Marinobacter genera, which are the two most popular genera hosting the almA gene. AlmA was also detected across a wide geographical range, as determined by the origin of the bacterial host. Our results demonstrate the diversity of almA and confirm its high rate of occurrence in hydrocarbon

  3. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

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    Pandey Sona

    2010-11-01

    Full Text Available Abstract Background Cytochrome P450 monooxygenases (P450s catalyze oxidation of various substrates using oxygen and NAD(PH. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of the soybean genome sequence allows us to identify and analyze soybean putative P450s at a genome scale. Co-expression analysis using an available soybean microarray and Illumina sequencing data provides clues for functional annotation of these enzymes. This approach is based on the assumption that genes that have similar expression patterns across a set of conditions may have a functional relationship. Results We have identified a total number of 332 full-length P450 genes and 378 pseudogenes from the soybean genome. From the full-length sequences, 195 genes belong to A-type, which could be further divided into 20 families. The remaining 137 genes belong to non-A type P450s and are classified into 28 families. A total of 178 probe sets were found to correspond to P450 genes on the Affymetrix soybean array. Out of these probe sets, 108 represented single genes. Using the 28 publicly available microarray libraries that contain organ-specific information, some tissue-specific P450s were identified. Similarly, stress responsive soybean P450s were retrieved from 99 microarray soybean libraries. We also utilized Illumina transcriptome sequencing technology to analyze the expressions of all 332 soybean P450 genes. This dataset contains total RNAs isolated from nodules, roots, root tips, leaves, flowers, green pods, apical meristem, mock-inoculated and Bradyrhizobium japonicum-infected root hair cells. The tissue-specific expression patterns of these P450 genes were analyzed and the expression of a representative set of genes were confirmed by qRT-PCR. We performed the co-expression analysis on many of the 108 P450 genes on the Affymetrix arrays. First we confirmed that CYP93C5 (an

  4. Auxin biosynthesis by the YUCCA6 flavin monooxygenase gene in woodland strawberry.

    Science.gov (United States)

    Liu, Hong; Xie, Wei-Fa; Zhang, Ling; Valpuesta, Victoriano; Ye, Zheng-Wen; Gao, Qing-Hua; Duan, Ke

    2014-04-01

    Auxin has been regarded as the main signal molecule coordinating the growth and ripening of fruits in strawberry, the reference genomic system for Rosaceae. The mechanisms regulating auxin biosynthesis in strawberry are largely elusive. Recently, we demonstrated that two YUCCA genes are involved in flower and fruit development in cultivated strawberry. Here, we show that the woodland strawberry (Fragaria vesca L.) genome harbors nine loci for YUCCA genes and eight of them encode functional proteins. Transcription pattern in different plant organs was different for all eight FvYUCs. Functionality of the FvYUC6 gene was studied in transgenic strawberry overexpressing FvYUC6, which showed typical high-auxin phenotypes. Overexpression of FvYUC6 also delayed flowering and led to complete male sterility in F. vesca. Additionally, specific repression of FvYUC6 expression by RNA interference significantly inhibited vegetative growth and reduced plant fertility. The development of leaves, roots, flowers, and fruits was greatly affected in FvYUC6-repressed plants. Expression of a subset of auxin-responsive genes was well correlated with the changes of FvYUC6 transcript levels and free indole-3-acetic acid levels in transgenic strawberry. These observations are consistent with an important role of FvYUC6 in auxin synthesis, and support a main role of the gene product in vegetative and reproductive development in woodland strawberry.

  5. Cloning and expression of three ladA-type alkane monooxygenase genes from an extremely thermophilic alkane-degrading bacterium Geobacillus thermoleovorans B23

    OpenAIRE

    2014-01-01

    An extremely thermophilic bacterium, Geobacillus thermoleovorans B23, is capable of degrading a broad range of alkanes (with carbon chain lengths ranging between C11 and C32) at 70 A degrees C. Whole-genome sequence analysis revealed that unlike most alkane-degrading bacteria, strain B23 does not possess an alkB-type alkane monooxygenase gene. Instead, it possesses a cluster of three ladA-type genes, ladA alpha(B23), ladA beta(B23), and ladB (B23), on its chromosome, whose protein products sh...

  6. Identification of the Pseudomonas stutzeri OX1 toluene-o-xylene monooxygenase regulatory gene (touR) and of its cognate promoter.

    Science.gov (United States)

    Arenghi, F L; Pinti, M; Galli, E; Barbieri, P

    1999-09-01

    Toluene-o-xylene monooxygenase is an enzymatic complex, encoded by the touABCDEF genes, responsible for the early stages of toluene and o-xylene degradation in Pseudomonas stutzeri OX1. In order to identify the loci involved in the transcriptional regulation of the tou gene cluster, deletion analysis and complementation studies were carried out with Pseudomonas putida PaW340 as a heterologous host harboring pFB1112, a plasmid that allowed regulated expression, inducible by toluene and o-xylene and their corresponding phenols, of the toluene-o-xylene monooxygenase. A locus encoding a positive regulator, designated touR, was mapped downstream from the tou gene cluster. TouR was found to be similar to transcriptional activators of aromatic compound catabolic pathways belonging to the NtrC family and, in particular, to DmpR (83% similarity), which controls phenol catabolism. By using a touA-C2,3O fusion reporter system and by primer extension analysis, a TouR cognate promoter (P(ToMO)) was mapped, which showed the typical -24 TGGC, -12 TTGC sequences characteristic of sigma(54)-dependent promoters and putative upstream activating sequences. By using the reporter system described, we found that TouR responds to mono- and dimethylphenols, but not the corresponding methylbenzenes. In this respect, the regulation of the P. stutzeri system differs from that of other toluene or xylene catabolic systems, in which the hydrocarbons themselves function as effectors. Northern analyses indicated low transcription levels of tou structural genes in the absence of inducers. Basal toluene-o-xylene monooxygenase activity may thus transform these compounds to phenols, which then trigger the TouR-mediated response.

  7. Identification of novel toluene monooxygenase genes in a hydrocarbon-polluted sediment using sequence- and function-based screening of metagenomic libraries.

    Science.gov (United States)

    Bouhajja, E; McGuire, M; Liles, M R; Bataille, G; Agathos, S N; George, I F

    2017-01-01

    The microbial potential for toluene degradation within sediments from a tar oil-contaminated site in Flingern, Germany, was assessed using a metagenomic approach. High molecular weight environmental DNA from contaminated sediments was extracted, purified, and cloned into fosmid and BAC vectors and transformed into Escherichia coli. The fosmid library was screened by hybridization with a PCR amplicon of the α-subunit of the toluene 4-monooxygenase gene to identify genes and pathways encoding toluene degradation. Fourteen clones were recovered from the fosmid library, among which 13 were highly divergent from known tmoA genes and several had the closest relatives among Acinetobacter species. The BAC library was transferred to the heterologous hosts Cupriavidus metallidurans (phylum Proteobacteria) and Edaphobacter aggregans (phylum Acidobacteria). The resulting libraries were screened for expression of toluene degradation in the non-degradative hosts. From expression in C. metallidurans, three novel toluene monooxygenase-encoding operons were identified that were located on IncP1 plasmids. The E. aggregans-hosted BAC library led to the isolation of a cloned genetic locus putatively derived from an Acidobacteria taxon that contained genes involved in aerobic and anaerobic toluene degradation. These data suggest the important role of plasmids in the spread of toluene degradative capacity and indicate putative novel tmoA genes present in this hydrocarbon-polluted environment.

  8. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

    Science.gov (United States)

    Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of soybean genome sequence allows us to ident...

  9. Intramitochondrial recombination - is it why some mitochondrial genes sleep around?

    Science.gov (United States)

    Dowton, M; Campbell, N J.H.

    2001-06-01

    A new paper by Kajander et al. undermines the general view that mitochondria do not recombine. The authors discovered the existence of 'sublimons', rearranged mitochondrial genomes present at very low levels in healthy human patients. Crucially, the different rearranged mitochondrial genomes can theoretically be interconverted through intramitochondrial recombination. The putative operation of intramitochondrial recombination should impact on our ideas of how mitochondrial genes evolve, particularly with respect to how mitochondrial genomes rearrange.

  10. Evaluation of bacterial communities by bacteriome analysis targeting 16S rRNA genes and quantitative analysis of ammonia monooxygenase gene in different types of compost.

    Science.gov (United States)

    Kitamura, Rika; Ishii, Kazuo; Maeda, Isamu; Kozaki, Toshinori; Iwabuchi, Kazunori; Saito, Takahiro

    2016-01-01

    Biofiltration technology based on microbial degradation and assimilation is used for the removal of malodorous compounds, such as ammonia. Microbes that degrade malodorous and/or organic substances are involved in composting and are retained after composting; therefore, mature composts can serve as an ideal candidate for a biofilter medium. In this study, we focused on different types of raw compost materials, as these are important factors determining the bacterial community profile and the chemical component of the compost. Therefore, bacterial community profiles, the abundance of the bacterial ammonia monooxygenase gene (amoA), and the quantities of chemical components were analyzed in composts produced from either food waste or cattle manure. The community profiles with the lowest beta diversity were obtained from single type of cattle manure compost. However, cattle manure composts showed greater alpha diversity, contained higher amounts of various rRNA gene fragments than those of food waste composts and contained the amoA gene by relative quantification, and Proteobacteria were abundantly found and nitrifying bacteria were detected in it. Nitrifying bacteria are responsible for ammonia oxidation and mainly belong to the Proteobacteria or Nitrospira phyla. The quantities of chemical components, such as salt, phosphorus, and nitrogen, differed between the cattle manure and food waste composts, indicating that the raw materials provided different fermentation environments that were crucial for the formation of different community profiles. The results also suggest that cattle manure might be a more suitable raw material for the production of composts to be used in the biofiltration of ammonia. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. The Origin and Evolution of Baeyer-Villiger Monooxygenases (BVMOs): An Ancestral Family of Flavin Monooxygenases.

    Science.gov (United States)

    Mascotti, Maria Laura; Lapadula, Walter Jesús; Juri Ayub, Maximiliano

    2015-01-01

    The Baeyer-Villiger Monooxygenases (BVMOs) are enzymes belonging to the "Class B" of flavin monooxygenases and are capable of performing exquisite selective oxidations. These enzymes have been studied from a biotechnological perspective, but their physiological substrates and functional roles are widely unknown. Here, we investigated the origin, taxonomic distribution and evolutionary history of the BVMO genes. By using in silico approaches, 98 BVMO encoding genes were detected in the three domains of life: Archaea, Bacteria and Eukarya. We found evidence for the presence of these genes in Metazoa (Hydra vulgaris, Oikopleura dioica and Adineta vaga) and Haptophyta (Emiliania huxleyi) for the first time. Furthermore, a search for other "Class B" monooxygenases (flavoprotein monooxygenases--FMOs--and N-hydroxylating monooxygenases--NMOs) was conducted. These sequences were also found in the three domains of life. Phylogenetic analyses of all "Class B" monooxygenases revealed that NMOs and BVMOs are monophyletic, whereas FMOs form a paraphyletic group. Based on these results, we propose that BVMO genes were already present in the last universal common ancestor (LUCA) and their current taxonomic distribution is the result of differential duplication and loss of paralogous genes.

  12. The Origin and Evolution of Baeyer-Villiger Monooxygenases (BVMOs: An Ancestral Family of Flavin Monooxygenases.

    Directory of Open Access Journals (Sweden)

    Maria Laura Mascotti

    Full Text Available The Baeyer-Villiger Monooxygenases (BVMOs are enzymes belonging to the "Class B" of flavin monooxygenases and are capable of performing exquisite selective oxidations. These enzymes have been studied from a biotechnological perspective, but their physiological substrates and functional roles are widely unknown. Here, we investigated the origin, taxonomic distribution and evolutionary history of the BVMO genes. By using in silico approaches, 98 BVMO encoding genes were detected in the three domains of life: Archaea, Bacteria and Eukarya. We found evidence for the presence of these genes in Metazoa (Hydra vulgaris, Oikopleura dioica and Adineta vaga and Haptophyta (Emiliania huxleyi for the first time. Furthermore, a search for other "Class B" monooxygenases (flavoprotein monooxygenases--FMOs--and N-hydroxylating monooxygenases--NMOs was conducted. These sequences were also found in the three domains of life. Phylogenetic analyses of all "Class B" monooxygenases revealed that NMOs and BVMOs are monophyletic, whereas FMOs form a paraphyletic group. Based on these results, we propose that BVMO genes were already present in the last universal common ancestor (LUCA and their current taxonomic distribution is the result of differential duplication and loss of paralogous genes.

  13. [Enzymatic regulatory processes in gene recombination].

    Science.gov (United States)

    Kovarskiĭ, V A; Profir, A V

    1988-01-01

    Recombination bistability in the system of genetic regulation in pro- and eucaryots is analysed on the basis of sigmoid kinetics of regulatory enzymes. It is shown that under an increase of either exogenic factors (temperature) or endogenic factors (concentration of molecules, which activate the enzymes) of crucial values, bistability solutions for recombination frequencies are possible. Histeresic character of the dependence of this value on the external parameters is pointed out. The role of fluctuation processes in distortion of the memory effects is discussed. On the basis of monostable solutions molecular account for the empiric Plau law is given for U-shaped dependence of recombination frequency on temperature.

  14. Diversity of alkane degrading bacteria associated with plants in a petroleum oil-contaminated environment and expression of alkane monooxygenase (alkB) genes

    Science.gov (United States)

    Andria, V.; Yousaf, S.; Reichenauer, T. G.; Smalla, K.; Sessitsch, A.

    2009-04-01

    Among twenty-six different plant species, Italian ryegrass (Lolium multiflorum var. Taurus), Birdsfoot trefoil (Lotus corniculatus var. Leo), and the combination of both plants performed well in a petroleum oil contaminated soil. Hydrocarbon degrading bacteria were isolated from the rhizosphere, root interior and shoot interior and subjected to the analysis of 16S rRNA, the 16S and 23S rRNA intergenic spacer region and alkane hydroxylase genes. Higher numbers of culturable, degrading bacteria were associated with Italian ryegrass, which were also characterized by a higher diversity, particularly in the plant interior. Only half of the isolated bacteria hosted known alkane hydroxylase genes (alkB and cytochrome P153-like). Our results indicated that alkB genes have spread through horizontal gene transfer, particularly in the Italian ryegrass rhizosphere, and suggested mobility of catabolic genes between Gram-negative and Gram-positive bacteria. We furthermore studied the colonization behaviour of selected hydrocarbon-degrading strains (comprising an endopyhte and a rhizosphere strain) as well as the expression of their alkane monooxygenase genes in association with Italian ryegrass. Results showed that the endophyte strain better colonized the plant, particularly the plant interior, and also showed higher expression of alkB genes suggesting a more efficient degradation of the pollutant. Furthermore, plants inoculated with the endophyte were better able to grow in the presence of diesel. The rhizosphere strain colonized primarily the rhizosphere and showed low alkB gene expression in the plant interior.

  15. Improved NADPH Regeneration for Fungal Cytochrome P450 Monooxygenase by Co-Expressing Bacterial Glucose Dehydrogenase in Resting-Cell Biotransformation of Recombinant Yeast.

    Science.gov (United States)

    Jeon, Hyunwoo; Durairaj, Pradeepraj; Lee, Dowoo; Ahsan, Md Murshidul; Yun, Hyungdon

    2016-12-28

    Fungal cytochrome P450 (CYP) enzymes catalyze versatile monooxygenase reactions and play a major role in fungal adaptations owing to their essential roles in the production avoid metabolites critical for pathogenesis, detoxification of xenobiotics, and exploitation avoid substrates. Although fungal CYP-dependent biotransformation for the selective oxidation avoid organic compounds in yeast system is advantageous, it often suffers from a shortage avoid intracellular NADPH. In this study, we aimed to investigate the use of bacterial glucose dehydrogenase (GDH) for the intracellular electron regeneration of fungal CYP monooxygenase in a yeast reconstituted system. The benzoate hydroxylase FoCYP53A19 and its homologous redox partner FoCPR from Fusarium oxysporum were co-expressed with the BsGDH from Bacillus subtilis in Saccharomyces cerevisiae for heterologous expression and biotransformations. We attempted to optimize several bottlenecks concerning the efficiency of fungal CYP-mediated whole-cell-biotransformation to enhance the conversion. The catalytic performance of the intracellular NADPH regeneration system facilitated the hydroxylation of benzoic acid to 4-hydroxybenzoic acid with high conversion in the resting-cell reaction. The FoCYP53A19+FoCPR+BsGDH reconstituted system produced 0.47 mM 4-hydroxybenzoic acid (94% conversion) in the resting-cell biotransformations performed in 50 mM phosphate buffer (pH 6.0) containing 0.5 mM benzoic acid and 0.25% glucose for 24 h at 30°C. The "coupled-enzyme" system can certainly improve the overall performance of NADPH-dependent whole-cell biotransformations in a yeast system.

  16. Mutation of glutamic acid 103 of toluene o-xylene monooxygenase as a means to control the catabolic efficiency of a recombinant upper pathway for degradation of methylated aromatic compounds.

    Science.gov (United States)

    Cafaro, Valeria; Notomista, Eugenio; Capasso, Paola; Di Donato, Alberto

    2005-08-01

    Toluene o-xylene monooxygenase (ToMO) and phenol hydroxylase (PH) of Pseudomonas stutzeri OX1 act sequentially in a recombinant upper pathway for the degradation of aromatic hydrocarbons. The catalytic efficiency and regioselectivity of these enzymes optimize the degradation of growth substrates like toluene and o-xylene. For example, the sequential monooxygenation of o-xylene by ToMO and PH leads to almost exclusive production of 3,4-dimethylcatechol (3,4-DMC), the only isomer that can be further metabolized by the P. stutzeri meta pathway. We investigated the possibility of producing ToMO mutants with modified regioselectivity compared with the regioselectivity of the wild-type protein in order to alter the ability of the recombinant upper pathway to produce methylcatechol isomers from toluene and to produce 3,4-DMC from o-xylene. The combination of mutant (E103G)-ToMO and PH increased the production of 4-methylcatechol from toluene and increased the formation of 3,4-DMC from o-xylene. These data strongly support the idea that the products and efficiency of the metabolic pathway can be controlled not only through mutations that increase the catalytic efficiency of the enzymes involved but also through tuning the substrate specificity and regioselectivity of the enzymes. These findings are crucial for the development of future metabolic engineering strategies.

  17. MPN- and Real-Time-Based PCR Methods for the Quantification of Alkane Monooxygenase Homologous Genes (alkB) in Environmental Samples

    Science.gov (United States)

    Pérez-de-Mora, Alfredo; Schulz, Stephan; Schloter, Michael

    Hydrocarbons are major contaminants of soil ecosystems as a result of uncontrolled oil spills and wastes disposal into the environment. Ecological risk assessment and remediation of affected sites is often constrained due to lack of suitable prognostic and diagnostic tools that provide information of abiotic-biotic interactions occurring between contaminants and biological targets. Therefore, the identification and quantification of genes involved in the degradation of hydrocarbons may play a crucial role for evaluating the natural attenuation potential of contaminated sites and the development of successful bioremediation strategies. Besides other gene clusters, the alk operon has been identified as a major player for alkane degradation in different soils. An oxygenase gene (alkB) codes for the initial step of the degradation of aliphatic alkanes under aerobic conditions. In this work, we present an MPN- and a real-time PCR method for the quantification of the bacterial gene alkB (coding for rubredoxin-dependent alkane monooxygenase) in environmental samples. Both approaches enable a rapid culture-independent screening of the alkB gene in the environment, which can be used to assess the intrinsic natural attenuation potential of a site or to follow up the on-going progress of bioremediation assays.

  18. Quasispecies theory for horizontal gene transfer and recombination

    Science.gov (United States)

    Muñoz, Enrique; Park, Jeong-Man; Deem, Michael W.

    2008-12-01

    We introduce a generalization of the parallel, or Crow-Kimura, and Eigen models of molecular evolution to represent the exchange of genetic information between individuals in a population. We study the effect of different schemes of genetic recombination on the steady-state mean fitness and distribution of individuals in the population, through an analytic field theoretic mapping. We investigate both horizontal gene transfer from a population and recombination between pairs of individuals. Somewhat surprisingly, these nonlinear generalizations of quasispecies theory to modern biology are analytically solvable. For two-parent recombination, we find two selected phases, one of which is spectrally rigid. We present exact analytical formulas for the equilibrium mean fitness of the population, in terms of a maximum principle, which are generally applicable to any permutation invariant replication rate function. For smooth fitness landscapes, we show that when positive epistatic interactions are present, recombination or horizontal gene transfer introduces a mild load against selection. Conversely, if the fitness landscape exhibits negative epistasis, horizontal gene transfer or recombination introduces an advantage by enhancing selection towards the fittest genotypes. These results prove that the mutational deterministic hypothesis holds for quasispecies models. For the discontinuous single sharp peak fitness landscape, we show that horizontal gene transfer has no effect on the fitness, while recombination decreases the fitness, for both the parallel and the Eigen models. We present numerical and analytical results as well as phase diagrams for the different cases.

  19. A collection of cytochrome P450 monooxygenase genes involved in modification and detoxification of herbicide atrazine in rice (Oryza sativa) plants.

    Science.gov (United States)

    Rong Tan, Li; Chen Lu, Yi; Jing Zhang, Jing; Luo, Fang; Yang, Hong

    2015-09-01

    Plant cytochrome P450 monooxygenases constitute one of the largest families of protein genes involved in plant growth, development and acclimation to biotic and abiotic stresses. However, whether these genes respond to organic toxic compounds and their biological functions for detoxifying toxic compounds such as herbicides in rice are poorly understood. The present study identified 201 genes encoding cytochrome P450s from an atrazine-exposed rice transcriptome through high-throughput sequencing. Of these, 69 cytochrome P450 genes were validated by microarray and some of them were confirmed by real time PCR. Activities of NADPH-cytochrome P450 reductase (CPR) and p-nitroanisole O-demethylase (PNOD) related to toxicity were determined and significantly induced by atrazine exposure. To dissect the mechanism underlying atrazine modification and detoxification by P450, metabolites (or derivatives) of atrazine in plants were analyzed by ultra performance liquid chromatography mass spectrometry (UPLC/MS). Major metabolites comprised desmethylatrazine (DMA), desethylatrazine (DEA), desisopropylatrazine (DIA), hydroxyatrazine (HA), hydroxyethylatrazine (HEA) and hydroxyisopropylatrazine (HIA). All of them were chemically modified by P450s. Furthermore, two specific inhibitors of piperonyl butoxide (PBO) and malathion (MAL) were used to assess the correlation between the P450s activity and rice responses including accumulation of atrazine in tissues, shoot and root growth and detoxification. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Seamless gene tagging by endonuclease-driven homologous recombination.

    Directory of Open Access Journals (Sweden)

    Anton Khmelinskii

    Full Text Available Gene tagging facilitates systematic genomic and proteomic analyses but chromosomal tagging typically disrupts gene regulatory sequences. Here we describe a seamless gene tagging approach that preserves endogenous gene regulation and is potentially applicable in any species with efficient DNA double-strand break repair by homologous recombination. We implement seamless tagging in Saccharomyces cerevisiae and demonstrate its application for protein tagging while preserving simultaneously upstream and downstream gene regulatory elements. Seamless tagging is compatible with high-throughput strain construction using synthetic genetic arrays (SGA, enables functional analysis of transcription antisense to open reading frames and should facilitate systematic and minimally-invasive analysis of gene functions.

  1. Recombination Rate Heterogeneity within Arabidopsis Disease Resistance Genes.

    Directory of Open Access Journals (Sweden)

    Kyuha Choi

    2016-07-01

    Full Text Available Meiotic crossover frequency varies extensively along chromosomes and is typically concentrated in hotspots. As recombination increases genetic diversity, hotspots are predicted to occur at immunity genes, where variation may be beneficial. A major component of plant immunity is recognition of pathogen Avirulence (Avr effectors by resistance (R genes that encode NBS-LRR domain proteins. Therefore, we sought to test whether NBS-LRR genes would overlap with meiotic crossover hotspots using experimental genetics in Arabidopsis thaliana. NBS-LRR genes tend to physically cluster in plant genomes; for example, in Arabidopsis most are located in large clusters on the south arms of chromosomes 1 and 5. We experimentally mapped 1,439 crossovers within these clusters and observed NBS-LRR gene associated hotspots, which were also detected as historical hotspots via analysis of linkage disequilibrium. However, we also observed NBS-LRR gene coldspots, which in some cases correlate with structural heterozygosity. To study recombination at the fine-scale we used high-throughput sequencing to analyze ~1,000 crossovers within the RESISTANCE TO ALBUGO CANDIDA1 (RAC1 R gene hotspot. This revealed elevated intragenic crossovers, overlapping nucleosome-occupied exons that encode the TIR, NBS and LRR domains. The highest RAC1 recombination frequency was promoter-proximal and overlapped CTT-repeat DNA sequence motifs, which have previously been associated with plant crossover hotspots. Additionally, we show a significant influence of natural genetic variation on NBS-LRR cluster recombination rates, using crosses between Arabidopsis ecotypes. In conclusion, we show that a subset of NBS-LRR genes are strong hotspots, whereas others are coldspots. This reveals a complex recombination landscape in Arabidopsis NBS-LRR genes, which we propose results from varying coevolutionary pressures exerted by host-pathogen relationships, and is influenced by structural heterozygosity.

  2. Recombination Rate Heterogeneity within Arabidopsis Disease Resistance Genes

    Science.gov (United States)

    Serra, Heïdi; Ziolkowski, Piotr A.; Yelina, Nataliya E.; Jackson, Matthew; Mézard, Christine; McVean, Gil; Henderson, Ian R.

    2016-01-01

    Meiotic crossover frequency varies extensively along chromosomes and is typically concentrated in hotspots. As recombination increases genetic diversity, hotspots are predicted to occur at immunity genes, where variation may be beneficial. A major component of plant immunity is recognition of pathogen Avirulence (Avr) effectors by resistance (R) genes that encode NBS-LRR domain proteins. Therefore, we sought to test whether NBS-LRR genes would overlap with meiotic crossover hotspots using experimental genetics in Arabidopsis thaliana. NBS-LRR genes tend to physically cluster in plant genomes; for example, in Arabidopsis most are located in large clusters on the south arms of chromosomes 1 and 5. We experimentally mapped 1,439 crossovers within these clusters and observed NBS-LRR gene associated hotspots, which were also detected as historical hotspots via analysis of linkage disequilibrium. However, we also observed NBS-LRR gene coldspots, which in some cases correlate with structural heterozygosity. To study recombination at the fine-scale we used high-throughput sequencing to analyze ~1,000 crossovers within the RESISTANCE TO ALBUGO CANDIDA1 (RAC1) R gene hotspot. This revealed elevated intragenic crossovers, overlapping nucleosome-occupied exons that encode the TIR, NBS and LRR domains. The highest RAC1 recombination frequency was promoter-proximal and overlapped CTT-repeat DNA sequence motifs, which have previously been associated with plant crossover hotspots. Additionally, we show a significant influence of natural genetic variation on NBS-LRR cluster recombination rates, using crosses between Arabidopsis ecotypes. In conclusion, we show that a subset of NBS-LRR genes are strong hotspots, whereas others are coldspots. This reveals a complex recombination landscape in Arabidopsis NBS-LRR genes, which we propose results from varying coevolutionary pressures exerted by host-pathogen relationships, and is influenced by structural heterozygosity. PMID:27415776

  3. Expression of recombination-activating genes and T cell receptor gene recombination in the human T cell leukemia cell line.

    Science.gov (United States)

    Zou, Hong-yun; Ma, Li; Meng, Min-jie; Yao, Xin-sheng; Lin, Ying; Wu, Zhen-qiang; He, Xiao-wei; Wang, Ju-fang; Wang, Xiao-ning

    2007-03-05

    Recent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes (RAG) and RAG-mediated V(D)J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether thebreceptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkathuman T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of Tcell receptor (TCR) gene recombination. TCR Dbeta-Jbeta signal joint T cell receptor excision DNA circles (sjTRECs) were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences (RSSs) in the TCRVbeta chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 (CDR3) size of the TCRVbeta chain was examined by the TCR GeneScan technique. RAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining (NHEJ) pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dbeta2-Jbeta2 signal joints and ds RSS breaks associated with the Dbeta2 5' and Dbeta 2 3' sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVbeta chain did not change during cell proliferation. RAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire.However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V(D)J recombination and as a "special" model of the coexistence of TCR gene rearrangements and "negative" receptor revision.

  4. Expression of recombination-activating genes and T cell receptor gene recombination in the human T cell leukemia cell line

    Institute of Scientific and Technical Information of China (English)

    ZOU Hong-yun; MA Li; MENG Min-jie; YAO Xin-sheng; LIN Ying; WU Zhen-qiang; HE Xiao-wei; WANG Ju-fang; WANG Xiao-ning

    2007-01-01

    Background Recent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes (RAG) and RAG-mediated V(D)J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether the receptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkat human T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of T cell receptor (TCR) gene recombination.Methods TCR Dβ-Jβ signal joint T cell receptor excision DNA circles (sjTRECs) were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences (RSSs) in the TCRVβ chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 (CDR3) size of the TCRVβ chain was examined by the TCR GeneScan technique.Results RAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining (NHEJ) pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dβ2-Jβ2 signal joints and ds RSS breaks associated with the Dβ25' and Dβ 23' sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVβ chain did not change during cell proliferation.Conclusions RAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire. However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V(D)J recombination and as a "special" model of the coexistence of TCR gene rearrangements and "negative" receptor revision.

  5. Diversity and distribution of 16S rRNA and phenol monooxygenase genes in the rhizosphere and endophytic bacteria isolated from PAH-contaminated sites

    Science.gov (United States)

    Peng, Anping; Liu, Juan; Ling, Wanting; Chen, Zeyou; Gao, Yanzheng

    2015-07-01

    This is the first investigation of the diversity and distribution of 16S rRNA and phenol monooxygenase (PHE) genes in endophytic and rhizosphere bacteria of plants at sites contaminated with different levels of PAHs. Ten PAHs at concentrations from 34.22 to 55.29 and 45.79 to 97.81 mg·kg-1 were measured in rhizosphere soils of Alopecurus aequalis Sobol and Oxalis corniculata L., respectively. The diversity of 16S rRNA and PHE genes in rhizosphere soils or plants changed with varying PAH pollution levels, as shown based on PCR-DGGE data. Generally, higher Shannon-Weiner indexes were found in mild or moderate contaminated areas. A total of 82 different bacterial 16S rRNA gene sequences belonging to five phyla; namely, Acfinobacteria, Proteobacteria, Chloroflexi, Cyanophyta, and Bacteroidetes, were obtained from rhizosphere soils. For the 57 identified PHE gene sequences, 18 were excised from rhizosphere bacteria and 39 from endophytic bacteria. The copy numbers of 16S rRNA and PHE genes in rhizosphere and endophytic bacteria varied from 3.83 × 103 to 2.28 × 106 and 4.17 × 102 to 1.99 × 105, respectively. The copy numbers of PHE genes in rhizosphere bacteria were significantly higher than in endophytic bacteria. Results increase our understanding of the diversity of rhizosphere and endophytic bacteria from plants grown in PAH-contaminated sites.

  6. Diversity and distribution of 16S rRNA and phenol monooxygenase genes in the rhizosphere and endophytic bacteria isolated from PAH-contaminated sites.

    Science.gov (United States)

    Peng, Anping; Liu, Juan; Ling, Wanting; Chen, Zeyou; Gao, Yanzheng

    2015-07-17

    This is the first investigation of the diversity and distribution of 16S rRNA and phenol monooxygenase (PHE) genes in endophytic and rhizosphere bacteria of plants at sites contaminated with different levels of PAHs. Ten PAHs at concentrations from 34.22 to 55.29 and 45.79 to 97.81 mg·kg(-1) were measured in rhizosphere soils of Alopecurus aequalis Sobol and Oxalis corniculata L., respectively. The diversity of 16S rRNA and PHE genes in rhizosphere soils or plants changed with varying PAH pollution levels, as shown based on PCR-DGGE data. Generally, higher Shannon-Weiner indexes were found in mild or moderate contaminated areas. A total of 82 different bacterial 16S rRNA gene sequences belonging to five phyla; namely, Acfinobacteria, Proteobacteria, Chloroflexi, Cyanophyta, and Bacteroidetes, were obtained from rhizosphere soils. For the 57 identified PHE gene sequences, 18 were excised from rhizosphere bacteria and 39 from endophytic bacteria. The copy numbers of 16S rRNA and PHE genes in rhizosphere and endophytic bacteria varied from 3.83 × 10(3) to 2.28 × 10(6) and 4.17 × 10(2) to 1.99 × 10(5), respectively. The copy numbers of PHE genes in rhizosphere bacteria were significantly higher than in endophytic bacteria. Results increase our understanding of the diversity of rhizosphere and endophytic bacteria from plants grown in PAH-contaminated sites.

  7. Monitoring the alkane monooxygenase gene alkB in different soil interfaces during plant litter degradation of C3 and C4 plants

    Science.gov (United States)

    Schulz, S.; Munch, J. C.; Schloter, M.

    2009-04-01

    Hydrocarbons like n-alkanes are ubiquitous in the environment as a result of anthropogenic contamination (e.g. oil spills) as well as a part of an ecosystem's biomass. For example n-alkanes become released during plant litter degradation; consequently they become a high abundant carbon source for microorganism. One possibility for the prokaryotic hydrocarbon metabolisation is an aerobic degradation pathway where the initial step is catalysed by the membrane bound alkane monooxygenase alkB. We analysed the influence of alkanes on the abundance of the alkB gene in different interfaces of the litter-soil system during the degradation of maize and pea litter. Therefore soil samples of a sandy and a loamy soil have been incubated with straw of maize and pea plants up to 30 weeks with constant soil moisture and temperature. Using quantitative real-time PCR we were able to monitor the changes of the abundance and the expression rates of alkB. In our experiments we focused on the straw layer, the litter/soil interface and the soil 1 cm below this interface (bulk soil). Our results clearly demonstrate time and space dependent abundance patterns of alkB genes and transcripts in the different layers studied, which are additionally shaped by the soil type used.

  8. Recombination facilitates neofunctionalization of duplicate genes via originalization

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    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.

  9. Cloning of Toluene 4-Monooxygenase Genes and Application of Two-Phase System to the Production of the Anticancer Agent, Indirubin.

    Science.gov (United States)

    Wongsaroj, Lampet; Sallabhan, Ratiboot; Dubbs, James M; Mongkolsuk, Skorn; Loprasert, Suvit

    2015-08-01

    Indirubin is a strong inhibitor of several eukaryotic cell signaling pathways and shows promise as a treatment for myelocytic leukemia and Alzheimer's disease. The tmoABCDEF operon, encoding the components of a novel toluene 4-monooxygenase from the paint factory soil isolate, Pseudomonas sp. M4, was cloned and expressed in Escherichia coli. E. coli::pKSR12 expressing the tmo genes was used to develop a two-phase [dioctyl phthalate (DOP)/aqueous medium] culture system that was optimized to obtain maximal yields of indirubin from the starting substrate, indole. DOP was used as the organic phase to solubilize and sequester the toxic indole substrate, making possible the use of high indole concentrations that would otherwise interfere with growth in aqueous media. A 50 % (v/v) DOP two-phase system using tryptophan medium containing 3 mM cysteine, 5 mM indole, and 1 mM isatin yielded 102.4 mg/L of indirubin with no conversion of indole to the less valuable alternate product, indigo.

  10. rec genes and homologous recombination proteins in Escherichia coli.

    Science.gov (United States)

    Clark, A J

    1991-04-01

    The twenty-five years since the first published report of recA mutants in Escherichia coli has seen the identification of more than 12 other recombination genes. The genes are usually grouped into three pathways named RecBCD, RecE and RecF for prominent genes which function in each. A proposal is made here that there are two RecF pathways, one sensitive and one resistant to exonuclease I, the SbcB enzyme. Five methods of grouping the genes functionally are discussed: 1) by enzyme activity, 2) by common indirect suppressor, 3) by common phenotype, 4) by common regulation and 5) by epistasis. Five classes of enzyme activities implicated in recombination are discussed according to their involvement in presynapsis, synapsis or postsynapsis: 1) nucleases 2) helicases 3) DNA-binding proteins 4) topoisomerases and 5) ligases. Plausible presynaptic steps for the RecBCD, RecF (SbcBS) and RecE pathways show the common feature of generating 3'-terminated single-stranded DNA (ssDNA). On this ssDNA it is proposed that a RecA protein filament is generated discontinuously. This implies the existence of nucleation and possibly measurement and 3' end protection proteins. Specific proposals are made for which recombination genes might encode such products. Finally the generality of the RecA-ssDNA-filament mechanism of synapsis in the cellular biological world is discussed.

  11. Cloning of Rabbit HPRT Gene Using the Recombineering System

    Institute of Scientific and Technical Information of China (English)

    Jianjun SHI; Donghui CAI; Xuejin CHEN; Huizheng SHENG

    2007-01-01

    Hypoxanthine phosphoribosyltransferase (HPRT) plays an important role in the metabolic salvage of purines, and been used as an alternative pathway for mutant selection in many studies. To facilitate its application in rabbits, we have cloned the cDNA and genomic DNA of the rabbit HPRT gene using an approach that combines bioinformatics and recombineering methods. The cDNA is comprised of 1449 bp containing a coding sequence for a protein of 218 amino acids. The deduced amino acid sequence of the rabbit HPRT gene shares 98%, 97%, 98% and 94% identity with human, mouse, pig and cattle HPRT genes, respectively. Reverse transcription-polymerase chain reaction analysis showed that this gene is ubiquitously expressed in tissues of adult rabbit. The rabbit HPRT gene spans approximately 48 kb in length and consists of nine exons. The cloning of the rabbit HPRT gene shows the usefulness of the recombineering system in cloning genes of large size. This system may facilitate the subcloning of DNA from bacterial artificial chromosomes for cloning genes of large size or filling big gaps in genomic sequencing.

  12. Three faces of recombination activating gene 1 (RAG1) mutations.

    Science.gov (United States)

    Patiroglu, Turkan; Akar, Himmet Haluk; Van Der Burg, Mirjam

    2015-12-01

    Severe combined immune deficiency (SCID) is a group of genetic disorder associated with development of T- and/or B-lymphocytes. Recombination-activating genes (RAG1/2) play a critical role on VDJ recombination process that leads to the production of a broad T-cell receptor (TCR) and B-cell receptor (BCR) repertoire in the development of T and B cells. RAG1/2 genes mutations result in various forms of primary immunodeficiency, ranging from classic SCID to Omenn syndrome (OS) to atypical SCID with such as granuloma formation and autoimmunity. Herein, we reported 4 patients with RAG1 deficiency: classic SCID was seen in two patients who presented with recurrent pneumonia and chronic diarrhoea, and failure to thrive. OS was observed in one patient who presented with chronic diarrhoea, skin rash, recurrent lower respiratory infections, and atypical SCID was seen in one patient who presented with Pyoderma gangrenosum (PG) and had novel RAG1 mutation.

  13. Detecting Key Structural Features within Highly Recombined Genes

    Science.gov (United States)

    Wertz, John E; McGregor, Karen F; Bessen, Debra E

    2007-01-01

    Many microorganisms exhibit high levels of intragenic recombination following horizontal gene transfer events. Furthermore, many microbial genes are subject to strong diversifying selection as part of the pathogenic process. A multiple sequence alignment is an essential starting point for many of the tools that provide fundamental insights on gene structure and evolution, such as phylogenetics; however, an accurate alignment is not always possible to attain. In this study, a new analytic approach was developed in order to better quantify the genetic organization of highly diversified genes whose alleles do not align. This BLAST-based method, denoted BLAST Miner, employs an iterative process that places short segments of highly similar sequence into discrete datasets that are designated “modules.” The relative positions of modules along the length of the genes, and their frequency of occurrence, are used to identify sequence duplications, insertions, and rearrangements. Partial alleles of sof from Streptococcus pyogenes, encoding a surface protein under host immune selection, were analyzed for module content. High-frequency Modules 6 and 13 were identified and examined in depth. Nucleotide sequences corresponding to both modules contain numerous duplications and inverted repeats, whereby many codons form palindromic pairs. Combined with evidence for a strong codon usage bias, data suggest that Module 6 and 13 sequences are under selection to preserve their nucleic acid secondary structure. The concentration of overlapping tandem and inverted repeats within a small region of DNA is highly suggestive of a mechanistic role for Module 6 and 13 sequences in promoting aberrant recombination. Analysis of pbp2X alleles from Streptococcus pneumoniae, encoding cell wall enzymes that confer antibiotic resistance, supports the broad applicability of this tool in deciphering the genetic organization of highly recombined genes. BLAST Miner shares with phylogenetics the

  14. Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase

    NARCIS (Netherlands)

    Kamerbeek, NM; Olsthoorn, AJJ; Fraaije, MW; Janssen, DB; Kamerbeek, Nanne M.; Olsthoorn, Arjen J.J.

    2003-01-01

    The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of ca

  15. 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.

  16. [Advances in biomolecular machine: methane monooxygenases].

    Science.gov (United States)

    Lu, Jixue; Wang, Shizhen; Fang, Baishan

    2015-07-01

    Methane monooxygenases (MMO), regarded as "an amazing biomolecular machine", catalyze the oxidation of methane to methanol under aerobic conditions. MMO catalyze the oxidation of methane elaborately, which is a novel way to catalyze methane to methanol. Furthermore, MMO can inspire the biomolecular machine design. In this review, we introduced MMO including structure, gene and catalytic mechanism. The history and the taxonomy of MMO were also introduced.

  17. Recombinant cells that highly express chromosomally-integrated heterologous gene

    Science.gov (United States)

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2007-03-20

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  18. Characterization of recombinant bacteriophages containing mosquito ribosomal RNA genes

    Energy Technology Data Exchange (ETDEWEB)

    Park, Y.J.

    1988-01-01

    A family of nine recombinant bacteriophages containing rRNA genes from cultured cells of the mosquito, Aedes albopictus, has been isolated by screening two different genomic DNA libraries - Charon 30 and EMBL 3 using {sup 32}P-labeled 18S and 28S rRNA as probes. These nine recombinant bacteriophages were characterized by restriction mapping, Southern blotting, and S1 nuclease analysis. The 18S rRNA coding region contains an evolutionarily conserved EcoRI site near the 3{prime}-end, and measures 1800 bp. The 28S rRNA genes were divided into {alpha} and {beta} coding regions measuring 1750 bp and 2000 bp, respectively. The gap between these two regions measures about 340 bp. No insertion sequences were found in the rRNA coding regions. The entire rDNA repeat unit had a minimum length of 15.6 kb, including a nontranscribed spacer region. The non-transcribed spacer region of cloned A. albopictus rDNA contained a common series of seven PvuI sites within a 1250 bp region upstream of the 18S rRNA coding region, and a proportion of this region also showed heterogeneity both in the length and in the restriction sites.

  19. Directed mutagenesis affects recombination in Azospirillum brasilense nif genes

    Directory of Open Access Journals (Sweden)

    C.P. Nunes

    2000-12-01

    Full Text Available In order to improve the gene transfer/mutagenesis system for Azospirillum brasilense, gene-cartridge mutagenesis was used to replace the nifD gene with the Tn5 kanamycin resistance gene. The construct was transferred to A. brasilense by electrotransformation. Of the 12 colonies isolated using the suicide plasmid pSUP202 as vector, only four did not show vector integration into the chromosome. Nevertheless, all 12 colonies were deficient in acetylene reduction, indicating an Nif- phenotype. Four Nif- mutants were analyzed by Southern blot, using six different probes spanning the nif and Km r genes and the plasmid vector. Apparently, several recombination events occurred in the mutant genomes, probably caused mainly by gene disruption owing to the mutagenesis technique used: resistance gene-cartridge mutagenesis combined with electrotransformation.Com o objetivo de melhorar os sistemas de transferência gênica e mutagênese para Azospirillum brasilense, a técnica de mutagênese através do uso de um gene marcador ("gene-cartridge mutagenesis" foi utilizada para substituir a região genômica de A. brasilense correspondente ao gene nifD por um segmento de DNA do transposon Tn5 contendo o gene que confere resistência ao antibiótico canamicina. A construção foi transferida para a linhagem de A. brasilense por eletrotransformação. Doze colônias transformantes foram isoladas com o plasmídeo suicida pSUP202 servindo como vetor. Dessas, somente quatro não possuíam o vetor integrado no cromossomo da bactéria. Independentemente da integração ou não do vetor, as 12 colônias foram deficientes na redução do gás acetileno, evidenciando o fenótipo Nif -. Quatro mutantes Nif - foram analisados através da técnica de Southern blot, utilizando-se seis diferentes fragmentos contendo genes nif, de resistência à canamicina e do vetor como sondas. Os resultados sugerem a ocorrência de eventos recombinacionais variados no genoma dos mutantes. A

  20. Effects of the rad52 gene on recombination in Saccharomyces cerevisiae. [Comparison of. gamma. -, uv-induced meiotic and spontaneous mitotic recombination

    Energy Technology Data Exchange (ETDEWEB)

    Prakash, S.; Prakash, L.; Burke, W.; Montelone, B.A.

    1979-01-01

    Effects of the rad52 mutation in Saccharomyces cerevisiae on meiotic, ..gamma..-ray-induced, uv-induced, and spontaneous mitotic recombination were studied. The rad52/rad52 diploids undergo premeiotic DNA synthesis; sporulation occurs but inviable spores are produced. Intra- and intergenic recombination during meiosis were examined in cells transferred from sporulation medium to vegetative medium at different time intervals. No intragenic recombination was observed at the hisl-1/hisl-315 and trp5-2/trp5-48 heteroalleles. Gene-centromere recombination was also not observed in rad52/rad52 diploids. No ..gamma..-ray-induced intragenic mitotic recombination is seen in rad52/rad52 diploids and uv-induced intragenic recombination is greatly reduced. However, spontaneous mitotic recombination is not similarly affected. The RAD52 gene thus functions in recombination in meiosis and in ..gamma..-ray and uv-induced mitotic recombination but not in spontaneous mitotic recombination.

  1. RNA structures facilitate recombination-mediated gene swapping in HIV-1.

    Science.gov (United States)

    Simon-Loriere, Etienne; Martin, Darren P; Weeks, Kevin M; Negroni, Matteo

    2010-12-01

    Many viruses, including retroviruses, undergo frequent recombination, a process which can increase their rate of adaptive evolution. In the case of HIV, recombination has been responsible for the generation of numerous intersubtype recombinant variants with epidemiological importance in the AIDS pandemic. Although it is known that fragments of genetic material do not combine randomly during the generation of recombinant viruses, the mechanisms that lead to preferential recombination at specific sites are not fully understood. Here we reanalyze recent independent data defining (i) the structure of a complete HIV-1 RNA genome and (ii) favorable sites for recombination. We show that in the absence of selection acting on recombinant genomes, regions harboring RNA structures in the NL4-3 model strain are strongly predictive of recombination breakpoints in the HIV-1 env genes of primary isolates. In addition, we found that breakpoints within recombinant HIV-1 genomes sampled from human populations, which have been acted upon extensively by natural selection, also colocalize with RNA structures. Critically, junctions between genes are enriched in structured RNA elements and are also preferred sites for generating functional recombinant forms. These data suggest that RNA structure-mediated recombination allows the virus to exchange intact genes rather than arbitrary subgene fragments, which is likely to increase the overall viability and replication success of the recombinant HIV progeny.

  2. RNA Structures Facilitate Recombination-Mediated Gene Swapping in HIV-1 ▿

    Science.gov (United States)

    Simon-Loriere, Etienne; Martin, Darren P.; Weeks, Kevin M.; Negroni, Matteo

    2010-01-01

    Many viruses, including retroviruses, undergo frequent recombination, a process which can increase their rate of adaptive evolution. In the case of HIV, recombination has been responsible for the generation of numerous intersubtype recombinant variants with epidemiological importance in the AIDS pandemic. Although it is known that fragments of genetic material do not combine randomly during the generation of recombinant viruses, the mechanisms that lead to preferential recombination at specific sites are not fully understood. Here we reanalyze recent independent data defining (i) the structure of a complete HIV-1 RNA genome and (ii) favorable sites for recombination. We show that in the absence of selection acting on recombinant genomes, regions harboring RNA structures in the NL4-3 model strain are strongly predictive of recombination breakpoints in the HIV-1 env genes of primary isolates. In addition, we found that breakpoints within recombinant HIV-1 genomes sampled from human populations, which have been acted upon extensively by natural selection, also colocalize with RNA structures. Critically, junctions between genes are enriched in structured RNA elements and are also preferred sites for generating functional recombinant forms. These data suggest that RNA structure-mediated recombination allows the virus to exchange intact genes rather than arbitrary subgene fragments, which is likely to increase the overall viability and replication success of the recombinant HIV progeny. PMID:20881047

  3. Molecular mechanisms of recombination restriction in the envelope gene of the human immunodeficiency virus.

    Directory of Open Access Journals (Sweden)

    Etienne Simon-Loriere

    2009-05-01

    Full Text Available The ability of pathogens to escape the host's immune response is crucial for the establishment of persistent infections and can influence virulence. Recombination has been observed to contribute to this process by generating novel genetic variants. Although distinctive recombination patterns have been described in many viral pathogens, little is known about the influence of biases in the recombination process itself relative to selective forces acting on newly formed recombinants. Understanding these influences is important for determining how recombination contributes to pathogen genome and proteome evolution. Most previous research on recombination-driven protein evolution has focused on relatively simple proteins, usually in the context of directed evolution experiments. Here, we study recombination in the envelope gene of HIV-1 between primary isolates belonging to subtypes that recombine naturally in the HIV/AIDS pandemic. By characterizing the early steps in the generation of recombinants, we provide novel insights into the evolutionary forces that shape recombination patterns within viral populations. Specifically, we show that the combined effects of mechanistic processes that determine the locations of recombination breakpoints across the HIV-1 envelope gene, and purifying selection acting against dysfunctional recombinants, can explain almost the entire distribution of breakpoints found within this gene in nature. These constraints account for the surprising paucity of recombination breakpoints found in infected individuals within this highly variable gene. Thus, the apparent randomness of HIV evolution via recombination may in fact be relatively more predictable than anticipated. In addition, the dominance of purifying selection in localized areas of the HIV genome defines regions where functional constraints on recombinants appear particularly strong, pointing to vulnerable aspects of HIV biology.

  4. Molecular mechanisms of recombination restriction in the envelope gene of the human immunodeficiency virus.

    Science.gov (United States)

    Simon-Loriere, Etienne; Galetto, Roman; Hamoudi, Meriem; Archer, John; Lefeuvre, Pierre; Martin, Darren P; Robertson, David L; Negroni, Matteo

    2009-05-01

    The ability of pathogens to escape the host's immune response is crucial for the establishment of persistent infections and can influence virulence. Recombination has been observed to contribute to this process by generating novel genetic variants. Although distinctive recombination patterns have been described in many viral pathogens, little is known about the influence of biases in the recombination process itself relative to selective forces acting on newly formed recombinants. Understanding these influences is important for determining how recombination contributes to pathogen genome and proteome evolution. Most previous research on recombination-driven protein evolution has focused on relatively simple proteins, usually in the context of directed evolution experiments. Here, we study recombination in the envelope gene of HIV-1 between primary isolates belonging to subtypes that recombine naturally in the HIV/AIDS pandemic. By characterizing the early steps in the generation of recombinants, we provide novel insights into the evolutionary forces that shape recombination patterns within viral populations. Specifically, we show that the combined effects of mechanistic processes that determine the locations of recombination breakpoints across the HIV-1 envelope gene, and purifying selection acting against dysfunctional recombinants, can explain almost the entire distribution of breakpoints found within this gene in nature. These constraints account for the surprising paucity of recombination breakpoints found in infected individuals within this highly variable gene. Thus, the apparent randomness of HIV evolution via recombination may in fact be relatively more predictable than anticipated. In addition, the dominance of purifying selection in localized areas of the HIV genome defines regions where functional constraints on recombinants appear particularly strong, pointing to vulnerable aspects of HIV biology.

  5. Molecular Mechanisms of Recombination Restriction in the Envelope Gene of the Human Immunodeficiency Virus

    Science.gov (United States)

    Simon-Loriere, Etienne; Galetto, Roman; Hamoudi, Meriem; Archer, John; Lefeuvre, Pierre; Martin, Darren P.; Robertson, David L.; Negroni, Matteo

    2009-01-01

    The ability of pathogens to escape the host's immune response is crucial for the establishment of persistent infections and can influence virulence. Recombination has been observed to contribute to this process by generating novel genetic variants. Although distinctive recombination patterns have been described in many viral pathogens, little is known about the influence of biases in the recombination process itself relative to selective forces acting on newly formed recombinants. Understanding these influences is important for determining how recombination contributes to pathogen genome and proteome evolution. Most previous research on recombination-driven protein evolution has focused on relatively simple proteins, usually in the context of directed evolution experiments. Here, we study recombination in the envelope gene of HIV-1 between primary isolates belonging to subtypes that recombine naturally in the HIV/AIDS pandemic. By characterizing the early steps in the generation of recombinants, we provide novel insights into the evolutionary forces that shape recombination patterns within viral populations. Specifically, we show that the combined effects of mechanistic processes that determine the locations of recombination breakpoints across the HIV-1 envelope gene, and purifying selection acting against dysfunctional recombinants, can explain almost the entire distribution of breakpoints found within this gene in nature. These constraints account for the surprising paucity of recombination breakpoints found in infected individuals within this highly variable gene. Thus, the apparent randomness of HIV evolution via recombination may in fact be relatively more predictable than anticipated. In addition, the dominance of purifying selection in localized areas of the HIV genome defines regions where functional constraints on recombinants appear particularly strong, pointing to vulnerable aspects of HIV biology. PMID:19424420

  6. Gene Disruption by Homologous Recombination in the Xylella fastidiosa Citrus Variegated Chlorosis Strain

    Science.gov (United States)

    Gaurivaud, Patrice; Souza, Leonardo C. A.; Virgílio, Andrea C. D.; Mariano, Anelise G.; Palma, Renê R.; Monteiro, Patrícia B.

    2002-01-01

    Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for β-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to β-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant. PMID:12200328

  7. Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates.

    Directory of Open Access Journals (Sweden)

    Senanu M Spring-Pearson

    Full Text Available The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is 'open', with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.

  8. Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates.

    Science.gov (United States)

    Spring-Pearson, Senanu M; Stone, Joshua K; Doyle, Adina; Allender, Christopher J; Okinaka, Richard T; Mayo, Mark; Broomall, Stacey M; Hill, Jessica M; Karavis, Mark A; Hubbard, Kyle S; Insalaco, Joseph M; McNew, Lauren A; Rosenzweig, C Nicole; Gibbons, Henry S; Currie, Bart J; Wagner, David M; Keim, Paul; Tuanyok, Apichai

    2015-01-01

    The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is 'open', with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.

  9. Construction of Recombinant Plasmid Containing S. Mutans F-ATPase β Subunit Gene

    Institute of Scientific and Technical Information of China (English)

    YU Dan-ni; JIANG Li

    2005-01-01

    objective: construct a homologous recombinant plasmid which was expected to be transformed into S. mutans Methods: a region at the 5' terminus of the S. mutans F-ATPase β subunit gene was amplified by PCR, the PCR product was inserted into vector pVA891, yielding recombinant plasmid. Results: the DNA sequence of the recombinant plasmid was identified correct in whole by restriction endonuclease and DNA sequence techniques. Conclusion: the recombinant plasmid of S. mutans DNA was cloned in effect ,it may assist in construction of homologues recombinant mutant.

  10. E. coli recA gene improves gene targeted homologous recombination in Mycoplasma hyorhinis.

    Science.gov (United States)

    Ishag, Hassan Z A; Xiong, Qiyan; Liu, Maojun; Feng, Zhixin; Shao, Guoqing

    2017-05-01

    Mycoplasma hyorhinis is an opportunistic pathogen of pigs. Recently, it has been shown to transform cell cultures, increasing the attention of the researchers. Studies on the pathogenesis require specific genetic tool that is not yet available for the pathogen. To address this limitation, we constructed two suicide plasmids pGEMT-tetM/LR and pGEMT-recA-tetM/LR having a tetracycline resistance marker flanked by two hemolysin gene arms. The latter plasmid encodes an E. coli recA, a gene involved in DNA recombination, repair and maintenance of DNA. Using inactivation of the hemolysin gene, which results in a detectable and measurable phenotype, we found that each plasmid can disrupt the hemolysin gene of M. hyorhinis through a double cross-over homologous recombination. However, inclusion of the E. coli recA gene in the construct resulted in 9-fold increase in the frequency of hemolysin gene mutants among the screened tetracycline resistance colonies. The resultant hemolysin mutant strain lacks the ability to lyse mouse bed blood cells (RBC) when tested in vitro (p<0.001). The host-plasmid system described in this study, has applications for the genetic manipulation of this pathogen and potentially other mycoplasmas.

  11. Lyophilization conditions for the storage of monooxygenases

    NARCIS (Netherlands)

    van Beek, Hugo L.; Beyer, Nina; Janssen, Dick B.; Fraaije, Marco W.

    2015-01-01

    Cyclohexanone monooxygenase (CHMO) was used as a model enzyme to find suitable freeze-drying conditions for long-term storage of an isolated monooxygenase. CHMO is a Baeyer-Villiger monooxygenase (BVMO) known for its ability to catalyze a large number of oxidation reactions. With a focus on establis

  12. Frequency and character of alternative somatic recombination fates of paralogous genes during T-DNA integration.

    Science.gov (United States)

    Jelesko, John G; Carter, Kristy; Kinoshita, Yuki; Gruissem, Wilhelm

    2005-09-01

    A synthetic RBCSB gene cluster was transformed into Arabidopsis in order to simultaneously evaluate the frequency and character of somatic illegitimate recombination, homologous recombination, and targeted gene replacement events associated with T-DNA-mediated transformation. The most frequent type of recombination event observed was illegitimate integration of the T-DNA without activation of the silent DeltaRBCS1B: LUC transgene. Sixteen luc(+) (firefly luciferase positive) T1 plants were isolated. Six of these were due to illegitimate recombination events resulting in a gene trapping effect. Nine resulted from homologous recombination between paralogous RBCSB sequences associated with T-DNA integration. The frequency of somatic homologous recombination associated with T-DNA integration was almost 200 times higher than previously reported rates of meiotic homologous recombination with the same genes. The distribution of (somatic homologous) recombination resolution sites generally fits a fractional interval length model. However, a small region adjacent to an indel showed a significant over-representation of resolution sites, suggesting that DNA mismatch recognition may also play an important role in the positioning of somatic resolution sites. The frequency of somatic resolution within exon-2 was significantly different from that previously observed during meiotic recombination.

  13. Mutants of Streptomyces roseosporus that express enhanced recombination within partially homologous genes.

    Science.gov (United States)

    Hosted, T J; Baltz, R H

    1996-10-01

    Streptomyces roseosporus mutants that express enhanced recombination between partially homologous (homeologous) sequences were isolated by selection for recombination between the bacteriophage phi C31 derivative KC570 containing the Streptomyces coelicolor glucose kinase (glk) gene and the S. roseosporus chromosome. The frequencies of homeologous recombination in the ehr mutants were determined by measuring the chromosomal insertion frequencies of plasmids containing S. coelicolor glnA or whiG genes. S. roseosporus ehr mutants showed 10(2)- to 10(4)-fold increases in homeologous recombination relative to Ehr+ strains, but no increase in homologous recombination. Southern hybridization analysis revealed single unique sites for the insertion of each of the plasmids, and the crossovers occurred in frame and in proper translational register, yielding functional chimeric glnA and whiG genes.

  14. Herpes simplex virus type 1 (HSV-1)-derived recombinant vectors for gene transfer and gene therapy.

    Science.gov (United States)

    Marconi, Peggy; Fraefel, Cornel; Epstein, Alberto L

    2015-01-01

    Herpes simplex virus type 1 (HSV-1 ) is a human pathogen whose lifestyle is based on a long-term dual interaction with the infected host, being able to establish both lytic and latent infections. The virus genome is a 153-kilobase pair (kbp) double-stranded DNA molecule encoding more than 80 genes. The interest of HSV-1 as gene transfer vector stems from its ability to infect many different cell types, both quiescent and proliferating cells, the very high packaging capacity of the virus capsid, the outstanding neurotropic adaptations that this virus has evolved, and the fact that it never integrates into the cellular chromosomes, thus avoiding the risk of insertional mutagenesis. Two types of vectors can be derived from HSV-1, recombinant vectors and amplicon vectors, and different methodologies have been developed to prepare large stocks of each type of vector. This chapter summarizes the approach most commonly used to prepare recombinant HSV-1 vectors through homologous recombination, either in eukaryotic cells or in bacteria.

  15. Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination

    Institute of Scientific and Technical Information of China (English)

    Qiang ZHANG; Qi-he CHEN; Ming-liang FU; Jin-ling WANG; Hong-bo ZHANG; Guo-qing HE

    2008-01-01

    The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone α-factor (MFals), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-βG) was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis ofgenome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/(h-ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.

  16. Accurate assessment of intragenic recombination frequency within the Duchenne muscular dystrophy gene.

    Science.gov (United States)

    Abbs, S; Roberts, R G; Mathew, C G; Bentley, D R; Bobrow, M

    1990-08-01

    Polymorphic loci that lie at the two extremities of the Duchenne/Becker muscular dystrophy (DMD/BMD) gene have been used to estimate intragenic recombination rates. Multipoint linkage analysis of the CEPH panel of families suggests a total intragenic recombination frequency of nearly 0.12 (confidence intervals 0.041-0.226) over the genomic length of approximately 2 Mb.

  17. Construction and function of recombinant AcMNPV with double copies of v-cath gene

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Two recombinant baculoviruses, dciAcMNPV and dcdAcMNPV in which another copy of the v-cath gene controlled by ie1 promoter and polh promoter was inserted, were respectively constructed by the Bac-to-Bac system. The expression of the v-cath gene of the recombinant baculoviruses in Sf9 cells at different phases was investigated by SDS- PAGE and Western blot. The results showed that only recombinant virus dciAcMNPV containing late gene v-cath driven by early gene promoter could express V-CATH protein, cathepsin encoded by virus genome, 12 h post-infection and dcdAcMNPV containing late gene v-cath driven by late and very late gene promoters could express more V-CATH protein. Negative control ncAcMNPV, a mutant deleted v- cath gene, could not express V-CATH protein at all. The Spodopera exigua larvae were infected with viruses respectively and the results showed that the toxicity was as follows: dcdAcMNPV>dciAcMNPV>wtAcMNPV>ncAcMNPV. The toxicity of recombinant viruses and the characters of dead larvae showed that the v-cath gene was relative to viral toxicity and host liquefaction. Recombinant baculovirus dcdAcMNPV might be used as a new kind of safe viral-pes- ticide, because of its high toxicity obtained by adding another gene copy and changing the expression level of its own gene relative to virulence.

  18. Recombination rates of Streptococcus pneumoniae isolates with both erm(B) and mef(A) genes.

    Science.gov (United States)

    Lee, Ji-Young; Song, Jae-Hoon; Ko, Kwan Soo

    2010-08-01

    Erythromycin-resistant Streptococcus pneumoniae isolates containing both erm(B) and mef(A) genes have a higher rate of multidrug resistance (MDR). We investigated the relationships between the presence of erythromycin resistance determinants and the recombination rate. We determined the mutation and recombination frequencies of 46 S. pneumoniae isolates, which included 19 with both erm(B) and mef(A), nine with only erm(B), six with only mef(A), and 11 erythromycin-susceptible isolates. Mutation frequency values were estimated as the number of rifampin-resistant colonies as a proportion of total viable count. Genotypes and serotypes of isolates with the hyper-recombination phenotype were determined. Twelve S. pneumoniae isolates were hypermutable and four isolates were determined to have hyper-recombination frequency. Streptococcus pneumoniae isolates with both erm(B) and mef(A) genes did not show a high mutation frequency. In contrast, all isolates with a hyper-recombination phenotype contained both erm(B) and mef(A) genes. In addition, the recombination rate of isolates with both erm(B) and mef(A) genes was statistically higher than the rate of other isolates. The dual presence of erm(B) and mef(A) genes in some pneumococcal isolates may be associated with high recombination frequency. This may be one of the reasons for the frequent emergence of MDR in certain pneumococcal isolates.

  19. Expression of alkane monooxygenase (alkB) genes by plant-associated bacteria in the rhizosphere and endosphere of Italian ryegrass (Lolium multiflorum L.) grown in diesel contaminated soil

    Energy Technology Data Exchange (ETDEWEB)

    Andria, Verania [AIT Austrian Institute of Technology GmbH, Bioresources Unit, A-2444 Seibersdorf (Austria); Reichenauer, Thomas G. [AIT Austrian Institute of Technology GmbH, Unit of Environmental Resources and Technologies, A-2444 Seibersdorf (Austria); Sessitsch, Angela, E-mail: angela.sessitsch@ait.ac.a [AIT Austrian Institute of Technology GmbH, Bioresources Unit, A-2444 Seibersdorf (Austria)

    2009-12-15

    For phytoremediation of organic contaminants, plants have to host an efficiently degrading microflora. To assess the role of endophytes in alkane degradation, Italian ryegrass was grown in sterile soil with 0, 1 or 2% diesel and inoculated either with an alkane degrading bacterial strain originally derived from the rhizosphere of Italian ryegrass or with an endophyte. We studied plant colonization of these strains as well as the abundance and expression of alkane monooxygenase (alkB) genes in the rhizosphere, shoot and root interior. Results showed that the endophyte strain better colonized the plant, particularly the plant interior, and also showed higher expression of alkB genes suggesting a more efficient degradation of the pollutant. Furthermore, plants inoculated with the endophyte were better able to grow in the presence of diesel. The rhizosphere strain colonized primarily the rhizosphere and showed low alkB gene expression in the plant interior. - Bacterial alkane degradation genes are expressed in the rhizosphere and in the plant interior.

  20. Reciprocal and nonreciprocal recombination at the glucocerebrosidase gene region: implications for complexity in Gaucher disease.

    Science.gov (United States)

    Tayebi, Nahid; Stubblefield, Barbara K; Park, Joseph K; Orvisky, Eduard; Walker, Jamie M; LaMarca, Mary E; Sidransky, Ellen

    2003-03-01

    Gaucher disease results from an autosomal recessive deficiency of the lysosomal enzyme glucocerebrosidase. The glucocerebrosidase gene is located in a gene-rich region of 1q21 that contains six genes and two pseudogenes within 75 kb. The presence of contiguous, highly homologous pseudogenes for both glucocerebrosidase and metaxin at the locus increases the likelihood of DNA rearrangements in this region. These recombinations can complicate genotyping in patients with Gaucher disease and contribute to the difficulty in interpreting genotype-phenotype correlations in this disorder. In the present study, DNA samples from 240 patients with Gaucher disease were examined using several complementary approaches to identify and characterize recombinant alleles, including direct sequencing, long-template polymerase chain reaction, polymorphic microsatellite repeats, and Southern blots. Among the 480 alleles studied, 59 recombinant alleles were identified, including 34 gene conversions, 18 fusions, and 7 downstream duplications. Twenty-two percent of the patients evaluated had at least one recombinant allele. Twenty-six recombinant alleles were found among 310 alleles from patients with type 1 disease, 18 among 74 alleles from patients with type 2 disease, and 15 among 96 alleles from patients with type 3 disease. Several patients carried two recombinations or mutations on the same allele. Generally, alleles resulting from nonreciprocal recombination (gene conversion) could be distinguished from those arising by reciprocal recombination (crossover and exchange), and the length of the converted sequence was determined. Homozygosity for a recombinant allele was associated with early lethality. Ten different sites of crossover and a shared pentamer motif sequence (CACCA) that could be a hotspot for recombination were identified. These findings contribute to a better understanding of genotype-phenotype relationships in Gaucher disease and may provide insights into the mechanisms

  1. Gene delivery into plant cells for recombinant protein production

    National Research Council Canada - National Science Library

    Chen, Qiang; Lai, Huafang

    2015-01-01

    .... 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...

  2. Gene Delivery into Plant Cells for Recombinant Protein Production

    OpenAIRE

    Qiang Chen; Huafang Lai

    2015-01-01

    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 d...

  3. Recombinant Gluconacetobacter diazotrophicus containing Cry1Ac gene codes for 130-kDa toxin protein.

    Science.gov (United States)

    Subashini, M; Moushumi Priya, A; Sundarakrishnan, B; Jayachandran, S

    2011-01-01

    Recombinant Gluconacetobacter diazotrophicus containing Cry1Ac gene from Bacillus thuringiensis var. kurstaki borne on pKT230, shuttle vector, was generated. PCR amplification of Cry1Ac gene present in recombinant G. diazotrophicus yielded a 278-bp DNA product. The nitrogenase assay has revealed that the recombinant G. diazotrophicus in sugarcane stem produced similar levels of nitrogenase compared to wild-type G. diazotrophicus. The presence of 130-kDa protein in apoplastic fluid from sugarcane stem harvested from pots inoculated with recombinant G. diazotrophicus shows that the translocated G. diazotrophicus produces 130-kDa protein which is recognized by the hyperimmune antiserum raised against 130-kDa protein. The first instar Eldana saccharina neonate larvae that fed on artificial medium containing recombinant G. diazotrophicus died within 72 h after incubation.

  4. DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

    DEFF Research Database (Denmark)

    Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas Salhøj

    2014-01-01

    -wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum......-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers...... of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens....

  5. Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca

    NARCIS (Netherlands)

    Schallmey, Anett; den Besten, Gijs; Teune, Ite G. P.; Kembaren, Roga F.; Janssen, Dick B.

    2011-01-01

    Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The enzym

  6. Construction and expression of SET gene and siRNA recombinant adenovirus vectors

    Institute of Scientific and Technical Information of China (English)

    Xu Bo-qun; Lu Pin-hong; Li Ying; Xue Kai; Li Mei; Ma Xiang; Diao Fei-yan; Cui Yu-gui; Liu Jia-yin

    2010-01-01

    Objective: To construct SET gene recombinant adenovirus vector and SET gene small interfering RNA (SiRNA) recombinant adenovirus vector for over-expression or knock-down of SET levels.Methods: The cDNA sequence of SET was cloned by reverse transcriptive polymerase chain reaction (RT-PCR) and the SET gene fragment was subcloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-SET. The shuttle plasmid pAdtrack-SET was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant Ad-CMV-SET and the recombinant Ad-CMV-SET was packaged and amplified in the AD293 cells. The expression of SET in AD293 cells was detected by Western blot. In addition, we constructed SET gene SiRNA recombinant adenovirus vector (Ad-H1-SiRNA/SET) and its efficacy of knockdown of SET protein was detected in infected GC-2spd(ts) cells by Western blot. Results: The recombinant adenovirus vectors, both SET gene recombinant adenovirus vector Ad-CMV-SET and SET gene SiRNA recombinant adenovirus vector Ad-H1-SiRNA/SET, were proven to be constructed successfully by the evidence of endonulease digestion and sequencing. AD293 cells infected with either recombinant adenovirus vector of Ad-CMV-SET or Ad-H1-SiRNA/SET were observed to express GFP. The expression of SET protein was up-regulated significantly in AD293 cells infected with SET gene recombinant adenovirus vector. On the contrast, SET protein was significantly down-regulated in the GC-2spd(ts) cells infected with Ad-H1-SiRNA/SET (P<0.05) and the knockdown efficiency was approximately 50%-70%. Conclusion: The recombinant adenovirus vector Ad-CMV-SET and Ad-H1-SiRNA/SET were successfully constructed and effectively expressed in germ cells and somatic cells. It provides an experimental tool for further study of SET gene in the physiological and pathophysiological mechanism of reproduction-related diseases.

  7. The population and evolutionary dynamics of homologous gene recombination in bacterial populations.

    Directory of Open Access Journals (Sweden)

    Bruce R Levin

    2009-08-01

    Full Text Available In bacteria, recombination is a rare event, not a part of the reproductive process. Nevertheless, recombination -- broadly defined to include the acquisition of genes from external sources, i.e., horizontal gene transfer (HGT -- plays a central role as a source of variation for adaptive evolution in many species of bacteria. Much of niche expansion, resistance to antibiotics and other environmental stresses, virulence, and other characteristics that make bacteria interesting and problematic, is achieved through the expression of genes and genetic elements obtained from other populations of bacteria of the same and different species, as well as from eukaryotes and archaea. While recombination of homologous genes among members of the same species has played a central role in the development of the genetics and molecular biology of bacteria, the contribution of homologous gene recombination (HGR to bacterial evolution is not at all clear. Also, not so clear are the selective pressures responsible for the evolution and maintenance of transformation, the only bacteria-encoded form of HGR. Using a semi-stochastic simulation of mutation, recombination, and selection within bacterial populations and competition between populations, we explore (1 the contribution of HGR to the rate of adaptive evolution in these populations and (2 the conditions under which HGR will provide a bacterial population a selective advantage over non-recombining or more slowly recombining populations. The results of our simulation indicate that, under broad conditions: (1 HGR occurring at rates in the range anticipated for bacteria like Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, and Bacillus subtilis will accelerate the rate at which a population adapts to environmental conditions; (2 once established in a population, selection for this capacity to increase rates of adaptive evolution can maintain bacteria-encoded mechanisms of recombination and prevent

  8. Engineered Zinc Finger Nuclease–Mediated Homologous Recombination of the Human Rhodopsin Gene

    Science.gov (United States)

    Greenwald, David L.; Cashman, Siobhan M.

    2010-01-01

    Purpose. Novel zinc finger nucleases (ZFNs) were designed to target the human rhodopsin gene and induce homologous recombination of a donor DNA fragment. Methods. Three-finger zinc finger nucleases were designed based on previously published guidelines. To assay for ZFN specificity, the authors generated human embryonic retinoblast cell lines stably expressing a Pro23His rhodopsin, the most common mutation associated with autosomal dominant retinitis pigmentosa in North America. They report quantification of these rhodopsin-specific ZFNs to induce a targeted double-strand break in the human genome, demonstrate their ability to induce homologous recombination of a donor DNA fragment, and report the quantification of the frequency of ZFN-mediated homologous recombination. Results. Compared with endogenous homologous recombination, the authors observed a 12-fold increase in homologous recombination and an absolute frequency of ZFN-directed homologous recombination as high as 17% in the human rhodopsin gene. Conclusions. ZFNs are chimeric proteins with significant potential for the treatment of inherited diseases. In this study, the authors report the design of novel ZFNs targeting the human rhodopsin gene. These ZFNs may be useful for the treatment of retinal diseases such as retinitis pigmentosa, one of the most common causes of inherited blindness in the developed world. Herein, they also report on several aspects of donor fragment design and in vitro conditions that facilitate ZFN-mediated homologous recombination. PMID:20671268

  9. Glyceryl ether monooxygenase resembles aromatic amino acid hydroxylases in metal ion and tetrahydrobiopterin dependence.

    Science.gov (United States)

    Watschinger, Katrin; Keller, Markus A; Hermetter, Albin; Golderer, Georg; Werner-Felmayer, Gabriele; Werner, Ernst R

    2009-01-01

    Glyceryl ether monooxygenase is a tetrahydrobiopterin-dependent membrane-bound enzyme which catalyses the cleavage of lipid ethers into glycerol and the corresponding aldehyde. Despite many different characterisation and purification attempts, so far no gene and primary sequence have been assigned to this enzyme. The seven other tetrahydrobiopterin-dependent enzymes can be divided in the family of aromatic amino acid hydroxylases - comprising phenylalanine hydroxylase, tyrosine hydroxylase and the two tryptophan hydroxylases - and into the three nitric oxide synthases. We tested the influences of different metal ions and metal ion chelators on glyceryl ether monooxygenase, phenylalanine hydroxylase and nitric oxide synthase activity to elucidate the relationship of glyceryl ether monooxygenase to these two families. 1,10-Phenanthroline, an inhibitor of non-heme iron-dependent enzymes, was able to potently block glyceryl ether monooxygenase as well as phenylalanine hydroxylase, but had no effect on inducible nitric oxide synthase. Two tetrahydrobiopterin analogues, N(5)-methyltetrahydrobiopterin and 4-aminotetrahydrobiopterin, had a similar impact on glyceryl ether monooxygenase activity, as has already been shown for phenylalanine hydroxylase. These observations point to a close analogy of the role of tetrahydrobiopterin in glyceryl ether monooxygenase and in aromatic amino acid hydroxylases and suggest that glyceryl ether monooxygenase may require a non-heme iron for catalysis.

  10. Recombination and population mosaic of a multifunctional viral gene, adeno-associated virus cap.

    Directory of Open Access Journals (Sweden)

    Yasuhiro Takeuchi

    Full Text Available Homologous recombination is a dominant force in evolution and results in genetic mosaics. To detect evidence of recombination events and assess the biological significance of genetic mosaics, genome sequences for various viral populations of reasonably large size are now available in the GenBank. We studied a multi-functional viral gene, the adeno-associated virus (AAV cap gene, which codes for three capsid proteins, VP1, VP2 and VP3. VP1-3 share a common C-terminal domain corresponding to VP3, which forms the viral core structure, while the VP1 unique N-terminal part contains an enzymatic domain with phospholipase A2 activity. Our recombinant detection program (RecI revealed five novel recombination events, four of which have their cross-over points in the N-terminal, VP1 and VP2 unique region. Comparison of phylogenetic trees for different cap gene regions confirmed discordant phylogenies for the recombinant sequences. Furthermore, differences in the phylogenetic tree structures for the VP1 unique (VP1u region and the rest of cap highlighted the mosaic nature of cap gene in the AAV population: two dominant forms of VP1u sequences were identified and these forms are linked to diverse sequences in the rest of cap gene. This observation together with the finding of frequent recombination in the VP1 and 2 unique regions suggests that this region is a recombination hot spot. Recombination events in this region preserve protein blocks of distinctive functions and contribute to convergence in VP1u and divergence of the rest of cap. Additionally the possible biological significance of two dominant VP1u forms is inferred.

  11. Biocatalytic conversion of ethylene to ethylene oxide using an engineered toluene monooxygenase

    Energy Technology Data Exchange (ETDEWEB)

    Carlin, DA; Bertolani, SJ; Siegel, JB

    2015-01-01

    Mutants of toluene o-xylene monooxygenase are demonstrated to oxidize ethylene to ethylene oxide in vivo at yields of >99%. The best mutant increases ethylene oxidation activity by >5500-fold relative to the native enzyme. This is the first report of a recombinant enzyme capable of carrying out this industrially significant chemical conversion.

  12. Biocatalytic conversion of ethylene to ethylene oxide using an engineered toluene monooxygenase.

    Science.gov (United States)

    Carlin, D A; Bertolani, S J; Siegel, J B

    2015-02-11

    Mutants of toluene o-xylene monooxygenase are demonstrated to oxidize ethylene to ethylene oxide in vivo at yields of >99%. The best mutant increases ethylene oxidation activity by >5500-fold relative to the native enzyme. This is the first report of a recombinant enzyme capable of carrying out this industrially significant chemical conversion.

  13. Protein engineering of the cytochrome P450 monooxygenase from bacillus megaterium

    OpenAIRE

    Urlacher, Vlada B.; Schmid, Rolf D

    2004-01-01

    The role and importance of cytochrome P450 enzymes (CYP) in drug development, biodegradation processes and biocatalysis has been widely acknowledged. P450 monooxygenases exhibit an extremely wide substrate spectrum which is the basis of their ability to activate or detoxify a large variety of target molecules. P450 monooxygenases have been isolated from bacteria, yeasts, insects, as well as mammalian and plant tissues. Currently, the enzyme family is one of the best known gene subfamilies wit...

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

    OpenAIRE

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

    2015-01-01

    The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT....

  15. Chromosomal Loop Domains Direct the Recombination of Antigen Receptor Genes.

    Science.gov (United States)

    Hu, Jiazhi; Zhang, Yu; Zhao, Lijuan; Frock, Richard L; Du, Zhou; Meyers, Robin M; Meng, Fei-long; Schatz, David G; Alt, Frederick W

    2015-11-05

    RAG initiates antibody V(D)J recombination in developing lymphocytes by generating "on-target" DNA breaks at matched pairs of bona fide recombination signal sequences (RSSs). We employ bait RAG-generated breaks in endogenous or ectopically inserted RSS pairs to identify huge numbers of RAG "off-target" breaks. Such breaks occur at the simple CAC motif that defines the RSS cleavage site and are largely confined within convergent CTCF-binding element (CBE)-flanked loop domains containing bait RSS pairs. Marked orientation dependence of RAG off-target activity within loops spanning up to 2 megabases implies involvement of linear tracking. In this regard, major RAG off-targets in chromosomal translocations occur as convergent RSS pairs at enhancers within a loop. Finally, deletion of a CBE-based IgH locus element disrupts V(D)J recombination domains and, correspondingly, alters RAG on- and off-target distributions within IgH. Our findings reveal how RAG activity is developmentally focused and implicate mechanisms by which chromatin domains harness biological processes within them. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Extensive Recombination Due to Heteroduplexes Generates Large Amounts of Artificial Gene Fragments during PCR

    Science.gov (United States)

    Liu, Jia; Song, Hongshuo; Liu, Donglai; Zuo, Tao; Lu, Fengmin; Zhuang, Hui; Gao, Feng

    2014-01-01

    Artificial recombinants can be generated during PCR when more than two genetically distinct templates coexist in a single PCR reaction. These recombinant amplicons can lead to the false interpretation of genetic diversity and incorrect identification of biological phenotypes that do not exist in vivo. We investigated how recombination between 2 or 35 genetically distinct HIV-1 genomes was affected by different PCR conditions using the parallel allele-specific sequencing (PASS) assay and the next generation sequencing method. In a standard PCR condition, about 40% of amplicons in a PCR reaction were recombinants. The high recombination frequency could be significantly reduced if the number of amplicons in a PCR reaction was below a threshold of 1013–1014 using low thermal cycles, fewer input templates, and longer extension time. Heteroduplexes (each DNA strand from a distinct template) were present at a large proportion in the PCR products when more thermal cycles, more templates, and shorter extension time were used. Importantly, the majority of recombinants were identified in heteroduplexes, indicating that the recombinants were mainly generated through heteroduplexes. Since prematurely terminated extension fragments can form heteroduplexes by annealing to different templates during PCR amplification, recombination has a better chance to occur with samples containing different genomes when the number of amplicons accumulate over the threshold. New technologies are warranted to accurately characterize complex quasispecies gene populations. PMID:25211143

  17. Extensive recombination due to heteroduplexes generates large amounts of artificial gene fragments during PCR.

    Directory of Open Access Journals (Sweden)

    Jia Liu

    Full Text Available Artificial recombinants can be generated during PCR when more than two genetically distinct templates coexist in a single PCR reaction. These recombinant amplicons can lead to the false interpretation of genetic diversity and incorrect identification of biological phenotypes that do not exist in vivo. We investigated how recombination between 2 or 35 genetically distinct HIV-1 genomes was affected by different PCR conditions using the parallel allele-specific sequencing (PASS assay and the next generation sequencing method. In a standard PCR condition, about 40% of amplicons in a PCR reaction were recombinants. The high recombination frequency could be significantly reduced if the number of amplicons in a PCR reaction was below a threshold of 10(13-10(14 using low thermal cycles, fewer input templates, and longer extension time. Heteroduplexes (each DNA strand from a distinct template were present at a large proportion in the PCR products when more thermal cycles, more templates, and shorter extension time were used. Importantly, the majority of recombinants were identified in heteroduplexes, indicating that the recombinants were mainly generated through heteroduplexes. Since prematurely terminated extension fragments can form heteroduplexes by annealing to different templates during PCR amplification, recombination has a better chance to occur with samples containing different genomes when the number of amplicons accumulate over the threshold. New technologies are warranted to accurately characterize complex quasispecies gene populations.

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

    Science.gov (United States)

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

    2015-11-02

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

  19. BF integrase genes of HIV-1 circulating in Sao Paulo, Brazil, with a recurrent recombination region.

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    Atila Iamarino

    Full Text Available Although some studies have shown diversity in HIV integrase (IN genes, none has focused particularly on the gene evolving in epidemics in the context of recombination. The IN gene in 157 HIV-1 integrase inhibitor-naïve patients from the São Paulo State, Brazil, were sequenced tallying 128 of subtype B (23 of which were found in non-B genomes, 17 of subtype F (8 of which were found in recombinant genomes, 11 integrases were BF recombinants, and 1 from subtype C. Crucially, we found that 4 BF recombinant viruses shared a recurrent recombination breakpoint region between positions 4900 and 4924 (relative to the HXB2 that includes 2 gRNA loops, where the RT may stutter. Since these recombinants had independent phylogenetic origin, we argue that these results suggest a possible recombination hotspot not observed so far in BF CRF in particular, or in any other HIV-1 CRF in general. Additionally, 40% of the drug-naïve and 45% of the drug-treated patients had at least 1 raltegravir (RAL or elvitegravir (EVG resistance-associated amino acid change, but no major resistance mutations were found, in line with other studies. Importantly, V151I was the most common minor resistance mutation among B, F, and BF IN genes. Most codon sites of the IN genes had higher rates of synonymous substitutions (dS indicative of a strong negative selection. Nevertheless, several codon sites mainly in the subtype B were found under positive selection. Consequently, we observed a higher genetic diversity in the B portions of the mosaics, possibly due to the more recent introduction of subtype F on top of an ongoing subtype B epidemics and a fast spread of subtype F alleles among the B population.

  20. Polymorphism, genetic exchange and intragenic recombination of the aureolysin gene among Staphylococcus aureus strains

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    Kowal Julia

    2008-07-01

    Full Text Available Abstract Background Staphylococcus aureus expresses several proteases, which are thought to contribute to the virulence of this bacterium. Here we focus on aureolysin, the major thermolysin-like metalloprotease. Despite the importance of aureolysin in the physiology and pathogenesis of S. aureus, relatively little information was so far available concerning the aur gene diversity and mobility within and between the major subdivisions of the S. aureus population. Therefore, an epidemiologically and genetically diverse collection of S. aureus strains was used to determine the range of aureolysin (aur gene polymorphism. Results Sequence analyses support the conclusion that the aur gene occurs in two distinct types of related sequences. The aur gene was much more polymorphic but, at the same time, showed higher purifying selection than genes utilized for multilocus sequence typing (MLST. Gene trees constructed from aur and concatenated MLST genes revealed several putative assortative recombination events (i.e. entire aur gene exchanges between divergent lineages of S. aureus. Evidence for intragenic recombination events (i.e. exchanges of internal aur segments across aur genes was also found. The biochemical properties and substrate specificity of the two types of aureolysin purified to homogeneity were studied, revealing minor differences in their affinity to low molecular weight synthetic substrates. Conclusion Although numerous nucleotide differences were identified between the aur alleles studied, our findings showed that a strong purifying selection is acting on the aur gene. Moreover, our study distinguishes between homologous exchanges of the entire aur gene (assortative recombination between divergent S. aureus lineages and recombination events within aur genes.

  1. A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs

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    Gong Shiaochin

    2009-03-01

    Full Text Available Abstract Background Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. Results We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1 subclone genes of interest into BAC linking vectors, (2 insert desired reporter genes into respective genes and (3 link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. Conclusion The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

  2. Recombination activating activity of XRCC1 analogous genes in X-ray sensitive and resistant CHO cell lines

    Science.gov (United States)

    Golubnitchaya-Labudová, O.; Portele, A.; Vaçata, V.; Lubec, G.; Rink, H.; Höfer, M.

    1997-10-01

    The XRCC1 gene (X-ray repair cross complementing) complements the DNA repair deficiency of the radiation sensitive Chinese hamster ovary (CHO) mutant cell line EM9 but the mechanism of the correction is not elucidated yet. XRCC1 shows substantial homology to the RAG2 gene (recombination activating gene) and we therefore tried to answer question, whether structural similarities (sequence of a putative recombination activating domain, aa 332-362 for XRCC1 and aa 286-316 in RAG2) would reflect similar functions of the homologous, putative recombination activating domain. PCR experiments revealed that no sequence homologous to the structural part of human XRCC1 was present in cDNA of CHO. Differential display demonstrated two putative recombination activating domains in the parental CHO line AA8 and one in the radiosensitive mutant EM9. Southern blot experiments showed the presence of several genes with partial homology to human XRCC1. Recombination studies consisted of expressing amplified target domains within chimeric proteins in recA - bacteria and subsequent detection of recombination events by sequencing the recombinant plasmids. Recombination experiments demonstrated recombination activating activity of all putative recombination activating domains amplified from AA8 and EM9 genomes as reflected by deletions within the insert of the recombinant plasmids. The recombination activating activity of XRCC1 analogues could explain a mechanism responsible for the correction of the DNA repair defect in EM9.

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

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    Marrero Luis

    2003-09-01

    Full Text Available Abstract Background Gene transfer into the amniotic fluid using recombinant adenovirus vectors was shown previously to result in high efficiency transfer of transgenes into the lungs and intestines. Adenovirus mediated in utero gene therapy, however, resulted in expression of the transgene for less than 30 days. Recombinant adenovirus associated viruses (rAAV have the advantage of maintaining the viral genome in daughter cells thus providing for long-term expression of transgenes. Methods Recombinant AAV2 carrying green fluorescent protein (GFP was introduced into the amniotic sac of fetal rodents and nonhuman primates. Transgene maintenance and expression was monitor. Results Gene transfer resulted in rapid uptake and long-term gene expression in mice, rats, and non-human primates. Expression and secretion of the reporter gene, GFP, was readily demonstrated within 72 hours post-therapy. In long-term studies in rats and nonhuman primates, maintenance of GFP DNA, protein expression, and reporter gene secretion was documented for over one year. Conclusions Because only multipotential stem cells are present at the time of therapy, these data demonstrated that in utero gene transfer with AAV2 into stem cells resulted in long-term systemic expression of active transgene roducts. Thus, in utero gene transfer via the amniotic fluid may be useful in treatment of gene disorders.

  4. Pathogen corruption and site-directed recombination at a plant disease resistance gene cluster

    Science.gov (United States)

    Nagy, Ervin D.; Bennetzen, Jeffrey L.

    2008-01-01

    The Pc locus of sorghum (Sorghum bicolor) determines dominant sensitivity to a host-selective toxin produced by the fungal pathogen Periconia circinata. The Pc region was cloned by a map-based approach and found to contain three tandemly repeated genes with the structures of nucleotide binding site–leucine-rich repeat (NBS–LRR) disease resistance genes. Thirteen independent Pc-to-pc mutations were analyzed, and each was found to remove all or part of the central gene of the threesome. Hence, this central gene is Pc. Most Pc-to-pc mutations were associated with unequal recombination. Eight recombination events were localized to different sites in a 560-bp region within the ∼3.7-kb NBS–LRR genes. Because any unequal recombination located within the flanking NBS–LRR genes would have removed Pc, the clustering of cross-over events within a 560-bp segment indicates that a site-directed recombination process exists that specifically targets unequal events to generate LRR diversity in NBS–LRR loci. PMID:18719093

  5. Genetic recombination within the human T-cell receptor. cap alpha. -chain gene complex

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-12-01

    Genetic analyses of the human T-cell receptor (TCR) ..cap alpha..-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 TCR..cap alpha.. 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 TCR..cap alpha.. constant region gene were observed in this study. A high recombination frequency for the TCR..cap alpha.. gene complex is further supported by the observation that two children, one in each of two families, inherited recombinant TCR..cap alpha.. haplotypes.

  6. PCR-based gene synthesis to produce recombinant proteins for crystallization

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    Byrne-Steele Miranda L

    2008-04-01

    Full Text Available Abstract Background Gene synthesis technologies are an important tool for structural biology projects, allowing increased protein expression through codon optimization and facilitating sequence alterations. Existing methods, however, can be complex and not always reproducible, prompting researchers to use commercial suppliers rather than synthesize genes themselves. Results A PCR-based gene synthesis method, referred to as SeqTBIO, is described to efficiently assemble the coding regions of two novel hyperthermophilic proteins, PAZ (Piwi/Argonaute/Zwille domain, a siRNA-binding domain of an Argonaute protein homologue and a deletion mutant of a family A DNA polymerase (PolA. The gene synthesis procedure is based on sequential assembly such that homogeneous DNA products can be obtained after each synthesis step without extensive manipulation or purification requirements. Coupling the gene synthesis procedure to in vivo homologous recombination techniques allows efficient subcloning and site-directed mutagenesis for error correction. The recombinant proteins of PAZ and PolA were subsequently overexpressed in E. coli and used for protein crystallization. Crystals of both proteins were obtained and they were suitable for X-ray analysis. Conclusion We demonstrate, by using PAZ and PolA as examples, the feasibility of integrating the gene synthesis, error correction and subcloning techniques into a non-automated gene to crystal pipeline such that genes can be designed, synthesized and implemented for recombinant expression and protein crystallization.

  7. THE ROLE OF RECOMBINANT Rb GENE ADENOVIRUS VECTOR IN THE GROWTH OF LUNG ADENOCARCINOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    Li Jian; Jiang Lei; Xia Yongjing; Li Hongxia; Hu Yajun; Hu Shixue; Xu Hongji

    1998-01-01

    Objective:To study the role of the most extensively studied tumor suppressor gene, retinoblastoma (Rb) gene,on the growth of lung adenocarcinoma cell line GLC-82 and explore a gene therapy approach for lung adenocarcinoma. Methods: The recombinant Rb gene adenovirus vector was constructed, the control virus which carries LacZ gene was producted by the same method. Infection effects were detected by biochemical staining of β-gal and immunohistochemical analysis of Rb protein. The Rb cDNA of infected cells were determined by PCR. The cell growth rate and cell cycle were observed by cell-counting and flow cytometry. Results: The constructed recombinant adenovirus vector could infect effectively the cells with high level expression of Rb cDNA and Rb protein. The transfection of wild-type Rb gene could suppress GLC-82 cell proliferation and decrease the cellular DNA synthesis. Conclusions: These results showed the possibility of using recombinant Rb gene adenovirus vector in the gene therapy of cancer to inhibit the growth of cancer.

  8. Cloning of Porcine Lactoferrin Gene and Construction of Expression System in Recombinant Lactobacillus

    Institute of Scientific and Technical Information of China (English)

    ZONG Xiaolin; HA Zhuo; ZHAO Lili; LIU Diqiu; QIAO Xinyuan; JIANG Yanping; GE Junwei; LI Yijing; TANG Lijie

    2011-01-01

    Lactobacillus was selected as a bacterial carrier for expression of N-lobe of porcine lactoferrin (PLFN). A pair of primers was designed with Oligo6.0 and used to amplify PLFN gene. It was in accordance with the characters of translational fusions from gene and expression vector plasmid. A 1 077 bp fragment of the gene from PLF was cloned from mammary gland tissue of the lactating sow on the third day by RT-PCR; the gene was connected with the vector plasmid pPG612.1 and transformed into the host strain JM109. The recombinant expression vector plasmid pPG612-PLFN was created and identified by using plasmid extraction, PCR, restriction enzyme digestion and sequence analysis. The recombinant plasmid was transformed into Lactobacillus casei ATCC393, Lactobacillus plantarum KLDS 1.0344, Lactobacillus paracasei KLDS 1.0652 and Lactobacillus pentosus KLDS 1.0413 by electroporation, and produced the recombinant strains of pPG612-PLFN/L, casei, pPG612-PLFN/L, plantarum, pPG612-PLFN/ L. paracasei and pPG612-PLFN/L, pentosus, respectively. The results indicated that PLFN gene had inserted into the expression vectors and achieved multiple Laetobacillus expression systems. It electes the base for the expression and production of recombinant porcine lactoferrin in Lactobaeillus

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

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    Penn Charles W

    2009-12-01

    Full Text Available Abstract Background Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains. Results Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the λ-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6 × His, 3 × FLAG, 4 × ProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the λ-Red system, which can lead

  10. Intratracheal administration of recombinant adenovirus containing IL-18 gene in treatment of experimental metastatic lung carcinoma

    Institute of Scientific and Technical Information of China (English)

    CHEN Ji-quan; GAO Xue-tao; XIU Qing-yu; YU Yi-zhi; LUO Wen-tong

    2001-01-01

    To study the treatment of experimental metastatic lung carcinoma by intratracheal injection of IL-18 gene recombinant adenovirus. Methods: (1)The mouse IL-18 mRNA was detected by RT-PCR, and the concentration of IL-18 and associated cytokines in lung lavages and blood were determined by ELISA at different time points after intratracheal injection of IL-18 recombinant adenovirus. (2)The lung metastasis nodes, mouse survival periods and survival rates were evaluated. NK activity and CTL activity were determined by 51Cr 4 h release method. Results: (1)IL-18 mRNA was detectable in lung tissue 6 h after intratracheal use of IL-18 recombinant adenovirus, and the concentration of IL-18 in lung lavage was higher than that in pelipheral blood. Neither IL-18 mRNA nor IL-18 was detectable in control group. (2) Intratracheal use of IL-18 recombinant adenovirus resulted in increased CTL and NK activity, longer survival time and higher survival rates compared with the control group, showing significant therapeutic effect on experimental lung metastasis. Conclusion: Intratracheal use of adenovirus vector containing IL-18 gene has therapeutic effect on the lung metastasis, denoting that gene therapy of lung diseases could be applied through airway directly with recombinant adenovirus.

  11. Direct evidence of recombination in the recA gene of Aeromonas bestiarum.

    Science.gov (United States)

    Sanglas, Ariadna; Albarral, Vicenta; Farfán, Maribel; Lorén, J Gaspar; Fusté, M Carmen

    2016-03-01

    Two hundred and twenty-one strains representative of all Aeromonas species were characterized using the recA gene sequence, assessing its potential as a molecular marker for the genus Aeromonas. The inter-species distance values obtained demonstrated that recA has a high discriminatory power. Phylogenetic analysis, based on full-length gene nucleotide sequences, revealed a robust topology with clearly separated clusters for each species. The maximum likelihood tree showed the Aeromonas bestiarum strains in a well-defined cluster, containing a subset of four strains of different geographical origins in a deep internal branch. Data analysis provided strong evidence of recombination at the end of the recA sequences in these four strains. Intergenomic recombination corresponding to partial regions of the two adjacent genes recA and recX (248 bp) was identified between A. bestiarum (major parent) and Aeromonas eucrenophila (minor parent). The low number of recombinant strains detected (1.8%) suggests that horizontal flow between recA sequences is relatively uncommon in this genus. Moreover, only a few nucleotide differences were detected among these fragments, indicating that recombination has occurred recently. Finally, we also determined if the recombinant fragment could have influenced the structure and basic functions of the RecA protein, comparing models reconstructed from the translated amino acid sequences of our A. bestiarum strains with known Escherichia coli RecA structures.

  12. Effects of virus and host genes on recombination among ultraviolet-irradiated bacteriophage T4

    Energy Technology Data Exchange (ETDEWEB)

    Priemer, M.M.; Chan, V.L.

    1978-07-15

    The influence of the polA, uvrA, and recA genes of Escherichia coli on recombination among ultraviolet-irradiated T4 bacteriophages was determined with respect to recombination between rII markers and phage yield. The polA and uvrA gene products have no effect on these two aspects of phage DNA metabolism. A recA mutation does not significantly alter rII recombination frequencies in irradiated phage crosses, nor does it greatly change the yield of infectious particles in wild-type phage crosses or crosses in which the phage strains possess the v mutation. However, the same cross experiment performed with a pair of T4x mutants in a recA host demonstrates an 84% reduction in the phage yield in an unirradiated control cross. Furthermore, with increasing doses of uv irradiation, phage productivity of the T4x mutant declines at an accelerated rate compared to T4x/sup +/ strains crossed in recA cells. Multiplicity reactivation experiments in which wild-type or recombination-deficient (x or y) T4 phages infect wild-type or recombination-deficient (recA) host cells show that irradiated phages can only be reactivated in recA/sup +/ hosts, regardless of the bacteriophage genotype. These results indicate the involvement of the E. coli recA gene product in normal T4 replication and multiplicity reactivation.

  13. A recombinant lactobacillus strain expressing genes coding for ...

    African Journals Online (AJOL)

    SERVER

    2007-08-06

    Aug 6, 2007 ... bacteria do not naturally produce restriction enzymes, but rather, have a number of putative alien genes of the type. ..... recurrent bacterial vaginosis. J. Med. ... ty than OBR alone in HIV treatment experienced patients. Program.

  14. Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes

    Directory of Open Access Journals (Sweden)

    Jofre Juan

    2006-09-01

    Full Text Available Abstract Background The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products. The aim of this study was to induce mutations in the genome of some temperate Shiga toxin encoding bacteriophages. When phage genes are in the prophage state, they behave like chromosomal genes. This enables marker genes, such as antibiotic resistance genes, to be incorporated into the stx gene. Once the phages' lytic cycle is activated, recombinant Shiga toxin converting phages are produced. These phages can transfer the marker genes to the bacteria that they infect and convert. As the Red system's effectiveness decreased when used for our purposes, we had to introduce significant variations to the original method. These modifications included: confirming the stability of the target stx gene increasing the number of cells to be transformed and using a three-step PCR method to produce the amplimer containing the antibiotic resistance gene. Results Seven phages carrying two different antibiotic resistance genes were derived from phages that are directly involved in the pathogenesis of Shiga toxin-producing strains, using this modified protocol. Conclusion This approach facilitates exploration of the transduction processes and is a valuable tool for studying phage-mediated horizontal gene transfer.

  15. Construction and Expression of Human PTEN Tumor Suppressor Gene Recombinant Adenovirus Vector

    Institute of Scientific and Technical Information of China (English)

    CHEN Qingyong; WANG Chunyou; CHEN Daoda; CHEN Jianying; JIANG Chunfang; ZHENG Hai

    2006-01-01

    The recombinant defective adenovirus vector carrying human PTEN tumor suppres sor gene was constructed by using AdEasy-1 system and its expression was detected in human breast cancer cell line MDA-MB-468. Human PTEN cDNA was cloned into adenovirus shuttle plasmid pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-PTEN, then homologeous recombination was carried out in the E. coli BJ5183 by contransforming linearized shuttle vector with adenovirus backbone plasmid pAdEasy-1. The newly recombined defective adenovirus vector AdPTEN containing green fluorescent protein (GFP) was packaged and propagated in 293 cells. After being purified by cesium chloride gradient centrifugation, the adenovirus was transfected into human breast cancer cell line MDA-MB-468 in vitro. The expression of PTEN mRNA and protein in infected human breast cancer cell line MDA-MB-468 was detected by RT-PCR and Western blot respectively. The recombinant defective adenovirus vector carrying PTEN gene was constructed successfully. The viral titer of purified adenovirus was 2.5×1010 pfu/mL, and about 70 % breast cancer cells were infected with Ad PTEN when multiplicity of infection (MOI) reached 50. The exogenous PTEN mRNA and protein were expressed in MDA-MB-468 cells infected with Ad-PTEN by RT-PCR and Western blot. The recombinant defective adenovirus vector of PTEN gene was constructed successfully using AdEasy-1 system rapidly, which paved a sound foundation for gene study of breast cancer.

  16. Recombinant vectors construction for cellobiohydrolase encoding gene constitutive expression

    Directory of Open Access Journals (Sweden)

    Leontina GURGU

    2012-12-01

    Full Text Available Cellobiohydrolases (EC 3.2.1.91 are important exo enzymes involved in cellulose hydrolysis alongside endoglucanases (EC 3.2.1.4 and β-glucosidases (EC 3.2.1.21. Heterologous cellobiohydrolase gene expression under constitutive promoter control using Saccharomyces cerevisiae as host system is of great importance for a successful SSF process. From this point of view, the main objective of the work was to use Yeplac181 expression vector as a recipient for cellobiohdrolase - cbhB encoding gene expression under the control of the actin promoter, in Saccharomyces cerevisiae. Two hybridvectors, YEplac-Actp and YEplac-Actp-CbhB, were generated usingEscherichia coli XLI Blue for the cloning experiments. Constitutive cbhB gene expression was checked by proteine gel electrophoresis (SDS-PAGE after insertion of these constructs into Saccharomyces cerevisiae.

  17. Cloning of human and mouse genes homologous to RAD52, a yeast gene involved in DNA repair and recombination.

    NARCIS (Netherlands)

    D.F.R. Muris; O.Y. Bezzubova (Olga); J-M. Buerstedde; K. Vreeken; A.S. Balajee; C.J. Osgood; C. Troelstra (Christine); J.H.J. Hoeijmakers (Jan); K. Ostermann; H. Schmidt (Henning); A.T. Natarajan; J.C.J. Eeken; P.H.M. Lohmann (Paul); A. Pastink (Albert)

    1994-01-01

    textabstractThe RAD52 gene of Saccharomyces cerevisiae is required for recombinational repair of double-strand breaks. Using degenerate oligonucleotides based on conserved amino acid sequences of RAD52 and rad22, its counterpart from Schizosaccharomyces pombe, RAD52 homologs from man and mouse were

  18. Problem-Solving Test: Conditional Gene Targeting Using the Cre/loxP Recombination System

    Science.gov (United States)

    Szeberényi, József

    2013-01-01

    Terms to be familiar with before you start to solve the test: gene targeting, knock-out mutation, bacteriophage, complementary base-pairing, homologous recombination, deletion, transgenic organisms, promoter, polyadenylation element, transgene, DNA replication, RNA polymerase, Shine-Dalgarno sequence, restriction endonuclease, polymerase chain…

  19. Evolution and homologous recombination of the hemagglutinin-esterase gene sequences from porcine torovirus

    Science.gov (United States)

    The objective of the present study was to gain new insights into the evolution, homologous recombination and selection pressures imposed on the porcine torovirus (PToV), by examining changes in the hemagglutinin-esterase (HE) gene. The most recent common ancestor of PToV was estimated to have emerge...

  20. Recombinant adenovirus vectors with knobless fibers for targeted gene transfer

    NARCIS (Netherlands)

    van Beusechem, VW; van Rijswijk, ALCT; van Es, HHG; Haisma, HJ; Pinedo, HM; Gerritsen, WR

    2000-01-01

    Adenoviral vector systems for gene therapy can be much improved by targeting vectors to specific cell types. This requires both the complete ablation of native adenovirus tropism and the introduction of a novel binding affinity in the viral capsid. We reasoned that these requirements could be fulfil

  1. Rapid recombination among transfected plasmids, chimeric episome formation and trans gene expression in Plasmodium falciparum.

    Science.gov (United States)

    Kadekoppala, M; Cheresh, P; Catron, D; Ji, D D; Deitsch, K; Wellems, T E; Seifert, H S; Haldar, K

    2001-02-01

    Although recombination is known to be important to generating diversity in the human malaria parasite P. falciparum, the low efficiencies of transfection and the fact that integration of transfected DNA into chromosomes is observed only after long periods (typically 12 weeks or more) have made it difficult to genetically manipulate the blood stages of this major human pathogen. Here we show that co-transfection of a P. falciparum line with two plasmids, one expressing a green fluorescent protein (gfp) reporter and the other expressing a drug resistance marker (Tgdhfr-ts M23), allowed selection of a population in which about approximately 30% of the parasites produce GFP. In these GFP-producing parasites, the transfected plasmids had recombined into chimeric episomes as large as 20 kb and could be maintained under drug pressure for at least 16 weeks. Our data suggest that chimera formation occurs early (detected by 7--14 days) and that it involves homologous recombination favored by presence of the same P. falciparum 5'hrp3 UTR promoting transcription from each plasmid. This indicates the presence of high levels of homologous recombination activity in blood stage parasites that can be used to drive rapid recombination of newly introduced DNA, study mechanisms of recombination, and introduce genes for trans expression in P. falciparum.

  2. Trypanosome Surface Antigen Genes: Analysis Using Recombinant DNA.

    Science.gov (United States)

    1984-06-15

    different VATs. These cDNAs were used to screen the genomic libraries . The cloned probes were examined by nucleotide sequence analysis and used to...examination of the consequence of - antigenic variation on the structure of the 1.1, 1.D and 1.11 VSG -, gene sequences. In addition, all genomic libraries have...immunoprecipitated by heterologous VAt specific antisera (preprint 2, figure 3; preprint 3, figure 1). These data presented in preprints 2 and Genomic

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

    Indian Academy of Sciences (India)

    Xianlei Wang; Xungang Tan; Pei-Jun Zhang; Yuqing Zhang; Peng Xu

    2014-12-01

    During the development of B and T lymphocytes, Ig and TCR variable region genes are assembled from germline V, D, and J gene segments by a site-specific recombination reaction known as V(D)J recombination. The process of somatic V(D)J recombination, mediated by the recombination-activating gene (RAG) products, is the most significant characteristic of adaptive immunity in jawed vertebrates. Flounder (Paralichthys olivaceus) RAG1 and RAG2 were isolated by Genome Walker and RT-PCR, and their expression patterns were analysed by RT-PCR and in situ hybridization on sections. RAG1 spans over 7.0 kb, containing 4 exons and 3 introns, and the full-length ORF is 3207 bp, encoding a peptide of 1068 amino acids. The first exon lies in the 5′-UTR, which is an alternative exon. RAG2 full-length ORF is 1062 bp, encodes a peptide of 533 amino acids, and lacks introns in the coding region. In 6-month-old flounders, the expression of RAG1 and RAG2 was essentially restricted to the pronephros (head kidney) and mesonephros (truck kidney). Additionally, both of them were mainly expressed in the thymus. These results revealed that the thymus and kidney most likely serve as the primary lymphoid tissues in the flounder.

  4. Homologous recombination and gene replacement at the dihydrofolate reductase-thymidylate synthase locus in Toxoplasma gondii.

    Science.gov (United States)

    Donald, R G; Roos, D S

    1994-02-01

    To investigate the feasibility of genomic transgene expression and gene targeting in Toxoplasma gondii, parasites have been transfected with constructs differing in the length of contiguous genomic sequence spanning the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. We have previously reported that vectors derived from a DHFR-TS cDNA 'minigene' containing mutations in the DHFR coding sequence confer pyrimethamine resistance to transfected parasites (Donald and Roos, 1993). Stably resistant parasite clones arise at high frequency, generally by virtue of transgene integration into parasite chromosomes at locations scattered throughout the genome. In contrast, using a vector which contains 8 kb of contiguous genomic sequence (vs. homologous recombination. Homologous recombination appears to occur at even higher frequency when a 16 kb genomic clone is used. Circular plasmids were more efficient than linearized molecules at producing homologous recombination in this system, integrating by reciprocal crossing-over to produce a duplication of the DHFR-TS locus. Double crossing-over (or gene conversion) was also observed at low frequency, resulting in complete allelic replacement in this haploid stage of the parasite. The ability to produce either homologous or non-homologous recombinants, by the selection of appropriate transformation constructs, has considerable genetic potential.

  5. IMGT/GeneInfo: enhancing V(D)J recombination database accessibility.

    Science.gov (United States)

    Baum, Thierry-Pascal; Pasqual, Nicolas; Thuderoz, Florence; Hierle, Vivien; Chaume, Denys; Lefranc, Marie-Paule; Jouvin-Marche, Evelyne; Marche, Patrice-Noël; Demongeot, Jacques

    2004-01-01

    IMGT/GeneInfo is a user-friendly online information system that provides information on data resulting from the complex mechanisms of immunoglobulin (IG) and T cell receptor (TR) V(D)J recombinations. For the first time, it is possible to visualize all the rearrangement parameters on a single page. IMGT/GeneInfo is part of the international ImMunoGeneTics information system (IMGT), a high-quality integrated knowledge resource specializing in IG, TR, major histocompatibility complex (MHC), and related proteins of the immune system of human and other vertebrate species. The IMGT/GeneInfo system was developed by the TIMC and ICH laboratories (with the collaboration of LIGM), and is the first example of an external system being incorporated into IMGT. In this paper, we report the first part of this work. IMGT/GeneInfo_TR deals with the human and mouse TRA/TRD and TRB loci of the TR. Data handling and visualization are complementary to the current data and tools in IMGT, and will subsequently allow the modelling of V(D)J gene use, and thus, to predict non-standard recombination profiles which may eventually be found in conditions such as leukaemias or lymphomas. Access to IMGT/GeneInfo is free and can be found at http://imgt.cines.fr/GeneInfo.

  6. [Constructing recombinant plasmid pSH-CUP and knockout of acid trehalase gene in baker's yeast].

    Science.gov (United States)

    He, Dongqin; Xiao, Dongguang; Lv, Ye

    2008-02-01

    The ATH1 gene encoded acid trehalase in Saccharomyces cerevisiae. The gene disruption cassette combined the heterologous dominant kan(r) resistance marker with a Cre/loxP-mediated marker removal procedure. The gene disruption cassette was produced by PCR using the same long oligonucleotides comprising 50 nucleotides that annealed to sites upstream or downstream of the genomic target sequence to be deleted. After transformation of the linear disruption cassettes with a Cre/loxP-mediated marker into the cells of Saccharomyces cerevisiae BY-6, selected transformants were checked by PCR for correct the integration of the cassette and concurrent deletion of the chromosomal target sequence. The copper-resistance gene (CUP1-MT1) was cloned into pSH47, which yielded pSH-CUP. The recombinant plasmid pSH-CUP was transformed into the cells of Saccharomyces cerevisiae BY-6(delta ATH1, G418(r)), and transformants were selected for copper resistance. Upon expression of the Cre recombinase results in removal of the kan(r) gene, leaving behind a single loxP site at the chromosomal locus. Construction of the recombinant plasmid pSH-CUP avoided inserting non-yeast gene and made the loxP - kanMX - loxP gene disruption cassette more conventional for eukaryotic organism gene disruption.

  7. Baculovirus expression of the glycoprotein gene of Lassa virus and characterization of the recombinant protein.

    Science.gov (United States)

    Hummel, K B; Martin, M L; Auperin, D D

    1992-09-01

    A recombinant baculovirus was constructed that expresses the glycoprotein gene of Lassa virus (Josiah strain) under the transcriptional control of the polyhedrin promoter. The expressed protein (B-LSGPC) comigrated with the authentic viral glycoprotein as observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), was reactive with monoclonal antibodies (MAbs) in Western blots, and was glycosylated. Although the recombinant protein was not processed into the mature glycoproteins, G1 and G2, it demonstrated reactivity with all known epitopes as measured by indirect immunofluorescence (IFA), and it was immunogenic, eliciting antisera in rabbits that recognized whole virus in IFAs. Regarding future applications to diagnostic assays, the recombinant glycoprotein proved to be an effective substitute for Lassa virus-infected mammalian cells in IFAs and it was able to distinguish sera from several human cases of Lassa fever, against a panel of known negative sera of African origin, in an enzyme immunoassay (EIA).

  8. ALTERATION OF GENE EXPRESSION IN LEUKOCYTES FROM RECOMBINANT SOMATOTROPIN TREATED ANIMALS: SEARCHING FOR INSPECTION INDICATORS

    Directory of Open Access Journals (Sweden)

    NR Brizioli

    2008-12-01

    Full Text Available Besides immunochemical approaches, biomolecular studies can be carried out in order to discover a greater number of biological indicators to be exploited for the identification of bovines treated with recombinant somatotropin (rbST. With this aim, we analysed the expression of a number of genes related to the somatotropic axis in leucocytes from rbST treated cows and non-treated animals. Significant differences were observed in the genes IGF-1,IGFBP-1, IGFBP-4 and the I- 5’UTR variant of the GHR gene.

  9. Targeting of human aFGF gene into silkworm,Bombyx mori L. through homologous recombination

    Institute of Scientific and Technical Information of China (English)

    吴小锋; 曹翠平

    2004-01-01

    The long-arm and short-arm genes of fibroin light chain (L-chain) of silkworm, Bombyx Mori L., and the gene of human acidic fibroblast growth factor were cloned respectively and subsequently inserted into a transfer vector pVL 1392 used as a tool to target the L-chain region of the silkworm genome. Genomic DNA from their offsprings was extracted and the expected targeting was detected using polymerase chain reaction and DNA sequencing, as well as protein analysis. The results showed that positive events occurred and that the FGF gene was integrated into the L-chain locus through homologous recombination.

  10. Targeting of human aFGF gene into silkworm, Bombyx mori L.Through homologous recombination

    Institute of Scientific and Technical Information of China (English)

    吴小锋; 曹翠平

    2004-01-01

    The long-arm and short-arm genes of fibroin light chain (L-chain) of silkworm, Bombyx Mori L., and the gene of human acidic fibroblast growth factor were cloned respectively and subsequently inserted into a transfer vector pVL1392 used as a tool to target the L-chain region of the silkworm genome. Genomic DNA from their offsprings was extracted and the expected targeting was detected using polymerase chain reaction and DNA sequencing, as well as protein analysis. The results showed that positive events occurred and that the FGF gene was integrated into the L-chain locus through homologous recombination.

  11. Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gen...

  12. Waste recombinant DNA: effectiveness of thermo-treatment to manage potential gene pollution.

    Science.gov (United States)

    Fu, Xiaohua; Li, Mengnan; Zheng, Guanghong; Le, Yiquan; Wang, Lei

    2009-01-01

    Heating at 100 degrees C for 5-10 min is a common method for treating wastewater containing recombinant DNA in many bio-laboratories in China. In this experiment, plasmid pET-28b was used to investigate decay efficiency of waste recombinant DNA during thermo-treatment. The results showed that the decay half-life of the plasmid was 2.7-4.0 min during the thermo-treatment, and even heating for 30 min the plasmids still retained some transforming activity. Low pH promoted the decay of recombinant DNA, but NaCl, bovine serum albumin and EDTA, which existed in the most wastewater from bio-laboratories, protected DNA from degradation. Thus, the decay half-life of plasmid DNA may be longer than 2.7-4.0 min practically. These results suggest that the effectiveness of heating at 100 degrees C for treating waste recombinant DNA is low and a gene pollution risk remains when those thermo-treated recombinant DNAs are discharged into the environment. Therefore other simple and effective methods should be developed.

  13. Germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian, fallopian tube, and peritoneal carcinomas.

    Science.gov (United States)

    Pennington, Kathryn P; Walsh, Tom; Harrell, Maria I; Lee, Ming K; Pennil, Christopher C; Rendi, Mara H; Thornton, Anne; Norquist, Barbara M; Casadei, Silvia; Nord, Alexander S; Agnew, Kathy J; Pritchard, Colin C; Scroggins, Sheena; Garcia, Rochelle L; King, Mary-Claire; Swisher, Elizabeth M

    2014-02-01

    Hallmarks of germline BRCA1/2-associated ovarian carcinomas include chemosensitivity and improved survival. The therapeutic impact of somatic BRCA1/2 mutations and mutations in other homologous recombination DNA repair genes is uncertain. Using targeted capture and massively parallel genomic sequencing, we assessed 390 ovarian carcinomas for germline and somatic loss-of-function mutations in 30 genes, including BRCA1, BRCA2, and 11 other genes in the homologous recombination pathway. Thirty-one percent of ovarian carcinomas had a deleterious germline (24%) and/or somatic (9%) mutation in one or more of the 13 homologous recombination genes: BRCA1, BRCA2, ATM, BARD1, BRIP1, CHEK1, CHEK2, FAM175A, MRE11A, NBN, PALB2, RAD51C, and RAD51D. Nonserous ovarian carcinomas had similar rates of homologous recombination mutations to serous carcinomas (28% vs. 31%, P = 0.6), including clear cell, endometrioid, and carcinosarcoma. The presence of germline and somatic homologous recombination mutations was highly predictive of primary platinum sensitivity (P = 0.0002) and improved overall survival (P = 0.0006), with a median overall survival of 66 months in germline homologous recombination mutation carriers, 59 months in cases with a somatic homologous recombination mutation, and 41 months for cases without a homologous recombination mutation. Germline or somatic mutations in homologous recombination genes are present in almost one third of ovarian carcinomas, including both serous and nonserous histologies. Somatic BRCA1/2 mutations and mutations in other homologous recombination genes have a similar positive impact on overall survival and platinum responsiveness as germline BRCA1/2 mutations. The similar rate of homologous recombination mutations in nonserous carcinomas supports their inclusion in PARP inhibitor clinical trials. ©2013 AACR.

  14. Gene targeting using homologous recombination in embryonic stem cells: The future for behavior genetics?

    Directory of Open Access Journals (Sweden)

    Robert eGerlai

    2016-04-01

    Full Text Available Gene targeting with homologous recombination in embryonic stem cells created a revolution in the analysis of the function of genes in behavioral brain research. The technology allowed unprecedented precision with which one could manipulate genes and study the effect of this manipulation on the central nervous system. With gene targeting, the uncertainty inherent in psychopharmacology regarding whether a particular compound would act only through a specific target was removed. Thus, gene targeting became highly popular. However, with this popularity came the realization that like other methods, gene targeting also suffered from some technical and principal problems. For example, two decades ago, issues about compensatory changes and about genetic linkage were raised. Since then, the technology developed, and its utility has been better delineated. This review will discuss the pros and cons of the technique along with these advancements from the perspective of the neuroscientist user. It will also compare and contrast methods that may represent novel alternatives to the homologous recombination based gene targeting approach, including the TALEN and the CRISPR/Cas9 systems. The goal of the review is not to provide detailed recipes, but to attempt to present a short summary of these approaches a behavioral geneticist or neuroscientist may consider for the analysis of brain function and behavior.

  15. Gene Targeting Using Homologous Recombination in Embryonic Stem Cells: The Future for Behavior Genetics?

    Science.gov (United States)

    Gerlai, Robert

    2016-01-01

    Gene targeting with homologous recombination in embryonic stem cells created a revolution in the analysis of the function of genes in behavioral brain research. The technology allowed unprecedented precision with which one could manipulate genes and study the effect of this manipulation on the central nervous system. With gene targeting, the uncertainty inherent in psychopharmacology regarding whether a particular compound would act only through a specific target was removed. Thus, gene targeting became highly popular. However, with this popularity came the realization that like other methods, gene targeting also suffered from some technical and principal problems. For example, two decades ago, issues about compensatory changes and about genetic linkage were raised. Since then, the technology developed, and its utility has been better delineated. This review will discuss the pros and cons of the technique along with these advancements from the perspective of the neuroscientist user. It will also compare and contrast methods that may represent novel alternatives to the homologous recombination based gene targeting approach, including the TALEN and the CRISPR/Cas9 systems. The goal of the review is not to provide detailed recipes, but to attempt to present a short summary of these approaches a behavioral geneticist or neuroscientist may consider for the analysis of brain function and behavior.

  16. Demonstration that menthofuran synthase of mint (Mentha) is a cytochrome P450 monooxygenase: cloning, functional expression, and characterization of the responsible gene.

    Science.gov (United States)

    Bertea, C M; Schalk, M; Karp, F; Maffei, M; Croteau, R

    2001-06-15

    (+)-Menthofuran is an undesirable monoterpenoid component of peppermint (Mentha x piperita) essential oil that is derived from the alpha,beta-unsaturated ketone (+)-pulegone. Microsomal preparations, from the oil gland secretory cells of a high (+)-menthofuran-producing chemotype of Mentha pulegium, transform (+)-pulegone to (+)-menthofuran in the presence of NADPH and molecular oxygen, implying that menthofuran is synthesized by a mechanism analogous to that of mammalian liver cytochrome P450s involving the hydroxylation of the syn-methyl group of (+)-pulegone, spontaneous intramolecular cyclization to the hemiketal, and dehydration to the furan. An abundant cytochrome P450 clone from a peppermint oil gland cell cDNA library was functionally expressed in Saccharomyces cerevisiae and Escherichia coli and shown to encode the (+)-menthofuran synthase (i.e., (+)-pulegone-9-hydroxylase). The full-length cDNA contains 1479 nucleotides, and encodes a protein of 493 amino acid residues of molecular weight 55,360, which bears all of the anticipated primary structural elements of a cytochrome P450 and most closely resembles (35% identity) a cytochrome P450 monoterpene hydroxylase, (+)-limonene-3-hydroxylase, from the same source. The availability of this gene permits transgenic manipulation of peppermint to improve the quality of the derived essential oil. Copyright 2001 Academic Press.

  17. Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement.

    Science.gov (United States)

    Cervera, Laura; Gutiérrez-Granados, Sonia; Berrow, Nicholas Simon; Segura, Maria Mercedes; Gòdia, Francesc

    2015-05-01

    Production of recombinant products in mammalian cell cultures can be achieved by stable gene expression (SGE) or transient gene expression (TGE). The former is based on the integration of a plasmid DNA into the host cell genome allowing continuous gene expression. The latter is based on episomal plasmid DNA expression. Conventional TGE is limited to a short production period of usually about 96 h, therefore limiting productivity. A novel gene expression approach termed extended gene expression (EGE) is explored in this study. The aim of EGE is to prolong the production period by the combination of medium exchange and repeated transfection of cell cultures with plasmid DNA to improve overall protein production. The benefit of this methodology was evaluated for the production of three model recombinant products: intracellular GFP, secreted GFP, and a Gag-GFP virus-like particles (VLPs). Productions were carried out in HEK 293 cell suspension cultures grown in animal-derived component free media using polyethylenimine (PEI) as transfection reagent. Transfections were repeated throughout the production process using different plasmid DNA concentrations, intervals of time, and culture feeding conditions in order to identify the best approach to achieve sustained high-level gene expression. Using this novel EGE strategy, the production period was prolonged between 192 and 240 h with a 4-12-fold increase in production levels, depending on the product type considered. © 2014 Wiley Periodicals, Inc.

  18. Recombination-dependent deletion formation in mammalian cells deficient in the nucleotide excision repair gene ERCC1.

    Science.gov (United States)

    Sargent, R G; Rolig, R L; Kilburn, A E; Adair, G M; Wilson, J H; Nairn, R S

    1997-11-25

    Nucleotide excision repair proteins have been implicated in genetic recombination by experiments in Saccharomyces cerevisiae and Drosophila melanogaster, but their role, if any, in mammalian cells is undefined. To investigate the role of the nucleotide excision repair gene ERCC1, the hamster homologue to the S. cerevisiae RADIO gene, we disabled the gene by targeted knockout. Partial tandem duplications of the adenine phosphoribosyltransferase (APRT) gene then were constructed at the endogenous APRT locus in ERCC1- and ERCC1+ cells. To detect the full spectrum of gene-altering events, we used a loss-of-function assay in which the parental APRT+ tandem duplication could give rise to APRT- cells by homologous recombination, gene rearrangement, or point mutation. Measurement of rates and analysis of individual APRT- products indicated that gene rearrangements (principally deletions) were increased at least 50-fold, whereas homologous recombination was affected little. The formation of deletions is not caused by a general effect of the ERCC1 deficiency on gene stability, because ERCC1- cell lines with a single wild-type copy of the APRT gene yielded no increase in deletions. Thus, deletion formation is dependent on the tandem duplication, and presumably the process of homologous recombination. Recombination-dependent deletion formation in ERCC1- cells is supported by a significant decrease in a particular class of crossover products that are thought to arise by repair of a heteroduplex intermediate in recombination. We suggest that the ERCC1 gene product in mammalian cells is involved in the processing of heteroduplex intermediates in recombination and that the misprocessed intermediates in ERCC1- cells are repaired by illegitimate recombination.

  19. 4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB as an oxidative biocatalyst in the synthesis of optically active sulfoxides

    NARCIS (Netherlands)

    Gonzalo, Gonzalo de; Torres Pazmiño, Daniel E.; Ottolina, Gianluca; Fraaije, Marco W.; Carrea, Giacomo

    2006-01-01

    Recombinant 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB has been tested as a catalyst in sulfoxidation reactions on a set of aromatic sulfides. With a few exceptions, excellent enantioselectivities in the synthesis of chiral phenyl and benzyl sulfoxides were achieved

  20. A recombinant varicella vaccine harboring a respiratory syncytial virus gene induces humoral immunity.

    Science.gov (United States)

    Murakami, Kouki; Matsuura, Masaaki; Ota, Megumi; Gomi, Yasuyuki; Yamanishi, Koichi; Mori, Yasuko

    2015-11-09

    The varicella-zoster virus (VZV) Oka vaccine strain (vOka) is highly efficient and causes few adverse events; therefore, it is used worldwide. We previously constructed recombinant vOka (rvOka) harboring the mumps virus gene. Immunizing guinea pigs with rvOka induced the production of neutralizing antibodies against the mumps virus and VZV. Here, we constructed recombinant vOka viruses containing either the respiratory syncytial virus (RSV) subgroup A fusion glycoprotein (RSV A-F) gene or RSV subgroup B fusion glycoprotein (RSV B-F) gene (rvOka-RSV A-F or rvOka-RSV B-F). Indirect immunofluorescence and Western blot analyses confirmed the expression of each recombinant RSV protein in virus-infected cells. Immunizing guinea pigs with rvOka-RSV A-F or rvOka-RSV B-F led to the induction of antibodies against RSV proteins. These results suggest that the current varicella vaccine genome can be used to generate custom-made vaccine vectors to develop the next generation of live vaccines.

  1. mRNA differential display in a microbial enrichment culture: simultaneous identification of three cyclohexanone monooxygenases from three species.

    Science.gov (United States)

    Brzostowicz, Patricia C; Walters, Dana M; Thomas, Stuart M; Nagarajan, Vasantha; Rouvière, Pierre E

    2003-01-01

    mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.

  2. Starch-degrading polysaccharide monooxygenases.

    Science.gov (United States)

    Vu, Van V; Marletta, Michael A

    2016-07-01

    Polysaccharide degradation by hydrolytic enzymes glycoside hydrolases (GHs) is well known. More recently, polysaccharide monooxygenases (PMOs, also known as lytic PMOs or LPMOs) were found to oxidatively degrade various polysaccharides via a copper-dependent hydroxylation. PMOs were previously thought to be either GHs or carbohydrate binding modules (CBMs), and have been re-classified in carbohydrate active enzymes (CAZY) database as auxiliary activity (AA) families. These enzymes include cellulose-active fungal PMOs (AA9, formerly GH61), chitin- and cellulose-active bacterial PMOs (AA10, formerly CBM33), and chitin-active fungal PMOs (AA11). These PMOs significantly boost the activity of GHs under industrially relevant conditions, and thus have great potential in the biomass-based biofuel industry. PMOs that act on starch are the latest PMOs discovered (AA13), which has expanded our perspectives in PMOs studies and starch degradation. Starch-active PMOs have many common structural features and biochemical properties of the PMO superfamily, yet differ from other PMO families in several important aspects. These differences likely correlate, at least in part, to the differences in primary and higher order structures of starch and cellulose, and chitin. In this review we will discuss the discovery, structural features, biochemical and biophysical properties, and possible biological functions of starch-active PMOs, as well as their potential application in the biofuel, food, and other starch-based industries. Important questions regarding various aspects of starch-active PMOs and possible economical driving force for their future studies will also be highlighted.

  3. Cellulosic Ethanol Production by Recombinant Cellulolytic Bacteria Harbouring pdc and adh II Genes of Zymomonas mobilis.

    Science.gov (United States)

    Piriya, P Sobana; Vasan, P Thirumalai; Padma, V S; Vidhyadevi, U; Archana, K; Vennison, S John

    2012-01-01

    The ethanol fermenting genes such as pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adh II) were cloned from Zymomonas mobilis and transformed into three different cellulolytic bacteria, namely Enterobacter cloacae JV, Proteus mirabilis JV and Erwinia chrysanthemi and their cellulosic ethanol production capability was studied. Recombinant E. cloacae JV was found to produce 4.5% and 3.5% (v/v) ethanol, respectively, when CMC and 4% NaOH pretreated bagasse were used as substrates, whereas recombinant P. mirabilis and E. chrysanthemi with the same substrates could only produce 4%, 3.5%, 1%, and 1.5 % of ethanol, respectively. The recombinant E. cloacae strain produced twofold higher percentage of ethanol than the wild type. The recombinant E. cloacae strain could be improved further by increasing its ethanol tolerance capability through media optimization and also by combining multigene cellulase expression for enhancing ethanol production from various types of lignocellulosic biomass so that it can be used for industrial level ethanol production.

  4. Cellulosic Ethanol Production by Recombinant Cellulolytic Bacteria Harbouring pdc and adh II Genes of Zymomonas mobilis

    Directory of Open Access Journals (Sweden)

    P. Sobana Piriya

    2012-01-01

    Full Text Available The ethanol fermenting genes such as pyruvate decarboxylase (pdc and alcohol dehydrogenase II (adh II were cloned from Zymomonas mobilis and transformed into three different cellulolytic bacteria, namely Enterobacter cloacae JV, Proteus mirabilis JV and Erwinia chrysanthemi and their cellulosic ethanol production capability was studied. Recombinant E. cloacae JV was found to produce 4.5% and 3.5% (v/v ethanol, respectively, when CMC and 4% NaOH pretreated bagasse were used as substrates, whereas recombinant P. mirabilis and E. chrysanthemi with the same substrates could only produce 4%, 3.5%, 1%, and 1.5 % of ethanol, respectively. The recombinant E. cloacae strain produced twofold higher percentage of ethanol than the wild type. The recombinant E. cloacae strain could be improved further by increasing its ethanol tolerance capability through media optimization and also by combining multigene cellulase expression for enhancing ethanol production from various types of lignocellulosic biomass so that it can be used for industrial level ethanol production.

  5. Identification of a DNA binding protein that recognizes the nonamer recombinational signal sequence of immunoglobulin genes.

    Science.gov (United States)

    Halligan, B D; Desiderio, S V

    1987-10-01

    Extracts of nuclei from B- and T-lymphoid cells contain a protein that binds specifically to the conserved nonamer DNA sequence within the recombinational signals of immunoglobulin genes. Complexes with DNA fragments from four kappa light-chain joining (J) segments have the same electrophoretic mobility. Nonamer-containing DNA fragments from heavy-chain and light-chain genes compete for binding. Within the 5'-flanking DNA of the J kappa 4 gene segment, the binding site has been localized to a 27-base-pair interval spanning the nonamer region. The binding activity is recovered as a single peak after ion-exchange chromatography. The site of binding of the protein and its presence in nuclei of lymphoid cells suggest that it may function in the assembly of immunoglobulin genes.

  6. Subcloning plus insertion (SPI)--a novel recombineering method for the rapid construction of gene targeting vectors.

    Science.gov (United States)

    Reddy, Thimma R; Kelsall, Emma J; Fevat, Léna M S; Munson, Sarah E; Cowley, Shaun M

    2015-01-08

    Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a 'targeting' vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4 kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

  7. Construction of recombinant adeno-associated viral vectors in human neurenergen-3 gene

    Institute of Scientific and Technical Information of China (English)

    Xiangli Wang; Haili Wang; Baojie Mi

    2007-01-01

    BACKGROUND: Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeutic gene.OBJECTIVE: To explore an effective way to construct recombinant adeno-associated viral vectors expression in human neurnnergen-3 gene. DESIGN: Gene directed cloning.SETTING: Central Laboratory of Northern China Coal Medical College.MATERIALS: DH5a competent bacillus coli strain was provided by Capital Medical University; pCDNA3-NT-3 by professor Chen from Bengbu Medical College; pAAV-Laze, pAAV-Helper, pAAV-RC and pAAV-MCS plasmids by Capital Medical University; HEK293 cells by Cell Center of Basic Medical College of Tongji Medical University.METHODS: NT-3 genes which were selected from pCDNA3-NT-3 plasmids were cloned in pAAV-MCS to form a recombinant adeno-associated viral plasmid (pAAV-NT-3). pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were extracted, purified and subjected to enzyme-shearing evaluation. In addition, pAAV-NT-3 and pAAV-LacZ were cotransfected with pHelper and pAAV-RC, respectively into AVV-293 cells with DNA mediated by calcium superphosphate transfection gene; and then, AVV-293 cells were packed into recombinant adeno-associated viral rAAV-NT-3 and rAAV-LacZ. After collection of viral particles, rAAV-LacZ viral stock solution was diluted based on ratio of 10:1 and the mixture was used to infect HT1080 cells. X-gal stain was used to measure virus liter.MAIN OUTCOME MEASURES: Size of targeted gene fragments, validity of vehicle construction and virus liter.RESULTS: Targeted gene NT-3 was successfully inserted into the relative vehicle pAAV and pAAV-NT-3 was correctly recongnized by enzyme-shearing evaluation. Enzyme-shearing electrophoresis demonstrated that pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were successfully extracted and purified.β-galactoside staining in situ indicated that LacZ genes were

  8. Regulation of Recombination between gtfB/gtfC Genes in Streptococcus mutans by Recombinase A

    Directory of Open Access Journals (Sweden)

    Satoko Inagaki

    2013-01-01

    Full Text Available Streptococcus mutans produces 3 types of glucosyltransferases (GTFs, whose cooperative action is essential for cellular adhesion. The recombinase A (RecA protein is required for homologous recombination. In our previous study, we isolated several strains with a smooth colony morphology and low GTF activity, characteristics speculated to be derived from the GTF fusions. The purpose of the present study was to investigate the mechanism of those fusions. S. mutans strain MT8148 was grown in the presence of recombinant RecA (rRecA protein, after which smooth colonies were isolated. The biological functions and sequences of the gtfB and gtfC genes of this as well as other clinical strains were determined. The sucrose-dependent adherence rates of those strains were reduced as compared to that of MT8148. Determination of the sequences of the gtfB and gtfC genes showed that an approximately 3500 bp region was deleted from the area between them. Furthermore, expression of the recA gene was elevated in those strains as compared to MT8148. These results suggest that RecA has an important role in fusions of gtfB and gtfC genes, leading to alteration of colony morphology and reduction in sucrose-dependent adhesion.

  9. 杜梨胆碱单加氧酶基因克隆及胁迫表达%Cloning and Expression Analysis of a Choline Monooxygenase Gene in Pyrus betulaefolia Bunge under Abiotic Stress

    Institute of Scientific and Technical Information of China (English)

    李慧; 丛郁; 常有宏; 蔺经; 盛宝龙

    2012-01-01

    To provide an insight into stress defense mechanism of birch-leaf pear,a choline monooxygenase gene (PbCMO) was cloned via reverse transcriptase PCR, the rapid-amplification of cDNA ends (RACE) and long PCR methods. At the same time,PbCMO sequence characteristics were analyzed by the bioinfor-matics software. Furthermore,semi-quantitative PCR with cross-intron primers was adopted to study the expression patterns of PbCMO gene under abiotic stresses. The results showed that: (1)PbCMO cDNA sequence contained a 1 227 bp length coding region which encoded 408 amino acid residues. PbCMO genomic DNA sequence length was 2 928 bp which consisted of 10 exons and 9 introns. Predicted isoelectric point and relative molecular mass of PbCMO were 6. 19 and 46. 27 kD,respectively. PbCMO encoded polypeptide included Rieske-type iron-sulfur [2Fe-2S] cluster-binding region and the conserved residues of the mono-nuclear Fe-binding motif. Homology analysis showed that the deduced PbCMO protein was highly similar with medlar CMO protein (71%). Phylogenetic analysis also indicated that PbCMO was more related to medlar CMO. (2)PbCMO gene expression was inducible and its transcription abundances climbed up quickly after 100 mmol/L NaCl,10% (W/V) PEG-6000,180 mmol/L mannitol or 20 μmol/L ABA treatment. It is showed that PbCMO expression was response to salt,drought,osmotic and ABA stress. Conclusively,PbCMO was involved in the defense mechanism against abiotic stress in birch-leaf pear.%为了解梨砧木—杜梨对非生物胁迫的防御机制,采用RT-PCR、RACE和长片段PCR技术从杜梨幼苗中获得1个甜菜碱合成相关的胆碱单加氧酶基因(PbCMO),运用生物信息学方法分析序列特点,并通过跨内含子引物进行半定量RT-PCR研究其在非生物胁迫下的表达情况.结果表明:(1) PbCMO基因cDNA序列编码区长1227bp,编码由408个氨基酸组成的多肽.其对应基因组DNA序列长2928 bp,由10个外显子和9个内含子组成.其推导

  10. Identification of dimethylamine monooxygenase in marine bacteria reveals a metabolic bottleneck in the methylated amine degradation pathway.

    Science.gov (United States)

    Lidbury, Ian; Mausz, Michaela A; Scanlan, David J; Chen, Yin

    2017-07-01

    Methylated amines (MAs) are ubiquitous in the marine environment and their subsequent flux into the atmosphere can result in the formation of aerosols and ultimately cloud condensation nuclei. Therefore, these compounds have a potentially important role in climate regulation. Using Ruegeria pomeroyi as a model, we identified the genes encoding dimethylamine (DMA) monooxygenase (dmmABC) and demonstrate that this enzyme degrades DMA to monomethylamine (MMA). Although only dmmABC are required for enzyme activity in recombinant Escherichia coli, we found that an additional gene, dmmD, was required for the growth of R. pomeroyi on MAs. The dmmDABC genes are absent from the genomes of multiple marine bacteria, including all representatives of the cosmopolitan SAR11 clade. Consequently, the abundance of dmmDABC in marine metagenomes was substantially lower than the genes required for other metabolic steps of the MA degradation pathway. Thus, there is a genetic and potential metabolic bottleneck in the marine MA degradation pathway. Our data provide an explanation for the observation that DMA-derived secondary organic aerosols (SOAs) are among the most abundant SOAs detected in fine marine particles over the North and Tropical Atlantic Ocean.

  11. Effect of recombinant adenovirus vector mediated human interleukin-24 gene transfection on pancreatic carcinoma growth

    Institute of Scientific and Technical Information of China (English)

    PAN Xin-ting; ZHU Qing-yun; LI De-chun; YANG Ji-cheng; ZHANG Zi-xiang; ZHU Xing-guo; ZHAO Hua

    2008-01-01

    Background Pancreatic cancer is a highly malignant tumor affecting an ever increasing number of patients with a mean 5-year survival rate below 4%. Therefore, gene therapy for cancer has become a potential novel therapeutic modality. In this study we sought to determine the inhibitory effects of adenovirus-mediated human interleukin-24 (AdhlL-24) on pancreatic cancer.Methods Human interleukin-24 gene was cloned into replication-defective adenovirus specific for patu8988 tumor cells by virus recombination technology. Reverse transcription-polymerase chain reaction and Western blotting analysis were used to determine the expression of human interleukin-24 mRNA in patu8988 cells in vitro. Induction of apoptosis by overexpression of human interleukin-24 in patu8988 cells was determined by flow cytometry. In vivo efficacy of adenoviral delivery of human interleukin-24 was assessed in nude mice (n=10 for each group) bearing patu8988 pancreatic cancer cell lines by determining inhibition of tumor growth, endothelial growth factor and CD34 expression, and intratumoral microvessel density (MVD).Results The recombinant adenovirus vector AdVGFP/IL-24 was constructed with a packaged recombinant retrovirus titer of 1.0x1010 pfu/ml and successfully expressed of both mRNA and protein in patu8988 cells. The AdVGFP/IL-24 induced apoptosis of patu8988 tumor cells in vitro and significantly inhibited tumor growth in vivo (P <0.05). The intratumoral MVD decreased significantly in the treated tumors (P <0.05).Conclusion The recombinant adenovirus AdGFP/IL-24 can effectively express biologically active human interleukin-24, which results in inhibition of pancreatic cancer growth.

  12. Cloning and characterization of a gene cluster for cyclododecanone oxidation in Rhodococcus ruber SC1.

    Science.gov (United States)

    Kostichka, K; Thomas, S M; Gibson, K J; Nagarajan, V; Cheng, Q

    2001-11-01

    Biological oxidation of cyclic ketones normally results in formation of the corresponding dicarboxylic acids, which are further metabolized in the cell. Rhodococcus ruber strain SC1 was isolated from an industrial wastewater bioreactor that was able to utilize cyclododecanone as the sole carbon source. A reverse genetic approach was used to isolate a 10-kb gene cluster containing all genes required for oxidative conversion of cyclododecanone to 1,12-dodecanedioic acid (DDDA). The genes required for cyclododecanone oxidation were only marginally similar to the analogous genes for cyclohexanone oxidation. The biochemical function of the enzymes encoded on the 10-kb gene cluster, the flavin monooxygenase, the lactone hydrolase, the alcohol dehydrogenase, and the aldehyde dehydrogenase, was determined in Escherichia coli based on the ability to convert cyclododecanone. Recombinant E. coli strains grown in the presence of cyclododecanone accumulated lauryl lactone, 12-hydroxylauric acid, and/or DDDA depending on the genes cloned. The cyclododecanone monooxygenase is a type 1 Baeyer-Villiger flavin monooxygenase (FAD as cofactor) and exhibited substrate specificity towards long-chain cyclic ketones (C11 to C15), which is different from the specificity of cyclohexanone monooxygenase favoring short-chain cyclic compounds (C5 to C7).

  13. Construction and characterization of recombinant flaviviruses bearing insertions between E and NS1 genes

    Directory of Open Access Journals (Sweden)

    Kubelka Claire F

    2007-10-01

    Full Text Available Abstract Background The yellow fever virus, a member of the genus Flavivirus, is an arthropod-borne pathogen causing severe disease in humans. The attenuated yellow fever 17D virus strain has been used for human vaccination for 70 years and has several characteristics that are desirable for the development of new, live attenuated vaccines. We described here a methodology to construct a viable, and immunogenic recombinant yellow fever 17D virus expressing a green fluorescent protein variant (EGFP. This approach took into account the presence of functional motifs and amino acid sequence conservation flanking the E and NS1 intergenic region to duplicate and fuse them to the exogenous gene and thereby allow the correct processing of the viral polyprotein precursor. Results YF 17D EGFP recombinant virus was grew in Vero cells and reached a peak titer of approximately 6.45 ± 0.4 log10 PFU/mL at 96 hours post-infection. Immunoprecipitation and confocal laser scanning microscopy demonstrated the expression of the EGFP, which was retained in the endoplasmic reticulum and not secreted from infected cells. The association with the ER compartment did not interfere with YF assembly, since the recombinant virus was fully competent to replicate and exit the cell. This virus was genetically stable up to the tenth serial passage in Vero cells. The recombinant virus was capable to elicit a neutralizing antibody response to YF and antibodies to EGFP as evidenced by an ELISA test. The applicability of this cloning strategy to clone gene foreign sequences in other flavivirus genomes was demonstrated by the construction of a chimeric recombinant YF 17D/DEN4 virus. Conclusion This system is likely to be useful for a broader live attenuated YF 17D virus-based vaccine development for human diseases. Moreover, insertion of foreign genes into the flavivirus genome may also allow in vivo studies on flavivirus cell and tissue tropism as well as cellular processes related

  14. Exploratory study of mechanical kinematic innovation design based on gene recombination operation

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Yixiong; Gao, Yicong; Tan, Jianrong [Zhejiang University, Hangzhou (China); Hagiwara, Ichiro [Tokyo Institute of Technology, Tokyo (Korea, Republic of)

    2013-03-15

    This paper analyzes the influencing factor of motion output of the inspired mechanism under the premise that the motion input is invariant. These factors are respectively expressed as kinematic pair chromosome number, kinematic pair feature gene and distance relationship vector gene by virtue of several concepts and principles in genetics, and then they are encoded. Mechanism chromosome model is established, which is constituted by mechanism chromosome relationship graph and mechanism chromosome matrix. Three kinematic pair chromosome gene recombination operations on mechanism chromosome model (dominance, translocation and metastasis), are proposed by using meiosis and chromosome variance in genetics for reference. Finally the paper takes shaper as the original mechanism and acquires its inspired mechanism, which proves the convenience and practicality of the methods.

  15. Generation and characterization of NV gene-knockout recombinant viral hemorrhagic septicemia virus (VHSV) genotype IVa.

    Science.gov (United States)

    Kim, Min Sun; Kim, Dong Soo; Kim, Ki Hong

    2011-11-03

    A recombinant viral hemorrhagic septicemia virus (rVHSV-deltaNV-EGFP) containing the enhanced green fluorescent protein (EGFP) gene instead of the NV gene was produced using the reverse-genetics method. For use as a positive control, another recombinant virus (rVHSV-wild) was also generated, which had an identical nucleotide sequence to the wild-type VHSV genome except for a few artificially replaced nucleotides. The rVHSVs were rescued using a system controlled by T7 RNA polymerase supplied by a retroviral vector. Generation of rVHSV-deltaNV-EGFP and rVHSV-wild was confirmed by sequencing of RT-PCR products, and rescue of infectious rVHSVs was confirmed by observation of plaque formation. Replication efficiency of rVHSV-wild was distinctly lower than that of wild-type VHSV, suggesting that the artificially replaced nucleotides, especially when immediately preceding the G or NV gene start codons, might affect the replication of the virus. Replication of rVHSV-deltaNV-EGFP was slightly lower than that of rVHSV-wild when epithelioma papulosum cyprini cells were infected with multiplicity of infection (MOI) 1.0, but much lower when cells were infected with MOI 0.00001. These results suggest that the NV gene plays an important role in VHSV replication through interactions with host-cell responses, and the lower replication ability of rVHSV-wild compared to wild-type VHSV might be caused by replaced nucleotides just before the NV gene open reading frame (ORF) rather than the G gene ORF. In olive flounder Paralichthys olivaceus, rVHSV-wild produced slower-progressing mortalities than wild-type VHSV, whereas rVHSV-deltaNV-EGFP pathogenesis was highly attenuated. These results suggest that the NV protein of VHSV may play an important role not only in viral replication but also in viral pathogenesis.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. The structure of HIV-1 genomic RNA in the gp120 gene determines a recombination hot spot in vivo.

    Science.gov (United States)

    Galetto, Román; Moumen, Abdeladim; Giacomoni, Véronique; Véron, Michel; Charneau, Pierre; Negroni, Matteo

    2004-08-27

    By frequently rearranging large regions of the genome, genetic recombination is a major determinant in the plasticity of the human immunodeficiency virus type I (HIV-1) population. In retroviruses, recombination mostly occurs by template switching during reverse transcription. The generation of retroviral vectors provides a means to study this process after a single cycle of infection of cells in culture. Using HIV-1-derived vectors, we present here the first characterization and estimate of the strength of a recombination hot spot in HIV-1 in vivo. In the hot spot region, located within the C2 portion of the gp120 envelope gene, the rate of recombination is up to ten times higher than in the surrounding regions. The hot region corresponds to a previously identified RNA hairpin structure. Although recombination breakpoints in vivo cluster in the top portion of the hairpin, the bias for template switching in this same region appears less marked in a cell-free system. By modulating the stability of this hairpin we were able to affect the local recombination rate both in vitro and in infected cells, indicating that the local folding of the genomic RNA is a major parameter in the recombination process. This characterization of reverse transcription products generated after a single cycle of infection provides insights in the understanding of the mechanism of recombination in vivo and suggests that specific regions of the genome might be prompted to yield different rates of evolution due to the presence of circumscribed recombination hot spots.

  18. Expression of Hepatitis B Virus Surface Antigen Gene in Cultured Cells by Using Recombinant Plasmid Vectors

    OpenAIRE

    Siddiqui, Aleem

    1983-01-01

    By using a new host-vector system, expression of the gene coding for hepatitis B surface antigen has been studied. A subgenomic fragment of cloned hepatitis B viral DNA was inserted into the plasmid vector pSV010. Transfection of COS cells with the recombinant plasmid vector containing hepatitis sequences leads to the synthesis of hepatitis B surface antigen, which is released in the culture medium in the form of 22-nm particles similar to those found in the sera of hepatitis carriers.

  19. Recombination events near the immunoglobulin Cmu gene join variable and constant region genes, switch heavy-chain expression, or inactivate the locus.

    Science.gov (United States)

    Cory, S; Webb, E; Gough, J; Adams, J M

    1981-04-28

    Immunoglobulin heavy-chain expression is initiated by recombination between a variable region (VH) gene and one of several joining region (JH) genes located near the mu constant region (Cmu) gene, and the active VH gene can subsequently switch to another CH gene. That the general mechanism for CH switching involves recombination between sites within the JH-Cmu intervening sequence and the 5' flanking region of another CH gene is supported here by Southern blot hybridization analysis of eight IgG- and IgA-secreting plasmacytomas. An alternative model requiring successive VH linkage to similar JH clusters near each CH gene is shown to be very unlikely since the mouse genome appears to contain only one complement of the JH locus and no JH gene was detectable within large cloned sequences flanking germline C gamma 3 and C gamma 1 genes. Thus, VH-JH joining and CH switching are mediated by separate regions of "the joining-switch" or J-S element. In each plasmacytoma examined, the J-S element had undergone recombination within both the JH locus and the switch region and was shown to be linked to the functional CH gene in an IgG3, and IgG1, and three IgA secretors. Both JH joining and CH switching occurred by deletion of DNA. Switch recombination occurred at more than one site within the J-S element in different lines, even for recombination with the same CH gene. Significantly, although heavy-chain expression is restricted to one allele ("allelic exclusion"), all rearranged in each plasmacytoma. Some rearrangements were aberrant, involving, for example, deletion of all JH genes from the allele. Hence, an error-prone recombination machinery may account for allelic exclusion in many plasmacytomas.

  20. Preliminary investigation of naringenin hydroxylation with recombinant E. coli expressing plant flavonoid hydroxylation gene.

    Science.gov (United States)

    Amor, Ilef Limem-Ben; Salem, Nidhal; Guedon, Emmanuel; Engasser, Jean-Marc; Chekir-Ghedrira, Leila; Ghoul, Mohamed

    2010-05-01

    Flavonoid hydroxylation is one way to increase the biological activities of these molecules and the number of hydroxyl groups needed for polymerization, esterification, alkylation, glycosylation and acylation reactions. These reactions have been suggested as a promising route to enhance flavonoid solubility and stability. In our preliminary study we hydroxylated naringenin (the first flavonoid core synthesized in plants) with recombinant E. coli harboring flavanone 3 hydroxylase (F3H). We demonstrated that recombinant E. coli harboring the F3H from Petroselinum crispum, can convert naringenin to dihydrokaempferol. The whole cell hydroxylase activity was often influenced by the stability of the plasmid harboring the cloned gene and the biomass yield. When the composition of the growth media became richer the amount of formed product decreased about twofold; the naringenin bioconversion yield in LB media was 70% and decreased to 33% in TB. However, the enrichment of culture media increased the biomass yield nearly threefold in LB media, only 0.5 g/L of bacteria was formed, but in TB there was 1.6 g/L. Thus, LB constitutes the best medium for naringenin bioconversion using the recombinant E. coli harboring the F3H; this allows for maximum bioconversion yield and plasmid stability when compared with the fourth tested culture medium. Consequently, E. coli harboring F3H from Petroselinum crispum can be used to produce flavonoids hydroxylated in position 3 that can serve in additional reactions like polymerization, glycosylation, and acylation,

  1. Immunogenicity of DNA and Recombinant Sendai Virus Vaccines Expressing the HIV-1 gag Gene

    Institute of Scientific and Technical Information of China (English)

    Xia FENG; Shuang-qing YU; Tsugumine Shu; Tetsuro Matano; Mamoru Hasegawa; Xiao-li WANG; Hong-tao MA; Hong-xia LI; Yi ZENG

    2008-01-01

    Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendal virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1(+)-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens.

  2. Human Jk recombination signal binding protein gene (IGKJRB): Comparison with its mouse homologue

    Energy Technology Data Exchange (ETDEWEB)

    Amakawa, Ryuichi; Jing, Wu; Matsunami, Norisada; Hamaguchi, Yasushi; Matsuda, Fumihiko; Kawaichi, Masashi; Honjo, Tasuku (Kyoto Univ., Sakyo-ku, Kyoto (Japan)); Ozawa, Kazuo (Tsukuba Life Science Center, Tsukuba, Ibraraki (Japan))

    1993-08-01

    The mouse Igkjrb protein specifically binds to the immunoglobulin Jk recombination signal sequence. The IGKJRB gene is highly conserved among many species such as human, Xenopus, and Drosophila. Using cDNA fragments of the mouse Igkjrb gene, the authors isolated its human counterpart, IGKJRB. The human genome contains one functional IGKJRB gene and two types of processed pseudogenes. In situ chromosome hybridization analysis demonstrated that the functional gene is localized at chromosome 3q25, and the pseudogenes (IGKJRBP1 and IGKJRBP2, respectively) are located at chromosomes 9p13 and 9q13. The functional gene is composed of 13 exons spanning at least 67 kb. Three types of cDNA with different 5[prime] sequences were isolated by rapid amplification of cDNA ends, suggesting the presence of three proteins. The aPCR-1 protein, which possessed the exon 1 sequence, was the counterpart of the mouse RBP-2 type protein. The aPCR-2 and 3 proteins may be specific to human cells because the mouse counterparts were not detected. The amino acid sequences of the human and mouse IGKJRB genes were 98% homologous in exons 2-11, whereas the homology of the human and mouse exon 1 sequences was 75%. 40 refs., 7 figs.

  3. Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi

    Directory of Open Access Journals (Sweden)

    In Sun Hwang

    2016-06-01

    Full Text Available Gene disruption by homologous recombination is widely used to investigate and analyze the function of genes in Fusarium fujikuroi, a fungus that causes bakanae disease and root rot symptoms in rice. To generate gene deletion constructs, the use of conventional cloning methods, which rely on restriction enzymes and ligases, has had limited success due to a lack of unique restriction enzyme sites. Although strategies that avoid the use of restriction enzymes have been employed to overcome this issue, these methods require complicated PCR steps or are frequently inefficient. Here, we introduce a cloning system that utilizes multi-fragment assembly by In-Fusion to generate a gene disruption construct. This method utilizes DNA fragment fusion and requires only one PCR step and one reaction for construction. Using this strategy, a gene disruption construct for Fusarium cyclin C1 (FCC1 , which is associated with fumonisin B1 biosynthesis, was successfully created and used for fungal transformation. In vivo and in vitro experiments using confirmed fcc1 mutants suggest that fumonisin production is closely related to disease symptoms exhibited by F. fujikuroi strain B14. Taken together, this multi-fragment assembly method represents a simpler and a more convenient process for targeted gene disruption in fungi.

  4. Integrase-mediated recombination of the veb1 gene cassette encoding an extended-spectrum β-lactamase.

    Directory of Open Access Journals (Sweden)

    Daniel Aubert

    Full Text Available The veb1 gene cassette encodes the extended spectrum β-lactamase, VEB-1 that is increasingly isolated from worldwide Gram-negative rods. Veb1 is commonly inserted into the variable region of different class 1 integrons in which it is always associated with a downstream-located aadB gene cassette encoding an aminoglycoside adenylyltransferase. In Pseudomonas aeruginosa, the majority of veb1-containing integrons also carry an insertion sequence, IS1999 that is inserted upstream of the veb1 gene cassette and disrupts the integron specific recombination site, attI1. Investigation of the recombination properties of the sites surrounding veb1 revealed that insertion of IS1999 reduces significantly the recombination frequency of attI1 and that veb1 attC is not efficient for recombination in contrast to aadB attC. Subsequent sequence optimisation of veb1 attC by mutagenesis, into a more consensual attC site resembling aadB attC, successfully improved recombination efficiency. Overall, this work gives some insights into the organisation of veb1-containing integrons. We propose that IS1999 and the nature of veb1 attC stabilize the veb1 gene cassette environment likely by impairing recombination events upstream or downstream of veb1, respectively.

  5. Highly diverse TCRα chain repertoire of pre-immune CD8+ T cells reveals new insights in gene recombination

    Science.gov (United States)

    Genolet, Raphael; Stevenson, Brian J; Farinelli, Laurent; Østerås, Magne; Luescher, Immanuel F

    2012-01-01

    Although the T-cell receptor αδ (TCRαδ) locus harbours large libraries of variable (TRAV) and junctional (TRAJ) gene segments, according to previous studies the TCRα chain repertoire is of limited diversity due to restrictions imposed by sequential coordinate TRAV-TRAJ recombinations. By sequencing tens of millions of TCRα chain transcripts from naive mouse CD8+ T cells, we observed a hugely diverse repertoire, comprising nearly all possible TRAV-TRAJ combinations. Our findings are not compatible with sequential coordinate gene recombination, but rather with a model in which contraction and DNA looping in the TCRαδ locus provide equal access to TRAV and TRAJ gene segments, similarly to that demonstrated for IgH gene recombination. Generation of the observed highly diverse TCRα chain repertoire necessitates deletion of failed attempts by thymic-positive selection and is essential for the formation of highly diverse TCRαβ repertoires, capable of providing good protective immunity. PMID:22373576

  6. BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector.

    Science.gov (United States)

    Yamamoto, H; Ishimura, M; Ochiai, M; Takada, H; Kusuhara, K; Nakatsu, Y; Tsuzuki, T; Mitani, K; Hara, T

    2016-02-01

    X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells.

  7. [Construction of a recombined adenovirus vector carrying pri-miR-21 gene and research on it's target gene TLR4].

    Science.gov (United States)

    Zhao, Jing; Xu, Guang-xian; Jia, Wei; Dong, Hui; Zhang, Yi-lin; Zhao, Zhi-jun; Wei, Jun

    2012-02-01

    To construct the recombined adenovirus vector carrying pri-miR-21 gene, which can express mature miR-21 efficiently, and to study the interaction of miR- 21 with its target gene TLR4. Using healthy mouse's gDNA as template, the primary miR-21 coding sequence was amplified by PCR and cloned into a shuttle vector pAdTrack-CMV. Constructed plasmid was sequenced and linearized for homologous recombination with pAdEasy-1 vector in BJ5183 bacteria. The recombined adenovirus vector carrying pri-miR-21 gene was used to challenge HeLa cell. The candidate target gene of miR-21 was determined by miRNA analysis databases. The expression level of TLR4 protein was detected by western blotting. Through the PCR, restriction enzyme digestion, DNA sequencing and expression of GFP, recombinant adenoviral vector pri-miR-21 gene was constructed successfully. Bioinformatic analysis suggested a few possible binding sites between miR-21 and TLR4. Results showed that miR-21 down-regulated TLR4 at protein levels. The recombinant adenoviral vector containing pri- miR-21 was successfully constructed. miR-21 gene interfered with the expression of TLR4 target gene.

  8. Gene duplication and fragment recombination drive functional diversification of a superfamily of cytoplasmic effectors in Phytophthora sojae.

    Science.gov (United States)

    Shen, Danyu; Liu, Tingli; Ye, Wenwu; Liu, Li; Liu, Peihan; Wu, Yuren; Wang, Yuanchao; Dou, Daolong

    2013-01-01

    Phytophthora and other oomycetes secrete a large number of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling and necrosis inducing proteins (CRN), or Crinkler. Here, we first investigated the evolutionary patterns and mechanisms of CRN effectors in Phytophthora sojae and compared them to two other Phytophthora species. The genes encoding CRN effectors could be divided into 45 orthologous gene groups (OGG), and most OGGs unequally distributed in the three species, in which each underwent large number of gene gains or losses, indicating that the CRN genes expanded after species evolution in Phytophthora and evolved through pathoadaptation. The 134 expanded genes in P. sojae encoded family proteins including 82 functional genes and expressed at higher levels while the other 68 genes encoding orphan proteins were less expressed and contained 50 pseudogenes. Furthermore, we demonstrated that most expanded genes underwent gene duplication or/and fragment recombination. Three different mechanisms that drove gene duplication or recombination were identified. Finally, the expanded CRN effectors exhibited varying pathogenic functions, including induction of programmed cell death (PCD) and suppression of PCD through PAMP-triggered immunity or/and effector-triggered immunity. Overall, these results suggest that gene duplication and fragment recombination may be two mechanisms that drive the expansion and neofunctionalization of the CRN family in P. sojae, which aids in understanding the roles of CRN effectors within each oomycete pathogen.

  9. Gene duplication and fragment recombination drive functional diversification of a superfamily of cytoplasmic effectors in Phytophthora sojae.

    Directory of Open Access Journals (Sweden)

    Danyu Shen

    Full Text Available Phytophthora and other oomycetes secrete a large number of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling and necrosis inducing proteins (CRN, or Crinkler. Here, we first investigated the evolutionary patterns and mechanisms of CRN effectors in Phytophthora sojae and compared them to two other Phytophthora species. The genes encoding CRN effectors could be divided into 45 orthologous gene groups (OGG, and most OGGs unequally distributed in the three species, in which each underwent large number of gene gains or losses, indicating that the CRN genes expanded after species evolution in Phytophthora and evolved through pathoadaptation. The 134 expanded genes in P. sojae encoded family proteins including 82 functional genes and expressed at higher levels while the other 68 genes encoding orphan proteins were less expressed and contained 50 pseudogenes. Furthermore, we demonstrated that most expanded genes underwent gene duplication or/and fragment recombination. Three different mechanisms that drove gene duplication or recombination were identified. Finally, the expanded CRN effectors exhibited varying pathogenic functions, including induction of programmed cell death (PCD and suppression of PCD through PAMP-triggered immunity or/and effector-triggered immunity. Overall, these results suggest that gene duplication and fragment recombination may be two mechanisms that drive the expansion and neofunctionalization of the CRN family in P. sojae, which aids in understanding the roles of CRN effectors within each oomycete pathogen.

  10. Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene

    Institute of Scientific and Technical Information of China (English)

    Shun-lin HU; Qin SUN; Qu-zhi WANG; Yul-iang LIU; Yan-tao WU; Xiu-fan LIU

    2007-01-01

    Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain,seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI,which contained the full-length cDNA of the NDV ZJI strain.The pNDV/ZJI,with three helper plasmids,pCIneoNP,pCIneoP and pCIneoL,were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase.After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock,an infectious NDV ZJI strain was successfully rescued.Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP.After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells,the recombinant NDV,NDV/ZJIGFP,was rescued.Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF) cells 48h post-infection,indicating that the GFP gene was expressed at a relatively high level.NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route.Four days post-infection,strong green fluorescence could be detected in the kidneys and tracheae,indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.

  11. Characterization of an endogenous gene expressed in Aedes aegypti using an orally infectious recombinant sindbis virus

    Directory of Open Access Journals (Sweden)

    L.L. Cheng

    2001-10-01

    Full Text Available Sindbis virus expression vectors have been used successfully to express and silence genes of interest in vivo in several mosquito species, including Aedes aegypti, Ae. albopictus, Ae. triseriatus, Culex pipiens, Armigeres subalbatus and Anopheles gambiae. Here we describe the expression of an endogenous gene, defensin, in Ae. aegypti using the orally infectious Sindbis virus, MRE/3'2J expression vector. We optimized conditions to infect mosquito larvae per os using C6/36 Ae. albopictus cells infected with the recombinant virus to maximize virus infection and expression of defensin. Infection with the parental Sindbis virus (MRE/3'2J did not induce defensin expression. Mosquito larvae infected by ingestion of recombinant Sindbis virus-infected C6/36 cells expressed defensin when they emerged as adults. Defensin expression was observed by western analysis or indirect fluorescent assay in all developmental stages of mosquitoes infected with MRE/3'2J virus that contained the defensin insert. The multiplicity of infection of C6/36 cells and the quantity of infected cells consumed by larvae played an important role in defensin expression. Parental viruses, missing the defensin insert, and/or other defective interfering virus may have contributed to these observations.

  12. The joint effects of background selection and genetic recombination on local gene genealogies.

    Science.gov (United States)

    Zeng, Kai; Charlesworth, Brian

    2011-09-01

    Background selection, the effects of the continual removal of deleterious mutations by natural selection on variability at linked sites, is potentially a major determinant of DNA sequence variability. However, the joint effects of background selection and genetic recombination on the shape of the neutral gene genealogy have proved hard to study analytically. The only existing formula concerns the mean coalescent time for a pair of alleles, making it difficult to assess the importance of background selection from genome-wide data on sequence polymorphism. Here we develop a structured coalescent model of background selection with recombination and implement it in a computer program that efficiently generates neutral gene genealogies for an arbitrary sample size. We check the validity of the structured coalescent model against forward-in-time simulations and show that it accurately captures the effects of background selection. The model produces more accurate predictions of the mean coalescent time than the existing formula and supports the conclusion that the effect of background selection is greater in the interior of a deleterious region than at its boundaries. The level of linkage disequilibrium between sites is elevated by background selection, to an extent that is well summarized by a change in effective population size. The structured coalescent model is readily extendable to more realistic situations and should prove useful for analyzing genome-wide polymorphism data.

  13. Regulated O2 activation in flavin-dependent monooxygenases.

    Science.gov (United States)

    Frederick, Rosanne E; Mayfield, Jeffery A; DuBois, Jennifer L

    2011-08-17

    Flavin-dependent monooxygenases (FMOs) are involved in important biosynthetic pathways in diverse organisms, including production of the siderophores used for the import and storage of essential iron in serious pathogens. We have shown that the FMO from Aspergillus fumigatus, an ornithine monooxygenase (Af-OMO), is mechanistically similar to its well-studied distant homologues from mammalian liver. The latter are highly promiscuous in their choice of substrates, while Af-OMO is unusually specific. This presents a puzzle: how do Af-OMO and other FMOs of the biosynthetic classes achieve such specificity? We have discovered substantial enhancement in the rate of O(2) activation in Af-OMO in the presence of L-arginine, which acts as a small molecule regulator. Such protein-level regulation could help explain how this and related biosynthetic FMOs manage to couple O(2) activation and substrate hydroxylation to each other and to the appropriate cellular conditions. Given the essentiality of Fe to Af and the avirulence of the Af-OMO gene knock out, inhibitors of Af-OMO are likely to be drug targets against this medically intractable pathogen.

  14. Recombination in pe/ppe genes contributes to genetic variation in Mycobacterium tuberculosis lineages

    KAUST Repository

    Phelan, Jody E.

    2016-02-29

    Background Approximately 10 % of the Mycobacterium tuberculosis genome is made up of two families of genes that are poorly characterized due to their high GC content and highly repetitive nature. The PE and PPE families are typified by their highly conserved N-terminal domains that incorporate proline-glutamate (PE) and proline-proline-glutamate (PPE) signature motifs. They are hypothesised to be important virulence factors involved with host-pathogen interactions, but their high genetic variability and complexity of analysis means they are typically disregarded in genome studies. Results To elucidate the structure of these genes, 518 genomes from a diverse international collection of clinical isolates were de novo assembled. A further 21 reference M. tuberculosis complex genomes and long read sequence data were used to validate the approach. SNP analysis revealed that variation in the majority of the 168 pe/ppe genes studied was consistent with lineage. Several recombination hotspots were identified, notably pe_pgrs3 and pe_pgrs17. Evidence of positive selection was revealed in 65 pe/ppe genes, including epitopes potentially binding to major histocompatibility complex molecules. Conclusions This, the first comprehensive study of the pe and ppe genes, provides important insight into M. tuberculosis diversity and has significant implications for vaccine development.

  15. Production of transgenic mice by random recombination of targeted genes in female germline stem cells

    Institute of Scientific and Technical Information of China (English)

    Yong Zhang; Ji Xiong; Jie Xiang; Ji Wu; Zhaojuan Yang; Yunze Yang; Shuzeng Wang; Lingjun Shi; Wenhai Xie; Kejing Sun; Kang Zou; Lei Wang

    2011-01-01

    Oocyte production in most mammalian species is believed to cease before birth. However, this idea has been challenged with the finding that postnatal mouse ovaries possess mitotically active germ cells. A recent study showed that female germline stem cells (FGSCs) from adult mice were isolated, cultured long term and produced oocytes and progeny after transplantation into infertile mice. Here, we demonstrate the successful generation of transgenic or gene knock-down mice using FGSCs. The FGSCs from ovaries of 5-day-old and adult mice were isolated and either infected with recombinant viruses carrying green fluorescent protein, Oocyte-G1 or the mouse dynein axonemal intermediate chain 2 gene, or transfected with the Oocyte-G1 specific shRNA expression vector (pRS shOocyte-G1 vector), and then transplanted into infertile mice. Transplanted cells in the ovaries underwent oogenesis and produced heterozygous offspring after mating with wild-type male mice. The offspring were genetically characterized and the biological functions of the transferred or knock-down genes were investigated. Efficiency of genetransfer or gene knock-down was 29%-37% and it took 2 months to produce transgenic offspring. Gene manipulation of FGSCs is a rapid and efficient method of animal transgenesis and may serve as a powerful tool for biomedical science and biotechnology.

  16. Bioresorbable microporous stents deliver recombinant adenovirus gene transfer vectors to the arterial wall.

    Science.gov (United States)

    Ye, Y W; Landau, C; Willard, J E; Rajasubramanian, G; Moskowitz, A; Aziz, S; Meidell, R S; Eberhart, R C

    1998-01-01

    The use of intravascular stents as an adjunct for percutaneous transluminal revascularization is limited by two principal factors, acute thrombosis and neointimal proliferation, resulting in restenosis. To overcome these limitations, we have investigated the potential of microporous bioresorbable polymer stents formed from poly(L-lactic acid) (PLLA)/poly(epsilon-caprolactone) (PCL) blends to function both to provide mechanical support and as reservoirs for local delivery of therapeutic molecules and particles to the vessel wall. Tubular PLLA/PCL stents were fabricated by the flotation-precipitation method, and helical stents were produced by a casting/winding technique. Hybrid structures in which a tubular sheath is deposited on a helical skeleton were also generated. Using a two-stage solvent swelling technique, polyethylene oxide has been incorporated into these stents to improve hydrophilicity and water uptake, and to facilitate the ability of these devices to function as drug carriers. Stents modified in this manner retain axial and radial mechanical strength sufficient to stabilize the vessel wall against elastic recoil caused by vasoconstrictive and mechanical forces. Because of the potential of direct gene transfer into the vessel wall to ameliorate thrombosis and neointimal proliferation, we have investigated the capacity of these polymer stents to function in the delivery of recombinant adenovirus vectors to the vessel wall. In vitro, virus stock was observed to readily absorb into, and elute from these devices in an infectious form, with suitable kinetics. Successful gene transfer and expression has been demonstrated following implantation of polymer stents impregnated with a recombinant adenovirus carrying a nuclear-localizing betaGal reporter gene into rabbit carotid arteries. These studies suggest that surface-modified polymer stents may ultimately be useful adjunctive devices for both mechanical support and gene transfer during percutaneous

  17. Recombinant Streptomyces clavuligerus strain including cas2 gene production and analysis its antibiotic overproduction by bioassay

    Directory of Open Access Journals (Sweden)

    Zohreh Hojati

    2014-03-01

    Full Text Available Background: Streptomyces clavuligerus is one of the most important strain that produce clavulanic acid that wildly used in combination of strong but sensitive to β-lactamase antibiotics in clinics. The cas2 is one of the important genes in the biosynthesis pathway of clavulanic acid. Materials and Methods: The recombinant construct pMTcas2 which contain cas2 gene is obtained from Isfahan University. Recombinant plasmid extracts from streptomyces lividans and confirm by enzyme digestion. The streptomyces clavuligerus protoplast was prepared and transformation was done by using polyethylene glycol. Transformation was confirmed by plasmid extraction and PCR using cas2 specific primers. Finally, bioassay method was used to survey the effect of extra copy of cas2 on clavulanic acid production. Result: Plasmid extraction was initially carried out and the structure of plasmid was confirmed by digestion. The typical white colony was seen on protoplast recovery culture containing thiostrepton antibiotic and gray spores were detected after one week. Plasmid extraction was done from transformed strain and transformation was confirmed by PCR. The results of the bioassay show that amplification of the cas2 gene in multicopy plasmids resulted in a 4.1 fold increase in clavulanic acid production. Conclusion: The bioassay was done and the diameters of zone of inhibition in control and sample were compared. The results of the bioassay show that amplification of the cas2 gene in multicopy plasmids resulted in a 4.1 fold increase in clavulanic acid production. Overproduction of clavulanic acid decreases the cost of its dependent drug production.

  18. Requirement of monooxygenase-mediated steps for sterigmatocystin biosynthesis by Aspergillus nidulans.

    Science.gov (United States)

    Keller, N P; Watanabe, C M; Kelkar, H S; Adams, T H; Townsend, C A

    2000-01-01

    Sterigmatocystin (ST) and aflatoxin B(1) (AFB(1)) are two polyketide-derived Aspergillus mycotoxins synthesized by functionally identical sets of enzymes. ST, the compound produced by Aspergillus nidulans, is a late intermediate in the AFB(1) pathway of A. parasiticus and A. flavus. Previous biochemical studies predicted that five oxygenase steps are required for the formation of ST. A 60-kb ST gene cluster in A. nidulans contains five genes, stcB, stcF, stcL, stcS, and stcW, encoding putative monooxygenase activities. Prior research showed that stcL and stcS mutants accumulated versicolorins B and A, respectively. We now show that strains disrupted at stcF, encoding a P-450 monooxygenase similar to A. parasiticus avnA, accumulate averantin. Disruption of either StcB (a putative P-450 monooxygenase) or StcW (a putative flavin-requiring monooxygenase) led to the accumulation of averufin as determined by radiolabeled feeding and extraction studies.

  19. Temporal Control of Cre Recombinase-mediated in Vitro DNA Recombination by Tet-on Gene Expression System

    Institute of Scientific and Technical Information of China (English)

    Zhong-Min GUO; Kang XU; Ying YUE; Bing HUANG; Xin-Yan DENG; Nü-Qi ZHONG; Xun HONG; Xi-Gu CHEN; Dong XIAO

    2005-01-01

    Conditional gene expression and gene deletion are important experimental approaches for examining the functions of particular gene products in mouse models. These strategies exploiting Cre-mediated site-specific DNA recombination have been incorporated into transgenic and gene-targeting procedures to allow in vivo manipulation of DNA in embryonic stem cells (ES cells) or living animals. The Cre/lox P system has become widely used in conditional gene targeting, conditional gene repair and activation, inducible chromosome translocation, and chromosome engineering. In this project, we have employed the universal transgenic system and the liver-specific promoter system for tightly temporal and liver-specific control of Cre gene expression in mice that (1) integrates the advantages of the Tet-on gene expression system and Cre/lox P site-mediated gene activation, and (2) simplifies the scheme of animal crosses through a combination of two control elements in a single transgene. A liver-specific apoE promoter was inserted into the promoter cloning site upstream of the rtTA cassette of pCore construct to generate the transgene construct pApoErtTAtetO-Cre, followed by demonstrating stringent regulation of doxycycline (Dox)-induced Cre-mediated recombination in the lox P-flanked transcription STOP cassette-modified BEL-7402 cells. That is to say, in the absence of Dox, the Cre gene is not expressed and will not induce site-specific recombination between two lox P sites, whereas on exposure to Dox, the Cre gene will be expressed and the recombination will occur.Together, these data indicate that the Tet-on gene expression system is able to successfully and stringently control Cre expression in vitro, which lays a solid foundation for efficient and spatio-temporal Cre gene activation in transgenic mice.

  20. The effects of recombination, mutation and selection on the evolution of the Rp1 resistance genes in grasses.

    Science.gov (United States)

    Jouet, Agathe; McMullan, Mark; van Oosterhout, Cock

    2015-06-01

    Plant immune genes, or resistance genes, are involved in a co-evolutionary arms race with a diverse range of pathogens. In agronomically important grasses, such R genes have been extensively studied because of their role in pathogen resistance and in the breeding of resistant cultivars. In this study, we evaluate the importance of recombination, mutation and selection on the evolution of the R gene complex Rp1 of Sorghum, Triticum, Brachypodium, Oryza and Zea. Analyses show that recombination is widespread, and we detected 73 independent instances of sequence exchange, involving on average 1567 of 4692 nucleotides analysed (33.4%). We were able to date 24 interspecific recombination events and found that four occurred postspeciation, which suggests that genetic introgression took place between different grass species. Other interspecific events seemed to have been maintained over long evolutionary time, suggesting the presence of balancing selection. Significant positive selection (i.e. a relative excess of nonsynonymous substitutions (dN /dS >1)) was detected in 17-95 codons (0.42-2.02%). Recombination was significantly associated with areas with high levels of polymorphism but not with an elevated dN /dS ratio. Finally, phylogenetic analyses show that recombination results in a general overestimation of the divergence time (mean = 14.3%) and an alteration of the gene tree topology if the tree is not calibrated. Given that the statistical power to detect recombination is determined by the level of polymorphism of the amplicon as well as the number of sequences analysed, it is likely that many studies have underestimated the importance of recombination relative to the mutation rate.

  1. Initiation of V(DJ recombination by Dbeta-associated recombination signal sequences: a critical control point in TCRbeta gene assembly.

    Directory of Open Access Journals (Sweden)

    Don-Marc Franchini

    Full Text Available T cell receptor (TCR beta gene assembly by V(DJ recombination proceeds via successive Dbeta-to-Jbeta and Vbeta-to-DJbeta rearrangements. This two-step process is enforced by a constraint, termed beyond (B12/23, which prohibits direct Vbeta-to-Jbeta rearrangements. However the B12/23 restriction does not explain the order of TCRbeta assembly for which the regulation remains an unresolved issue. The initiation of V(DJ recombination consists of the introduction of single-strand DNA nicks at recombination signal sequences (RSSs containing a 12 base-pairs spacer. An RSS containing a 23 base-pairs spacer is then captured to form a 12/23 RSSs synapse leading to coupled DNA cleavage. Herein, we probed RSS nicks at the TCRbeta locus and found that nicks were only detectable at Dbeta-associated RSSs. This pattern implies that Dbeta 12RSS and, unexpectedly, Dbeta 23RSS initiate V(DJ recombination and capture their respective Vbeta or Jbeta RSS partner. Using both in vitro and in vivo assays, we further demonstrate that the Dbeta1 23RSS impedes cleavage at the adjacent Dbeta1 12RSS and consequently Vbeta-to-Dbeta1 rearrangement first requires the Dbeta1 23RSS excision. Altogether, our results provide the molecular explanation to the B12/23 constraint and also uncover a 'Dbeta1 23RSS-mediated' restriction operating beyond chromatin accessibility, which directs Dbeta1 ordered rearrangements.

  2. Initiation of V(D)J recombination by Dbeta-associated recombination signal sequences: a critical control point in TCRbeta gene assembly.

    Science.gov (United States)

    Franchini, Don-Marc; Benoukraf, Touati; Jaeger, Sébastien; Ferrier, Pierre; Payet-Bornet, Dominique

    2009-01-01

    T cell receptor (TCR) beta gene assembly by V(D)J recombination proceeds via successive Dbeta-to-Jbeta and Vbeta-to-DJbeta rearrangements. This two-step process is enforced by a constraint, termed beyond (B)12/23, which prohibits direct Vbeta-to-Jbeta rearrangements. However the B12/23 restriction does not explain the order of TCRbeta assembly for which the regulation remains an unresolved issue. The initiation of V(D)J recombination consists of the introduction of single-strand DNA nicks at recombination signal sequences (RSSs) containing a 12 base-pairs spacer. An RSS containing a 23 base-pairs spacer is then captured to form a 12/23 RSSs synapse leading to coupled DNA cleavage. Herein, we probed RSS nicks at the TCRbeta locus and found that nicks were only detectable at Dbeta-associated RSSs. This pattern implies that Dbeta 12RSS and, unexpectedly, Dbeta 23RSS initiate V(D)J recombination and capture their respective Vbeta or Jbeta RSS partner. Using both in vitro and in vivo assays, we further demonstrate that the Dbeta1 23RSS impedes cleavage at the adjacent Dbeta1 12RSS and consequently Vbeta-to-Dbeta1 rearrangement first requires the Dbeta1 23RSS excision. Altogether, our results provide the molecular explanation to the B12/23 constraint and also uncover a 'Dbeta1 23RSS-mediated' restriction operating beyond chromatin accessibility, which directs Dbeta1 ordered rearrangements.

  3. Molecular Analysis of the pmo (Particulate Methane Monooxygenase) Operons from Two Type II Methanotrophs

    OpenAIRE

    Gilbert, Bettina; McDonald, Ian R.; Finch, Ruth; Stafford, Graham P.; Nielsen, Allan K.; Murrell, J. Colin

    2000-01-01

    The particulate methane monooxygenase gene clusters, pmoCAB, from two representative type II methanotrophs of the α-Proteobacteria, Methylosinus trichosporium OB3b and Methylocystis sp. strain M, have been cloned and sequenced. Primer extension experiments revealed that the pmo cluster is probably transcribed from a single transcriptional start site located 300 bp upstream of the start of the first gene, pmoC, for Methylocystis sp. strain M. Immediately upstream of the putative start site, co...

  4. Restricted expression of recombination activating gene (RAG-1) in mouse lymphoid tissues

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, Akihito; Fujinaga, Hiroyuki; Hamatani, Kiyohiro [Radiation Effects Research Foundation, Nagasaki (Japan). Nagasaki Branch; Atsuta, Mitsuru

    1993-03-01

    In an attempt to determine the distribution of recombinase activity in the mouse thymus, spleen, and lymph nodes, we used the in situ hybridization method to examine the expression of the recombination activating genes RAG-1 and RAG-2. Expression of RAG-1 was found in most cortical thymocytes but not in the majority of medullary thymocytes. Although hybridization signals of RAG-2 were not as intense as those of RAG-1, the localization of RAG-2 transcripts was similar to that of RAG-1. In the spleen, expression of RAG-1 was found only in limited cells near the splenic sinus, and the majority of the cells within the follicle were negative for RAG-1 transcript. In nude mice, RAG-1-expressing cells were detected in the same regions, which suggests that in situ hybridization signals of RAG-1 in the spleen are due to the cells of B-cell origin. In the lymph nodes, expression of RAG-1 was found only in the medullary region. Expression of RAG-2 transcript in the spleen and the lymph nodes, if any, was too faint to allow determination of the specific localization. These results suggest that most of the cortical thymocytes and some cells in the spleen are capable of rearranging T-cell receptor genes and immunoglobulin genes, respectively, but the possible involvement of the RAG-1 transcript in RAG-1-positive cells of the spleen and the lymph nodes in functions other than the rearrangement of genes could not be ruled out. (author).

  5. Over-expression of brain-derived neurotrophic factor in mesenchymal stem cells transfected with recombinant lentivirus BDNF gene.

    Science.gov (United States)

    Zhang, X; Zhu, J; Zhang, K; Liu, T; Zhang, Z

    2016-12-30

    This study was aimed at investigating the expression of brain-derived neurotrophic factor (BDNF) in mesenchymal stem cells (MSCs) modified with recombinant lentivirus bearing BDNF gene. Lentivirus vectors bearing BDNF gene were constructed. MSCs were isolated from rats and cultured. The lentiviral vectors containing BDNF gene were transfected into the MSCs, and BDNF gene and protein expressions were monitored with enhanced green fluorescent protein (EGFP). RT-PCR and Western blot were used to measure gene and protein expressions, respectibvely in MSCs, MSCs-EGFP and MSCs-EGFP-BDNF groups. Green fluorescence assay confirmed successful transfection of BDNF gene recombinant lentivirus into MSCs. RT-PCR and Western blot revealed that BDNF gene and protein expressions in the MSCs-EGFP-BDNF group were significantly higher than that in MSCs group and MSCs-EGFP group. There were no statistically significant differences in gene expression between MSCs and MSCs-EGFP groups. MSCs can over-express BDNF when transfected with recombinant lentivirus bearing BDNF gene.

  6. 1,3-Propanediol production by new recombinant Escherichia coli containing genes from pathogenic bacteria.

    Science.gov (United States)

    Przystałowska, Hanna; Zeyland, Joanna; Szymanowska-Powałowska, Daria; Szalata, Marlena; Słomski, Ryszard; Lipiński, Daniel

    2015-02-01

    1,3-Propanediol (1,3-PDO) is an organic compound, which is a valuable intermediate product, widely used as a monomer for synthesizing biodegradable polymers, increasing their strength; as well as an ingredient of textile, cosmetic and medical products. 1,3-PDO is mostly synthesized chemically. Global companies have developed technologies for 1,3-PDO synthesis from petroleum products such as acrolein and ethylene oxide. A potentially viable alternative is offered by biotechnological processes using microorganisms capable of synthesizing 1,3-PDO from renewable substrates (waste glycerol, a by-product of biofuel production, or glucose). In the present study, genes from Citrobacter freundii and Klebsiella pneumoniae were introduced into Escherichia coli bacteria to enable the synthesis of 1,3-PDO from waste glycerol. These strains belong to the best 1,3-PDO producers, but they are pathogenic, which restricts their application in industrial processes. The present study involved the construction of two gene expression constructs, containing a total of six heterologous glycerol catabolism pathway genes from C. freundii ATCC 8090 and K. pneumoniae ATCC 700721. Heterologous genes encoding glycerol dehydratase (dhaBCE) and the glycerol dehydratase reactivation factor (dhaF, dhaG) from C. freundii and gene encoding 1,3-PDO oxidoreductase (dhaT) from K. pneumoniae were expressed in E. coli under the control of the T7lac promoter. An RT-PCR analysis and overexpression confirmed that 1,3-PDO synthesis pathway genes were expressed on the RNA and protein levels. In batch fermentation, recombinant E. coli bacteria used 32.6gl(-1) of glycerol to produce 10.6 gl(-1) of 1,3-PDO, attaining the efficiency of 0.4 (mol₁,₃-PDO molglycerol(-1)). The recombinant E. coli created is capable of metabolizing glycerol to produce 1,3-PDO, and the efficiency achieved provides a significant research potential of the bacterium. In the face of shortage of fossil fuel supplies and climate warming

  7. Arabidopsis RAD51C gene is important for homologous recombination in meiosis and mitosis.

    Science.gov (United States)

    Abe, Kiyomi; Osakabe, Keishi; Nakayama, Shigeki; Endo, Masaki; Tagiri, Akemi; Todoriki, Setsuko; Ichikawa, Hiroaki; Toki, Seiichi

    2005-10-01

    Rad51 is a homolog of the bacterial RecA recombinase, and a key factor in homologous recombination in eukaryotes. Rad51 paralogs have been identified from yeast to vertebrates. Rad51 paralogs are thought to play an important role in the assembly or stabilization of Rad51 that promotes homologous pairing and strand exchange reactions. We previously characterized two RAD51 paralogous genes in Arabidopsis (Arabidopsis thaliana) named AtRAD51C and AtXRCC3, which are homologs of human RAD51C and XRCC3, respectively, and described the interaction of their products in a yeast two-hybrid system. Recent studies showed the involvement of AtXrcc3 in DNA repair and functional role in meiosis. To determine the role of RAD51C in meiotic and mitotic recombination in higher plants, we characterized a T-DNA insertion mutant of AtRAD51C. Although the atrad51C mutant grew normally during vegetative developmental stage, the mutant produced aborted siliques, and their anthers did not contain mature pollen grains. Crossing of the mutant with wild-type plants showed defective male and female gametogeneses as evidenced by lack of seed production. Furthermore, meiosis was severely disturbed in the mutant. The atrad51C mutant also showed increased sensitivity to gamma-irradiation and cisplatin, which are known to induce double-strand DNA breaks. The efficiency of homologous recombination in somatic cells in the mutant was markedly reduced relative to that in wild-type plants.

  8. Two DNA repair and recombination genes in Saccharomyces cerevisiae, RAD52 and RAD54, are induced during meiosis

    Energy Technology Data Exchange (ETDEWEB)

    Cole, G.M.; Mortimer, R.K. (Univ. of California, Berkeley (United States)); Schild, D. (Lawrence Berkeley Lab., CA (United States))

    1989-07-01

    The DNA repair and recombination genes of Saccharomyces cerevisiae, RAD52 and RAD54, were transcriptionally induced approximately 10- to 15-fold in sporulating MATa/{alpha} cells. Congenic MATa/a cells, which did not sporulate, did not show similar increases. Assays of {beta}-galactosidase activity in strains harboring either a RAD52- or RAD54-lacZ gene fusion indicated that this induction occurred at a time concomitant with a commitment to meiotic recombination, as measured by prototroph formation from his1 heteroalleles.

  9. Direct estimation of the recombination frequency between the RB1 gene and two closely linked microsatellites using sperm typing.

    Science.gov (United States)

    Girardet, A; Lien, S; Leeflang, E P; Beaufrère, L; Tuffery, S; Munier, F; Arnheim, N; Claustres, M; Pellestor, F

    1999-01-01

    In this study, single sperm typing has been used for high-resolution recombination analysis between the retinoblastoma gene and two closely linked extragenic microsatellites (D13S284 and D13S1307). The analysis of 1198 single sperm from three donors allowed the determination of recombination fractions between RB1.20 and D13S284 and RB1.20 and D13S1307 of 0.022 and 0.033, respectively. These results show that RB1 gene and the two microsatellites are closely linked, which validates their potential use in indirect genetic diagnosis of retinoblastoma.

  10. Alleles of the homologous recombination gene, RAD59, identify multiple responses to disrupted DNA replication in Saccharomyces cerevisiae.

    Science.gov (United States)

    Liddell, Lauren C; Manthey, Glenn M; Owens, Shannon N; Fu, Becky X H; Bailis, Adam M

    2013-10-14

    In Saccharomyces cerevisiae, Rad59 is required for multiple homologous recombination mechanisms and viability in DNA replication-defective rad27 mutant cells. Recently, four rad59 missense alleles were found to have distinct effects on homologous recombination that are consistent with separation-of-function mutations. The rad59-K166A allele alters an amino acid in a conserved α-helical domain, and, like the rad59 null allele diminishes association of Rad52 with double-strand breaks. The rad59-K174A and rad59-F180A alleles alter amino acids in the same domain and have genetically similar effects on homologous recombination. The rad59-Y92A allele alters a conserved amino acid in a separate domain, has genetically distinct effects on homologous recombination, and does not diminish association of Rad52 with double-strand breaks. In this study, rad59 mutant strains were crossed with a rad27 null mutant to examine the effects of the rad59 alleles on the link between viability, growth and the stimulation of homologous recombination in replication-defective cells. Like the rad59 null allele, rad59-K166A was synthetically lethal in combination with rad27. The rad59-K174A and rad59-F180A alleles were not synthetically lethal in combination with rad27, had effects on growth that coincided with decreased ectopic gene conversion, but did not affect mutation, unequal sister-chromatid recombination, or loss of heterozygosity. The rad59-Y92A allele was not synthetically lethal when combined with rad27, stimulated ectopic gene conversion and heteroallelic recombination independently from rad27, and was mutually epistatic with srs2. Unlike rad27, the stimulatory effect of rad59-Y92A on homologous recombination was not accompanied by effects on growth rate, cell cycle distribution, mutation, unequal sister-chromatid recombination, or loss of heterozygosity. The synthetic lethality conferred by rad59 null and rad59-K166A alleles correlates with their inhibitory effect on association

  11. A Simplified Method for Gene Knockout and Direct Screening of Recombinant Clones for Application in Paenibacillus polymyxa.

    Directory of Open Access Journals (Sweden)

    Seong-Bin Kim

    Full Text Available Paenibacillus polymyxa is a bacterium widely used in agriculture, industry, and environmental remediation because it has multiple functions including nitrogen fixation and produces various biologically active compounds. Among these compounds are the antibiotics polymyxins, and the bacterium is currently being reassessed for medical application. However, a lack of genetic tools for manipulation of P. polymyxa has limited our understanding of the biosynthesis of these compounds.To facilitate an understanding of the genetic determinants of the bacterium, we have developed a system for marker exchange mutagenesis directly on competent cells of P. polymyxa under conditions where homologous recombination is enhanced by denaturation of the suicide plasmid DNA. To test this system, we targeted P. polymyxa α-and β-amylase genes for disruption. Chloramphenicol or erythromycin resistance genes were inserted into the suicide plasmid pGEM7Z-f+ (Promega. To mediate homologous recombination and replacement of the targeted genes with the antibiotic resistance genes nucleotide sequences of the α-and β-amylase genes were cloned into the plasmid flanking the antibiotic resistance genes.We have created a simple system for targeted gene deletion in P. polymyxa E681. We propose that P. polymyxa isogenic mutants could be developed using this system of marker exchange mutagenesis. α-and β-amylase genes provide a useful tool for direct recombinant screening in P. polymyxa.

  12. Generation of recombinant Orf virus using an enhanced green fluorescent protein reporter gene as a selectable marker

    Directory of Open Access Journals (Sweden)

    Ning Zhangyong

    2011-12-01

    Full Text Available Abstract Background Reporter genes are often used as a selectable marker for generation of recombinant viruses in order to investigate the mechanism of pathogenesis and to obtain candidate vaccine viruses. Routine selection of the recombinant parapoxvirus is time-consuming and labor intensive. Therefore, developing a novel method for selection is critical. Results In this study, we developed a rapid method to generate recombinant Orf viruses (ORFV based on the enhanced green fluorescent protein (EGFP reporter gene as a selectable marker. The coding sequence of EGFP gene was amplified from pEGFP-N1 vector and subcloned into the pZIPPY-neo/gus plasmid under the control of the early-late vaccinia virus (VACV VV7.5 promoter and flanked by two multiple cloning sites (MCS to generate a novel transfer vector pSPV-EGFP. Using the pSPV-EGFP, two recombination cassettes pSPV-113LF-EGFP-113RF and pSPV-116LF-EGFP-116RF were constructed by cloning the flanking regions of the ORFV113 and ORFV116 and inserted into two MCS flanking the EGFP gene. Using this novel system, two single gene deletion mutants OV-IA82Δ113 and OV-IA82Δ116 were successfully generated. Conclusions This approach shortens the time needed to generate recombinant ORFVs (rORFVs. Thus, the pSPV-EGFP vector provides a direct, fast, and convenient way to manipulate the recombinant viruses, indicating that it is highly suited for its designed purpose.

  13. Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

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    Olivier J Becherel

    2013-04-01

    Full Text Available Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2, plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI. Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops, and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

  14. Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

    Directory of Open Access Journals (Sweden)

    Olivier J Becherel

    2013-04-01

    Full Text Available Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2, plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI. Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops, and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

  15. Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

    Science.gov (United States)

    Becherel, Olivier J; Yeo, Abrey J; Stellati, Alissa; Heng, Evelyn Y H; Luff, John; Suraweera, Amila M; Woods, Rick; Fleming, Jean; Carrie, Dianne; McKinney, Kristine; Xu, Xiaoling; Deng, Chuxia; Lavin, Martin F

    2013-04-01

    Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

  16. Gene transfer to the nonhuman primate retina with recombinant feline immunodeficiency virus vectors.

    Science.gov (United States)

    Lotery, Andrew J; Derksen, Todd A; Russell, Stephen R; Mullins, Robert F; Sauter, Sybille; Affatigato, Louisa M; Stone, Edwin M; Davidson, Beverly L

    2002-04-10

    We hypothesize that recombinant feline immunodeficiency viral (rFIV) vectors may be useful for gene transfer to the nonhuman primate retina. We performed vitrectomies and subretinal injections in the right eyes of 11 cynomolgus monkeys. Vesicular stomatitis virus glycoprotein-pseudotyped rFIV that expressed the Escherichia coli beta-galactosidase gene was injected into eight eyes. Sham vehicle or lactose buffer injections were also performed in two of these eight study eyes. rFIV pseudotyped with an amphotropic envelope was used in two eyes, and in one animal injections of lactose buffer only were given. After surgery the animals were clinically evaluated by retinal photography and electroretinography. beta-Galactosidase expression was evaluated, at a final end point, in histological sections. We found photoreceptor and Müller cells to have the greatest transgene expression. Focal inflammatory responses localized to the injection site were seen histologically in all eyes. No difference in transduction efficiency was seen between injections near the macula and more peripheral injections. Visual function as assessed by electroretinography was not significantly affected by vector or vehicle injections. We conclude that rFIV vectors administered beneath the retina can transduce a variety of retinal cells in the nonhuman primate retina. rFIV vectors have therapeutic potential and could be exploited to develop gene therapy for the human eye.

  17. Effects of recombinant adenoviral vector containing IRE1α gene on proliferation and apoptosis of ATDC5 stem cells

    Directory of Open Access Journals (Sweden)

    Xiang-zhu LI

    2013-09-01

    Full Text Available Objective To construct the recombinant adenoviral vector containing human IRE1α (type I transmembrane protein kinase/endoribonucleasegene, and investigate its effects on proliferation and apoptosis of ATDC5 stem cells. Methods  By using pAdEasyTM adenovirus vector system, the recombinant shuttle vectors of IRE1α full-length gene(pAdTrack-IRE1αand RNase+Kinasedomain(pAdTrack-R+Kwere constructed, and then transferred with pAdEasy-1 to generate recombinant adenovirus plasmid pAd-IRE1α and pAd-R+K by electroporation method. Subsequently, the plasmids were transfected into HEK-293 cells to pack and amplify the recombinant adenovirus Ad-IRE1α and Ad-R+K. The expression of recombinant adenovirus was detected by PCR. The ATDC5 cells wereinfected in vitro by recombinant adenovirus Ad-IRE1α and Ad-R+K, the infection efficiency of green fluorescent protein(GFPwas observed, and the influence of Ad-IRE1α and Ad-R+K on the proliferation and apoptosis of ATDC5 cells under endoplasmic reticulum stress(ERS or non-ERS was detected by flow cytometry(FCM. Results Restriction endonuclease digestion analysis and PCR indicated that the recombinant adenovirus vector Ad-IRE1α andAd-R+K was successfully constructed. FCM detection showed that under ERS conditions, the G1 phasedcreased and S phase increased in ATDC5 cells after transfected by Ad-IRE1α and Ad-R+K, meanwhile the apoptosis rate increased significantly(P<0.05. Conclusion Infection of recombinant adenovirus containing IRE1α gene may promote the proliferation and apoptosis of ATDC5cells.

  18. A new short-term toxicity assay using Aspergillus awamori with recombinant aequorin gene

    Directory of Open Access Journals (Sweden)

    Zwinderman Mark

    2005-07-01

    Full Text Available Abstract Background Most currently available short-term toxicity assays are based on bacterial cells. Therefore there is a need for novel eukaryotic microbial bioassays that will be relevant to higher eukaryotes such as animals and plants. Ca2+ is a universal intracellular signalling molecule found in all organisms from prokaryotes to highly specialized animal cells. In fungi calcium has been demonstrated to be involved in control of many important processes. The recombinant aequorin gene from the jellyfish Aequorea victoria responsible for the expression of the Ca2+-sensitive aequorin photoprotein has been cloned in the filamentous fungus Aspergillus awamori. This has allowed real life monitoring of [Ca2+]c changes in living fungal cells. When subjected to different physico-chemical stimuli fungal cells respond by transiently changing the concentration of free Ca2+ in the cytosol ([Ca2+]c and the pattern of these changes (Ca2+ signature is specific to each particular stimulus. Therefore it was interesting to investigate whether different environmental toxicants would be able to affect the pattern of [Ca2+]c changes in a reproducible and dose dependant manner. Results Toxicity bioassay has been developed to monitor changes [Ca2+]c of the recombinant fungus in the presence of toxicants representing heavy metals – Cr6+ and Zn2+ and a phenolic polar narcotic -3,5-DCP. The fungus responds to toxicants by a decrease in the amplitude of [Ca2+]c response to 5 mM external CaCl2 and an increase in Ca2+ final resting levels and recovery time. Conclusion A novel toxicity bioassay utilizing eukaryotic cells has been developed based on filamentous fungi transformed with the recombinant aequorin gene. A range of parameters characterising changes in [Ca2+]c has been identified, e.g. Amplitude, Length of Transient, Final Resting Level and Recovery Time. These parameters can be used to determine the toxicity of a range of chemicals to eukaryotic cells in a 96

  19. StyA1 and StyA2B from Rhodococcus opacus 1CP: a Multifunctional Styrene Monooxygenase System

    NARCIS (Netherlands)

    Tischler, D.; Kermer, R.; Groning, J.A.D.; Kaschabek, S.R.; Berkel, van W.J.H.; Schlomann, M.

    2010-01-01

    Two-component flavoprotein monooxygenases are emerging biocatalysts that generally consist of a monooxygenase and a reductase component. Here we show that Rhodococcus opacus 1CP encodes a multifunctional enantioselective flavoprotein monooxygenase system composed of a single styrene monooxygenase (S

  20. Two genes, rif15 and rif16, of the rifamycin biosynthetic gene cluster in Amycolatopsis mediterranei likely encode a transketolase and a P450 monooxygenase,respectively, both essential for the conversion of rifamycin SV into B

    Institute of Scientific and Technical Information of China (English)

    Hua Yuan; Wei Zhao; Yi Zhong; Jin Wang; Zhongiun Qin; Xiaoming Ding; Guo-Ping Zhao

    2011-01-01

    Amycolatopsis mediterranei produces an important antibiotic rifamycin,the biosynthesis of which involves many unusual modifications.Previous work suggested a putative P450 enzyme encoded by rif16 within the rifamycin biosynthetic gene cluster (rif) was required for the conversion of the intermediate rifamycin SV into the end product rifamycin B.In this study,we genetically proved that a putative transketolase encoded by rif15 is another essential enzyme for this conversion.Expression of merely rif15 and rif16 in a rif cluster null mutant ofA.mediterranei U32 was able to convert rifamycin SV into B.However,this Rifl5- and Rifl6-mediated conversion was only detected in intact cells of A.meidterranei,but not in Streptomyce coelicolor or Mycobacterium smegmatis,suggesting that yet-characterized gene(s) in A.mediterranei other than those encoded by the rif cluster should be involved in this process.

  1. A sensitive and rapid assay for homologous recombination in mosquito cells: impact of vector topology and implications for gene targeting

    Directory of Open Access Journals (Sweden)

    Zhao Yuguang

    2001-12-01

    Full Text Available Abstract Background Recent progress in insect transgenesis has been dramatic but existing transposon-based approaches are constrained by position effects and potential instability. Gene targeting would bring a number of benefits, however progress requires a better understanding of the mechanisms involved. Much can be learned in vitro since extrachromosomal recombination occurs at high frequency, facilitating the study of multiple events and the impact of structural changes among the recombining molecules. We have investigated homologous recombination in mosquito cells through restoration of luciferase activity from deleted substrates. The implications of this work for the construction of insect gene targeting vectors are discussed. Results We show that linear targeting vectors are significantly more efficient than circular ones and that recombination is stimulated by introducing double-strand breaks into, or near, the region of homology. Single-strand annealing represents a very efficient pathway but may not be feasible for targeting unbroken chromosomes. Using circular plasmids to mimic chromosomal targets, one-sided invasion appears to be the predominant pathway for homologous recombination. Non-homologous end joining reactions also occur and may be utilised in gene targeting if double-strand breaks are first introduced into the target site. Conclusions We describe a rapid, sensitive assay for extrachromosomal homologous recombination in mosquito cells. Variations in substrate topology suggest that single-strand annealing and one-sided invasion represent the predominant pathways, although non-homologous end joining reactions also occur. One-sided invasion of circular chromosomal mimics by linear vectors might therefore be used in vitro to investigate the design and efficiency of gene targeting strategies.

  2. Phylogenetic conservation of the 3' cryptic recombination signal sequence (3'cRSS) in the VH genes of jawed vertebrates.

    Science.gov (United States)

    Sun, Yi; Liu, Zhancai; Li, Zhaoyong; Lian, Zhengxing; Zhao, Yaofeng

    2012-01-01

    The VH replacement process is a RAG-mediated secondary recombination in which the variable region of a rearranged VHDJH is replaced by a different germline VH gene. In almost all human and mouse VH genes, two sequence features appear to be crucial for VH replacement. First, an embedded heptamer, which is located near the 3' end of the rearranged VH gene, serves as a cryptic recombination signal sequence (3'cRSS) for the VH replacement process. Second, a short stretch of nucleotides located downstream of the 3'cRSS serve as a footprint of the original VH region, frequently encoding charged amino acids. In this review, we show that both of these two features are conserved in the VH genes of all jawed vertebrates, which suggests that the VH replacement process may be a conserved mechanism.

  3. Crystal structure of a phenol-coupling P450 monooxygenase involved in teicoplanin biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhi; Rupasinghe, Sanjeewa G.; Schuler, Mary A.; Nair, Satish K. (UIUC)

    2012-02-08

    The lipoglycopeptide antibiotic teicoplanin has proven efficacy against gram-positive pathogens. Teicoplanin is distinguished from the vancomycin-type glycopeptide antibiotics, by the presence of an additional cross-link between the aromatic amino acids 1 and 3 that is catalyzed by the cytochrome P450 monooxygenase Orf6* (CYP165D3). As a goal towards understanding the mechanism of this phenol-coupling reaction, we have characterized recombinant Orf6* and determined its crystal structure to 2.2-{angstrom} resolution. Although the structure of Orf6* reveals the core fold common to other P450 monooxygenases, there are subtle differences in the disposition of secondary structure elements near the active site cavity necessary to accommodate its complex heptapeptide substrate. Specifically, the orientation of the F and G helices in Orf6* results in a more closed active site than found in the vancomycin oxidative enzymes OxyB and OxyC. In addition, Met226 in the I helix replaces the more typical Gly/Ala residue that is positioned above the heme porphyrin ring, where it forms a hydrogen bond with a heme iron-bound water molecule. Sequence comparisons with other phenol-coupling P450 monooxygenases suggest that Met226 plays a role in determining the substrate regiospecificity of Orf6*. These features provide further insights into the mechanism of the cross-linking mechanisms that occur during glycopeptide antibiotics biosynthesis.

  4. Gene synthesis, expression in Escherichia coli, purification and characterization of the recombinant bovine acyl-CoA-binding protein

    DEFF Research Database (Denmark)

    Mandrup, S; Højrup, P; Kristiansen, K

    1991-01-01

    -initiation codon were chosen to allow efficient expression in Escherichia coli as well as in yeast. The synthetic gene was inserted into the expression vector pKK223-3 and expressed in E. coli. In maximally induced cultures, recombinant ACBP constitutes 12-15% of total cellular protein. A fraction highly enriched...

  5. Comparisons of Maize pericarp color1 Alleles Reveal Paralogous Gene Recombination and an Organ-Specific Enhancer Region

    National Research Council Canada - National Science Library

    Feng Zhang; Thomas Peterson

    2005-01-01

    ... (for red pericarp/white cob) alleles, P1-rw1077 and P1-rw751::Ac. Structural analysis of P1-rw1077 indicated that this allele was generated by recombination between p1 and the tightly linked paralogous gene, p2...

  6. New recombinant bacterium comprises a heterologous gene encoding glycerol dehydrogenase and/or an up-regulated native gene encoding glycerol dehydrogenase, useful for producing ethanol

    DEFF Research Database (Denmark)

    2010-01-01

    from Geobacillus. It is selected from SEQ ID NO. 1-17. Sequences not defined here may be found at ftp://ftp.wipo.int/pub/publishedpctsequences/publication. The heterologous gene encoding glycerol dehydrogenase has been incorporated into the chromosome of the bacterium, or is inserted into a lactate...... glycerol dehydrogenase; and/or (ii) up-regulating a native gene encoding glycerol dehydrogenase; and (b) obtaining the recombinant bacterium. Preferred Bacterium: In the recombinant bacterium above, the inserted heterologous gene and/or the up-regulated native gene is encoding a glycerol dehydrogenase...... selected from glycerol dehydrogenase (E.C 1.1.1.6); glycerol dehydrogenase (NADP(+)) (E.C. 1.1.1.72); glycerol 2-dehydrogenase (NADP(+)) (E.C. 1.1.1.156); and glycerol dehydrogenase (acceptor) (E.C. 1.1.99.22). The heterologous gene encoding a glycerol dehydrogenase is derived from Thermotoga or is derived...

  7. Analysis of two lysozyme genes and antimicrobial functions of their recombinant proteins in Asian seabass.

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    Gui Hong Fu

    Full Text Available Lysozymes are important proteins of the innate immune system for the defense against bacterial infection. We cloned and analyzed chicken-type (c-type and goose-type (g-type lysozymes from Asian seabass (Lates calcarifer. The deduced amino acid sequence of the c-type lysozyme contained 144 residues and possessed typical structure residues, conserved catalytic residues (Glu(50 and Asp(67 and a "GSTDYGIFQINS" motif. The deduced g-type lysozyme contained 187 residues and possessed a goose egg white lysozyme (GEWL domain containing three conserved catalytic residues (Glu(71, Asp(84, Asp(95 essential for catalytic activity. Real time quantitative PCR (qRT-PCR revealed that the two lysozyme genes were constitutively expressed in all the examined tissues. The c-type lysozyme was most abundant in liver, while the g-type lysozyme was predominantly expressed in intestine and weakly expressed in muscle. The c-type and g-type transcripts were up-regulated in the kidney, spleen and liver in response to a challenge with Vibrio harveyi. The up-regulation of the c-type lysozyme was much stronger than that of the g-type lysozyme in kidney and spleen. The recombinant proteins of the c-type and g-type lysozymes showed lytic activities against the bacterial pathogens Vibrio harveyi and Photobacterium damselae in a dosage-dependent manner. We identified single nucleotide polymorphisms (SNPs in the two lysozyme genes. There were significant associations of these polymorphisms with resistance to the big belly disease. These results suggest that the c- and g-type genes play an important role in resistance to bacterial pathogens in fish. The SNP markers in the two genes associated with the resistance to bacterial pathogens may facilitate the selection of Asian seabass resistant to bacterial diseases.

  8. Increase of Clavulanic acid production by using recombinant Streptomyces clavuligerus strain including claR gene

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    Maryam Kay

    2014-07-01

    Full Text Available   Introduction : Clavulanic acid is a major β-lactam antibiotic which is produced by Streptomyces clavuligerus. Clavulanic acid is used in combination of strong but sensitive to β-lactamase antibiotics. The claR gene has an important role in regulation of clavulanic acid production and is needed for the expression of the genes in final step of clavulanic acid biosynthesis.   Materials and methods: The recombinant construct pMTclaR which contains claR gene is obtained from Isfahan University and plasmid extraction was done from Streptomyces lividans for next steps. The Streptomyces clavuligerus protoplast was prepared and transformation was done by using polyethylene glycol. Transformation was confirmed by plasmid extraction and PCR. Finally, bioassay method was used to survey the effect of extra copy of claR on clavulanic acid production .   Results : The typical chalky white colony of Streptomyces clavuligerus was seen on GYME plates containing thiostrepton antibiotic. Plasmid extraction was initially carried out. Furthermore, PCR reaction was done by claR specific primers and the 1334 bp band which was belonging to claR was detected. Finally, the bioassay was done and the diameters of zone of inhibition in control and sample were compared. The results of the bioassay show that amplification of the claR gene in multicopy plasmids resulted in a 2.5 fold increase in clavulanic acid production .   Discussion and conclusion : In this study the 3.3 fold increase in clavulanic acid production was obtained by using an expression vector containing claR. According to the clinical use of clavulanic acid, production of bacterial strains which are able to produce high level of antibiotic can help significantly in customization of antibiotic production.

  9. Recombinant hepatitis B surface antigen production in Aspergillus niger: evaluating the strategy of gene fusion to native glucoamylase

    CSIR Research Space (South Africa)

    James, ER

    2012-10-01

    Full Text Available Microbiology and Biotechnology October 2012/ Vol. 96, No.2 Recombinant hepatitis B surface antigen production in Aspergillus niger: evaluating the strategy of gene fusion to native glucoamylase ER James a,c & WH van Zyl b & PJ van Zyl c & JF Görgens..., Pretoria 0001, South Africa Abstract This study demonstrates the potential of Aspergillus niger as a candidate expression system for virus- like particle production using gene fusion. Hepatitis B surface antigen (HBsAg) production, targeted...

  10. Change of gene structure and function by non-homologous end-joining, homologous recombination, and transposition of DNA.

    OpenAIRE

    Wolfgang Goettel; Joachim Messing

    2009-01-01

    An important objective in genome research is to relate genome structure to gene function. Sequence comparisons among orthologous and paralogous genes and their allelic variants can reveal sequences of functional significance. Here, we describe a 379-kb region on chromosome 1 of maize that enables us to reconstruct chromosome breakage, transposition, non-homologous end-joining, and homologous recombination events. Such a high-density composition of various mechanisms in a small chromosomal int...

  11. Allelic effects on starch structure and properties of six starch biosynthetic genes in a rice recombinant inbred line population

    OpenAIRE

    2015-01-01

    Background The genetic diversity of six starch biosynthetic genes (Wx, SSI, SSIIa, SBEI, SBEIIa and SBEIIb) in indica and japonica rices opens an opportunity to produce a new variety with more favourable grain starch quality. However, there is limited information about the effects of these six gene allele combinations on starch structure and properties. A recombinant inbred line population from a cross between indica and japonica varieties offers opportunities to combine specific alleles of t...

  12. Base composition, selection, and phylogenetic significance of indels in the recombination activating gene-1 in vertebrates

    Directory of Open Access Journals (Sweden)

    Vences Miguel

    2009-12-01

    Full Text Available Abstract Background The Recombination Activating Proteins, RAG1 and RAG2, play a crucial role in the immune response in vertebrates. Among the nuclear markers currently used for phylogenetic purposes, Rag1 has especially enjoyed enormous popularity, since it successfully contributed to elucidating the relationships among and within a large variety of vertebrate lineages. We here report on a comparative investigation of the genetic variation, base composition, presence of indels, and selection in Rag1 in different vertebrate lineages (Actinopterygii, Amphibia, Aves, Chondrichthyes, Crocodylia, Lepidosauria, Mammalia, and Testudines through the analysis of 582 sequences obtained from Genbank. We also analyze possible differences between distinct parts of the gene with different type of protein functions. Results In the vertebrate lineages studied, Rag1 is over 3 kb long. We observed a high level of heterogeneity in base composition at the 3rd codon position in some of the studied vertebrate lineages and in some specific taxa. This result is also paralleled by taxonomic differences in the GC content at the same codon position. Moreover, positive selection occurs at some sites in Aves, Lepidosauria and Testudines. Indels, which are often used as phylogenetic characters, are more informative across vertebrates in the 5' than in the 3'-end of the gene. When the entire gene is considered, the use of indels as phylogenetic character only recovers one major vertebrate clade, the Actinopterygii. However, in numerous cases insertions or deletions are specific to a monophyletic group. Conclusions Rag1 is a phylogenetic marker of undoubted quality. Our study points to the need of carrying out a preliminary investigation on the base composition and the possible existence of sites under selection of this gene within the groups studied to avoid misleading resolution. The gene shows highly heterogeneous base composition, which affects some taxa in particular and

  13. Initiation of V(D)J Recombination by Dβ-Associated Recombination Signal Sequences: A Critical Control Point in TCRβ Gene Assembly

    Science.gov (United States)

    Franchini, Don-Marc; Benoukraf, Touati; Jaeger, Sébastien; Ferrier, Pierre; Payet-Bornet, Dominique

    2009-01-01

    T cell receptor (TCR) β gene assembly by V(D)J recombination proceeds via successive Dβ-to-Jβ and Vβ-to-DJβ rearrangements. This two-step process is enforced by a constraint, termed beyond (B)12/23, which prohibits direct Vβ-to-Jβ rearrangements. However the B12/23 restriction does not explain the order of TCRβ assembly for which the regulation remains an unresolved issue. The initiation of V(D)J recombination consists of the introduction of single-strand DNA nicks at recombination signal sequences (RSSs) containing a 12 base-pairs spacer. An RSS containing a 23 base-pairs spacer is then captured to form a 12/23 RSSs synapse leading to coupled DNA cleavage. Herein, we probed RSS nicks at the TCRβ locus and found that nicks were only detectable at Dβ-associated RSSs. This pattern implies that Dβ 12RSS and, unexpectedly, Dβ 23RSS initiate V(D)J recombination and capture their respective Vβ or Jβ RSS partner. Using both in vitro and in vivo assays, we further demonstrate that the Dβ1 23RSS impedes cleavage at the adjacent Dβ1 12RSS and consequently Vβ-to-Dβ1 rearrangement first requires the Dβ1 23RSS excision. Altogether, our results provide the molecular explanation to the B12/23 constraint and also uncover a ‘Dβ1 23RSS-mediated’ restriction operating beyond chromatin accessibility, which directs Dβ1 ordered rearrangements. PMID:19238214

  14. Impact of recombination on polymorphism of genes encoding Kunitz-type protease inhibitors in the genus Solanum.

    Science.gov (United States)

    Speranskaya, Anna S; Krinitsina, Anastasia A; Kudryavtseva, Anna V; Poltronieri, Palmiro; Santino, Angelo; Oparina, Nina Y; Dmitriev, Alexey A; Belenikin, Maxim S; Guseva, Marina A; Shevelev, Alexei B

    2012-08-01

    The group of Kunitz-type protease inhibitors (KPI) from potato is encoded by a polymorphic family of multiple allelic and non-allelic genes. The previous explanations of the KPI variability were based on the hypothesis of random mutagenesis as a key factor of KPI polymorphism. KPI-A genes from the genomes of Solanum tuberosum cv. Istrinskii and the wild species Solanum palustre were amplified by PCR with subsequent cloning in plasmids. True KPI sequences were derived from comparison of the cloned copies. "Hot spots" of recombination in KPI genes were independently identified by DnaSP 4.0 and TOPALi v2.5 software. The KPI-A sequence from potato cv. Istrinskii was found to be 100% identical to the gene from Solanum nigrum. This fact illustrates a high degree of similarity of KPI genes in the genus Solanum. Pairwise comparison of KPI A and B genes unambiguously showed a non-uniform extent of polymorphism at different nt positions. Moreover, the occurrence of substitutions was not random along the strand. Taken together, these facts contradict the traditional hypothesis of random mutagenesis as a principal source of KPI gene polymorphism. The experimentally found mosaic structure of KPI genes in both plants studied is consistent with the hypothesis suggesting recombination of ancestral genes. The same mechanism was proposed earlier for other resistance-conferring genes in the nightshade family (Solanaceae). Based on the data obtained, we searched for potential motifs of site-specific binding with plant DNA recombinases. During this work, we analyzed the sequencing data reported by the Potato Genome Sequencing Consortium (PGSC), 2011 and found considerable inconsistence of their data concerning the number, location, and orientation of KPI genes of groups A and B. The key role of recombination rather than random point mutagenesis in KPI polymorphism was demonstrated for the first time. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  15. Genealogy-based methods for inference of historical recombination and gene flow and their application in Saccharomyces cerevisiae.

    Science.gov (United States)

    Jenkins, Paul A; Song, Yun S; Brem, Rachel B

    2012-01-01

    Genetic exchange between isolated populations, or introgression between species, serves as a key source of novel genetic material on which natural selection can act. While detecting historical gene flow from DNA sequence data is of much interest, many existing methods can be limited by requirements for deep population genomic sampling. In this paper, we develop a scalable genealogy-based method to detect candidate signatures of gene flow into a given population when the source of the alleles is unknown. Our method does not require sequenced samples from the source population, provided that the alleles have not reached fixation in the sampled recipient population. The method utilizes recent advances in algorithms for the efficient reconstruction of ancestral recombination graphs, which encode genealogical histories of DNA sequence data at each site, and is capable of detecting the signatures of gene flow whose footprints are of length up to single genes. Further, we employ a theoretical framework based on coalescent theory to test for statistical significance of certain recombination patterns consistent with gene flow from divergent sources. Implementing these methods for application to whole-genome sequences of environmental yeast isolates, we illustrate the power of our approach to highlight loci with unusual recombination histories. By developing innovative theory and methods to analyze signatures of gene flow from population sequence data, our work establishes a foundation for the continued study of introgression and its evolutionary relevance.

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

    Directory of Open Access Journals (Sweden)

    Vassetzky Yegor S

    2008-12-01

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

  17. Gene delivery to dendritic cells by orally administered recombinant Saccharomyces cerevisiae in mice.

    Science.gov (United States)

    Kiflmariam, Meron G; Yang, Hanjiang; Zhang, Zhiying

    2013-02-27

    DNA vaccination has caught the attention of many for triggering humoral as well as cellular immune responses. And delivering DNA into the antigen presenting cells (APCs) in order to induce efficient immunoresponse has become the backbone of this field. It has been confirmed that Saccharomyces cerevisiae, though non-pathogenic, is being engulfed by the dendritic cells and macrophages and delivers not only proteins, but also DNA materials (already confirmed in vitro). In this research, S. cerevisiae is used to deliver green fluorescent protein (GFP) reporter gene controlled under cytomegalovirus (CMV) promoter in living organism (mice). The recombinant yeast, transfected with the plasmid containing the GFP gene, was heat killed and orally administered to mice. After 60 h of yeast administration, mice were sacrificed and intestine was separated, washed and frozen in liquid nitrogen. Tissues were cut at the size of 10 μm using Cryostat machine, and GFP expression was successfully detected under a fluorescence microscope. After 45 days Western blot was able to detect GFP antibody in the blood of mice. These results imply that S. cerevisiae, being non-pathogenic, cheap, and easy to culture could be a good candidate to deliver DNA materials to the immune cells for vaccination.

  18. MHC-restricted antigen presentation and recognition: constraints on gene, recombinant and peptide vaccines in humans

    Directory of Open Access Journals (Sweden)

    Cunha-Neto E.

    1999-01-01

    Full Text Available The target of any immunization is to activate and expand lymphocyte clones with the desired recognition specificity and the necessary effector functions. In gene, recombinant and peptide vaccines, the immunogen is a single protein or a small assembly of epitopes from antigenic proteins. Since most immune responses against protein and peptide antigens are T-cell dependent, the molecular target of such vaccines is to generate at least 50-100 complexes between MHC molecule and the antigenic peptide per antigen-presenting cell, sensitizing a T cell population of appropriate clonal size and effector characteristics. Thus, the immunobiology of antigen recognition by T cells must be taken into account when designing new generation peptide- or gene-based vaccines. Since T cell recognition is MHC-restricted, and given the wide polymorphism of the different MHC molecules, distinct epitopes may be recognized by different individuals in the population. Therefore, the issue of whether immunization will be effective in inducing a protective immune response, covering the entire target population, becomes an important question. Many pathogens have evolved molecular mechanisms to escape recognition by the immune system by variation of antigenic protein sequences. In this short review, we will discuss the several concepts related to selection of amino acid sequences to be included in DNA and peptide vaccines.

  19. Constructionof the recombinant adenovirus vectors of CALB2 gene and small interfering RNA, and application in testicular Leydig cells

    Institute of Scientific and Technical Information of China (English)

    Luo Jian; Wang Jing; Liu Shan; Sun Xue-ping; Gao Chao; Gao Li; Yang Xiao-yu; Liu Jia-yin; Cui Yu-gui

    2011-01-01

    Objective:To construct the recombinant adenovirus vectors of calretinin (CALB2) gene and small interfering RNA (siRNA),for over-expression or knock-down of CALB2,as the basis of functional investigation of CALB2 in testicular Leydig cells.Methods:The cDNA sequence of CALB2 was cloned by the reverse transcriptive polymerase chain reaction (RT-PCR).A CALB2 gene fragment was sub-cloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-CALB2.Then it was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant AdCMV-CALB2.The recombinant AdCMV-CALB2 was further packaged and amplificated in AD293 cells.The expression of CALB2 protein in AD293 cells was detected by Western blotting.CALB2 protein was over-expressed in mouse Leydig cell line (MLTC-1 cells) by the constructed AdCMVCALB2.CALB2 gene siRNA recombinant adenovirus vector (Ad-H1-siRNA/CALB2 was also constructed simultaneously.Its efficacy was detected in AD293 cells by Western blotting.Results:The CALB2 gene recombinant adenovirus vector AdCMV-CALB2 and the CALB2 gene siRNA recombinant adenovirus vector Ad-H1-siRNA/CALB2 were constructed successfully by endonulease digestion and sequencing.AD293 cells infected with AdCMV-CALB2 or Ad-H1-SiRNA/CALB2 significantly expressed GFP protein.The expression of CALB2 protein was significantly up-regulated in AD293 cells infected with AdCMV-CALB2 plasmids,while the expression of CALB2 protein was down-regulated by 60% in the CALB2 cells infected with Ad-H1SiRNA/CALB2.MLTC-1 cells did not markedly express CALLB2 protein,while MLTC-1 cells infected with AdCMV-CALB2 expressed CALB2 protein at a high level.Conclusions:The recombinant adenovirus vectors of AdCMV-CALB2 and Ad-H1-SiRNA/CALB2 were successfully constructed.Both vectors effectively expressed in AD293.CALB2 protein was over-expressed in the cultured MLTC-1 cells by AdCMV-CALB2.These vectors of CALB2 gene and Leydig cell line are

  20. Protection and synergism by recombinant fowl pox vaccines expressing multiple genes from Marek's disease virus.

    Science.gov (United States)

    Lee, Lucy E; Witter, R L; Reddy, S M; Wu, P; Yanagida, N; Yoshida, S

    2003-01-01

    Recombinant fowl poxviruses (rFPVs) were constructed to express genes from serotype 1 Marek's disease virus (MDV) coding for glycoproteins B, E, I, H, and UL32 (gB1, gE, gI, gH, and UL32). An additional rFPV was constructed to contain four MDV genes (gB1, gE, gI, and UL32). These rFPVs were evaluated for their ability to protect maternal antibody-positive chickens against challenge with highly virulent MDV isolates. The protection induced by a single rFPV/gB1 (42%) confirmed our previous finding. The protection induced by rFPV/gI (43%), rFPV/gB1UL32 (46%), rFPV/gB1gEgI (72%), and rFPV/gB1gEgIUL32 (70%) contributed to additional knowledge on MDV genes involved in protective immunity. In contrast, the rFPV containing gE, gH, or UL32 did not induce significant protection compared with turkey herpesvirus (HVT). Levels of protection by rFPV/gB1 and rFPV/gl were comparable with that of HVT. Only gB1 and gI conferred synergism in rFPV containing these two genes. Protection by both rFPV/gB1gEgI (72%) and rFPV/gB1gEgIUL32(70%) against Marek's disease was significantly enhanced compared with a single gB1 or gI gene (40%). This protective synergism between gB1 and gI in rFPVs may be the basis for better protection when bivalent vaccines between serotypes 2 and 3 were used. When rFPV/gB1gIgEUL32 + HVT were used as vaccine against Md5 challenge, the protection was significantly enhanced (94%). This synergism between rFPV/gB1gIgEUL32 and HVT indicates additional genes yet to be discovered in HVT may be responsible for the enhancement.

  1. Differential gene expression in human granulosa cells from recombinant FSH versus human menopausal gonadotropin ovarian stimulation protocols

    Directory of Open Access Journals (Sweden)

    Bietz Mandi G

    2010-03-01

    Full Text Available Abstract Background The study was designed to test the hypothesis that granulosa cell (GC gene expression response differs between recombinant FSH and human menopausal gonadotropin (hMG stimulation regimens. Methods Females Results After exclusions, 1736 genes exhibited differential expression between groups. Over 400 were categorized as signal transduction genes, ~180 as transcriptional regulators, and ~175 as enzymes/metabolic genes. Expression of selected genes was confirmed by RT-PCR. Differentially expressed genes included A kinase anchor protein 11 (AKAP11, bone morphogenetic protein receptor II (BMPR2, epidermal growth factor (EGF, insulin-like growth factor binding protein (IGFBP-4, IGFBP-5, and hypoxia-inducible factor (HIF-1 alpha. Conclusions Results suggest that major differences exist in the mechanism by which pure FSH alone versus FSH/LH regulate gene expression in preovulatory GC that could impact oocyte maturity and developmental competence.

  2. Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase

    NARCIS (Netherlands)

    Romero, Elvira; Castellanos, J Rubén Gómez; Mattevi, Andrea; Fraaije, Marco W

    2016-01-01

    Cyclohexanone monooxygenase (CHMO) is a promising biocatalyst for industrial reactions owing to its broad substrate spectrum and excellent regio-, chemo-, and enantioselectivity. However, the low stability of many Baeyer-Villiger monooxygenases is an obstacle for their exploitation in industry. Char

  3. The role of germline promoters and I exons in cytokine-induced gene-specific class switch recombination.

    Science.gov (United States)

    Dunnick, Wesley A; Shi, Jian; Holden, Victoria; Fontaine, Clinton; Collins, John T

    2011-01-01

    Germline transcription precedes class switch recombination (CSR). The promoter regions and I exons of these germline transcripts include binding sites for activation- and cytokine-induced transcription factors, and the promoter regions/I exons are essential for CSR. Therefore, it is a strong hypothesis that the promoter/I exons regions are responsible for much of cytokine-regulated, gene-specific CSR. We tested this hypothesis by swapping the germline promoter and I exons for the murine γ1 and γ2a H chain genes in a transgene of the entire H chain C-region locus. We found that the promoter/I exon for γ1 germline transcripts can direct robust IL-4-induced recombination to the γ2a gene. In contrast, the promoter/I exon for the γ2a germline transcripts works poorly in the context of the γ1 H chain gene, resulting in expression of γ1 H chains that is type level. Nevertheless, the small amount of recombination to the chimeric γ1 gene is induced by IFN-γ. These results suggest that cytokine regulation of CSR, but not the magnitude of CSR, is regulated by the promoter/I exons.

  4. Construction and characterization of a recombinant fowlpox virus containing HIV-1 multi-epitope-p24 chimeric gene in mice

    Institute of Scientific and Technical Information of China (English)

    ZHANG LiShu; JIN NingYi; SONG YingJin; WANG Hong; MA HeWen; LI ZiJian; JIANG WenZheng

    2007-01-01

    The epidemic of HIV/AIDS is sweeping across the world. It is of great importance to figure out new ways to curb this disease. Epitope-based vaccine is one of these solutions. In this study, a chimeric gene was obtained by combination of a designed HIV-1 multi-epitope gene (MEG) and HIV-1 p24 gene. A recombinant plasmid pUTA2-MEGp24 was then constructed by inserting MEGp24 gene into the down-stream of the promoter (ATI-P7.5×20) of fowlpox virus (FPV) transfer vector pUTA2. The recombinant plasmid and wild-type FPV 282E4 strain were then co-transfected into CEF cells and homologous recombination occurred. A recombinant virus expressing HIV-1 protein MEGp24 was screened by genome PCR and Western blot assay. Large scale preparation and purification of the recombinant fowlpox virus (rFPV) were then carried out. BALB/c mice were immunized intramuscularly with the rFPV for three times on day 0, 14 and 42. Mice were executed and sampled one week after the third inoculation.Anti-HIV-1 antibody in serum and Th1 cytokines in the supernatant of cultured spleen cells were assayed by ELISA. The count of T lymphocyte subsets and the CTL activity of spleen lymphocytes were analyzed by flow cytometry and lactate dehydrogenase (LDH) release assay, respectively. The results showed that HIV-1 specific antibody in serum and increased T lymphocyte subsets (CD4+ T, CD8+ T)were detected in the immunization group. CTL target-killing activity and higher secretion of Th1 cytokines (IFN-Y and IL-2) of spleen lymphocytes stimulated by H-2d-restricted CTL peptide were observed in immunized mice.We concluded that the rFPV may induce HIV-1 specific immunity especially cellular immunity in mice.

  5. Gene synthesis, expression in Escherichia coli, purification and characterization of the recombinant bovine acyl-CoA-binding protein

    DEFF Research Database (Denmark)

    Mandrup, S; Højrup, P; Kristiansen, K;

    1991-01-01

    A synthetic gene encoding the 86 amino acid residues of mature acyl-CoA-binding protein (ACBP), and the initiating methionine was constructed. The synthetic gene was assembled from eight partially overlapping oligonucleotides. Codon usage and nucleotides surrounding the ATG translation......-initiation codon were chosen to allow efficient expression in Escherichia coli as well as in yeast. The synthetic gene was inserted into the expression vector pKK223-3 and expressed in E. coli. In maximally induced cultures, recombinant ACBP constitutes 12-15% of total cellular protein. A fraction highly enriched...

  6. Microbial flavoprotein monooxygenases as mimics of mammalian flavin-containing monooxygenases for the enantioselective preparation of drug metabolites

    NARCIS (Netherlands)

    Gul, Turan; Krzek, Marzena; Permentier, Hjalmar; Fraaije, Marco; Bischoff, Rainer

    2016-01-01

    Mammalian flavin-containing monooxygenases are difficult to obtain and study while they play a major role in detoxifying various xenobiotics. In order to provide alternative biocatalytic tools to generate FMO-derived drug metabolites, a collection of microbial flavoprotein monooxygenases, sequence-r

  7. Construction of the recombinant vector carrying herpes simplex virus thymidine kinase and cytokine genes expressed in cell line Tca8113

    Institute of Scientific and Technical Information of China (English)

    JI Guang-hui; ZOU Jing-zhi; QU Le; YUE Ying; KUAI Jian-ke

    2004-01-01

    Objective: To construct expression vector containing fusion genes of herpes simplex virus thymidine kinase(Hsv-tk), Interleukin-2(IL-2) with internal ribosome entry sites(IRES), and to assess their expression in cell lineTca8113. Methods: IL-2 cDNA was obtained by reverse transcription. Hsv-tk, IL-2 and IRES genes were amplified by PCR. The purified amplification products were inserted into pGEM-T-Easy, and transformed into E. coli JM109. The purified recombinant plasmids were identified by restriction endonucleases. The recombinant plasmids were digested and pEGFPN3 were linearized, DNA fragments of Hsv-tk, IRES and IL-2 were ligated into linearized pEGFP-N3, and then transferred into E. coli JM109. The recombinant tk-IL-2 genes were cloned separately and introduced into the expression vector pEGFPN3 containing GFP. The recombinant vectors were identified by their restriction sites through PCR. The plasmids pEGFP-TI was also transfected into Tca8113 cells by calcium phosphate method for the expression of fusion proteins. Fusion genes expressing vector PL(TI)SN was generated by the fusion of HSV-tk, IRES and IL-2 with the use of DNA recombination technology. The recombinant retroviruses were transferred into Tca8113 cells by lipofectamine. The positive clones were obtained after G418 selection and named Tca/TI respectively. Results: The pEGFP-TI pasmid was identified respectively by restriction endonucleases, and their fragment sizes were 1 120 bp and 450 bp. The pEGFP-TI pasmid as templates were amplified respectively by PCR, and their PCR products were 1 120 bp and 450 bp. The pEGFP-TI vectors were used to transfect Tca8113 cell, and the cells with fluorescence accounted for 60 % of the total amount. Conclusion: pFGFP- tk- IRES- IL-2 expressing vector is easy to assess the expression of tk-IRES-IL-2-GFP fusion protein localization in transfected cells. The successful construction of expressing vector containing fusion genes of Hsv-tk, IRES and IL-2 may be

  8. Construction and identification of recombinant adenovirus-mediated gene transfer system for rat vascular endothelial growth factor

    Institute of Scientific and Technical Information of China (English)

    Hongyu Yang; Hong Qi; Junjie Zou; Xiwei Zhang

    2008-01-01

    Objective: To construct the recombinant adenovirus vector carrying rat vascular endothelial growth factor(VEGF), as preparation for genetic transfection that follows. Methods: Rat VEGF was obtained by using RT-PCR amplification and then cloned into the shutter plasmid pDC316. Subsequently, this newly constructed plasmid pDC316-VEGF, after identification by nuclease digestion analysis and sequencing analysis, was transfected into human embryonic kidney cells HEK293 by Lipofectamine 2000 mediation, together with adenovirus-packaging plasmid pBHGE3. Based on the homologous recombination of the two plasmids within HEK293 cells, the recombinant adenovirus vector carrying VEGF and VDC316-VEGF was created. VDC316-VEGF was subsequently identified using PCR, purified using repeated plaque passages, proliferated using freezing and melting within HEK293 cells, and titrated using 50% Tissue Culture Infective Dose(TCID50) assay. Results:The newly constructed recombinant adenovirus was confirmed to carry rat VEGF based on PCR results, and its titration value determined based on TCID50 assay was 3×109 pfu/ml. Conclusion:The recombinant adenovirus carrying rat VEGF was successfully constructed. The newly constructed adenovirus can produce a sufficiently high titration value within HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches.

  9. Gene targeting and transgene stacking using intra genomic homologous recombination in plants.

    Science.gov (United States)

    Kumar, Sandeep; Barone, Pierluigi; Smith, Michelle

    2016-01-01

    Modern agriculture has created a demand for plant biotechnology products that provide durable resistance to insect pests, tolerance of herbicide applications for weed control, and agronomic traits tailored for specific geographies. These transgenic trait products require a modular and sequential multigene stacking platform that is supported by precise genome engineering technology. Designed nucleases have emerged as potent tools for creating targeted DNA double strand breaks (DSBs). Exogenously supplied donor DNA can repair the targeted DSB by a process known as gene targeting (GT), resulting in a desired modification of the target genome. The potential of GT technology has not been fully realized for trait deployment in agriculture, mainly because of inefficient transformation and plant regeneration systems in a majority of crop plants and genotypes. This challenge of transgene stacking in plants could be overcome by Intra-Genomic Homologous Recombination (IGHR) that converts independently segregating unlinked donor and target transgenic loci into a genetically linked molecular stack. The method requires stable integration of the donor DNA into the plant genome followed by intra-genomic mobilization. IGHR complements conventional breeding with genetic transformation and designed nucleases to provide a flexible transgene stacking and trait deployment platform.

  10. Precision control of recombinant gene transcription for CHO cell synthetic biology.

    Science.gov (United States)

    Brown, Adam J; James, David C

    2016-01-01

    The next generation of mammalian cell factories for biopharmaceutical production will be genetically engineered to possess both generic and product-specific manufacturing capabilities that may not exist naturally. Introduction of entirely new combinations of synthetic functions (e.g. novel metabolic or stress-response pathways), and retro-engineering of existing functional cell modules will drive disruptive change in cellular manufacturing performance. However, before we can apply the core concepts underpinning synthetic biology (design, build, test) to CHO cell engineering we must first develop practical and robust enabling technologies. Fundamentally, we will require the ability to precisely control the relative stoichiometry of numerous functional components we simultaneously introduce into the host cell factory. In this review we discuss how this can be achieved by design of engineered promoters that enable concerted control of recombinant gene transcription. We describe the specific mechanisms of transcriptional regulation that affect promoter function during bioproduction processes, and detail the highly-specific promoter design criteria that are required in the context of CHO cell engineering. The relative applicability of diverse promoter development strategies are discussed, including re-engineering of natural sequences, design of synthetic transcription factor-based systems, and construction of synthetic promoters. This review highlights the potential of promoter engineering to achieve precision transcriptional control for CHO cell synthetic biology. Copyright © 2015. Published by Elsevier Inc.

  11. FSHbeta gene mutation in a female with delayed puberty and hypogonadism: response to recombinant human FSH.

    Directory of Open Access Journals (Sweden)

    S Alain

    2010-01-01

    Full Text Available We report a woman with primary amenorrhoea and infertility associated with an isolated deficiency of pituitary FSH that does not respond to GnRH administration. Serum inhibin B was undetectable and antimullerian hormone (AMH was within the normal range. Ultra sound examination revealed a small uterus and small ovaries with few small follicles. We identified an homozygous 1-bp (G deletion at codon 79 in FSHbeta gene suggesting a complete loss of function. The patient underwent studies of ovarian responsiveness to recombinant human FSH according to the following protocol: 150UI/d for five days following by 75 UI/d for 10 days. Estradiol plasma level started to increase from day 5 associated to a sharp increase of inhibine B and a decrease of LH. During the same time, we observed an excessive development of multiple follicles resulting in an arrest of the treatment to avoid hyperstimulation. The present study confirm that follicles up to 5 mm in diameter had developed in the absence of FSH and that FSH is required for the growth of follicles beyond the two-layer granulose stage.

  12. Gene therapy for allergic rhinitis with recombinant adenovirus vector containing CTLA4Ig in mice

    Institute of Scientific and Technical Information of China (English)

    朱瑾; 吴军; 陈希炜; 易绍萱; 罗高兴; 贺伟峰

    2003-01-01

    Objective: To examine the role of recombinant adenovirus vector containing CTLA4Ig gene(Ad-CTLA4Ig) in the treatment of induced allergic rhinitis in mice.Methods: Allergic rhinitis was induced by sensitizing and challenging with ovalbumin(OVA).Ad-CTLA4Ig was intraperitoneally injected 30 min before OVA challenge.Adenovirus vector without inserted CTLA4Ig cDNA served as the control.The symptoms and morphological changes of nasal mucosa of each group were observed, and the serum levels of IgE against OVA were detected with ELISA.Results: There were no obvious symptoms and pathological changes in Ad-CTLA4Ig treated group, in which the serum OVA-specific IgE levels were significantly lower than that in control groups(P< 0.05).Conclusion: Ad-CTLA4Ig prevents and treats allergic rhinitis of mice,implying the possibility of the usage of Ad-CTLA4Ig against allergic rhinitis in clinic in future.

  13. Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy

    Science.gov (United States)

    Powell, C.; Shansky, J.; Del Tatto, M.; Forman, D. E.; Hennessey, J.; Sullivan, K.; Zielinski, B. A.; Vandenburgh, H. H.

    1999-01-01

    Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, postmitotic myofibers. This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantable living protein secretory devices for therapeutic use.

  14. Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy

    Science.gov (United States)

    Powell, C.; Shansky, J.; Del Tatto, M.; Forman, D. E.; Hennessey, J.; Sullivan, K.; Zielinski, B. A.; Vandenburgh, H. H.

    1999-01-01

    Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, postmitotic myofibers. This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantable living protein secretory devices for therapeutic use.

  15. Construction of recombinant baculovirus Ac-CMV-hSox9 for gene therapy of intervertebral disc degeneration

    Institute of Scientific and Technical Information of China (English)

    LIU Xiao-yun; YANG Shu-hua; LIANG Chang-yong; SONG Jian-hua; LI Kang-hua; CHEN Xin-wen

    2007-01-01

    Objective: To construct the recombinant baculovirus Ac-cytomegalovirus (CMV)-hSox9 for gene therapy of intervertebral disc degeneration. Methods: Bac-to-Bac system was used for the construction of baculovirus Ac-CMV-hSox9. The cDNA of hSox9 was first cloned into a plasmid vector under the control of CMV promotor to generate the donor plasmid pFastBacDul-green fluorescene protein (GFP)-CMV (pFGC)-hSox9.The resultant plasmid was transformed into DH10Bac cells and then the transformation mixture was spread on Luria-Bertani (LB) agarose culture medium containing isopropyl-β-D-thiogalactoside (IPTG), X-gal, gentamicin, kanamycin and tetracycline.The white colonies were selected and cultured for amplification, and the hSox9Bacmid DNA was extracted. After verification, recombinant baculovirus Ac-CMV-hSox9 was obtained through transfecting Sf 21 cells.The expression of hSox9 gene in the intervertebral disc cells in rabbits was determined by Western blotting and immunohistochemical staining.Results: Polymerase chain reaction (PCR) confirmed the presence of hSox9 gene in the recombinant baculovirus and the Sf 21 cells transfected by the baculovirus showed the expression of fluorescence protein.Western blotting and immunohistochemical staining analysis indicated that exogenous hSox9 gene was expressed in the disc cells.Conclusions: The successful construction of the recombinant baculovirus Ac-CMV-hSox9 and the confirmation of the target gene expression provides a novel expression vector system for basic research and clinical treatment of intervertebral degenerative disc disease.

  16. Identification, Characterization and Down-Regulation of Cysteine Protease Genes in Tobacco for Use in Recombinant Protein Production.

    Directory of Open Access Journals (Sweden)

    Kishor Duwadi

    Full Text Available Plants are an attractive host system for pharmaceutical protein production. Many therapeutic proteins have been produced and scaled up in plants at a low cost compared to the conventional microbial and animal-based systems. The main technical challenge during this process is to produce sufficient levels of recombinant proteins in plants. Low yield is generally caused by proteolytic degradation during expression and downstream processing of recombinant proteins. The yield of human therapeutic interleukin (IL-10 produced in transgenic tobacco leaves was found to be below the critical level, and may be due to degradation by tobacco proteases. Here, we identified a total of 60 putative cysteine protease genes (CysP in tobacco. Based on their predicted expression in leaf tissue, 10 candidate CysPs (CysP1-CysP10 were selected for further characterization. The effect of CysP gene silencing on IL-10 accumulation was examined in tobacco. It was found that the recombinant protein yield in tobacco could be increased by silencing CysP6. Transient expression of CysP6 silencing construct also showed an increase in IL-10 accumulation in comparison to the control. Moreover, CysP6 localizes to the endoplasmic reticulum (ER, suggesting that ER may be the site of IL-10 degradation. Overall results suggest that CysP6 is important in determining the yield of recombinant IL-10 in tobacco leaves.

  17. A heuristic Bayesian method for segmenting DNA sequence alignments and detecting evidence for recombination and gene conversion.

    Science.gov (United States)

    Kedzierska, Anna; Husmeier, Dirk

    2006-01-01

    We propose a heuristic approach to the detection of evidence for recombination and gene conversion in multiple DNA sequence alignments. The proposed method consists of two stages. In the first stage, a sliding window is moved along the DNA sequence alignment, and phylogenetic trees are sampled from the conditional posterior distribution with MCMC. To reduce the noise intrinsic to inference from the limited amount of data available in the typically short sliding window, a clustering algorithm based on the Robinson-Foulds distance is applied to the trees thus sampled, and the posterior distribution over tree clusters is obtained for each window position. While changes in this posterior distribution are indicative of recombination or gene conversion events, it is difficult to decide when such a change is statistically significant. This problem is addressed in the second stage of the proposed algorithm, where the distributions obtained in the first stage are post-processed with a Bayesian hidden Markov model (HMM). The emission states of the HMM are associated with posterior distributions over phylogenetic tree topology clusters. The hidden states of the HMM indicate putative recombinant segments. Inference is done in a Bayesian sense, sampling parameters from the posterior distribution with MCMC. Of particular interest is the determination of the number of hidden states as an indication of the number of putative recombinant regions. To this end, we apply reversible jump MCMC, and sample the number of hidden states from the respective posterior distribution.

  18. Dengue-1 Virus Envelope Glycoprotein Gene Expressed in Recombinant Baculovirus Elicits Virus-Neutralizing Antibody in Mice and Protects them from Virus Challenge

    Science.gov (United States)

    1991-01-01

    8217 terminus of E. When the recombinant virus was grown in Spodoptera frugiperda cells. about I mg of E antigen was made per 10’ cells. Recombinant E antigen...assay with DEN-I virus coprotein gene and its expression in Spodoptera hyperimmune mouse ascitic fluid. This heat-in- frugiperda cells activated...immunization, S. frugiperda cells infected with tion with BstNI (cuts at nucleotides 801 and recombinant baculovirus were pelleted. lysed by 2150). The

  19. Gene therapy for pathological scar with hepatocyte growth factor mediated by recombinant adenovirus vector

    Institute of Scientific and Technical Information of China (English)

    哈小琴; 苑宾; 李元敏; 劳妙芬; 吴祖泽

    2003-01-01

    A complementary DNA (cDNA) encoding human hepatocyte growth factor wasintroduced into a replication-defective type 5 adenovirus (lacking E1, E3 domains) vector by homologous recombination of intracellular plasmid DNA, thus a recombinant vector containing HGF (Ad-HGF) was obtained. Ad-HGF and Ad-GFP (adenovirus vector carrying green fluorescence protein gene) were expanded in 293 cells and purified by cesium chloride gradient centrifugation for large-scale preparation, then were infected to the primarily cultured scar fibroblast of rabbit ear to observe the transfer efficiency and expression level of HGF in vitro. To evaluate the effect of Ad-HGF on established scar Ad-HGF solution was injected into excessively formed scar, which bears some clinical and histologic similarities tohuman hypertrophic scars. The results showed that: (i) the transfer efficiency was 36.8%±14.1% on day 3 in primarily cultured scar fibroblasts treated with Ad-GFP and lasted more than 20 d; (ii) high-level expression of HGF protein was detected by means of ELISA in supernatant of scar fibroblasts treated with Ad-HGF,the amount of expression was 76 ng/4.0×105 cells on day 3; (iii) on day 32 after a single intradermal injection of Ad-HGF at different doses (8.6×109 pfu, 8.6×108 pfu, 8.6×107 pfu, 8.6×106 pfu) per scar, most of the scars in the former two dose groups were dramatically flattened, some were even similar to that ofthe normal skin. The value of HI (hypertrophic index) showed that there was a therapeutic effect of Ad-HGF on scars at the dose of 109 pfu and 108 pfu. Whereasno therapeutic effects were seen at lower dose (107 pfu and 106 pfu of Ad-HGF) groups. In addition, clusters of hair were observed to different extent on healed wound treated with Ad-HGF. Histopathologic examination revealed that in most healed wounds of Ad-HGF treated group, the dermal layer was thinner, the amount of fibrous tissue was much fewer, and hair follicles growth and sebaceous glands were observed

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.

    Science.gov (United States)

    Montiel, Daniel; Kang, Hahk-Soo; Chang, Fang-Yuan; Charlop-Powers, Zachary; Brady, Sean F

    2015-07-21

    Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters.

  2. Specific targeted gene repair using single-stranded DNA oligonucleotides at an endogenous locus in mammalian cells uses homologous recombination.

    Science.gov (United States)

    McLachlan, Jennifer; Fernandez, Serena; Helleday, Thomas; Bryant, Helen E

    2009-12-03

    The feasibility of introducing point mutations in vivo using single-stranded DNA oligonucleotides (ssON) has been demonstrated but the efficiency and mechanism remain elusive and potential side effects have not been fully evaluated. Understanding the mechanism behind this potential therapy may help its development. Here, we demonstrate the specific repair of an endogenous non-functional hprt gene by a ssON in mammalian cells, and show that the frequency of such an event is enhanced when cells are in S-phase of the cell cycle. A potential barrier in using ssONs as gene therapy could be non-targeted mutations or gene rearrangements triggered by the ssON. Both the non-specific mutation frequencies and the frequency of gene rearrangements were largely unaffected by ssONs. Furthermore, we find that the introduction of a mutation causing the loss of a functional endogenous hprt gene by a ssON occurred at a similarly low but statistically significant frequency in wild type cells and in cells deficient in single strand break repair, nucleotide excision repair and mismatch repair. However, this mutation was not induced in XRCC3 mutant cells deficient in homologous recombination. Thus, our data suggest ssON-mediated targeted gene repair is more efficient in S-phase and involves homologous recombination.

  3. Construction of recombinant eukaryotic expression plasmid containing murine CD40 ligand gene and its expression in H22 cells

    Institute of Scientific and Technical Information of China (English)

    Yong-Fang Jiang; Yan He; Guo-Zhong Gong; Jun Chen; Chun-Yan Yang; Yun Xu

    2005-01-01

    AIM: To construct a recombinant murine CD40 ligand (mCD40L) eukaryotic expression vector for gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: mCD40L cDNA was synthesized by RT-PCR with the specific primers and directly cloned into T vector to generate middle recombinant. After digestion with restriction endonuclease, the target fragment was subcloned into the multi-clone sites of the eukaryotic vector. The constructed vector was verified by enzyme digestion and sequencing,and the product expressed was detected by RT-PCR and immunofluorescence methods.RESULTS: The full-length mCD40L-cDNA was successfully cloned into the eukaryotic vector through electrophoresis,and mCD40L gene was integrated into the genome of infected H22 cells by RT-PCR. Murine CD40L antigen molecule was observed in the plasma of mCD40L-H22 by indirect immuno-fluorescence staining.CONCLUSION: The recombined mCD40L eukaryotic expression vector can be expressed in H22 cell line. It providesexperimental data for gene therapy and target therapy ofhepatocellular carcinoma.

  4. New single nucleotide polymorphisms (SNPs) in homologous recombination repair genes detected by microarray analysis in Polish breast cancer patients.

    Science.gov (United States)

    Romanowicz, Hanna; Strapagiel, Dominik; Słomka, Marcin; Sobalska-Kwapis, Marta; Kępka, Ewa; Siewierska-Górska, Anna; Zadrożny, Marek; Bieńkiewicz, Jan; Smolarz, Beata

    2016-11-30

    Breast cancer is the most common cause of malignancy and mortality in women worldwide. This study aimed at localising homologous recombination repair (HR) genes and their chromosomal loci and correlating their nucleotide variants with susceptibility to breast cancer. In this study, authors analysed the association between single nucleotide polymorphisms (SNPs) in homologous recombination repair genes and the incidence of breast cancer in the population of Polish women. Blood samples from 94 breast cancer patients were analysed as test group. Individuals were recruited into the study at the Department of Oncological Surgery and Breast Diseases of the Institute of the Polish Mother's Memorial Hospital in Lodz, Poland. Healthy controls (n = 500) were obtained from the Biobank Laboratory, Department of Molecular Biophysics, University of Lodz. Then, DNA of breast cancer patients was compared with one of the disease-free women. The test was supported by microarray analysis. Statistically significant correlations were identified between breast cancer and 3 not described previously SNPs of homologous recombination repair genes BRCA1 and BRCA2: rs59004709, rs4986852 and rs1799950. Further studies on larger groups are warranted to support the hypothesis of correlation between the abovementioned genetic variants and breast cancer risk.

  5. Risk Assessment of Synergism and Recombination on the Transgenic Plants Containing Viral Movement Protein and Replicase Genes

    Institute of Scientific and Technical Information of China (English)

    NIU Yan-bing; LI Gui-xin; WEN Rui; ZHOU Xue-ping

    2003-01-01

    The transgenic tobacco plants transformed with movement protein gene of Tomato mosaic virus (ToMV) or Tobacco mosaic virus (TMV) and partial replicase gene of Cucumber mosaic virus (CMV) P1 isolate (CMV-P1), were inoculated with Potato virus X, Potato virus Y, TMV and CMV isolate RB (CMVRB), respectively. Symptom observation showed there were no symptom differences in transgenic tobacco plants as compared with those in non-transgenic tobacco plants. ELISA also illustrated that the virus concentrations in the transgenic plants were similar to those in non-transgenic plants, indicating that no synergism is found in these plants. The transgenic tobacco plants expressing movement protein gene of ToMV or partial replicase gene of CMV-P1 were inoculated with TMV and CMV-RB, respectively. The local or systemic infected leaves were then used for elucidation of the possible virus recombination in transgenic plants with biological infectivity test, ELISA and immuno-capture RT-PCR. Within 16 months, no recombination was found between transformed genes and inoculated virus genomes. The research provides fundamental data for understanding of the possible risk of the transgenic plants expressing viral sequences.

  6. Integration of a transfected gene into the genome of Babesia bovis occurs by legitimate homologous recombination mechanisms.

    Science.gov (United States)

    Suarez, Carlos E; Johnson, Wendell C; Herndon, David R; Laughery, Jacob M; Davis, William C

    2015-08-01

    This study examines the patterns of gene integration of gfp-bsd upon stable transfection into the T3Bo strain of Babesia bovis using a plasmid designed to integrate homologous sequences of the parasite's two identical ef-1α A and B genes. While the transfected BboTf-149-6 cell line displayed two distinct patterns of gene integration, clonal lines derived from this strain by cell sorting contained only single gfp-bsd insertions. Whole genome sequencing of two selected clonal lines, E9 and C6, indicated two distinct patterns of gfp-bsd insertion occurring by legitimate homologous recombination mechanisms: one into the expected ef-1α orf B, and another into the ef-1α B promoter. The data suggest that expression of the ef-1α orf B is not required for development of B. bovis in cultured erythrocyte stages. Use of legitimate homologous recombination mechanisms in transfected B. bovis supports the future use of transfection methods for developing efficient gene function assignment experiments using gene knockout techniques. Published by Elsevier B.V.

  7. Requirement of heterogeneous nuclear ribonucleoprotein C for BRCA gene expression and homologous recombination.

    Directory of Open Access Journals (Sweden)

    Rachel W Anantha

    Full Text Available BACKGROUND: Heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C is a core component of 40S ribonucleoprotein particles that bind pre-mRNAs and influence their processing, stability and export. Breast cancer tumor suppressors BRCA1, BRCA2 and PALB2 form a complex and play key roles in homologous recombination (HR, DNA double strand break (DSB repair and cell cycle regulation following DNA damage. METHODS: PALB2 nucleoprotein complexes were isolated using tandem affinity purification from nuclease-solubilized nuclear fraction. Immunofluorescence was used for localization studies of proteins. siRNA-mediated gene silencing and flow cytometry were used for studying DNA repair efficiency and cell cycle distribution/checkpoints. The effect of hnRNP C on mRNA abundance was assayed using quantitative reverse transcriptase PCR. RESULTS AND SIGNIFICANCE: We identified hnRNP C as a component of a nucleoprotein complex containing breast cancer suppressor proteins PALB2, BRCA2 and BRCA1. Notably, other components of the 40S ribonucleoprotein particle were not present in the complex. hnRNP C was found to undergo significant changes of sub-nuclear localization after ionizing radiation (IR and to partially localize to DNA damage sites. Depletion of hnRNP C substantially altered the normal balance of repair mechanisms following DSB induction, reducing HR usage in particular, and impaired S phase progression after IR. Moreover, loss of hnRNP C strongly reduced the abundance of key HR proteins BRCA1, BRCA2, RAD51 and BRIP1, which can be attributed, at least in part, to the downregulation of their mRNAs due to aberrant splicing. Our results establish hnRNP C as a key regulator of BRCA gene expression and HR-based DNA repair. They also suggest the existence of an RNA regulatory program at sites of DNA damage, which involves a unique function of hnRNP C that is independent of the 40S ribonucleoprotein particles and most other hnRNP proteins.

  8. Curative effect of transplantation of mesenchymal stem cells transfected with recombinant lentiviral vectors carrying brain-derived neurotrophic factor gene on intracerebral hemorrhage in rats

    Institute of Scientific and Technical Information of China (English)

    任瑞芳

    2013-01-01

    Objective To observe the curative effect of transplantation of mesenchymal stem cells(MSCs) transfected with recombinant lentiviral vectors carrying brain-derived neurotrophic factor(BDNF) gene on intracerebral

  9. Molecular cloning, recombinant gene expression, and antifungal activity of cystatin from taro (Colocasia esculenta cv. Kaosiung no. 1).

    Science.gov (United States)

    Yang, A H; Yeh, K W

    2005-06-01

    A cDNA clone, designated CeCPI, encoding a novel phytocystatin was isolated from taro corms (Colocasia esculenta) using both degenerated primers/RT-PCR amplification and 5'-/3'-RACE extension. The full-length cDNA gene is 1,008 bp in size, encodes 206 amino acid residues, with a deduced molecular weight of 29 kDa. It contains a conserved reactive site motif Gln-Val-Val-Ser-Gly of cysteine protease inhibitors, and another consensus ARFAV sequence for phytocystatin. Sequence analysis revealed that CeCPI is phylogenetically closely related to Eudicots rather than to Monocots, despite taro belonging to Monocot. Recombinant GST-CeCPI fusion protein was overexpressed in Escherichia coli and its inhibitory activity against papain was identified on gelatin/SDS-PAGE. These results confirmed that recombinant CeCPI protein exhibited strong cysteine protease inhibitory activity. Investigation of its antifungal activity clearly revealed a toxic effect on the mycelium growth of phytopathogenic fungi, such as Sclerotium rolfsii Sacc. etc., at a concentration of 80 microg recombinant CeCPI/ ml. Moreover, mycelium growth was completely inhibited and the sclerotia lysed at a concentration of 150-200 microg/ml. Further studies have demonstrated that recombinant CeCPI is capable of acting against the endogenous cysteine proteinase in the fungal mycelium.

  10. Recombination and Mutation of some Genes with Gene Splicing by Overlap Extension%利用套叠PCR技术进行基因突变和拼接

    Institute of Scientific and Technical Information of China (English)

    周鹏; 董克家

    2001-01-01

    利用套叠PCR技术(又称重叠区扩增基因拼接法) 对hGM-CSF基因内第28位氨基酸处的糖基化位点进行突变和进行人促性腺激素基因、腺苷酸激酶短肽与胰岛素样生长因子-I基因三者之间的拼接,结果表明采用该技术能在体外实行有效的基因重组和定点突变,其成功率为100%;这一技术不需要内切酶消化和连接酶处理,技术操作简单易行,在基因拼接、基因内部突变方面具有良好的应用价值。%The glycosylation site (at the 28th amino acid residue) of human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene was mutated by gene SOEing.Follicle stimulating hormone gene (FSH),short peptide of adenine nucleotide kinase and insulin-like gowth factor-I gene (IGF-I) were recombined together by gene splicing by overlap extension (gene SOEing). Results showed that gene SOEing was a successful specific PCR technique for gene recombination and gene mutation in vitro. The technique didnt need restrict enzymes and ligases,which could be developed as a simple and useful technique in the studies of gene splicing and gene mutation.

  11. Construction of prokaryotic expression system of TGF-β1 epitope gene and identification of recombinant fusion protein immunity

    Institute of Scientific and Technical Information of China (English)

    Yong-Hong Guo; Zhi-Ming Hao; Jin-Yan Luo; Jun-Hong Wang

    2005-01-01

    AIM: To insert the constructed TGF-β1the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1METHODS: The TGF-β1mature TGF-β1TGF-32) was amplified by polymerase chain reaction from the recombinant pGEM-7z/TGF-β1HBcAg gene fragments (encoding HBcAg from 1-71 and 89-144 amino acid residues) were amplified from PYTA1-HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-β1into restrictive endonuclease enzyme and ligated with T4ligase. The fusion gene fragments HBcAg1-71-TGF-β1HBcAg89-144 were recloned to pET28a(+) and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a (+)/CTC was transformed and expressed in E.. Coli BL21 (DE3)under induction of IPTG. After purification with Ni+2-NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope.RESULTS: Enzyme digestion analysis and sequencing showed that TGF-β1loop of C-terminus of truncated hepatitis B core antigen.SDS-PAGE analysis showed that relative molecular mass(Mr) of the expressed product by pET28a (+)/CTC was Mr 24 600.The output of the target recombinant protein was approximately 34.8% of the total bacterial protein,mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-β1not with anti-HBcAg. The purity of protein was about 90% and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-β1could be used as anti-TGF-β1CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target TGF- epitope gene was successfully established.The fusion protein is in the form of self-assembling particles

  12. Recombinant feline leukemia virus genes detected in naturally occurring feline lymphosarcomas.

    OpenAIRE

    Sheets, R L; Pandey, R.; Jen, W C; Roy-Burman, P

    1993-01-01

    Using a polymerase chain reaction strategy aimed at detecting recombinant feline leukemia virus (FeLV) genomes with 5' env sequences originating from an endogenous source and 3' env sequences resulting from FeLV subgroup A (FeLV-A), we detected recombinant proviruses in approximately three-fourths of naturally occurring thymic and alimentary feline lymphosarcomas (LSAs) and one-third of the multicentric LSAs from cats determined to be FeLV capsid antigen positive by immunofluorescence assay. ...

  13. Ocular gene therapy: an evaluation of recombinant adeno-associated virus-mediated gene therapy interventions for the treatment of ocular disease.

    Science.gov (United States)

    Roy, Kamolika; Stein, Linda; Kaushal, Shalesh

    2010-08-01

    Both gene replacement therapy and alteration of host gene expression are playing increasingly important roles in the treatment of ocular diseases. Ocular gene therapy may provide alternatives to current treatments for eye diseases that are either greatly invasive and thus run the risk of complications, that offer only short-term relief from disease symptoms, or that are unable to directly treat vision loss. The success of three separate phase I clinical trials investigating a gene therapy intervention for the treatment of the retinal degenerative disorder Leber's congenital amaurosis (LCA) has unveiled the therapeutic potential of gene therapy. Preliminary results have demonstrated ocular gene transfer, using nonpathogenic recombinant adeno-associated viral (rAAV) vectors specifically, to be a safe, effective, and long-term treatment for LCA, a previously untreatable disorder. Nonpathogenic rAAV vectors offer the potential for long-term treatment. Many of the genes implicated in human ocular diseases have been identified, and animal models for such diseases have been developed, which have greatly facilitated the application of experimental rAAV-mediated gene therapy. This review highlights the key features of rAAV-mediated gene therapy that make it the most suitable gene therapy treatment approach for ocular diseases. Furthermore, it summarizes the current progress of rAAV-mediated gene therapy interventions/applications for a wide variety of ophthalmologic disorders.

  14. Recombinant adeno-associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult microglia.

    Science.gov (United States)

    Su, Wei; Kang, John; Sopher, Bryce; Gillespie, James; Aloi, Macarena S; Odom, Guy L; Hopkins, Stephanie; Case, Amanda; Wang, David B; Chamberlain, Jeffrey S; Garden, Gwenn A

    2016-01-01

    Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. Neonatal microglia are functionally distinct from adult microglia, although the majority of in vitro studies utilize rodent neonatal microglia cultures because of difficulties of culturing adult cells. In addition, cultured microglia are refractory to most methods for modifying gene expression. Here, we developed a novel protocol for culturing adult microglia and evaluated the feasibility and efficiency of utilizing Recombinant Adeno-Associated Virus (rAAV) to modulate gene expression in cultured microglia.

  15. Putative recombination events and evolutionary history of five economically important viruses of fruit trees based on coat protein-encoding gene sequence analysis.

    Science.gov (United States)

    Boulila, Moncef

    2010-06-01

    To enhance the knowledge of recombination as an evolutionary process, 267 accessions retrieved from GenBank were investigated, all belonging to five economically important viruses infecting fruit crops (Plum pox, Apple chlorotic leaf spot, Apple mosaic, Prune dwarf, and Prunus necrotic ringspot viruses). Putative recombinational events were detected in the coat protein (CP)-encoding gene using RECCO and RDP version 3.31beta algorithms. Based on RECCO results, all five viruses were shown to contain potential recombination signals in the CP gene. Reconstructed trees with modified topologies were proposed. Furthermore, RECCO performed better than the RDP package in detecting recombination events and exhibiting their evolution rate along the sequences of the five viruses. RDP, however, provided the possible major and minor parents of the recombinants. Thus, the two methods should be considered complementary.

  16. Alteration of a recombinant protein N-glycan structure in silkworms by partial suppression of N-acetylglucosaminidase gene expression.

    Science.gov (United States)

    Kato, Tatsuya; Kikuta, Kotaro; Kanematsu, Ayumi; Kondo, Sachiko; Yagi, Hirokazu; Kato, Koichi; Park, Enoch Y

    2017-09-01

    To synthesize complex type N-glycans in silkworms, shRNAs against the fused lobe from Bombyx mori (BmFDL), which codes N-acetylglucosaminidase (GlcNAcase) in the Golgi, was expressed by recombinant B. mori nucleopolyhedrovirus (BmNPV) in silkworm larvae. Expression was under the control of the actin promoter of B. mori or the U6-2 and i.e.-2 promoters from Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). The reduction of specific GlcNAcase activity was observed in Bm5 cells and silkworm larvae using the U6-2 promoter. In silkworm larvae, the partial suppression of BmFDL gene expression was observed. When shRNA against BmFDL was expressed under the control of U6-2 promoter, the Man3GlcNAc(Fuc)GlcNAc structure appeared in a main N-glycans of recombinant human IgG. These results suggested that the control of BmFDL expression by its shRNA in silkworms caused the modification of its N-glycan synthetic pathway, which may lead to the alteration of N-glycans in the expressed recombinant proteins. Suppression of BmFDL gene expression by shRNA is not sufficient to synthesize complex N-glycans in silkworm larvae but can modify the N-glycan synthetic pathway.

  17. Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Ivanov Ivan

    2011-03-01

    Full Text Available Abstract Background Segregation of expression plasmids leads to loss of recombinant DNA from transformed bacterial cells due to the irregular distribution of plasmids between the daughter cells during cell division. Under non-selective conditions this segregational instability results in a heterogeneous population of cells, where the non-productive plasmid-free cells overgrow the plasmid-bearing cells thus decreasing the yield of recombinant protein. Amongst the factors affecting segregational plasmid instability are: the plasmid design, plasmid copy-number, host cell genotype, fermentation conditions etc. This study aims to investigate the influence of transcription and translation on the segregation of recombinant plasmids designed for constitutive gene expression in Escherichia coli LE392 at glucose-limited continuous cultivation. To this end a series of pBR322-based plasmids carrying a synthetic human interferon-gamma (hIFNγ gene placed under the control of different regulatory elements (promoter and ribosome-binding sites were used as a model. Results Bacterial growth and product formation kinetics of transformed E. coli LE392 cells cultivated continuously were described by a structured kinetic model proposed by Lee et al. (1985. The obtained results demonstrated that both transcription and translation efficiency strongly affected plasmid segregation. The segregation of plasmid having a deleted promoter did not exceed 5% after 190 h of cultivation. The observed high plasmid stability was not related with an increase in the plasmid copy-number. A reverse correlation between the yield of recombinant protein (as modulated by using different ribosome binding sites and segregational plasmid stability (determined by the above model was also observed. Conclusions Switching-off transcription of the hIFNγ gene has a stabilising effect on ColE1-like plasmids against segregation, which is not associated with an increase in the plasmid copy

  18. Relative contributions of recombination and mutation to the diversification of the opa gene repertoire of Neisseria gonorrhoeae.

    Science.gov (United States)

    Bilek, Nicole; Ison, Catherine A; Spratt, Brian G

    2009-03-01

    To understand the rates and mechanisms of Neisseria gonorrhoeae opa gene variation, the 11 opa genes were amplified independently so that an opa allelic profile could be defined for any isolate from the sequences at each locus. The opa allelic profiles from 14 unrelated isolates were all different, with no opa alleles shared between isolates. Examination of very closely related isolates from sexual contacts and sexual networks showed that these typically shared most opa alleles, and the mechanisms by which recent changes occurred at individual opa loci could be determined. The great majority of changes were due to recombination among existing alleles that duplicated an opa allele present at another locus or resulted in a mosaic of existing opa alleles. Single nucleotide changes or insertion/deletion of a single codon also occurred, but few of these events were assigned to mutation, the majority being assigned to localized recombination. Introduction of novel opa genes from coinfecting strains was rare, and all but one were observed in the same sexual network. Changes at opa loci occurred at a greater rate than those at the porin locus, and the opa11 locus changed more rapidly than other opa loci, almost always differing even between recent sexual contacts. Examination of the neighboring pilE gene showed that changes at opa11 and pilE often occurred together, although this linkage may not be a causal one.

  19. Genes encoding homologous antigens in taeniid cestode parasites: Implications for development of recombinant vaccines produced in Escherichia coli.

    Science.gov (United States)

    Gauci, Charles; Lightowlers, Marshall W

    2013-01-01

    Recombinant vaccine antigens are being evaluated for their ability to protect livestock animals against cysticercosis and related parasitic infections. Practical use of some of these vaccines is expected to reduce parasite transmission, leading to a reduction in the incidence of neurocysticercosis and hydatid disease in humans. We recently showed that an antigen (TSOL16), expressed in Escherichia coli, confers high levels of protection against Taenia solium cysticercosis in pigs, which provides a strategy for control of T. solium parasite transmission. Here, we discuss the characteristics of this antigen that may affect the utility of TSOL16 and related antigens for development as recombinant vaccines. We also report that genes encoding antigens closely related to TSOL16 from T. solium also occur in other related species of parasites. These highly homologous antigens have the potential to be used as vaccines and may provide protection against related species of Taenia that cause infection in other hosts.

  20. Phylogenetic analysis of VP1 gene sequences of waterfowl parvoviruses from the Mainland of China revealed genetic diversity and recombination.

    Science.gov (United States)

    Wang, Shao; Cheng, Xiao-Xia; Chen, Shao-Ying; Lin, Feng-Qiang; Chen, Shi-Long; Zhu, Xiao-Li; Wang, Jin-Xiang; Huang, Mei-Qing; Zheng, Min

    2016-03-01

    To determine the origin and evolution of goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) in the Mainland of China, phylogenetic and recombination analyses in the present study were performed on 32 complete VP1 gene sequences from China and other countries. Based on the phylogenetic analysis of the VP1 gene, GPV strains studied here from Mainland China (PRC) could be divided into three genotypes, namely PRC-I, PRC-II and PRC-III. Genotype PRC-I is indigenous to Mainland China. Only one GPV strain from Northeast China was of Genotype PRC-II and was thought to be imported from Europe. Genotype PRC-III, which was the most isolated genotype during 1999-2012, is related to GPVs in Taiwan and has been the predominant pathogen responsible for recent Derzy's disease outbreaks in Mainland China. Current vaccine strains used in Mainland China belong to Genotype PRC-I that is evolutionary distant from Genotypes PRC-II and PRC-III. In comparison, MDPV strains herein from Mainland China are clustered in a single group which is closely related to Taiwanese MDPV strains, and the full-length sequences of the VP1 gene of China MDPVs are phylogenetic closely related to the VP1 sequence of a Hungarian MDPV strain. Moreover, We also found that homologous recombination within VP1 gene plays a role in generating genetic diversity in GPV evolution. The GPV GDFSh from Guangdong Province appears to be the evolutionary product of a recombination event between parental GPV strains GD and B, while the major parent B proved to be a reference strain for virulent European GPVs. Our findings provide valuable information on waterfowl parvoviral evolution in Mainland China.

  1. Construction and characterization of a recombinant fowlpox virus containing HIV-1 multi-epitope-p24 chimeric gene in mice

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The epidemic of HIV/AIDS is sweeping across the world. It is of great importance to figure out new ways to curb this disease. Epitope-based vaccine is one of these solutions. In this study, a chimeric gene was obtained by combination of a designed HIV-1 multi-epitope gene (MEG) and HIV-1 p24 gene. A re- combinant plasmid pUTA2-MEGp24 was then constructed by inserting MEGp24 gene into the down- stream of the promoter (ATI-P7.5×20) of fowlpox virus (FPV) transfer vector pUTA2. The recombinant plasmid and wild-type FPV 282E4 strain were then co-transfected into CEF cells and homologous re- combination occurred. A recombinant virus expressing HIV-1 protein MEGp24 was screened by ge- nome PCR and Western blot assay. Large scale preparation and purification of the recombinant fowl- pox virus (rFPV) were then carried out. BALB/c mice were immunized intramuscularly with the rFPV for three times on day 0, 14 and 42. Mice were executed and sampled one week after the third inoculation. Anti-HIV-1 antibody in serum and Th1 cytokines in the supernatant of cultured spleen cells were as- sayed by ELISA. The count of T lymphocyte subsets and the CTL activity of spleen lymphocytes were analyzed by flow cytometry and lactate dehydrogenase (LDH) release assay, respectively. The results showed that HIV-1 specific antibody in serum and increased T lymphocyte subsets (CD4+ T, CD8+ T) were detected in the immunization group. CTL target-killing activity and higher secretion of Th1 cyto- kines (IFN-γ and IL-2) of spleen lymphocytes stimulated by H-2d-restricted CTL peptide were observed in immunized mice. We concluded that the rFPV may induce HIV-1 specific immunity especially cellular immunity in mice.

  2. Change of gene structure and function by non-homologous end-joining, homologous recombination, and transposition of DNA.

    Science.gov (United States)

    Goettel, Wolfgang; Messing, Joachim

    2009-06-01

    An important objective in genome research is to relate genome structure to gene function. Sequence comparisons among orthologous and paralogous genes and their allelic variants can reveal sequences of functional significance. Here, we describe a 379-kb region on chromosome 1 of maize that enables us to reconstruct chromosome breakage, transposition, non-homologous end-joining, and homologous recombination events. Such a high-density composition of various mechanisms in a small chromosomal interval exemplifies the evolution of gene regulation and allelic diversity in general. It also illustrates the evolutionary pace of changes in plants, where many of the above mechanisms are of somatic origin. In contrast to animals, somatic alterations can easily be transmitted through meiosis because the germline in plants is contiguous to somatic tissue, permitting the recovery of such chromosomal rearrangements. The analyzed region contains the P1-wr allele, a variant of the genetically well-defined p1 gene, which encodes a Myb-like transcriptional activator in maize. The P1-wr allele consists of eleven nearly perfect P1-wr 12-kb repeats that are arranged in a tandem head-to-tail array. Although a technical challenge to sequence such a structure by shotgun sequencing, we overcame this problem by subcloning each repeat and ordering them based on nucleotide variations. These polymorphisms were also critical for recombination and expression analysis in presence and absence of the trans-acting epigenetic factor Ufo1. Interestingly, chimeras of the p1 and p2 genes, p2/p1 and p1/p2, are framing the P1-wr cluster. Reconstruction of sequence amplification steps at the p locus showed the evolution from a single Myb-homolog to the multi-gene P1-wr cluster. It also demonstrates how non-homologous end-joining can create novel gene fusions. Comparisons to orthologous regions in sorghum and rice also indicate a greater instability of the maize genome, probably due to diploidization

  3. Origin of the CMS gene locus in rapeseed cybrid mitochondria: active and inactive recombination produces the complex CMS gene region in the mitochondrial genomes of Brassicaceae.

    Science.gov (United States)

    Oshima, Masao; Kikuchi, Rie; Imamura, Jun; Handa, Hirokazu

    2010-01-01

    CMS (cytoplasmic male sterile) rapeseed is produced by asymmetrical somatic cell fusion between the Brassica napus cv. Westar and the Raphanus sativus Kosena CMS line (Kosena radish). The CMS rapeseed contains a CMS gene, orf125, which is derived from Kosena radish. Our sequence analyses revealed that the orf125 region in CMS rapeseed originated from recombination between the orf125/orfB region and the nad1C/ccmFN1 region by way of a 63 bp repeat. A precise sequence comparison among the related sequences in CMS rapeseed, Kosena radish and normal rapeseed showed that the orf125 region in CMS rapeseed consisted of the Kosena orf125/orfB region and the rapeseed nad1C/ccmFN1 region, even though Kosena radish had both the orf125/orfB region and the nad1C/ccmFN1 region in its mitochondrial genome. We also identified three tandem repeat sequences in the regions surrounding orf125, including a 63 bp repeat, which were involved in several recombination events. Interestingly, differences in the recombination activity for each repeat sequence were observed, even though these sequences were located adjacent to each other in the mitochondrial genome. We report results indicating that recombination events within the mitochondrial genomes are regulated at the level of specific repeat sequences depending on the cellular environment.

  4. Inhibition of tumor growth in xenografted nude mice with adenovirus-mediated endostatin gene comparison with recombinant endostatin protein

    Institute of Scientific and Technical Information of China (English)

    梁志慧; 吴沛宏; 李立; 薛刚; 曾益新; 黄文林

    2004-01-01

    Background Inhibition of tumor growth by endostatin has been shown to be an effective strategy in cancer therapy in mice. However, its widespread application has been hampered by difficulties in a large-scale production of the recombinant endostatin protein, rapid loss bioactivity of the protein, and the cumbersome daily administration. These limitations could be resolved by in vivo delivery and expression of the endostatin gene. In this study, we observed the effect and advantage of endostatin gene therapy mediated by a recombinant adenoviral vector (Ad/hEndo) on the growth of hepatocellular carcinoma BEL-7402 xenografted tumors, comparison with recombinant endostatin protein.Results After 4 courses of treatment, the tumor growth rates of high-dose treated group with 1×109 pfu of Ad/hEndo were inhibited by 42.26% compared with the Ad/LacZ control group (P=0.001) and by 46.26% compared with the NIH buffer control group (P=0.003), respectively. However, in this study, Ad/hEndo at low dose of 5×108 pfu failed to demonstrate significant inhibition of tumor growth, compared with control groups. After daily administration of recombinant human endostatin protein (rhEndo) for 9 days, the ratio of T/C (rhEndo group versus PBS group) was less than 47%. However, two days after rhEndo treatment ceased, the ratio of T/C was more than 50%. The peak of expression of endostatin mRNA in tumor tissue was at 2 or 3 days after administration intratumorally with Ad/hEndo of 1×109 pfu and gradually dropped undetectable by day 7. Dynamic analysis of endostatin concentration in tumor tissue showed that the highest level of mRNA is up at the third day after injection, and dropped to basal level three weeks later.Conclusions Endostatin gene therapy mediated by a recombinant adenoviral vector had significantly inhibited the growth of hepatocellular carcinoma BEL-7402 xenografted tumors at a high dose of 1×109 pfu compared with other groups. The analysis of dynamic expression of

  5. A tale of two methane monooxygenases

    Science.gov (United States)

    Ross, Matthew O.

    2017-01-01

    Methane monooxygenase (MMO) enzymes activate O2 for oxidation of methane. Two distinct MMOs exist in nature, a soluble form that uses a diiron active site (sMMO) and a membrane-bound form with a catalytic copper center (pMMO). Understanding the reaction mechanisms of these enzymes is of fundamental importance to biologists and chemists, and is also relevant to the development of new biocatalysts. The sMMO catalytic cycle has been elucidated in detail, including O2 activation intermediates and the nature of the methane-oxidizing species. By contrast, many aspects of pMMO catalysis remain unclear, most notably the nuclearity and molecular details of the copper active site. Here, we review the current state of knowledge for both enzymes, and consider pMMO O2 activation intermediates suggested by computational and synthetic studies in the context of existing biochemical data. Further work is needed on all fronts, with the ultimate goal of understanding how these two remarkable enzymes catalyze a reaction not readily achieved by any other metalloenzyme or biomimetic compound. PMID:27878395

  6. A concept of eliminating nonhomologous recombination for scalable and safe AAV vector generation for human gene therapy.

    Science.gov (United States)

    Dong, Biao; Moore, Andrea R; Dai, Jihong; Roberts, Sean; Chu, Kirk; Kapranov, Philipp; Moss, Bernard; Xiao, Weidong

    2013-07-01

    Scalable and efficient production of high-quality recombinant adeno-associated virus (rAAV) for gene therapy remains a challenge despite recent clinical successes. We developed a new strategy for scalable and efficient rAAV production by sequestering the AAV helper genes and the rAAV vector DNA in two different subcellular compartments, made possible by using cytoplasmic vaccinia virus as a carrier for the AAV helper genes. For the first time, the contamination of replication-competent AAV particles (rcAAV) can be completely eliminated in theory by avoiding ubiquitous nonhomologous recombination. Vector DNA can be integrated into the host genomes or delivered by a nuclear targeting vector such as adenovirus. In suspension HeLa cells, the achieved vector yield per cell is similar to that from traditional triple-plasmid transfection method. The rcAAV contamination was undetectable at the limit of our assay. Furthermore, this new concept can be used not only for production of rAAV, but also for other DNA vectors.

  7. Analysis of the L1 gene product of human papillomavirus type 16 by expression in a vaccinia virus recombinant.

    Science.gov (United States)

    Browne, H M; Churcher, M J; Stanley, M A; Smith, G L; Minson, A C

    1988-06-01

    The L1 open reading frame of human papillomavirus type 16 (HPV16) has been expressed in vaccinia virus under the control of both the 7.5K early and late promoter, and the 4b major late promoter. Antibodies to a beta-galactosidase fusion protein containing a C-terminal portion of the HPV16 L1 gene product were used to compare the levels of L1 expression in the two recombinants, and showed that greater levels of expression were obtained when the gene was placed under the control of the 4b late promoter. Immunofluorescence studies revealed a nuclear location of the L1 gene product when expressed in vaccinia virus. Antibodies to the beta-galactosidase fusion protein detected a major polypeptide species of 57K and a minor species of 64K in Western blots of recombinant-infected cell lysates. The 64K species was not detected when cells were infected in the presence of tunicamycin, indicating that the primary translation product of the HPV16 L1 open reading frame is modified by N-linked glycosylation when expressed in vaccinia virus. Whereas antibodies to HPV16 L1 fusion proteins and to a peptide containing amino acids from the C terminus of HPV16 L1 reacted well in Western blots with the HPV16 L1 target expressed in vaccinia virus, no reactivity was observed with antibodies to bovine papillomavirus type 1 particles or to a HPV6b fusion protein.

  8. Induction of Epstein-Barr Virus Lytic Replication by Recombinant Adenoviruses Expressing the Zebra Gene with EBV Specific Promoters

    Institute of Scientific and Technical Information of China (English)

    Lu CHEN; Juan YIN; Yi CHEN; Jiang ZHONG

    2005-01-01

    The latent Epstein-Barr virus (EBV) is found in the cells of many tumors. For example, EBV is detectable in almost all cases, and in almost all tumor cells, of non-keratinizing nasopharyngeal carcinoma.Activating the latent virus, which will result in its lytic replication and the death of tumor cells, is a potential approach for the treatment of EBV-associated cancers. In this study, three recombinant adenoviruses were constructed to express the Zebra gene, an EBV gene responsible for switching from the latent state to lytic replication. EBV-specific promoters were used in order to limit Zebra expression in EBV-positive cells, and reduce the potential side effects. The EBV promoters used were Cp, Zp and a dual promoter combining both promoters, CpZp. The Zebra protein was detected in HEK293 cells as well as the EBV-positive D98-HR1 cells infected with recombinant viruses. An EBV lytic replication early antigen, EA-D, was also detected in infected D98-HR1, implying the initiation of lytic replication. In the cell viability assay, Zebra-expressing adenoviruses had little effect on EBV-negative HeLa cells, while significantly reducing the cell viability and proliferation of D98-HR1 cells. The results indicate that EBV virus promoters can be used in adenovirus vectors to express the Zebra gene and induce EBV lytic replication in D98-HR1 cells.

  9. Gene transfer into primary cultures of fetal neural stem cells by a recombinant adenovirus carrying the gene for green fluorescent protein

    Institute of Scientific and Technical Information of China (English)

    Yong FU; Shen-qing WANG; Ying-peng LIU; Guo-peng WANG; Jian-ting WANG; Shu-sheng GONG

    2008-01-01

    Objective: To evaluate the transduction efficiency of a recombinant adenovirus carrying the gene for green fluorescent protein (Ad-GFP) into the primary cultures of fetal neural stem cells (NSCs) by the expression of GFP. Methods: The Ad-GFP was constructed by homologous recombination in bacteria with the AdEasy system; NSCs were isolated from rat fetal hippocampus and cultured as neurosphere suspensions. After infection with the recombinant Ad-GFP, NSCs were examined with a fluorescent microscopy and a flow cytometry for their expression of GFP. Results: After the viral infection, flow cytometry analysis revealed that the percentage of GFP-positive cells was as high as 97.05%. The infected NSCs sustained the GFP expression for above 4 weeks. After differentiated into astrocytes or neurons, they continued to express GFP efficiently. Conclusion: We have successfully constructed a viral vector Ad-GFP that can efficiently infect the primary NSCs. The reporter gene was showed fully and sustained expression in the infected cells as well as their differentiated progenies.

  10. Meiotic homoeologous recombination-based alien gene introgression in the genomics era of wheat

    Science.gov (United States)

    Wheat (Triticum spp.) has a narrow genetic basis due to its allopolyploid origin. However, wheat has numerous wild relatives usable for expanding genetic variability of its genome through meiotic homoeologous recombination. Traditionally, laborious cytological analyses have been employed to detect h...

  11. Base composition, selection, and phylogenetic significance of indels in the recombination activating gene-1 in vertebrates

    NARCIS (Netherlands)

    Chiari, Y.; Meijden, van der A.; Madsen, O.; Vences, M.; Meyer, A.

    2009-01-01

    Background: The Recombination Activating Proteins, RAG1 and RAG2, play a crucial role in the immune response in vertebrates. Among the nuclear markers currently used for phylogenetic purposes, Rag1 has especially enjoyed enormous popularity, since it successfully contributed to elucidating the relat

  12. Productivity of cyclohexanone oxidation of the recombinant Corynebacterium glutamicum expressing chnB of Acinetobacter calcoaceticus.

    Science.gov (United States)

    Doo, Eun-Hee; Lee, Won-Heong; Seo, Hyo-Seel; Seo, Jin-Ho; Park, Jin-Byung

    2009-06-15

    The biocatalytic efficiency of recombinant Corynebacterium glutamicum expressing the chnB gene encoding cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871 was investigated. Optimization of an expression system and induction conditions enabled the recombinant biocatalyst to produce CHMO to a specific activity of ca. 0.5 U mg(-1) protein. Tight control of feeding of an energy source (i.e., glucose) and dissolved oxygen tension during fed-batch culture-based biotransformation allowed the cells to produce epsilon-caprolactone to a concentration of 16.0 g l(-1). The specific and volumetric productivity for cyclohexanone oxidation were 0.12 g g drycells(-1)h(-1) (17.5 U g(-1) of dry cells) and 2.3 g l(-1)h(-1) (330 U l(-1)), respectively. These values correspond to over 5.4- and 2.7-fold of recombinant Escherichia coli expressing the same gene under similar reaction conditions. It could be concluded that the recombinant C. glutamicum is a promising biocatalyst for Baeyer-Villiger oxidations.

  13. Towards practical Baeyer-Villiger-monooxygenases: design of cyclohexanone monooxygenase mutants with enhanced oxidative stability.

    Science.gov (United States)

    Opperman, Diederik J; Reetz, Manfred T

    2010-12-10

    Baeyer-Villiger monooxygenases (BVMOs) catalyze the conversion of ketones and cyclic ketones into esters and lactones, respectively. Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 is known to show an impressive substrate scope as well as exquisite chemo-, regio-, and enantioselectivity in many cases. Large-scale synthetic applications of CHMO are hampered, however, by the instability of the enzyme. Oxidation of cysteine and methionine residues contributes to this instability. Designed mutations of all the methionine and cysteine residues in the CHMO wild type (WT) showed that the amino acids labile towards oxidation are mostly either surface-exposed or located within the active site, whereas the two methionine residues identified for thermostabilization are buried within the folded protein. Combinatorial mutations gave rise to two stabilized mutants with either oxidative or thermal stability, without compromising the activity or stereoselectivity of the enzyme. The most oxidatively stabilized mutant retained nearly 40 % of its activity after incubation with H(2)O(2) (0.2 M), whereas the wild-type enzyme's activity was completely abolished at concentrations as low as 5 mM H(2)O(2). We propose that oxidation-stable mutants might well be a "prerequisite" for thermostabilization, because laboratory-evolved thermostability in CHMO might be masked by a high degree of oxidation instability.

  14. Genetic control of the immune response to staphylococcal nuclease. IX. Recombination between genes determining BALB/c antinuclease idiotypes and the heavy chain allotype locus.

    Science.gov (United States)

    Pisetsky, D S; Riordan, S E; Sachs, D H

    1979-03-01

    The genetic linkage relationship of two antinuclease idiotypes produced by the BALB/c strain was investigated in the backcross (BALB/c x CB.20) X CB.20. These two idiotypes were detected by Lewis rat anti-idiotypic antisera prepared against affinity-purified A/J and SJL antinuclease antibodies, termed the A/J and SJL idiotypes, respectively. Both idiotypes were found to be linked to the IgCHa immunoglobulin heavy chain allotype locus. There was, however, a high frequency of recombination observed between both markers and the IgCHa locus, with eight of 83 backcross animals recombinant for the A/J idiotype and five of 83 recombinant for the SJL idiotype. All such recombinant animals were IgCHb/b homozygotes that had gained one or both idiotypes. These results are consistent with a genetic map of VHr region genes in the BALB/c strain in which genes determining the SJL idiotype are closer to the IgCHa allotype locus than are genes determining the A/J idiotype. This high frequency of recombination may indicate that the chromosome segment containing VH region genes is very large or that it has structural features that promote recombination.

  15. Differential pathogenicity of two different recombinant PVYNTN isolates in Physalis floridana is likely determined by the coat protein gene

    Directory of Open Access Journals (Sweden)

    Xiong Xingyao

    2011-05-01

    Full Text Available Abstract A previous study has identified two types of recombinant variants of Potato virus Y strain NTN (PVYNTN in China and sequenced the complete genome of the variant PVYNTN-HN2. In this study, the complete genome of isolate PVYNTN-HN1 was fully sequenced and analyzed. The most striking difference between the two variants was the location of recombinant joint three (RJ3. In PVYNTN-HN1, like other typical European-PVYNTN isolates such as PVYNTN-Hun, the RJ3 was located at nucleotide (nt 9183, namely the 3' proximal end of the CP gene (nt. 8571-9371, thus leading to most (the first 613 nucleotides from the 5' proximal end of the CP gene (801 bp with a PVYN origin and PVYN-serotype; whereas in contrast, the RJ3 in PVYNTN-HN2 was located at nt 8572, consequently leading to a CP gene of PVYO origin and PVYO-serotype. The varied genome composition among PVYO, PVYN, PVYN:O, PVYNTN-HN1 and PVYNTN-HN2 made them useful for the investigation of possible roles of gene segment(s in symptom formation on host plants. When Physalis floridana plants were infected with different PVY isolates, two types of symptoms were induced. PVYN and PVYNTN-HN1 induced mild symptoms (mainly mild mottling whereas PVYO, PVYN:O and PVYNTN-HN2 induced serve symptoms including leaf and stem necrosis, leaf-drop and stunting. These results, together with a previous study using artificial PVY chimeras, demonstrate that the CP gene, especially the 5' proximal segment (nt 8572-9183, and/or CP likely determine the pathogenicity of PVY in P. floridana.

  16. Recombination Rate Variation Modulates Gene Sequence Evolution Mainly via GC-Biased Gene Conversion, Not Hill-Robertson Interference, in an Avian System.

    Science.gov (United States)

    Bolívar, Paulina; Mugal, Carina F; Nater, Alexander; Ellegren, Hans

    2016-01-01

    The ratio of nonsynonymous to synonymous substitution rates (ω) is often used to measure the strength of natural selection. However, ω may be influenced by linkage among different targets of selection, that is, Hill-Robertson interference (HRI), which reduces the efficacy of selection. Recombination modulates the extent of HRI but may also affect ω by means of GC-biased gene conversion (gBGC), a process leading to a preferential fixation of G:C ("strong," S) over A:T ("weak," W) alleles. As HRI and gBGC can have opposing effects on ω, it is essential to understand their relative impact to make proper inferences of ω. We used a model that separately estimated S-to-S, S-to-W, W-to-S, and W-to-W substitution rates in 8,423 avian genes in the Ficedula flycatcher lineage. We found that the W-to-S substitution rate was positively, and the S-to-W rate negatively, correlated with recombination rate, in accordance with gBGC but not predicted by HRI. The W-to-S rate further showed the strongest impact on both dN and dS. However, since the effects were stronger at 4-fold than at 0-fold degenerated sites, likely because the GC content of these sites is farther away from its equilibrium, ω slightly decreases with increasing recombination rate, which could falsely be interpreted as a consequence of HRI. We corroborated this hypothesis analytically and demonstrate that under particular conditions, ω can decrease with increasing recombination rate. Analyses of the site-frequency spectrum showed that W-to-S mutations were skewed toward high, and S-to-W mutations toward low, frequencies, consistent with a prevalent gBGC-driven fixation bias.

  17. The promastigote surface antigen gene family of the Leishmania parasite: differential evolution by positive selection and recombination

    Directory of Open Access Journals (Sweden)

    Bañuls Anne-Laure

    2008-10-01

    Full Text Available Abstract Background PSA (promastigote surface antigen is one of the major classes of membrane proteins present at the surface of the parasitic protozoan Leishmania. While it harbours leucine rich repeats, which are suggestive of its involvement in parasite-to-host physical interactions, its exact role is largely unknown. Furthermore, the extent of diversity of this gene family, both in copy number and sequence has not been established. Results From the newly available complete genome sequences of L. major, L. infantum and L. braziliensis, we have established the complete list of PSA genes, based on the conservation of specific domain architecture. The latter includes an array of leucine rich repeats of unique signature flanked by conserved cysteine-rich domains. All PSA genes code either for secreted or membrane-anchored surface proteins. Besides the few previously identified PSA genes, which are shown here to be part of a relatively large subclass of PSA genes located on chromosome 12, this study identifies seven other PSA subtypes. The latter, whose genes lie on chromosomes 5, 9, 21 and 31 in all three species, form single gene (two genes in one instance subfamilies, which phylogenetically cluster as highly related orthologs. On the other hand, genes found on chromosome 12 generally show high diversification, as reflected in greater sequence divergence between species, and in an extended set of divergent paralogs. Moreover, we show that the latter genes are submitted to strong positive selection. We also provide evidence that evolution of these genes is driven by intra- and intergenic recombination, thereby modulating the number of LRRs in protein and generating chimeric genes. Conclusion PSA is a Leishmania family of membrane-bound or secreted proteins, whose main signature consists in a specific LRR sequence. All PSA genes found in the genomes of three sequenced Leishmania species unambiguously distribute into eight subfamilies of orthologs

  18. Highly diverse TCRα chain repertoire of pre-immune CD8⁺ T cells reveals new insights in gene recombination.

    Science.gov (United States)

    Genolet, Raphael; Stevenson, Brian J; Farinelli, Laurent; Osterås, Magne; Luescher, Immanuel F

    2012-04-04

    Although the T-cell receptor αδ (TCRαδ) locus harbours large libraries of variable (TRAV) and junctional (TRAJ) gene segments, according to previous studies the TCRα chain repertoire is of limited diversity due to restrictions imposed by sequential coordinate TRAV-TRAJ recombinations. By sequencing tens of millions of TCRα chain transcripts from naive mouse CD8(+) T cells, we observed a hugely diverse repertoire, comprising nearly all possible TRAV-TRAJ combinations. Our findings are not compatible with sequential coordinate gene recombination, but rather with a model in which contraction and DNA looping in the TCRαδ locus provide equal access to TRAV and TRAJ gene segments, similarly to that demonstrated for IgH gene recombination. Generation of the observed highly diverse TCRα chain repertoire necessitates deletion of failed attempts by thymic-positive selection and is essential for the formation of highly diverse TCRαβ repertoires, capable of providing good protective immunity.

  19. Development of wheat-Aegilops speltoides recombinants and simple PCR-based markers for stem rust resistance genes on the 2S#1 chromosome

    Science.gov (United States)

    Wild relatives of wheat are important but underutilized resources for new rust resistance genes because linked negative traits often hinder deployment of these genes in commercial wheats. Here we report reduced alien chromatin recombinants derived from E.R. Sears' wheat-Aegilops speltoides transloca...

  20. Recombinant Newcastle disease virus (NDV) with inserted gene coding for GM-CSF as a new vector for cancer immunogene therapy

    NARCIS (Netherlands)

    Janke, M.; Peeters, B.P.H.; Leeuw, de O.S.; Moormann, R.J.M.; Arnold, A.; Fournier, P.; Schirrmacher, V.

    2007-01-01

    This is the first report describing recombinant (rec) Newcastle disease virus (NDV) as vector for gene therapy of cancer. The gene encoding granulocyte/macrophage colony-stimulating factor (GM-CSF) was inserted as an additional transcription unit at two different positions into the NDV genome. The r

  1. The p450 monooxygenase BcABA1 is essential for abscisic acid biosynthesis in Botrytis cinerea

    DEFF Research Database (Denmark)

    Siewers, V.; Smedsgaard, Jørn; Tudzynski, P.

    2004-01-01

    The phytopathogenic ascomycete Botrytis cinerea is known to produce abscisic acid (ABA), which is thought to be involved in host-pathogen interaction. Biochemical analyses had previously shown that, in contrast to higher plants, the fungal ABA biosynthesis probably does not proceed via carotenoids...... but involves direct cyclization of farnesyl diphosphate and subsequent oxidation steps. We present here evidence that this "direct" pathway is indeed the only one used by an ABA-overproducing strain of B. cinerea. Targeted inactivation of the gene bccpr1 encoding a cytochrome P450 oxidoreductase reduced...... the ABA production significantly, proving the involvement of P450 monooxygenases in the pathway. Expression analysis of 28 different putative P450 monooxygenase genes revealed two that were induced under ABA biosynthesis conditions. Targeted inactivation showed that one of these, bcaba1, is essential...

  2. In vitro Recombination and Identification of Mutated Fragment Corresponding to Regulation Region of mtrR Gene of Neisseria Gonorrhoeae

    Institute of Scientific and Technical Information of China (English)

    HUANG Changzheng; LIN Nengxing; TU Yating; LIAN Xin; KANG Jian; ZHU Li

    2007-01-01

    A site-directed mutant DNA fragment was synthesized and transfected into clinical Neisseria Gonorrhoeae (NG) stains to construct the transformants that contained the corresponding mutagenesis of regulation region of mtrR gene. According to the technique of gene splicing by over-lap extension (SOEing), a DNA segment with specific mutagenesis was constructed by two-step po-lymerase chain reaction (PCR). The mutation fragments EF could be used for the next experiment in which the mutation NG strains were induced. By comparing the recombinant EF fragments to the corresponding DNA fragments of clinical NG strains, 2 of these were not compatible completely. The results of sequencing revealed that there was a 9 bp deletion between the 45 to 54 inverted repeat se-quence localized within the mtrR promoter. It can be confirmed that the fragments EF are the specifi-cally designed mutant fragments.

  3. Recombinant receptor/reporter gene bioassays for assessing the estrogenic and dioxin-like activities of xenobiotics and complex mixtures

    Energy Technology Data Exchange (ETDEWEB)

    Zacharewski, T. [Univ. of Western Ontario, London, Ontario (Canada). Dept. of Pharmacology and Toxicology

    1995-12-31

    Exposure to naturally occurring or synthetic substances that possess sex steroid and/or dioxin-like activity may have long range effects on human health, reproductive fitness and environmental quality. Results from recent epidemiological studies have suggested that xenobiotics with sex steroid activity may contribute to the development of hormone-dependent cancers and disorders in the male reproductive tract as well as attenuate sperm production. However, most of these compounds, which are referred to as endocrine disruptors, are structurally dissimilar to sex steroids. Yet, based upon ambiguous assays, it has been conceded that the effects of these compounds are mediated by receptors. The authors have taken advantage of the mechanism of action of these compounds to develop recombinant receptor/reporter gene bioassays for environmental estrogens and dioxin-like compounds. The assays use an easily measurable enzyme activity (i.e. firefly luciferase), exhibit improved sensitivity and selectivity and are amenable to automation. Data will be presented demonstrating that phytoestrogens (e.g. genistein) and xenobiotics such as pesticides (e.g. DDT, Kepone), nonionic surfactants (e.g. p-nonylphenol), and precursors used in the manufacture of plastics (e.g. Bisphenol A) exhibit estrogenic activity. In addition, the assays have been used to detect estrogenic and dioxin-like activity in complex mixtures such as pulp and paper mill black liquor and effluent. These results demonstrate the utility of recombinant receptor/reporter gene bioassays for identifying substances or complex mixtures with estrogenic and/or dioxin-like activity.

  4. Characterization of Alu and recombination-associated motifs mediating a large homozygous SPG7 gene rearrangement causing hereditary spastic paraplegia.

    Science.gov (United States)

    López, Eva; Casasnovas, Carlos; Giménez, Javier; Matilla-Dueñas, Antoni; Sánchez, Ivelisse; Volpini, Víctor

    2015-04-01

    Spastic paraplegia type 7 (SPG7) is one of the most common forms of autosomal recessive hereditary spastic paraplegia (AR-HSP). Although over 77 different mutations have been identified in SPG7 patients, only 9 gross deletions have been reported with only a few of them being fully characterized. Here, we present a detailed description of a large homozygous intragenic SPG7 gene rearrangement involving a 5144-base pair (bp) genomic loss (c. 1450-446_1779 + 746 delinsAAAGTGCT) encompassing exons 11 to 13, identified in a Spanish AR-HSP family. Analysis of the deletion junction sequences revealed that the 5' breakpoint of this SPG7 gene deletion was located within highly homologous Alu sequences where the 3' breakpoint appears to be flanked by the core crossover hotspot instigator (chi)-like sequence (GCTGG). Furthermore, an 8-bp (AAAGTTGCT) conserved sequence at the breakpoint junction was identified, suggesting that the most likely mechanism for the occurrence of this rearrangement is by Alu microhomology and chi-like recombination-associated motif-mediated multiple exon deletion. Our results are consistent with non-allelic homologous recombination and non-homologous end joining in deletion mutagenesis for the generation of rearrangements. This study provides more evidence associating repeated elements as a genetic mechanism underlying neurodegenerative disorders, highlighting their importance in human diseases.

  5. Antigen Gene Transfer to Human Plasmacytoid Dendritic Cells Using Recombinant Adenovirus and Vaccinia Virus Vectors

    Directory of Open Access Journals (Sweden)

    Hetty J. Bontkes

    2005-01-01

    Full Text Available Recombinant adenoviruses (RAd and recombinant vaccinia viruses (RVV expressing tumour-associated antigens (TAA are used as anti-tumour vaccines. It is important that these vaccines deliver the TAA to dendritic cells (DC for the induction of a strong immune response. Infection of myeloid DC (MDC with RAd alone is relatively inefficient but CD40 retargeting significantly increases transduction efficiency and DC maturation. Infection with RVV is efficient without DC maturation. Plasmacytoid dendritic cells (PDC play a role in the innate immune response to viral infections through the secretion of IFNα but may also play a role in specific T-cell induction. The aim of our study was to investigate whether PDC are better targets for RAd and RVV based vaccines. RAd alone hardly infected PDC (2% while CD40 retargeting did not improve transduction efficiency, but it did increase PDC maturation (25% CD83 positive cells. Accordingly, specific CTL activation by RAd infected PDC was limited (the number of IFNγ producing CTL was reduced by 75% compared to stimulation with peptide loaded PDC. RVV infected PDC specifically stimulated CTL but PDC were not activated. These Results indicate that PDC are not ideal targets for RAd and RVV based vaccines. However, PDC induced specific CTL activation after pulsing with recombinant protein, indicating that PDC can also cross-present antigens released from surrounding infected cells.

  6. Supplementary Material for: Recombination in pe/ppe genes contributes to genetic variation in Mycobacterium tuberculosis lineages

    KAUST Repository

    Phelan, Jody

    2016-01-01

    Abstract Background Approximately 10 % of the Mycobacterium tuberculosis genome is made up of two families of genes that are poorly characterized due to their high GC content and highly repetitive nature. The PE and PPE families are typified by their highly conserved N-terminal domains that incorporate proline-glutamate (PE) and proline-proline-glutamate (PPE) signature motifs. They are hypothesised to be important virulence factors involved with host-pathogen interactions, but their high genetic variability and complexity of analysis means they are typically disregarded in genome studies. Results To elucidate the structure of these genes, 518 genomes from a diverse international collection of clinical isolates were de novo assembled. A further 21 reference M. tuberculosis complex genomes and long read sequence data were used to validate the approach. SNP analysis revealed that variation in the majority of the 168 pe/ppe genes studied was consistent with lineage. Several recombination hotspots were identified, notably pe_pgrs3 and pe_pgrs17. Evidence of positive selection was revealed in 65 pe/ppe genes, including epitopes potentially binding to major histocompatibility complex molecules. Conclusions This, the first comprehensive study of the pe and ppe genes, provides important insight into M. tuberculosis diversity and has significant implications for vaccine development.

  7. An in vitro recombination-based reverse genetic system for rapid mutagenesis of structural genes of the Japanese encephalitis virus

    Institute of Scientific and Technical Information of China (English)

    Ruikun; Du; Manli; Wang; Zhihong; Hu; Hualin; Wang; Fei; Deng

    2015-01-01

    Japanese encephalitis virus(JEV) is one of the most common pathogens of severe viral encephalitis, which is a severe threat to human health. Despite instability of the JEV genome in bacteria, many strategies have been developed to establish molecular clone systems of JEV, providing convenient tools for studying the virus life cycle and virus–host interactions. In this study, we adapted an In-Fusion enzyme-based in vitro recombination method to construct a reverse genetic system of JEV, thereby providing a rapid approach to introduce mutations into the structural genes. A truncated genome without the structural genes was constructed as the backbone, and the complementary segment containing the structural genes was recombined in vitro, which was then transfected directly into virus-permissive cells. The progeny of the infectious virus was successfully detected in the supernatant of the transfected cells, and showed an identical phenotype to its parental virus. To provide a proof-of-principle, the 12 conserved cysteine residues in the envelope(E) protein of JEV were respectively mutated using this approach, and all mutations resulted in a complete failure to generate infectious virus. However, a leucine-tophenylanine mutation at amino acid 107 of the E protein did not interfere with the production of the infectious virus. These results suggested that all 12 cysteines in the E protein are essential for the JEV life cycle. In summary, a novel reverse genetic system of JEV was established for rapidly introducing mutations into structural genes, which will serve as a useful tool for functional studies.

  8. Comparative Analysis of the Conventional and Novel pmo (Particulate Methane Monooxygenase) Operons from Methylocystis Strain SC2

    OpenAIRE

    Ricke, Peter; Erkel, Christoph; Kube, Michael; Reinhardt, Richard; Liesack, Werner

    2004-01-01

    In addition to the conventional pmoA gene (pmoA1) encoding the active site polypeptide of particulate methane monooxygenase, a novel pmoA gene copy (pmoA2) is widely distributed among type II methanotrophs (methane-oxidizing bacteria [MOB]) (M. Tchawa Yimga, P. F. Dunfield, P. Ricke, J. Heyer, and W. Liesack, Appl. Environ. Microbiol. 69:5593-5602, 2003). Here we report that the pmoA1 and pmoA2 gene copies in the type II MOB Methylocystis strain SC2 are each part of a complete pmoCAB gene clu...

  9. Untangling the multiple monooxygenases of Mycobacterium chubuense strain NBB4, a versatile hydrocarbon degrader.

    Science.gov (United States)

    Coleman, Nicholas V; Yau, Sheree; Wilson, Neil L; Nolan, Laura M; Migocki, Margaret D; Ly, Mai-Anh; Crossett, Ben; Holmes, Andrew J

    2011-06-01

    Mycobacterium strain NBB4 was isolated on ethene as part of a bioprospecting study searching for novel monooxygenase (MO) enzymes of interest to biocatalysis and bioremediation. Previous work indicated that strain NBB4 contained an unprecedented diversity of MO genes, and we hypothesized that each MO type would support growth on a distinct hydrocarbon substrate. Here, we attempted to untangle the relationships between MO types and hydrocarbon substrates. Strain NBB4 was shown to grow on C2 -C4 alkenes and C2 -C16 alkanes. Complete gene clusters encoding six different monooxygenases were recovered from a fosmid library, including homologues of ethene MO (etnABCD), propene MO (pmoABCD), propane MO (smoABCD), butane MO (smoXYB1C1Z), cytochrome P450 (CYP153; fdx-cyp-fdr) and alkB (alkB-rubA1-rubA2). Catabolic enzymes involved in ethene assimilation (EtnA, EtnC, EtnD, EtnE) and alkane assimilation (alcohol and aldehyde dehydrogenases) were identified by proteomics, and we showed for the first time that stress response proteins (catalase/peroxidase, chaperonins) were induced by growth on C2 -C5 alkanes and ethene. Surprisingly, none of the identified MO genes could be specifically associated with oxidation of small alkanes, and thus the nature of the gaseous alkane MO in NBB4 remains mysterious.

  10. Extensive substrate profiling of cyclopentadecanone monooxygenase as Baeyer-Villiger biocatalyst reveals novel regiodivergent oxidations

    NARCIS (Netherlands)

    Fink, Michael J.; Fischer, Thomas C.; Rudroff, Florian; Dudek, Hanna; Fraaije, Marco W.; Mihovilovic, Marko D.

    2011-01-01

    Cyclopentadecanone monooxygenase (CPDMO) is one of the latest additions to the established library of Baeyer-Villiger monooxygenases. Desymmetrizations of substituted cyclobutanones and -hexanones as well as kinetic resolutions of racemic cycloketones are efficiently catalyzed by CPDMO. Moreover the

  11. Genetic mapping on chromosome 20 near the MODY gene: Is there suppression of recombination?

    Energy Technology Data Exchange (ETDEWEB)

    Howard, T.D.; Rothschild, C.B.; Bowden, D.W. [Wake Forest Univ., Winston-Salem, NC (United States)] [and others

    1994-09-01

    Maturity onset diabetes of the young (MODY) is a rare, autosomal dominant form of non-insulin-dependent diabetes mellitus. We are evaluating a chromosome 20-linked form of MODY in a large, well-characterized pedigree known as the R-W family. DNA from 97 family members, including spouses, have been genotyped with nine microsatellite markers and four RFLPs from 20q12-13.1. The highest likelihood order of loci in this 9.8 cM (sex-average) interval is: cen-ADA-D20S119-D20S17-PPGB-D20S178-D20S197-D20S213-D20S16A-D20S22-D20S16B-qter. Two polymorphic loci separable by recombination comprise D20S16 (called A and B here). The highest twopoint LOD scores were observed with D20S16 (Zmax = 17.0, {theta} = 0.001) and ADA (Zmax = 16.4, {theta} = 0.001). Multipoint analysis limits MODY to a sex-average interval of approximately 10 cM (calculated from CEPH family data) defined by ADA and D20S16. The maximum multipoint LOD score observed in this region was 17.22. No recombination events have been observed within this 10 cM interval in affected individuals. The probability of this occurring by chance is P = (1-{theta}){sup n}, where n = number of meioses. With the present data there are 57 (equivalent) informative meioses, so the probability of finding no recombination within the 10 cM inteval is 0.002. In the R-W family, however, there is a 3:1 ratio of male:female meioses. When this is taken into consideration, and the sex-specific genetic distances are used (15.9 cM for female map, 6.8 cM for male map), the probability of observing no recombination in this region is still quite low: P = 0.004, suggesting the possibility that recombination is suppressed in this family.

  12. Construction and Expression of Periplanete fuliginosa densovirus Recombinant Plasmid Which Contains Luciferase Gene%含荧光素酶基因的黑胸大蠊浓核病毒重组质粒的构建与表达

    Institute of Scientific and Technical Information of China (English)

    杨娟; 张珈敏; 蒋洪; 邓晓军; 胡建芳; 胡远扬

    2003-01-01

    A luciferase gene has been inserted into the recombinant plasmid PfDNV-pUC119 which contained partly deletion of genome of Periplanete fuliginosa densovirus(PfDNV.)The recombinant plasmid with luciferase gene was co-transfrected with PfDNV-pUC 119 into Periplanele fuliginosa larvae and had a high luciferase gene expression in enteron of the transfected larvae.

  13. The cylindrospermopsin gene cluster of Aphanizomenon sp. strain 10E6: organization and recombination.

    Science.gov (United States)

    Stüken, Anke; Jakobsen, Kjetill S

    2010-08-01

    Cylindrospermopsin (CYN), a potent hepatoxin, occurs in freshwaters worldwide. Several cyanobacterial species produce the toxin, but the producing species vary between geographical regions. Aphanizomenon flos-aquae, a common algae species in temperate fresh and brackish waters, is one of the three well-documented CYN producers in European waters. So far, no genetic information on the CYN genes of this species has been available. Here, we describe the complete CYN gene cluster, including flanking regions from the German Aphanizomenon sp. strain 10E6 using a full genome sequencing approach by 454 pyrosequencing and bioinformatic identification of the gene cluster. In addition, we have sequenced a approximately 7 kb fragment covering the genes cyrC (partially), cyrA and cyrB (partially) of the same gene cluster in the CYN-producing Aphanizomenon sp. strains 10E9 and 22D11. Comparisons with the orthologous gene clusters of the Australian Cylindrospermopsis raciborskii strains AWT205 and CS505 and the partial gene cluster of the Israeli Aphanizomenon ovalisporum strain ILC-146 revealed a high gene sequence similarity, but also extensive rearrangements of gene order. The high sequence similarity (generally higher than that of 16S rRNA gene fragments from the same strains), atypical GC-content and signs of transposase activities support the suggestion that the CYN genes have been horizontally transferred.

  14. Construction the hairpin RNA recombinant plasmids targeting human Pokemon gene%靶向人Pokemon基因的shRNA重组质粒的构建

    Institute of Scientific and Technical Information of China (English)

    Wenmiao Cao; Tao Zhang; Buhai Wang; Xizhi Zhang; Jinghua Ren; Jie Yao

    2011-01-01

    Objective:The aim of this study was to establish the foundation for studying the role of pokemon gene in tumorigenesis and development by constructing recombinant plasmids that can express small interfering RNA (siRNA) targeting human Pokemon gene. Methods: Hairpin siRNA templates targeting Pokemon gene were synthesized and cloned into plasmid vector psiRNA-H1neo. Three vectors derived siRNAs (psiRNA1, 2, 3) and one mocking psiRNAc (as control) were constructed.The recombinant Pokemon siRNA plasmids were constructed and identified using restrictive enzyme analysis and DNA sequencing. Results: Restrictive enzyme analysis and DNA sequencing revealed the successful construction of siRNA expression plasmids. Conclusion: Constructing siRNA templates targeting Pokemon gene may provide us with practical tools for further study the role of Pokemon gene in the development of some diseases and gene therapy of tumor.

  15. Change of gene structure and function by non-homologous end-joining, homologous recombination, and transposition of DNA.

    Directory of Open Access Journals (Sweden)

    Wolfgang Goettel

    2009-06-01

    Full Text Available An important objective in genome research is to relate genome structure to gene function. Sequence comparisons among orthologous and paralogous genes and their allelic variants can reveal sequences of functional significance. Here, we describe a 379-kb region on chromosome 1 of maize that enables us to reconstruct chromosome breakage, transposition, non-homologous end-joining, and homologous recombination events. Such a high-density composition of various mechanisms in a small chromosomal interval exemplifies the evolution of gene regulation and allelic diversity in general. It also illustrates the evolutionary pace of changes in plants, where many of the above mechanisms are of somatic origin. In contrast to animals, somatic alterations can easily be transmitted through meiosis because the germline in plants is contiguous to somatic tissue, permitting the recovery of such chromosomal rearrangements. The analyzed region contains the P1-wr allele, a variant of the genetically well-defined p1 gene, which encodes a Myb-like transcriptional activator in maize. The P1-wr allele consists of eleven nearly perfect P1-wr 12-kb repeats that are arranged in a tandem head-to-tail array. Although a technical challenge to sequence such a structure by shotgun sequencing, we overcame this problem by subcloning each repeat and ordering them based on nucleotide variations. These polymorphisms were also critical for recombination and expression analysis in presence and absence of the trans-acting epigenetic factor Ufo1. Interestingly, chimeras of the p1 and p2 genes, p2/p1 and p1/p2, are framing the P1-wr cluster. Reconstruction of sequence amplification steps at the p locus showed the evolution from a single Myb-homolog to the multi-gene P1-wr cluster. It also demonstrates how non-homologous end-joining can create novel gene fusions. Comparisons to orthologous regions in sorghum and rice also indicate a greater instability of the maize genome, probably due to

  16. Effects of recombinant retroviral vector mediated human insulin like growth factor-1 gene transfection on skeletal muscle growth in rat

    Institute of Scientific and Technical Information of China (English)

    RONG Shu-Ling; LU Yong-Xin; LIAO Yu-Hua; WANG Xiao-Lin; GUO He-Ping; CHANG Chao; GAO Yan-Zhang; MI Shao-Hua; Wan Jian-Ping

    2006-01-01

    Background This study transferred a recombinant gene encoding human insulin like growth factor-1 (hIGF-1)into modified primary skeletal myoblasts with a retroviral vector (pLgXSN) and determined whether the hIGF-1 promoted growth of skeletal muscle in rat.Methods hIGF-lcDNA was amplified in vitro from normal human liver cells by using RT-PCR and cloned into plasmid vector pLgXSN. The recombinant vector pLghIGF-1SN and control vector pLgGFPSN were transfected into packaging cell PT67 and G418 was used to select positive colony. Myoblasts were infected with a high titre viral supernatant and transduction efficiency was evaluated as GFP expression. The expression of hIGF-1 mRNA in myoblasts was investigated by immunocytochemistry and RT-PCR. MTT assays detected the growth of myoblasts in vitro. Myoblasts transduced with pLghIGF-1SN were injected into hind limb muscles of 10-12 week male SD rats. Formed tissues were harvested 4 weeks later. Myocyte diameter, mean weight of hind limb and body were measured to evaluate the skeletal muscle growth.Results Recombinant retroviral plasmid vector pLghIGF-1SN was constructed successfully. The titre of the packaged recombinant retrovirus was 1 × 106 cfu/ml. The transfection rate of PT67 cells reached 100% after G418 screening. hIGF-1 expression was positive in myoblast-IGF-1. The proliferation rate of myoblast-IGF-1 in vitro was higher than GFP-myoblast or myoblast (P< 0.05). The mean weights of hind limb and body of rats injected myoblast-IGF-1 were higher than those of the rats injected with myoblast-GFP or myoblast (P< 0.05). Myocyte diameter had a significant increase in IGF-1 group compared to GFP group and myoblast group (P< 0.05).Conclusions The transfection of the human IGF- 1 gene mediated by a retroviral vector can promote the growth of skeletal muscle in rats. Genetically modified primary skeletal myoblasts provide a possibly effective approach to treat some skeletal muscle diseases.

  17. Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase.

    Science.gov (United States)

    Chang, Shu-Wei; Lee, Guan-Chiun; Shaw, Jei-Fu

    2006-02-08

    An important industrial enzyme, Candida rugosa lipase (CRL) possesses several different isoforms encoded by the lip gene family (lip1-lip7), in which the recombinant LIP1 is the major form of the CRL multigene family. Previously, 19 of the nonuniversal serine codons (CTG) of the lip1 gene hav been successfully converted into universal serine codons (TCT) by overlap extension PCR-based multiple-site-directed mutagenesis to express an active recombinant LIP1 in the yeast Pichia pastoris. To improve the expression efficiency of recombinant LIP1 in P. pastoris, a regional synthetic gene fragment of lip1 near the 5' end of a transcript has been constructed to match P. pastoris-preferred codon usage for simple scale-up fermentation. The present results show that the production level (152 mg/L) of coLIP1 (codon-optimized LIP1) has an overall improvement of 4.6-fold relative to that (33 mg/L) of non-codon-optimized LIP1 with only half the cultivation time of P. pastoris. This finding demonstrates that the regional codon optimization the lip1 gene fragment at the 5' end can greatly increase the expression level of recombinant LIP1 in the P. pastoris system. More distinct biochemical properties of the purified recombinant LIP1 for further industrial applications are also determined and discussed in detail.

  18. Cloning of BMP-2 Gene and Its Expression Study in E. coli in Order to Produce a Recombinant Drug

    Directory of Open Access Journals (Sweden)

    N. Mohammadi

    2014-10-01

    Full Text Available Introduction & Objective: Bone morphogenetic proteins are a group of cytokines that belongs to superfamily TGF?. These proteins play an important role in evolution of many of organs and tissues through germinal period followed by amending and rebuilding of bone tissue and car-tilage. The aim of this study was to clone and expression analysis of BMP-2 gene in E. coli bacteria. Materials & Methods: In this experimental study the sequence of cDNA related to the mature peptide of human morphogenetic protein-2 (BMP-2 in E.coli was synthesized and cloned in a PET system. After sequencing, recombinant plasmid pET28a/BMP-2 was transformed into the expression host, E.coli BL21 (DE3. The transformed bacteria were cultured in LB me-dium containing kanamaycin antibiotic at 37° C for O/N. Then, induction with IPTG took place. The expression was evaluated by reverse transcriptase PCR and SDS-PAGE followed by western blotting to confirm its identity. The observed band on SDS-PSGE showed the presence of the expressed protein at the 14k Dalton segment which was confirmed by west-ern blotting technique. Results: The gene sequence was amplified by PCR. After gene and plasmid preparation, lega-tion was performed. Sequencing confirmed accuracy of cloning. Protein expression was demonstrated by RT-PCR and SDS-PAGE. Results were confirmed by western blotting. Conclusion: In this study over-expression of this recombinant protein was achieved in a pro-karyotic system. Different concentrations of inducer were applied and harvesting was per-formed in different times after induction. The best expression was detected in 4 hours after induction with a concentration of 1mM IPTG. (Sci J Hamadan Univ Med Sci 2014; 21 (3: 196-202

  19. Construction and anti-tumor effects of recombinant fowlpox virus expressing Newcastle disease virus hemagglutinin-neuramidinase gene

    Institute of Scientific and Technical Information of China (English)

    LI Xiao; JIN Ningyi; LIAN Hai; GUAN Goufang; SUN Lili; LI Xuemei; ZHENG Hongling

    2006-01-01

    Hemagglutinin-neuramidinase (HN), aNewcastle disease virus-derived protein, not only mediates receptor recognition but also possesses neuraminidase (NA) activity, the ability to cleave a component of those receptors, N-acetylneuraminic acid (NAcneu, sialic acid). It is known that this protein in mammalian species, including human beings, has interesting anti-neoplastic as well as immune stimulating properties. To explore the use of the HN gene in cancer gene therapy, we constructed a recombinant fowlpox virus expressing the HN protein (vFV-HN) and compared the anti-tumor activity of the recombinant virus with that of wild-type fowlpox virus (FPV) in vivo and in vitro. Here we found that although B16 cells were somewhat resistant to the basal cytotoxic effect of wild-type fowlpox virus, infection with vFV-HN caused a pronounced cytotoxic effect and, the survival of tumor-bearing mice immunized with vFV-HN was significantly increased compared with the survival of mice immunized with the FPV alone. Furthermore, the immunization of mice with vFV-HN elicited a B16 tumor-specific cytotoxic T lymphocyte (CTL) response and clonal expansion of both CD4+ and CD8+ T cell populations in vivo. In addition, T cells from lymph nodes of mice vaccinated with vFV-HN secreted high levels of the Th1 cytokine IL-2 and IFN-y, indicating that the regression of tumor cells is related to a Th1-type dominant immune response. These results demonstrate that vaccination with vFV-HN may be a potential strategy for cancer gene therapy.

  20. Recombinant adenovirus-mediated shRNA silencing of midkine gene in BxPC-3 cells

    Institute of Scientific and Technical Information of China (English)

    Mingyue Xiong; Kunzheng Wang

    2009-01-01

    Objective:To investigate the silencing effects of recombinant adenovirus Ad-shRNA-MK on midkine(MK) gene in pancreatic cancer cells. Methods:Ad-shRNA-MK was used to infect pancreatic cancer BxPC-3 cells. Assays were conducted for knockdown of the MK gene on the day of infection and on the 1a, 3rd, 5th, 7th, and 9th days post-infection by using immunocytochemistry, real-time RT-PCR, and Western blot analysis. Results:The adenoviral Ad-shRNA-PTN was constructed successfully, and infection was confirmed by electron microscopic observation. By using real-time RT-PCR, the inhibition rates of MK mRNA expression in the BxPC-3 cells were 20%, 80%, 55%, and 23% on the 1st, 3rd, 5th, and 7th days post-infection. Immunocytochemistry and Western blot analysis confirmed this effect at the gene product level. Conclusion:Efficient and specific knockdown of MK in pancreatic cancer cells by adenoviral Ad-shRNA-PTN is a potentially powerful tool for the study of gene therapy of pancreatic cancer nerve infiltration.

  1. Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris.

    Science.gov (United States)

    Chang, S W; Shieh, C J; Lee, G C; Shaw, J F

    2005-04-01

    Candida rugosa lipase, a significant catalyst, had been widely employed to catalyze various chemical reactions such as non-specific, stereo-specific hydrolysis and esterification for industrial biocatalytic applications. Several isozymes encoded by the lip gene family, namely lip1 to lip7, possess distinct thermal stability and substrate specificity, among which the recombinant LIP1 showed a distinguished catalytic characterization. In this study, we utilized PCR to remove an unnecessary linker of pGAPZalphaC vector and used overlap extension PCR-based multiple site-directed mutagenesis to convert the 19 non-universal CTG-serine codons into universal TCT-serine codons and successfully express a highly active recombinant C. rugosa LIP1 in the Pichia expression system. Response surface methodology and 4-factor-5-level central composite rotatable design were adopted to evaluate the effects of growth parameters, such as temperature (21.6-38.4 degrees C), glucose concentration (0.3-3.7%), yeast extract (0.16-1.84%), and pH (5.3-8.7) on the lipolytic activity of LIP1 and biomass of P. pastoris. Based on ridge max analysis, the optimum LIP1 production conditions were temperature, 24.1 degrees C; glucose concentration, 2.6%; yeast extract, 1.4%; and pH 7.6. The predicted value of lipolytic activity was 246.9+/-39.7 U/ml, and the actual value was 253.3+/-18.8 U/ml. The lipolytic activity of the recombinant LIP1 resulting from the present work is twofold higher than that achieved by a methanol induction system.

  2. Proteomic Analysis of Recombinant Escherichia coli Expressing Zymomonas mobilis pdc and adh Genes

    Institute of Scientific and Technical Information of China (English)

    Xiaoqin Wang; Xuefeng Li; Hui Wang; Mingfeng Yang; Xiushan Yang; Yikun He

    2012-01-01

    Pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are efficient enzymes for ethanol production in Zymomonas mobilis.These two enzymes were over-expressed in Escherichia coli,which was a better candidate for industrial ethanol production,resulting in a recombinant Escherichia coli strain efficient for ethanol production.To investigate the underlying mechanism,2-DE and LC-MS/MS were preformed.More than 1000 protein spots were reproducibly detected in the gel by image analysis,and 99 protein spots showed significant changes in recombinant E.coli,in which 46 were down-regulated and 53 were up-regulated.These proteins were mainly involved in energy metabolism,small molecule biosynthesis and degradation,transport,and stress.Except for the expected significant upregulation of PDC and ADH,most proteins involved in energy metabolism,purine/pyrimidine ribonucleotide biosynthesis,amino acid biosynthesis,and transport were up-regulated.It suggested that in response to a significant up-regulation of foreign proteins,E.coli could readjust other pathways to reach a new balance in cells,and these up-regulated proteins and pathways cooperated in ethanol production efficiently.

  3. Failure to induce a DNA repair gene, RAD54, in Saccharomyces cerevisiae does not affect DNA repair or recombination phenotypes

    Energy Technology Data Exchange (ETDEWEB)

    Cole, G.M.; Mortimer, R.K. (Univ. of California, Berkeley (USA))

    1989-08-01

    The Saccharomyces cerevisiae RAD54 gene is transcriptionally regulated by a broad spectrum of DNA-damaging agents. Induction of RAD54 by DNA-damaging agents is under positive control. Sequences responsible for DNA damage induction (the DRS element) lie within a 29-base-pair region from -99 to -70 from the most proximal transcription start site. This inducible promoter element is functionally separable from a poly(dA-dT) region immediately downstream which is required for constitutive expression. Deletions which eliminate induction of RAD54 transcription by DNA damage but do not affect constitutive expression have no effect on growth or survival of noninducible strains relative to wild-type strains in the presence of DNA-damaging agents. The DRS element is also not required for homothallic mating type switching, transcriptional induction of RAD54 during meiosis, meiotic recombination, or spontaneous or X-ray-induced mitotic recombination. We find no phenotype for a lack of induction of RAD54 message via the damage-inducible DRS, which raises significant questions about the physiology of DNA damage induction in S. cerevisiae.

  4. A simple, economical method of converting gene expression products of insulin into recombinant insulin and its application

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhou; CHEN Hui; TANG Yuehua; FENG Youmin

    2003-01-01

    A method, by which the gene expression product of recombinant single chain insulin can be converted into insulin by directly digesting with trypsin, has been established. This method has been used in process of porcine insulin precursor (PIP), [B16Ala]PIP and [B26Ala]PIP into (desB30)insulin, (desB30)[B16Ala]insulin and (desB30)[B26Ala]insulin, respectively, and all of them retain full biological activity of that of their corresponding parent, recombinant human insulin, [B16Ala]insulin and [B26Ala]insulin. The results further demonstrate that the C-terminal residue of B chain is not necessary for insulin's biological activity. Compared with the method of transpeptidation, this method is simple, with a high yield, and avoids the use of organic reagents, and in comparison with the trypsin/carboxypeptidase method, it omits the use of carboxylpeptidase. Besides, (desB30)[B16Ala]insulin and (desB30)[B26Ala]insulin still remained without self-association property as that of their parents, which demonstrate that they are monomeric insulin. So they can be used for substituting for monomeric insulin, [B16Ala]insulin and [B26Ala]insulin, in clinical applications.

  5. [Construction of eukaryotic recombinant vector and expression in COS7 cell of LipL32-HlyX fusion gene from Leptospira serovar Lai].

    Science.gov (United States)

    Huang, Bi; Bao, Lang; Zhong, Qi; Zhang, Huidong; Zhang, Ying

    2009-04-01

    This study was conducted to construct eukaryotic recombinant vector of LipL32-HlyX fusion gene from Leptospira serovar Lai and express it in mammalian cell. Both of LipL32 gene and HlyX gene were amplified from Leptospira strain O17 genomic DNA by PCR. Then with the two genes as template, LipL32-HlyX fusion gene was obtained by SOE PCR (gene splicing by overlap extension PCR). The fusion gene was then cloned into pcDNA3.1 by restriction nuclease digestion. Having been transformed into E. coli DH5alpha, the recombiant plasmid was identified by restriction nuclease digestion, PCR analysis and sequencing. The recombinant plasmid was then transfected into COS7 cell whose expression was detected by RT-PCR and Western blotting analysis. RT-PCR amplified a fragment about 2000 bp and Western blotting analysis found a specific band about 75 KD which was consistent with the expected fusion protein size. In conclusion, the successful construction of eukaryotic recombinant vector containing LipL32-HlyX fusion gene and the effective expression in mammalian have laid a foundation for the application of Leptospira DNA vaccine.

  6. Increased antibody responses to human papillomavirus type 16 L1 protein expressed by recombinant vaccinia virus lacking serine protease inhibitor genes.

    Science.gov (United States)

    Zhou, J; Crawford, L; McLean, L; Sun, X Y; Stanley, M; Almond, N; Smith, G L

    1990-09-01

    The L1 gene of human papillomavirus type 16 (HPV-16) driven by the vaccinia virus major late 4b gene promoter has been inserted into three different sites of the vaccinia virus genome. Insertion into the thymidine kinase (TK) gene was achieved by selection of TK- mutants in BUdR on TK- cells. Insertion into two vaccinia virus serine protease inhibitor (serpin) genes was achieved by co-insertion of the Escherichia coli xanthine guanine phosphoribosyltransferase gene linked to the vaccinia virus 7.5K promoter and selection of mycophenolic acid-resistant recombinant viruses. Each recombinant virus expressed a 57K L1 protein at similar levels and with similar kinetics. However, immunization of mice with these recombinant viruses induced different levels of antibody to the L1 protein. Viruses lacking serpin genes B13R and B24R induced significantly higher antibody levels than did viruses lacking the TK gene. The presence of functional B13R and B24R gene products is therefore somehow immunosuppressive at least for antibody responses to the L1 protein of HPV-16.

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

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Hyun Suk; Park, Seong-Wook [Department of Internal Medicine (Cardiology), Asan Medical Center, University of Ulsan College of Medicine, 388-1 Pungnap-dong, Songpa-gu, 138-736, Seoul (Korea); Lee, Heuiran; Kim, Sung Jin [Department of Microbiology, University of Ulsan College of Medicine, Seoul (Korea); Lee, Won Woo [Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seongnam (Korea); Department of Nuclear Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea); Yang, You-Jung; Moon, Dae Hyuk [Department of Nuclear Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea)

    2004-09-01

    We evaluated the feasibility of non-invasive imaging of recombinant adenovirus-mediated human sodium-iodide symporter (hNIS) gene expression by {sup 99m}TcO{sub 4}{sup -} scintigraphy in skeletal muscle of rats. Replication-defective recombinant adenovirus encoding hNIS gene [Rad-CMV-hNIS 5 x 10{sup 7}, 2 x 10{sup 8} or 1 x 10{sup 9} plaque forming units (pfu)] or {beta}-galactosidase gene (Rad-CMV-LacZ 1 x 10{sup 9} pfu) was injected into the right biceps femoris muscle of rats (n=5-6 for each group). Three days after gene transfer, scintigraphy was performed using a gamma camera 30 min after injection of {sup 99m}TcO{sub 4}{sup -} (1.85 MBq). An additional two rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS underwent {sup 99m}TcO{sub 4}{sup -} scintigraphy with sodium perchlorate. After the imaging studies, rats were sacrificed for assessment of the biodistribution of {sup 99m}TcO{sub 4}{sup -} and measurement of hNIS mRNA expression. In all the rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS, hNIS expression was successfully imaged by {sup 99m}TcO{sub 4}{sup -} scintigraphy, while rats injected with Rad-CMV-LacZ or lower doses of Rad-CMV-hNIS failed to show uptake. The biodistribution studies indicated that a significantly different amount of {sup 99m}TcO{sub 4}{sup -} was retained in the liver (p<0.001) and the right muscle (p<0.05), with the highest uptake in rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS. The muscular hNIS mRNA level quantified by real-time reverse transcription-polymerase chain reaction was significantly higher in rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS (p<0.05), with a positive correlation with the imaging counts (r=0.810, p<0.05) and the biodistribution (r=0.847, p<0.001). Hot spots in rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS were specifically inhibited by sodium perchlorate. This study illustrated that {sup 99m}TcO{sub 4}{sup -} scintigraphy can monitor Rad-CMV-hNIS-mediated gene expression in

  8. Removal of nonhomologous DNA ends in double-strand break recombination: The role of the yeast ultraviolet repair gene RAD1

    Energy Technology Data Exchange (ETDEWEB)

    Fishman-Lobell, J.; Habert, J.E. (Brandeis Univ., Waltham, MA (United States))

    1992-10-15

    Double-strand breaks (DSBs) in Saccharomyces cerevisiae can be repaired by gene conversions or by deletions resulting from single-strand annealing between direct repeats of homologous sequences. Although rad1 mutants are resistant to x-rays and can complete DSB-mediated mating-type switching, they could not complete recombination when the ends of the break contained approximately 60 base pairs of nonhomology. Recombination was restored when the ends of the break were made homologous to donor sequences. Additionally, the absence of RAD1 led to the frequent appearance of a previously unobserved type of recombination product. These data suggest RAD1 is required to remove nonhomologous DNA from the 3{prime} ends of recombining DNA, a process analogous to the excision of photodimers during repair of ultraviolet-damaged DNA.

  9. Functional assembly of camphor converting two-component Baeyer-Villiger monooxygenases with a flavin reductase from E. coli.

    Science.gov (United States)

    Kadow, Maria; Balke, Kathleen; Willetts, Andrew; Bornscheuer, Uwe T; Bäckvall, J-E

    2014-05-01

    The major limitation in the synthetic application of two-component Baeyer-Villiger monooxygenases was addressed by identifying the 28-kDa flavin-reductase Fre from Escherichia coli as a suitable supplier of reduced FMN for these enzymes. Coexpression of Fre with either 2,5- or 3,6-diketocamphane monooxygenase from Pseudomonas putida NCIMB 10007 significantly enhanced the conversion of camphor and norcamphor serving as representative ketones. With purified enzymes, full conversion was achieved, while only slight amounts of product were formed in the absence of this flavin reductase. Fusion of the genes of Fre and DKCMOs into single open reading frame constructs resulted in unstable proteins exhibiting flavin reducing, but poor oxygenating activity, which led to overall decreased conversion of camphor.

  10. Conditions for gene disruption by homologous recombination of exogenous DNA into the Sulfolobus solfataricus genome

    Directory of Open Access Journals (Sweden)

    Sonja-Verena Albers

    2008-01-01

    Full Text Available The construction of directed gene deletion mutants is an essential tool in molecular biology that allows functional studies on the role of genes in their natural environment. For hyperthermophilic archaea, it has been difficult to obtain a reliable system to construct such mutants. However, during the past years, systems have been developed for Thermococcus kodakarensis and two Sulfolobus species, S. acidocaldarius and derivatives of S. solfataricus 98/2. Here we describe an optimization of the method for integration of exogenous DNA into S. solfataricus PBL 2025, an S. solfataricus 98/2 derivative, based on lactose auxotrophy that now allows for routine gene inactivation.

  11. Canada continues use prohibition of gene recombination cattle somatotropin; Kanada wa idenshikumikaegyu seicho horumon no shiyo kinshi wo keizoku

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1999-04-05

    Canada continued use prohibition of gene recombination cattle somatotropin (BST) which stimulated milk increased production of this company in spite of request of Monsanto Co.. The cow that BST was administered is based on the report of veterinary surgeon that causes reproductive function and lowering in the life, dysbasia and increase in the mastitis on this. From the research report that BST causes thyroid gland cyst of the male rat in U.S.A., consumer organization and environment group petition FDA for the elimination of BST from the U.S.A. market. In the meantime, Monsanto Co. in the report which Canada adopted, chemical structure is different, and it reports that it uses BST of other company in which dosing method and dosing frequency differ and disregards the report of Cornell university in which the life of the cow administered more and more does not lower BST. (translated by NEDO)

  12. Enhancement of cytidine production by coexpression of gnd, zwf, and prs genes in recombinant Escherichia coli CYT15.

    Science.gov (United States)

    Fang, Haitian; Xie, Xixian; Xu, Qingyang; Zhang, Chenglin; Chen, Ning

    2013-02-01

    Cytidine is a precursor of several antiviral drugs. The pentose phosphate pathway (PPP) is primarily responsible for NADPH and 5-phospho-α-D-ribose 1-diphosphate as an important precursor of cytidine biosynthesis in Escherichia coli. To enhance cytidine production, we obtained the recombinant E. coli CYT15-gnd-prs-zwf that co-expressed the prs, zwf, and gnd genes encoding phosphoribosylpyrophosphate synthetase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (three key enzymes in PPP) respectively. In fermentation experiments, strain CYT15-gnd-prs-zwf produced 735 mg cytidine/l using glucose as substrate, which was approx. 128 % higher than the cytidine production by the parental strain (CYT15). Co-expression of zwf, gnd, and prs decreased growth (3.2 %) slightly and increased glucose uptake (72 %). This is the first study to report increased cytidine production by increasing metabolic flux through the PPP in E. coli.

  13. A Novel Neurotoxin Gene ar1b Recombination Enhances the Efficiency of Helicoverpa armigera Nucleopolyhedrovirus as a Pesticide by Inhibiting the Host Larvae Ability to Feed and Grow.

    Directory of Open Access Journals (Sweden)

    Huan Yu

    Full Text Available A recombinant Helicoverpa armigera nucleopolyhedrovirus (HearNPV, Ar1b-HearNPV, was constructed and identified as an improved bio-control agent of Helicoverpa armigera larvae. The HearNPV polyhedrin promoter was used to express the insect-specific neurotoxin gene, ar1b, which was originally isolated from the Australian funnel-web spider (Atrax robustus. RT-PCR and Western blotting analysis showed that both the ar1b transcript and protein were produced successfully in Ar1b-HearNPV-infected HzAM1 cells. In order to investigate the influence of foreign gene insertion in HearNPV, including the ar1b gene, chloramphenicol resistance gene, lacZ, kanamycin resistance gene, and the gentamicin resistance gene, two virus strains (HZ8-HearNPV and wt-HearNPV were used as controls in the cell transfection analysis. As expected, foreign gene insertion had no impact on budded virus production and viral DNA replication. Both optical microscopy and electron microscopy observations indicated that the formation of the occlusion bodies of recombinant virus was similar to wild type virus. The Ar1b-HearNPV-infected H. armigera larvae exhibited paralysis and weight loss before dying. This recombinant virus also showed a 32.87% decrease in LT50 assays compared with the wild type virus. Besides, Ar1b-HearNPV also inhibited host larval growth and diet consumption. This inhibition was still significant in the older instar larvae treated with the recombinant virus. All of these positive properties of this novel recombinant HearNPV provide a further opportunity to develop this virus strain into a commercial product to control the cotton bollworm.

  14. A Novel Neurotoxin Gene ar1b Recombination Enhances the Efficiency of Helicoverpa armigera Nucleopolyhedrovirus as a Pesticide by Inhibiting the Host Larvae Ability to Feed and Grow.

    Science.gov (United States)

    Yu, Huan; Meng, Jiao; Xu, Jian; Liu, Tong-Xian; Wang, Dun

    2015-01-01

    A recombinant Helicoverpa armigera nucleopolyhedrovirus (HearNPV), Ar1b-HearNPV, was constructed and identified as an improved bio-control agent of Helicoverpa armigera larvae. The HearNPV polyhedrin promoter was used to express the insect-specific neurotoxin gene, ar1b, which was originally isolated from the Australian funnel-web spider (Atrax robustus). RT-PCR and Western blotting analysis showed that both the ar1b transcript and protein were produced successfully in Ar1b-HearNPV-infected HzAM1 cells. In order to investigate the influence of foreign gene insertion in HearNPV, including the ar1b gene, chloramphenicol resistance gene, lacZ, kanamycin resistance gene, and the gentamicin resistance gene, two virus strains (HZ8-HearNPV and wt-HearNPV) were used as controls in the cell transfection analysis. As expected, foreign gene insertion had no impact on budded virus production and viral DNA replication. Both optical microscopy and electron microscopy observations indicated that the formation of the occlusion bodies of recombinant virus was similar to wild type virus. The Ar1b-HearNPV-infected H. armigera larvae exhibited paralysis and weight loss before dying. This recombinant virus also showed a 32.87% decrease in LT50 assays compared with the wild type virus. Besides, Ar1b-HearNPV also inhibited host larval growth and diet consumption. This inhibition was still significant in the older instar larvae treated with the recombinant virus. All of these positive properties of this novel recombinant HearNPV provide a further opportunity to develop this virus strain into a commercial product to control the cotton bollworm.

  15. Hydroxylation of methane through component interactions in soluble methane monooxygenases.

    Science.gov (United States)

    Lee, Seung Jae

    2016-04-01

    Methane hydroxylation through methane monooxygenases (MMOs) is a key aspect due to their control of the carbon cycle in the ecology system and recent applications of methane gas in the field of bioenergy and bioremediation. Methanotropic bacteria perform a specific microbial conversion from methane, one of the most stable carbon compounds, to methanol through elaborate mechanisms. MMOs express particulate methane monooxygenase (pMMO) in most strains and soluble methane monooxygenase (sMMO) under copper-limited conditions. The mechanisms of MMO have been widely studied from sMMO belonging to the bacterial multicomponent monooxygenase (BMM) superfamily. This enzyme has diiron active sites where different types of hydrocarbons are oxidized through orchestrated hydroxylase, regulatory and reductase components for precise control of hydrocarbons, oxygen, protons, and electrons. Recent advances in biophysical studies, including structural and enzymatic achievements for sMMO, have explained component interactions, substrate pathways, and intermediates of sMMO. In this account, oxidation of methane in sMMO is discussed with recent progress that is critical for understanding the microbial applications of C-H activation in one-carbon substrates.

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

    Science.gov (United States)

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

    2013-09-01

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

  17. p21 is dispensable for AID-mediated class switch recombination and mutagenesis of immunoglobulin genes during somatic hypermutation.

    Science.gov (United States)

    Shansab, Maryam; Selsing, Erik

    2011-03-01

    In B cells, activation-induced cytidine deaminase (AID) induces somatic hypermutation (SHM) at rearranged immunoglobulin (Ig) variable (V) regions. Previous studies have shown that both monoubiquitination of proliferating cell nuclear antigen (PCNA) and translesional DNA polymerase activity are important for inducing mutagenesis during SHM. Regulation of PCNA ubiquitination by p21, also known as Cdkn1a and p21(Cip1/Waf1), is an important mechanism that controls mutation loads in mammalian cells. In this study, we have assessed whether p21 has an in vivo function in regulating mutagenesis in B cells by analyzing SHM frequency in p21-deficient mice. Our results show that p21 is dispensable for SHM. This suggests that, during SHM of Ig genes, p21 does not act to regulate mutagenesis load. We also show that p21 transcript levels are the same in both wildtype and AID-deficient B cells during B cell activation, and that AID-mediated class switch recombination (CSR) is not affected by p21 deficiency; thereby indicating that p21 regulation in B cells is not altered by AID-induced DNA damage and that p21 has no affect on AID-dependent Ig gene diversification. Our results suggest that regulation of p21 in activated B cells is probably more important for maintaining proper cell cycle progression as opposed to promoting SHM of Ig genes.

  18. Foreign gene transfer in termite cells using a recombinant vesicular stomatitis virus

    Science.gov (United States)

    The Formosan subterranean termite, Coptotermes formosanus Shiraki, and the eastern subterranean termite, Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae), are well known for their destruction of human dwellings and flora in the tropics and subtropics. A method to deliver foreign genes in...

  19. ama1 genes of sympatric Plasmodium vivax and P. falciparum from Venezuela differ significantly in genetic diversity and recombination frequency.

    Directory of Open Access Journals (Sweden)

    Rosalynn L Ord

    Full Text Available BACKGROUND: We present the first population genetic analysis of homologous loci from two sympatric human malaria parasite populations sharing the same human hosts, using full-length sequences of ama1 genes from Plasmodium vivax and P. falciparum collected in the Venezuelan Amazon. METHODOLOGY/PRINCIPAL FINDINGS: Significant differences between the two species were found in genetic diversity at the ama1 locus, with 18 distinct haplotypes identified among the 73 Pvama1 sequences obtained, compared to 6 unique haplotypes from 30 Pfama1 sequences, giving overall diversity estimates of h = 0.9091, and h = 0.538 respectively. Levels of recombination were also found to differ between the species, with P. falciparum exhibiting very little recombination across the 1.77 kb sequence. In contrast, analysis of patterns of nucleotide substitutions provided evidence that polymorphisms in the ama1 gene of both species are maintained by balancing selection, particularly in domain I. The two distinct population structures observed are unlikely to result from different selective forces acting upon the two species, which share both human and mosquito hosts in this setting. Rather, the highly structured P. falciparum population appears to be the result of a population bottleneck, while the much less structured P. vivax population is likely to be derived from an ancient pool of diversity, as reflected in a larger estimate of effective population size for this species. Greatly reduced mosquito transmission in 1997, due to low rainfall prior to the second survey, was associated with far fewer P. falciparum infections, but an increase in P. vivax infections, probably due to hypnozoite activation. CONCLUSIONS/SIGNIFICANCE: The relevance of these findings to putative competitive interactions between these two important human pathogen species is discussed. These results highlight the need for future control interventions to employ strategies targeting each of the parasite

  20. Characterization of a defensin from the oyster Crassostrea gigas. Recombinant production, folding, solution structure, antimicrobial activities, and gene expression.

    Science.gov (United States)

    Gueguen, Yannick; Herpin, Amaury; Aumelas, André; Garnier, Julien; Fievet, Julie; Escoubas, Jean-Michel; Bulet, Philippe; Gonzalez, Marcelo; Lelong, Christophe; Favrel, Pascal; Bachère, Evelyne

    2006-01-06

    In invertebrates, defensins were found in arthropods and in the mussels. Here, we report for the first time the identification and characterization of a defensin (Cg-Def) from an oyster. Cg-def mRNA was isolated from Crassostrea gigas mantle using an expressed sequence tag approach. To gain insight into potential roles of Cg-Def in oyster immunity, we produced the recombinant peptide in Escherichia coli, characterized its antimicrobial activities, determined its solution structure by NMR spectroscopy, and quantified its gene expression in vivo following bacterial challenge of oysters. Recombinant Cg-Def was active in vitro against Gram-positive bacteria but showed no or limited activities against Gram-negative bacteria and fungi. The activity of Cg-Def was retained in vitro at a salt concentration similar to that of seawater. The Cg-Def structure shares the so-called cystine-stabilized alpha-beta motif (CS-alphabeta) with arthropod defensins but is characterized by the presence of an additional disulfide bond, as previously observed in the mussel defensin (MGD-1). Nevertheless, despite a similar global fold, the Cg-Def and MGD-1 structures mainly differ by the size of their loops and by the presence of two aspartic residues in Cg-Def. Distribution of Cg-def mRNA in various oyster tissues revealed that Cg-def is mainly expressed in mantle edge where it was detected by mass spectrometry analyses. Furthermore, we observed that the Cg-def messenger concentration was unchanged after bacterial challenge. Our results suggest that Cg-def gene is continuously expressed in the mantle and would play a key role in oyster by providing a first line of defense against pathogen colonization.

  1. Recombination and Heterologous Expression of Allophycocyanin Gene in the Chloroplast of Chlamydomonas reinhardtii

    Institute of Scientific and Technical Information of China (English)

    Zhong-Liang SU; Kai-Xian QIAN; Cong-Ping TAN; Chun-Xiao MENG; Song QIN

    2005-01-01

    Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires the introduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins or metabolic pathways. In order to accomplish the expression of multiple genes in a single transformation event, we inserted both large and small subunits of allophycocyanin gene (apcA and apcB) into Chlamydomonas reinhardtii chloroplast expression vector, resulting in papc-S. The constructed vector was then introduced into the chloroplast of C. reinhardtii by micro-particle bombardment. Polymerase chain reaction and Southern blot analysis revealed that the two genes had integrated into the chloroplast genome. Western blot and enzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria could be correctly expressed in the chloroplasts of C. reinhardtii. The expressed foreign protein in transformants accounted for about 2%-3% of total soluble proteins. These findings pave the way to the reconstitution of multi-subunit proteins or metabolic pathways in transgenic C. reinhardtii chloroplasts in a single transformation event.

  2. Controlled oxidation of aliphatic CH bonds in metallo-monooxygenases: mechanistic insights derived from studies on deuterated and fluorinated hydrocarbons.

    Science.gov (United States)

    Chen, Yao-Sheng; Luo, Wen-I; Yang, Chung-Ling; Tu, Yi-Jung; Chang, Chun-Wei; Chiang, Chih-Hsiang; Chang, Chi-Yao; Chan, Sunney I; Yu, Steve S-F

    2014-05-01

    The control over the regio- and/or stereo-selective aliphatic CH oxidation by metalloenzymes is of great interest to scientists. Typically, these enzymes invoke host-guest chemistry to sequester the substrates within the protein pockets, exploiting sizes, shapes and specific interactions such as hydrogen-bonding, electrostatic forces and/or van der Waals interactions to control the substrate specificity, regio-specificity and stereo-selectivity. Over the years, we have developed a series of deuterated and fluorinated variants of these hydrocarbon substrates as probes to gain insights into the controlled CH oxidations of hydrocarbons facilitated by these enzymes. In this review, we illustrate the application of these designed probes in the study of three monooxygenases: (i) the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath), which oxidizes straight-chain C1-C5 alkanes and alkenes to form their corresponding 2-alcohols and epoxides, respectively; (ii) the recombinant alkane hydroxylase (AlkB) from Pseudomonas putida GPo1, which oxidizes the primary CH bonds of C5-C12 linear alkanes; and (iii) the recombinant cytochrome P450 from Bacillus megaterium, which oxidizes C12-C20 fatty acids at the ω-1, ω-2 or ω-3 CH positions.

  3. Genetic Diversity of Spike, 3a, 3b and E Genes of Infectious Bronchitis Viruses and Emergence of New Recombinants in Korea

    Directory of Open Access Journals (Sweden)

    Chang-Seon Song

    2013-01-01

    Full Text Available The nucleotide sequences of a region including S1, S2, 3a, 3b and E genes of twenty-seven infectious bronchitis virus (IBV isolates in Korea between 1990–2011 were determined and phylogenetic and computational recombination analyses were conducted. The sizes of coding regions of some genes varied among IBV isolates due to deletion or insertion of nucleotides; the nucleotide similarities of S1, S2, 3a, 3b and E genes among the 27 isolates were 75.9%–100.0%, 85%–100.0%, 64.0%–100.0%, 60.4%–100.0% and 83.1%–100.0%, respectively. According to phylogenetic analysis of S1 gene, the 27 isolates were divided into five genotypes, Mass, Korean-I (K-I, QX-like, KM91-like and New cluster 1. The phylogenetic trees based on the S2, 3a, 3b, E genes and S1-S2-3a-3b-E (S1-E region nucleotide sequences did not closely follow the clustering based on the S1 sequence. The New cluster 1 prevalent during 2009 and 2010 was not found in 2011 but QX-like viruses became prevalent in 2011. The recombination analysis revealed two new S gene recombinants, 11036 and 11052 which might have been derived from recombinations between the New cluster 1 and QX-like viruses and between the K-I and H120 (vaccine viruses, respectively. In conclusion, multiple IBV genotypes have co-circulated; QX-like viruses have recurred and new recombinants have emerged in Korea. This has enriched molecular epidemiology information of IBV and is useful for the control of IB in Korea.

  4. Recombination analysis and structure prediction show correlation between breakpoint clusters and RNA hairpins in the pol gene of human immunodeficiency virus type 1 unique recombinant forms

    DEFF Research Database (Denmark)

    Galli, Andrea; Lai, Alessia; Corvasce, Stefano

    2008-01-01

    Recombination is recognized as a primary force in human immunodeficiency virus type 1 (HIV-1) evolution, increasing viral diversity through reshuffling of genomic portions. The strand-switching activity of reverse transcriptase is required to complete HIV-1 replication and can occur randomly thro...

  5. Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

    Institute of Scientific and Technical Information of China (English)

    YUAN Ye; WANG Xiuli; GUO Sheping; QIU xuemei

    2011-01-01

    Gram-negative vibrio parahaemolyticus is a common pathogen in humans and marine animals.The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host.Thus,the outer membrane proteins are an ideal target for vaccines.We amplified a complete outer membrane protein gene (ompW) from V.parahaemolyticus ATCC 17802.We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells.The gene coded for a protein that was 42.78 kDa.We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting,respectively.Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V.parahaemolyticus.In addition,the purified OmpW protein can be used for further functional and structural studies.

  6. Gene cloning and prokaryotic expression of recombinant flagellin A from Vibrio parahaemolyticus

    Science.gov (United States)

    Yuan, Ye; Wang, Xiuli; Guo, Sheping; Liu, Yang; Ge, Hui; Qiu, Xuemei

    2010-11-01

    The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin proteins are an ideal target for vaccines. We amplified the complete flagellin subunit gene ( flaA) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 62.78 kDa. We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus. In addition, the purified FlaA protein can be used for further functional and structural studies.

  7. Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

    Science.gov (United States)

    Yuan, Ye; Wang, Xiuli; Guo, Sheping; Qiu, Xuemei

    2011-06-01

    Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V. parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.

  8. Overexpressions of Lambda Phage Lysis Genes and Biosynthetic Genes of Poly-β-hydroxybutyrate in Recombinant E.coli

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    A plasmid (pTU9) containing the lambda (λ) phage lysis genes S(-)RRz and the biosynthetic genes phbCAB of poly-β-hydroxybutyrate (PHB) was constructed and transformed into E.coli JM109. Cultured in Luria-Bertani (LB) medium with 20 g/L glucose, E.coli JM109 (pTU9) could accumulate PHB in cells up to 40% (g PHB per g dry cells). A chelating agent EDTA was applied to induce a complete cell lysis and PHB granules were released. This method has a potential application in PHB separation.

  9. Flavin-dependent monooxygenases as a detoxification mechanism in insects: new insights from the arctiids (lepidoptera.

    Directory of Open Access Journals (Sweden)

    Sven Sehlmeyer

    Full Text Available Insects experience a wide array of chemical pressures from plant allelochemicals and pesticides and have developed several effective counterstrategies to cope with such toxins. Among these, cytochrome P450 monooxygenases are crucial in plant-insect interactions. Flavin-dependent monooxygenases (FMOs seem not to play a central role in xenobiotic detoxification in insects, in contrast to mammals. However, the previously identified senecionine N-oxygenase of the arctiid moth Tyria jacobaeae (Lepidoptera indicates that FMOs have been recruited during the adaptation of this insect to plants that accumulate toxic pyrrolizidine alkaloids. Identification of related FMO-like sequences of various arctiids and other Lepidoptera and their combination with expressed sequence tag (EST data and sequences emerging from the Bombyx mori genome project show that FMOs in Lepidoptera form a gene family with three members (FMO1 to FMO3. Phylogenetic analyses suggest that FMO3 is only distantly related to lepidopteran FMO1 and FMO2 that originated from a more recent gene duplication event. Within the FMO1 gene cluster, an additional gene duplication early in the arctiid lineage provided the basis for the evolution of the highly specific biochemical, physiological, and behavioral adaptations of these butterflies to pyrrolizidine-alkaloid-producing plants. The genes encoding pyrrolizidine-alkaloid-N-oxygenizing enzymes (PNOs are transcribed in the fat body and the head of the larvae. An N-terminal signal peptide mediates the transport of the soluble proteins into the hemolymph where PNOs efficiently convert pro-toxic pyrrolizidine alkaloids into their non-toxic N-oxide derivatives. Heterologous expression of a PNO of the generalist arctiid Grammia geneura produced an N-oxygenizing enzyme that shows noticeably expanded substrate specificity compared with the related enzyme of the specialist Tyria jacobaeae. The data about the evolution of FMOs within lepidopteran insects

  10. Genome engineering and direct cloning of antibiotic gene clusters via phage ϕBT1 integrase-mediated site-specific recombination in Streptomyces.

    Science.gov (United States)

    Du, Deyao; Wang, Lu; Tian, Yuqing; Liu, Hao; Tan, Huarong; Niu, Guoqing

    2015-03-04

    Several strategies have been used to clone large DNA fragments directly from bacterial genome. Most of these approaches are based on different site-specific recombination systems consisting of a specialized recombinase and its target sites. In this study, a novel strategy based on phage ϕBT1 integrase-mediated site-specific recombination was developed, and used for simultaneous Streptomyces genome engineering and cloning of antibiotic gene clusters. This method has been proved successful for the cloning of actinorhodin gene cluster from Streptomyces coelicolor M145, napsamycin gene cluster and daptomycin gene cluster from Streptomyces roseosporus NRRL 15998 at a frequency higher than 80%. Furthermore, the system could be used to increase the titer of antibiotics as we demonstrated with actinorhodin and daptomycin, and it will be broadly applicable in many Streptomyces.

  11. The membrane-associated monooxygenase in the butane-oxidizing Gram-positive bacterium Nocardioides sp. strain CF8 is a novel member of the AMO/PMO family.

    Science.gov (United States)

    Sayavedra-Soto, Luis A; Hamamura, Natsuko; Liu, Chih-Wen; Kimbrel, Jeffrey A; Chang, Jeff H; Arp, Daniel J

    2011-06-01

    The Gram-positive bacterium Nocardioides sp. strain CF8 uses a membrane-associated monooxygenase (pBMO) to grow on butane. The nucleotide sequences of the genes encoding this novel monooxygenase were revealed through analysis of a de novo assembled draft genome sequence determined by high-throughput sequencing of the whole genome. The pBMO genes were in a similar arrangement to the genes for ammonia monooxygenase (AMO) from the ammonia-oxidizing bacteria and for particulate methane monooxygenase (pMMO) from the methane-oxidizing bacteria. The pBMO genes likely constitute an operon in the order bmoC, bmoA and bmoB. The nucleotide sequence was less than 50% similar to the genes for AMO and pMMO. The operon for pBMO was confirmed to be a single copy in the genome by Southern and computational analyses. In an incubation on butane the increase of transcriptional activity of the pBmoA gene was congruent with the increase of pBMO activity and suggested correspondence between gene expression and the utilization of butane. Phylogenetic comparison revealed distant but significant similarity of all three pBMO subunits to homologous members of the AMO/pMMO family and indicated that the pBMO represents a deeply branching third lineage of this group of particulate monooxygenases. No other bmoCAB-like genes were found to cluster with pBMO lineage in phylogenetic analysis by database searches including genomic and metagenomic sequence databases. pBMO is the first example of the AMO/pMMO-like monooxygenase from Gram-positive bacteria showing similarities to proteobacterial pMMO and AMO sequences. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

  12. Effects of NV gene knock-out recombinant viral hemorrhagic septicemia virus (VHSV) on Mx gene expression in Epithelioma papulosum cyprini (EPC) cells and olive flounder (Paralichthys olivaceus).

    Science.gov (United States)

    Kim, Min Sun; Kim, Ki Hong

    2012-03-01

    To determine whether the NV gene of viral hemorrhagic septicemia virus (VHSV) is related to the type I interferon response of hosts, expression of Mx gene in Epithelioma papulosum cyprini (EPC) cells and in olive flounder (Paralichthys olivaceus) in response to infection with either wild-type VHSV or recombinant VHSVs (rVHSV-ΔNV-EGFP and rVHSV-wild) was investigated. A reporter vector was constructed for measuring Mx gene expression using olive flounder Mx promoter, in which the reporter Metridia luciferase was designed to be excreted to culture medium to facilitate measurement. The highest increase of luciferase activity was detected from supernatant of cells infected with rVHSV-ΔNV-EGFP. In contrast cells infected with wild-type VHSV showed a slight increase of the luciferase activity. Interestingly, cells infected with rVHSV-wild that has artificially changed nucleotides just before and after the NV gene ORF, also showed highly increased luciferase activity, but the increased amplitude was lower than that by rVHSV-ΔNV-EGFP. These results strongly suggest that the NV protein of VHSV plays an important role in suppressing interferon response in host cells, which provides a condition for the viruses to efficiently proliferate in host cells. In an in vivo experiment, the Mx gene expression in olive flounder challenged with the rVHSV-ΔNV-EGFP was clearly higher than fish challenged with rVHSV-wild or wild-type VHSV, suggesting that lacking of the NV gene in the genome of rVHSV-ΔNV-EGFP brought to strong interferon response that subsequently inhibit viral replication in fish.

  13. Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage phiC31 site-specific recombination system.

    Science.gov (United States)

    Thomason, L C; Calendar, R; Ow, D W

    2001-08-01

    The site-specific recombination system used by the Streptomyces bacteriophage phiC31 was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the phiC31 integrase gene. High-efficiency transformation to the Ura+ phenotype occurred when the integrase gene was expressed. Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site. Sequence analysis showed that the attBxattP recombination was precise. In another approach, DNA with a ura4+ marker flanked by two attB sites in direct orientation was used to transform S. pombe cells bearing an attP duplication. The phiC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement. The site-specific insertions are stable, as no excision (the reverse reaction) was observed on maintenance of the integrase gene in the integrant lines. The irreversibility of the phiC31 site-specific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily. Deployment of the phiC31 recombination provides new opportunities for directing transgene and chromosome rearrangements in eukaryotic systems.

  14. Phylogeny of Mycobacterium tuberculosis Beijing strains constructed from polymorphisms in genes involved in DNA replication, recombination and repair.

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    Olga Mestre

    Full Text Available BACKGROUND: The Beijing family is a successful group of M. tuberculosis strains, often associated with drug resistance and widely distributed throughout the world. Polymorphic genetic markers have been used to type particular M. tuberculosis strains. We recently identified a group of polymorphic DNA repair replication and recombination (3R genes. It was shown that evolution of M. tuberculosis complex strains can be studied using 3R SNPs and a high-resolution tool for strain discrimination was developed. Here we investigated the genetic diversity and propose a phylogeny for Beijing strains by analyzing polymorphisms in 3R genes. METHODOLOGY/PRINCIPAL FINDINGS: A group of 3R genes was sequenced in a collection of Beijing strains from different geographic origins. Sequence analysis and comparison with the ones of non-Beijing strains identified several SNPs. These SNPs were used to type a larger collection of Beijing strains and allowed identification of 26 different sequence types for which a phylogeny was constructed. Phylogenetic relationships established by sequence types were in agreement with evolutionary pathways suggested by other genetic markers, such as Large Sequence Polymorphisms (LSPs. A recent Beijing genotype (Bmyc10, which included 60% of strains from distinct parts of the world, appeared to be predominant. CONCLUSIONS/SIGNIFICANCE: We found SNPs in 3R genes associated with the Beijing family, which enabled discrimination of different groups and the proposal of a phylogeny. The Beijing family can be divided into different groups characterized by particular genetic polymorphisms that may reflect pathogenic features. These SNPs are new, potential genetic markers that may contribute to better understand the success of the Beijing family.

  15. Clinical utility of recombinant adenoviral human p53 gene therapy: current perspectives

    Directory of Open Access Journals (Sweden)

    Chen GX

    2014-10-01

    Full Text Available Guang-xia Chen,1,* Shu Zhang,2–4,* Xiao-hua He,1 Shi-yu Liu,1 Chao Ma,2–4 Xiao-Ping Zou2–4 1Department of Gastroenterology, First People’s Hospital of Xuzhou, Xuzhou, Jiangsu Province, People’s Republic of China; 2Department of Gastroenterology, Drum Tower Hospital, 3Medical School of Nanjing University, 4Jiangsu Clinical Medical Center of Digestive Disease, Nanjing, People’s Republic of China *These authors have contributed equally to the paperAbstract: Gene therapy has promised to be a highly effective antitumor treatment by introducing a tumor suppressor gene or the abrogation of an oncogene. Among the potential therapeutic transgenes, the tumor suppressor gene p53 serves as an attractive target. Restoration of wild-type p53 function in tumors can be achieved by introduction of an intact complementary deoxyribonucleic acid copy of the p53 gene using a suitable viral vector, in most cases an adenoviral vector (Adp53. Preclinical in vitro and in vivo studies have shown that Adp53 triggers a dramatic tumor regression response in various cancers. These viruses are engineered to lack certain early proteins and are thus replication defective, including Gendicine, SCH-58500, and Advexin. Several types of tumor-specific p53-expressing conditionally replicating adenovirus vectors (known as replication-competent CRAdp53 vectors have been developed, such as ONYX 015, AdDelta24-p53, SG600-p53, OBP-702, and H101. Various clinical trials have been conducted to investigate the safety and efficiency of these adenoviral vectors. In this review we will talk about the biological mechanisms, clinical utility, and therapeutic potentials of the replication-deficient Adp53-based and replication-competent CRAdp53-based gene therapy.Keywords: adenovirus, Adp53, CRAdp53

  16. Both FMNH2 and FADH2 can be utilized by the dibenzothiophene monooxygenase from a desulfurizing bacterium Mycobacterium goodii X7B.

    Science.gov (United States)

    Li, Jingchen; Feng, Jinhui; Li, Qian; Ma, Cuiqing; Yu, Bo; Gao, Chao; Wu, Geng; Xu, Ping

    2009-05-01

    To investigate the flavin utilization by dibenzothiophene monooxygenase (DszC), DszC of a desulfurizing bacterium Mycobacterium goodii X7B was purified from the recombinant Escherichia coli. It was shown to be able to utilize either FMNH(2) or FADH(2) when coupled with a flavin reductase that reduces either FMN or FAD. Sequence analysis indicated that DszC was similar to the C(2) component of p-hydroxyphenylacetate hydroxylase from Acinetobacter baumannii, which can use both FADH(2) and FMNH(2) as substrates. Both flavins at high concentrations could inhibit the activity of DszC due to autocatalytic oxidation of reduced flavins. The results suggest that DszC should be reclassified as an FMNH(2) and FADH(2) both-utilizing monooxygenase component and the flavins should be controlled at properly reduced levels to obtain optimal biodesulfurization results.

  17. Rules of donor preference in saccharomyces mating-type gene switching revealed by a competition assay involving two types of recombination.

    Science.gov (United States)

    Wu, X; Wu, C; Haber, J E

    1997-10-01

    Mating type (MAT) switching in Saccharomyces cerevisiae is initiated by a double-strand break (DSB) created at MAT by HO endonuclease. MATa cells activate the entire left arm of chromosome III; thus MATa preferentially recombines with the silent donor HML. In contrast, MAT alpha cells inactivate the left arm, including HML, and thus preferentially recombine with HMR, 100 kb to the right of MAT. We present a novel competition assay, in which the DSB at MAT can be repaired either by MAT switching or by single-strand annealing (SSA) between two URA3 genes flanking MAT. With preferred donors, MATa or MAT alpha switching occurs 65-70% of the time in competition with SSA. When HML is deleted, 40% of MATa cells recombine with the "wrong" donor HMR; however, when HMR is deleted, only 18% of MAT alpha cells recombine with HML. In interchromosomal switching, with donors on chromosome III and MAT on chromosome V, MATa retains its strong preference for HML and switching is efficient, when the chromosome III recombination enhancer is present. However, MAT alpha donor preference is lost and interchromosomal switching is very inefficient. These experiments demonstrate the utility of using competition between two outcomes to measure the relative efficiency of recombination.

  18. NIH oversight of human gene transfer research involving retroviral, lentiviral, and adeno-associated virus vectors and the role of the NIH recombinant DNA advisory committee.

    Science.gov (United States)

    O'Reilly, Marina; Shipp, Allan; Rosenthal, Eugene; Jambou, Robert; Shih, Tom; Montgomery, Maureen; Gargiulo, Linda; Patterson, Amy; Corrigan-Curay, Jacqueline

    2012-01-01

    In response to public and scientific concerns regarding human gene transfer research, the National Institutes of Health (NIH) developed a transparent oversight system that extends to human gene transfer protocols that are either conducted with NIH funding or conducted at institutions that receive NIH funding for recombinant DNA research. The NIH Recombinant DNA Advisory Committee (RAC) has been the primary advisory body to NIH regarding the conduct of this research. Human gene transfer research proposals that are subject to the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) must be submitted to the NIH Office of Biotechnology Activities (OBA), and protocols that raise novel scientific, safety, medical, ethical, or social issues are publicly discussed at the RAC's quarterly public meetings. OBA also convenes gene transfer safety symposia and policy conferences to provide a public forum for scientific experts to discuss emerging issues in the field. This transparent system of review promotes the rapid exchange of important scientific information and dissemination of data. The goal is to optimize the conduct of individual research protocols and to advance gene transfer research generally. This process has fostered the development of retroviral, lentiviral, and adeno-associated viral vector mediated gene delivery.

  19. Targeting Human α-Lactalbumin Gene Insertion into the Goat β-Lactoglobulin Locus by TALEN-Mediated Homologous Recombination.

    Directory of Open Access Journals (Sweden)

    Hongmei Zhu

    Full Text Available Special value of goat milk in human nutrition and well being is associated with medical problems of food allergies which are caused by milk proteins such as β-lactoglobulin (BLG. Here, we employed transcription activator-like effector nuclease (TALEN-assisted homologous recombination in goat fibroblasts to introduce human α-lactalbumin (hLA genes into goat BLG locus. TALEN-mediated targeting enabled isolation of colonies with mono- and bi-allelic transgene integration in up to 10.1% and 1.1%, respectively, after selection. Specifically, BLG mRNA levels were gradually decreasing in both mo- and bi-allelic goat mammary epithelial cells (GMECs while hLA demonstrated expression in GMECs in vitro. Gene-targeted fibroblast cells were efficiently used in somatic cell nuclear transfer, resulting in production of hLA knock-in goats directing down-regulated BLG expression and abundant hLA secretion in animal milk. Our findings provide valuable background for animal milk optimization and expedited development for agriculture and biomedicine.

  20. CBP and p300 histone acetyltransferases contribute to homologous recombination by transcriptionally activating the BRCA1 and RAD51 genes.

    Science.gov (United States)

    Ogiwara, Hideaki; Kohno, Takashi

    2012-01-01

    Histone acetylation at DNA double-strand break (DSB) sites by CBP and p300 histone acetyltransferases (HATs) is critical for the recruitment of DSB repair proteins to chromatin. Here, we show that CBP and p300 HATs also function in DSB repair by transcriptionally activating the BRCA1 and RAD51 genes, which are involved in homologous recombination (HR), a major DSB repair system. siRNA-mediated depletion of CBP and p300 impaired HR activity and downregulated BRCA1 and RAD51 at the protein and mRNA levels. Chromatin immunoprecipitation assays showed that CBP and p300 bind to the promoter regions of the BRCA1 and RAD51 genes, and that depletion of CBP and/or p300 reduces H3 and H4 acetylation and inhibits binding of the transcription factor E2F1 to these promoters. Depletion of CBP and p300 impaired DNA damage-induced phosphorylation and chromatin binding of the single-strand DNA-binding protein RPA following BRCA1-mediated DNA end resection. Consistent with this, subsequent phosphorylation of CHK1 and activation of the G2/M damage checkpoint were also impaired. These results indicate that the HATs CBP and p300 play multiple roles in the activation of the cellular response to DSBs.

  1. Characterization of the highly active fragment of glyceraldehyde-3-phosphate dehydrogenase gene promoter for recombinant protein expression in Pleurotus ostreatus.

    Science.gov (United States)

    Yin, Chaomin; Zheng, Liesheng; Zhu, Jihong; Chen, Liguo; Ma, Aimin

    2015-03-01

    Developing efficient native promoters is important for improving recombinant protein expression by fungal genetic engineering. The promoter region of glyceraldehyde-3-phosphate dehydrogenase gene in Pleurotus ostreatus (Pogpd) was isolated and optimized by upstream truncation. The activities of these promoters with different lengths were further confirmed by fluorescence, quantitative real-time PCR and Western blot analysis. A truncated Pogpd-P2 fragment (795 bp) drove enhanced green fluorescence protein (egfp) gene expression in P. ostreatus much more efficiently than full-length Pogpd-P1. Further truncating Pogpd-P2 to 603, 403 and 231 bp reduced the eGFP expression significantly. However, the 403-bp fragment between -356 bp and the start codon was the minimal but sufficient promoter element for eGFP expression. Compact native promoters for genetic engineering of P. ostreatus were successfully developed and validated in this study. This will broaden the preexisting repertoire of fungal promoters for biotechnology application. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. CBP and p300 histone acetyltransferases contribute to homologous recombination by transcriptionally activating the BRCA1 and RAD51 genes.

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    Hideaki Ogiwara

    Full Text Available Histone acetylation at DNA double-strand break (DSB sites by CBP and p300 histone acetyltransferases (HATs is critical for the recruitment of DSB repair proteins to chromatin. Here, we show that CBP and p300 HATs also function in DSB repair by transcriptionally activating the BRCA1 and RAD51 genes, which are involved in homologous recombination (HR, a major DSB repair system. siRNA-mediated depletion of CBP and p300 impaired HR activity and downregulated BRCA1 and RAD51 at the protein and mRNA levels. Chromatin immunoprecipitation assays showed that CBP and p300 bind to the promoter regions of the BRCA1 and RAD51 genes, and that depletion of CBP and/or p300 reduces H3 and H4 acetylation and inhibits binding of the transcription factor E2F1 to these promoters. Depletion of CBP and p300 impaired DNA damage-induced phosphorylation and chromatin binding of the single-strand DNA-binding protein RPA following BRCA1-mediated DNA end resection. Consistent with this, subsequent phosphorylation of CHK1 and activation of the G2/M damage checkpoint were also impaired. These results indicate that the HATs CBP and p300 play multiple roles in the activation of the cellular response to DSBs.

  3. Bioremediation of cadmium contaminated soil using symbiosis between leguminous plant and recombinant rhizobia with the MTL4 and the PCS genes.

    Science.gov (United States)

    Ike, Akiko; Sriprang, Rutchadaporn; Ono, Hisayo; Murooka, Yoshikatsu; Yamashita, Mitsuo

    2007-01-01

    Cadmium contamination in rice grains is one of the important issues in Asian countries. We have developed a novel bio-remediation system based on the symbiosis between leguminous plant and genetically engineered rhizobia. We designed two types of recombinant rhizobia, carrying two genes, synthetic tetrameric metallothionein (MTL4) and cDNA encoding phytochelatin synthase from Arabidopsis thaliana (AtPCS). The MTL4 and AtPCS genes were transferred to Mesorhizobium huakuii subsp. rengei B3, which can infect and form nodules on Chinese milk vetch, Astragalus sinicus. The two genes were fused to the nolB or nifH promoter, which generated nodule specific expression of these genes in strain B3. The two recombinant strains, B3(pMPnolBMTL4nifHPCS) and B3::nifHMTL4(pMPnifHPCS), showed 25 and 12-fold increase in Cd concentration, in the free-living cells, respectively. When these recombinant strains established the symbiotic relationship with A. sinicus, the symbionts increased Cd accumulation in nodules by two-fold in hydroponic culture. The expression of the both MTL4 and AtPCS genes showed additive effect on cadmium accumulation in nodules. We also applied these recombinant bacteria to rice paddy soil polluted with Cd (1mgkg(-1) dry weight soil). The accumulation of Cd increased not only in nodules but also in the roots of A. sinicus infected by the recombinant rhizobia. The accumulation of Cd in the plant roots infected by B3(pMPnolBMTL4nifHPCS) achieved three-fold than that by the wild-type B3. After two months of cultivation of the symbiont, a maximum of 9% of Cd in paddy soil was removed. Thus, the symbiosis will be useful in phytoremediation for heavy metals.

  4. A nuclear mutant of Chlamydomonas that exhibits increased sensitivity to UV irradiation, reduced recombination of nuclear genes, and altered transmission of chloroplast genes.

    Science.gov (United States)

    Rosen, H; Newman, S M; Boynton, J E; Gillham, N W

    1991-01-01

    Meiotic progeny of Chlamydomonas reinhardtii normally receive chloroplast genomes only from the mt+ parent. However, exceptional zygotes, which transmit the chloroplast genomes of both parents or, more rarely, only those of the mt- parent, arise at a low frequency. Mutations at the mt(+)-linked mat-3 locus were found previously to elevate the transmission of chloroplast genomes from the mt- parent, resulting in a much higher than normal frequency of exceptional zygotes. In this paper we demonstrate that an ultraviolet-sensitive nuclear mutation mapping at the uvsE1 locus, which is unlinked to mating type, also promotes chloroplast genome transmission from the mt- parent. This mutant, which was previously shown to reduce recombination of nuclear genes in meiosis, acts synergistically with the mat-3-3 mutation to produce an extremely high frequency of exceptional zygotes. Through the use of restriction fragment length polymorphisms existing in the chloroplast genomes of C. reinhardtii and the interfertile strain C. smithii, we show that chloroplast DNA fragments from the mt- parent normally begin to disappear shortly after zygote formation. However, this process appears to be blocked totally in the absence of wild-type uvsE1 and mat-3 gene products. Our findings are consistent with the hypothesis that both gene products contribute to the mechanism responsible for uniparental inheritance of the chloroplast genome from the mt+ parent.

  5. Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: modular construction of multiple cDNA expression elements using recombinant cloning.

    Science.gov (United States)

    Sone, Takefumi; Yahata, Kazuhide; Sasaki, Yukari; Hotta, Junko; Kishine, Hiroe; Chesnut, Jonathan D; Imamoto, Fumio

    2008-09-10

    Much attention has been focused on manipulating multiple genes in living cells for analyzing protein function. In order to perform high-throughput generation of multi-gene expression clones, gateway cloning technology (which represents a high-throughput DNA transfer from vector to vector) can be anticipated. In the conventional strategy for gateway cloning, the construction of two or more expression elements into tandem elements on a single plasmid requires the recombination of multiple entry clones with a destination vector in a single reaction mixture. Use of increasing numbers of entry clones in a single reaction is inefficient due to the difficulty in successfully recognizing multiple pairs of matched att signals simultaneously. To address this problem, a "Modular Destination" vector has been devised and constructed, whereby cDNA inserts are sequentially introduced, resulting in a tandem structure with multiple inserts. Whereas the standard destination vector contains only Cm(R) and ccdB genes flanked by two attR signals, this destination vector contains, in addition, one or two cDNA expression elements. Here, we show the rapid construction of expression vectors containing three or four tandemly arrayed cDNA expression elements and their expression in mammalian cells.

  6. The Toluene o-Xylene Monooxygenase Enzymatic Activity for the Biosynthesis of Aromatic Antioxidants.

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    Giuliana Donadio

    Full Text Available Monocyclic phenols and catechols are important antioxidant compounds for the food and pharmaceutic industries; their production through biotransformation of low-added value starting compounds is of major biotechnological interest. The toluene o-xylene monooxygenase (ToMO from Pseudomonas sp. OX1 is a bacterial multicomponent monooxygenase (BMM that is able to hydroxylate a wide array of aromatic compounds and has already proven to be a versatile biochemical tool to produce mono- and dihydroxylated derivatives of aromatic compounds. The molecular determinants of its regioselectivity and substrate specificity have been thoroughly investigated, and a computational strategy has been developed which allows designing mutants able to hydroxylate non-natural substrates of this enzyme to obtain high-added value compounds of commercial interest. In this work, we have investigated the use of recombinant ToMO, expressed in cells of Escherichia coli strain JM109, for the biotransformation of non-natural substrates of this enzyme such as 2-phenoxyethanol, phthalan and 2-indanol to produce six hydroxylated derivatives. The hydroxylated products obtained were identified, isolated and their antioxidant potential was assessed both in vitro, using the DPPH assay, and on the rat cardiomyoblast cell line H9c2. Incubation of H9c2 cells with the hydroxylated compounds obtained from ToMO-catalyzed biotransformation induced a differential protective effect towards a mild oxidative stress induced by the presence of sodium arsenite. The results obtained confirm once again the versatility of the ToMO system for oxyfunctionalization reactions of biotechnological importance. Moreover, the hydroxylated derivatives obtained possess an interesting antioxidant potential that encourages the use of the enzyme for further functionalization reactions and their possible use as scaffolds to design novel bioactive molecules.

  7. The biochemical mechanism of auxin biosynthesis by an arabidopsis YUCCA flavin-containing monooxygenase.

    Science.gov (United States)

    Dai, Xinhua; Mashiguchi, Kiyoshi; Chen, Qingguo; Kasahara, Hiroyuki; Kamiya, Yuji; Ojha, Sunil; DuBois, Jennifer; Ballou, David; Zhao, Yunde

    2013-01-18

    Auxin regulates every aspect of plant growth and development. Previous genetic studies demonstrated that YUCCA (YUC) flavin-containing monooxygenases (FMOs) catalyze a rate-limiting step in auxin biosynthesis and that YUCs are essential for many developmental processes. We proposed that YUCs convert indole-3-pyruvate (IPA) to indole-3-acetate (IAA). However, the exact biochemical mechanism of YUCs has remained elusive. Here we present the biochemical characterization of recombinant Arabidopsis YUC6. Expressed in and purified from Escherichia coli, YUC6 contains FAD as a cofactor, which has peaks at 448 nm and 376 nm in the UV-visible spectrum. We show that YUC6 uses NADPH and oxygen to convert IPA to IAA. The first step of the YUC6-catalyzed reaction is the reduction of the FAD cofactor to FADH(-) by NADPH. Subsequently, FADH(-) reacts with oxygen to form a flavin-C4a-(hydro)peroxy intermediate, which we show has a maximum absorbance at 381 nm in its UV-visible spectrum. The final chemical step is the reaction of the C4a-intermediate with IPA to produce IAA. Although the sequences of the YUC enzymes are related to those of the mammalian FMOs, which oxygenate nucleophilic substrates, YUC6 oxygenates an electrophilic substrate (IPA). Nevertheless, both classes of enzymes form quasi-stable C4a-(hydro)peroxyl FAD intermediates. The YUC6 intermediate has a half-life of ∼20 s whereas that of some FMOs is >30 min. This work reveals the catalytic mechanism of the first known plant flavin monooxygenase and provides a foundation for further investigating how YUC activities are regulated in plants.

  8. Novel recombinant human lactoferrin: differential activation of oxidative stress related gene expression.

    Science.gov (United States)

    Kruzel, Marian L; Actor, Jeffrey K; Zimecki, Michał; Wise, Jasen; Płoszaj, Paulina; Mirza, Shaper; Kruzel, Mark; Hwang, Shen-An; Ba, Xueqing; Boldogh, Istvan

    2013-12-01

    Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins. The aim of this study was to scale-up expression and purification of rhLF in a CHO expression system, verify its glycan primary structure, and assess its biological properties in cell culture models. A stable CHO cell line producing >200mg/L of rhLF was developed and established. rhLF was purified by a single-step cation-exchange chromatography procedure. The highly homogenous rhLF has a molecular weight of approximately 80 kDa. MALDI-TOF mass spectrometric analysis revealed N-linked, partially sialylated glycans at two glycosylation sites, typical for human milk LF. This novel rhLF showed a protective effect against oxidative stress in a similar manner to its natural counterpart. In addition, rhLF revealed a modulatory effect on cellular redox via upregulation of key antioxidant enzymes. These data imply that the CHO-derived rhLF is fully compatible with the native molecule, thus it has promise for human therapeutic applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Immunogenicity of the recombinant adenovirus type 5 vector with type 35 fiber containing HIV-1 gag gene

    Institute of Scientific and Technical Information of China (English)

    XIN LEI LIU; SHUNAG QING YU; XIA FENG; XIAO LI WANG; HONG MEI LIU; XIAO MEI ZHANG; HONG XIA LI; LING ZHOU; YI ZENG

    2006-01-01

    The immune efficiency of a recombinant adenovirus type 5 with type 35 fiber containing HIV-1 gag gene (rAd5/F35-mod.gag) was investigated in BALB/c mice, in which the rAd5/F35-mod. gag was firstly identified with PCR, then transfected to 293 cells and the in vitro expression level of Gag protein was determined by Western blotting and indirect immuno-fluorescent assay. Mice were immunized with intramuscular injections of rAd5/F35-mod. gag, rAd5-mod. gag or DNA and were boosted after 3 weeks.To test the effect of pre-existing anti-viral immunity on immunization, mice were also injected with Ad5-GFP vector and then immunized 4 and 7 weeks later with Ad5/F35-mod. gag vector. The P24-specific IgG antibody in sera of immunized mice was determined by ELISA and the specific cytotoxic T lymphocyte (CTL) response was assayed by intracellular cytokine staining. It was demonstrated that the rAd5/F35-mod. gag vector could express efficiently the HIV Gag protein in 293 cells in vitro and induce strong HIVspecific immune responses in vivo. The strongest CTL and serum IgG response occurred when mice were immunized twice with injection of rAd5/F35 alone, but the anti-Ad5 antibody after primary infection with adenovirus could inhibit the specific immune responses induced by rAd5/F35 vector. It is concluded that single immunization with recombinant adenovirus rAd5/F35-mod. gag can induce specific CTL and serum IgG antibody responses in mice, but the immunogenicity of rAd5/F35 is comparably weaker than that of rAd5.

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

    Science.gov (United States)

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

    2016-01-01

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

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

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    Choi Jin-Ok

    2010-04-01

    Full Text Available Abstract Background Enzyme replacement therapy (ERT with α-galactosidase A (α-Gal A is currently the most effective therapeutic strategy for patients with Fabry disease, a lysosomal storage disease. However, ERT has limitations of a short half-life, requirement for frequent administration, and limited efficacy for patients with renal failure. Therefore, we investigated the efficacy of recombinant adeno-associated virus (rAAV vector-mediated gene therapy for a Fabry disease mouse model and compared it with that of ERT. Methods A pseudotyped rAAV2/8 vector encoding α-Gal A cDNA (rAAV2/8-hAGA was prepared and injected into 18-week-old male Fabry mice through the tail vein. The α-Gal A expression level and globotriaosylceramide (Gb3 levels in the Fabry mice were examined and compared with Fabry mice with ERT. Immunohistochemical and ultrastructural studies were conducted. Results Treatment of Fabry mice with rAAV2/8-hAGA resulted in the clearance of accumulated Gb3 in tissues such as liver, spleen, kidney, heart, and brain with concomitant elevation of α-Gal A enzyme activity. Enzyme activity was elevated for up to 60 weeks. In addition, expression of the α-Gal A protein was identified in the presence of rAAV2/8-hAGA at 6, 12, and 24 weeks after treatment. α-Gal A activity was significantly higher in the mice treated with rAAV2/8-hAGA than in Fabry mice that received ERT. Along with higher α-Gal A activity in the kidney of the Fabry mice treated with gene therapy, immunohistochemical studies showed more α-Gal A expression in the proximal tubules and glomerulus, and less Gb3 deposition in Fabry mice treated with this gene therapy than in mice given ERT. The α-gal A gene transfer significantly reduced the accumulation of Gb3 in the tubules and podocytes of the kidney. Electron microscopic analysis of the kidneys of Fabry mice also showed that gene therapy was more effective than ERT. Conclusions The rAAV2/8-hAGA mediated α-Gal A gene

  12. TOL PLASMID-SPECIFIED XYLENE OXYGENASE IS A WIDE SUBSTRATE RANGE MONOOXYGENASE CAPABLE OF OLEFIN EPOXIDATION

    NARCIS (Netherlands)

    Wubbolts, Marcel; REUVEKAMP, P; Witholt, Bernard

    1994-01-01

    Xylene oxygenase, which is encoded on the TOL plasmid pWWO of Pseudomonas putida mt-2, is a key enzyme system in the degradation of toluene and xylenes by this organism. It was expressed in an Escherichia coli recombinant strain carrying the xy1MA structural genes. This recombinant, which expressed

  13. Bioproduction of 4-vinylphenol from corn cob alkaline hydrolyzate in two-phase extractive fermentation using free or immobilized recombinant E. coli expressing pad gene

    OpenAIRE

    Salgado, José M.; Rodríguez-Solana, R.; Curiel, José Antonio; Rivas, Blanca de las; Muñoz, Rosario; Domínguez, José Manuel

    2014-01-01

    In situ extractive fermentation was used to produce 4-vinyl derivatives from hydroxycinnamic acids extracted from corn cobs by recombinant Escherichia coli cells expressing Lactobacillus plantarum phenolic acid descarboxylase (PAD) gene. This microorganism mainly produced 4-vinylphenol (4VP) from p-coumaric acid (p-CA). In the first study , we observed that the concentrations of 4VP are higher than 1g/L which had a negative impact on decarboxylation of p-CA to 4VP by recombinant E. coli cells...

  14. Cloning, expression analysis and recombinant expression of a gene encoding a polygalacturonase-inhibiting protein from tobacco, Nicotiana tabacum

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    Chengsheng Zhang

    2016-05-01

    Full Text Available Polygalacturonase inhibiting proteins (PGIPs are major defensive proteins produced by plant cell walls that play a crucial role in pathogen resistance by reducing polygalacturonase (PG activity. In the present study, a novel PGIP gene was isolated from tobacco (Nicotiana tabacum, hereafter referred as NtPGIP. A full-length NtPGIP cDNA of 1,412 bp with a 186 bp 5′-untranslated region (UTR, and 209 bp 3′-UTR was cloned from tobacco, NtPGIP is predicted to encode a protein of 338 amino acids. The NtPGIP sequence from genomic DNA showed no introns and sequence alignments of NtPGIP’s deduced amino acid sequence showed high homology with known PGIPs from other plant species. Moreover, the putative NtPGIP protein was closely clustered with several Solanaceae PGIPs. Further, the expression profile of NtPGIP was examined in tobacco leaves following stimulation with the oomycete Phytophthora nicotianae and other stressors, including salicylic acid (SA, abscisic acid (ABA, salt, and cold treatment. The results showed that all of the treatments up-regulated the expression of NtPGIP at different times. To understand the biochemical activity of NtPGIP gene, a full-length NtPGIP cDNA sequence was subcloned into a pET28a vector and transformed into E. coli BL21 (DE3. Recombinant proteins were successfully induced by 1.0 nmol/L IPTG and the purified proteins effectively inhibited Phytophthora capsici PG activity. The results of this study suggest that NtPGIP may be a new candidate gene with properties that could be exploited in plant breeding.

  15. Quantitative Trait Loci and Candidate Genes for Neutrophil Recruitment in Sterile Inflammation Mapped in AXB-BXA Recombinant Inbred Mice.

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    Quyen Cheng

    Full Text Available Neutrophil recruitment (NR to sites of sterile inflammation plays a key role in tissue damage and healing potential of lesions characteristic to non-infectious inflammatory diseases. Previous studies suggested significant genetic control of neutrophil survival, function, and migration in inflammatory responses to endogenous and exogenous stimuli. We have mapped the murine genome for quantitative trait loci (QTLs harbouring genetic determinants that regulate NR in SI using a murine model of chemically-induced peritonitis. NR was quantified in 16 AXB-BXA recombinant inbred strains and their progenitors, A/J (A and C57BL/6J (B. A continuous distribution of NR was found among the strains, with parent B showing higher NR and parent A showing lower NR (3.0-fold difference, p=0.05. Within the progeny strains, a 5.5-fold difference in NR was observed between the lowest, BXA1, and the highest responders AXB19 (p<0.001. This data was analyzed using GeneNetwork, which linked NR to one significant QTL on chromosome 12 (Peritoneal Neutrophil Recruitment 1, PNR1 and two suggestive QTLs (PNR2, PNR3 on chromosomes 12 and 16 respectively. Sixty-four candidate genes within PNR1 were cross-referenced with currently published data, mRNA expression from two NR microarrays, and single nucleotide polymorphism analysis. The present study brings new light into the genetics of NR in response to cell injury and highlights potential candidate genes Hif1α, Fntb, and Prkch and their products for further studies on neutrophil infiltration and inflammation resolution in sterile inflammation.

  16. Biocatalytic synthesis of flavones and hydroxyl-small molecules by recombinant Escherichia coli cells expressing the cyanobacterial CYP110E1 gene

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    Makino Takuya

    2012-07-01

    Full Text Available Abstract Background Cyanobacteria possess several cytochrome P450s, but very little is known about their catalytic functions. CYP110 genes unique to cyanaobacteria are widely distributed in heterocyst-forming cyanobacteria including nitrogen-fixing genera Nostoc and Anabaena. We screened the biocatalytic functions of all P450s from three cyanobacterial strains of genus Nostoc or Anabaena using a series of small molecules that contain flavonoids, sesquiterpenes, low-molecular-weight drugs, and other aromatic compounds. Results Escherichia coli cells carrying each P450 gene that was inserted into the pRED vector, containing the RhFRed reductase domain sequence from Rhodococcus sp. NCIMB 9784 P450RhF (CYP116B2, were co-cultured with substrates and products were identified when bioconversion reactions proceeded. Consequently, CYP110E1 of Nostoc sp. strain PCC 7120, located in close proximity to the first branch point in the phylogenetic tree of the CYP110 family, was found to be promiscuous for the substrate range mediating the biotransformation of various small molecules. Naringenin and (hydroxyl flavanones were respectively converted to apigenin and (hydroxyl flavones, by functioning as a flavone synthase. Such an activity is reported for the first time in prokaryotic P450s. Additionally, CYP110E1 biotransformed the notable sesquiterpene zerumbone, anti-inflammatory drugs ibuprofen and flurbiprofen (methylester forms, and some aryl compounds such as 1-methoxy and 1-ethoxy naphthalene to produce hydroxylated compounds that are difficult to synthesize chemically, including novel compounds. Conclusion We elucidated that the CYP110E1 gene, C-terminally fused to the P450RhF RhFRed reductase domain sequence, is functionally expressed in E. coli to synthesize a robust monooxygenase, which shows promiscuous substrate specificity (affinity for various small molecules, allowing the biosynthesis of not only flavones (from flavanones but also a variety of

  17. Constructing the recombinant plasmid-pLXSN-SjYF-of Yolk Ferritin gene of Schistosoma japonicum

    Institute of Scientific and Technical Information of China (English)

    ZhouJunmei; YuXinbing; WuZhongdao; ZhengYinan; LiYan

    1999-01-01

    TO construct the recotabinant plasmid —— pLXSN-SjYF to prepare for expression and DNA vaccine of Schistosoma japonicum gene. Methods :Mnpliffing DNA fragment coding yolk fereltin from a female adult Sehistosoma japonicum DNAs by PCR. The fragment was inseted into pLXSN retrovirus vector by digesting with restrictive enzymes and linking reactions. The positive clone was screened on LB plates contmaining amplcillin asld identified by restrictive enzymes digestion and PCR amplification, Resttlts The specific DNA fragmem SjYF was amplified from the female adult SjDNAs. IaLXSN-SjYF was constructed successfully and the further research will be carried out.

  18. [Analysis of the meiotic recombination frequency in transgenic tomato hybrids expressing recA and NLS-recA-licBM3 genes].

    Science.gov (United States)

    Komakhin, R A; Komakhina, V V; Miliukova, N A; Zhuchenko, A A

    2012-01-01

    To study and induce meiotic recombination in plants, we generated and analyzed transgenic tomato hybrids F1-RecA and F1-NLS-recA-LicBM3 expressing, respectively, the recA gene of Escherichia coli and the NLS-recA-licBM3 gene. It was found that the recA and NLS-recA-licBM3 genes are inherited through the maternal and paternal lineages, they have no selective influence on the pollen and are contained in tomato F1-RecA and F1-NLS-RecA-LicBM3 hybrids outside the second chromosome in the hemizygous state. The comparative analysis of the meiotic recombination frequency (rf) in the progenies of the transgenic and nontransgenic hybrids showed that only the expression of the recA gene of E. coli in cells of the F1-RecA plants produced a 1.2-1.5-fold increase in the frequency of recombination between some linked marker genes of the second chromosome of tomato.

  19. Immunogenicity of Hepavax-Gene recombinant hepatitis B vaccine in INML workers, Colombia.

    Directory of Open Access Journals (Sweden)

    Heber Siachoque

    2009-11-01

    Full Text Available Objetivo: Evaluar la inmunogenicidad de la vacuna recombinante Hepavax-Gene para hepatitis B desde la última dosis administrada en trabajadores del INML. Materiales y métodos: Estudio de corte transversal en 603 trabajadores de la salud con mínimo 3 dosis de vacuna recombinante (0, 1, 6 meses donde se midieron los niveles de anticuerpos anti-HBs con la técnica de ELISA entre diciembre de 2000 y enero de 2001 desde la aplicación de la última dosis de la vacuna, que varió entre 1 y 6 años. Resultados: El grupo de estudio lo conformaron 344 hombres y 259 mujeres, con un promedio de edad de 38.8±7.3 años. El nivel de protección fue 90.7% (>10 U/l que disminuyó significativamente con el tiempo de aplicación de la última dosis de la vacuna (p45 (RRI=3.58, IC 95%:1.83, 6.99 con respecto a Conclusión: La vacuna recombinante Hepavax-Gene anti-HBs tiene alta efectividad en los trabajadores de la salud (90.7% aunque presenta disminución de protección a mayor tiempo de aplicación de la última dosis y al aumentar la edad del trabajador.

  20. Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase.

    Science.gov (United States)

    Romero, Elvira; Castellanos, J Rubén Gómez; Mattevi, Andrea; Fraaije, Marco W

    2016-12-19

    Cyclohexanone monooxygenase (CHMO) is a promising biocatalyst for industrial reactions owing to its broad substrate spectrum and excellent regio-, chemo-, and enantioselectivity. However, the low stability of many Baeyer-Villiger monooxygenases is an obstacle for their exploitation in industry. Characterization and crystal structure determination of a robust CHMO from Thermocrispum municipale is reported. The enzyme efficiently converts a variety of aliphatic, aromatic, and cyclic ketones, as well as prochiral sulfides. A compact substrate-binding cavity explains its preference for small rather than bulky substrates. Small-scale conversions with either purified enzyme or whole cells demonstrated the remarkable properties of this newly discovered CHMO. The exceptional solvent tolerance and thermostability make the enzyme very attractive for biotechnology.

  1. Cloning and expression analysis of recombination activating genes (RAG1/2) in red snapper (Lutjanus sanguineus).

    Science.gov (United States)

    Zhang, X L; Lu, Y S; Jian, J C; Wu, Z H

    2012-04-01

    Recombination activating genes (RAG1 and RAG2), involved in the V(D)J recombination of immunoglobulin and T-cell receptor genes play a crucial role in the adaptive immune response in vertebrates. The expression of these genes was required for the proper development and maturity of lymphocytes so that they can be used as useful markers to evaluate the development of lymphoid organ. In this paper, the cDNA of RAG1 and RAG2 in red snapper, Lutjanus sanguineus were cloned by homological cloning and rapid amplification of cDNA ends (RACE) methods. Results showed the full length of RAG1 cDNA was 3944 bp, containing a 5' untranslated region (UTR) of 200 bp, a 3'-UTR of 561 bp and an open reading frame of 3183 bp encoding 1060 amino acids. Three important structural motifs, a RING/U-box domain, a RING/FYVE/PHD-type domain and a RAG Nonamer-binding domain were detected in the deduced amino acid sequence of RAG1 by InterProScan analysis. The full length of RAG2 cDNA was 2200 bp, consisting of a 141 bp 5'-UTR, a 457 bp 3'-UTR and an open reading frame of 1602 bp encoding 533 amino acids. Two important structural motifs, a Galactose oxidase/kelch, beta-propeller domain and a kelch-type beta-propeller domain were detected in the deduced amino acid sequence of RAG2 by InterProScan analysis. BLAST analysis revealed that the RAG1 and RAG2 in red snapper shared a high homology with other known RAG1 and RAG2 genes, while the greatest degree of identity was observed with Hippoglossus hippoglossus RAG1 at 82% and Takifugu rubripes RAG2 at 87%, respectively. The differential expressions of RAG1 and RAG2 in various tissues of red snapper were analyzed by fluorescent quantitative real-time PCR. The overall expression pattern of the two genes was quite similar. In healthy red snappers, the RAGs transcripts were mainly detected in thymus, following head kidney, spleen, intestine, liver and brain. After vaccinated with inactivated Vibrio alginolyticus 48 h later, the RAGs m

  2. Two families of rep-like genes that probably originated by interspecies recombination are represented in viral, plasmid, bacterial, and parasitic protozoan genomes.

    Science.gov (United States)

    Gibbs, Mark J; Smeianov, Vladimir V; Steele, James L; Upcroft, Peter; Efimov, Boris A

    2006-06-01

    Two families of genes related to, and including, rolling circle replication initiator protein (Rep) genes were defined by sequence similarity and by evidence of intergene family recombination. The Rep genes of circoviruses were the best characterized members of the "RecRep1 family." Other members of the RecRep1 family were Rep-like genes found in the genomes of the Canarypox virus, Entamoeba histolytica, and Giardia duodenalis and in a plasmid, p4M, from the Gram-positive bacterium, Bifidobacterium pseudocatenulatum. The "RecRep2 family" comprised some previously identified Rep-like genes from plasmids of phytoplasmas and similar Rep-like genes from the genomes of Lactobacillus acidophilus, Lactococcus lactis, and Phytoplasma asteris. Both RecRep1 and RecRep2 proteins have a nucleotide-binding domain significantly similar to the helicases (2C proteins) of picorna-like viruses. On the N-terminal side of the nucleotide binding domain, RecRep1 proteins have a domain significantly similar to one found in nanovirus Reps, whereas RecRep2 proteins have a domain significantly similar to one in the Reps of pLS1 plasmids. We speculate that RecRep genes have been transferred from viruses or plasmids to parasitic protozoan and bacterial genomes and that Rep proteins were themselves involved in the original recombination events that generated the ancestral RecRep genes.

  3. Tropine forming tropinone reductase gene from Withania somnifera (Ashwagandha: biochemical characteristics of the recombinant enzyme and novel physiological overtones of tissue-wide gene expression patterns.

    Directory of Open Access Journals (Sweden)

    Amit Kumar Kushwaha

    Full Text Available Withania somnifera is one of the most reputed medicinal plants of Indian systems of medicine synthesizing diverse types of secondary metabolites such as withanolides, alkaloids, withanamides etc. Present study comprises cloning and E. coli over-expression of a tropinone reductase gene (WsTR-I from W. somnifera, and elucidation of biochemical characteristics and physiological role of tropinone reductase enzyme in tropane alkaloid biosynthesis in aerial tissues of the plant. The recombinant enzyme was demonstrated to catalyze NADPH-dependent tropinone to tropine conversion step in tropane metabolism, through TLC, GC and GC-MS-MS analyses of the reaction product. The functionally active homodimeric ~60 kDa enzyme catalyzed the reaction in reversible manner at optimum pH 6.7. Catalytic kinetics of the enzyme favoured its forward reaction (tropine formation. Comparative 3-D models of landscape of the enzyme active site contours and tropinone binding site were also developed. Tissue-wide and ontogenic stage-wise assessment of WsTR-I transcript levels revealed constitutive expression of the gene with relatively lower abundance in berries and young leaves. The tissue profiles of WsTR-I expression matched those of tropine levels. The data suggest that, in W. somnifera, aerial tissues as well possess tropane alkaloid biosynthetic competence. In vivo feeding of U-[(14C]-sucrose to orphan shoot (twigs and [(14C]-chasing revealed substantial radiolabel incorporation in tropinone and tropine, confirming the de novo synthesizing ability of the aerial tissues. This inherent independent ability heralds a conceptual novelty in the backdrop of classical view that these tissues acquire the alkaloids through transportation from roots rather than synthesis. The TR-I gene expression was found to be up-regulated on exposure to signal molecules (methyl jasmonate and salicylic acid and on mechanical injury. The enzyme's catalytic and structural properties as well as gene

  4. Endostatin gene therapy for liver cancer by a recombinant adenovirus delivery

    Institute of Scientific and Technical Information of China (English)

    Li Li; Jia-Ling Huang; Qi-Cai Liu; Pei-Hong Wu; Ran-Yi Liu; Yi-Xin Zeng; Wen-Lin Huang

    2004-01-01

    AIM: To investigate the expression of adenovirus-mediated human endostatin (Ad/hEndo) gene transfer and its effect on the growth of hepatocellular carcinoma (HCC) BEL-7402xenografted tumors.METHODS: Immunohistochemistry analysis with an anti endostatin antibody was preformed to detect endostatin protein expression in HCC BEL-7402 cells infected with Ad/hEndo. MTT assay was used to investigate the effects of Ad/hEndo on proliferation of human umbilical vein endothelial cells (HUVEC). Intra-tumoral injections of 1×109 pfu Ad/hEndo was given to treat BEL-7402 xenografted tumors in nude mice once weekly for 6 wk. Mice received injections of Ad/LacZ and DMEM were regarded as control groups. After intra-turmoral administration with Ad/hEndo, the endostatin mRNA expression in tumor tissue was analyzed by Northern blotting, and plasma endostatin levels were determined using enzyme-linked immunosorbent assay (ELTSA).RESULTS: High level expression of endostatin gene was detected in the infected HCC BEL-7402 cells. Ad/hEndo significantly inhibited HUVEC cell proliferation by 57.2% at a multiplicity of infection (MOI) of 20. After 6-week treatment with Ad/hEndo, the growth of treated tumors was inhibited by 46.50% compared to the Ad/ LacZ control group (t=2.729, P<0.05) and by 48.56% compared to the DMEM control group (t=2.485, P<0.05). The ratio of mean tumor volume in treated animals to mean tumor volume in the control animals (T:C ratio) was less than 50% after 24 d of treatment. Endostatin mRNA in tumor tissue was clearly demonstrated as a band of approximately 1.2 kb, which was the expected size of intact and functional endostatin.Plasma endostatin levels peaked at 87.52±8.34 ng/mL at d 3 after Ad/hEndo injection, which was significantly higher than the basal level (12.23±2.54 ng/mL). By d 7,plasma levels dropped to nearly half the peak level(40.34±4.80 ng/mL).CONCLUSION: Adenovirus-mediated human endostatin gene can successfully express endogenous

  5. Polymorphisms of the Homologous Recombination Gene RAD51 in Keratoconus and Fuchs Endothelial Corneal Dystrophy

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    Ewelina Synowiec

    2013-01-01

    Full Text Available Purpose. We investigated the association between genotypes and haplotypes of the c.-61G>T (rs 1801320 and c.-98G>C (rs 1801321 polymorphisms of the RAD51 gene and the occurrence of keratoconus (KC and Fuchs endothelial corneal dystrophy (FECD in dependence on some environmental factors. Methods. The polymorphisms were genotyped in peripheral blood lymphocytes of 100 KC and 100 FECD patients as well as 150 controls with PCR-RFLP. Results. The G/T genotype of the c.-61G>T polymorphism was associated with significantly increased frequency occurrence of KC (crude OR 2.99, 95% CI 1.75–5.13. On the other hand, the G/G genotype of this polymorphism was positively correlated with a decreased occurrence of this disease (crude OR 0.52, 95% CI 0.31–0.88. We did not find any correlation between genotypes/alleles of the c.-98G>C polymorphism and the occurrence of KC. We also found that the G/G genotype and G allele of the c.-98G>C polymorphism had a protective effect against FECD (crude OR 0.51, 95% CI 0.28–0.92; crude OR 0.53, 95% CI 0.30–0.92, resp., while the G/C genotype and the C allele increased FECD occurrence (crude OR 1.85, 95% CI 1.01–3.36; crude OR 1.90, 95% CI 1.09–3.29, resp.. Conclusions. The c.-61T/T and c.-98G>C polymorphisms of the RAD51 gene may have a role in the KC and FECD pathogenesis and can be considered as markers in these diseases.

  6. Iron-dependent hydrogenases of Entamoeba histolytica and Giardia lamblia: activity of the recombinant entamoebic enzyme and evidence for lateral gene transfer.

    Science.gov (United States)

    Nixon, Julie E J; Field, Jessica; McArthur, Andrew G; Sogin, Mitchell L; Yarlett, Nigel; Loftus, Brendan J; Samuelson, John

    2003-02-01

    Entamoeba histolytica and Spironucleus barkhanus have genes that encode short iron-dependent hydrogenases (Fe-hydrogenases), even though these protists lack hydrogenosomes. To understand better the biochemistry of the protist Fe-hydrogenases, we prepared a recombinant E. histolytica short Fe-hydrogenase and measured its activity in vitro. A Giardia lamblia gene encoding a short Fe-hydrogenase was identified from shotgun genomic sequences, and RT-PCR showed that cultured entamoebas and giardias transcribe short Fe-hydrogenase mRNAs. A second E. histolytica gene, which encoded a long Fe-hydrogenase, was identified from shotgun genomic sequences. Phylogenetic analyses suggested that the short Fe-hydrogenase genes of entamoeba and diplomonads share a common ancestor, while the long Fe-hydrogenase gene of entamoeba appears to have been laterally transferred from a bacterium. These results are discussed in the context of competing ideas for the origins of genes encoding fermentation enzymes of these protists.

  7. Immune Efficacy of a Recombinant Fowlpox Virus Co-Ex-pressing HA and NA Genes of Avian Influenza Virus in SPF Chickens

    Institute of Scientific and Technical Information of China (English)

    QIAO Chuan-ling; JIANG Yong-ping; YU Kang-zhen; TIAN Guo-bin; CHEN Hua-lan

    2004-01-01

    A recombinant fowlpox virus co-expressing Haemagglutinin(HA)and Neuraminidase(NA)named as rFPV-HA-NA was produced by HA and NA gene of A/Goose/Guangdong/3/96(H5N1)isolate of avian influenza virus recombined into the genome of fowlpox virus. In this study,to evaluate its ability of protecting chickens against challenge with a lethal dose of highly pathogenic isolates of avian influenza virus,eight-week-old specificpathogenic-free(SPF)chickens were vaccinated with recombinant virus or the wildtypefowlpox virus by wing-web puncture. After challenge 4 weeks with 10 LD50 highly pathogenic avian influenza virus H5N1 and H7N1 isolate,all chickens vaccinated with recombinant virus were protected,while the chickens vaccinated with the wildtype fowlpox virus or unvaccinated controls experienced 100% mortality respectively following challenge. This complete protection was accompanied by the high levels of specific antibody response to the respectivecomponents of the recombinant virus.

  8. Molecular expression of l-asparaginase gene from Nocardiopsis alba NIOT-VKMA08 in Escherichia coli: A prospective recombinant enzyme for leukaemia chemotherapy.

    Science.gov (United States)

    Meena, Balakrishnan; Anburajan, Lawrance; Vinithkumar, Nambali Valsalan; Shridhar, Divya; Raghavan, Rangamaran Vijaya; Dharani, Gopal; Kirubagaran, Ramalingam

    2016-09-30

    l-Asparaginase is an antineoplastic agent that selectively reduces the level of l-asparagine in blood and diminishes the proliferation of cancerous cells. Studies were carried out on the cloning and heterologous expression of l-asparaginase biosynthesis gene (ansA) from Nocardiopsis alba NIOT-VKMA08 to achieve the stable inducible system that overproduces the glutaminase-free recombinant l-asparaginase. Overexpression of recombinant l-asparaginase was achieved with an optimized final concentration of 1.5mM of isopropyl-β-d-thiogalactoside (IPTG) and the enzyme was expressed as a soluble protein. The recombinant enzyme was purified using nickel-nitrilotriacetic acid (Ni-NTA) chromatography and the purified enzyme disclosed an elevated level of asparaginase activity (158.1IU/mL). Optimum pH and temperature of the purified l-asparaginase for the hydrolysis of l-asparagine were 8.0 and 37°C and it was very specific for its natural substrate, l-asparagine. Detailed studies were carried out on the kinetics of enzyme reaction, catalytic activity, temperature and ionic strength and the thermostability of the l-asparaginase enzyme. The functional characterisation of the recombinant l-asparaginase was studied through Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), in silico sequence analysis and protein structural modelling. Glutaminase activity was not detected in the recombinant l-asparaginase, which could reduce the probable side effects during leukaemia therapy.

  9. Engineering the cellular protein secretory pathway for enhancement of recombinant tissue plasminogen activator expression in Chinese hamster ovary cells: effects of CERT and XBP1s genes.

    Science.gov (United States)

    Rahimpour, Azam; Vaziri, Behrouz; Moazzami, Reza; Nematollahi, Leila; Barkhordari, Farzaneh; Kokabee, Leila; Adeli, Ahmad; Mahboudi, Fereidoun

    2013-08-01

    Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERTS132A- based secretion engineering could be an effective strategy for enhancing recombinant t- PA production in CHO cells.

  10. Gene therapy that inhibits NF-κB results in apoptosis of human hepatocarcinoma by recombinant adenovirus

    Institute of Scientific and Technical Information of China (English)

    Tie-Jun Li; Li-Ping Jia; Xiao-Ling Gao; Ai-Long Huang

    2006-01-01

    AIM: To investigate whether the recombinant adenovirus induces the TNF-α-mediated apoptosis in vivo.METHODS: Human hepatocarcinoma cell line (HepG2)cells were transfected into BALB/c nude mice, and the tumor growth curve was drawn. We analyzed apoptosis in HepG2 cells by TUNEL, HE staining and electron microscopy.RESULTS: AdIκBαM was expressed stably and efficiently in HepG2 and could not be degraded by induction of TNF-α. Tumor growth in mice could be reduced remarkably if treated by AdIκBαM plus TNF-α. There was apoptosis of > 70% of cells treated with AdIκBαM plus TNF-α and about 50% of cells treated with AdIκBαM. In contrast, there was few cell apoptosis in HepG2 cells treated with phosphate buffered saline and AdIκBα. HepG2 cells in mice also exhibited a high level of apoptosis after in vivo injection with AdIκBαM. The tumor growth curve indicated the tumor transfected with AdIκBαM could be restrained.CONCLUSION: AdIκBαM gene therapy greatly enhances apoptosis due to inhibition of an NF-κB-mediated antiapoptosis signaling pathway.

  11. Mutations in Recombination Activating Gene 1 and 2 in patients with severe combined immunodeficiency disorders in Egypt.

    Science.gov (United States)

    Meshaal, Safa; El Hawary, Rabab; Elsharkawy, Marwa; Mousa, Reem K; Farid, Reem J; Abd Elaziz, Dalia; Alkady, Radwa; Galal, Nermeen; Massaad, Michel J; Boutros, Jeannette; Elmarsafy, Aisha

    2015-06-01

    The Recombination Activating Genes (RAG) 1/2 are important for the development and function of T and B cells. Loss of RAG1/2 function results in severe combined immunodeficiency (SCID), which could lead to early death. We studied the prevalence of RAG1/2 mutations in ten SCID patients in Egypt. We identified two novel homozygous nonsense mutations in RAG1, a novel homozygous deletion, and a previously reported homozygous missense mutation from four patients, as well as two homozygous mutations in RAG2 from the same patient. Prenatal diagnosis performed in the mother of a patient with RAG1 deficiency determined that the fetus was heterozygous for the same mutation. This represents the first report on RAG1/2 mutations in SCID patients in Egypt. The early diagnosis dramatically affects the outcome of the disease by allowing bone marrow transplantation at an early age, and providing prenatal diagnosis and genetic counseling for families with a history of SCID.

  12. Genetic transformation of marine Actinomycete sp. Isolate M048 and expression of a recombinant plasmid carrying the apc gene

    Institute of Scientific and Technical Information of China (English)

    HOU Yanhua; LI Fuchao; QIN Song; WANG Quanfu

    2006-01-01

    Optimal conditions for protoplasts formation of marine Actinomycete sp. isolate M048 were described, dense and disperse mycelia were cultured in SGGP medium, 0.5% glycine, lysozyme exposure (2 mg/cm3, 37 ℃, 40 min), and the concentration of sucrose in protoplast buffer was 0.4 mol/dm3 for keeping the balance of osmotic pressure. Using PEG-mediated protoplasts transformation, the transformation frequency was 89 transformants per microgramme of pIJ702. Meanwhile, an effective transformation procedure was established based on intergeneric conjugation from E. coli ET12567 (pUZ8002) using shuttle vectors pPM801, pPM803 and a(ψ)C31-derived integration vector pIJ8600 containing oriT and attP fragments. Transformation frequencies were 5.30×10-4±0.26×10-4, 8.92×10-4±0.19×10-4 and 6.38×10-5±0.41×10-5, respectively. Further, the heterologous expression of the allophycocyanin gene (apc) in the strain M048 was used to demonstrate this transformation system. SDS-PAGE and Western blot analysis confirmed the expression of recombinant APC (rAPC).

  13. Exploring the 5'-UTR DNA region as a target for optimizing recombinant gene expression from the strong and inducible Pm promoter in Escherichia coli.

    Science.gov (United States)

    Berg, Laila; Kucharova, Veronika; Bakke, Ingrid; Valla, Svein; Brautaset, Trygve

    2012-04-30

    By using the strong and inducible Pm promoter as a model, we recently reported that the β-lactamase production (encoded by bla) can be stimulated up to 20-fold in Escherichia coli by mutating the DNA region corresponding to the 5'-untranslated region of mRNA (UTR). One striking observation was the unexpected large stimulatory effect some of these UTR variants had on the bla transcript production level. We here demonstrate that such UTR variants can also be used to improve the expression level of the alternative genes celB (encoding phosphoglucomutase) and inf-α2b (encoding human cytokine interferon α2b), which both can be expressed to high levels even with the wild-type Pm UTR DNA sequence. Our data indicated some degree of context dependency between the UTR DNA and concomitant recombinant gene sequences. By constructing and using a synthetic operon, we demonstrated that UTR variants optimized for high-level expression of probably any recombinant gene can be efficiently selected from large UTR mutant libraries. The stimulation affected both the transcript production and translational level, and such modified UTR sequences therefore clearly have a significant applied potential for improvement of recombinant gene expression processes.

  14. A functional analysis of N-glycosylation-related genes on sialylation of recombinant erythropoietin in six commonly used mammalian cell lines.

    Science.gov (United States)

    Zhang, Peiqing; Tan, Diana Lifen; Heng, Desmond; Wang, Tianhua; Mariati; Yang, Yuansheng; Song, Zhiwei

    2010-11-01

    Significant efforts have been made to improve the sialylation of recombinant glycoproteins with the aim of extending their in vivo circulation time. Here, we report a systematic functional analysis of 31 N-glycosylation-related genes on sialylation of recombinant EPO in six cell lines. BHK and CHO cells were found to sialylate recombinant EPO most effectively. None of the 31 genes, individually or in combination, was able to improve EPO sialylation in these cells. HEK293, Cos-7 and 3T3 cells showed intermediate sialylation capabilities, whereas NS0 cells sialylated recombinant EPO poorly. Overexpression of ST6GalI, ST3GalIII or ST3GalIV, but not ST3GalVI, was able to improve EPO sialylation in these four cell lines. qRT-PCR experiments revealed that ST3GalIII and ST3GalIV are indeed under expressed in HEK293, 3T3 and NS0 cells. Co-expression of upstream glycogenes failed to synergize with these sialyltransferases to further enhance sialylation, suggesting that the upstream glycogenes are all expressed at sufficient levels.

  15. Kynurenine 3-monooxygenase polymorphisms: relevance for kynurenic acid synthesis in patients with schizophrenia and healthy controls

    DEFF Research Database (Denmark)

    Holtze, Maria; Saetre, Peter; Engberg, Göran;

    2012-01-01

    on the activity of kynurenine 3-monooxygenase (KMO), the enzyme converting kynurenine to 3-hydroxykynurenine. Methods: We analyzed the association between KMO gene polymorphisms and CSF concentrations of KYNA in patients with schizophrenia and healthy controls. Fifteen single nucleotide polymorphisms (SNPs) were......Background: Patients with schizophrenia show increased brain and cerebrospinal fluid (CSF) concentrations of the endogenous N-methyl-D-aspartate receptor antagonist kynurenic acid (KYNA). This compound is an end-metabolite of the kynurenine pathway, and its formation indirectly depends...... selected covering KMO and were analyzed in UNPHASED. Results: We included 17 patients with schizophrenia and 33 controls in our study. We found an association between a KMO SNP (rs1053230), encoding an amino acid change of potential importance for substrate interaction, and CSF concentrations of KYNA...

  16. High-level recombinant protein production in CHO cells using an adenoviral vector and the cumate gene-switch.

    Science.gov (United States)

    Gaillet, Bruno; Gilbert, Rénald; Amziani, Rachid; Guilbault, Claire; Gadoury, Christine; Caron, Antoine W; Mullick, Alaka; Garnier, Alain; Massie, Bernard

    2007-01-01

    To facilitate and accelerate the production of eukaryotic proteins with correct post-translational modifications, we have developed a protein production system based on the transduction of Chinese hamster ovary (CHO) cells using adenovirus vectors (AdVs). We have engineered a CHO cell line (CHO-cTA) that stably expresses the transactivator (cTA) of our newly developed cumate gene-switch transcription system. This cell line is adapted to suspension culture and can grow in serum-free and protein-free medium. To increase the transduction level of AdVs, we have also generated a cell line (CHO-cTA-CAR) that expresses additional amounts of the coxackievirus and adenovirus receptor (CAR) on its surface. Recombinant protein production was tested using an AdV carrying the secreted alkaline phosphatase (SEAP) under the control of the CR5 promoter, which is strongly and specifically activated by binding to cTA. The SEAP expression was linked to the expression of the green fluorescent protein (GFP) through an internal ribosome entry site (IRES) to facilitate titration of the AdV. We monitored SEAP expression on a daily basis for 9 days after transduction of CHO-cTA and CHO-cTA-CAR using different quantities of AdVs at 37 and 30 degrees C. Incubation at the latter temperature increased the production of SEAP at least 10-fold, and the presence of CAR increased the transduction level of the AdV. Maximum SEAP production (63 mg/L) was achieved at 6-7 days post-infection at 30 degrees C by transducing CHO-cTA-CAR with 500 infectious particles/cell. Because numerous AdVs can now be generated within a few weeks and large-scale production of AdVs is now a routine procedure, this system could be used to produce rapidly milligram quantities of a battery of recombinant proteins as well as for large-scale protein production.

  17. Independent recruitment of a flavin-dependent monooxygenase for safe accumulation of sequestered pyrrolizidine alkaloids in grasshoppers and moths.

    Directory of Open Access Journals (Sweden)

    Linzhu Wang

    Full Text Available Several insect lineages have developed diverse strategies to sequester toxic pyrrolizidine alkaloids from food-plants for their own defense. Here, we show that in two highly divergent insect taxa, the hemimetabolous grasshoppers and the holometabolous butterflies, an almost identical strategy evolved independently for safe accumulation of pyrrolizidine alkaloids. This strategy involves a pyrrolizidine alkaloid N-oxygenase that transfers the pyrrolizidine alkaloids to their respective N-oxide, enabling the insects to avoid high concentrations of toxic pyrrolizidine alkaloids in the hemolymph. We have identified a pyrrolizidine alkaloid N-oxygenase, which is a flavin-dependent monooxygenase, of the grasshopper Zonocerus variegatus. After heterologous expression in E. coli, this enzyme shows high specificity for pyrrolizidine alkaloids of various structural types and for the tropane alkaloid atropine as substrates, a property that has been described previously for a pyrrolizidine alkaloid N-oxygenase of the arctiid moth Grammia geneura. Phylogenetic analyses of insect flavin-dependent monooxygenase sequences suggest that independent gene duplication events preceded the establishment of this specific enzyme in the lineages of the grasshoppers and of arctiid moths. Two further flavin-dependent monooxygenase sequences have been identified from Z. variegatus sharing amino acid identities of approximately 78% to the pyrrolizidine alkaloid N-oxygenase. After heterologous expression, both enzymes are also able to catalyze the N-oxygenation of pyrrolizidine alkaloids, albeit with a 400-fold lower specific activity. With respect to the high sequence identity between the three Z. variegatus sequences this ability to N-oxygenize pyrrolizidine alkaloids is interpreted as a relict of a former bifunctional ancestor gene of which one of the gene copies optimized this activity for the specific adaptation to pyrrolizidine alkaloid containing food plants.

  18. Independent recruitment of a flavin-dependent monooxygenase for safe accumulation of sequestered pyrrolizidine alkaloids in grasshoppers and moths.

    Science.gov (United States)

    Wang, Linzhu; Beuerle, Till; Timbilla, James; Ober, Dietrich

    2012-01-01

    Several insect lineages have developed diverse strategies to sequester toxic pyrrolizidine alkaloids from food-plants for their own defense. Here, we show that in two highly divergent insect taxa, the hemimetabolous grasshoppers and the holometabolous butterflies, an almost identical strategy evolved independently for safe accumulation of pyrrolizidine alkaloids. This strategy involves a pyrrolizidine alkaloid N-oxygenase that transfers the pyrrolizidine alkaloids to their respective N-oxide, enabling the insects to avoid high concentrations of toxic pyrrolizidine alkaloids in the hemolymph. We have identified a pyrrolizidine alkaloid N-oxygenase, which is a flavin-dependent monooxygenase, of the grasshopper Zonocerus variegatus. After heterologous expression in E. coli, this enzyme shows high specificity for pyrrolizidine alkaloids of various structural types and for the tropane alkaloid atropine as substrates, a property that has been described previously for a pyrrolizidine alkaloid N-oxygenase of the arctiid moth Grammia geneura. Phylogenetic analyses of insect flavin-dependent monooxygenase sequences suggest that independent gene duplication events preceded the establishment of this specific enzyme in the lineages of the grasshoppers and of arctiid moths. Two further flavin-dependent monooxygenase sequences have been identified from Z. variegatus sharing amino acid identities of approximately 78% to the pyrrolizidine alkaloid N-oxygenase. After heterologous expression, both enzymes are also able to catalyze the N-oxygenation of pyrrolizidine alkaloids, albeit with a 400-fold lower specific activity. With respect to the high sequence identity between the three Z. variegatus sequences this ability to N-oxygenize pyrrolizidine alkaloids is interpreted as a relict of a former bifunctional ancestor gene of which one of the gene copies optimized this activity for the specific adaptation to pyrrolizidine alkaloid containing food plants.

  19. Immunogenicity of recombinant attenuated Salmonella enterica serovar Typhimurium vaccine strains carrying a gene that encodes Eimeria tenella antigen SO7.

    Science.gov (United States)

    Konjufca, Vjollca; Jenkins, Mark; Wang, Shifeng; Juarez-Rodriguez, Maria Dolores; Curtiss, Roy

    2008-12-01

    Recombinant attenuated Salmonella vaccines against avian coccidiosis were developed to deliver Eimeria species antigens to the lymphoid tissues of chickens via the type 3 secretion system (T3SS) and the type 2 secretion system (T2SS) of Salmonella. For antigen delivery via the T3SS, the Eimeria tenella gene encoding sporozoite antigen SO7 was cloned downstream of the translocation domain of the Salmonella enterica serovar Typhimurium sopE gene in the parental pYA3868 and pYA3870 vectors to generate pYA4156 and pYA4157. Newly constructed T3SS vectors were introduced into host strain chi8879 (Delta phoP233 Delta sptP1033::xylE Delta asdA16), an attenuated derivative of the highly virulent UK-1 strain. The SopE-SO7 fusion protein was secreted by the T3SS of Salmonella. The vector pYA4184 was constructed for delivery of the SO7 antigen via the T2SS. The SO7 protein was toxic to Salmonella when larger amounts were synthesized; thus, the synthesis of this protein was placed under the control of the lacI repressor gene, whose expression in turn was dependent on the amount of available arabinose in the medium. The pYA4184 vector was introduced into host strain chi9242 (Delta phoP233 Delta asdA16 Delta araBAD23 Delta relA198::araC P(BAD) lacI TT [TT is the T4ipIII transcription terminator]). In addition to SO7, for immunization and challenge studies we used the EAMZ250 antigen of Eimeria acervulina, which was previously shown to confer partial protection against E. acervulina challenge when it was delivered via the T3SS. Immunization of chickens with a combination of the SO7 and EAMZ250 antigens delivered via the T3SS induced superior protection against challenge by E. acervulina. In contrast, chickens immunized with SO7 that was delivered via the T2SS of Salmonella were better protected from challenge by E. tenella.

  20. Osteogenic differentiation of recombinant adeno-associated virus 2-transduced murine mesenchymal stem cells and development of an immunocompetent mouse model for ex vivo osteoporosis gene therapy.

    Science.gov (United States)

    Kumar, Sanjay; Mahendra, Gandham; Nagy, Tim R; Ponnazhagan, Selvarangan

    2004-12-01

    Gene therapy for osteopenic conditions including osteoporosis is a potential alternative to pharmacotherapy for cost effectiveness, long-term viability, and the ability to enhance bone mass by anabolic approaches. Increased understanding of mesenchymal stem cell (MSC) lineage differentiation during osteogenesis, and of the molecular pathways involved in bone cell production, provides an opportunity for the advancement of gene therapy approaches for osteopenic conditions. The potential of MSCs in osteoblast differentiation and the relative ease of MSC isolation and culturing offer a promising resource for the development of ex vivo gene therapy for bone defects. In an effort to develop ex vivo gene therapy for osteoporosis, we used gene-modified MSCs in a preclinical mouse model to determine the efficiency of transduction of murine MSCs by recombinant adeno-associated virus 2 (AAV) vectors carrying reporter genes and determined their osteogenic potential after recombinant AAV-mediated expression of bone morphogenic protein 2, known to induce osteoblast differentiation. Although surgical ovariectomy is believed to induce progressive bone loss in mouse models, similar to an osteoporosis-like phenotype in humans, several factors, including hormonal alteration and dietary habits, significantly affect both the onset and progression of the disease. Thus, in the present study, we determined the influence of these factors and developed an immunocompetent mouse model of osteoporosis with degenerative bone loss as in the human pathology.

  1. Immunoglobulin genes undergo legitimate repair in human B cells not only after cis- but also frequent trans-class switch recombination.

    Science.gov (United States)

    Laffleur, B; Bardet, S M; Garot, A; Brousse, M; Baylet, A; Cogné, M

    2014-01-01

    Immunoglobulin (Ig) genes specifically recruit activation-induced deaminase (AID) for 'on-target' DNA deamination, initiating either variable (V) region somatic hypermutation, or double-strand break intermediates of class switch recombination (CSR). Such breaks overwhelmingly undergo legitimate intra-Ig repair rather than rare illegitimate and potentially oncogenic junctions outside of Ig loci. We show that in human B cells, legitimate synapsis and repair efficiently join Ig genes whether physically linked on one chromosome or located apart on both alleles. This indicates mechanisms faithfully recognizing and/or pairing loci with homology in structure and accessibility, thus licensing interchromosomal trans-CSR junctions while usually preventing illegitimate interchromosomal recombination with AID off-target genes. Physical linkage of IgH genes in cis on the same allele just increases the likelihood of legitimate repair by another fourfold. The strongest force driving CSR might thus be recognition of legitimate target genes. Formation of IgH intra-allelic loops along this process would then constitute a consequence rather than a pre-requisite of this gene-pairing process.

  2. Molecular mapping and intra-cluster recombination between anthracnose race-specific resistance genes in the common bean differential cultivars Mexico 222 and Widusa.

    Science.gov (United States)

    Rodríguez-Suárez, Cristina; Ferreira, Juan José; Campa, Ana; Pañeda, Astrid; Giraldez, Ramón

    2008-04-01

    Resistance to races 19, 31, 38, 65, 73, 102, and 449, of the pathogenic fungus Colletotrichum lindemuthianum (anthracnose) was evaluated in F(3) families derived from the cross between the anthracnose differential bean cultivars Mexico 222 (resistant to races 19, 31, and 38) and Widusa (resistant to races 38, 65, 73, 102, and 449). Molecular marker analyses were carried out in the corresponding F(2) individuals in order to identify the genes for anthracnose resistance present in these two differential cultivars. The results of the combined segregation indicate that the resistance to anthracnose races 19, 31, and 38, present in Mexico 222, is conferred by single dominant race-specific genes organized in a cluster located in B4 linkage group, corresponding to the previously described Co-3/Co-9 locus. The resistance to anthracnose races 65, 73, 102, and 449, present in Widusa, is conferred by a dominant gene (or genes) representing a different haplotype of the same Co-3/Co-9 cluster. A single dominant gene located in a position independent from cluster Co-3/Co-9 (probably at the Co-1 locus) confers specific resistance to race 38 in Widusa. Recombinants for closely linked resistance specificities belonging to the Co-3/Co-9 cluster have been detected. The possibility of pyramiding race-specific resistance genes by means of intra-cluster recombination, and its potential use in plant breeding, is indicated.

  3. Differences in gene expression of granulosa cells from women undergoing controlled ovarian hyperstimulation with either recombinant follicle-stimulating hormone or highly purified human menopausal gonadotropin

    DEFF Research Database (Denmark)

    Grøndahl, Marie Louise; Borup, Rehannah; Lee, Young Bae

    2009-01-01

    OBJECTIVE: To investigate the differences in the gene expression profile of granulosa cells from preovulatory follicles after controlled ovarian hyperstimulation (COH) with recombinant follicle-stimulating hormone (FSH) or urinary human menopausal gonadotropin (hMG) FSH. DESIGN: Prospective rando...... be important for periovulatory events, which suggests that the preparation used for COH is important for granulosa cell function and may influence the developmental competence of the oocyte and the function of corpus luteum....

  4. Prevalence of Germline Mutations in Genes Engaged in DNA Damage Repair by Homologous Recombination in Patients with Triple-Negative and Hereditary Non-Triple-Negative Breast Cancers.

    Directory of Open Access Journals (Sweden)

    Pawel Domagala

    Full Text Available This study sought to assess the prevalence of common germline mutations in several genes engaged in the repair of DNA double-strand break by homologous recombination in patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers. Tumors deficient in this type of DNA damage repair are known to be especially sensitive to DNA cross-linking agents (e.g., platinum drugs and to poly(ADP-ribose polymerase (PARP inhibitors.Genetic testing was performed for 36 common germline mutations in genes engaged in the repair of DNA by homologous recombination, i.e., BRCA1, BRCA2, CHEK2, NBN, ATM, PALB2, BARD1, and RAD51D, in 202 consecutive patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers.Thirty five (22.2% of 158 patients in the triple-negative group carried mutations in genes involved in DNA repair by homologous recombination, while 10 (22.7% of the 44 patients in the hereditary non-triple-negative group carried such mutations. Mutations in BRCA1 were most frequent in patients with triple-negative breast cancer (18.4%, and mutations in CHEK2 were most frequent in patients with hereditary non-triple-negative breast cancers (15.9%. In addition, in the triple-negative group, mutations in CHEK2, NBN, and ATM (3.8% combined were found, while mutations in BRCA1, NBN, and PALB2 (6.8% combined were identified in the hereditary non-triple-negative group.Identifying mutations in genes engaged in DNA damage repair by homologous recombination other than BRCA1/2 can substantially increase the proportion of patients with triple-negative breast cancer and hereditary non-triple-negative breast cancer who may be eligible for therapy using PARP inhibitors and platinum drugs.

  5. Cello-Oligosaccharide Oxidation Reveals Differences between Two Lytic Polysaccharide Monooxygenases (Family GH61) from Podospora anserina

    OpenAIRE

    Bey, Mathieu; Zhou, Simeng; Poidevin, Laetitia; Henrissat, Bernard; Coutinho, Pedro M.; Berrin, Jean-Guy; Sigoillot, Jean-Claude

    2013-01-01

    The genome of the coprophilic ascomycete Podospora anserina encodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserina GH61A [PaGH61A] and PaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced in Pichia pastoris. Synergistic cooperation between PaGH61A or PaGH61B with the cellobiose dehydrogenase (CDH) of Pycnoporus cinnab...

  6. RAD1 and RAD10, but not other excision repair genes, are required for double-strand break-induced recombination in Saccharomyces cerevisiae.

    Science.gov (United States)

    Ivanov, E L; Haber, J E

    1995-04-01

    HO endonuclease-induced double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae can be repaired by the process of gap repair or, alternatively, by single-strand annealing if the site of the break is flanked by directly repeated homologous sequences. We have shown previously (J. Fishman-Lobell and J. E. Haber, Science 258:480-484, 1992) that during the repair of an HO-induced DSB, the excision repair gene RAD1 is needed to remove regions of nonhomology from the DSB ends. In this report, we present evidence that among nine genes involved in nucleotide excision repair, only RAD1 and RAD10 are required for removal of nonhomologous sequences from the DSB ends. rad1 delta and rad10 delta mutants displayed a 20-fold reduction in the ability to execute both gap repair and single-strand annealing pathways of HO-induced recombination. Mutations in RAD2, RAD3, and RAD14 reduced HO-induced recombination by about twofold. We also show that RAD7 and RAD16, which are required to remove UV photodamage from the silent HML, locus, are not required for MAT switching with HML or HMR as a donor. Our results provide a molecular basis for understanding the role of yeast nucleotide excision repair gene and their human homologs in DSB-induced recombination and repair.

  7. Evaluation of a novel strain of infectious bronchitis virus emerged as a result of spike gene recombination between two highly diverged parent strains.

    Science.gov (United States)

    Hewson, Kylie A; Noormohammadi, Amir H; Devlin, Joanne M; Browning, Glenn F; Schultz, Bridie K; Ignjatovic, Jagoda

    2014-01-01

    The emergence of new variant strains of the poultry pathogen infectious bronchitis virus (IBV) is continually reported worldwide, owing to the labile nature of the large single-stranded RNA IBV genome. High resolution melt curve analysis previously detected a variant strain, N1/08, and the present study confirmed that this strain had emerged as a result of recombination between Australian subgroup 2 and 3 strains in the spike gene region, in a similar manner reported for turkey coronaviruses. The S1 gene for N1/08 had highest nucleotide similarity with subgroup 2 strains, which is interesting considering subgroup 2 strains have not been detected since the early 1990s. SimPlot analysis of the 7.2-kb 3' end of the N1/08 genome with the same region for other Australian reference strains identified the sites of recombination as immediately upstream and downstream of the S1 gene. A pathogenicity study in 2-week-old chickens found that N1/08 had similar pathogenicity for chicken respiratory tissues to that reported for subgroup 2 strains rather than subgroup 3 strains. The results of this study demonstrate that recombination is a mechanism utilized for the emergence of new strains of IBV, with the ability to alter strain pathogenicity in a single generation.

  8. Therapeutic targeting of liver cancer with a recombinant DNA vaccine containing the hemagglutinin-neuraminidase gene of Newcastle disease virus via apoptotic-dependent pathways.

    Science.gov (United States)

    Chen, Li-Gang; Liu, Yuan-Sheng; Zheng, Tang-Hui; Chen, Xu; Li, Ping; Xiao, Chuan-Xing; Ren, Jian-Lin

    2016-11-01

    A total of ~38.6 million mortalities occur due to liver cancer annually, worldwide. Although a variety of therapeutic methods are available, the efficacy of treatment at present is extremely limited due to an increased risk of malignancy and inherently poor prognosis of liver cancer. Gene therapy is considered a promising option, and has shown notable potential for the comprehensive therapy of liver cancer, in keeping with advances that have been made in the development of cancer molecular biology. The present study aimed to investigate the synergistic effects of the abilities of the hemagglutinin neuraminidase protein of Newcastle disease virus (NDV), the pro-apoptotic factor apoptin from chicken anaemia virus, and the interferon-γ inducer interleukin-18 (IL-18) in antagonizing liver cancer. Therefore, a recombinant DNA plasmid expressing the three exogenous genes, VP3, IL-18 and hemagglutinin neuraminidase (HN), was constructed. Flow cytometry, acridine orange/ethidium bromide staining and analysis of caspase-3 activity were performed in H22 cell lines transfected with the recombinant DNA plasmid. In addition, 6-week-old C57BL/6 mice were used to establish a H22 hepatoma-bearing mouse model. Mice tumor tissue was analyzed by immunohistochemistry and scanning electron microscopy. The results of the present study revealed that the recombinant DNA vaccine containing the VP3, IL-18 and HN genes inhibited cell proliferation and induced autophagy via the mitochondrial pathway in vivo and in vitro.

  9. Replacement of Imu-Cmu intron by NeoR gene alters Imu germ-line expression but has no effect on V(D)J recombination.

    Science.gov (United States)

    Haddad, Dania; Dougier, Hei-Lanne; Laviolette, Nathalie; Puget, Nadine; Khamlichi, Ahmed Amine

    2010-02-01

    The NeoR gene has often been used to unravel the mechanisms underlying long-range interactions between promoters and enhancers during V(D)J assembly and class switch recombination (CSR) in the immunoglobulin heavy chain (IgH) locus. This approach led to the notion that CSR is regulated through competition of germ-line (GL) promoters for activities displayed by the 3' regulatory region (3'RR). This polarized long-range effect of the 3'RR is disturbed upon insertion of NeoR gene in the IgH constant (C(H)) region, where only GL transcription derived from upstream GL promoters is impaired. In the context of V(D)J recombination, replacement of Emu enhancer or Emu core enhancer (cEmu) by NeoR gene fully blocked V(D)J recombination and mu0 GL transcription which originates 5' of DQ52 and severely diminished Imu GL transcription derived from Emu/Imu promoter, suggesting a critical role for cEmu in the regulation of V(D)J recombination and of mu0 and Imu expression. Here we focus on the effect of NeoR gene on mu0 and Imu GL transcription in a mouse line in which the Imu-Cmu intron was replaced by a NeoR gene in the sense-orientation. B cell development was characterized by a marked but incomplete block at the pro-B cell stage. However, V(D)J recombination was unaffected in sorted pro-B and pre-B cells excluding an interference with the accessibility control function of Emu. mu0 GL transcription initiation was relatively normal but the maturation step seemed to be affected most likely through premature termination at NeoR polyadenylation sites. In contrast, Imu transcription initiation was impaired suggesting an interference of NeoR gene with the IgH enhancers that control Imu expression. Surprisingly, in stark contrast with the NeoR effect in the C(H) region, LPS-induced NeoR expression restored Imu transcript levels to normal. The data suggest that Emu enhancer may be the master control element that counteracts the down-regulatory "Neo effect" on Imu expression upon LPS

  10. Construction of prokaryotic expression system of ureB gene from a clinical Helicobacter pylori strain and identification of the recombinant protein immunity

    Institute of Scientific and Technical Information of China (English)

    Ya-Fei Mao; Jie Yan

    2004-01-01

    AIM: To clone ureB gene from a clinical isolate ofHelicobacter pyloriand construct a prokaryotic expression system of the gene and identify immunity of the expressed recombinant protein.METHODS: ureB gene from a clinical H pyloristrain Y06 was amplified by the high fidelity polymerase chain reaction technique. The target DNA fragment amplified from ureB gene was sequenced after T-A cloning. Prokaryotic recombinant expression vector pET32a inserted with ureB gene (pET32a-ureB) was constructed. The expression of recombinant UreB protein (rUreB) in E. coliBL21DE3 induced by isopropylthio-β-D-galactoside (IPTG) at different concentrations was examined by SDS-PAGE. Western blot using commercial antibodies against whole cell of Hpylori and an immunodiffusion assay using a self-prepared rabbit anti-rUreB antibody were applied to determine immunity of the target recombinant protein. ELISA was used to detect the antibody against rUreB in sera of 125 Hpyloriinfected patients and to examine rUreB expression in 109 Hpylori isolates.RESULTS: In comparison with the reported corresponding sequences, the nucleotide sequence homology of the cloned ureBgene was from 96.88-97.82% while the homology of its putative amino acid sequence was as high as 99.65-99.82%.The rUreB output expressed by pET32a-ureB-BL21DE3 was approximate 30% of the total bacterial proteins. rUreB specifically combined with the commercial antibodies against whole cell of H pyloriand strongly induced rabbits to produce antibody with a 1:8 immunodiffusion titer after the animals were immunized with the recombinant protein.Serum samples from all H pyloriinfected patients were positive for UreB antibody and UreB expression were detectable in all tested H pyloriisolates.CONCLUSION: A prokaryotic expression system with high expression efficiency of H pylori ureB gene was successfully established. The expressed rUreB showed qualified immunoreactivity and antigenicity. High frequencies of UreB expression in different H

  11. Recombination and horizontal transfer of nodulation and ACC deaminase (acdS) genes within Alpha- and Betaproteobacteria nodulating legumes of the Cape Fynbos biome.

    Science.gov (United States)

    Lemaire, Benny; Van Cauwenberghe, Jannick; Chimphango, Samson; Stirton, Charles; Honnay, Olivier; Smets, Erik; Muasya, A Muthama

    2015-11-01

    The goal of this work is to study the evolution and the degree of horizontal gene transfer (HGT) within rhizobial genera of both Alphaproteobacteria (Mesorhizobium, Rhizobium) and Betaproteobacteria (Burkholderia), originating from South African Fynbos legumes. By using a phylogenetic approach and comparing multiple chromosomal and symbiosis genes, we revealed conclusive evidence of high degrees of horizontal transfer of nodulation genes among closely related species of both groups of rhizobia, but also among species with distant genetic backgrounds (Rhizobium and Mesorhizobium), underscoring the importance of lateral transfer of symbiosis traits as an important evolutionary force among rhizobia of the Cape Fynbos biome. The extensive exchange of symbiosis genes in the Fynbos is in contrast with a lack of significant events of HGT among Burkholderia symbionts from the South American Cerrado and Caatinga biome. Furthermore, homologous recombination among selected housekeeping genes had a substantial impact on sequence evolution within Burkholderia and Mesorhizobium. Finally, phylogenetic analyses of the non-symbiosis acdS gene in Mesorhizobium, a gene often located on symbiosis islands, revealed distinct relationships compared to the chromosomal and symbiosis genes, suggesting a different evolutionary history and independent events of gene transfer. The observed events of HGT and incongruence between different genes necessitate caution in interpreting topologies from individual data types.

  12. Environment control to improve recombinant protein yields in plants based on Agrobacterium-mediated transient gene expression

    Directory of Open Access Journals (Sweden)

    Naomichi eFujiuchi

    2016-03-01

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

  13. Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization

    Energy Technology Data Exchange (ETDEWEB)

    La Ferla, Marco; Mercatanti, Alberto; Rocchi, Giulia; Lodovichi, Samuele; Cervelli, Tiziana; Pignata, Luca [Yeast Genetics and Genomics, Institute of Clinical Physiology, National Council of Research (CNR), via Moruzzi 1, 56122 Pisa (Italy); Caligo, Maria Adelaide [Section of Genetic Oncology, University Hospital and University of Pisa, via Roma 57, 56125 Pisa (Italy); Galli, Alvaro, E-mail: alvaro.galli@ifc.cnr.it [Yeast Genetics and Genomics, Institute of Clinical Physiology, National Council of Research (CNR), via Moruzzi 1, 56122 Pisa (Italy)

    2015-04-15

    Highlights: • The human poly (ADP-ribose) polymerase 1 (PARP-1) gene affects growth and UV-induced homologous recombination in yeast. • PARP-1 chemical inhibition impacts yeast growth and UV-induced recombination. • A genome-wide screen identifies 99 yeast genes that suppress the growth defect inferred by PARP-1. • Bioinformatics analysis identifies 41 human orthologues that may have a role in PARP-1 intracellular localization. • The findings suggest that PARP-1 nuclear localization may affect the response to PARP inhibitors in cancer therapy. - Abstract: The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen. We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the

  14. Recombinant methods and materials

    Energy Technology Data Exchange (ETDEWEB)

    Roizman, B.; Post, L.E.

    1988-09-06

    This patent describes a method for stably effecting the insertion or deletion of a selected DNA sequence at a specific site in a viral genome. The method consists of: (1) isolating from the genome a linear DNA fragment comprising both (a) the specific site determined for insertion or deletion of selected DNA sequence and (b) flanking DNA sequences normally preceding and following the site; (2) preparing first and second altered genome fragments from the fragment isolated in step (1). (a) the first altered fragment comprising the fragment comprising a thymidine kinase gene in a position intermediate the ends of the fragment, and (b) the second altered fragment comprising the fragment having the selected DNA sequence inserted therein or deleted therefrom; (3) contacting the genome with the first altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome comprising the thymidine kinase gene, and isolating the recombinant genome; and (4) contacting the recombinant genome isolated in step (3) with the second altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome lacking the thymidine kinase gene, and isolating the recombinant genome product.

  15. Construction of prokaryotic expression system of ItB-ureB fusion gene and identification of the recombinant protein immunity and adjuvanticity

    Institute of Scientific and Technical Information of China (English)

    Jie Yan; Yuan Wang; Shi-He Shao; Ya-Fei Mao; Hua-Wen Li; Yi-Hui Luo

    2004-01-01

    AIM: To construct ItB-ureB fusion gene and its prokaryotic expression system and identify immunity and adjuvanticity of the expressed recombinant protein.METHODS: The ureB gene from a clinical Helicobacterpylori(Hpylori) strain Y06 and the ItB gene from Escherichiacoli(E coli) strain 44851 were linked into ItB-ureB fusiongene by PCR. The fusion gene sequence was analyzedafter T-A cloning. A prokaryotic recombinant expressionvector pET32a inserted with ItB-ureB fusion gene (pET32aItB-ureB) was constructed. Expression of the recombinantLTB-UreB protein (rLTB-UreB)in E. coliBL21DE3 inducedby isopropylthio-β-D-galactoside (IPTG) at differentconcentrations was detected by SDS-PAGE. Western blot assays were used to examine the immunoreaction of rLTBUreB by a commercial antibody against whole cell of H pylori and a self-prepared rabbit anti-rUreB serum, respectively, and determine the antigenicity of the recombinant proteinon inducing specific antibody in rabbits. GM1-ELISA wasused to demonstrate the adjuvanticity of rLTB-UreB. Immunoreaction of rLTB-UreB to the UreB antibody positivesera from 125 gastric patients was determined by using ELISA. RESULTS: In comparison with the corresponding sequences of original genes, the nucleotide sequence homologies of the cloned ItB-ureB fusion gene were 100%. IPTG withdifferent dosages of 0.1-1.0 mmol/L could efficiently inducepET32a-ItB-ureB-E.coli BL21DE3 to express the rLTB-UreB. The output of the target recombinant protein expressed by pET32a-ureB-E. coli BL21DE3 was approximately 35%of the total bacterial proteins. rLTB-UreB mainly presented in the form of inclusion body. Western blotting results demonstrated that rLTB-UreB could combine with the commercial antibody against whole cell of H pylori andanti-rUreB serum as well as induce rabbit to produce specific antibody. The strong ability of rLTB-UreB bindingbovine GM1 indicated the existence of adjuvanticity of the recombinant protein. All the UreB antibody positive sera from

  16. Heterologous expression of phaC2 gene and poly-3-hydroxyalkanoate production by recombinant Cupriavidus necator strains using canola oil as carbon source.

    Science.gov (United States)

    Valdés, J; Kutralam-Muniasamy, G; Vergara-Porras, B; Marsch, R; Pérez-Guevara, F; López-Cuellar, M R

    2017-08-19

    Many heterologous transformation studies have been carried out using the Cupriavidus necator PHB(-4) strain to investigate the expression characteristics of various polyhydroxyalkanoate (PHA) synthase enzymes. In this study, we generated a recombinant C. necator PHB(-4) strain by transforming a plasmid (pMRC03) harbouring the synthetic phaC2 gene of Pseudomonas putida CA-3. Under conditions favourable for expression of the phaC2 P.putCA-3 gene, canola oil was used as carbon source for the synthesis of PHAs. The expressed synthase polymerised monomers of 3-hydroxybutyrate (3-HB), 3-hydroxyvalerate (3-HV) and 3-hydroxyhexanoate (3-HHx) in the recombinant C. necator PHB(-4) (pMRC03) strain. We then co-expressed the phaC2P.putCA-3 gene with the native phaC1C.ne gene in wild type Cupriavidus necator H16 (C. necator H16 (pMRC03)). This co-expression produced a PHA blend of 3-HB, 3-HV, 3-HHx and 3-hydroxyoctanoate (3-HO) monomers in the presence of canola oil. Gas chromatography analysis revealed the presence of 94mol% 3-HB, 1mol% 3-HV, 4mol% 3-HHx and 1mol% 3-HO in a tetra-polymer. Thus, we confirmed that a synthetic phaC2 gene encoding the synthase enzyme is functionally active with substrates ranging from short to medium chain length PHAs. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Characterization of in vitro metabolites of methylenedioxypyrovalerone (MDPV): An N-oxide metabolite formation mediated by flavin monooxygenase.

    Science.gov (United States)

    Kim, In Sook; Rehman, Shaheed Ur; Choi, Min Sun; Jang, Moonhee; Yang, Wonkyung; Kim, Eunmi; Yoo, Hye Hyun

    2016-11-30

    Methylenedioxypyrovalerone (MDPV) has emerged in recent years as a recreational substance with psychostimulant properties. In this study, in vitro metabolites of MDPV were characterized based on liquid chromatography/quadrupole-time-of-flight mass spectrometry (LC/QTOF MS). MDPV was incubated with human liver microsomes, human recombinant cDNA-expressed cytochrome P450 enzymes and flavin monooxygenase (FMO). MDPV was metabolized to yield eight metabolites (M1-M8) with major metabolic reactions such as demethylenation and oxidation. Among them, M6 was assigned as an N-oxide metabolite. FMO was found to be a principal enzyme responsible for the formation of M6; FMO1 and FMO3 were the main enzymes involved in N-oxidation of MDPV. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Exposing the Alkanesulfonate Monooxygenase Protein-Protein Interaction Sites.

    Science.gov (United States)

    Dayal, Paritosh V; Singh, Harsimran; Busenlehner, Laura S; Ellis, Holly R

    2015-12-29

    The alkanesulfonate monooxygenase enzymes (SsuE and SsuD) catalyze the desulfonation of diverse alkanesulfonate substrates. The SsuE enzyme is an NADPH-dependent FMN reductase that provides reduced flavin to the SsuD monooxygenase enzyme. Previous studies have highlighted the presence of protein-protein interactions between SsuE and SsuD thought to be important in the flavin transfer event, but the putative interaction sites have not been identified. Protected sites on specific regions of SsuE and SsuD were identified by hydrogen-deuterium exchange mass spectrometry. An α-helix on SsuD containing conserved charged amino acids showed a decrease in percent deuteration in the presence of SsuE. The α-helical region of SsuD is part of an insertion sequence and is adjacent to the active site opening. A SsuD variant containing substitutions of the charged residues showed a 4-fold decrease in coupled assays that included SsuE to provide reduced FMN, but there was no activity observed with an SsuD variant containing a deletion of the α-helix under similar conditions. Desulfonation by the SsuD deletion variant was only observed with an increase in enzyme and substrate concentrations. Although activity was observed under certain conditions, there were no protein-protein interactions observed with the SsuD variants and SsuE in pull-down assays and fluorimetric titrations. The results from these studies suggest that optimal transfer of reduced flavin from SsuE to SsuD requires defined protein-protein interactions, but diffusion can occur under specified conditions. A basis is established for further studies to evaluate the structural features of the alkanesulfonate monooxygenase enzymes that promote desulfonation.

  19. Recombinant adeno-associated virus-mediated delivery of antisense angiotensin Ⅱ receptor 1 gene attenuates hypertension development

    Institute of Scientific and Technical Information of China (English)

    Xu-guang LI; Jiang-tao YAN; Xi-zheng XU; Jia-ning WANG; Li-ming CHENG; Tao WANG; Ping ZUO; Dao-wen WANG

    2007-01-01

    Aim:The renin-angiotensin system plays a crucial role in the development and establishment of hypertension,and the pharmacological blockade of the system results in a reduction in blood pressure. In the present study,we investigated whether the effects of a novel,double-stranded,recombinant adeno-associated virus vector (rAAV)-mediated antisense angiotensin Ⅱ receptor l (AT1R) gene efficiently prevents the development of hypertension induced by a high-salt diet in adult,male Sprague-Dawley (SD) rats. Methods:A rAAV was prepared with a cassette containing a cytomegalovirus promoter and partial cDNA (660 base pairs) for the AT1R inserted in the antisense direction (rAAV-AT1AS). A single tail vein injection of the rAAV-AT1-AS or rAAV-GFP (green fluorescent protein,a reporter gene) was performed in adult,male SD rats. Two weeks after injection,the animals were fed a diet containing 8% NaCI,and the systolic blood pressure was measured weekly using the tail-cuff method for 12 weeks. Results:The high-salt diet induced a significant rise in systolic blood pressure in the rAAV-GFP-treated animals;however,the rAAV-AT:AS treatment attenuated the rise in blood pressure (142.7±4.5 mmHg vs 117±3.8 mmHg,P<0.01),and the hypotensive effect was maintained until the experiments ended at 12 weeks. In the rAAV-GFP-treated animals AT1 was overexpressed in various tissues,especially in the aorta and kidney at mRNA levels;in contrast,rAAV-AT:AS treatment markedly attenuated AT1 expression. Furthermore,rAAV-AT:AS treatment prevented target organ damages from hypertension,including cardiac dysfunction and renal injury compared to the rAAV-GFP group. Conclusion:These results suggest that rAAVmediated anti-AT1 delivery attenuates the development of hypertension and protects against renal injury and cardiac remodeling.

  20. Analysis of the src gene of sarcoma viruses generated by recombination between transformation-defective mutants and quail cellular sequences.

    Science.gov (United States)

    Wang, L H; Moscovici, C; Karess, R E; Hanafusa, H

    1979-01-01

    Tumors were produced in quails about 2 months after injection with a transformation-defective mutant of the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup A (SR-A), that retains a small portion of the src gene. Sarcoma viruses were isolated from each of five such tumors. A transformation-defective mutant which has a nearly complete deletion of the src gene was unable to induce tumors. The avian sarcoma viruses recovered from quail tumors (rASV-Q) had biological properties similar to those of the avian sarcoma viruses previously acquired from chicken tumors (rASV-C); these chicken tumors had been induced by the same transformation-defective mutants. Both rASV-Q and rASV-C transformed cells in culture with similar focus morphology and produced tumors within 7 to 14 days after injection into chickens or quails. The size of rASV-Q genomic RNA was indistinguishable from that of SR-A by polyacrylamide gel electrophoresis. The sequences of rASV-Q RNA genomes were analyzed and compared with those of the parental transformation-defective virus, SR-A and of rASV-C by RNase T1 fingerprinting and oligonucleotide mapping. We found that the src sequences of all five isolates of rASV-Q were identical to each other but different from those of SR-A and rASV-C. Of 13 oligonucleotides of rASV-Q identified as src specific, two were not found in either SR-A or rASV-C RNA. Furthermore, some oligonucleotides present in SR-A or rASV-C or both were absent in rASV-Q. No differences were found for the sequences outside the src region in any of the viruses examined. In addition, rASV-Q-infected cells possessed a 60,000-dalton protein specifically precipitable by rabbit serum raised against SR-D-induced tumors. The facts that the src sequences are essentially the same for rASV's recovered from one animal species and different for rASV's obtained from different species provide conclusive evidence that cellular sequences of normal birds were inserted into the viral genome and supplied to

  1. Expression, purification and characterization of human Dopamine ß-monooxygenase

    DEFF Research Database (Denmark)

    Vendelboe, Trine Vammen

    This thesis deals with expression, purification and characterization of the copper containing enzyme dopamine ß-monooxygenase (DBM). DBM is an ascorbate dependent protein that requires Cu in the active site in order to be functional. DBM is made of four domains; An Nterminal DOMON domain, the two...... others, one of the reasons why these proteins are considered to follow the same mechanism. DBM converts dopamine (DA) into Norepinphrine (NE). Both substrate and product functions as neurotransmitters and the levels of these are involved in many different disorders such as depression and hypertension...

  2. Expression, purification and characterization of human Dopamine ß-monooxygenase

    DEFF Research Database (Denmark)

    Vendelboe, Trine Vammen

    This thesis deals with expression, purification and characterization of the copper containing enzyme dopamine ß-monooxygenase (DBM). DBM is an ascorbate dependent protein that requires Cu in the active site in order to be functional. DBM is made of four domains; An Nterminal DOMON domain, the two...... others, one of the reasons why these proteins are considered to follow the same mechanism. DBM converts dopamine (DA) into Norepinphrine (NE). Both substrate and product functions as neurotransmitters and the levels of these are involved in many different disorders such as depression and hypertension...

  3. Structural characterization of Lytic Polysaccharide MonoOxygenases

    DEFF Research Database (Denmark)

    Frandsen, Kristian Erik Høpfner

    Lytic polysaccharide monooxygenases (LPMOs) are a new class of copper-containingmetalloenzymes that have been found to oxidatively degrade polysaccharides (and recently alsooligosaccharides). They dependent on redox partners to provide them with electrons and they utilizemolecular oxygen to cleave......) and their interaction with substratehave been structurally characterized. A number of structures of LsAA9A have been obtained in complexwith a range of cellulosic- and hemicellulosic substrates and with the active site Cu in different redox state.Two of the LsAA9A structures with the active site Cu in essentially a Cu...

  4. Polymorphisms of Homologous Recombination RAD51, RAD51B, XRCC2, and XRCC3 Genes and the Risk of Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Maria Nowacka-Zawisza

    2015-01-01

    Full Text Available Genetic polymorphisms in DNA repair genes may induce individual variations in DNA repair capacity, which may in turn contribute to the risk of cancer developing. Homologous recombination repair (HRR plays a critical role in maintaining chromosomal integrity and protecting against carcinogenic factors. The aim of the present study was to evaluate the relationship between prostate cancer risk and the presence of single nucleotide polymorphisms (SNPs in the genes involved in HRR, that is, RAD51 (rs1801320 and rs1801321, RAD51B (rs10483813 and rs3784099, XRCC2 (rs3218536, and XRCC3 (rs861539. Polymorphisms were analyzed by PCR-RFLP and Real-Time PCR in 101 patients with prostate adenocarcinoma and 216 age- and sex-matched controls. A significant relationship was detected between the RAD51 gene rs1801320 polymorphism and increased prostate cancer risk. Our results indicate that the RAD51 gene rs1801320 polymorphism may contribute to prostate cancer susceptibility in Poland.

  5. Polymorphisms of Homologous Recombination RAD51, RAD51B, XRCC2, and XRCC3 Genes and the Risk of Prostate Cancer

    Science.gov (United States)

    Nowacka-Zawisza, Maria; Wiśnik, Ewelina; Wasilewski, Andrzej; Skowrońska, Milena; Forma, Ewa; Bryś, Magdalena; Różański, Waldemar; Krajewska, Wanda M.

    2015-01-01

    Genetic polymorphisms in DNA repair genes may induce individual variations in DNA repair capacity, which may in turn contribute to the risk of cancer developing. Homologous recombination repair (HRR) plays a critical role in maintaining chromosomal integrity and protecting against carcinogenic factors. The aim of the present study was to evaluate the relationship between prostate cancer risk and the presence of single nucleotide polymorphisms (SNPs) in the genes involved in HRR, that is, RAD51 (rs1801320 and rs1801321), RAD51B (rs10483813 and rs3784099), XRCC2 (rs3218536), and XRCC3 (rs861539). Polymorphisms were analyzed by PCR-RFLP and Real-Time PCR in 101 patients with prostate adenocarcinoma and 216 age- and sex-matched controls. A significant relationship was detected between the RAD51 gene rs1801320 polymorphism and increased prostate cancer risk. Our results indicate that the RAD51 gene rs1801320 polymorphism may contribute to prostate cancer susceptibility in Poland. PMID:26339569

  6. Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production.

    Directory of Open Access Journals (Sweden)

    Chiemi Noguchi

    Full Text Available Amplification of the dihydrofolate reductase gene (Dhfr by methotrexate (Mtx exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR and a matrix attachment region (MAR, which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.

  7. Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production.

    Science.gov (United States)

    Noguchi, Chiemi; Araki, Yoshio; Miki, Daisuke; Shimizu, Noriaki

    2012-01-01

    Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.

  8. Selective Oxidations of Organoboron Compounds Catalyzed by Baeyer-Villiger Monooxygenases

    NARCIS (Netherlands)

    Brondani, Patricia B.; de Gonzalo, Gonzalo; Fraaije, Marco W.; Andrade, Leandro H.

    2011-01-01

    The applicability of Baeyer-Villiger monooxygenases (BVMOs) in organoboron chemistry has been explored through testing chemo-and enantioselective oxidations of a variety of boron-containing aromatic and vinylic compounds. Several BVMOs, namely: phenylacetone monooxygenase (PAMO), M446G PAMO mutant,

  9. Altering the substrate specificity and enantioselectivity of phenylacetone monooxygenase by structure-inspired enzyme redesign

    NARCIS (Netherlands)

    Torres Pazmino, Daniel E.; Snajdrova, Radka; Rial, Daniela V.; Mihovilovic, Marko D.; Fraaije, Marco W.

    Of all presently available Baeyer-Villiger monooxygenases, phenylacetone monooxygenase (PAMO) is the only representative for which a structure has been determined. While it is an attractive biocatalyst because of its thermostability, it is only active with a limited number of substrates. By means of

  10. Polycyclic Ketone Monooxygenase (PockeMO): A Robust Biocatalyst for the Synthesis of Optically Active Sulfoxides

    NARCIS (Netherlands)

    de Gonzalo, Gonzalo; Fürst, Maximilian; Fraaije, Marco

    2017-01-01

    A recently discovered, moderately thermostable Baeyer-Villiger monooxygenase, polycyclic ketone monooxygenase (PockeMO), from Thermothelomyces thermophila has been employed as a biocatalyst in a set of asymmetric sulfoxidations. The enzyme was able to catalyze the oxidation of various alkyl aryl

  11. Molecular analysis of the pmo (particulate methane monooxygenase) operons from two type II methanotrophs.

    Science.gov (United States)

    Gilbert, B; McDonald, I R; Finch, R; Stafford, G P; Nielsen, A K; Murrell, J C

    2000-03-01

    The particulate methane monooxygenase gene clusters, pmoCAB, from two representative type II methanotrophs of the alpha-Proteobacteria, Methylosinus trichosporium OB3b and Methylocystis sp. strain M, have been cloned and sequenced. Primer extension experiments revealed that the pmo cluster is probably transcribed from a single transcriptional start site located 300 bp upstream of the start of the first gene, pmoC, for Methylocystis sp. strain M. Immediately upstream of the putative start site, consensus sequences for sigma(70) promoters were identified, suggesting that these pmo genes are recognized by sigma(70) and negatively regulated under low-copper conditions. The pmo genes were cloned in several overlapping fragments, since parts of these genes appeared to be toxic to the Escherichia coli host. Methanotrophs contain two virtually identical copies of pmo genes, and it was necessary to use Southern blotting and probing with pmo gene fragments in order to differentiate between the two pmoCAB clusters in both methanotrophs. The complete DNA sequence of one copy of pmo genes from each organism is reported here. The gene sequences are 84% similar to each other and 75% similar to that of a type I methanotroph of the gamma-Proteobacteria, Methylococcus capsulatus Bath. The derived proteins PmoC and PmoA are predicted to be highly hydrophobic and consist mainly of transmembrane-spanning regions, whereas PmoB has only two putative transmembrane-spanning helices. Hybridization experiments showed that there are two copies of pmoC in both M. trichosporium OB3b and Methylocystis sp. strain M, and not three copies as found in M. capsulatus Bath.

  12. Analysis of HIV-1 protease gene reveals frequent multiple infections followed by recombination among drug treated individuals living in Sao Paulo and Santos, Brazil.

    Directory of Open Access Journals (Sweden)

    Edsel Renata De Morais Nunes

    Full Text Available The present study investigated the prevalence of HIV-1 multiple infections in a population composed by 47 patients under HAART failure and enrolled at the National DST/AIDS, Program, Ministry of Health, Brazil.Detection of multiple infections was done using a previously published RFLP assay for the HIV-1 protease gene, which is able of distinguishing between infections caused by a single or multiple HIV-1 subtypes. Samples with multiple infections were cloned, and sequence data submitted to phylogenetic analysis. We were able to identify 17 HIV-1 multiple infections out of 47 samples. Multiple infections were mostly composed by a mixture of recombinant viruses (94%, with only one case in which protease gene pure subtypes B and F were recovered. This is the first study that reports the prevalence of multiple infections and intersubtype recombinants in a population undergoing HAART in Brazil. Based on the data there was a steep increase of multiple infections after the introduction of the combined antiretroviral therapy in Brazil. Cases of multiple infections may be associated with HIV-1 genetic diversity through recombination allowing for the generation of viruses showing a combination of resistance mutations.

  13. Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation, gene and protein expression.

    Science.gov (United States)

    Manira, Maarof; Khairul Anuar, Khairoji; Seet, Wan Tai; Ahmad Irfan, Abd Wahab; Ng, Min Hwei; Chua, Kien Hui; Mohd Heikal, Mohd Yunus; Aminuddin, Bin Saim; Ruszymah, Bt Hj Idrus

    2014-03-01

    Animal-derivative free reagents are preferred in skin cell culture for clinical applications. The aim of this study was to compare the performance and effects between animal-derived trypsin and recombinant trypsin for skin cells culture and expansion. Full thickness human skin was digested in 0.6 % collagenase for 6 h to liberate the fibroblasts, followed by treatment with either animal-derived trypsin; Trypsin EDTA (TE) or recombinant trypsin; TrypLE Select (TS) to liberate the keratinocytes. Both keratinocytes and fibroblasts were then culture-expanded until passage 2. Trypsinization for both cell types during culture-expansion was performed using either TE or TS. Total cells yield was determined using a haemocytometer. Expression of collagen type I, collagen type III (Col-III), cytokeratin 10, and cytokeratin 14 genes were quantified via RT-PCR and further confirmed with immunocytochemical staining. The results of our study showed that the total cell yield for both keratinocytes and fibroblasts treated with TE or TS were comparable. RT-PCR showed that expression of skin-specific genes except Col-III was higher in the TS treated group compared to that in the TE group. Expression of proteins specific to the two cell types were confirmed by immunocytochemical staining in both TE and TS groups. In conclusion, the performance of the recombinant trypsin is comparable with the well-established animal-derived trypsin for human skin cell culture expansion in terms of cell yield and expression of specific cellular markers.

  14. Protective immunization of horses with a recombinant canarypox virus vectored vaccine co-expressing genes encoding the outer capsid proteins of African horse sickness virus.

    Science.gov (United States)

    Guthrie, Alan J; Quan, Melvyn; Lourens, Carina W; Audonnet, Jean-Christophe; Minke, Jules M; Yao, Jiansheng; He, Ling; Nordgren, Robert; Gardner, Ian A; Maclachlan, N James

    2009-07-16

    We describe the development and preliminary characterization of a recombinant canarypox virus vectored (ALVAC) vaccine for protective immunization of equids against African horse sickness virus (AHSV) infection. Horses (n=8) immunized with either of two concentrations of recombinant canarypox virus vector (ALVAC-AHSV) co-expressing synthetic genes encoding the outer capsid proteins (VP2 and VP5) of AHSV serotype 4 (AHSV-4) developed variable titres (horse immunized with a commercial recombinant canarypox virus vectored vaccine expressing the haemagglutinin genes of two equine influenza H3N8 viruses was seronegative to AHSV and following infection with virulent AHSV-4 developed pyrexia, thrombocytopenia and marked oedema of the supraorbital fossae typical of the "dikkop" or cardiac form of African horse sickness. AHSV was detected by virus isolation and quantitative reverse transcriptase polymerase chain reaction in the blood of the control horse from 8 days onwards after challenge infection whereas AHSV was not detected at any time in the blood of the ALVAC-AHSV vaccinated horses. The control horse seroconverted to AHSV by 2 weeks after challenge infection as determined by both virus neutralization and ELISA assays, whereas six of eight of the ALVAC-AHSV vaccinated horses did not seroconvert by either assay following challenge infection with virulent AHSV-4. These data confirm that the ALVAC-AHSV vaccine will be useful for the protective immunization of equids against African horse sickness, and avoids many of the problems inherent to live-attenuated AHSV vaccines.

  15. Recombinant HAP Phytase of the Thermophilic Mold Sporotrichum thermophile: Expression of the Codon-Optimized Phytase Gene in Pichia pastoris and Applications.

    Science.gov (United States)

    Ranjan, Bibhuti; Satyanarayana, T

    2016-02-01

    The codon-optimized phytase gene of the thermophilic mold Sporotrichum thermophile (St-Phy) was expressed in Pichia pastoris. The recombinant P. pastoris harboring the phytase gene (rSt-Phy) yielded a high titer of extracellular phytase (480 ± 23 U/mL) on induction with methanol. The recombinant phytase production was ~40-fold higher than that of the native fungal strain. The purified recombinant phytase (rSt-Phy) has the molecular mass of 70 kDa on SDS-PAGE, with K m and V max (calcium phytate), k cat and k cat/K m values of 0.147 mM and 183 nmol/mg s, 1.3 × 10(3)/s and 8.84 × 10(6)/M s, respectively. Mg(2+) and Ba(2+) display a slight stimulatory effect, while other cations tested exert inhibitory action on phytase. The enzyme is inhibited by chaotropic agents (guanidinium hydrochloride, potassium iodide, and urea), Woodward's reagent K and 2,3-bunatedione, but resistant to both pepsin and trypsin. The rSt-Phy is useful in the dephytinization of broiler feeds efficiently in simulated gut conditions of chick leading to the liberation of soluble inorganic phosphate with concomitant mitigation in antinutrient effects of phytates. The addition of vanadate makes it a potential candidate for generating haloperoxidase, which has several applications.

  16. Comparison on virulence and immunogenicity of two recombinant vaccinia vaccines, Tian Tan and Guang9 strains, expressing the HIV-1 envelope gene.

    Science.gov (United States)

    Zhu, Rong; Huang, Weijin; Wang, Wenbo; Liu, Qiang; Nie, Jianhui; Meng, Shufang; Yu, Yongxin; Wang, Youchun

    2012-01-01

    The vaccinia virus Guang9 strain (VG9), derived from the vaccinia virus Tian Tan strain (VTT) has been found to be less virulent than VTT. To investigate whether VG9 could be a potential replicating virus vector, the TK genes in VG9 and VTT were replaced with the HIV-1 envelope gene via homologous recombination, resulting in the recombinant viruses, VG9-E and VTT-E. The biology, virulence, humoral and cellular immunological responses of VG9-E and VTT-E were evaluated. Our results indicated no obvious difference in range of host cells and diffusion between two recombinant viruses. Neurovirulence for VG9-E in weanling and suckling mice, and skin virulence in rabbits, were lower than that of VTT-E. The humoral immune responses, including binding antibody and neutralizing antibody responses, induced by VG9-E were not significantly different from those for VTT-E whilst IFN-γ response which represented cellular immune response induced by VG9-E was significantly higher than that did by VTT-E. Our results indicated that VG9-E was less virulent, yet induced higher cellular immune response than VTT-E. Therefore, it could be an ideal replicating vaccinia vector for HIV vaccine research and development.

  17. Mechanism Analysis of Kynurenine 3-monooxygenase GeneBcKMO in Regulation of Pathogenicity inBotrytis cinerea%灰葡萄孢犬尿氨酸单氧酶基因BcKMO调控病菌致病力的机制分析

    Institute of Scientific and Technical Information of China (English)

    李培芬; 赵福鑫; 董丽萍; 郑会欣; 赵斌; 韩建民; 邢继红; 董金皋

    2014-01-01

    而影响病菌的致病力。%[Objective]The objective of this study is to reveal the molecular mechanism of kynurenine 3-monooxygenase gene BcKMO in regulating pathogenicity ofBotrytis cinerea and lay a foundation for clarifing the pathogenic mechanism ofB. cinereain the future.[Method]The activities of polymethylgalacturonase (PMG), endopolygalacturonase (PG), cellulase (CX), polygalacturonic acid transeliminase (PGTE) and pectin methyl transelimination enzyme (PMTE) were analyzed in the wild-type strain (WT), the BcKMO gene ATMT mutant (BCG183) and gene complement mutant (BCG183/BcKMO) by DNS and Hoffman methods. The toxin was isolated from WT, BCG183 and BCG183/BcKMO and the activity of toxin was analyzed. Acid production assays was performed on PDA medium with bromothymol blue. Penetrability of WT, BCG183 and BCG183/BcKMO was detected with onion epidermis spread on PDA medium. Real-time PCR was used to measure the transcription levels of pathogenicity-related genes, e.g.,Bac, Bcg2,Bcg3, PkaR,Bmp1,Sak1,Bcreg1,Bos1,Bcp1,Ras2,Bcpg1, andSod1 in WT, BCG183 and BCG183/BcKMO.[Result]The activity of PMG and PG in mutant BCG183 were significantly higher than that of WT and BCG183/BcKMO. The CX, PGTE and PMTE activities were no significant difference while compared with WT and BCG183/BcKMO. The toxin activity of mutant BCG183 was significantly higher than that of WT and BCG183/BcKMO. The acid production of the mutant BCG183 significantly decreased. The penetrability of mutant BCG183 appeared no significant difference compared to WT and BCG183/BcKMO. The expression level of pathogenicity-related genes, i.e.,Bac,Bcg2,Bcg3, PkaR,Bmp1,Sak1,Bcreg1,Bos1,Bcp1,Ras2,Bcpg1, and Sod1, was obviously up-regulated in mutant BCG183.[Conclusion]TheBcKMO gene is involved in regulating cell wall degradation enzyme activity, toxin activity, acid production, penetrability, and the expression of pathogenicity- related genes inB. cinerea.

  18. Mutation of the mouse Rad17 gene leads to embryonic lethality and reveals a role in DNA damage-dependent recombination.

    Science.gov (United States)

    Budzowska, Magda; Jaspers, Iris; Essers, Jeroen; de Waard, Harm; van Drunen, Ellen; Hanada, Katsuhiro; Beverloo, Berna; Hendriks, Rudolf W; de Klein, Annelies; Kanaar, Roland; Hoeijmakers, Jan H; Maas, Alex

    2004-09-01

    Genetic defects in DNA repair mechanisms and cell cycle checkpoint (CCC) genes result in increased genomic instability and cancer predisposition. Discovery of mammalian homologs of yeast CCC genes suggests conservation of checkpoint mechanisms between yeast and mammals. However, the role of many CCC genes in higher eukaryotes remains elusive. Here, we report that targeted deletion of an N-terminal part of mRad17, the mouse homolog of the Schizosaccharomyces pombe Rad17 checkpoint clamp-loader component, resulted in embryonic lethality during early/mid-gestation. In contrast to mouse embryos, embryonic stem (ES) cells, isolated from mRad17(5'Delta/5'Delta) embryos, produced truncated mRad17 and were viable. These cells displayed hypersensitivity to various DNA-damaging agents. Surprisingly, mRad17(5'Delta/5'Delta) ES cells were able to arrest cell cycle progression upon induction of DNA damage. However, they displayed impaired homologous recombination as evidenced by a strongly reduced gene targeting efficiency. In addition to a possible role in DNA damage-induced CCC, based on sequence homology, our results indicate that mRad17 has a function in DNA damage-dependent recombination that may be responsible for the sensitivity to DNA-damaging agents.

  19. Improvement of manganese peroxidase production by the hyper lignin-degrading fungus Phanerochaete sordida YK-624 by recombinant expression of the 5-aminolevulinic acid synthase gene.

    Science.gov (United States)

    Hirai, Hirofumi; Misumi, Kenta; Suzuki, Tomohiro; Kawagishi, Hirokazu

    2013-12-01

    The manganese peroxidase (MnP) gene (mnp4) promoter of Phanerochaete sordida YK-624 was used to drive expression of 5-aminolevulinic acid synthase (als), which is a key heme biosynthesis enzyme. The expression plasmid pMnP4pro-als was transformed into P. sordida YK-624 uracil auxotrophic mutant UV-64, and 14 recombinant als expressing-transformants were generated. Average cumulative MnP activities in the transformants were 1.18-fold higher than that of control transformants. In particular, transformants A-14 and A-61 showed significantly higher MnP activity (approximately 2.8-fold) than wild type. RT-PCR analysis indicated that the increased MnP activity was caused by elevated recombinant als expression. These results suggest that the production of MnP is improved by high expression of als.

  20. Two Novel Flavin-Containing Monooxygenases Involved in Biosynthesis of Aliphatic Glucosinolates

    Directory of Open Access Journals (Sweden)

    Wenwen Kong

    2016-08-01

    Full Text Available Glucosinolates (GSLs, a class of secondary metabolites from cruciferous plants, are derived from amino acids and have diverse biological activities, such as in biotic defense, depending on their side chain modification. The first structural modification step in the synthesis of aliphatic (methionine-derived GSLs—S-oxygenation of methylthioalkyl GSLs to methylsulfinylalkyl GSLs—was found to be catalyzed by five flavin-containing monooxygenases (FMOs, FMOGS-OX1-5. Here, we report two additional FMOGS-OX enzymes, FMOGS-OX6 and FMOGS-OX7, encoded by At1g12130 and At1g12160, respectively. The overexpression of both FMOGS-OX6 and FMOGS-OX7 decreased the ratio of MT GSL to the sum of MT and MS GSL, suggesting that the introduction of the two genes converted MT GSL into MS GSL. Analysis of expression pattern revealed that the spatial expression of the two genes is quite similar and partially overlapped with the other FMOGS-OX genes, which are primarily expressed in vascular tissue. We further analyzed the responsive expression pattern of all the seven FMOGS-OX genes to exogenous treatment with abscisic acid (ABA, 1-aminocyclopropane-1-carboxylic acid (ACC, jasmonic acid (JA, salicylic acid (SA, indole-3-acetic acid (IAA, and low and high temperatures. Although these genes showed same tendency toward the changing stimulus, the sensitivity of each gene was quite different. The variety in spatial expression among the FMOGS-OX genes while responding to environmental stimulus indicated a complex and finely tuned regulation of GSL modifications. Identification of these two novel FMOGS-OX enzymes will enhance the understanding of GSL modifications and the importance of evolution of these duplicated genes.

  1. Identiifcation of genes upregulated by recombinant inter feron-alpha in HepG2 cells by suppressive subtractive hybridization analysis

    Institute of Scientific and Technical Information of China (English)

    Jian-Hui Qu; Jun Cheng; Ling-Xia Zhang; Li-Ying Zhang; Yan-Wei Zhong; Yan Liu; Lin Wang; Jiu-Zeng Dai; Dong-Ping Xu

    2007-01-01

    BACKGROUND:Interferon-alpha (IFN-α) is an important cytokine with multiple functions, but the target genes transactivated by IFN-αremain largely unknown. A study of such genes will help to understand the mechanism of function of IFN-α. To isolate the gene transcripts speciifcally upregulated by IFN-α in HepG2 cells, we conducted suppressive subtractive hybridization (SSH) analysis. METHODS: SSH was used to analyze the target genes transactivated by recombinant IFN-α protein, and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-α (rIFN-α, 2000 IU/ml) for 16 hours as tester, and cells not treated with rIFN-αas driver. The SSH PCR products from the library were cloned into pGEM-T easy vector and with BLASTX, the positive clones were randomly selected, sequenced and compared to the database in GenBank of the 35 differentially expressed gene fragments from the library, 6 clones showed signiifcant homology to other known proteins. RESULTS: The subtractive cDNA library of genes upregulated by IFN-α was constructed successfully. rIFN-α upregulated the expression of the RAN binding protein 5 (RANBP5), NADH dehydrogenase, exosome component 3 (EXOSC3), zinc ifnger RNA binding protein, Dickkopf homolog 1 (DKK1) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). CONCLUSIONS: These results suggest that rIFN-α can upregulate the expression of important genes to exert its functions, and provide new clues for discovering the molecular mechanisms of action of IFN-α.

  2. Homoeologous recombination-based transfer and molecular cytogenetic mapping of a wheat streak mosaic virus and Triticum mosaic virus resistance gene Wsm3 from Thinopyrum intermedium to wheat.

    Science.gov (United States)

    Danilova, Tatiana V; Zhang, Guorong; Liu, Wenxuan; Friebe, Bernd; Gill, Bikram S

    2017-03-01

    Here, we report the production of a wheat- Thinopyrum intermedium recombinant stock conferring resistance to wheat streak mosaic virus and Triticum mosaic virus. Wheat streak mosaic caused by the wheat streak mosaic virus (WSMV) is an important disease of bread wheat (Triticum aestivum) worldwide. To date, only three genes conferring resistance to WSMV have been named and two, Wsm1 and Wsm3, were derived from the distantly related wild relative Thinopyrum intermedium. Wsm3 is only available in the form of a compensating wheat-Th. intermedium whole-arm Robertsonian translocation T7BS·7S#3L. Whole-arm alien transfers usually suffer from linkage drag, which prevents their use in cultivar improvement. Here, we report ph1b-induced homoeologous recombination to shorten the Th. intermedium segment and recover a recombinant chromosome consisting of the short arm of wheat chromosome 7B, part of the long arm of 7B, and the distal 43% of the long arm derived from the Th. intermedium chromosome arm 7S#3L. The recombinant chromosome T7BS·7BL-7S#3L confers resistance to WSMV at 18 and 24 °C and also confers resistance to Triticum mosaic virus, but only at 18 °C. Wsm3 is the only gene conferring resistance to WSMV at a high temperature level of 24 °C. We also developed a user-friendly molecular marker that will allow to monitor the transfer of Wsm3 in breeding programs. Wsm3 is presently being transferred to adapted hard red winter wheat cultivars and can be used directly in wheat improvement.

  3. A new method for rapidly generating gene-targeting vectors by engineering BACs through homologous recombination in bacteria.

    Science.gov (United States)

    Cotta-de-Almeida, Vinicius; Schonhoff, Susan; Shibata, Tomoyuki; Leiter, Andrew; Snapper, Scott B

    2003-09-01

    Generating knockout mice is still an expensive and highly time-consuming process. Target construct generation, the first labor-intensive step in this process, requires the manipulation of large fragments of DNA and numerous, and often cumbersome, cloning steps. Here we show the development of a rapid approach for generating targeting constructs that capitalizes on efficient homologous recombination between linear DNA fragments and circular plasmids in Escherichia coli ("recombineering"), the availability of bacterial artificial chromosomes (BACs), and the accessibility of the sequence of the mouse genome. Employing recombineering, we demonstrate with only 1-2 template plasmids, short homologies (40-50bp) between donor and target DNA, and one subcloning step that we can efficiently manipulate BACs in situ to generate a complicated targeting vector. This procedure avoids the need to construct or screen genomic libraries and permits the generation of most standard, conditional, or knock-in targeting vectors, often within two weeks.

  4. Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay.

    Science.gov (United States)

    Kittl, Roman; Kracher, Daniel; Burgstaller, Daniel; Haltrich, Dietmar; Ludwig, Roland

    2012-10-26

    Recent studies demonstrate that enzymes from the glycosyl hydrolase family 61 (GH61) show lytic polysaccharide monooxygenase (PMO) activity. Together with cellobiose dehydrogenase (CDH) an enzymatic system capable of oxidative cellulose cleavage is formed, which increases the efficiency of cellulases and put PMOs at focus of biofuel research. Large amounts of purified PMOs, which are difficult to obtain from the native fungal producers, are needed to study their reaction kinetics, structure and industrial application. In addition, a fast and robust enzymatic assay is necessary to monitor enzyme production and purification. Four pmo genes from Neurospora crassa were expressed in P. pastoris under control of the AOX1 promoter. High yields were obtained for the glycosylated gene products PMO-01867, PMO-02916 and PMO-08760 (>300 mg L-1), whereas the yield of non-glycosylated PMO-03328 was moderate (~45 mg L-1). The production and purification of all four enzymes was specifically followed by a newly developed, fast assay based on a side reaction of PMO: the production of H2O2 in the presence of reductants. While ascorbate is a suitable reductant for homogeneous PMO preparations, fermentation samples require the specific electron donor CDH. P. pastoris is a high performing expression host for N. crassa PMOs. The pmo genes under control of the native signal sequence are correctly processed and active. The novel CDH-based enzyme assay allows fast determination of PMO activity in fermentation samples and is robust against interfering matrix components.

  5. Evolutionary recruitment of a flavin-dependent monooxygenase for stabilization of sequestered pyrrolizidine alkaloids in arctiids.

    Science.gov (United States)

    Langel, Dorothee; Ober, Dietrich

    2011-09-01

    Pyrrolizidine alkaloids are secondary metabolites that are produced by certain plants as a chemical defense against herbivores. They represent a promising system to study the evolution of pathways in plant secondary metabolism. Recently, a specific gene of this pathway has been shown to have originated by duplication of a gene involved in primary metabolism followed by diversification and optimization for its specific function in the defense machinery of these plants. Furthermore, pyrrolizidine alkaloids are one of the best-studied examples of a plant defense system that has been recruited by several insect lineages for their own chemical defense. In each case, this recruitment requires sophisticated mechanisms of adaptations, e.g., efficient excretion, transport, suppression of toxification, or detoxification. In this review, we briefly summarize detoxification mechanism known for pyrrolizidine alkaloids and focus on pyrrolizidine alkaloid N-oxidation as one of the mechanisms allowing insects to accumulate the sequestered toxins in an inactivated protoxic form. Recent research into the evolution of pyrrolizidine alkaloid N-oxygenases of adapted arctiid moths (Lepidoptera) has shown that this enzyme originated by the duplication of a gene encoding a flavin-dependent monooxygenase of unknown function early in the arctiid lineage. The available data suggest several similarities in the molecular evolution of this adaptation strategy of insects to the mechanisms described previously for the evolution of the respective pathway in plants.

  6. Recombinant hybrid infectious hematopoietic necrosis virus (IHNV) carrying viral haemorrhagic septicaemia virus (VHSV) G or NV genes show different virulence properities

    DEFF Research Database (Denmark)

    Einer-Jensen, Katja; Biacchesi, S.; Stegmann, Anders

    Viral haemorrhagic septicaemia virus (VHSV) is the economically most important viral disease in European rainbow trout farming. The virus was introduced to fresh water farms in the 1950ies from a reservoir of VHSV in the marine environment. Isolates from wild marine fish and fresh water farms....... By a reverse genetics approach using the related novirrhabdovirus infectious hematopoietic necrosis virus (IHNV) as basis, four hybrid IHNV-VHSV variants were generated. These chimeric variants included substitution of the IHNV glyco(G) or nonstrutrual (Nv) protein with the corresponding G or Nv-protein from...... or fresh water VHSV. Recombinant IHNV gained higher virulence following substitution of the homologous G gene with the VHSV G gene, while the opposite was the case following substitution of the Nv gene. These findings suggest that higher virulence of VHSV compared to IHNV might be related to the G protein...

  7. Analysis of crossover breakpoints yields new insights into the nature of the gene conversion events associated with large NF1 deletions mediated by nonallelic homologous recombination.

    Science.gov (United States)

    Bengesser, Kathrin; Vogt, Julia; Mussotter, Tanja; Mautner, Victor-Felix; Messiaen, Ludwine; Cooper, David N; Kehrer-Sawatzki, Hildegard

    2014-02-01

    Large NF1 deletions are mediated by nonallelic homologous recombination (NAHR). An in-depth analysis of gene conversion operating in the breakpoint-flanking regions of large NF1 deletions was performed to investigate whether the rate of discontinuous gene conversion during NAHR with crossover is increased, as has been previously noted in NAHR-mediated rearrangements. All 20 germline type-1 NF1 deletions analyzed were mediated by NAHR associated with continuous gene conversion within the breakpoint-flanking regions. Continuous gene conversion was also observed in 31/32 type-2 NF1 deletions investigated. In contrast to the meiotic type-1 NF1 deletions, type-2 NF1 deletions are predominantly of post-zygotic origin. Our findings therefore imply that the mitotic as well as the meiotic NAHR intermediates of large NF1 deletions are processed by long-patch mismatch repair (MMR), thereby ensuring gene conversion tract continuity instead of the discontinuous gene conversion that is characteristic of short-patch repair. However, the single type-2 NF1 deletion not exhibiting continuous gene conversion was processed without MMR, yielding two different deletion-bearing chromosomes, which were distinguishable in terms of their breakpoint positions. Our findings indicate that MMR failure during NAHR, followed by post-meiotic/mitotic segregation, has the potential to give rise to somatic mosaicism in human genomic rearrangements by generating breakpoint heterogeneity.

  8. Nonallelic homologous recombination of the FCGR2/3 locus results in copy number variation and novel chimeric FCGR2 genes with aberrant functional expression.

    Science.gov (United States)

    Nagelkerke, S Q; Tacke, C E; Breunis, W B; Geissler, J; Sins, J W R; Appelhof, B; van den Berg, T K; de Boer, M; Kuijpers, T W

    2015-09-01

    The human FCGR2/3 locus, containing five highly homologous genes encoding the major IgG receptors, shows extensive copy number variation (CNV) associated with susceptibility to autoimmune diseases. Having genotyped >4000 individuals, we show that all CNV at this locus can be explained by nonallelic homologous recombination (NAHR) of the two paralogous repeats that constitute the majority of the locus, and describe four distinct CNV regions (CNRs) with a highly variable prevalence in the population. Apart from CNV, NAHR events also created several hitherto unidentified chimeric FCGR2 genes. These include an FCGR2A/2C chimeric gene that causes a decreased expression of FcγRIIa on phagocytes, resulting in a decreased production of reactive oxygen species in response to immune complexes, compared with wild-type FCGR2A. Conversely, FCGR2C/2A chimeric genes were identified to lead to an increased expression of FCGR2C. Finally, a rare FCGR2B null-variant allele was found, in which a polymorphic stop codon of FCGR2C is introduced into one FCGR2B gene, resulting in a 50% reduction in protein expression. Our study on CNRs and the chimeric genes is essential for the correct interpretation of association studies on FCGR genes as a determinant for disease susceptibility, and may explain some as yet unidentified extreme phenotypes of immune-mediated disease.

  9. Raw starch-degrading α-amylase from Bacillus aquimaris MKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme.

    Science.gov (United States)

    Puspasari, F; Radjasa, O K; Noer, A S; Nurachman, Z; Syah, Y M; van der Maarel, M; Dijkhuizen, L; Janeček, S; Natalia, D

    2013-01-01

    The aims were to isolate a raw starch-degrading α-amylase gene baqA from Bacillus aquimaris MKSC 6.2, and to characterize the gene product through in silico study and its expression in Escherichia coli. A 1539 complete open reading frame of a starch-degrading α-amylase gene baqA from B. aquimaris MKSC 6·2 has been determined by employing PCR and inverse PCR techniques. Bioinformatics analysis revealed that B. aquimaris MKSC 6.2 α-amylase (BaqA) has no starch-binding domain, and together with a few putative α-amylases from bacilli may establish a novel GH13 subfamily most closely related to GH13_1. Two consecutive tryptophans (Trp201 and Trp202, BaqA numbering) were identified as a sequence fingerprint of this novel GH13 subfamily. Escherichia coli cells produced the recombinant BaqA protein as inclusion bodies. The refolded recombinant BaqA protein degraded raw cassava and corn starches, but exhibited no activity with soluble starch. A novel raw starch-degrading B. aquimaris MKSC 6.2 α-amylase BaqA is proposed to be a member of new GH13 subfamily. This study has contributed to the overall knowledge and understanding of amylolytic enzymes that are able to bind and digest raw starch directly. © 2012 The Society for Applied Microbiology.

  10. Impact of alg3 gene deletion on growth, development, pigment production, protein secretion, and functions of recombinant Trichoderma reesei cellobiohydrolases in Aspergillus niger

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Ziyu; Aryal, Uma K.; Shukla, Anil; Qian, Wei-Jun; Smith, Richard D.; Magnuson, Jon K.; Adney, William S.; Beckham, Gregg T.; Brunecky, Roman; Himmel, Michael E.; Decker, Stephen R.; Ju, Xiaohui; Zhang, Xiao; Baker, Scott E.

    2013-12-01

    ALG3 is a Family 58 glycosyltransferase enzyme involved in early N-linked glycan synthesis. Here, we investigated the effect of the alg3 gene disruption on growth, development, metabolism, and protein secretion in Aspergillus niger. The alg3 gene deletion resulted in a significant reduction of growth on complete (CM) and potato dextrose agar (PDA) media and a substantial reduction of spore production on CM. It also delayed spore germination in the liquid cultures of both CM and PDA media, but led to a significant accumulation of red pigment on both CM and liquid modified minimal medium (MM) supplemented with yeast extract. The relative abundance of 55 proteins of the total 190 proteins identified in the secretome was significantly different as a result of alg3 gene deletion. Comparison of a Trichoderma reesei cellobiohydrolase (Cel7A) heterologously expressed in A. niger parental and Δalg3 strains showed that the recombinant Cel7A expressed in the mutant background was smaller in size than that from the parental strains. This study suggests that ALG3 is critical for growth and development, pigment production, and protein secretion in A. niger. Functional analysis of recombinant Cel7A with aberrant glycosylation demonstrates the feasibility of this alternative approach to evaluate the role of N-linked glycosylation in glycoprotein secretion and function.

  11. Development, evaluation, and laboratory validation of immunoassays for the diagnosis of equine infectious anemia (EIA) using recombinant protein produced from a synthetic p26 gene of EIA virus.

    Science.gov (United States)

    Singha, Harisankar; Goyal, Sachin K; Malik, Praveen; Khurana, Sandip K; Singh, Raj K

    2013-12-01

    Equine infectious anemia (EIA)-a retroviral disease caused by equine infectious anemia virus (EIAV)-is a chronic, debilitating disease of horses, mules, and donkeys. EIAV infection has been reported worldwide and is recognized as pathogen of significant economic importance to the horse industry. This disease falls under regulatory control program in many countries including India. Control of EIA is based on identification of inapparent carriers by detection of antibodies to EIAV in serologic tests and "Stamping Out" policy. The current internationally accepted test for diagnosis of EIA is the agar gel immune-diffusion test (AGID), which detects antibodies to the major gag gene (p26) product. The objective of this study was to develop recombinant p26 based in-house immunoassays [enzyme linked immunosorbent assays (ELISA), and AGID] for EIA diagnosis. The synthetic p26 gene of EIAV was expressed in Escherichia coli and diagnostic potential of recombinant p26 protein were evaluated in ELISA and AGID on 7,150 and 1,200 equine serum samples, respectively, and compared with commercial standard AGID kit. The relative sensitivity and specificity of the newly developed ELISA were 100 and 98.6 %, respectively. Whereas, relative sensitivity and specificity of the newly developed AGID were in complete agreement in respect to commercial AGID kit. Here, we have reported the validation of an ELISA and AGID on large number of equine serum samples using recombinant p26 protein produced from synthetic gene which does not require handling of pathogenic EIAV. Since the indigenously developed reagents would be economical than commercial diagnostic kit, the rp26 based-immunoassays could be adopted for the sero-diagnosis and control of EIA in India.

  12. Induced change of formative processes in pepper (Capsicum annuum L. ). I. Effect of mutagenic treatment on the crossingover frequency of the linked and recombination of unlinked marker genes

    Energy Technology Data Exchange (ETDEWEB)

    Samovol, A.P.

    1986-05-01

    The effect of mutagenic treatment of the F/sub 1/ seeds of pepper on the crossingover frequency in the al/sub 2/-b segment, monohybrid and dihybrid segregation for the unlinked marker genes al/sub 2/ and pi was studied. It has been demonstrated that treatment leads to a significant reduction in the crossover frequency in the al/sub 2/-b zone. Highly significant differences between the control and individual treatment of the hybrid seeds indicated reduction in recombinations due to the mutagens used. A case of induced deviation in independent segregation of al/sub 2/ and pi, i.e., quasilinkage has been recorded.

  13. Deletion of C7L and K1L Genes Leads to Significantly Decreased Virulence of Recombinant Vaccinia Virus TianTan

    OpenAIRE

    Zheng Liu; Shuhui Wang; Qicheng Zhang; Meijuan Tian; Jue Hou; Rongmin Wang; Chang Liu; Xu Ji; Ying Liu; Yiming Shao

    2013-01-01

    The vaccinia virus TianTan (VTT) has been modified as an HIV vaccine vector in China and has shown excellent performance in immunogenicity and safety. However, its adverse effects in immunosuppressed individuals warrant the search for a safer vector in the following clinic trails. In this study, we deleted the C7L and K1L genes of VTT and constructed six recombinant vaccinia strains VTT△C7L, VTT△K1L, VTT△C7LK1L, VTKgpe△C7L, VTKgpe△K1L and VTT△C7LK1L-gag. The pathogenicity and immunogenicity o...

  14. A Novel Neurotoxin Gene ar1b Recombination Enhances the Efficiency of Helicoverpa armigera Nucleopolyhedrovirus as a Pesticide by Inhibiting the Host Larvae Ability to Feed and Grow

    OpenAIRE

    Huan Yu; Jiao Meng; Jian Xu; Tong-Xian Liu; Dun Wang

    2015-01-01

    A recombinant Helicoverpa armigera nucleopolyhedrovirus (HearNPV), Ar1b-HearNPV, was constructed and identified as an improved bio-control agent of Helicoverpa armigera larvae. The HearNPV polyhedrin promoter was used to express the insect-specific neurotoxin gene, ar1b, which was originally isolated from the Australian funnel-web spider (Atrax robustus). RT-PCR and Western blotting analysis showed that both the ar1b transcript and protein were produced successfully in Ar1b-HearNPV-infected H...

  15. Acetylcholinesterase of Rhipicephalus (Boophilus) microplus and Phlebotomus papatasi: Gene Identification, Expression, and Biochemical Properties of Recombinant Proteins

    Science.gov (United States)

    2013-01-01

    inspection of all imported cattle has prevented re-establish- ment of ‘‘cattle fever ticks’’ within the United States. The Texas Ani- mal Health...Q86GC8], and Aedes aegypti [GenBank: XP_001656977]. Recombinant acetylcholinesterase1 of P. papatasi expressed and biochemically characterized in

  16. DNA-binding factor CTCF and long-range gene interactions in V(D)J recombination and oncogene activation

    NARCIS (Netherlands)

    C. Ribeiro de Almeida (Claudia); R. Stadhouders (Ralph); S. Thongjuea (Supat); E. Soler (Eric); R.W. Hendriks (Rudi)

    2012-01-01

    textabstractRegulation of V(D)J recombination events at immunoglobulin (Ig) and T-cell receptor loci in lymphoid cells is complex and achieved via changes in substrate accessibility. Various studies over the last year have identified the DNA-binding zinc-finger protein CCCTC-binding factor (CTCF) as

  17. The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus

    DEFF Research Database (Denmark)

    Torres-Rosell, Jordi; Sunjevaric, Ivana; De Piccoli, Giacomo;

    2007-01-01

    at an extranucleolar site. The nucleolar exclusion of Rad52 recombination foci entails Mre11 and Smc5-Smc6 complexes and depends on Rad52 SUMO (small ubiquitin-related modifier) modification. Remarkably, mutations that abrogate these activities result in the formation of Rad52 foci within the nucleolus and cause r...

  18. THE CONSTRUCTION AND EXPRESSION OF RECOMBINANT SHUTTLE PLASMID WITH OMPL1 GENE FROM LEPTOSPIRA INTERROGANS SEROVAR LAI STRAIN 017 IN BACILLE CALMETTE GUERIN

    Institute of Scientific and Technical Information of China (English)

    鲍朗; 邱洪宇; 晏菊芳; 谢勇恩; 陈玮

    2002-01-01

    Objective.To construct recombinant BCG against leptospirosis.Methods.We amplified the entire open reading frame of the OmpL1 gene from the genome of the leptospire serovar Lai strain 017.Two recombinant plasmids pBQ1 and pBQ2 were constructed by oriented ligation based on the E.coli BCG shuttle plasmids pMV261 and pMV361 respectively.The recombinant plasmids were transformed into BCG by electroporation.The rBCGs bearing pBQ1 and pBQ2 were induced by high temperature of 45℃ .Results.The expressed product,a 35kD protein was detected by SDS PAGE.The result indicates that pBQ1 and pBQ2 can express OmpL1 in rBCG.Conclusion.The technical methods in this study may help detect the immunogenicity and immunoprotection of OmpL1 and develop more safe,highly effective rBCG bearing leptospiral antigen with long lasting protection.

  19. Cloning and expression of synthetic genes encoding the broad antimicrobial spectrum bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by recombinant Pichia pastoris.

    Science.gov (United States)

    Arbulu, Sara; Jiménez, Juan J; Gútiez, Loreto; Cintas, Luis M; Herranz, Carmen; Hernández, Pablo E

    2015-01-01

    We have evaluated the cloning and functional expression of previously described broad antimicrobial spectrum bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by recombinant Pichia pastoris. Synthetic genes, matching the codon usage of P. pastoris, were designed from the known mature amino acid sequence of these bacteriocins and cloned into the protein expression vector pPICZαA. The recombinant derived plasmids were linearized and transformed into competent P. pastoris X-33, and the presence of integrated plasmids into the transformed cells was confirmed by PCR and sequencing of the inserts. The antimicrobial activity, expected in supernatants of the recombinant P. pastoris producers, was purified using a multistep chromatographic procedure including ammonium sulfate precipitation, desalting by gel filtration, cation exchange-, hydrophobic interaction-, and reverse phase-chromatography (RP-FPLC). However, a measurable antimicrobial activity was only detected after the hydrophobic interaction and RP-FPLC steps of the purified supernatants. MALDI-TOF MS analysis of the antimicrobial fractions eluted from RP-FPLC revealed the existence of peptide fragments of lower and higher molecular mass than expected. MALDI-TOF/TOF MS analysis of selected peptides from eluted RP-FPLC samples with antimicrobial activity indicated the presence of peptide fragments not related to the amino acid sequence of the cloned bacteriocins.

  20. Cloning and Expression of Synthetic Genes Encoding the Broad Antimicrobial Spectrum Bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by Recombinant Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Sara Arbulu

    2015-01-01

    Full Text Available We have evaluated the cloning and functional expression of previously described broad antimicrobial spectrum bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by recombinant Pichia pastoris. Synthetic genes, matching the codon usage of P. pastoris, were designed from the known mature amino acid sequence of these bacteriocins and cloned into the protein expression vector pPICZαA. The recombinant derived plasmids were linearized and transformed into competent P. pastoris X-33, and the presence of integrated plasmids into the transformed cells was confirmed by PCR and sequencing of the inserts. The antimicrobial activity, expected in supernatants of the recombinant P. pastoris producers, was purified using a multistep chromatographic procedure including ammonium sulfate precipitation, desalting by gel filtration, cation exchange-, hydrophobic interaction-, and reverse phase-chromatography (RP-FPLC. However, a measurable antimicrobial activity was only detected after the hydrophobic interaction and RP-FPLC steps of the purified supernatants. MALDI-TOF MS analysis of the antimicrobial fractions eluted from RP-FPLC revealed the existence of peptide fragments of lower and higher molecular mass than expected. MALDI-TOF/TOF MS analysis of selected peptides from eluted RP-FPLC samples with antimicrobial activity indicated the presence of peptide fragments not related to the amino acid sequence of the cloned bacteriocins.

  1. Cloning and Expression of Synthetic Genes Encoding the Broad Antimicrobial Spectrum Bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by Recombinant Pichia pastoris

    Science.gov (United States)

    Jiménez, Juan J.; Gútiez, Loreto; Cintas, Luis M.; Herranz, Carmen; Hernández, Pablo E.

    2015-01-01

    We have evaluated the cloning and functional expression of previously described broad antimicrobial spectrum bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by recombinant Pichia pastoris. Synthetic genes, matching the codon usage of P. pastoris, were designed from the known mature amino acid sequence of these bacteriocins and cloned into the protein expression vector pPICZαA. The recombinant derived plasmids were linearized and transformed into competent P. pastoris X-33, and the presence of integrated plasmids into the transformed cells was confirmed by PCR and sequencing of the inserts. The antimicrobial activity, expected in supernatants of the recombinant P. pastoris producers, was purified using a multistep chromatographic procedure including ammonium sulfate precipitation, desalting by gel filtration, cation exchange-, hydrophobic interaction-, and reverse phase-chromatography (RP-FPLC). However, a measurable antimicrobial activity was only detected after the hydrophobic interaction and RP-FPLC steps of the purified supernatants. MALDI-TOF MS analysis of the antimicrobial fractions eluted from RP-FPLC revealed the existence of peptide fragments of lower and higher molecular mass than expected. MALDI-TOF/TOF MS analysis of selected peptides from eluted RP-FPLC samples with antimicrobial activity indicated the presence of peptide fragments not related to the amino acid sequence of the cloned bacteriocins. PMID:25821820

  2. Roles of Breast Cancer Genes in DNA Homologous Recombination and Cellular Sensitivity to Radiation and Anticancer Drugs

    Science.gov (United States)

    2002-06-01

    cells using GenePorter transfection reagent ( GeneTherapy Sys- tems). Forty-eight hours after transfection, cells were collected, treated A C-terminal...into HeLa cells using GenePorter transfection reagent ( GeneTherapy Systems). Forty-eight hours after transfection, cells were collected, treated with

  3. Isolation of a novel gene from Photobacterium damselae subsp. piscicida and analysis of the recombinant antigen as promising vaccine candidate.

    Science.gov (United States)

    Andreoni, Francesca; Boiani, Romina; Serafini, Giordano; Amagliani, Giulia; Dominici, Sabrina; Riccioni, Giulia; Zaccone, Renata; Mancuso, Monique; Scapigliati, Giuseppe; Magnani, Mauro

    2013-01-21

    Photobacterium damselae subsp. piscicida (PDP) is the causative agent of fish pasteurellosis, a bacterial disease causing important losses in marine aquaculture. Vaccines against the pathogen can be a way to control the infection and avoid antibiotic treatments. However, a satisfactory protective vaccine against fish pasteurellosis is not commercially available. In this study, a biotechnogical approach based on reverse vaccinology has been used to identify potential vaccine candidates for the development of a recombinant subunit vaccine. Genome sequencing of clones from a genomic cosmid library of PDP and in silico selection of the surface exposed proteins were the initial steps in vaccine candidate identification. From 370 open reading frames (ORF) eight potential antigens were selected, expressed as recombinant proteins and purified. These vaccine candidates were used to generate specific polyclonal antibodies in mice. Each antibody was then screened in vitro by inhibition adherence assay of live PDP on chinook salmon embryo cells (CHSE-214). A lipoprotein, found to be involved in the adherence of the bacterium to epithelial cells and annotated as PDP_0080, was then selected. The recombinant protein was further investigated in fish vaccination and challenge experiments to assess its ability to protect sea bass, Dicentrarchus labrax, against PDP infection. Immunisation with PDP_0080 recombinant protein elicited high specific antibody titres. Furthermore, the survival rate of fish immunized with the 25 μg dose of protein was significantly higher compared to the control group. The results of the study suggest that the PDP_0080 protein could be a promising candidate for the design of a recombinant vaccine against pasteurellosis.

  4. Induction of genetic recombination in the lambda bacteriophage by ultraviolet irradiation of the Escherichia Coli cells. III. Role of the ruvA and recN genes; Induccion de recombinacion genetica en el bacteriofago lambda por irradiacion ultravioleta de las celulas de Escherichia Coli. III. Papel de los genes ruvA and recN

    Energy Technology Data Exchange (ETDEWEB)

    Alcantara D, D. [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)

    1987-05-15

    The objective of this work is to determine the paper of the genes ruvA and recN in the stimulation of the recombination of Lambda for UV irradiation of Escherichia Coli, taking into account that both genes are inducible, they belong to the group of genes that participate in the SOS response and that a deficiency in its expression reduces the capacity to repair and recombiner the DNA. (Author)

  5. Conservation of recombination hotspots in yeast

    OpenAIRE

    Tsai, Isheng J.; Burt, Austin; Koufopanou, Vassiliki

    2010-01-01

    Meiotic recombination does not occur randomly along a chromosome, but instead tends to be concentrated in small regions, known as “recombination hotspots.” Recombination hotspots are thought to be short-lived in evolutionary time due to their self-destructive nature, as gene conversion favors recombination-suppressing alleles over recombination-promoting alleles during double-strand repair. Consistent with this expectation, hotspots in humans are highly dynamic, with little correspondence in ...

  6. Characterization of opaque2 modifier QTLs and candidate genes in recombinant inbred lines derived from the K0326Y quality protein maize inbred

    KAUST Repository

    Holding, David R.

    2010-11-13

    Quality protein maize (QPM) is a high lysine-containing corn that is based on genetic modification of the opaque2 (o2) mutant. In QPM, modifier genes convert the starchy endosperm of o2 to the vitreous phenotype of wild type maize. There are multiple, unlinked o2 modifier loci (Opm) in QPM and their nature and mode of action are unknown. We previously identified seven Opm QTLs and characterized 16 genes that are differentially up-regulated at a significant level in K0326Y QPM, compared to the starchy endosperm mutant W64Ao2. In order to further characterize these Opm QTLs and the genes up-regulated in K0326Y QPM, we created a population of 314 recombinant inbred lines (RILs) from a cross between K0326Y QPM and W64Ao2. The RILs were characterized for three traits associated with endosperm texture: vitreousness, density and hardness. Genetic linkage analysis of the RIL population confirmed three of the previously identified QTLs associated with o2 endosperm modification in K0326Y QPM. Many of the genes up-regulated in K0326Y QPM showed substantially higher levels of expression in vitreous compared with opaque RILs. These included genes associated with the upstream regulation of the ethylene response pathway, and a gene encoding a regulatory subunit of pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase, an adaptive enzyme of the glycolytic pathway. © 2010 Springer-Verlag.

  7. Informational redundancy of tRNA(4Ser) and tRNA(7Ser) genes in Drosophila melanogaster and evidence for intergenic recombination.

    Science.gov (United States)

    Leung, J; Sinclair, D A; Hayashi, S; Tener, G M; Grigliatti, T A

    1991-05-20

    Variant tRNA genes have been widely observed in multicellular eukaryotes. Recent biochemical studies have shown that some of them are expressed in a tissue- or a stage-specific manner. These findings would thus imply that certain modified tRNAs may be crucial for the development of the organism. Using Drosophila melanogaster as a model, we have taken a combined genetic and molecular approach to examine critically the possible biological functions of tRNA(4, 7Ser) genes. We showed that at least 50% of the total templates can be deleted from the genome without inducing abnormal phenotypes such as Minute, or a decrease in viability. In addition, two of the tRNASer variant genes that are unique in sequence are also completely dispensable. This strongly implies that even though they may be expressed in vivo, they play no essential role in the development of the fruitfly. By comparison with some of the corresponding tRNA genes in another sibling species, Drosophila erecta, our results suggest strongly that the variants are products non-reciprocal exchanges among the tRNA(4, 7Ser), genes. Such intergenic recombination events may have a major influence in the concerted evolution of the two gene families.

  8. Aryl hydrocarbon mono-oxygenase activity in human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Griffin, G.D.; Schuresko, D.D.

    1981-06-01

    Aryl hydrocarbon mono-oxygenase (AHM), an enzyme of key importance in metabolism of xenobiotic chemicals such as polynuclear aromatic hydrocarbons (PNA), is present in human lymphocytes. Studies investing the relation of activity of AHM in human lymphocytes to parameters such as disease state, PNA exposure, in vitro mitogen stimulation, etc. have been summarized in this report. Some studies have demonstrated increased AHM activity in lymphocytes from cigarette smokers (compared to nonsmokers), and in lung cancer patients when compared to appropriate control groups. These observations are confused by extreme variability in human lymphocyte AHM activities, such variability arising from factors such as genetic variation in AHM activity, variation in in vitro culture conditions which affect AHM activity, and the problematical relationship of common AHM assays to actual PNA metabolism taking place in lymphocytes. If some of the foregoing problems can be adequately addressed, lymphocyte AHM activity could hold the promise of being a useful biomarker system for human PNA exposure.

  9. Lytic polysaccharide monooxygenases disrupt the cellulose fibers structure

    Science.gov (United States)

    Villares, Ana; Moreau, Céline; Bennati-Granier, Chloé; Garajova, Sona; Foucat, Loïc; Falourd, Xavier; Saake, Bodo; Berrin, Jean-Guy; Cathala, Bernard

    2017-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that breakdown recalcitrant polysaccharides such as cellulose. Here we investigate the action of LPMOs on cellulose fibers. After enzymatic treatment and dispersion, LPMO-treated fibers show intense fibrillation. Cellulose structure modifications visualized at different scales indicate that LPMO creates nicking points that trigger the disintegration of the cellulose fibrillar structure with rupture of chains and release of elementary nanofibrils. Investigation of LPMO action using solid-state NMR provides direct evidence of modification of accessible and inaccessible surfaces surrounding the crystalline core of the fibrils. The chains breakage likely induces modifications of the cellulose network and weakens fibers cohesion promoting their disruption. Besides the formation of new initiation sites for conventional cellulases, this work provides the first evidence of the direct oxidative action of LPMOs with the mechanical weakening of the cellulose ultrastructure. LPMOs can be viewed as promising biocatalysts for enzymatic modification or degradation of cellulose fibers. PMID:28071716

  10. Structural characterization of Lytic Polysaccharide MonoOxygenases

    DEFF Research Database (Denmark)

    Frandsen, Kristian Erik Høpfner

    Lytic polysaccharide monooxygenases (LPMOs) are a new class of copper-containingmetalloenzymes that have been found to oxidatively degrade polysaccharides (and recently alsooligosaccharides). They dependent on redox partners to provide them with electrons and they utilizemolecular oxygen to cleave......) and their interaction with substratehave been structurally characterized. A number of structures of LsAA9A have been obtained in complexwith a range of cellulosic- and hemicellulosic substrates and with the active site Cu in different redox state.Two of the LsAA9A structures with the active site Cu in essentially a Cu......(II) state show differences in thenature of the Cu-ligand with and without cellulosic substrate bound and provide structural insight into themechanistic action of LPMOs. Interestingly, for an LsAA9A complex structure with a hemicellulosicsubstrate (xylooligosaccharide) a corresponding difference...

  11. Removal of bacterial suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a Nicotiana benthamiana transient gene expression system.

    Science.gov (United States)

    Fujiuchi, Naomichi; Matsuda, Ryo; Matoba, Nobuyuki; Fujiwara, Kazuhiro

    2016-04-01

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory.

  12. Novel substrates and inhibitors of peptidylglycine alpha-amidating monooxygenase.

    Science.gov (United States)

    Katopodis, A G; May, S W

    1990-05-15

    Peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3) catalyzes the formation of alpha-amidated peptides from their glycine-extended precursors, thus playing a key role in the processing of peptide neurohormones. We now report that PAM readily catalyzes three alternate monooxygenase reactions--sulfoxidation, amine N-dealkylation, and O-dealkylation. Thus, (4-nitrobenzyl)thioacetic acid is converted to the analogous sulfoxide, N-(4-nitrobenzyl)glycine is converted to 4-nitrobenzylamine and glyoxylate, and [(4-nitrobenzyl)oxy]acetic acid is converted to 4-nitrobenzyl alcohol and glyoxylate. All these new activities display the characteristics expected for the normal PAM-catalyzed reductive oxygenation pathway and produce an equimolar amount of glyoxylate together with the heteroatom-containing dealkylation products. The ester [(4-methoxybenzoyl)oxy]acetic acid is not a PAM substrate, but is instead a good competitive inhibitor (KI = 0.48 mM). In addition, we report that the olefinic substrate analogues trans-benzoylacrylic acid and 4-phenyl-3-butenoic acid are potent time-dependent inactivators of PAM, with inactivation exhibiting the characteristics expected for mechanism-based inhibition. Monoethyl fumarate is also a time-dependent inactivator of PAM. Finally, we introduce several small non-peptide substrates for PAM by demonstrating that PAM catalyzes the transformation of hippuric acid and several ring-substituted derivatives to the corresponding benzamides and glyoxylic acid, with the most facile substrate of this class being 4-nitrohippuric acid. These compounds are the smallest amide substrates yet reported for PAM, and it is thus apparent that only the minimal structure of an acylglycine is required for PAM-catalyzed oxygenative amidation.

  13. Search for genes essential for pneumococcal transformation: the RADA DNA repair protein plays a role in genomic recombination of donor DNA.

    Science.gov (United States)

    Burghout, Peter; Bootsma, Hester J; Kloosterman, Tomas G; Bijlsma, Jetta J E; de Jongh, Christa E; Kuipers, Oscar P; Hermans, Peter W M

    2007-09-01

    We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counterselection of mutants in 21 different genes during the challenge. Eight of the counterselected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants of seven of the eight remaining genes, i.e., spr0459-spr0460, spr0777, spr0838, spr1259-spr1260, and spr1357, resulted in reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation with chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and in DNA damage repair in S. pneumoniae.

  14. Increased expression and secretion of recombinant hIFNγ through amino acid starvation-induced selective pressure on the adjacent HIS4 gene in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Razaghi Ali

    2015-12-01

    Full Text Available Transcriptional co-regulation of adjacent genes has been observed for prokaryotic and eukaryotic organisms, alike. High levels of gene adjacency were also found in a wide variety of yeast species with a high frequency of co-regulated gene sets. The aim of this research was to study how selective pressure on the Histidinol dehydrogenase gene (HIS4, using amino acid starvation, affects the level of expression and secretion of the adjacent human interferon gamma gene (hIFNγ in the recombinant Pichia pastoris GS115 strain, a histidine-deficient mutant. hIFNγ was cloned into the pPIC9 vector adjacent to the HIS4 gene, a gene essential for histidine biosynthesis, which was then transformed into P. pastoris. The transformed P. pastoris was cultured under continuous amino acid starvation in amino acid-free minimal medium for ten days, with five inoculations into unspent medium every second day. Under these conditions, only successfully transformed cells (hIFNγ -HIS4+ are able to synthesise histidine and therefore thrive. As shown by ELISA, amino acid starvation-induced selective pressure on HIS4 improved expression and secretion of the adjacent hIFNγ by 55% compared to unchallenged cells. RT-qPCR showed that there was also a positive correlation between duration of amino acid starvation and increased levels of the hIFNγ RNA transcripts. According to these results, it is suggested that these adjacent genes (hIFNγ and HIS4 in the transformed P. pastoris are transcriptionally co-regulated and their expression is synchronised. To the best of the knowledge of the authors; this is the first study demonstrating that amino acid starvationinduced selective pressure on HIS4 can alter the regulation pattern of adjacent genes in P. pastoris.

  15. Molecular phylogeny, long-term evolution, and functional divergence of flavin-containing monooxygenases.

    Science.gov (United States)

    Hao, Da Cheng; Chen, Shi Lin; Mu, Jun; Xiao, Pei Gen

    2009-11-01

    Flavin-containing monooxygenases (FMOs) metabolize xenobiotic compounds, many of which are clinically important, as well as endogenous substrates as part of a discrete physiological process. The FMO gene family is conserved and ancient with representatives present in all phyla so far examined. However, there is a lack of information regarding the long-term evolution and functional divergence of these proteins. This study represents the first attempt to characterize the long-term evolution followed by the members of this family. Our analysis shows that there is extensive silent divergence at the nucleotide level suggesting that this family has been subject to strong purifying selection at the protein level. Invertebrate FMOs have a polyphyletic origin. The functional divergence of FMOs 1-5 started before the split between amphibians and mammals. The vertebrate FMO5 is more ancestral than other four FMOs. Moreover, the existence of higher levels of codon bias was detected at the N-terminal ends, which can be ascribed to the critical role played by the FAD binding motif in this region. Finally, critical amino acid residues for FMO functional divergence (type I & II) after gene duplication were detected and characterized.

  16. A bovine herpesvirus 5 recombinant defective in the thymidine kinase (TK gene and a double mutant lacking TK and the glycoprotein E gene are fully attenuated for rabbits

    Directory of Open Access Journals (Sweden)

    S.C. Silva

    2010-02-01

    Full Text Available Bovine herpesvirus 5 (BoHV-5, the agent of herpetic meningoencephalitis in cattle, is an important pathogen of cattle in South America and several efforts have been made to produce safer and more effective vaccines. In the present study, we investigated in rabbits the virulence of three recombinant viruses constructed from a neurovirulent Brazilian BoHV-5 strain (SV507/99. The recombinants are defective in glycoprotein E (BoHV-5gEΔ, thymidine kinase (BoHV-5TKΔ and both proteins (BoHV-5gEΔTKΔ. Rabbits inoculated with the parental virus (N = 8 developed neurological disease and died or were euthanized in extremis between days 7 and 13 post-infection (pi. Infectivity was detected in several areas of their brains. Three of 8 rabbits inoculated with the recombinant BoHV-5gEΔ developed neurological signs between days 10 and 15 pi and were also euthanized. A more restricted virus distribution was detected in the brain of these animals. Rabbits inoculated with the recombinants BoHV-5TKΔ (N = 8 or BoHV-5gEΔTKΔ (N = 8 remained healthy throughout the experiment in spite of variable levels of virus replication in the nose. Dexamethasone (Dx administration to rabbits inoculated with the three recombinants at day 42 pi did not result in viral reactivation, as demonstrated by absence of virus shedding and/or increase in virus neutralizing titers. Nevertheless, viral DNA was detected in the trigeminal ganglia or olfactory bulbs of all animals at day 28 post-Dx, demonstrating they were latently infected. These results show that recombinants BoHV-5TKΔ and BoHV-5gEΔTKΔ are attenuated for rabbits and constitute potential vaccine candidates upon the confirmation of this phenotype in cattle.

  17. Expression of a chimeric human/salmon calcitonin gene integrated into the Saccharomyces cerevisiae genome using rDNA sequences as recombination sites.

    Science.gov (United States)

    Sun, Hengyi; Zang, Xiaonan; Liu, Yuantao; Cao, Xiaofei; Wu, Fei; Huang, Xiaoyun; Jiang, Minjie; Zhang, Xuecheng

    2015-12-01

    Calcitonin participates in controlling homeostasis of calcium and phosphorus and plays an important role in bone metabolism. The aim of this study was to endow an industrial strain of Saccharomyces cerevisiae with the ability to express chimeric human/salmon calcitonin (hsCT) without the use of antibiotics. To do so, a homologous recombination plasmid pUC18-rDNA2-ura3-P pgk -5hsCT-rDNA1 was constructed, which contains two segments of ribosomal DNA of 1.1 kb (rDNA1) and 1.4 kb (rDNA2), to integrate the heterologous gene into host rDNA. A DNA fragment containing five copies of a chimeric human/salmon calcitonin gene (5hsCT) under the control of the promoter for phosphoglycerate kinase (P pgk ) was constructed to express 5hsCT in S. cerevisiae using ura3 as a selectable auxotrophic marker gene. After digestion by restriction endonuclease HpaI, a linear fragment, rDNA2-ura3-P pgk -5hsCT-rDNA1, was obtained and transformed into the △ura3 mutant of S. cerevisiae by the lithium acetate method. The ura3-P pgk -5hsCT sequence was introduced into the genome at rDNA sites by homologous recombination, and the recombinant strain YS-5hsCT was obtained. Southern blot analysis revealed that the 5hsCT had been integrated successfully into the genome of S. cerevisiae. The results of Western blot and ELISA confirmed that the 5hsCT protein had been expressed in the recombinant strain YS-5hsCT. The expression level reached 2.04 % of total proteins. S. cerevisiae YS-5hsCT decreased serum calcium in mice by oral administration and even 0.01 g lyophilized S. cerevisiae YS-5hsCT/kg decreased serum calcium by 0.498 mM. This work has produced a commercial yeast strain potentially useful for the treatment of osteoporosis.

  18. Hydroxylation of Longiborneol by a Clm2-Encoded CYP450 Monooxygenase to Produce Culmorin in Fusarium graminearum.

    Science.gov (United States)

    Bahadoor, Adilah; Schneiderman, Danielle; Gemmill, Larissa; Bosnich, Whynn; Blackwell, Barbara; Melanson, Jeremy E; McRae, Garnet; Harris, Linda J

    2016-01-22

    A second structural gene required for culmorin biosynthesis in the plant pathogen Fus