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Results: 1 to 10 of 27

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An improved reverse genetics system for mammalian orthoreoviruses.
Kobayashi T, Ooms LS, Ikizler M, Chappell JD, Dermody TS
(2010) Virology 398: 194-200
MeSH Terms: Animals, Cell Line, Cricetinae, DNA, Recombinant, DNA, Viral, Electrophoresis, Polyacrylamide Gel, Fluorescent Antibody Technique, Genetic Vectors, Orthoreovirus, Mammalian, Plasmids, Reoviridae Infections, Transfection
Show Abstract · Added December 10, 2013
Mammalian orthoreoviruses (reoviruses) are highly useful models for studies of double-stranded RNA virus replication and pathogenesis. We previously developed a strategy to recover prototype reovirus strain T3D from cloned cDNAs transfected into murine L929 fibroblast cells. Here, we report the development of a second-generation reovirus reverse genetics system featuring several major improvements: (1) the capacity to rescue prototype reovirus strain T1L, (2) reduction of required plasmids from 10 to 4, and (3) isolation of recombinant viruses following transfection of baby hamster kidney cells engineered to express bacteriophage T7 RNA polymerase. The efficiency of virus rescue using the 4-plasmid strategy was substantially increased in comparison to the original 10-plasmid system. We observed full compatibility of T1L and T3D rescue vectors when intermixed to produce a panel of T1LxT3D monoreassortant viruses. Improvements to the reovirus reverse genetics system enhance its applicability for studies of reovirus biology and clinical use.
Copyright 2009 Elsevier Inc. All rights reserved.
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12 MeSH Terms
Covalent modification at Cys151 dissociates the electrophile sensor Keap1 from the ubiquitin ligase CUL3.
Rachakonda G, Xiong Y, Sekhar KR, Stamer SL, Liebler DC, Freeman ML
(2008) Chem Res Toxicol 21: 705-10
MeSH Terms: Animals, COS Cells, Cell Cycle Proteins, Cell Line, Tumor, Cells, Cultured, Chlorocebus aethiops, Circular Dichroism, Cullin Proteins, Cysteine, DNA, Recombinant, Humans, Immunoprecipitation, Intracellular Signaling Peptides and Proteins, Kelch-Like ECH-Associated Protein 1, Kinetics, Mass Spectrometry, Recombinant Proteins
Show Abstract · Added March 5, 2014
The regulation of cellular stress responses to electrophiles and oxidants is mediated by the transcription factor NF-E2-related factor 2 (Nrf2), which, in turn, is regulated by CUL-E3 (CUL3) ligase-mediated ubiquitylation. The Kelch-like ECH-associated protein 1 (Keap1) serves as an adapter between CUL3 and Nrf2. We used the model electrophile N-iodoacetyl- N-biotinylhexylenediamine (IAB) to define the relationship among the adduction of Keap1 cysteine residues, structure, and function. Exposure of Keap1 to IAB in vitro was accompanied by progressive loss of protein secondary structure, as monitored by CD spectroscopy and a loss of the ability to associate with recombinant CUL3. Dissociation of Keap1 from CUL3 in vitro was dependent upon C151 in Keap1. A quantitative mass spectrometry-based kinetic analysis of adduction in HEK293 cells expressing FLAG-Keap1 revealed that Cys151 was one of the most reactive residues in vivo and that it was required for IAB-mediated dissociation of the Keap1-CUL3 interaction. These results demonstrate that Cys151 adduction confers a critical alkylation sensor function upon Keap1, making Keap1 unique among BTB CUL3 adapter proteins.
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17 MeSH Terms
The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.
Kwan KM, Fujimoto E, Grabher C, Mangum BD, Hardy ME, Campbell DS, Parant JM, Yost HJ, Kanki JP, Chien CB
(2007) Dev Dyn 236: 3088-99
MeSH Terms: Animals, Animals, Genetically Modified, Cloning, Molecular, DNA Transposable Elements, DNA, Recombinant, Gene Transfer Techniques, Genetic Techniques, Genetic Vectors, Green Fluorescent Proteins, Plasmids, Recombination, Genetic, Transposases, Zebrafish
Show Abstract · Added May 25, 2016
Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter]-[coding sequence]-[3' tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and internal ribosome entry sequence-driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large-scale projects testing the functions of libraries of regulatory or coding sequences.
Copyright 2007 Wiley-Liss, Inc.
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13 MeSH Terms
Generation of recombinant human monoclonal antibodies to rotavirus from single antigen-specific B cells selected with fluorescent virus-like particles.
Weitkamp JH, Kallewaard N, Kusuhara K, Feigelstock D, Feng N, Greenberg HB, Crowe JE
(2003) J Immunol Methods 275: 223-37
MeSH Terms: Adult, Antibodies, Monoclonal, Antibodies, Viral, Antigens, Viral, B-Lymphocytes, Base Sequence, Capsid Proteins, Cell Separation, DNA Primers, DNA, Recombinant, Gastroenteritis, Genes, Immunoglobulin, Genetic Vectors, Humans, Immunoglobulin Fab Fragments, Immunoglobulin Heavy Chains, Immunoglobulin Light Chains, Infant, Polymerase Chain Reaction, Recombinant Proteins, Rotavirus, Rotavirus Infections
Show Abstract · Added February 27, 2014
Technical difficulties have severely limited the yield of methods for the generation of human antiviral monoclonal antibodies (Mabs) in the past. We describe here a novel method for the efficient development of human Mabs against viruses. Rotavirus (RV) is a major cause of gastroenteritis in infants and adults worldwide. We generated fluorescent virus-like particles (VLPs) to identify and physically sort single RV-specific B cells from healthy adult blood donors, or RV-infected infants or adults. We expanded the sorted single B cells in culture, tested for RV-specific antibody secretion, and cloned and sequenced the antibody heavy and light chain variable region (VH and VL) genes. The percentage of wells that produced antibodies after sorting and expanding RV-specific adult B cell clones was high at 23%. The overall efficiency of RV-specific antibody gene recovery after the isolation, confirmation, and cloning of RV-specific VH segments was 1.3% of sorted cells in adults. RV-specific variable gene segments also were obtained from acutely infected infants, although infant B cells did not proliferate and differentiate in culture as well as adult B cells. We expressed recombinant Fabs incorporating the VH and VL genes from RV-specific B cell clones using a new modified bacterial Fab expression vector that we describe. Finally, we demonstrated binding of purified Fabs to RV proteins by immunofluorescence and ELISA. This method for the generation of recombinant human Mabs to RV from single antigen-specific B cell clones selected with fluorescent VLPs could be used to generate human Mabs to many other viruses whose proteins can self-assemble into VLPs.
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22 MeSH Terms
Echoviruses 1 and 8 are closely related genetically, and bind to similar determinants within the VLA-2 I domain.
Ohman T, King SL, Krithivas A, Cunningham J, Dickeson SK, Santoro SA, Bergelson JM
(2001) Virus Res 76: 1-8
MeSH Terms: Amino Acid Sequence, Animals, Binding Sites, Capsid, Cloning, Molecular, DNA, Complementary, DNA, Recombinant, Enterovirus B, Human, HeLa Cells, Humans, Mice, Models, Molecular, Molecular Sequence Data, Phylogeny, Protein Binding, Protein Structure, Tertiary, Receptors, Very Late Antigen, Receptors, Virus, Recombinant Fusion Proteins, Sequence Homology, Amino Acid
Show Abstract · Added March 5, 2014
Echoviruses (EV) 1 and 8 were originally considered to be distinct serotypes, but more recently have been considered strains of the same virus. In experiments with chimeric recombinant fusion proteins, both viruses bound to the I domain of the integrin VLA-2, and both required the same receptor residues for attachment. A full-length, infectious cDNA clone encoding EV1 was obtained; its nucleotide sequence was determined, as were the sequences encoding the EV8 capsid. EV1 and 8 show 94% amino acid identity within the capsid region and are more similar to each other than to any other human picornavirus.
1 Communities
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20 MeSH Terms
The evolutionarily conserved Dim1 protein defines a novel branch of the thioredoxin fold superfamily.
Zhang YZ, Gould KL, Dunbrack RL JR, Cheng H, Roder H, Golemis EA
(1999) Physiol Genomics 1: 109-18
MeSH Terms: Amino Acid Sequence, Cell Cycle, Cell Cycle Proteins, Conserved Sequence, DNA, Recombinant, Evolution, Molecular, Fungal Proteins, G2 Phase, Green Fluorescent Proteins, HeLa Cells, Humans, Luminescent Proteins, Magnetic Resonance Spectroscopy, Microscopy, Fluorescence, Molecular Sequence Data, Mutation, Plasmids, Protein Folding, Protein Structure, Secondary, Recombinant Fusion Proteins, Schizosaccharomyces, Schizosaccharomyces pombe Proteins, Sequence Alignment, Sequence Homology, Amino Acid, Thioredoxins, Transfection, Transformation, Genetic
Show Abstract · Added March 5, 2014
Dim1 is a small evolutionarily conserved protein essential for G2/M transition that has recently been implicated as a component of the mRNA splicing machinery. To date, the mechanism of Dim1 function remains poorly defined, in part because of the absence of informative sequence homologies between Dim1 and other functionally defined proteins or protein domains. We have used a combination of molecular modeling and NMR structural analysis to demonstrate that approximately 125 of the 142 amino acids of human Dim1 (hDim1) define a novel branch of the thioredoxin fold superfamily. Mutational analysis of Dim1 based on the predicted fold indicates that alterations in the region corresponding to the thioredoxin active site do not affect Dim1 activity. However, removal of a very short carboxy-terminal extension generates a dominant negative form of the protein [hDim1-(1-128)] that when overproduced induces cell cycle arrest in G2, via a mechanism likely to involve alteration of Dim1 association with partner molecules. In sum, this study identifies the Dim1 proteins as a novel sixth branch of the thioredoxin superfamily involved in cell cycle.
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27 MeSH Terms
In vitro evolution of preferred topoisomerase II DNA cleavage sites.
Burden DA, Osheroff N
(1999) J Biol Chem 274: 5227-35
MeSH Terms: Animals, Base Sequence, Cloning, Molecular, DNA Primers, DNA Topoisomerases, Type II, DNA, Recombinant, Drosophila melanogaster, Genome, Hydrolysis, Saccharomyces cerevisiae, Substrate Specificity
Show Abstract · Added March 5, 2014
Topoisomerase II is an essential enzyme that is the target for several clinically important anticancer drugs. Although this enzyme must create transient double-stranded breaks in the genetic material in order to carry out its indispensable DNA strand passage reaction, the factors that underlie its nucleotide cleavage specificity remain an enigma. Therefore, to address the critical issue of enzyme specificity, a modified systematic evolution of ligands by exponential enrichment (SELEX) protocol was employed to select/evolve DNA sequences that were preferentially cleaved by Drosophila melanogaster topoisomerase II. Levels of DNA scission rose substantially (from 3 to 20%) over 20 rounds of SELEX. In vitro selection/evolution converged on an alternating purine/pyrmidine sequence that was highly AT-rich (TATATATACATATATATA). The preference for this sequence was more pronounced for Drosophila topoisomerase II over other species and was increased in the presence of DNA cleavage-enhancing anticancer drugs. Enhanced cleavage appeared to be based on higher rates of DNA scission rather than increased binding affinity or decreased religation rates. The preferred sequence for topoisomerase II-mediated DNA cleavage is dramatically overrepresented ( approximately 10,000-fold) in the euchromatic genome of D. melanogaster, implying that it may be a site for the physiological action of this enzyme.
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11 MeSH Terms
The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes.
Liu Q, Li MZ, Leibham D, Cortez D, Elledge SJ
(1998) Curr Biol 8: 1300-9
MeSH Terms: Amino Acid Sequence, Artificial Gene Fusion, Base Sequence, Cloning, Molecular, DNA Restriction Enzymes, DNA, Recombinant, Escherichia coli, Evaluation Studies as Topic, Gene Expression, Gene Library, Gene Transfer Techniques, Genetic Techniques, Genetic Vectors, Molecular Sequence Data, Open Reading Frames, Plasmids, Polymerase Chain Reaction, Recombinant Fusion Proteins, Recombination, Genetic
Show Abstract · Added March 5, 2014
BACKGROUND - . Modern biological research is highly dependent upon recombinant DNA technology. Conventional cloning methods are time-consuming and lack uniformity. Thus, biological research is in great need of new techniques to rapidly, systematically and uniformly manipulate the large sets of genes currently available from genome projects.
RESULTS - . We describe a series of new cloning methods that facilitate the rapid and systematic construction of recombinant DNA molecules. The central cloning method is named the univector plasmid-fusion system (UPS). The UPS uses Cre-lox site-specific recombination to catalyze plasmid fusion between the univector - a plasmid containing the gene of interest - and host vectors containing regulatory information. Fusion events are genetically selected and place the gene under the control of new regulatory elements. A second UPS-related method allows for the precise transfer of coding sequences only from the univector into a host vector. The UPS eliminates the need for restriction enzymes, DNA ligases and many in vitro manipulations required for subcloning, and allows for the rapid construction of multiple constructs for expression in multiple organisms. We demonstrate that UPS can also be used to transfer whole libraries into new vectors. Additional adaptations are described, including directional PCR cloning and the generation of 3' end gene fusions using homologous recombination in Escherichia coli.
CONCLUSIONS - . Together, these recombination-based cloning methods constitute a new comprehensive approach for the rapid and efficient generation of recombinant DNA that can be used for parallel processing of large gene sets, a feature that will facilitate future genomic analysis.
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19 MeSH Terms
Construction of vectors and a genomic library for use with his3-deficient strains of Schizosaccharomyces pombe.
Ohi R, Feoktistova A, Gould KL
(1996) Gene 174: 315-8
MeSH Terms: Cloning, Molecular, DNA, Recombinant, Gene Library, Genetic Complementation Test, Genetic Markers, Genetic Variation, Genetic Vectors, Hydro-Lyases, Plasmids, Schizosaccharomyces
Show Abstract · Added March 5, 2014
The construction of vectors for use in Schizosaccharomyces pombe using the his3+ gene as a selectable marker is described. In addition, we report the construction of a genomic library in a his3(+)-containing shuttle vector to facilitate the cloning of genes by complementation of mutant function in strains defective for His3 activity.
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2 Members
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10 MeSH Terms
Use of the yeast one-hybrid system to screen for mutations in the ligand-binding domain of the estrogen receptor.
Bush SM, Folta S, Lannigan DA
(1996) Steroids 61: 102-9
MeSH Terms: Animals, Base Sequence, Cell Line, Cricetinae, DNA, Recombinant, Estradiol, Humans, Molecular Sequence Data, Mutagenesis, Mutation, Protein Binding, Receptors, Estrogen, Saccharomyces cerevisiae, Sequence Deletion, Transcriptional Activation
Show Abstract · Added January 20, 2015
This report describes a novel yeast one-hybrid system which easily allows for the detection of mutations in the ligand-binding domain of the estrogen receptor. This screen is based on the observation that a fusion protein consisting of the GAL4 DNA-binding domain and the estrogen receptor can interact with a GAL4 upstream activating sequence and induce the expression of an integrated GAL1-lacZ gene only in the presence of estradiol. Various deletion mutants of the estrogen receptor were tested in this assay and activating function 1 which is present in the N-terminus of the estrogen receptor was found to be responsible for the transactivation produced in the assay. To test if the screen could be used to detect random mutants in the ligand-binding domain of the estrogen receptor the region of the human receptor between amino acids 381 to 403 was mutated by oligonucleotide saturation mutagenesis. Two of the mutants generated by this mutagenesis were characterized to demonstrate that the results obtained from the screen in the yeast screen are relevant to mammalian systems. One of the mutants which has a valine at position number 388 instead of a glycine was able to transactivate in both the yeast and a mammalian system. This mutant was a more potent activator of transcription and also appeared to have a higher affinity for [3H]estradiol in vivo than the wild type receptor. The other mutant which was characterized has five amino acid changes from amino acids 390 through 400. This mutant was nonfunctional in the yeast and mammalian transcription assays and did not bind [3H]estradiol in vivo or in vitro.
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15 MeSH Terms