Enhanced operator binding by trp superrepressors of Escherichia coli.
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The trp repressor of Escherichia coli binds to the operators of three operons concerned with tryptophan biosynthesis and regulates their expression. trp superrepressors can repress expression of the trp operon in vivo at lower tryptophan concentrations than those required by the wild-type repressor. The five known superrepressors have been purified and characterized using a modified filter binding assay. In four of the five superrepressors, EK13, EK18, DN46 and EK49, negatively charged wild-type residues located on the surface of the repressor that faces the operator are replaced by positively charged or neutral residues. Each of these proteins has higher affinity for the trp operator than wild-type repressor. Decreased rates of dissociation of the repressor-operator complex were found to be responsible for the higher affinities. The fifth superrepressor, AV77, has an amino acid substitution in the turn of the helix-turn-helix DNA-binding motif. This superrepressor was indistinguishable from wild-type repressor in our filter binding assay. We conclude that rapid dissociation of repressor from operator is important for trp repressor function in vivo. The negatively charged wild-type residues that are replaced in superrepressors are probably responsible for the characteristic rapid dissociation of the trp repressor from the trp operator. (+info)
Controlled reagent transport in disposable 2D paper networks.
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Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein.
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We have expressed the human immunodeficiency virus type 1 (HIV-1) gag polyprotein (Pr55gag) in bacteria under the control of the T7 phage gene 10 promoter. When the gene encoding the viral protease is included in cis, in the -1 reading frame, the expected proteolytic cleavage products MA and CA are produced. Disruption of the protease-coding sequence prevents proteolytic processing, and full-length polyprotein is produced. Pr55gag, separated from bacterial proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immobilized on nitrocellulose membranes, binds RNA containing sequences from the 5' end of the HIV-1 genome. This binding is tolerant of a wide range of pH and temperature but has distinct salt preferences. Conditions were identified which prevented nonspecific binding of RNA to bacterial proteins but still allowed binding to Pr55gag. Under these conditions, irrelevant RNA probes lacking HIV-1 sequences bound Pr55gag less efficiently. Quantitation of binding to Pr55gag by HIV-1 RNA probes with deletions mutations demonstrated that there are two regions lying within the HIV-1 gag gene which independently promote binding of RNA to Pr55gag. (+info)
Development of a membrane-bound random DNA sequence combinatorial array recognition surface (CARS).
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A partially overlapping population of random sequence 60mer DNA molecules consisting of many concatamers of varied lengths was spatially separated in one and two dimensions by electrophoresis in polyacrylamide and transferred to nitrocellulose membranes. The spatially separated library serves as a potential sensor interface on which many different molecular recognition events or target analyte-binding patterns may emerge, thereby theoretically representing a "universal sensor" surface. The separated DNA library has been referred to as a DNA combinatorial array recognition surface or "CARS." After UV baking and various fluorescence staining or fluorescent probe interactions, the one-dimensional (1-D) and 2-D membrane-bound CARS were digitally photographed and subjected to image analysis with National Institutes of Health Image-Java software. Image analysis demonstrated relatively consistent and more similar spatial fluorescence patterns within CARS analyte treatment groups but noteworthy pattern differences before and after analyte addition and between different analyte treatments. Taken together, these data suggest a potential role for CARS as a novel, inexpensive, self-assembling universal molecular recognition surface that could be coupled to sophisticated Bayesian or other pattern recognition algorithms to classify analytes or make specific identifications, much like the senses of smell or taste. (+info)
A method for MS(E) differential proteomic analysis of archival formalin-fixed celloidin-embedded human inner ear tissue.
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Identification of multiple ral gene products in human platelets that account for some but not all of the platelet Gn-proteins.
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Polyclonal antibodies raised against specific recombinant low molecular mass GTP-binding proteins were tested for their ability to recognize partially purified human platelet membrane Gn-proteins (i.e. proteins that bind [alpha-32P]GTP on nitrocellulose blots of SDS/polyacrylamide gels). An antiserum against simian ralA protein recognized a 27 kDa human platelet protein with the same apparent molecular mass as the major platelet Gn-protein (Gn27). In further analysis by two-dimensional polyacrylamide gel electrophoresis, the isoelectric focusing step permitted resolution of 12 major Gn-protein forms, seven of 27 kDa (Gn27a-g), one of 26 kDa (Gn26) and four of 24 kDa (Gn24a-d). The ralA antibody reacted strongly with the five most basic Gn27 species (a-e), weakly with Gn26 and not at all with Gn27f, Gn27g or Gn24a-d. We conclude that ral gene products account for some but probably not for all of the platelet Gn-proteins. (+info)
Western blotting: sample preparation to detection.
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