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Upstream activating sequence
About: Upstream activating sequence is a research topic. Over the lifetime, 1633 publications have been published within this topic receiving 100112 citations.
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TL;DR: In this article, an in vivo RNAi screening method was developed to identify flow-sensitive genes that regulate endothelial inflammation, including Bap60, psmd12, ERN1, and NF-κB.
Abstract: Disturbed blood flow (d-flow) induces atherosclerosis by altering the expression of mechanosensitive genes in the arterial endothelium. Previously, we identified >580 mechanosensitive genes in the mouse arterial endothelium, but their role in endothelial inflammation is incompletely understood. From this set, we obtained 84 Drosophila RNAi lines that silences the target gene under the control of upstream activation sequence (UAS) promoter. These lines were crossed with C564-GAL4 flies expressing GFP under the control of drosomycin promoter, an NF-κB target gene and a marker of pathogen-induced inflammation. Silencing of psmd12 or ERN1 decreased infection-induced drosomycin expression, while Bap60 silencing significantly increased the drosomycin expression. Interestingly, knockdown of Bap60 in adult flies using temperature-inducible Bap60 RNAi (C564ts-GAL4-Bap60-RNAi) enhanced drosomycin expression upon Gram-positive bacterial challenge but the basal drosomycin expression remained unchanged compared to the control. In the mammalian system, smarcd3 (mammalian ortholog of Bap60) expression was reduced in the human- and mouse aortic endothelial cells exposed to oscillatory shear in vitro as well as in the d-flow regions of mouse arterial endothelium in vivo. Moreover, siRNA-mediated knockdown of smarcd3 induced endothelial inflammation. In summary, we developed an in vivo Drosophila RNAi screening method to identify flow-sensitive genes that regulate endothelial inflammation.
7 citations
TL;DR: It is shown that Saccharomyces cerevisiae cells possess a 33-41-kilodalton protein with DNA-binding properties remarkably similar to those of the immunoglobulin enhancer-binding protein NF-muE3, suggesting that the yeast protein, designated YEB-3, and NF- muE3 are functionally related and perhaps evolutionarily conserved.
Abstract: We demonstrate that Saccharomyces cerevisiae cells possess a 33-41-kilodalton protein with DNA-binding properties remarkably similar to those of the immunoglobulin enhancer-binding protein NF-muE3. We further show that the muE3-binding site functions as an upstream activating sequence in yeast cells, stimulating transcription from a truncated CYC1 promoter. These data suggest that the yeast protein, designated YEB-3, and NF-muE3 are functionally related and perhaps evolutionarily conserved.
7 citations
TL;DR: Results indicate that activator binding to the upstream region of P2 late promoters compensates in part for poor sigma(70) contacts and helps to recruit RNA polymerase holoenzyme.
Abstract: We have carried out a mutational scan of the upstream region of the bacteriophage P2 FETUD late operon promoter, PF, which spans an element of hyphenated dyad symmetry that is conserved among all six of the P2 and P4 late promoters. All mutants were assayed for activation by P4 Delta in vivo, by using a lacZ reporter plasmid, and a subset of mutants was assayed in vitro for Delta binding. The results confirm the critical role of the three complementary nucleotides in each half site of the upstream element for transcription factor binding and for activation of transcription. A trinucleotide DNA recognition site is consistent with a model in which these transcription factors bind via a zinc finger motif. The mutational scan also led to identification of the −35 region of the promoter. Introduction of a σ70 −35 consensus sequence resulted in increased constitutive expression, which could be further stimulated by Delta. These results indicate that activator binding to the upstream region of P2 late promoters compensates in part for poor σ70 contacts and helps to recruit RNA polymerase holoenzyme.
7 citations
TL;DR: This protocol describes the generation of driver and reporter lines for use with the GAL4 system in Drosophila and an alternative strategy for integration using the phiC31 system is provided.
Abstract: INTRODUCTIONThe GAL4 system is a method for ectopic gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. This protocol describes the generation of driver and reporter lines for use with the GAL4 system in Drosophila. A promoter-GAL4 fusion is constructed using a P-element transformable vector, and a GAL4-responsive target gene is created via generation of an upstream activation sequence (UAS)-reporter construct. An alternative strategy for integration using the phiC31 system is also provided. Transformant lines are generated using standard procedures for microinjection.
7 citations
TL;DR: By deletion analysis, a 39-bp element in the proximal region of murine CD34 promoter that is critical for promoter activity is identified and this transcription factor binds specifically to the 39-BP region and stimulates promoter activity in both hematopoietic cells and in Sp1 null Drosophila S2 cells.
7 citations