Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Epstein-Barr virus nuclear antigen EBNA-2 is essential for Epstein-Barr virus-induced immortalization of B cells. EBNA-2 is a transcriptional activator capable of modifying the expression of specific viral and cellular genes. However, the mechanism of EBNA-2 transactivation has been an enigma. We used a fractionated extract of CA46 lymphoblastoid cells and bacterially expressed EBNA-2 polypeptides to demonstrate that EBNA-2 is targeted to the Epstein-Barr virus latency C promoter (Cp) through interaction with a cellular DNA binding protein designated Cp binding factor 1 (CBF1). A glutathione S-transferase-EBNA-2 fusion protein containing aa 252-425 of EBNA-2 interacted with CBF1 to yield a slowly migrating complex in an electrophoretic mobility shift assay. Mutation of EBNA-2 aa 323 and 324, which lie within a highly conserved amino acid motif, abolished the interaction with CBF1. This same mutation also abolished the ability of EBNA-2 to activate the Cp in a cotransfection assay. The binding site for CBF1 was localized to residues -359 to -388 of the Cp by using an electrophoretic mobility shift assay and DNase I footprinting. Introduction of multiple copies of the CBF1 binding site upstream of a minimal heterologous promoter conferred EBNA-2 responsiveness on that promoter. Mutation of a core sequence CNGTGGGAA abolished CBF1 binding, and the mutated sequence was unable to mediate EBNA-2 transactivation. The CBF1 core sequence also occurs in other EBNA-2-responsive promoters suggesting that CBF1 may mediate EBNA-2 transactivation of both cellular and viral targets.
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PMID:The Epstein-Barr virus immortalizing protein EBNA-2 is targeted to DNA by a cellular enhancer-binding protein. 841 84

We describe a rapid and sensitive method to isolate sets of high-affinity binding sites for sequence-specific DNA binding proteins. The DNA binding domain of the protein is expressed in Escherichia coli as a fusion with glutathione S-transferase (GST). Binding reactions are set up with total soluble extract from induced bacteria and a double-stranded oligonucleotide for which the central 32 bp have been randomized. To ensure stringent conditions, binding is done in the presence of high levels of poly(dI:C). The GST fusion protein is recovered by the addition of glutathione-Sepharose. Following extensive washing of the Sepharose beads, the bound oligonucleotides are rescued by polymerase chain reaction amplification. The amplified material is used in the next cycle of selection and amplification. Approximately five cycles are needed to obtain a pure population of high-affinity sites, which are then cloned and sequenced. This procedure should be applicable to any sequence-specific DNA binding protein for which the cDNA is available and which can be expressed in bacteria in a functional form.
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PMID:Selection of high-affinity binding sites for sequence-specific, DNA binding proteins from random sequence oligonucleotides. 853 2

Octopine synthase (ocs) elements are a group of promoter elements that have been exploited by plant pathogens to express genes in plants. ocs elements are components of the promoters of certain plant glutathione S-transferase genes and may function as oxidative stress response elements. Genes for ocs element binding factors (OBFs), which belong to a specific class of highly conserved, plant basic domain-leucine zipper transcription factors, have been isolated and include the Arabidopsis OBF4 and OBF5 genes. To characterize proteins that modulate the activity of the OBF proteins, we screened an Arabidopsis cDNA library with the labeled OBF4 protein and isolated OBP1 (for OBF binding protein). OBP1 contains a 51-amino acid domain that is highly conserved with two plant DNA binding proteins, which we refer to as the MOA domain. OBP1 is also a DNA binding protein and binds to the cauliflower mosaic virus 35S promoter at a site distinct from the ocs element in the 35S promoter. OBP1 specifically increased the binding of the OBF proteins to ocs element sequences, raising the possibility that interactions between these proteins are important for the activity of the 35S promoter.
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PMID:Interactions between distinct types of DNA binding proteins enhance binding to ocs element promoter sequences. 871 29

Ethylene transcriptionally activates a glutathione S-transferase gene (GST1) at the onset of the senescence program in carnation (Dianthus caryophyllus L.) flower petals. A 126 bp region of the GST1 promoter sequence has been identified as an ethylene-responsive enhancer element (ERE). In this paper, we demonstrate the ability of nuclear proteins from senescing petals to recognize a 22 bp sequence within the ERE (ERE oligonucleotide). Mutation of the ERE oligonucleotide sequence significantly alters the strength of this nuclear protein-DNA association. The wild-type ERE oligonucleotide sequence was used to isolate a cDNA clone encoding a sequence-specific DNA binding protein. Nucleotide sequencing and deduced amino acid sequence analysis of this cDNA predicted a 32 kDa protein which we have designated carnation ethylene-responsive element-binding protein-1 (CEBP-1). The mRNA expression pattern of CEBP-1 suggests that it is not transcriptionally regulated by ethylene. The amino acid sequence homology of CEBP-1 with other plant nucleic acid binding proteins indicates a conserved nucleic acid binding domain. Within this domain are two highly conserved RNA-binding motifs, RNP-1 and RNP-2. An acidic region and a putative nuclear localization signal are also identified.
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PMID:Cloning of a DNA-binding protein that interacts with the ethylene-responsive enhancer element of the carnation GST1 gene. 880 6

All the known members of the mouse Notch receptor family were examined for their biochemical function by interaction with a DNA binding protein RBP-Jkappa. mNotch2, mNotch3 and int3 (= mNotch4) were shown to interact with RBP-Jkappa by the GST-fusion pull down assay and dominant negative competition with Epstein Barr virus nuclear antigen 2. Furthermore the intracellular region of int3 was shown to transactivate the Epstein Barr virus TP1 promoter. These results indicate that all mouse Notch family members have biochemical functions similar to mNotch1, which transduces proliferative signal by direct interaction with the DNA binding protein RBP-Jkappa.
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PMID:Functional conservation of mouse Notch receptor family members. 889

EBNA2 is essential for Epstein-Barr virus (EBV) immortalization of B lymphocytes. EBNA2 functions as a transcriptional activator and targets responsive promoters through interaction with the cellular DNA binding protein CBF1. We have examined the mechanism whereby EBNA2 overcomes CBF1-mediated transcriptional repression. A yeast two-hybrid screen performed using CBF1 as the bait identified a protein, SKIP, which had not previously been recognized as a CBF1-associated protein. Protein-protein interaction assays demonstrated contacts between SKIP and the SMRT, CIR, Sin3A, and HDAC2 proteins of the CBF1 corepressor complex. Interestingly, EBNA2 also interacted with SKIP in glutathione S-transferase affinity and mammalian two-hybrid assays and colocalized with SKIP in immunofluorescence assays. Interaction with SKIP was not affected by mutation of EBNA2 conserved region 6, the CBF1 interaction region, but was abolished by mutation of conserved region 5. Mutation of conserved region 5 also severely impaired EBNA2 activation of a reporter containing CBF1 binding sites. Thus, interaction with both CBF1 and SKIP is necessary for efficient promoter activation by EBNA2. A model is presented in which EBNA2 competes with the SMRT-corepressor complex for contacts on SKIP and CBF1.
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PMID:A role for SKIP in EBNA2 activation of CBF1-repressed promoters. 1064 67

At present, only a little is known about the transcriptional regulation in chondrocytes submitted to various physicomechanical factors known to exist in articular cartilage. Recently, we have investigated the effects of hydrostatic pressure on transcriptional control in chondrocytes using human chondrosarcoma and immortalized chondrocyte cell lines for the experiments. Hydrostatic pressure was applied on the cells in a special computer-controlled, water-filled pressure chamber, where cyclic and static pressures up to 32 MPa can be created. Differential display RT-PCR and probing of cDNA arrays are the methods we have used to study differential gene expression due to hydrostatic pressure. By differential display RT-PCR experiments, we have observed several differentially expressed cDNA bands under continuous 30 MPa hydrostatic pressure, while 30 MPa cyclic pressure at 1 Hz produced much fewer changes. In the first phase of our studies, we have focused on the effects of 30 MPa hydrostatic pressure because it causes a unique hsp70-mediated stress response in immortalized chondrocytes. Differential display RT-PCR screening provided us with several clones that derive from low-abundance mRNAs, such as death-associated protein 3 (DAP3), a nucleotide-binding protein which increases due to interferon-gamma induced cell death; PTZ-17 (or p311), a seizure-related protein; H-NUC, a nuclear DNA binding protein; and one new gene of unknown function. In Northern blots, an induction was confirmed for the new gene, DAP3 and PTZ-17 were down-regulated in some but not in all parallel experiments; however, basal level of H-NUC mRNA was too low to be detected in Northern blots. We then chose to widen our screening to a number of known genes arrayed as cDNA blots. Under 30 MPa continuous hydrostatic pressure, four different time points were chosen (0, 3, 6 and 24 h) for the experiments. The screening of 588 cDNAs showed 15 up-regulated and 6 down-regulated genes. Consistently with our previous results hsp70 was highly induced, as well as hsp40, a chaperone protein functioning together with hsp70. Gadd45 and to a lesser extent Gadd153 (stress genes induced by, e.g., ionizing radiation and ischaemia) were up-regulated, as well as p21waf1,cip1, a protein participating in cell cycle regulation that can interact with Gadd45. Northern blots confirmed Gadd45 induction. Down-regulated transcripts included, e.g., DAD-1, glutathione S-transferase pI, DNA-binding inhibitor ID-1H, and cytoplasmic dynein light chain.
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PMID:Transcriptional activation in chondrocytes submitted to hydrostatic pressure. 1091 81

DNA sequence coding for a portion of DNA binding protein (amino acids 3-58) of bovine adenovirus type-3 (BAV-3) was cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase. The fusion protein was affinity purified and used to immunize rabbits. Immunoprecipitation and Western blot analysis showed that the antiserum could specifically recognize a protein of 48 kDa in BAV-3-infected cells, which was produced both in early and late phases of BAV-3 life cycle. Based on the ability of antiserum to recognize DNA binding protein, a novel assay for BAV-3 quantitation was established. The assay is less time consuming and can be performed on a wide variety of bovine cells. In addition, virus titers determined by this assay are comparable to the standard plaque assay.
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PMID:Determination of bovine adenovirus-3 titer based on immunohistochemical detection of DNA binding protein in infected cells. 1133 49

To identify and characterize the protein encoded by the E2A region of porcine adenovirus (PAV)-3, DNA sequence coding for a portion (amino acids 102-457) of the DNA binding protein (DBP) open reaching frame was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase protein of Schistosoma japonica. The affinity-purified fusion protein was used to immunize rabbits. Immunoprecipitation/Western blot analysis demonstrated that the antisera specifically recognized a protein of 50 kD in PAV-3-infected cells. Immunoperoxidase staining detected the DBP protein predominantly in the nucleus of the cells. Western blot analysis demonstrated that DBP was detected as early as 6 h after infection and remained detectable throughout the infection. Based on these results, a novel assay for quantitation of PAV-3 was established. The assay is less time consuming and can be performed in different porcine cells. In addition, virus titers determined by this assay are comparable to the standard plaque assay.
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PMID:Characterization of DNA binding protein of porcine adenovirus type 3. 1180 41

Previous studies have indicated that the U(L)6, U(L)15, U(L)17, U(L)28, U(L)32, and U(L)33 genes are required for the cleavage and packaging of herpes simplex viral DNA. To identify proteins that interact with the U(L)28-encoded DNA binding protein of herpes simplex virus type 1 (HSV-1), a previously undescribed rabbit polyclonal antibody directed against the U(L)28 protein fused to glutathione S-transferase was used to immunopurify U(L)28 and the proteins with which it associated. It was found that the antibody specifically coimmunoprecipitated proteins encoded by the genes U(L)28, U(L)15, and U(L)33 from lysates of both HEp-2 cells infected with HSV-1(F) and insect cells infected with recombinant baculoviruses expressing these three proteins. In reciprocal reactions, antibodies directed against the U(L)15- or U(L)33-encoded proteins also coimmunoprecipitated the U(L)28 protein. The coimmunoprecipitation of the three proteins from HSV-infected cells confirms earlier reports of an association between the U(L)28 and U(L)15 proteins and represents the first evidence of the involvement of the U(L)33 protein in this complex.
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PMID:DNA cleavage and packaging proteins encoded by genes U(L)28, U(L)15, and U(L)33 of herpes simplex virus type 1 form a complex in infected cells. 1196 95


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