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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)

A DNA-binding site selection and enrichment procedure revealed a sequence-specific DNA-binding activity selectively associated with glutathione S-transferase-retinoblastoma protein chimeras (GST-RB) that had been incubated with a human cell extract. Appropriate mutant forms of GST-RB, incubated in equivalent extracts, did not associate with this specific DNA-binding activity, and a peptide replica of the HPV E7 RB-binding segment selectively inhibited the association of GST-RB with the sequence-specific DNA-binding protein(s). Sequence analysis of oligonucleotides with high affinity for GST-RB complexes, as well as the results of competition binding studies, strongly suggest that RB can associate specifically with the transcription factor E2F or with a protein having closely related DNA-binding properties.
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PMID:The T/E1A-binding domain of the retinoblastoma product can interact selectively with a sequence-specific DNA-binding protein. 182 94

We have previously described a colorimetric test, designated an amplified DNA assay (ADA), for specific segments of DNA amplified by polymerase chain reactions (PCRs), suited to diagnostic applications. This relied on binding the amplified DNA via a sequence in one oligodeoxyribonucleotide (oligo) to the DNA-binding protein GCN4 coated on the wells of a microtiter dish. Avidin-peroxidase was then bound to biotin at the 5' end of the other oligo and detected colorimetrically. Two successive PCRs with nested oligos were utilized. We describe here several modifications that greatly simplify the ADA. First, we bind the DNA to a glutathione S-transferase-GCN4 fused polypeptide (GST-GCN4) and avidin-peroxidase simultaneously, rather than successively. Second, we carry out the two successive PCRs in the one reaction mixture, using the thermal stabilities of oligos of differing lengths to separate the two reactions. Third, PCRs can be performed in the wells of a microtiter dish and the amplified DNA captured and detected via GST-GCN4 immobilized on beads attached to the lid of the microtiter dish. Hence it is only necessary to pipette the DNA sample once, and up to 96 samples can then be handled simultaneously.
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PMID:Simplified colorimetric analysis of polymerase chain reactions: detection of HIV sequences in AIDS patients. 225 54

We have previously shown that a 30 kDa DNA-binding protein isolated from rat cell nuclei exhibits the chemical and immunological properties of glutathione S-transferase Yb subunits [Bennett, Spector & Yeoman (1986) J. Cell Biol. 102, 600-609]. It was of interest, therefore, to determine whether Yb subunits isolated from rat liver nuclei would return to nuclear fractions upon reintroduction to cell cytoplasms via red-blood-cell-mediated fusion. Labelled Yb subunits were associated with nuclear fractions 60 min after cell fusion. The microinjected protein remained associated with the nuclei for 18 h and was not extractable with low-salt washes. In addition, injected Yb subunits were found to equally distribute between extractable (56%) and residual (44%) nuclear fractions. These experiments demonstrate that glutathione S-transferase Yb subunits isolated from nuclei rapidly translocate to nuclei upon reintroduction into cell cytoplasms.
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PMID:Microinjected glutathione S-transferase Yb subunits translocate to the cell nucleus. 368 37

FlbD is a transcriptional regulatory protein that negatively autoregulates fliF, and it is required for expression of other Caulobacter crescentus flagellar genes, including flaN and flbG. In this report we have investigated the interaction between carboxy-terminal fragments of FlbD protein and enhancer-like ftr sequences in the promoter regions of fliF, flaN, and flbG. FlbDc87 is a glutathione S-transferase (GST)-FlbD fusion protein that carries the carboxy-terminal 87 amino acids of FlbD, and FlbDc87 binds to restriction fragments containing the promoter regions of fliF, flaN, and flbG, whereas a GST-FlbD fusion protein carrying the last 48 amino acids of FlbD failed to bind to these promoter regions. DNA footprint analysis demonstrated that FlbDc87 is a sequence-specific DNA-binding protein that makes close contact with 11 nucleotides in ftr4, and 6 of these nucleotides were shown previously to function in negative regulation of fliF transcription in vivo (S. M. Van Way, A. Newton, A. H. Mullin, and D. A. Mullin, J. Bacteriol. 175:367-376, 1993). Three DNA fragments, each carrying an ftr4 mutation that resulted in elevated fliF transcript levels in vivo, were defective in binding to FlbDc87 in vitro. We also found that a missense mutation in the recognition helix of the putative helix-turn-helix DNA-binding motif of FlbDc87 resulted in defective binding to ftr4 in vitro. These data suggest that the binding of FlbDc87 to ftr4 is relevant to negative transcriptional regulation of fliF and that FlbD functions directly as a repressor. Footprint analysis showed that FlbDc87 also makes close contacts with specific nucleotides in ftr1, ftr2, and ftr3 in the flaN-flbG promoter region, and some of these nucleotides were shown previously to be required for regulated transcription of flaN and flbG (D. A. Mullin and A. Newton, J. Bacteriol. 175:2067-2076, 1993). Footprint analysis also revealed a new ftr-like sequence, ftr5, at -136 from the transcription start site of flbG. Our results demonstrate that FlbD contains a sequence-specific DNA-binding activity within the 87 amino acids at its carboxy terminus, and the results suggest that FlbD exerts its effect as a positive and negative regulator of C. crescentus flagellar genes by binding to ftr sequences.
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PMID:FlbD has a DNA-binding activity near its carboxy terminus that recognizes ftr sequences involved in positive and negative regulation of flagellar gene transcription in Caulobacter crescentus. 792 58

Recently, we demonstrated that a DNA-binding protein(s) is involved in transcriptional repression of the rat serine dehydratase gene in fetal liver. Here, we report that a GAT(A/T) motif is the target sequence for the DNA-binding protein. By screening a fetal liver cDNA library, we isolated a rat homolog of GATA-1. Rat GATA-1 expressed as a GST-fusion protein in E. coli bound to the GAT(A/T) motif in the serine dehydratase gene. Northern analysis show that GATA-1 and GATA-4 mRNAs are expressed in fetal hepatocytes.
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PMID:Expression of GATA-binding transcription factors in rat hepatocytes. 795 72

A partial-length A-myb complementary DNA recently cloned by low-stringency hybridization with a c-myb probe to complementary DNA libraries derived from human cell lines showed a high degree of homology with the DNA-binding domain of c-myb and B-myb, suggesting that A-myb also encoded a DNA-binding protein. We report here the sequence of the entire coding region of A-myb complementary DNA and show that the full-length GST-A-myb fusion protein or a truncated derivative corresponding only to the putative DNA-binding domain interacts specifically with Myb-binding sites of the c-myb responsive promoters, MIM-1 and CD34. In transient transfection assays, A-myb transactivated the bound promoters. These results suggest that, analogous to the other members of the Myb family, the A-myb gene encodes a bona fide transactivator. The distinct function of A-myb might derive from its pattern of expression and/or its relative potency as a transactivator of myb target genes.
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PMID:DNA binding and transactivation activity of A-myb, a c-myb-related gene. 798 50

The pGEX system for protein production in E. coli is widely used in molecular biology. A bacterial expression vector, pETGEXCT, which incorporates features of the pGEX and pET expression systems was designed. pETGEXCT allows the production of N- and C-terminal fusions to glutathione S-transferase (GST) under the tight control of the T7 promoter. Use of this vector can circumvent problems associated with unstable or inactive fusions to the N terminus of GST. Indeed, it is demonstrated that fusions to the N terminus of the eukaryotic DNA-binding protein, RSRFC4, cannot be tolerated. Fusion of RSRFC4 to the N terminus of GST in the pETGEXCT vector is a prerequisite to purify the RSRFC4 DNA-binding domain in an active form using glutathione-agarose affinity chromatography.
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PMID:A T7 expression vector for producing N- and C-terminal fusion proteins with glutathione S-transferase. 812 85

The Epstein-Barr virus gene BRLF1 encodes the transcription factor R, which is a sequence-specific DNA-binding protein important for the switch from latency to a productive cycle. We have defined a repertoire of specific R-binding sites using a GST-R fusion protein and a pool of 23 bp random DNA sequences. The R-bound sequences were selected by several rounds of Electrophoretic Mobility Shift Assay (EMSA) and amplification by PCR. Among the 45 sites selected, some positions in the sequences were highly conserved, i.e., 5'-GTGCC N7GTGGTG-3'. The guanine methylation assay revealed that R simultaneously contacts guanines in the two conserved cores, defining the consensus binding site 5'-GNCC N9 GGNG-3', and 30 sites among the 45 selected have this sequence. This last result also suggests that R binds two adjacent major grooves of the DNA. As shown by EMSA assay, R binds to all the sites tested with a comparable affinity, and they all mediate R-induced transcriptional activation in a transient expression assay.
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PMID:Characterization of the DNA-binding site repertoire for the Epstein-Barr virus transcription factor R. 816 30

Increases in transforming growth factor beta1 (TGF-beta1) mRNA and biological activity in the early phase of human cytomegalovirus (CMV) infection in fibroblasts are paralleled by increased TGF-beta1-chloramphenicol acetyltransferase (CAT) reporter gene activity. To determine how CMV infection transactivates the TGF-beta1 promoter, we examined the effects of the cotransfected IE2 regulatory protein of human CMV on 5'-deleted TGF-beta1 promoter-CAT reporter genes in transient DNA transfection assays. Two upstream TGF-beta1 promoter regions each containing an Egr-1 consensus site were shown to be important for IE2-induced transactivation in a cell type that displayed greatly reduced nonspecific activity. Furthermore, transfer of an Egr-l site from between positions -125 and -98, but not point mutant versions of this site, to a heterologous promoter also conveyed IE2 responsiveness. Addition of an IE2 expression vector or use of the U373 A45 astrocytoma cell line expressing IE2 also produced synergistic stimulation of GAL4-Egr-l-mediated activation of a target promoter containing GAL4 binding sites. The 80-kDa IE2 protein present in A45 cells proved to selectively bind to glutathione S-transferase (GST)-Egr-1 beads. The results of in vitro protein binding assays also revealed that an intact in vitro-translated IE2 protein bound directly to the GST-Egr-1 fusion protein through the zinc finger domain of the Egr-1 protein and that this binding activity was abolished by deletion of parts of the zinc finger DNA-binding domain. Similarly, the Egr-1 protein was found to associate preferentially with a small region within the C-terminal half of the IE2 protein adjacent to the DNA-binding and dimerization domains that are important for both transactivation and downregulation. We conclude from these observations that IE2 may regulate transcription of the TGF-beta1 gene as well as other potential cellular targets by virtue of its ability to interact with the Egr-1 DNA-binding protein.
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PMID:The IE2 regulatory protein of human cytomegalovirus induces expression of the human transforming growth factor beta1 gene through an Egr-1 binding site. 879 51

In Pseudomonas aeruginosa, the activator protein LasR and a cognate autoinducer (AI) are required for expression of the elastase gene (lasB). In the present study, we investigated the binding properties of the P. aeruginosa lasR gene product. The LasR protein was overexpressed and purified as a glutathione S-transferase (GST) fusion protein. Using gel retardation and UV cross-linking analysis, we demonstrated that the GST-LasR could bind to a separate site in the lasB upstream operator regions 1 and 3 in the presence of the autoinducer. Regions 1 and 3 are located at 105 and 42 base pairs upstream, respectively, from the lasB transcriptional start site. Our present results clearly demonstrate that LasR is a specific DNA-binding protein that regulates the transcription of the lasB gene in the presence of an autoinducer.
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PMID:Purification and characterization of LasR as a DNA-binding protein. 881 May 14


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