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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe an assay employing the competitive binding of estrogen receptor (ER) with basal transcription factors on a constitutive promoter (cytomegalovirus-hormone response element[s]-chloramphenicol acetyltransferase [CMV-(HRE)n-CAT, containing a hormone response element(s) between the TATA box and the start site of transcription]) to examine the DNA-binding ability of the human ER in whole cells. We used this promoter interference assay to examine the DNA binding of ER in cell lines containing high and low levels of endogenous ER, as well as in CHO cells expressing wild-type and mutant ERs from cotransfected expression vectors. The ER is capable of binding to the promoter interference constructs in the absence of added ligand, and estrogen (estradiol) or antiestrogen (trans-hydroxytamoxifen or ICI 164,384) enhances or stabilizes this interaction. The binding of unoccupied ER to reporter gene activation plasmids results in ligand-independent transactivation, presumably due to the TAF-1 function of the receptor. DNA binding of ER in the absence of ligand is observed in cells containing endogenous ER, or expressed ER, and occurs in cells with high or low receptor contents. Although estrogen- and antiestrogen-occupied ER complexes bind to DNA and reduce the template promoter activity, the extent of suppression achieved by ICI-bound ERs is consistently less than that achieved with the other ligands, presumably caused by the fact that ICI rapidly reduces the level of ER in most of the cells examined. However, the ICI-ER complexes that remain are in sufficient quantity to bind to gene activation reporter constructs, and in these cells, ICI still behaves as a pure antagonist of gene transcription and does not activate reporter genes. Hence, obstruction of ER DNA binding or reduction of ER in target cells may contribute to, but cannot fully explain, the pure antagonist character of the antiestrogen ICI 164,384. In addition, DNA binding by the ER alone is clearly not sufficient for ensuring full activation of transcription and argues for an intermediate in the receptor activation of promoters.
Mol Cell Biol 1992 Oct
PMID:Examination of the DNA-binding ability of estrogen receptor in whole cells: implications for hormone-independent transactivation and the actions of antiestrogens. 140 42

DNA encoding the N-terminal 415 residues of the human thyrotrophin receptor (predicted to code for the large extracellular region) was introduced into Chinese hamster ovary (CHO) cells using the glutamine synthetase/cytomegalovirus amplifiable expression system, and into E. coli using the pGEX-3X expression vector. Substantial quantities of insoluble fusion protein product resulted from bacterial expression; by Western blot analysis, this was shown to be reactive with anti-receptor antibodies raised against a peptide corresponding to residues 313-330. Immunoreactivity was not retained by the solubilized protein. In eukaryotic expression, several successful CHO transfectants were observed and one (ExG2) was characterized thoroughly. Using agarose-bound Concanavalin A, a glycoprotein with an M(r) of approximately 60,000 was detected in a detergent extract of metabolically labelled ExG2 cells, agreeing with the predicted molecular size of 45,000, plus carbohydrate. The same protein could also be detected by immunoprecipitation using the experimental anti-peptide antisera and also sera from patients with Graves' disease. The protein was immunoreactive in Western blot analyses of ExG2 cells using the experimental antisera but not the pathological sera, supporting the view that linear sequences are not sufficient for autoantibody binding. These are the first studies in which visualization of eukaryotically expressed recombinant receptor by such immunological techniques has been possible, presumably because of the higher expression of the glutamine synthetase system. Surprisingly, the recombinant protein was retained within the cells rather than being secreted. The recombinant protein was very effective at absorbing the adenylate cyclase-stimulating activity of the sera from patients with Graves' disease, but not that of thyrotrophin. This suggests that the large N-terminal extracellular region contains epitopes for stimulatory autoantibodies, but that high affinity thyrotrophin binding requires additional components.
J Mol Endocrinol 1992 Dec
PMID:Characterization of the extracellular region of the human thyrotrophin receptor expressed as a recombinant protein. 147 10

Using homologous probes for the cloning of related genes within the family of guanine nucleotide-binding protein-coupled receptors, we have cloned the gene for the rhesus macaque D1 dopamine receptor. By using the rat D1 receptor coding sequence as a probe under high stringency conditions, the rhesus D1 receptor gene was isolated from a lambda EMBL3 rhesus genomic DNA library. The rhesus D1 dopamine receptor gene is intronless and encodes a 446-amino acid protein that contains two consensus sites for asparagine-linked glycosylation (Asn-5 and Asn-176) and two consensus sites for cAMP-dependent protein kinase phosphorylation (Thr-136 and Thr-268). The primary amino acid sequence of the rhesus D1 dopamine receptor shows an extremely high degree of similarity (99.6%) to the human D1 receptor. Genomic DNA analyses conducted with high and reduced stringency hybridizations indicate that the rhesus macaque D1 receptor is a member of a large multigene family. Like the human D1 receptor mRNA, the rhesus D1 receptor mRNA is approximately 4 kilobases in size and is localized predominantly in the caudate, with lesser amounts in the hippocampus and cortex. The rhesus D1 receptor coding region was inserted into the cytomegalovirus promoter-driven expression vector pcDNA-1, and the recombinant (pcDNA-D1) was cotransfected with the selectable marker pRSVneo, conferring G418 resistance, into D1 receptor-deficient C6 glioma cells. Analyses of the selected transfectant demonstrate the expression of a high affinity, functional D1 dopamine receptor. The D1 receptor radioligand [3H]SCH 23390 bound transfectant membranes with an affinity (Kd), of 0.3 nM; the D2-selective ligand spiperone, the dopamine receptor ligand clozapine, and the serotonin receptor antagonist ketanserin bound with considerably lower affinities (102, 80, and 95 nM, respectively). Both dopamine and the D1-selective agonist SKF 38393 inhibited the binding of [3H]SCH 23390 to transfectant cell membranes; the binding of these agonists was sensitive to GTP. Dopamine potently stimulated the accumulation of cAMP in transfected C6 cells, whereas SKF 38393 was a partial agonist in these cells. Also, the density of recombinant D1 receptors on the transfectant cells was decreased 40% upon treatment with 10 microM dopamine, indicating that occupation of recombinant D1 receptors by agonists alters surface expression of the receptors.
Mol Pharmacol 1992 Apr
PMID:Molecular cloning and expression of the rhesus macaque D1 dopamine receptor gene. 153 68

Prointerleukin 1 beta (IL-1 beta) is a cytokine that mediates a broad range of biological activities. Genomic sequences that regulate IL-1 beta transcription include both inducible regulatory elements located more than 2,700 bp upstream of the transcriptional start site (cap site) and proximal elements located near the TATA box of this gene. In this study, we focused on the identification and characterization of trans-acting nuclear regulatory proteins that bind to the cap site-proximal region of the human IL-1 beta gene. We identified a protein, termed NFIL-1 beta A (NF beta A), that binds to a highly conserved 12-bp DNA sequence (-49 to -38) located upstream of the TATA box motif in both the human and murine IL-1 beta genes. The IL-1 alpha gene, which lacks a TATA motif, does not possess an NF beta A-binding sequence within the promoter region, suggesting that NF beta A may selectively regulate IL-1 beta expression. Using electrophoretic mobility shift assays, we identified several distinct DNA-protein complexes that are expressed in a cell-type-specific manner. In monocytic cell lines, the relative abundance of these complexes varies rapidly following stimulation of the cells with phorbol esters or lipopolysaccharide. UV cross-linking analysis identified two distinct DNA-binding polypeptides that comprise distinct complexes. The functional role of NF beta A was assessed in transient transfection assays. These data indicate that NF beta A is required for both basal and inducible promoter activity in monocytic cells. Furthermore, the human cytomegalovirus immediate-early 1 gene product requires the presence of NF beta A in order to trans-activate the proximal IL-1 beta promoter in a monocytic cell line. We propose that NF beta A is a factor that mediates either direct or indirect activation by the immediate-early 1 gene product. The proximity of this essential factor to the TATA motif suggests a possible role in transcriptional initiation.
Mol Cell Biol 1992 Aug
PMID:The functional importance of a cap site-proximal region of the human prointerleukin 1 beta gene is defined by viral protein trans-activation. 163 Apr 55

Human cytomegalovirus (HCMV) immediate early (IE) genes act as trans-acting factors to upregulate various viral promoters. We used various IE plasmid constructs in transient transfection assays and demonstrated that the HCMV IE2 gene product upregulated expression from the interleukin (IL)-2 and IL-2 receptor (IL-2R) promoters and increased amounts of endogenous, steady-state IL-2 and IL-2R RNA. In marked contrast, the IE1 gene product, which can upregulate the major IE promoter and the IL-1 beta promoter, had no effect on the IL-2 and IL-2R promoters. These studies suggest a role for the HCMV IE2 gene product as a modulator of the inflammatory response associated with HCMV infection.
Am J Respir Cell Mol Biol 1991 Sep
PMID:The immediate early genes of human cytomegalovirus upregulate expression of the interleukin-2 and interleukin-2 receptor genes. 165 52

Cyclic AMP regulates a variety of cellular responses through activation of the catalytic subunit of cAMP-dependent protein kinase. The cDNAs for two protein isoforms of the catalytic subunit, C alpha and C beta, were placed into expression vectors, and their ability to stimulate cAMP-dependent transcription of the human enkephalin promoter was examined in transiently transfected CV-1 cells. Expression vectors for C alpha and C beta that were directed by the human cytomegalovirus promoter produced up to 350- and 200-fold increases in chloramphenicol acetyltransferase activity, respectively, when cotransfected with the ENKAT-12 reporter plasmid. Transcriptional activation was shown to be dependent upon functional kinase activity by point mutations in catalytic subunit vectors which eliminated activation. Transcriptional activation by C alpha and C beta was eliminated when the cAMP response elements (CREs) were deleted from the native enkephalin promoter, but activation was recovered when this region was replaced with an oligonucleotide containing two copies of the somatostatin CRE consensus TGACGTCA. C alpha expression vectors were found to produce 2-fold greater transcriptional activation than C beta expression vectors. These results were most likely due to the cellular kinase activity produced by the catalytic subunit expression vectors and did not appear to be dependent on CRE motif or substrate specificity. In vitro mutagenesis indicates that neither C alpha nor C beta requires N-terminal myristylation for transcriptional activation, but threonine-197 is critical to subunit function.
Mol Endocrinol 1991 Jul
PMID:Regulation of the human enkephalin promoter by two isoforms of the catalytic subunit of cyclic adenosine 3',5'-monophosphate-dependent protein kinase. 165 33

Eight recombinant clones were obtained by insertion of BamHI fragments of herpes simplex type I viral DNA into a vector plasmid pUC19o. Of the obtained clones 5 were found to hybridize with herpes simplex type I and 2 viral DNA while 3 clones revealed a positive reaction with the Vero cells DNA. A constructed DNA-probe possessing the highest level of activity was selected for further studies. The probe is a BamHI fragment of herpes simplex type I viral DNA labelled with 32P dTTP. Probe sensitivity in blot hybridization is 10 pg for identification of type I viral DNA and 50 pg for type 2 viral DNA. The DNAs of cytomegalovirus and herpes zoster virus do not show positive signals with the probe. The increased sensitivity of the used dot hybridization as compared with biological or IEA antigen identification of the virus was confirmed with the clinical material from 59 patients with the different clinical manifestations of the herpes viral infection.
Mol Gen Mikrobiol Virusol 1991 Oct
PMID:[Detection of herpes simplex virus by DNA-DNA hybridization method]. 166 48

Fifty AIDS patients were investigated for human cytomegalovirus (HCMV) viraemia when potentially HCMV-related clinical symptoms or syndromes were observed. Nine patients underwent prolonged virologic follow-up, while 41 additional patients were examined only once or sporadically. Concentrated preparations of polymorphonuclear leukocytes (PMNL) from 153 blood samples were obtained for monitoring: (1) early virus isolation in cell cultures 24 h p.i. (viraemia); (2) early structural antigen detection in cytospin preparations (antigenemia); and (3) HCMV DNA in blood (DNAemia) through DNA amplification by the polymerase chain reaction (PCR). Viraemia and antigenemia were quantitated, whereas evaluation of DNAemia was only qualitative. A good correlation between levels of viraemia and antigenemia was consistently found except during ganciclovir treatment. HCMV-related clinical symptoms were observed when the number of infected PMNL was greater than 100 per 2 x 10(5) cells examined. All 56 blood samples positive for viraemia and antigenemia were also PCR-positive, whereas 44 samples (39 of which taken from patients with ascertained HCMV infection in blood) were positive by PCR only. Viraemia and antigenemia were often unrelated to HCMV organ syndromes, such as retinitis, in which only DNAemia was often detected. Prolonged ganciclovir treatment kept viraemia, antigenemia and even DNAemia at a low or negative level, yet drug discontinuation led to rapid progression of HCMV infection in blood. In addition, prolonged antiviral treatment could induce appearance of ganciclovir-resistant HCMV strains, requiring alternative foscarnet therapy. In conclusion, determination of viraemia and antigenemia appears essential for correct clinical management and antiviral treatment of disseminated HCMV infections in AIDS patients. However, PCR is the most sensitive method for diagnosis and monitoring of HCMV infections in blood at a pre-clinical stage.
Mol Cell Probes 1991 Oct
PMID:Early virus isolation, early structural antigen detection and DNA amplification by the polymerase chain reaction in polymorphonuclear leukocytes from AIDS patients with human cytomegalovirus viraemia. 166 8

The macrolide FK-506, like the cyclic undecapeptide cyclosporin A (CsA), is a potent immunosuppressant that interferes with the transcriptional activation of several early-phase genes in T lymphocytes, including that for interleukin-2 (IL-2). We compared the effects of FK-506 and CsA on transcription from the 5' upstream activating sequences (UAS) of the human IL-2 gene and several cellular and viral UAS to define cis-acting sites which may be responsive to FK-506. The UAS surveyed included the human IL-2 receptor alpha-chain, human metallothionein II, simian virus 40 early, human cytomegalovirus immediate-early, adenovirus major late, and Rous sarcoma virus long terminal repeat UAS. In addition, we studied multimers of several defined promoter elements (NFIL-2A, NF-kappa B, or NF-AT1) which are found in the UAS of the human IL-2 gene and which have been reported to be responsive to CsA when linked to a minimal promoter element (TATA box and transcription start site). Each promoter-regulatory region was fused to the bacterial chloramphenicol acetyltransferase gene and used to transiently transfect Jurkat cells. Quantitative chloramphenicol acetyltransferase assay determinations indicated that the transcriptional activity of each UAS induced upon T-cell activation was (i) completely sensitive, (ii) partially sensitive, or (iii) resistant to inhibition by CsA and FK-506. The induced transcription driven by the IL-2 promoter elements NF-AT1 and NFIL-2A could be blocked completely by FK-506 or CsA. Gel mobility shift assays indicated that the binding activities of the factors specifically interacting with these sequences were detected in activated cells regardless of whether the cells were treated with FK-506 or CsA. The results suggest that FK-506 or CsA inhibits a transacting mechanism(s) without disrupting the binding activities of these transcription factors. The degree to which each UAS was resistant to FK-506 was consistent with the level of transcription induced by phorbol myristate acetate, while UAS which were sensitive to inhibition by FK-506 were dependent on the presence of both phorbol myristate acetate and ionomycin.
Mol Cell Biol 1991 Aug
PMID:The immunosuppressant FK-506 specifically inhibits mitogen-induced activation of the interleukin-2 promoter and the isolated enhancer elements NFIL-2A and NF-AT1. 171 1

Chicken blastodermal cells (CBCs) and primary chicken fibroblasts (PCFs) have been lipofected with a variety of lacZ constructs encoding Escherichia coli beta-galactosidase (beta-gal). A reporter construct (phspPTlacZpA) containing a mouse heat-shock protein 68 gene (hsp 68) promoter was used to establish conditions for efficient lipofection. The construct, in circular or linear plasmid form or as reporter sequences alone, was transferred efficiently by incubating the cells for 3.5 h in a mixture of 6.2 micrograms Lipofectin (a cationic liposome preparation from Bethesda Research Laboratories) and 1.55-3.1 micrograms DNA per mL DMEM. These lipofection conditions were used to transfer a reporter construct (pCBcMtlacZ) containing a Zn(2+)-inducible chicken metallothionein (cMt) promoter, and constructs showing constitutive expression due to Rous sarcoma virus plus chicken beta-actin (pmiwZ) or cytomegalovirus (pMaori3) promoters. Endogenous chicken beta-gal and transferred bacterial beta-gal activity could be distinguished clearly by incubating the cells with the substrate, Xgal, at pH 4.3 or 7.4, respectively. Expression of phspPTlacZpA in chicken cells did not appear to require specific induction of the mouse hsp68 promoter, whereas expression of pCBcMtlacZ required treatment of the cells for 6-12 h with 150 microM ZnCl2. Bacterial beta-gal activity was observed following lipofection of CBCs that were cultured in suspension or plated. The efficiency of lipofection was at least 1 in 25 for CBCs, judging by the proportion of cells shown to have beta-gal activity 16-24 h after lipofection treatment began; these events could represent transient or stable incorporation of the construct.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1991 Dec
PMID:Efficient transfection of chicken cells by lipofection, and introduction of transfected blastodermal cells into the embryo. 175 Oct 34


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