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:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

At least two genes encode isoenzymes of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Alternative splicing of one of these genes generates a skeletal muscle-specific transcript from an upstream promoter and a liver-specific transcript from a downstream promoter. A potent glucocorticoid response element was identified in the first intron of the gene, i.e. between liver exon I and exon II. The element is approximately 3.5 kilobase pairs (kb) downstream of the liver isoenzyme transcription start site and 13 kb upstream of exon II of the gene and confers dexamethasone-sensitive expression of chloramphenicol acetyltransferase (CAT) activity from a heterologous thymidine kinase promoter and from both homologous 5'-flanking regions of the gene. This glucocorticoid response element also exhibits androgen- but not estrogen-sensitive expression of CAT activity in HeLa cells cotransfected with the appropriate receptor expression vector. DNase footprint and sequence analysis revealed that the element is comprised minimally of two adjacent 15-mer glucocorticoid receptor dimer binding sites situated in opposite orientations. Glucocortcoid regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in liver and skeletal muscle is mediated by a single complex glucocorticoid response element located in the first intron of the skeletal muscle/liver gene.
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PMID:Regulation of gene expression of rat skeletal muscle/liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Isolation and characterization of a glucocorticoid response element in the first intron of the gene. 133 34

Expression of prostate-specific antigen (PA) mRNA was tested at various time periods after incubation of the human prostate tumor cell line LNCaP with the synthetic androgen R1881. Androgen-stimulated expression was observed within 6 h after addition of R1881 to the cells. Run-on experiments with nuclei isolated from LNCaP cells showed that expression of the PA gene could be regulated by R1881 on the level of transcription. DNase I footprints of the promoter region of the PA gene (-320 to +12) with nuclear protein extracts from LNCaP cells showed at least four protected regions. The protected areas include the TATA-box, a GC-box sequence, and a sequence AGAACAgcaAGTGCT at position -170 to -156, which closely resembles the reverse complement of the consensus sequence GGTACAnnnTGTTCT for binding of the glucocorticoid receptor and the progesterone receptor. Fragments of the PA promoter region were cloned in front of the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with an androgen receptor expression plasmid into COS cells in a transient expression assay. CAT activity of COS cells grown in the presence of 1 nM R1881 was compared to untreated controls. A 110-fold induction of CAT activity was found if a -1600 to +12 PA promoter fragment was used in the construct. By further deletion mapping of the PA promoter a minimal region (-320 to -155) was identified as being essential for androgen-regulated gene expression. Mutation of the sequence AGAACAgcaAGTGCT (at -170 to -156) to AAAAAAgcaAGTGCT almost completely abolished androgen inducibility of the reporter gene constructs. One or more copies of the sequence AGAACAgcaAGTGCT cloned in front of a thymidine kinase promoter-CAT reporter gene confers androgen regulation to the reporter gene. These findings provide strong evidence for transcription regulation of the PA gene by androgens via the sequence AGAACAgcaAGTGCT. Interestingly, in addition to the AGAACAgcaAGTGCT element, an upstream region (-539 to -320) is needed for optimal androgen inducibility of the PA promoter.
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PMID:The promoter of the prostate-specific antigen gene contains a functional androgen responsive element. 172 87

Transfection of HeLa cells with cDNA vectors expressing the wild-type human glucocorticoid receptor (GR) enabled dexamethasone to strongly repress cytokine- and second messenger-induced expression of cotransfected chimeric reporter genes containing transcription regulatory DNA elements from the human interleukin 6 (IL-6) promoter. Deletion of the DNA-binding domain or of the second Zn finger or a point mutation in the Zn catenation site in the second finger blocked the ability of GR to mediate repression of the IL-6 promoter. Unexpectedly, deletion of the first Zn finger, a point mutation in the Zn-catenation site in the first finger, or one in the steroid-specificity domain at the base of the first finger converted GR into a dexamethasone-responsive activator that enhanced basal and interleukin 1-induced IL-6 promoter function. These first-finger mutants of GR also mediated dexamethasone-responsive enhancement of expression of the herpesvirus thymidine kinase-chloramphenicol acetyltransferase (TK-105-CAT and TK-80-CAT) reporter genes but not of the murine mammary tumor virus long terminal repeat-CAT or the c-fos-CAT (pFC700) reporter genes. Wild-type GR was able to specifically bind to DNA fragments containing glucocorticoid response element sequences in both the murine mammary tumor virus and IL-6 promoters, albeit weakly to the latter, in a sequential DNA-binding immunoprecipitation assay. The first-finger mutants of GR, however, were inactive in this assay. Thus, mutations in the first Zn finger unmask unusual promoter-specific activation properties of GR that may not require direct high-affinity binding of the mutant GR to target DNA.
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PMID:Repressor to activator switch by mutations in the first Zn finger of the glucocorticoid receptor: is direct DNA binding necessary? 187 Nov 24

Steroid receptors have been reported to stimulate transcription in a manner synergistic with other transcription factors. We have examined this synergism or functional cooperativity between glucocorticoid receptors and basal transcription factors in a variety of promoter and reporter gene contexts. A fragment containing a hormone response element from mouse mammary tumor virus was fused to well characterized promoters from the herpes virus thymidine kinase and mouse beta-globin genes and to related mutant promoters altered by inactivation of transcription factor-binding sites through point mutagenesis or deletion. These constructs were transfected into glucocorticoid-sensitive fibroblasts, and reporter gene activity was assessed with or without hormonal stimulation. In contrast to previous studies, we found little indication of synergistic interaction between elements mediating a hormone response and adjacent basal promoters. In fact, we observed that inactivating basal factor-binding sites, thereby decreasing promoter strength, actually increased hormone inducibility. We suggest that the inverse relationship between basal promoter strength and the induction ratio attained upon hormonal stimulation may be due to limitation of a common factor, an "adaptor" through which glucocorticoid receptor and basal transcription factors interact with the components of the RNA polymerase II complex to stimulate rates of transcription.
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PMID:Concerted stimulation of transcription by glucocorticoid receptors and basal transcription factors: limited transcriptional synergism suggests mediation by coactivators/adaptors. 207 21

Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells. In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA. Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility. This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids. In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexamethasone. A similar result was obtained with fibroblasts that contain endogenous glucocorticoid receptors. When the distance between receptor-binding sites or between the binding sites and the promoter was increased, the dependence of progestin induction on DNA topology was more pronounced. In contrast to the original plasmid, these constructs also revealed a similar topological dependence for induction by glucocorticoids. The differential influence of DNA topology is not due to differences in the affinity of the two hormone receptors for DNA of various topologies, but probably reflects an influence of DNA topology on the interaction between different DNA-bound receptor molecules and between receptors and other transcription factors.
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PMID:Hormonal induction of transfected genes depends on DNA topology. 215 20

Immortalization of B lymphocytes by Epstein-Barr virus (EBV) is complex and poorly understood. However, some evidence suggests that glucocorticoids influence this process. We identified a glucocorticoid-responsive element in the BamHI C fragment of EBV which we call ES-1. In glucocorticoid-treated cells, ES-1 enhanced chloramphenicol acetyltransferase gene expression from the herpes simplex virus thymidine kinase promoter, as well as the EBV Bam-C promoter, from which several latent viral gene products are transcribed. By Northern blot analysis, glucocorticoid treatment enhanced transcription from the Bam-C promoter in Jijoye cells, a Burkitt's lymphoma cell line. In addition, the DNA-binding domain of the glucocorticoid receptor bound specifically to the ES-1 region. These glucocorticoid effects on the Bam-C promoter region may provide some insight into the process of EBV immortalization.
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PMID:Identification of a glucocorticoid-responsive element in Epstein-Barr virus. 215 66

The feedback inhibition of interleukin-6 (IL-6) gene expression by glucocorticoids represents a regulatory link between the endocrine and immune systems. The mechanism of the efficient repression of the IL-6 promoter by dexamethasone (Dex) was investigated in HeLa cells transiently transfected with plasmid constructs containing different IL-6 promoter elements linked to the herpesvirus thymidine kinase gene (tk) promoter and the bacterial chloramphenicol acetyltransferase gene (cat) and cotransfected with cDNA vectors constitutively expressing either the active wild-type or inactive mutant human glucocorticoid receptor (GR). The induction by interleukin-1, tumor necrosis factor, phorbol ester, or forskolin of IL-6-tk-cat chimeric constructs containing a single copy of the IL-6 DNA segment from -173 to -151 (MRE I) or from -158 to -145 (MRE II), which derive from within the multiple cytokine- and second-messenger-responsive enhancer (MRE) region, was strongly repressed by Dex in a wild-type GR-dependent fashion irrespective of the inducer used. The induction by pseudorabies virus of an IL-6 construct containing the IL-6 TATA box and the RNA start site ("initiator" or Inr element) but not the MRE region was also repressed by Dex in the presence of wild-type GR. DNase I footprinting showed that the purified DNA-binding fragment of GR bound across the MRE, the TATA box, and the Inr site in the IL-6 promoter; this footprint overlapped that produced by proteins present in nuclear extracts from uninduced or induced HeLa cells. Imperfect palindromic nucleotide sequence motifs moderately related to the consensus GR-responsive element (GRE) motif were present at the Inr, the TATA box, and the MRE II site in the IL-6 promoter; although MRE I and a GR-binding site between -201 and -210 in IL-6 both lacked a discernible inverted repeat motif, their sequences showed considerable similarity with negative GRE sequences in other Dex-repressed genes. Surprisingly, chimeric genes containing MRE II, which lacks a recognizable GACGTCA cyclic AMP- and phorbol ester-responsive motif, were strongly induced by both phorbol ester and forskolin, suggesting that MRE II (ACATTGCACAATCT) may be the prototype of a novel cyclic AMP- and phorbol ester-responsive element. Taken together, these observations suggest that ligand-activated GR represses the IL-6 gene by occlusion not only of the inducible IL-6 MRE enhancer region but also of the basal IL-6 promoter elements.
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PMID:On the mechanism for efficient repression of the interleukin-6 promoter by glucocorticoids: enhancer, TATA box, and RNA start site (Inr motif) occlusion. 223 15

Steroid responsive elements (SRE) have been mapped at variable positions relative to the transcription start site and are often adjacent to binding sites of transcription regulatory proteins. In order to define the role of these transcriptional control sequences in the induction process, we inserted the previously defined 15-bp glucocorticoid response element (GRE) or 15-bp estrogen response element (ERE) immediately upstream of the TATA box of the thymidine kinase promoter, deleting all distal promoter elements. Both ERE and GRE confer inducibility by the respective hormone to the truncated promoter. These data suggest that the steroid receptor protein, possibly in conjunction with the TATA box binding protein, is able to form an active transcription complex. In contrast, the GRE when inserted 351 bp upstream of the start site of transcription of the tyrosine aminotransferase gene (TAT) is not capable of mediating hormone inducibility. Inducibility can be attained at this position by either two GREs or a single GRE in combination with a CCAAT motif. A cluster of point mutations in the CCAAT box abolishes hormone inducibility, strongly suggesting a synergistic action between the glucocorticoid receptor and the factor recognizing the CCAAT motif. The CCAAT box can be replaced by a CACCC box, an NF I and an SP1 binding site, thus demonstrating that synergistic action is not restricted to the CCAAT box binding protein. These combinations of a GRE with different transcription factor binding sites show a pronounced cell-type-dependent glucocorticoid induction of expression.
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PMID:Synergistic action of the glucocorticoid receptor with transcription factors. 246 58

Glucocorticoid receptor (GR) induces transcriptional activation of specific genes by binding to cis-acting enhancer sequences termed glucocorticoid responsive elements (GREs). Insulin regulates gene transcription part of which is mediated sequences dominated by GREs. By transient expression in HTC cells of fusion gene consist MMTV 5' flanking sequences containing the GREs on the thymidine kinase promoter (TK) and the coding region of the chloramphenicol acetyl transferase gene (CAT), we demonstrate that insulin increases 2-fold the produced induction from dexamethasone, while insulin alone does not exceed any significant effect. This synergistic effect of insulin is sequence specific since, expression of the glucocorticoid unresponsive gene RSV-CAT at the same cell line is affected neither by insulin nor dexamethasone or both. Transient expression of CAT constructs with progressive 5' deletion mutants of mouse mammary tumor virus (MMTV) promoter, fail to detect a cis-acting insulin responsive DNA element, that participates in increased dexamethasone mediated induction. We believe that insulin activates an intracellular mediator-transcription factor, that stimulates gene expression indirectly, by interaction with the glucocorticoid receptor.
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PMID:Insulin enhances glucocorticoid induction of gene expression in a sequence specific manner. 256 May 16

We have previously described the inhibition of glucocorticoid-dependent transcription from the mouse mammary tumor virus long terminal repeat promoter by products of the H-ras and v-mos oncogenes. We have studied the effects of conditional oncogenes on expression of glucocorticoid-dependent indicator genes. Expression of the glucocorticoid-dependent transcription of the tyrosine aminotransferase gene was monitored in FTO-2B rat hepatoma cells during Mr 21,000 protein (p21) H-ras induction. A strong transcriptional repression of the tyrosine aminotransferase gene followed p21 H-ras expression. The sequences in a glucocorticoid-dependent promoter which are responsible for the oncogene-mediated repression could be localized to the glucocorticoid response element; a construct in which a 15-base pair glucocorticoid response element was inserted 5' of the thymidine kinase promoter exhibited the oncogene-mediated repression of transcription. We observed a strong repression of glucocorticoid-dependent promoters and promoter constructs not only in the presence of p21 H-ras and p37 v-mos but also with p60 v-src. p57 v-myc, however, had no effect. Oncogene expression is not a sufficient prerequisite for an initial repression of glucocorticoid hormone-dependent gene transcription, since even in the presence of constitutively high levels of oncogene product a transient stimulation of glucocorticoid-dependent gene expression was found. Protein synthesis inhibition experiments revealed that no hormonally induced cellular protein is needed for the oncogene-mediated repression. It seemed reasonable that this phenomenon might reflect oncogene effects on the glucocorticoid receptor. We, therefore, made measurements of the glucocorticoid receptor protein. In the presence of glucocorticoid hormone the receptor translocated rapidly from the cytoplasm to the nucleus. In normal NIH 3T3 cells, after 24-h treatments the nuclear receptor levels had declined to about 50% of those determined at 2 h and in the presence of p21 H-ras they declined to 15%. The levels of cytoplasmic receptor were not affected by p21 H-ras expression.
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PMID:Oncogene mediated repression of glucocorticoid hormone response elements and glucocorticoid receptor levels. 256 9


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