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:1.14.14.3 (luciferase)
38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Synthesis and activity of the enzymatic equivalent of the sodium pump, Na,K-ATPase, are regulated by thyroid hormone in responsive tissues. The purpose of this study was to determine whether triiodothyronine (T3) regulates the level of the messenger RNA (mRNA) coding for Na,K-ATPase alpha- and beta-subunits in the heart. The expression of Na,K-ATPase mRNAs in in vitro myocardial cells was directly assayed by Northern and slot blot hybridization using Na,K-ATPase alpha- and beta-isoform-specific cDNA probes. Exposure of cultured neonatal rat cardiocytes to 10(-8) M T3 resulted in 1) threefold to fourfold increase in alpha 1- and beta 1-mRNA accumulation, with a maximum elevation at 48 hours, 2) sevenfold increase in alpha 2-mRNA accumulation with a peak elevation at 72 hours, and 3) transient threefold increase in alpha 3-mRNA within the first 24 hours followed by a deinduction thereafter. The increase in alpha 1-mRNA accumulation by T3 occurred over the physiological T3 concentration range with an EC50 of 5 x 10(-10) M. This was associated with a twofold increase in alpha 1-subunit protein accumulation and an increase in Na,K-ATPase transport activity. The half-life of alpha 1-mRNA analyzed by actinomycin D chase was less than 3 hours and was not affected by T3. Transfection experiments with the luciferase reporter gene revealed that thyroid hormone response sequences are located within the 5'-flanking regions of each alpha-isoform gene.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of Na,K-ATPase gene expression by thyroid hormone in rat cardiocytes. 133 Mar 58

Endocrine factors involved in the transcriptional regulation of the oxytocin (OT) gene were investigated in heterologous expression systems. Plasmids having a 5'-flanking region of the rat OT gene (-363/+16) or the human OT gene (-382/+41) cloned in front of the firefly luciferase gene were co-transfected with an expression vector for the rat thyroid hormone receptor alpha in P19 embryonal carcinoma (EC) cells. Thyroid hormone (T3) stimulated the activity of the rat and human OT promoters about 10-fold. In MCF-7 breast tumor cells transfected with the human OT promoter-luciferase fusion gene, T3 stimulation through endogenous thyroid hormone receptors was about 5-fold. Co-transfection experiments in P19EC cells using 5' deletion mutants of the rat OT gene showed that thyroid hormone responsiveness was located in two regions, one located between nucleotides -195 and -172, the other between nucleotides -172 and -148. Each region accounted for about 3-fold T3 stimulation. Gel retardation analysis using extracts from HeLa cells over-producing the c-erbA/TR alpha protein showed specific binding to the -172/-148 element, while no binding occurred on the -195/-172 element. The -172/-148 element which contains the imperfect estrogen response element, GGTGACCTTGACC, has inverted as well as direct repeats of the TGACC motif. Mutagenesis of TGACC motifs separately reduced thyroid hormone responsiveness by about 50%. However, simultaneous mutation of two TGACC motifs abolished the responsiveness to T3 completely. There was no cooperativity between the activated thyroid hormone and estrogen receptors in transfected MCF-7 cells nor in thyroid hormone receptor and estrogen receptor co-transfected P19EC cells. Negative interactions between these two receptors were observed and gel retardation assays showed interaction between the two receptors proteins. It was shown in an in vivo experiment that treatment of rats with thyroid hormone increased hypothalamic OT mRNA levels, the pituitary OT content, as well as OT levels in blood. The results reveal thyroid hormone as a physiological regulator of OT gene expression, which stimulates OT promoter activity directly through interaction with a thyroid hormone-response element in the OT gene.
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PMID:Thyroid hormone regulates the oxytocin gene. 137 Dec 78

A recombinant adenovirus system has been designed that confers glucocorticoid responsiveness upon infected cells in culture. Two mutually dependent viruses are required: a trans-activator virus containing the human glucocorticoid receptor transcription unit and a second receptor virus harboring a glucocorticoid response element linked to the firefly luciferase gene. Another reciprocal pair of viruses has been generated; one member expresses the rat thyroid hormone receptor alpha, while the other contains the luciferase gene regulated by a thyroid hormone-responsive DNA element. Corticosteroid- or thyroid hormone-induced transcription can be efficiently and accurately quantitated from cells coinfected with the appropriate complementary virus pair 20 h after infection in 96-well microtiter plates. This coinfection assay offers a convenient way to measure transcriptional activation by nuclear receptors and has certain key advantages over the commonly used cotransfection method. Its sensitivity and precision make it a practical approach to rapidly identify substances extracted from complex biological samples activating candidate "orphan" nuclear receptor molecules.
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PMID:An adenoviral vector system for functional identification of nuclear receptor ligands. 164 57

The first exon of the human thyrotropin-beta (hTSH beta) gene has been demonstrated in our laboratory to contain a major thyroid hormone inhibitory element. In order to characterize fully this element, we have performed a detailed functional and structural scanning mutational analysis of this element. Various -1192 to +37 (base pairs) bp fragments of the hTSH beta gene containing consecutive five deoxythymidine substitution mutations of the first exon were inserted into a luciferase reporter plasmid and transiently transfected into human embryonal cells (293) and stably transfected into rat pituitary cells (GH3). Two domains (domain 1 and 2) were identified by scanning mutations that were essential for function of the thyroid hormone inhibitory element: +3 to +13 bp and +28 to +37 bp. Biotinylated DNA fragments containing -12 to +43 bp of the hTSH beta gene and the identical scanning mutations demonstrate that in vitro synthesized c-erbA-beta binding is disrupted as much as 95% by mutations from -3 to +17 bp and to a lesser extent (20-30%) by mutations from +23 to +27 bp and from +33 to +43 bp. Domain 1 displayed a higher affinity for c-erbA-beta than domain 2 in avidin-biotin complex DNA-binding and gel-mobility assays. Using increasing amounts of in vitro synthesized c-erbA-beta, we were unable to demonstrate more than one protein-DNA complex in gel-mobility assays. However, using the avidin-biotin complex DNA-binding assay and the cross-linking reagent, 1,6-bismaleimidohexane, we were able to demonstrate thyroid hormone receptor dimer formation on domain 1 but not to any significant extent on domain 2. In conclusion, functional and DNA-binding studies suggest that the thyroid hormone receptor binds to two distinct regions in the first exon of the hTSH beta gene. The upstream site (domain 1) binds c-erbA-beta with higher affinity and is capable of binding c-erbA-beta as a dimer under some conditions, while the downstream site (domain 2) appears to bind a single molecule of c-erbA-beta with lower affinity. These results suggest that thyroid hormone receptor, binding to at least two sites in the first exon, act in conjunction to mediate T3 inhibition of hTSH beta expression.
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PMID:A detailed functional and structural analysis of a major thyroid hormone inhibitory element in the human thyrotropin beta-subunit gene. 165 75

The alpha-subunit gene of the glycoprotein hormones is normally expressed in pituitary thyrotropes and gonadotropes and in placental cells. Thus, this gene must contain elements that mediate expression and hormonal responses in different cell types. The localization of DNA regions important for expression and regulation of the alpha-subunit gene in thyrotrope cells has not previously been reported. In these studies luciferase expression constructs containing 1700 basepairs of 5' flanking DNA derived from the mouse alpha-subunit gene were introduced by electroporation into freshly dispersed cells from TSH-producing mouse pituitary tumors (TtT 97). This promoter functioned with greater efficiency in thyrotropes than in nonthyrotrope pituitary GH4 cells and L-cell fibroblasts. Primer extension confirmed that transcription from the alpha-subunit constructs initiated at the same site as the endogenous gene. Studies using 5' truncations showed a progressive loss of alpha-subunit promoter activity in thyrotropes between -480 and -120, with regions upstream of -254 contributing substantially to expression in thyrotrope cells. Thyroid hormone inhibited alpha-subunit promoter activity in a dose-dependent fashion, although in vivo treatment of tumors with thyroid hormone before transfection was necessary to achieve maximal inhibition. Thyroid hormone inhibition of alpha-subunit promoter activity also occurred in GH4 cells, but no effect was observed in L-cells. Studies using 5' truncations localized a region responsible for thyroid hormone inhibition between -62 and +43, encompassing the TATA sequence and the transcriptional initiation site. When this region was compared to the thyroid hormone inhibitory regions of the alpha-subunit genes from other species and the mouse TSH beta-subunit gene, a 6-basepair motif, 5' (G/A)GTG(G/A)G 3', emerged as a possible consensus sequence for a thyroid hormone inhibitory element.
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PMID:Thyrotrope expression and thyroid hormone inhibition map to different regions of the mouse glycoprotein hormone alpha-subunit gene promoter. 169 83

Retinoic acid receptors are members of the steroid/thyroid hormone receptor superfamily. Pursuant to the discovery that dexamethasone increases complement factor H expression, we examined the effects of retinoic acid on this gene. Both H mRNA and protein levels are increased by retinoic acid in L cells. Using the luciferase reporter gene system we have identified a region of the H promoter required for the retinoic acid response. This region contains an imperfect palindrome of the TGACC motif, present in thyroid hormone and estrogen-responsive elements. We demonstrate specific binding of the retinoic acid receptor beta to this sequence of the H gene by DNA-protein gel retardation assay. Therefore, these studies extend the sphere of influence of the retinoids to complement, an intrinsic component of the humoral immune system.
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PMID:Mapping of a retinoic acid-responsive element in the promoter region of the complement factor H gene. 170 Jul 80

We and others have previously reported that transforming growth factor-alpha (TGF alpha) expression is hormonally responsive and its expression is coregulated with that of its receptor [the epidermal growth factor (EGF) receptor]. The 5'-flanking region of the TGF alpha gene was characterized to determine whether it could confer hormone responsiveness to a reporter gene (luciferase) in human mammary carcinoma cells (MDA468). This segment of the gene is GC rich and contains an element strikingly similar to the core element of the EGF receptor gene that has been shown to mediate both basal and hormone-stimulated expression of the EGF receptor. We now report that a 313-basepair (bp) proximal element of the TGF alpha 5'-flanking region (-373 to -59 relative to the TGF alpha translation start codon) is capable of conferring responses to phorbol ester and EGF. This gene segment does not contain the EGF receptor gene homolog or potential AP-2-binding sites, suggesting that these elements are not necessary for basal and EGF- or phorbol ester-responsive TGF alpha gene expression. This 313-bp proximal element also confers proper transcriptional initiation to the chimeric TGF alpha-luciferase reporter construct, indicating it is the TGF alpha promoter. A 1.1-kilobase segment of the TGF alpha 5'-flanking region also confers retinoic acid, thyroid hormone, and glucocorticoid responsiveness despite the absence of recognizable steroid hormone receptor-binding sites. These hormones stimulate reporter expression 1.5- to 2-fold in a dose-dependent manner. Extension of the 5'-flanking region to -3500 results in marked suppression of reporter gene expression. These results indicate that the TGF alpha gene 5'-flanking sequence contains the elements responsible for hormonal responsiveness of this gene and that these elements are distinct from those that regulate the expression of the EGF receptor gene.
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PMID:Transcriptional regulation of the human transforming growth factor-alpha gene. 192 84

The murine thyrotropic MGH101A tumor is characterized by absent thyrotropin (TSH) beta gene expression and altered thyroid hormone (T3) regulation of the alpha-subunit. Comparison of the promoter structures of both alpha and TSH beta subunit genes from MGH101A with the promoter in expressing TtT-97 thyrotropes revealed no detectable differences. Transfection of the TSH beta promoter from MGH101A linked to luciferase showed minimal expression in primary or cloned MGH101A cells, or L-cells. However, a 6- to 10-fold increase in expression was exhibited in transfected thyrotropes. For the alpha gene, promoter activity was highest in thyrotropes and in cloned MGH101A cells, 5-fold lower in MGH101A tumors, and 10-fold lower in L-cells. Both promoters were not substantially affected by T3 treatment in MGH101A cells. In thyrotropes, promoter activity was inhibited 62.5% and 57.7% by 10 nM T3 treatment for the TSH beta and alpha genes, respectively. DNase I protection showed that factors from TtT-97 but not from MGH101A cells interacted with regions in the TSH beta promoter, while nuclear extracts from each tumor demonstrated at least one protein-DNA interaction with the alpha-subunit promoter. These studies suggest that the molecular defects in the MGH101A tumor are related to the absence of trans-acting factors and are not a result of altered primary gene structure.
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PMID:TSH subunit gene promoters from a murine alpha-subunit producing tumor function normally. 237 87

In TtT 97 cells, a thyrotropin-producing mouse pituitary tumor, thyroid hormone rapidly inhibits the transcription rate of both the thyrotropin alpha- and beta-subunit (TSH beta) genes, and this closely parallels the increase in nuclear thyroid hormone receptor occupancy. In this study, we have identified regions of the mouse TSH beta gene which are involved in mediating tissue-specific and thyroid hormone-regulated expression. Transient expression studies were performed using a series of chimeric plasmids in which 5'-flanking DNA was ligated to the firefly luciferase gene. Following transfection by electroporation, efficient expression of TSH beta 5'-flanking luciferase constructs occurred only in cells derived from TtT 97 tumors which express the endogenous TSH beta gene. Deletion analysis demonstrated that the region of the 5'-flanking DNA between positions -271 and -80 relative to the major transcriptional start site is important for TSH beta promoter activity in thyrotropes. No expression was measurable in mouse L cells, a fibroblast line, whereas a low level of expression was seen in MGH 101A cells derived from a thyrotropic tumor which no longer expresses the TSH beta gene. Reduced expression of TSH beta constructs was also found in GH3 and GH4 pituitary tumor lines. Addition of thyroid hormone effectively inhibited the level of transient TSH beta promoter activity in TtT 97 cells in a dose-dependent manner. The inhibitory effect was more pronounced and more accurately reflected the transcription rate data when transfected cells were derived from tumors treated with thyroid hormone for 5 days prior to transfection. Deletion of all but 46 base pairs of TSH beta gene 5'-flanking DNA and 3 base pairs of the first exon had no effect on thyroid hormone inhibition. This indicates that signals sufficient for transcriptional regulation of the TSH beta gene by thyroid hormone reside in the vicinity of the proximal promoter and may act by interfering with basal transcriptional factors.
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PMID:Thyroid hormone regulates the mouse thyrotropin beta-subunit gene promoter in transfected primary thyrotropes. 276 43

In MC3T3-E1 mouse osteoblastic cells, the adenylate cyclase activator forskolin increases osteocalcin (OC) mRNA levels. We have analyzed the effects of forskolin and 8-Br cAMP on the transcriptional activity of the rat OC promoter (with luciferase reporter) in MC3T3-E1 cells. Both forskolin and 8-Br cAMP activate the rat OC promoter 2- to 5-fold. By 5' deletion analysis, we have mapped the cAMP response to the region -121 to -92. The 48-base pair rat OC promoter region -121 to -74 (hence denoted ROCRR) can confer cAMP responsiveness to an unresponsive heterologous minimal promoter. Crude nuclear extracts prepared from MC3T3-E1 cells form three complexes with the ROCRR by gel shift analysis. No specific change in nuclear factor binding in response to cellular forskolin treatment could be demonstrated. Intriguingly, two nuclear factor complexes bound to the ROCRR also recognized the thyroid hormone response element palindrome (AGGTCATGACCT) but did not bind the classic cAMP (TGACGTCA) or glucocorticoid (AGAACANNNTGTTCT) response elements. The rat OC promoter possesses two directly repeated PuGGTCA steroid hormone response element hexamer motifs (bottom strand) in the region -114 to -93 within the ROCRR, separated by a 10 nucleotide spacer. Oligos encoding the individual rat OC hexamer sites compete for the ROCRR DNA:protein complexes recognized by the thyroid hormone response element palindrome. Removal of the up-stream hexamer site by 5' deletion (-121 to -100) in the context of the native OC promoter abrogates cAMP responsiveness. Taken together, these data suggest that this novel rat OC cAMP response region assembles a protein:DNA complex containing member(s) of the steroid hormone receptor superfamily. Transcriptional activity, but not DNA binding, is regulated by cAMP.
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PMID:Identification of a rat osteocalcin promoter 3',5'-cyclic adenosine monophosphate response region containing two PuGGTCA steroid hormone receptor binding motifs. 786 63


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