Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.13.12.7 (firefly luciferase)
 2,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

We have studied the thyroid-hormone responsiveness of the alpha-myosin heavy chain (MHC) gene in vivo by directly injecting an expression vector containing the alpha-MHC 5' regulatory sequences (-613 to +421 base pairs) into the rat heart. In the expression vector pAM1Luc the alpha-MHC promoter elements direct the synthesis of firefly luciferase. Although thyroxine administration of both euthyroid and thyroidectomized rats for 5 days increased alpha-MHC promoter activity, the pAM1Luc gene construct did not mimic expression of the endogenous gene. These studies of direct gene transfer into mammalian myocardium suggest that additional cis-acting elements necessary for the in vivo response to thyroid hormone reside outside the -613 to +421 region.
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PMID:Thyroid hormone regulation of alpha-myosin heavy chain promoter activity assessed by in vivo DNA transfer in rat heart. 193 Jan 72

We demonstrate gene transfer into rat heart in vivo by the direct injection of plasmid DNA. Injection of gene constructs driven by retroviral and cellular promoters resulted in detectable levels of reporter gene activities. The cellular promoter and 5' flanking sequence (positions -613 to +32) were derived from the rat alpha-myosin heavy chain gene whose expression in vivo is restricted to cardiac muscle and is positively regulated by thyroid hormone. After DNA injection, activity of the firefly luciferase gene coupled to the myosin heavy chain promoter and regulatory sequence was detected in heart but not in skeletal muscle and was significantly increased in response to thyroid hormone treatment. Consequently, expression of injected genes can be targeted to specific cell types in vivo and can be modulated by the hormonal status of the animal. This approach provides a means of mapping the elements of genes that regulate their responses to complex stimuli that cannot be modeled in vitro.
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PMID:Hormonal modulation of a gene injected into rat heart in vivo. 203 60

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

A deletion analysis of the human insulin gene extending to 2 kb upstream of the transcription start site provided evidence of regulatory sequences located upstream of the insulin-linked polymorphic region (ILPR). Within this ILPR-distal region is a sequence (Ink, for insulin kilobase upstream) which contains three potential nuclear hormone-receptor half-sites, closely matching the consensus sequence AGGTCA. These sequences are arranged as a palindromic element with zero spacing over-lapping a direct repeat with 2 bp spacing. The Ink sequence was used in electrophoretic mobility-shift assays within nuclear extracts from COS-7 cells overexpressing the vitamin D, thyroid hormone or retinoic acid receptors, or from an insulin-expressing hamster cell line, HIT-T15. These studies suggest that the insulin-expressing cell line contains thyroid hormone and retinoic acid receptors at least, and that these receptors are able to recognize the Ink sequence. Three copies of the Ink sequence were placed upstream of the thymidine kinase promoter and firefly luciferase reporter gene. In COS-7 cells expressing the appropriate nuclear hormone receptor, this construct was responsive to both thyroid hormone (18-fold) and all-trans-retinoic acid (31-fold). In HIT-T15 cells the same construct responded to all-trans-retinoic acid, but not to thyroid hormone. Within the context of a 2 kb insulin gene fragment, the Ink sequence was shown to be activated by retinoic acid and by the retinoic acid receptor, but acted as a negative element in the presence of both retinoic acid and the retinoic acid receptor. Mutagenesis studies demonstrated that the palindromic sequence was important for the retinoic acid response, and for binding of complexes containing retinoic acid receptor. In human islets of Langerhans, retinoic acid was shown to stimulate insulin mRNA levels. These results demonstrate that a functional nuclear hormone-receptor-response element is located upstream of the human ILPR. As retinoic acid and thyroid hormone are frequently involved in developmental regulatory processes, it is possible that this element may be important in the process of islet cell differentiation.
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PMID:Identification and characterization of a functional retinoic acid/thyroid hormone-response element upstream of the human insulin gene enhancer. 763 3

We constructed a recombinant adenovirus carrying the firefly luciferase gene driven by the thymidine kinase promoter and controlled by the palindromic thyroid hormone/retinoic acid-responsive element. The same adenovirus vector without the hormone-responsive element was used as control. Regulation of the luciferase gene expression was tested in pituitary-derived GH cells and hepatoma-derived HepG2 cells infected with the recombinant adenoviruses. Administration of triiodothyronine to GH cells and all-trans-retinoic acid to HepG2 cells resulted in 8.0 +/- 0.3-fold and 4.6 +/- 0.5-fold induction of luciferase activity, respectively. No significant increase was observed with the control adenovirus. Hormonal regulation was also examined in adult mice. Mice depleted of thyroid hormone were injected intravenously with the recombinant adenoviruses and given 4 times the replacement dose of triiodothyronine or vehicle only for 4 days. Hormone administration resulted in 4.2-fold increase of luciferase activity in liver homogenates. No significant effect was observed in animals injected with the control adenovirus. This recombinant adenovirus provides a new experimental system in the study of thyroid hormone and retinoic acid action and offers the potential to regulate by physiological means the expression of genes transferred for the purpose of therapy.
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PMID:Expression of a thyroid hormone-responsive recombinant gene introduced into adult mice livers by replication-defective adenovirus can be regulated by endogenous thyroid hormone receptor. 792 32

The human Na,K-ATPase beta 1 subunit gene promoter activity is stimulated by thyroid hormone (T3) in the human intestinal Caco-2 cells. To identify potential cis-acting transcriptional regulatory elements involved in this process, chimeric plasmids containing varying lengths of the 5' flanking region of the human beta 1 Na,K-ATPase gene linked to the firefly luciferase reporter gene were introduced into Caco-2 cells by transient transfection. Analysis of T3-regulated luciferase activity of cells carrying these plasmids, and subsequent use of site-directed mutagenesis revealed that a region from -459 to -438 (relative to the transcriptional start site) is required for the induction of the beta 1 Na,K-ATPase gene by T3. An oligonucleotide containing this sequence from -465 to -433 confers T3 responsiveness to a heterologous promoter. Gel mobility shift assays showed specific binding of nuclear proteins of Caco-2 cells to this region and immunoreactive T3 receptor was identified in one of these complexes. These data demonstrate that there is a cis-acting thyroid hormone responsive element in the 5' flanking region of the human beta 1 Na,K-ATPase gene and induction of transcription of this gene by T3 involves specific binding of the thyroid hormone receptor to the TRE located at position -459 to -438.
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PMID:Identification of a functional thyroid hormone response element in the upstream flanking region of the human Na,K-ATPase beta 1 gene. 839 3

Cardiac myocytes have the unique ability to express exogenous genes that have been injected directly into the heart tissue in vivo. This technique makes it possible to identify cis-acting DNA sequences responsible for the regulation of myocyte-specific genes in a working heart. In these studies we introduced recombinant plasmids containing 5'-flanking sequences of the alpha-myosin heavy chain (alpha MHC) gene into the rat myocardium in order to identify sufficient promoter/enhancer sequences that faithfully reproduced the activity of the endogenous gene. The transcriptional activity of the alpha MHC promoter sequence was measured by the level of activity of the firefly luciferase reporter gene and was reported as the activity relative to a coinjected constitutively active viral promoter construct (pRSVCAT) which corrected for variations in DNA uptake and posttranscriptional events. We report that a recombinant plasmid containing 5'-flanking sequences -2560 to +421 basepairs of the transcriptional start site of the alpha MHC gene was appropriately inactive in the hypothyroid rat heart, in which expression of the endogenous gene was also inhibited. The activity of this promoter sequence was increased 44-fold by thyroid hormone in the hearts of thyroidectomized rats. In contrast, although this recombinant plasmid was appropriately active in the euthyroid myocardium, its activity could not be further stimulated by thyroid hormone. The observation that regulation of the transcriptional activity of the alpha MHC promoter by thyroid hormone was different in euthyroid and hypothyroid hearts suggests that the participation of nuclear regulatory factors, including the thyroid hormone/retinoid family of receptors, may differ according to thyroid status.
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PMID:In vivo regulation of recombinant cardiac myosin heavy chain gene expression by thyroid hormone. 844 Jan 68

The activity of the apical membrane Na+/H+ exchanger NHE3 isoform of renal or intestinal epithelial cells is chronically regulated by a wide variety of stimuli, including acidosis, cAMP, glucocorticoids, and thyroid hormone. To understand the molecular mechanisms responsible for long term regulation of this cation transporter, we have isolated and determined the structure of this gene from a rat genomic library. The Nh3 gene spans > 40 kilobases and contains 17 exons that are flanked by typical splice donor and acceptor sequences at the exon-intron boundaries. The transcription initiation site was mapped by S1 nuclease protection analyses of mRNA from rat kidney and intestine. Multiple start sites were clustered between nucleotides -100 and -96 relative to the translation initiation codon. An atypical TATA-box and CCAAT-box are centered 30 and 147 nucleotides, respectively, upstream of the predominant transcription initiation site. Sequence analysis of approximately 1.4 kilobases of the 5'-flanking promoter region also revealed the presence of other putative cis-acting elements recognized by various transcription factors (e.g. AP-1, AP-2, C/EBP, NF-I, OCT-1/OTF-1, PEA3, Sp1, glucocorticoid, and thyroid hormone receptors), some of which may participate in the chronic regulation of this gene. The glucocorticoid responsiveness of the Nhe3 gene was assessed by fusing its 5' regulatory region to the firefly luciferase reporter gene and then by measuring the expression of the chimeric gene in transiently transfected renal epithelial OK and LLC-PK1 cells. Glucocorticoid treatment significantly increased the luciferase activity of the chimeric gene in both cell lines, thereby indicating that glucocorticoid regulation of Nhe3 is mediated primarily by a transcriptional mechanism.
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PMID:Genomic organization and glucocorticoid transcriptional activation of the rat Na+/H+ exchanger Nhe3 gene. 863 55


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