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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To understand the molecular basis of the phosphorylation-enhanced transcriptional activity of human thyroid hormone nuclear receptor subtype beta 1 (hTR beta 1), we studied the effect of phosphorylation on the interaction of hTR beta 1 with the retinoid X receptor beta (RXR beta), we studied the effect of phosphorylation on the interaction of hTR beta 1 with the retinoid X receptor beta (RXR beta). In vitro, the extent of hTR beta 1.RXR beta heterodimer bound to various thyroid hormone response elements (TREs) was compared before and after phosphorylation of hTR beta 1. Without phosphorylation, hTR beta 1.RXR beta heterodimer was barely detectable under the experimental conditions. After phosphorylation of hTR beta 1, heterodimer bound to (i) the chicken lysozyme gene TRE, (ii) a TRE consisting of direct repeats of half-site binding motifs separated by four gaps, and (iii) a palindromic TRE was enhanced by approximately 10-, 7-, and 6-fold, respectively. The effect of phosphorylation on hTR beta 1.RXR beta heterodimerization was reversible. Dephosphorylation of the phosphorylated hTR beta 1 by alkaline phosphatase led to loss of the ability of hTR beta 1 to form a heterodimer with RXR beta in either the absence or the presence of DNA. These results indicate that the heterodimerization is enhanced by phosphorylation. To evaluate the effect of phosphorylation on the interaction of hTR beta 1 with RXR beta in vivo, we cotransfected hTR beta 1, RXR beta and TRE-chloramphenicol acetyltransferase (CAT) expression plasmids into CV-1 cells. CAT activity was assessed in the presence or absence of okadaic acid. Okadaic acid is a potent inhibitor of phosphatases 1 and 2A and increases the in vivo phosphorylation of hTR beta 1 by approximately 10-fold. Using the CAT reporter gene under control of the TRE from the malic enzyme gene, we found that RXR beta increased the okadaic acid-enhanced hTR beta 1-mediated CAT activity by 2- to 3-fold in the presence of 3,3',5-triiodo-L-thyronine. However, 9-cis-retinoic acid did not enhance the effect of okadaic acid. Our results indicate that phosphorylation is essential for the interaction of hTR beta 1 with RXR beta. Thus, phosphorylation plays a pivotal role in the gene-regulating activity of hTR beta 1.
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PMID:Phosphorylation enhances the target gene sequence-dependent dimerization of thyroid hormone receptor with retinoid X receptor. 805 36

Cloning of human thyroxine-binding globulin (TBG) cDNA and gene revealed that the primary structure of TBG and its gene organization are homologous to those of serine protease inhibitors (serpin). The transfection study of the TBG promoter linked to chloramphenicol acetyltransferase gene demonstrated that the putative hepatocyte nuclear factor-1 site (located at -77 approximately -65) is required for the transcription of the TBG gene. In addition, the sequence located at -218 approximately -102 is responsible for liver-specific expression of the gene. Estrogen, thyroid hormone and glucocorticoid had little effect on the promoter activity, suggesting that the alteration of serum TBG concentration by these hormones is due to their effect on posttranslational steps in TBG synthesis and secretion.
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PMID:[Cloning of human thyroxine-binding globulin cDNA, isolation of the gene, and its transcriptional regulation]. 819 73

T4-binding globulin (TBG) is a glycoprotein of hepatic origin which transports thyroid hormone in serum. To characterize the human TBG (hTBG) gene, we studied its genomic organization, promoter activity, and regulation. To this purpose, we isolated from liver a complete hTBG cDNA clone containing the 5'-untranslated region and localized the transcription start site (TSS). The analysis of genomic clones revealed that the hTBG gene consists of five exons and that its exon-intron organization is similar to that of other members of the serine protease inhibitor family. The first exon (exon 0) is a short noncoding sequence located 1.62 kilobase pairs (kbp) upstream from exon 1. Potential cis-acting transcriptional regulatory elements including a TATA box, a CAAT box, and a hepatocyte nuclear factor-1 binding motif were identified in the upstream region. A reporter gene in which 3.2 kbp of the 5'-flanking region, including exon 0, was inserted upstream of the bacterial chloramphenicol acetyltransferase gene showed significant activity when transfected into a hepatblastoma-derived (HepG2) cell line. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, down-regulated the promoter activity by more than 80% and completely inhibited hTBG synthesis, whereas thyroid hormone, glucocorticoid, estrogen, and nicotinic acid had little, if any, effect. A series of 5'-deletions revealed that the fragment -218 to +4 from the TSS had the highest promoter activity, nearly 1000-fold greater than the promoterless chloramphenicol acetyltransferase construct. When nonhepatocyte-derived cell lines (CV-1 and CHO) were tested, promoter activity was reduced by a factor of 100, showing that the promoter works in liver-specific manner. The region -218 to -102 contains liver-specific enhancer elements, since deletion to nucleotide -101 resulted in a profound reduction of the promoter activity in HepG2 cells but not in CV-1 or CHO cells. On the other hand, mutational disruption of the putative hepatocyte nuclear factor-1 site (located 65 bp upstream of the TSS) completely abolished the promoter activity in all cell lines, indicating that this site is absolutely required for the transcription of the hTBG gene.
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PMID:Human thyroxine-binding globulin gene: complete sequence and transcriptional regulation. 823 4

Expression of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) gene family in rat intestinal epithelial cells was examined using RNA blot hybridization analyses. Rat intestinal epithelial cells express only the alpha 1- and beta 1-subunit mRNAs. A gradient in expression of alpha 1- and beta 1-subunit mRNA was seen along the villus-crypt unit in both jejunum and ileum, i.e., villus tip >> crypt cells. Regional differences in expression were observed along the intestine. alpha 1- and beta 1-subunit mRNA abundance was similar in jejunum, ileum, and colon while enzymatic activity was highest in the jejunum and lowest in the ileum. Administration of thyroid hormone to thyroidectomized rats increased the expression of alpha 1- and beta 1-subunit mRNAs in jejunum but not in colon. Hypothyroidism had no effect on subunit mRNA expression. The human intestinal cell line Caco-2 was also studied. These cells also expressed only the alpha 1- and beta 1-isoform mRNAs and demonstrated a developmental profile in both mRNA and enzymatic activity. Furthermore, in Caco-2 cells both alpha 1- and beta 1-mRNAs and Na(+)-K(+)-ATPase enzymatic activity were stimulated by thyroid hormone. Caco-2 cells transfected with 5' flanking regions of the human Na(+)-K(+)-ATPase beta 1-gene linked to the chloramphenicol acetyltransferase (CAT) reporter gene responded to 3,5,3'-triiodothyronine (T3) treatment with increased expression of CAT activity. This suggests that the 5' flanking region of the beta 1-gene contains a thyroid hormone response element and that T3 upregulation occurs at the transcriptional level.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Na(+)-K(+)-ATPase gene expression in rat intestine and Caco-2 cells: response to thyroid hormone. 823 61

C-erbA receptors and v-erbA have been shown to functionally interact with 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-inducible gene expression. These proteins enhance trans-activation by c-jun, and the c-erbA receptors in the presence of thyroid hormone repress TPA and c-jun induction of transcription. Also, v-erbA can abrogate T3-mediated repression. We have examined how dominant negative (S and CL) and nondominant negative (G-H) receptors cloned from various patients with thyroid hormone resistance syndromes affect expression of the collagenase promoter induced with TPA. The CL receptor (ARG315HIS mutation) has a 2-fold reduction in T3-binding affinity compared with human c-erbA beta 1 wild-type (WT) receptor, whereas the G-H receptor (ARG311HIS) and S receptor (deletion, THR codon 332) have T3-binding affinities reduced by 100-fold and greater than 100-fold, respectively. These mutant receptors were cotransfected with a collagenase promoter (-1200 to +63 base pairs) chloramphenicol acetyltransferase reporter gene (Col-CAT) into COS-7 cells. Levels of CAT reporter gene expression after transient transfection were determined in the presence or absence of 3-10 nM T3 and the presence or absence of 100 nM TPA. Unoccupied CL receptor and G-H and S receptors stimulated TPA-induced Col-CAT expression 1.5- to 9-fold. The CL receptor with thyroid hormone totally repressed TPA induction of the collagenase receptor. In the presence of thyroid hormone, the enhancing effects by S and G-H receptors on TPA-induced Col-CAT expression were unaffected and minimally diminished, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Dominant and nondominant negative C-erbA beta 1 receptors associated with thyroid hormone resistance syndromes augment 12-O-tetradecanoyl-phorbol-13-acetate induction of the collagenase promoter and exhibit defective 3,5,3'-triiodothyronine-mediated repression. 824 13

Although the important role of thyroid hormones in regulating metamorphosis of amphibian larvae is well known, it has not been clearly established if thyroid hormones have any function in the activities of adult amphibian tissues. We now describe a strong effect of 3,3',5-triiodothyronine (T3) on adult Xenopus liver cells. Low doses of T3 rapidly (within 6-12 h) potentiate the activation of vitellogenin (Vit) genes by estradiol-17 beta (E2) in primary cultures of adult male and female Xenopus hepatocytes. This effect is developmentally regulated and is first manifested during metamorphic climax. In an attempt to explain this potentiation, we find that T3 also upregulates thyroid hormone receptor beta, but not alpha, transcripts and rapidly enhances the autoinduction of estrogen receptor (ER) mRNA in adult Xenopus hepatocytes. In transient transfection of the Xenopus cell line XTC-2 with an estrogen response element--chloramphenicol transacetylase (ERE-CAT) construct T3 was found to potentiate the transcription by E2 from the transfected ERE, thus suggesting that it enhances the accumulation of functional ER. We conclude that T3 can function in adult amphibian tissues, and discuss the significance of thyroid hormone potentiation of responses to estrogen in reproductive processes.
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PMID:Thyroid hormone potentiates estrogen activation of vitellogenin genes and autoinduction of estrogen receptor in adult Xenopus hepatocytes. 827 36

Metamorphosis in amphibians is marked by dramatic thyroid hormone-induced changes that include tail regression. To examine thyroid hormone effects on gene transcription during the early stages of tail resorption, we injected exogenous genes directly into the caudal skeletal muscle of Xenopus tadpoles and followed their expression in vivo. Gene expression was both strong and reproducible, and it correlated with the amount of DNA injected. Moreover, expression continued as long as the animals were blocked in prometamorphosis by antithyroid drugs (for up to 4 months). Thyroid hormone-dependent effects on transcription were examined by using a palindromic thyroid hormone response element linked to a chloramphenicol acetyltransferase reporter gene. Reporter gene expressions were normalized for transfection efficiency by using a constitutively expressed luciferase construct. Physiological concentrations of 3,5,3' triiodo-L-thyronine (1 nM), applied for 120 hr, produced a 5-fold increase in transcription (P < 0.05) from the thyroid hormone response element but did not modify transcription from constitutive viral promoters. This study thus demonstrates that by directly expressing genes in Xenopus tadpole muscle in vivo, one can exploit the powerful experimental advantages of gene transfer systems in an intact, physiologically normal animal.
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PMID:Thyroid hormone-dependent transcriptional regulation of exogenous genes transferred into Xenopus tadpole muscle in vivo. 834 51

It has been shown that the mouse histone H10 promoter contains a DNA element, composed of a direct repeat of the sequence GGTGACC separated by 7 nt, which is able to bind retinoic acid receptors and to modulate transcription of reporter genes following treatment with retinoic acid. We have now investigated whether this DNA motif is also responsive to thyroid hormone. We co-transfected CV-1 monkey kidney cells with chloramphenicol acetyltransferase (CAT) expression plasmids containing either 740 bp of the H10 wild-type promoter or five copies of the repeat element cloned in front of the thymidine kinase promoter and expression vectors for human thyroid hormone receptors (TRs) alpha or beta and retinoid X receptor alpha (RXR alpha). Treatment of transfected cells with triiodothyronine led to a dose-dependent increase in CAT activity. Transfection experiments with increasing amounts of expression vectors for either TR alpha or RXR alpha resulted in up to 6-fold enhancement of CAT transcription. Furthermore, point mutations within the half-sites of the response element of the H10 promoter, as well as deletions within the interspace region, lowered CAT activity to 60-80% of that of the wild-type control. Electrophoretic mobility shift assays showed that the repeat element was able to form retarded complexes with TR alpha homodimers, as well as with TR alpha-RXR alpha heterodimers. Our results suggest that thyroid hormone receptors are involved in the regulation of mouse histone H10 expression.
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PMID:Thyroid hormone receptors bind to the promoter of the mouse histone H10 gene and modulate its transcription. 855 62

We describe a dominant-negative approach in vivo to assess the strong, early upregulation of thyroid hormone receptor beta (TR beta) gene in response to thyroid hormone, characteristic of the onset of natural and thyroid hormone-induced amphibian metamorphosis, 3,3',5-Triiodo-thyronine (T3) treatment of organ cultures of premetamorphic Xenopus tadpole tails coinjected in vivo with the wild-type Xenopus TR beta (wt-xTR beta) and three different thyroid responsive element chloramphenicol acetyltransferase (TRE-CAT) reporter constructs, including a direct repeat +4 (DR +4) element in the -200/+87 fragment of the xTR beta promoter, resulted in a 4- to 8-fold enhancement of CAT activity. Two human C-terminal TR beta 1 mutants (delta-hTR beta 1 and Ts-hTR beta 1), an artificial Xenopus C-terminal deletion mutant (mt-xTR beta), and the oncogenic viral homology v-erbA, none of which binds T3, inhibited this T3 response of the endogenous wt-xTR in Xenopus XTC-2 cells cotransfected with the -1600/+87 xTR beta promoter-CAT construct, the potency of the dominant-negative effect of these mutant TRs being a function of the strength of their heterodimerization with Xenopus retinoid X receptor gamma. Coinjection of the dominant-negative Xenopus and human mutant TR beta s into Xenopus tadpole tails totally abolished the T3 responsiveness of the wt-xTR beta with different TREs, including the natural DR +4 TRE of the xTR beta promoter.
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PMID:Dominant-negative mutant thyroid hormone receptors prevent transcription from Xenopus thyroid hormone receptor beta gene promoter in response to thyroid hormone in Xenopus tadpoles in vivo. 857 41

Thyroid hormone exerts marked effects on cardiovascular function. Expression of cardiac alpha- and beta-myosin heavy chain (MHC) isoforms can be altered in response to thyroid hormone as well as by hemodynamic changes imposed on the heart. The molecular mechanisms that mediate these changes are not completely known. We studied the contractile and thyroid hormone responsiveness of the betaMHC promoter in both cultured cardiac myocytes and in vivo by direct DNA transfer. Using transient transfection of neonatal rat cardiomyocytes, the activities of recombinant reporter plasmids containing betaMHC 5'-flanking sequences terminating at positions -2250, -1145, -670, and -354 were decreased significantly in cultures containing L-T3 (50 nM). Similar deletion analysis showed that 5'-flanking regions terminating within -2250 to -151 bp were contractility responsive; however, deletion to position -126 attenuated this response. In vivo betaMHC promoter activity, determined by injecting the recombinant plasmid into the myocardium, was significantly higher by 2-fold in hyperthyroid than in euthyroid ventricles (2.47 +/- 0.41 vs. 1.33 +/- 0.25 luciferase/ chloramphenicol acetyltransferase; P<0.05). Increased ventricular workload, produced by aortic coarctation for 5 days, resulted in ventricular hypertrophy (heart/body weight, 4.05 +/- 0.19 vs. 3.42 +/- 0.16 mg/g; P < 0.02) and a 3.4-fold increase in betaMHC messenger RNA content. However, betaMHC promoter activity in vivo was not significantly different between rats experiencing aortic coarctation and sham-operated rats (1.49 +/- 0.41 vs. 0.96 +/- 0.27 luciferase chloramphenicol acetyltransferase, respectively) and was similar to that in euthyroid animals. These results show that betaMHC promoter activity is T3 responsive in cultured myocytes and in vivo, but that the increase in betaMHC messenger RNA observed in the in vivo pressure overloaded myocardium cannot be explained entirely by transcription control mechanisms.
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PMID:Thyroid hormone and hemodynamic regulation of beta-myosin heavy chain promoter in the heart. 860 87


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