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)

Expression of the human chorionic gonadotropin (hCG)-alpha gene in placental trophoblasts is markedly stimulated by cAMP, a property preserved in a reporter plasmid containing its cAMP response elements (CREs) linked to the chloramphenicol acetyltransferase coding sequence (CRE alpha CAT). In search of a potential physiologic regulator of hCG gene expression via cAMP, we found that JEG-3 syncytial trophoblast cells have specific binding sites for vasoactive intestinal peptide (VIP) with dissociation constant of 1 nM. VIP maximally increased the transient expression of CRE alpha CAT and the expression of endogenous hCG-alpha mRNA in JEG-3 cells by 4- and 9-fold, respectively. Exposure of JEG-3 cells to 30 nM VIP increased cAMP levels 60-fold after 10-30 min, but cAMP rapidly declined thereafter. As a consequence of this desensitization, the effect of VIP on stimulation of both CRE alpha CAT and endogenous hCG-alpha and hCG-beta mRNA levels more closely resembled that of forskolin or 8-br-cAMP at time points much less than 24 h. Moreover, transient exposure to 8-br-cAMP was much less effective than 24 h of continuous incubation on CRE alpha CAT activity. We conclude that VIP rapidly increases cAMP content and activates hCG-alpha gene expression in JEG-3 cells, but sustained elevations in cAMP are necessary for maximal accumulation of this CRE-regulated gene product.
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PMID:Vasoactive intestinal peptide increases intracellular cAMP and gonadotropin-alpha gene activity in JEG-3 syncytial trophoblasts. Constraints posed by desensitization. 169 18

Gonadotropins (follicle-stimulating hormone (FSH), luteinizing hormone, and human chorionic gonadotropin) and beta-adrenergic agonists have been shown to stimulate expression of the proopiomelanocortin (POMC) gene in ovarian granulosa cells. The current studies investigate the intracellular mechanisms by which gonadotropins regulate gene expression. Primary cultures of rat granulosa cells were transfected with the plasmid POMC-CAT-150, which expresses the chloramphenicol acetyltransferase (CAT) reporter gene under the regulation of the rat POMC 5'-flanking region. CAT activity was stimulated by treatment of the cells with either 20 ng/ml FSH or 1 microM isoproterenol. To assess the role of protein kinase A (ATP:protein phosphotransferase; EC 2.7.1.37) in the gonadotropin and adrenergic response, an expression vector, MtR-AB, encoding a mutant RI regulatory subunit was cotransfected with POMC-CAT-150. The mutant protein kinase A regulatory subunit encoded by MtR-AB lacks functional cAMP-binding sites but effectively binds and specifically inhibits the catalytic activity of protein kinase A. The results of this analysis demonstrated that gonadotropin and adrenergic agonist stimulation of the POMC-CAT reporter construct in primary cultures of rat granulosa cells were abolished by cotransfection with MtR-AB; whereas a control SV40-promoter construct was unaffected by either gonadotropin treatment or cotransfection with MtR-AB. Basal expression directed by the POMC promoter was also decreased by cotransfection with the MtR-AB, implying that basal expression from the POMC promoter may also depend on protein kinase A. Deletion analysis of the POMC sequence indicated regions (-40 to -33 and +4 to +63) important for basal and FSH-stimulated expression. These studies suggest that both gonadotropin and adrenergic stimulation of the POMC promoter are mediated by protein kinase A and that regions proximal to the promoter are essential for gonadotropin-regulated expression from the promoter.
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PMID:Specific inhibition of protein kinase A in granulosa cells abolishes gonadotropin regulation of the proopiomelanocortin promoter. 190 60

Multiple forms of human thyroid hormone (T3) receptor have been identified, including true receptors that bind T3 (alpha 1 and beta) and a splicing variant (alpha 2) that does not bind T3. The alpha 1- and beta-receptors activate transcription through interactions with positive thyroid response elements (TREs). The alpha 2 variant is unable to activate transcription and has been reported to inhibit alpha 1 or beta stimulation of positive TREs, a property referred to as dominant negative activity. In this report we have performed studies to assess the functional properties of different members of the thyroid receptor family with regard to both positive and negative transcriptional regulation. The alpha 1-, alpha 2-, and beta-receptors were each coexpressed in JEG-3 cells with either TreTKCAT (CAT = chloramphenicol acetyltransferase), a reporter gene that contains a positive TRE, or TSH alpha CAT, a negatively regulated reporter gene. The alpha 1 and beta isoforms stimulated transcription of TreTKCAT and inhibited TSH alpha CAT transcription in a T3-dependent manner, whereas the alpha 2 variant was inactive. When coexpressed with alpha 1- or beta-receptors, alpha 2 inhibited regulation of positive TREs, but the effects of alpha 2 were modest and only occurred when relatively high doses of receptor were transfected. The alpha 2-receptor variant did not affect negative regulation by alpha 1- or beta-receptors. Thus, in both positive and negative regulation, thyroid hormone receptor isoforms that bind T3 (alpha 1, beta) are functional, whereas the alpha 2 isoform, which does not bind T3, is not functional.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Negative and positive transcriptional regulation by thyroid hormone receptor isoforms. 228

cAMP regulates transcription of the gene encoding the alpha-subunit of human chorionic gonadotropin (hCG) in choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, we inserted fragments from the 5' flanking region of the alpha-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between positions -146 and -111. In the absence of cAMP, the alpha-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. We localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element. The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the alpha-subunit gene.
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PMID:Cyclic AMP regulation of the human glycoprotein hormone alpha-subunit gene is mediated by an 18-base-pair element. 243 26

The alpha and beta subunit genes encoding chorionic gonadotropin (CG) are regulated transcriptionally in placental cells by cyclic AMP (cAMP). The regulatory response sequences of the alpha gene have been studied extensively. Similar studies of the CG beta subunit (CG beta) gene have not been possible because transcriptionally active sequences have not been identified in the clones isolated to date. The CG beta subunit genes form a complex cluster of seven structurally similar genes that include six CG beta-like genes and a single luteinizing hormone beta subunit (LH beta) gene. We isolated overlapping clones containing the entire CG beta/LH beta gene cluster (68 kilobases) from a human genomic cosmid library. The organization of the gene cluster was similar to that found in previous analyses, as determined by Southern blots of genomic DNA, but differed from some of the gene assignments, as determined by fragments cloned in lambda phage. The 5'-flanking sequence of the most active CG beta gene (CG beta 5) was linked to the chloramphenicol acetyltransferase (CAT) coding sequence for analyses of transient expression in different cell types. CG beta CAT was expressed preferentially in JEG-3 choriocarcinoma cells, and expression was markedly stimulated by treatment with 8-bromo-cAMP. Deletion mutagenesis of the CG beta 5'-flanking sequence revealed that multiple regions were required for maximal expression. The kinetics for cAMP stimulation of alpha CAT and CG beta CAT expression were different, suggesting that different pathways may be involved in cAMP-stimulated expression of the alpha and CG beta genes.
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PMID:Isolation and characterization of the human chorionic gonadotropin beta subunit (CG beta) gene cluster: regulation of transcriptionally active CG beta gene by cyclic AMP. 246 94

The alpha subunit of the placental hormone chorionic gonadotropin is regulated by cyclic AMP (cAMP) at the transcriptional level. A cAMP-responsive fusion gene (alpha-CAT) containing 1.5 kilobases of the alpha gene 5'-flanking sequence linked to the chloramphenicol acetyltransferase (CAT) gene was used as a transcriptional reporter in competition assays in transfected JEG-3 choriocarcinoma cells. Expression of the alpha-CAT fusion gene increased linearly with increasing amounts of transfected plasmid and was maximal at the same amount of alpha-CAT DNA (2 micrograms) with or without cAMP treatment. Various amounts of different competitor DNA sequences were cotransfected with the alpha-CAT reporter plasmid to examine the interactions of intracellular trans-acting factors with the regulatory elements of the alpha gene promoter. An 800-base-pair fragment of alpha gene 5'-flanking sequence inhibited both basal and cAMP-stimulated transcription of the alpha-CAT reporter plasmid in a dose-dependent manner, indicative of interactions with one or more trans-acting factors that activate alpha gene expression. The alpha gene sequences that interact with intracellular regulatory factors were defined by using several discrete regions of the 5'-flanking sequence as competitors for alpha-CAT expression. A proximal promoter sequence (-99 to +44) containing the CCAAT box, TATA box, and transcriptional initiation site was a relatively ineffective competitor of alpha-CAT transcription. In contrast, an upstream sequence between -236 and -100 was an effective competitor for transcriptional activators of alpha-CAT expression. Competition for alpha-CAT expression by this regulatory sequence did not require cis interactions with downstream promoter elements and was equally effective with or without cAMP treatment. An 18-base-pair repeated sequence within this region of the alpha gene (-146 to -111) greatly enhanced both basal gene expression and cAMP responsivity and also competed for limiting cellular transcription factors. These findings suggest that JEG-3 cells contain trans-acting factors that interact with a cAMP response element to activate alpha gene transcription. The chorionic gonadotropin beta gene 5'-flanking sequence also competed for alpha-CAT expression, suggesting that a common trans-acting factor is shared by the regulatory sequences of the alpha and beta genes.
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PMID:trans-acting factors interact with a cyclic AMP response element to modulate expression of the human gonadotropin alpha gene. 282 16

Fusion genes containing segments of the promoter region of the human LDL receptor gene and the coding sequence of the bacterial enzyme, chloramphenicol acetyltransferase (CAT), were introduced into JEG-3 human choriocarcinoma cells. Constructs containing 177 base pairs of 5'-flanking DNA (pLDLR-CAT 234) or 6500 base pairs (pLDLR-CAT 6500) promoted CAT activity in transient expression assays. Although both pLDLR-CAT 234 and pLDLR-CAT 6500 contain sequences related to the recently reported consensus sequence for cyclic AMP responsiveness, treatment of the transfected JEG-3 cells with 8-bromo-cAMP did not increase CAT activity. The cyclic AMP analog did, however, stimulate steroidogenesis and hCG secretion and increase CAT activity in cells transfected with p18X2SV1CAT, which contains two copies of an 18 base pair sequence corresponding to the cAMP-responsive element of the human alpha chorionic gonadotropin gene.
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PMID:The upstream promoter of the human LDL receptor gene does not contain a cyclic AMP response element. 283 85

The human chorionic gonadotropin-alpha (CG-alpha) gene is transcriptionally activated by cAMP. Sequencing the CG-alpha 5'-flanking region identified two copies of a palindrome, 5'-TGACGTCA-3', homologous to sequences in other cAMP-responsive genes. The two palindromes are contained within two identical 18-base pair (bp) sequences arranged as adjacent direct repeats. One or two synthetic copies of the 18-bp sequences were inserted into plasmids containing either the CG-alpha promoter or the SV40 promoter directing transcription of the chloramphenicol acetyltransferase gene. The 36-bp (double) element markedly enhanced chloramphenicol acetyltransferase activity in placental choriocarcinoma (JEG-3) cells when inserted in either orientation both 5' to the cap site or 3' of the coding sequence, thus defining it as an enhancer. Moreover, 8-br-cAMP stimulated the enhancer activity 30-40-fold. A single 18-bp element also stimulated chloramphenicol acetyltransferase activity, although 5-fold less than the double element, but still imparted a 35-fold transcriptional cAMP responsivity. The enhancer activates its homologous promoter much more efficiently than the SV40 promoter in JEG-3 cells. The alpha-promoter is not nearly as receptive to activation by the enhancer in baby hamster kidney fibroblasts, whereas the more modest enhancement of the SV40 promoter is less cell-specific. These studies suggest that the interaction of a 36-bp enhancer-like element with the homologous promoter represents part of the mechanism of cell-specific expression of the CG-alpha gene and that the enhancer is co-localized with a highly effective cAMP-response element.
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PMID:Cyclic AMP responsiveness of human gonadotropin-alpha gene transcription is directed by a repeated 18-base pair enhancer. Alpha-promoter receptivity to the enhancer confers cell-preferential expression. 304 Jul 32

The promoter of the rat prostaglandin endoperoxide synthase 2 (PGS-2) gene has recently been shown to confer gonadotropic hormone (follicle-stimulating hormone (FSH), luteinizing hormone (LH), cAMP) inducibility when ligated to a CAT (chloramphenicol acetyltransferase) reporter gene and transfected into primary cultures of differentiated granulosa cells. To delineate cis-acting elements and trans-activating factors mediating this response, deletions of the active promoter region (-192/-53 base pairs upstream of the transcriptional start site) were tested for their ability to bind protein of granulosa cell nuclear extracts and activate reporter gene activity. Electrophoretic mobility shift assays revealed that the DNA subregion -142/-120 inhibited protein/DNA binding observed between granulosa cell nuclear extracts and the labeled PGS-2 fragment -192/-53. The subregion -142/-120 acting element C/EBP beta, 5'-TTATGCAAT-3'. Point mutations within the C/EBP beta element abolished protein/DNA binding and resulted in a 50% loss of forskolin/LH/FSH inducibility of reporter gene activity. C/EBP beta mRNA and protein were induced rapidly in granulosa cells in vivo by an ovulatory dose of human chorionic gonadotropin (hCG). Collectively, these results indicate that C/EPB beta appears to play a key role in regulating induction of the PGS-2 gene in granulosa cells prior to ovulation.
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PMID:Transcriptional regulation of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. Evidence for the role of a cis-acting C/EBP beta promoter element. 840 49

The transcription factor Oct-3/4 may be important in maintaining embryonic cells in an undifferentiated state. It is probably down-regulated at about the time that human chorionic gonadotropin (hCG) is first expressed in embryonic trophectoderm. Here we report that Oct-3/4 strongly inhibits the hCGbeta subunit (hCGbeta) promoter in JAr choriocarcinoma cells. Oct-3/4 reduced chloramphenicol acetyltransferase (CAT) reporter expression from the -305hCGbeta promoter by about 90% in transient co-transfection assays, but had no effect on expression from the -249hCGbeta promoter. The -305/-249 hCGbeta fragment specifically bound synthetic Oct-3/4 protein as measured in electrophoretic mobility shift assays, and the Oct-3/4-binding site was localized around -270 by methylation interference footprinting. Site-directed mutagenesis of this binding site abolished Oct-3/4 repression. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGbeta messenger RNA and hCG protein to less than 10% of controls. We suggest that silencing of Oct-3/4 in trophectoderm is a prerequisite for hCG up-regulation in early human embryos at the time of maternal recognition of pregnancy.
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PMID:Silencing of the gene for the beta subunit of human chorionic gonadotropin by the embryonic transcription factor Oct-3/4. 866 60


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