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)

The gene encoding PTH-related peptide (PTHrP) is expressed in a wide variety of normal and neoplastic tissues. Increased PTHrP gene expression in and secretion of PTHrP by specific tumors directly contributes to the development of malignancy-associated hypercalcemia in vivo. To define the genetic elements important for the control of PTHrP gene transcription, we used the reverse transcription polymerase chain reaction to delineate the control of promoter utilization and the splicing patterns of the exons encoding 5'-untranslated sequences. The majority of normal and neoplastic human tissues contained PTHrP mRNA transcripts initiating from both the up-stream (P1) and down-stream (P2) human PTHrP promoters. Furthermore, the downstream promoter was preferentially used by a factor of more than 30-fold. P1-initiated transcripts contained RNA species both with and without exon 2 (E2) sequences, except in the pancreas, adrenal, and stomach, where E2-containing sequences predominated. The transcriptional activities of P1, P2, and P1 + P2 were assessed by transfection of the corresponding PTHrP-chloramphenicol acetyltransferase (CAT) fusion genes into heterologous cell lines. Fusion genes containing P2 sequences were more transcriptionally active than fusion genes containing P1 sequences. The transcriptional activities of P1 + P2 in their natural tandem orientation were additive in rat keratinocytes and human JEG choriocarcinoma cells. In contrast, the activity of P1 + P2 was less than that of P2 alone in hamster BHK fibroblasts and InR1-G9 cells, and human HeLa cells. Analysis of the transcriptional properties of 5'-deleted human PTHrP-CAT constructs revealed the presence of multiple positive and negative DNA sequences (within both P1 and P2) functionally important for human PTHrP gene transcription. Distinct positive and negative DNA elements were also identified from analysis of 5'-deleted rat PTHrP-CAT fusion genes. The results of these experiments provide evidence for cell- and tissue-specific utilization of 1) distinct human PTHrP transcription start sites and specific patterns of 5'-exon splicing and 2) multiple positive and negative DNA control elements, important for the regulation of human and rat PTHrP gene transcription.
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PMID:Regulation of parathyroid hormone-related peptide (PTHrP) gene transcription: cell- and tissue-specific promoter utilization mediated by multiple positive and negative cis-acting DNA elements. 128 Mar 27

The regulation of human corticotropin-releasing hormone (hCRH) gene promoter activity by inducers of cAMP was investigated by transient transfection with a construct containing the hCRH gene promoter fused to the chloramphenicol acetyltransferase gene. Expression of hCRH-chloramphenicol acetyltransferase was strongly enhanced by forskolin in the neuroblastoma SK-N-MC and choriocarcinoma JAR cell lines. Overexpression of the catalytic subunit of protein kinase A dispensed the need for forskolin, and cotransfection of cAMP-responsive element-binding protein cDNAs enhanced forskolin-dependent expression of the hCRH promoter. Progressive 5'-end deletions of the hCRH promoter delineated a cAMP- responsive region between -226 and -164 base pairs. This fragment contained the sequence TGACGTCA at -221 base pairs, consistent with the consensus motif for a CRE. A homologous oligonucleotide responded to cAMP when cloned in either orientation in front of the thymidine kinase promoter. However, the level of constitutive and inductive cAMP expression was dependent on the cell line and on intrinsic properties of the promoter. Mutation of the wild type CRH-CRE sequence into an AP-1 site (TGAGTCA) completely abolished stimulation by cAMP. In contrast, coexpression of the catalytic subunit of protein kinase A dispensed the need for stimulation with forskolin, which showed that the CRH-CRE oligonucleotide served as a functional equivalent of the native CRE element.
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PMID:Identification and characterization of a 3',5'-cyclic adenosine monophosphate-responsive element in the human corticotropin-releasing hormone gene promoter. 148 Jan 79

The 5' flanking region of the mouse renin genes (Ren-1d and Ren-2d) contains two motifs that are homologous to known negative regulatory elements (NREs). Ren-2d has a 150-base-pair (bp) insertion 5' to the upstream putative NRE (NRE-1), which is lacking in Ren-1d. We tested the functionality of these sequences by using site-directed mutagenesis to delete individually each putative NRE from Ren-1d and to delete the 150-bp insertion from Ren-2d. We examined the effect of these mutations on the expression of the reporter gene chloramphenicol acetyltransferase, which was expressed from a truncated thymidine kinase promoter fused to the renin regulatory region. This plasmid was transfected into human choriocarcinoma JEG-3 cells. Only the upstream NRE (positions -619 to -597) was found to be functional in Ren-1d. The deletion of a 150-bp insertion from Ren-2d resulted in the suppression of chloramphenicol acetyltransferase activity to the level of Ren-1d expression. These data suggest that the upstream NRE that is functional in Ren-1d, but not in Ren-2d, may be partly responsible for differential expression of the renin genes in various tissues. The molecular mechanism of the NRE was examined by studying its interaction with nuclear proteins in submandibular gland and JEG-3 cells by gel-mobility-shift assays. Specific nuclear protein binding was observed only to the upstream NRE and the molecular mass of this protein was approximately 72 kDa as determined by Southwestern blot analysis. Thus our results suggest that both Ren-1d and Ren-2d conserve a cis-acting NRE in the 5' flanking region. In Ren-1d, this NRE could bind a specific nuclear protein resulting in the inhibition of Ren-1d expression in these tissues. On the other hand, the NRE in Ren-2d is nonfunctional due to interference by an adjacent 150-bp insertion.
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PMID:Identification of a negative regulatory element involved in tissue-specific expression of mouse renin genes. 173 3

Expression of the gene for the porcine transplacental iron transport protein uteroferrin (UF) is largely restricted to the uterus, where it is differentially regulated by estrogen (E) and progesterone (P). To study the regulatory mechanisms subserving these effects, a 2-kilobase genomic fragment corresponding to -2005 to 48 nucleotides of the UF gene was ligated up-stream to the reporter gene chloramphenicol acetyltransferase (CAT). This construct (UF-CAT) was transiently transfected into rabbit endometrial (HRE-H9), mouse fibroblastic (AKR-2B), and human choriocarcinoma (JEG-3) cells. The basal gene promoter activity of UF-CAT was exhibited in H9 cells, but not in AKR-2B or JEG-3 cells. In contrast, a simian virus-40 early promoter (SV2) was functional in all three cell lines. The H9 cells were used to examine steroid regulation of the UF gene promoter. The CAT expression in H9 cells primed with E and PRL, but not with E or PRL alone, was stimulated by P. In contrast, basal activity of SV2 in these cells was unaffected by hormones, singly or in combination. To examine the basis for the E/PRL-dependent response to P, levels of P and E receptors in H9 cells were quantified. PRL and E plus PRL increased the number of high affinity sites for P, but had little effect on levels of high affinity sites for E in treated vs. untreated H9 cells. In vivo administration of PRL to cyclic gilts had no effect on levels of endometrial UF mRNA and secreted UF protein; however, E- plus PRL-treated gilts had higher (P less than 0.05) levels of endometrial UF mRNA and luminal UF than PRL-treated gilts. These results demonstrate in vitro functional activity of the UF gene promoter and associated 5' flanking region and suggest that sequences within this region may mediate tissue-specific and steroid hormone-regulated expression of the UF gene. Moreover, interactions among E, PRL, and P modulate UF gene expression in vivo and in vitro.
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PMID:Regulation of the uteroferrin gene promoter in endometrial cells: interactions among estrogen, progesterone, and prolactin. 185 67

This study is an attempt to determine whether estrogen could directly regulate human gonadotropin-releasing hormone (GnRH) gene expression. Human GnRH expression vectors were constructed by fusing various 5' flanking regions of the human GnRH gene upstream of the luciferase reporter gene (LUC) or the thymidine kinase promoter linked to the chloramphenicol acetyltransferase reporter gene (CAT). These constructs were transiently transfected into a human choriocarcinoma cell line (JEG-3) and LUC or CAT activity was measured after either no treatment or treatment with various concentrations of estradiol. A stimulatory estrogen response element (ERE) was localized to a 32-bp region between -547 and -516 bp. To determine whether estrogen receptor bound to this region of the gene, we performed DNase I footprinting using purified calf uterine estrogen receptor. DNase I footprinting demonstrates a strong footprint between -567 and -514 bp of the human GnRH gene. In addition, an avidin-biotin complex DNA-binding assay demonstrated that a biotinylated DNA fragment containing -541 to -517 bp of the human GnRH gene bound 35S-labeled estrogen receptor as well as a biotinylated DNA fragment containing the xenopus vitellogenin ERE. On the other hand, the negative control biotinylated DNA fragment derived from adenovirus 5 bound insignificant amounts of 35S-labeled estrogen receptor. Both the GnRH ERE and vitellogenin ERE bound 35S-labeled estrogen receptor with high affinity (approximately 1 nM). These data indicate that the human GnRH gene contains an ERE sufficient to mediate a stimulatory response to estrogen in heterologous cells. Based upon these data we hypothesize that the human GnRH gene might also be directly regulated by estrogen in the hypothalamus, and that this regulation may explain the GnRH hypersecretion observed at the time of the preovulatory luteinizing hormone (LH) surge.
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PMID:Evidence for direct estrogen regulation of the human gonadotropin-releasing hormone gene. 193 51

To identify regulatory elements in the promoter of a human placental lactogen gene (hPL3) that are important for its transcriptional activation, sequences 5' to the start of transcription were linked to the reporter gene chloramphenicol acetyltransferase (CAT) and transiently transfected into JEG-3 cells, a human placental choriocarcinoma cell line. In the presence of the hPL3 enhancer, deletion of the promoter sequence between -142 and -129 basepairs resulted in an 8-fold decrease in CAT activity. Similar results were seen with the SV40 enhancer and the hPL3 promoter in HepG2 liver cells. Nuclear proteins from HepG2, HeLa, and JEG-3 cells formed specific binding complexes with this region of the hPL3 promoter by a gel mobility shift assay, indicating that the DNA-binding protein was not tissue specific. The -142 to -129 basepair region contains a sequence similar to that of a variant binding site for the transcription factor Sp1. An oligonucleotide containing Sp1-binding sites specifically competes for proteins binding the hPL3 promoter, and the methylation interference pattern is similar to that for an Sp1-binding site. This suggests that the hPL3 promoter binds Sp1- or an Sp1-like trans-acting factor, and this binding site is important for transcriptional regulation by the hPL3 enhancer in PL-producing cells.
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PMID:DNA sequences involved in the transcriptional activation of a human placental lactogen gene. 196 88

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

To identify the promoter sequence(s) of the CG beta gene, genomic fragments derived from a cosmid containing the CG beta gene family were transfected into mouse Y1 adrenal cortical cells. Using this system, we showed that the CG beta genes 5, 3, and 8 have functional promoters, the basal element of which in the case of CG beta 5, was within 78 base pairs 5' ward from the CAP site. The size of the CG beta transcripts and identity of the transcription start site was the same for CG beta mRNA synthesized in Y1 cells as in first trimester placenta. The promoter region identified by this system was also capable of driving the chloramphenicol acetyltransferase gene when transfected stably into choriocarcinoma cells. Chloramphenicol acetyltransferase constructs bearing variable length of 5'-flanking sequences from CG beta 5 and transfected into trophoblast cells suggest the presence of regulatory sequences within 700 base pairs from the CAP site. The information obtained here provide a foundation for studies of analyzing trans-acting placental and pituitary proteins to the defined CG beta promoter region.
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PMID:Identification of a promoter region in the CG beta gene cluster. 245 22

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 5' flanking regions of the mouse renin genes (Ren1d and Ren2d) contain putative negative control and cAMP responsive elements. Sequence analysis shows additionally that these putative control elements in the Ren2d gene are interrupted by a 160-base-pair insertion. To document the functions of these elements, we isolated these regions and fused them to the reporter gene chloramphenicol acetyltransferase (CAT), which was linked upstream to a thymidine kinase (TK) promoter (pUTKAT1). The chimeric constructs were transfected into mouse pituitary tumor AtT-20 and human choriocarcinoma JEG-3 cells. At the basal unstimulated condition, Ren1d 5' flanking sequence in the sense orientation inhibited basal CAT expression from the TK promoter of pUTKAT1, whereas the same sequence in the antisense orientation did not. The 5' flanking region of Ren2d had no inhibitory effect on basal CAT expression. These data demonstrate that the negative control element is functional in Ren1d but is nonfunctional in Ren2d, suggesting that the 160-base-pair insertion in Ren2d interferes with the function of the negative control elements. In response to 8-bromo-cAMP, both renin genes increased transcription 3-fold, suggesting a functional cis action of the cAMP responsive element in both genes. These data may be important in the understanding of the regulation of the tissue-specific expression of mouse renin genes.
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PMID:Negative control elements and cAMP responsive sequences in the tissue-specific expression of mouse renin genes. 253 60


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