Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Activin A is a potent growth and differentiation factor related to transforming growth factor beta. In somatotrophs, activin suppresses the biosynthesis and secretion of growth hormone (GH) and cellular proliferation. We report here that, in MtTW15 somatotrophic tumor cells, activin decreased GH mRNA levels and inhibited expression of transfected GH promoter--chloramphenicol acetyltransferase fusion genes. Deletion mapping of nucleotide sequences mediating this inhibition led to the identification of a region that has previously been characterized as binding the pituitary-specific transcription factor Pit-1/GHF-1. Characterization of nuclear factor binding to this region demonstrated that binding of Pit-1 to the GH promoter is lost on activin treatment. These results indicate that activin-induced repression of GH biosynthesis is mediated by the loss of tissue-specific transcription factor binding to the GH promoter and suggest a possible general mechanism for other activin responses, whereby activin regulates the function of other POU- or homeodomain-containing transcription factors.
Proc Natl Acad Sci U S A 1992 Dec 01
PMID:Activin inhibits binding of transcription factor Pit-1 to the growth hormone promoter. 145 33

The cytokine tumor necrosis factor alpha (TNF-alpha) alone does not induce class II major histocompatibility complex (MHC) expression in most primary cells but can regulate ongoing class II expression in either a positive or negative fashion. The mechanism(s) by which TNF-alpha enhances interferon gamma (IFN-gamma)-induced class II expression was examined in a primary cell type, the astrocyte, by transient transfection of the HLA-DRA promoter linked to a chloramphenicol acetyltransferase reporter gene (DRA-CAT). We show that TNF-alpha, while having no effect on its own, can synergize with IFN-gamma to increase the level of promoter activity of a DRA-CAT construct. Three known sequences--W, X, and Y--are required for TNF-alpha enhancement of IFN-gamma-induced promoter activity. The corollary effect of TNF-alpha on DNA-binding proteins specific for these elements was examined. A previous report described a DNA-binding protein, IFN-gamma-enhanced factor X (IFNEX), which is upregulated by IFN-gamma in astrocytes and is specific for the X box of the DRA promoter. In this study, we found that TNF-alpha alone did not induce any nuclear proteins; however, combined treatment of astrocytes with both IFN-gamma and TNF-alpha induced a DNA-protein complex of slower electrophoretic mobility than IFNEX. The TNF-alpha-induced complex (TIC-X) has specificity for the X element of the DRA promoter. These results suggest a mechanism by which TNF-alpha enhances IFN-gamma-induced class II MHC expression via the formation of TIC-X.
Proc Natl Acad Sci U S A 1992 Dec 01
PMID:Tumor necrosis factor alpha response elements in the HLA-DRA promoter: identification of a tumor necrosis factor alpha-induced DNA-protein complex in astrocytes. 145 41

Oncogenic activation of ras results in changes in the transcription of several genes leading to uncontrolled cell growth. In this paper, we demonstrate that transformation of fibroblast cells by the ras oncogene leads to transcriptional repression of the smooth muscle alpha-actin promoter. Transient transfection analysis of plasmids containing the 5' upstream region of the human alpha-actin gene fused to human growth hormone or bacterial chloramphenicol acetyltransferase coding sequences into Rat-2 and ras-transformed Rat-2 (HO6) cells indicates that alpha-actin promoter is repressed in ras-transformed cells. In addition, stable rat fibroblast cell lines expressing human growth hormone or beta-galactosidase under the control of alpha-actin promoter exhibit repressed reporter gene activity following transformation by the ras oncogene. alpha-Actin promoter-driven beta-galactosidase activity is derepressed in revertants of ras-transformed stable cell lines. This revertant cell line expresses elevated levels of ras p21 protein and is resistant to retransformation by Ki and Ha-ras oncogenes. The revertant may have either a defective target protein whose activity is essential for the transforming activity of ras or an activated tumor suppressor gene which can suppress the activity of ras. These results indicate that smooth muscle alpha-actin promoter activity is a sensitive marker to follow phenotypic changes following transformation by ras and subsequent reversion. The advantages of this alpha-actin promoter-reporter gene assay system to screen for drugs that inhibit the transforming activity of ras, either directly or indirectly, are discussed.
Cancer Res 1992 Dec 15
PMID:Regulation of smooth muscle alpha-actin promoter in ras-transformed cells: usefulness for setting up reporter gene-based assay system for drug screening. 145 76

The effects of addition of exogenous spermidine and spermine and of two inhibitors of polyamine biosynthesis, alpha-difluoromethylornithine (DFMO), which decreases spermidine concentrations, and n-butyl-1,3-diaminopropane, which depletes spermine, on the expression of S-adenosylmethionine decarboxylase (AdoMetDC) activity were studied in mammalian cell lines (HT29, CHO and COS-7). AdoMetDC levels were inversely related to the polyamine content, and spermine was the more potent repressor of AdoMetDC activity, but only spermidine affected the amount of AdoMetDC mRNA. Transfection of COS-7 cells or CHO cells with plasmid constructs containing a chloramphenicol acetyltransferase (CAT) reporter gene driven by portions of the AdoMetDC promoter region indicated that CAT expression was altered by spermidine, but not by spermine, suggesting that there is a spermidine-responsive element in this promoter. Transient transfection of COS-7 cells with pSAMh1, a plasmid containing the AdoMetDC cDNA in a vector with the SV40 promoter and origin of replication, led to a large increase in AdoMetDC expression. Although treatment of COS-7 cells with n-butyl-1,3-diaminopropane greatly increased endogenous AdoMetDC activity, the spermine depletion brought about by this inhibitor did not stimulate AdoMetDC expression from pSAMh1. The pSAMh1 cDNA is missing 72 nucleotides from the 5' end of the AdoMetDC mRNA, and it is possible that translational regulation by spermine involves this region. The expression of AdoMetDC from pSAMh1 in COS-7 cells was greatly inhibited by DFMO treatment, although endogenous AdoMetDC activity was increased. The expression of other plasmids containing the SV40 origin of replication was also inhibited by DFMO in COS-7 cells, but not in CHO cells. DFMO treatment did not interfere with the expression of plasmids driven by the RSV promoter. These results suggest that low spermidine levels interfere with the replication of plasmids containing the SV40 origin of replication.
Biochem J 1992 Dec 01
PMID:Regulation of S-adenosylmethionine decarboxylase activity by alterations in the intracellular polyamine content. 146 54

The human prothrombin gene is expressed predominantly in hepatocytes. Previous work indicated that this tissue specificity is transcriptionally regulated. In order to identify the cis-acting regulatory elements in the 5' flanking region of the human prothrombin gene which may direct the expression of prothrombin in hepatocytes, a series of hybrid plasmids were constructed linking portions of the 5' flanking region of the human prothrombin gene to the bacterial chloramphenicol acetyltransferase gene. Expression of these hybrid plasmids was examined in calcium phosphate-mediated transient transfections of HepG2 cells, a human hepatoblastoma cell line which expresses prothrombin, and HeLa cells, an adenocarcinoma cell line which does not express detectable amounts of prothrombin. Both the prothrombin promoter and an upstream regulatory region containing sequence homologous to the hepatocyte nuclear factor 1 (HNF-1) binding site (nucleotides -919 to -790 relative to the prothrombin transcription initiation site) were required for expression in HepG2 cells. The upstream region also exhibited non-tissue-specific enhancer activity. Gel mobility shift assays confirmed cell-type-specific differences in the protein-DNA interactions between proteins in HepG2 or HeLa nuclear extracts and either the promoter region or the upstream regulatory region of the gene.
Biochemistry 1992 Dec 15
PMID:The human prothrombin gene: transcriptional regulation in HepG2 cells. 146 33

RNA molecules were prepared that consisted of an mRNA encoding chloramphenicol acetyltransferase embedded within the sequence of midivariant RNA, which is a template for the RNA-directed RNA polymerase Q beta replicase. These recombinant RNAs were shown to be bifunctional: they are amplified exponentially by incubation with Q beta replicase, and the replicated RNA serves as template for the cell-free synthesis of enzymatically active chloramphenicol acetyltransferase. The availability of amplifiable mRNAs will enable relatively large amounts of protein to be synthesized in vitro.
Proc Natl Acad Sci U S A 1992 Dec 15
PMID:Amplifiable messenger RNA. 146 96

We have isolated the avian gene T64 corresponding to the mammalian clusterin, on the basis of high accumulation of its template mRNA in cells infected with oncogenic retroviruses. Since the clusterin was shown to have a protective effect against the immune system, its induction by oncogenic viruses is of major biological importance. The unique, short 5 kb-long T64 genomic locus is inactive in normal quail embryo fibroblasts in primary culture whereas it shows a high transcriptional activity after transformation by the Rous sarcoma virus. The 963 bp-long 5' flanking region is sufficient to drive the transcription of the chloramphenicol acetyltransferase reporter gene in a thermodependent manner when a thermosensitive version of pp60v-src is used. Deletion and point mutation analyses of the promoter show that the v-src response requires at least two separate elements: PUR and AP-1, located respectively at positions -167 to -152 and -25 to -19 relative to the single transcription initiation site. In addition, the binding of specific nuclear factors to these responsive elements correlates with the T64 promoter activation.
Nucleic Acids Res 1992 Dec 11
PMID:V-src-induced-transcription of the avian clusterin gene. 147 99

We have recently isolated a functional promoter encoding the human polypeptide-binding protein (BiP) gene from Burkitt's lymphoma cells by polymerase chain reaction (The EMBL Data Library accession number X59969, 1991). This promoter DNA segment (termed BiP670) was fused to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and expressed in NIH3T3 cells. BiP670 retains basal and Ca2+ ionophore A23187-inducible activities. Using 5' deletion assay, we found three basal expression elements (BEE) in the BiP670. Removal of the distal BBE (BBE3), which is contained in a segment spanning -368/-170, caused a 50% loss of the basal activity; removal together with the middle BBE (BBE2), which is contained in a segment spanning -170/-107, resulted in a further 30% loss of the activity. Further removal of the proximal BBE (BBE1), which spans -107/-39, abolished greater than 95% of the basal expression. In addition, an A23187-inducible element (AIE) appeared to be associated with the BBE1. At least a six-fold inducibility remained as long as the BiP promoter retained the sequences -107/-39. Using an in vitro gel mobility shift assay, an A23187-inducible nuclear factor (AINF) was detected from NIH3T3 cells. DNA binding competition experiments indicate that the -107/-39 segment contains a sequence motif which interacts with this cellular factor. Further analysis showed that the two direct repeats, ranging -108/-73 and -72/-40, are the target for AINF binding. A 3-4 fold increase of the AINF binding to both repeated sequences was detected from induced cells. Similar results were also demonstrated in HeLa cells, suggesting that transcriptional control of BiP gene expression in mammalian cells is conserved. These findings also imply that the identified nuclear factor may be important in mediating transcriptional activation of the BiP gene.
Nucleic Acids Res 1992 Dec 25
PMID:A direct-repeat sequence of the human BiP gene is required for A23187-mediated inducibility and an inducible nuclear factor binding. 148 Apr 70

We have studied the 5' upstream sequences required for the transcriptional regulation of the hamster gene encoding the intermediate filament protein, vimentin. Although vimentin is regarded as the intermediate filament protein of mesothelial tissue, it is also produced in most cultured cells. The human mammary carcinoma cell line, MCF-7, belongs to the exceptions. It contains no vimentin, and the complete upstream promoter region is inactive in this particular cell line. By using transient transfection of chimeric constructs into MCF-7 and HeLa cells, and subsequent chloramphenicol acetyltransferase assays, we were able to show the presence of two negative control regions flanking a double AP-1 enhancer element. Our data indicate that these elements exert their effect irrespective of orientation and position, suggesting that they are silencers. In vitro footprinting assays, gel mobility assays and Southwestern (protein-DNA) blotting revealed the presence of trans-acting factors interacting with both silencer elements. The silencing effect was particularly pronounced in MCF-7 cells, although DNA-binding proteins are present in HeLa cells as well.
Gene 1992 Dec 15
PMID:Identification of two silencers flanking an AP-1 enhancer in the vimentin promoter. 148 48

The effect of glucocorticoids on the regulation of stably transfected human glucocorticoid receptors has been examined. Exposure of a Chinese hamster ovary-derived cell line containing stably transfected human glucocorticoid receptor genes and glucocorticoid-responsive dihydrofolate reductase genes to 5 nM dexamethasone resulted in a rapid, time-dependent reduction in the level of glucocorticoid receptor protein to 50% of control levels within 5 h of steroid treatment. This decrease in receptor protein was persistent, with a maximal 70% reduction observed even after 4 weeks of dexamethasone treatment. Immunocytochemical analysis of the influence of dexamethasone on stably transfected glucocorticoid receptors revealed efficient translocation of receptors to the nucleus within 1 h of hormone treatment. However, upon longer exposure to dexamethasone (5 h), immunoreactive glucocorticoid receptors were localized primarily to the cytoplasm. By 24 h of treatment, glucocorticoid receptors were absent from the cytoplasm and the nucleus, suggesting that the ligand-induced loss of glucocorticoid receptors may be a cytoplasmic event. The decrease in transfected glucocorticoid receptor protein was largely reflected by similar changes in steady state levels of human glucocorticoid receptor mRNA; however, the effects of hormone on receptor protein levels were more profound than on receptor mRNA. There was an initial rapid reduction in transfected glucocorticoid receptor mRNA to 50% of control levels within 2 h of dexamethasone treatment. This reduction was followed by a transient rise in mRNA expression after 12 h of hormone treatment. With prolonged exposure to dexamethasone (> 12 h) a second, more gradual decline in human glucocorticoid receptor mRNA was observed. This biphasic pattern of glucocorticoid receptor gene expression was not reflected at the level of receptor protein, suggesting that both transcriptional and translational control mechanisms may be involved in ligand-dependent receptor regulation. When cells were removed from dexamethasone after up to 48 h of treatment, glucocorticoid receptor mRNA levels fully recovered within 12 h. Receptor protein recovered only partially during this same time period. Down-regulation of glucocorticoid receptor protein and mRNA levels by dexamethasone in stably transfected cells led to corresponding reductions in the hormone sensitivity to two glucocorticoid-regulated genes: a transiently transfected chloramphenicol acetyltransferase receptor gene and a stably integrated dihydrofolate reductase gene. These results demonstrate that stably transfected human glucocorticoid receptors are subject to ligand-induced down-regulation in a heterologous cell line. Moreover, glucocorticoid receptor autoregulation appears to be a highly conserved mechanism for attenuating cellular responsiveness to hormone.
Mol Endocrinol 1992 Dec
PMID:Ligand-dependent down-regulation of stably transfected human glucocorticoid receptors is associated with the loss of functional glucocorticoid responsiveness. 149 90


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