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)

Transcription of the gene coding for serine dehydratase (SDH, EC 4.2.1.13) in the rat in vivo is dramatically increased by glucocorticoid hormones. To identify DNA elements mediating the glucocorticoid-regulated expression of the SDH gene, we transiently transfected 7AD-7 rat hepatoma cells with fusion genes consisting of various regions of the SDH 5' flanking sequence linked to the coding sequence of the gene for chloramphenicol acetyltransferase (CAT). Analysis of the CAT activities from these 5' deletion mutants identified three closely associated glucocorticoid-responsive elements (GREs), located more than 5 kb upstream relative to the cap site. Two distal GREs act synergistically to confer strong glucocorticoid inducibility to the gene, whereas the proximal GRE functions independently of the distal GREs and confers only a weak hormone response to the gene. The purified DNA-binding domain of rat glucocorticoid receptor binds to the sequence of each GRE as shown by footprinting experiments. However, only one of these sequences contains the TGTTCT consensus sequence reportedly associated with many other GREs.
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PMID:Location and characterization of multiple glucocorticoid-responsive elements in the rat serine dehydratase gene. 149 43

We have replaced the polyomavirus (Py) enhancer, which is an essential component of the Py origin of DNA replication (ori), with five repeats of a 17-bp oligonucleotide including the yeast GAL4 upstream activating sequence (5xGAL4 sites). Plasmids containing this modified Py ori, designated test plasmids, and plasmids encoding either the GAL4 transcriptional activator protein or various derivatives of this protein were cotransfected into mouse cells which constitutively synthesize a temperature-sensitive Py large tumor antigen (T-Ag). Replication of the test plasmids was monitored by Southern blot determinations of the amounts of plasmid DNA that became resistant to cleavage by the enzyme DpnI. These studies showed that in the presence of a functional T-Ag, the GAL4 protein, and hybrid proteins including the GAL4 DNA-binding domain and the activating domain of the adenovirus E1a or herpesvirus VP16 protein transactivated the modified Py ori. A truncated protein including just the GAL4 DNA-binding domain was inactive in these assays. The authentic GAL4 protein was found to be a more efficient replication transactivator than the hybrid proteins. In contrast, chloramphenicol acetyltransferase assays showed that the hybrid proteins were more efficient transcriptional activators than the GAL4 protein. The extent of the GAL4-dependent replication of a plasmid in which the Py early promoter was deleted was 55% lower than that of a plasmid including the promoter. However, the extents of replication of plasmids including two tandem repeats of the remaining Py origin core and 5xGAL4 sites or two origin cores flanking a single cluster of 5xGAL4 sites were 4.8- and 1.6-fold higher than that of the plasmid including a single copy of each element. The replication of a plasmid including two clusters of 5xGAL4 sites flanking a single origin core was below the limit of detection of our assays. These results indicate that the GAL4 and hybrid transactivators do not activate the Py ori by virtue of their interactions with transcription factors that bind promoter elements. Rather, it appears that these activator proteins may interact with the replication initiation complexes, thereby facilitating or inhibiting the initiation of replication.
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PMID:The yeast GAL4 protein transactivates the polyomavirus origin of DNA replication in mouse cells. 164 81

H-2RIIBP is a member of the nuclear hormone receptor superfamily that binds to the region II enhancer of major histocompatibility complex (MHC) class I genes. The binding occurs through the GG(T/A)CA motif present also in many other genes. The role of H-2RIIBP in developmental regulation of MHC class I genes has been studied in undifferentiated N-Tera2 embryonal carcinoma cells by transient cotransfection of an expressible H-2RIIBP plasmid and a chloramphenicol acetyltransferase reporter gene linked to the MHC class I promoter. Transfection of the expression plasmid led to production of H-2RIIBP transcripts and enhanced MHC class I promoter activity in cells that were treated with retinoic acid but not yet differentiated. Retinoic acid concentrations required for transactivation overlapped with those capable of inducing morphological differentiation and expression of endogenous MHC class I genes in these cells. This enhancement was mediated by region II, as a heterologous thymidine kinase promoter driven by region II also served as a target for H-2RIIBP transactivation. Deletion of the bulk of the DNA-binding domain or the ligand-binding domain of H-2RIIBP, but not of the N-terminal domain, abolished transactivation, indicating that the former two domains are critical for the enhancement. Moreover, H-2RIIBP transactivation exhibited a strict cell-type restriction. As observed in other cell lines, N-Tera2 cells that had undergone differentiation failed to elicit transactivation, suggesting that H-2RIIBP acts in concert with a cofactor expressed in undifferentiated N-Tera2 cells that requires retinoic acid for its function. These results suggest that H-2RIIBP can function as a developmentally specific transcription factor for MHC class I genes.
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PMID:Retinoic acid-dependent transactivation of major histocompatibility complex class I promoters by the nuclear hormone receptor H-2RIIBP in undifferentiated embryonal carcinoma cells. 173 9

We present evidence that CRE-BP1 binding to the cyclic AMP (cAMP) response element (CRE) is a transcriptional activator. Transcriptional activation was assayed by cotransfection into CV-1 cells of a CRE-BP1 expression plasmid together with a reporter plasmid in which the thymidine kinase promoter and four tandem repeats of CRE were linked to the chloramphenicol acetyltransferase (CAT) gene. Cotransfection with the CRE-BP1 expression plasmid caused an 8-fold stimulation of CAT activity, while cotransfection with the plasmids to express CRE-BP1 and c-Jun induced a 32-fold stimulation of CAT activity, suggesting that a heterodimer of CRE-BP1 with c-Jun is a stronger trans-activator than a homodimer of CRE-BP1. By using a series of deletion and point mutants of CRE-BP1 in this cotransfection assay, two functional domains of CRE-BP1 were identified: the putative metal finger structure in the amino-terminal region and the leucine zipper motif linked to a cluster of basic amino acids in the carboxyl-terminal region. The former was a transcriptional activation domain in the absence of c-Jun. The latter was a DNA-binding domain, and was essential in both the presence and absence of c-Jun.
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PMID:Identification of the functional domains of the transcriptional regulator CRE-BP1. 183 93

TR3 receptor is a human homolog of mouse Nurr77 and N10 protein and the rat NGFI-B protein. A cDNA encoding a chimeric nuclear receptor composed of the N-terminal domain and C-terminal putative ligand-binding domain of the orphan receptor TR3 receptor and the DNA-binding domain of the androgen receptor was constructed. The chimeric receptor, called TR3/AR/TR3 receptor, when expressed in COS-1 monkey kidney cells or PC-3 human prostate tumor cells, cotransfected with an ARE-containing mouse mammary tumor virus long terminal repeat-linked reporter gene encoding chloramphenicol acetyltransferase (CAT), activated CAT expression in the absence of any added factor. The activation was dependent on the amount of expression vector transfected and appeared to be independent of the concentration of serum supplement. Intact TR3 receptor was not active in this system. A TR3/AR/TR3 receptor protein truncated in the putative ligand-binding domain also induced CAT activity. TR3 receptor appears to be a transcriptional factor that activates transcription independently of ligand or binds an endogenous ligand present constitutively in cultured cells.
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PMID:Transcriptional activation by TR3 receptor, a member of the steroid receptor superfamily. 184 11

Transfection of HeLa cells with cDNA vectors expressing the wild-type human glucocorticoid receptor (GR) enabled dexamethasone to strongly repress cytokine- and second messenger-induced expression of cotransfected chimeric reporter genes containing transcription regulatory DNA elements from the human interleukin 6 (IL-6) promoter. Deletion of the DNA-binding domain or of the second Zn finger or a point mutation in the Zn catenation site in the second finger blocked the ability of GR to mediate repression of the IL-6 promoter. Unexpectedly, deletion of the first Zn finger, a point mutation in the Zn-catenation site in the first finger, or one in the steroid-specificity domain at the base of the first finger converted GR into a dexamethasone-responsive activator that enhanced basal and interleukin 1-induced IL-6 promoter function. These first-finger mutants of GR also mediated dexamethasone-responsive enhancement of expression of the herpesvirus thymidine kinase-chloramphenicol acetyltransferase (TK-105-CAT and TK-80-CAT) reporter genes but not of the murine mammary tumor virus long terminal repeat-CAT or the c-fos-CAT (pFC700) reporter genes. Wild-type GR was able to specifically bind to DNA fragments containing glucocorticoid response element sequences in both the murine mammary tumor virus and IL-6 promoters, albeit weakly to the latter, in a sequential DNA-binding immunoprecipitation assay. The first-finger mutants of GR, however, were inactive in this assay. Thus, mutations in the first Zn finger unmask unusual promoter-specific activation properties of GR that may not require direct high-affinity binding of the mutant GR to target DNA.
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PMID:Repressor to activator switch by mutations in the first Zn finger of the glucocorticoid receptor: is direct DNA binding necessary? 187 Nov 24

The vRel oncoprotein is member of a family of related proteins that also includes cRel, NF-kappa B, and Dorsal. We investigated the transcriptional regulatory properties of several Rel proteins in cotransfection assays with chicken embryo fibroblasts (CEF). Retroviral vectors expressing hybrid proteins that contain the DNA-binding domain of LexA fused to portions of the viral oncoprotein vRel or chicken, mouse, human, or Drosophila melanogaster (Dorsal) cRel proteins were cotransfected with a reporter plasmid that contains the DNA sequence recognized by LexA, a promoter, and the assayable gene for chloramphenicol acetyltransferase. In transient assays, a LexA-vRel protein did not activate transcription in CEF. Full-length chicken cRel, mouse cRel, and Dorsal fusion proteins all activated transcription weakly; however, deletion of N-terminal Rel sequences from each of these proto-oncogene encoded proteins resulted in strong activation by LexA fusion proteins containing only C-terminal sequences. Inhibition of the C-terminal chicken cRel gene activation domain by N-terminal sequences was seen in CEF and mouse and monkey fibroblasts. These results show that cRel proteins from different species have the same general organization: an N-terminal inhibitory domain and a C-terminal activation domain. Sequence comparison suggests that the inhibitory domain is conserved but the activation domain is species specific. In contrast, vRel lacks a strong C-terminal gene activation function, since a LexA fusion protein containing C-terminal vRel sequences alone only weakly activated transcription. In addition, the wild-type vRel protein (lacking LexA sequences) repressed transcription from reporter plasmids containing NF-kappa B target sequences; nontransforming vRel mutants did not repress transcription from these plasmids. Our results suggest that vRel transforms cells by interfering with transcriptional activation by cellular Rel proteins.
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PMID:vRel is an inactive member of the Rel family of transcriptional activating proteins. 190 56

Immortalization of B lymphocytes by Epstein-Barr virus (EBV) is complex and poorly understood. However, some evidence suggests that glucocorticoids influence this process. We identified a glucocorticoid-responsive element in the BamHI C fragment of EBV which we call ES-1. In glucocorticoid-treated cells, ES-1 enhanced chloramphenicol acetyltransferase gene expression from the herpes simplex virus thymidine kinase promoter, as well as the EBV Bam-C promoter, from which several latent viral gene products are transcribed. By Northern blot analysis, glucocorticoid treatment enhanced transcription from the Bam-C promoter in Jijoye cells, a Burkitt's lymphoma cell line. In addition, the DNA-binding domain of the glucocorticoid receptor bound specifically to the ES-1 region. These glucocorticoid effects on the Bam-C promoter region may provide some insight into the process of EBV immortalization.
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PMID:Identification of a glucocorticoid-responsive element in Epstein-Barr virus. 215 66

The syndrome of hereditary resistance to 1,25-dihydroxyvitamin D3 is due to defective function of the vitamin D receptor (VDR). The recent cloning and nucleotide sequence determination of the human VDR chromosomal gene have enabled a direct evaluation of the genetic basis for this disease in affected patients. In this report we employed polymerase chain reaction techniques to amplify the gene exons that encode the DNA-binding domain of the VDR from two 1,25-dihydroxyvitamin D3-resistant patients whose receptors displayed defective binding to nonspecific DNA. Although their families were apparently unrelated, each patient displayed an identical homozygous point mutation within the third exon, a mutation that causes substitution of a glutamine for an arginine residue highly conserved within the entire steroid receptor superfamily. We introduced this base change into the normal VDR cDNA via site-directed mutagenesis, transfected an expression vector containing this cDNA into cells, and examined the functional properties of the resultant VDR expression product. The produced mutant receptor bound 1,25-dihydroxyvitamin D3 with normal affinity, but displayed weak affinity for the nuclear fraction and for heterologous DNA. More importantly, the protein was inactive in promoting transcription in a cotransfection assay employing a chloramphenicol acetyltransferase gene reporter fused down-stream of the VDR-inducible osteocalcin gene promoter-enhancer. These results provide the genetic and functional basis for the phenotype of rickets in this inherited disease.
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PMID:A unique point mutation in the human vitamin D receptor chromosomal gene confers hereditary resistance to 1,25-dihydroxyvitamin D3. 217 43

The human vitamin D receptor (VDR) has been cloned recently. Two cDNAs comprising the full-length VDR were spliced, cloned into a mammalian expression vector, and transiently expressed in COS-1 cells. The protein product exhibited properties consistent with that observed for receptor in human cells. A series of 5'- and 3'-deletions of the full-length VDR cDNA was prepared and evaluated. Native DNA binding was localized to a peptide fragment (residues 1-114) whose most prominent feature is the cysteine rich region proven to represent the DNA binding domain in other steroid receptors. Steroid binding-competence required synthesis of a peptide that initiated C-terminal to the DNA-binding domain at residue 114 and which contained the remaining 313 residues. To determine the location of elements within the receptor necessary for transcription, an osteocalcin gene promoter-chloramphenicol acetyltransferase reporter gene was cotransfected together with wild type or mutant VDR cDNAs and the latter's effect on chloramphenicol acetyltransferase activity was assessed. Cotransfection of wild type receptor alone resulted in efficient transcription of the reporter plasmid. However, synthesis of a peptide containing the DNA binding domain as well as 76 residues carboxy terminal to this region exhibited some degree of activity, albeit constitutive. These results suggest that the functional domains of the VDR are similar to that of other steroid receptors and that these domains participate in the transcriptional regulation of the human osteocalcin gene.
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PMID:Functional domains of the human vitamin D3 receptor regulate osteocalcin gene expression. 254 79


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