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 ability of the glucocorticosteroid receptor to bind mineralocorticosteroids suggests that spironolactone, a potent aldosterone antagonist, may also interact with the glucocorticosteroid receptor, resulting in an agonist or antagonist glucocorticosteroid activity. We have investigated the effect of this drug on the activity of the glucocorticosteroid-regulated mouse mammary tumor virus (MMTV) promoter. For these studies we used the mouse fibroblast cell line 1471.1. It contains about 200 copies of a permanently established chimeric DNA construct comprising a transcription unit [MMTV long terminal repeat (LTR)] driving the reporter gene chloramphenicol acetyltransferase linked to the 69% transforming fragment of the bovine papilloma virus genome. This cell line has a high level of glucocorticosteroid receptor (1200 fmol/mg protein) and no detectable mineralocorticosteroid receptor. Competition experiments showed a binding of spironolactone to glucocorticosteroid receptor, with an affinity 50-fold lower than that of dexamethasone. In these cells, spironolactone behaves as an antiglucocorticosteroid, inhibiting in a dose-dependent fashion dexamethasone-induced chloramphenicol acetyltransferase activity, with an ED50 of 8 microM. The absence of agonist activity, even at a high concentration of this compound (10 microM), demonstrates that spironolactone is a pure antiglucocorticosteroid in this cell line. MMTV LTR DNase-I hypersensitivity studies demonstrated that spironolactone, when administered in combination with dexamethasone, inhibits formation of the hormone-induced hypersensitive site located about 160 basepairs up-stream of the MMTV cap site. Furthermore, spironolactone alone failed to induce this DNase-I-hypersensitive site, suggesting that the antagonist-receptor complex does not interact productively with MMTV LTR chromatin.
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PMID:Spironolactone, an aldosterone antagonist, acts as an antiglucocorticosteroid on the mouse mammary tumor virus promoter. 130 41

The long control region (LCR) of bovine papilloma-virus type 4 demonstrated enhancer activity when cloned upstream of a bacterial chloramphenicol acetyltransferase reporter gene under thymidine kinase promoter control. Deletion analysis of the LCR revealed the presence of several positive and negative control elements, all of which could function independently of the viral E2 trans-activator. Each of the three positive elements present appeared to be paired with a negative element which modulated its activity. DNase I footprinting was used to identify protein binding sites within the LCR, which might represent these control elements. The results suggest a highly complex and finely tuned control of viral gene expression.
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PMID:Positive and negative E2-independent regulatory elements in the long control region of bovine papillomavirus type 4. 184 70

To investigate whether DNA viruses can augment gene expression of the human immunodeficiency virus (HIV), cotransfection experiments were carried out in which a recombinant plasmid containing the HIV long terminal repeat (LTR) linked to the chloramphenicol acetyltransferase (CAT) gene was transfected into cultured cells along with plasmids containing DNA from various distinct classes of DNA viruses. Molecular clones containing JC virus, BK virus, lymphotropic papovavirus, bovine papilloma virus, type 1 herpes simplex virus (HSV-1), and varicella-zoster virus sequences increased CAT expression directed by the HIV LTR. Trans-activation of the HIV LTR varied in different cell lines, but in each case the HIV tat gene product elicited the greatest stimulation. Primer-extension assays specific for HIV LTR mRNA revealed increased levels of steady-state RNA following transfection with HIV tat as well as with several of the DNA viruses. Virus-specific RNA expression paralleled the stimulation of CAT activity. More-than-additive effects were observed at both the RNA and protein levels when tat plus type 1 herpes simplex virus DNAs or tat plus JC virus DNAs were transfected into cells with the HIV LTR-CAT plasmid. These data suggest that coinfection of cells by HIV and some DNA viruses can stimulate the expression of HIV.
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PMID:Trans-activation of the human immunodeficiency virus long terminal repeat sequence by DNA viruses. 243 2

The upstream regulatory region of the human papilloma virus-16 (HPV-16) genomic DNA contains a sequence element with a large degree of homology to the partially palindromic sequence GGTACANNNTGTTCT, which is the consensus sequence of the glucocorticoid responsive elements of known genes regulated by this steroid hormone. DNase I and dimethylsulfate protection experiments reveal the binding of this sequence by rat glucocorticoid receptor protein. A 400-bp DNA segment centrally containing this sequence confers strong inducibility by dexamethasone to the promoter p97 of HPV-16 and to the Herpes simplex virus thymidine kinase promoter, as judged by chloramphenicol acetyltransferase activity and RNase protection assays. The same DNA segment, that does not contain the consensus sequences of all papilloma viruses relevant for E2 protein-mediated transcription enhancement, functions in an enhancer-like fashion in addition to its glucocorticoid responsive action. This hormone-independent transcription enhancement is absent in human MCF7 cells, but is strong in human HeLa cells where the combined activity of the constitutive and the steroid hormone-dependent enhancer elements stimulate transcription by a factor of 500. This cell type specificity of the HPV-16 enhancer may be responsible for the tissue tropism of the virus. These observations and the presence of numerous homologies to known enhancers of cellular and viral genes suggest a complex pattern of activation of the human papilloma virus-16 promoters.
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PMID:The upstream regulatory region of the human papilloma virus-16 contains an E2 protein-independent enhancer which is specific for cervical carcinoma cells and regulated by glucocorticoid hormones. 282 35

We have previously reported the presence of a variant of human papillomavirus (HPV) type 11 in a nasal inverting papilloma [Respler et al., 1987]. In the present study, we have cloned molecularly the DNA of this variant at its unique restriction enzyme Bam HI site into lambda BF101 phage. Restriction enzyme mapping and DNA sequencing revealed that the genome of this virus contained an extra 531 base pair (bp) which was the repeat of most of the noncoding region (ncr) of HPV 11. Insertion of transcriptional control elements, including the repeated sequence, in front of the chloramphenicol acetyltransferase (CAT) gene resulted in a 5- to 30-fold increase in expression in transfected cells, as compared to the constructs containing a single ncr of HPV 11. This increased expression was due to enhanced levels of CAT RNA the synthesis of which is initiated by the viral promoter element.
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PMID:Isolation and characterization of a variant of human papillomavirus type 11 from a nasal inverting (Schneiderian) papilloma. 283 8

We have mapped a transcriptional regulatory sequence within the 1.0 kb noncoding region of the bovine papilloma virus (BPV-1) genome, using an enhancer dependent expression vector for chloramphenicol acetyltransferase. This transcriptional regulatory element works independently of position and orientation, and its function is significantly augmented in BPV-1 transformed C127 cells and in monkey CV-1 cells acutely transfected with plasmids expressing BPV-1 early gene products. Using defined deletion mutants of the BPV-1 DNA and full-length viral cDNAs expressed from an SV40 early promoter, we demonstrate that the expression of this trans-activating factor maps to the 3' open reading frames of the viral transforming region. A premature termination codon engineered into the E2 ORF eliminates expression of this diffusible transactivation function establishing the E2 gene product as the diffusible trans-activating factor.
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PMID:Transactivation of a bovine papilloma virus transcriptional regulatory element by the E2 gene product. 299 Jul 24

We have studied the extrachromosomal maintenance and the transcription regulation of two glucocorticoid-inducible genes on bovine papilloma virus (BPV) vectors in c127 mouse fibroblasts. These genetic elements were the rat tryptophan oxygenase (TOase) gene promoter, which is active in vivo only in hepatocytes, and the long terminal repeat of the mouse mammary tumor virus (MMTV-LTR). From both genes, fusions of the 5'-flanking region of the transcription unit to the bacterial gene for chloramphenicol acetyltransferase (CATase) were constructed. These fusion genes were inserted either into pCGBPV9, a BPV vector encoding G418 resistance, into pBPV-BV1, a vector containing "stabilizing" segments of the human beta-globin gene, or into a BPV construct, whose bacterial plasmid sequences could be removed before transfection. Five constructs of the two latter groups, selectable in c127 cells only as foci, were normally maintained in the extrachromosomal state. In contrast, three out of five constructs based on pCGBPV9 and selectable for resistance against G418 were maintained in a high molecular weight form, most probably of intrachromosomal concatemeric nature, while the remaining two G418-resistant constructs appeared alternatively in this or the extrachromosomal monomeric form. In contrast to its absence of expression in fibroblasts in vivo, the TOase gene element present on BPV vectors was found to be active in fibroblasts in these transfection experiments. As judged by CATase activities and for TOase also by mapping of the transcription start sites, transcription of both genes was under hormonal regulation. All BPV vectors proved to be useful tools in the study of these regulated genes, and in only one out of ten constructs was regulation atypical, possibly due to effects from flanking vector sequences.
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PMID:Physical state, expression and regulation of two glucocorticoid-controlled genes on bovine papilloma virus vectors. 301 94

The retinoic acid (RA) signaling pathway was investigated by transient transfection of a chloramphenicol acetyltransferase (CAT) reporter gene construct containing the RA response element (RARE) of the murine (m) RARbeta2 gene into murine primary epidermal keratinocytes (PEK), papilloma-derived SP1 cells, and carcinoma-derived 3P2 cells. Murine PEK transfected in a low-Ca2+ medium (0.05 mM Ca2+) exhibited a strong transactivation of the CATgene after exposure of the cells to 0.1 microM RA. Transactivation of the CATgene could, however, also be achieved by shifting RAREbeta2-transfected low-Ca2+ PEK to high-Ca2+ conditions (0.15-1.2 mM Ca2+). Concomitantly, the Ca2+ raise also led to the induction of both cellular retinol (ROL)-binding protein I (CRBPI) and cellular RA-binding protein II (CRABPII), whereas expression of cellular RA-binding protein I (CRABPI) was not observed. Moreover, induction of in vitro differentiation also activated the ROL-->RA converting enzyme system in PEK. These findings suggest the following sequence of events involved in the high Ca2+-mediated activation of RAREbeta2. First, high Ca2+ induces the synthesis of mCRBPI, which binds ROL released from retinyl ester stores and makes it accessible to the ROL-RA converting enzyme system. Enzymatically generated RA is taken over by mCRABPII and transported to the nucleus, where it acts as ligand for nuclear receptors, which complex with RAREbeta2 to activate the reporter gene. This hypothetical cascade of RA signaling was supported by our findings that inhibition of the ROL-->RA converting enzyme system by citral abolished the Ca2+-mediated transactivation of the CAT gene in a nontoxic manner. Studies in transformed murine cell lines revealed that Ca2+-induced activation of RAREbeta2 was essentially maintained in papilloma-derived SP1 cells, although all parameters of the Ca2+-dependent RAREbeta2 activation cascade were induced to a much lower extent. In contrast, strong RAREbeta2 activity was already observed in low-Ca2+ carcinoma-derived 3P2 cells. Low-Ca2+ 3P2 cells also expressed high levels of both mCRBPI and mCRABPII and possessed a highly active ROL-->RA converting enzyme system. Again, inhibition of the enzyme by citral abolished RAREbeta2 activity in low-Ca2+ 3P2 cells. Our data show that Ca2+-induced differentiation in cultured murine PEK entails a series of events that ultimately lead to the activation of RARE-containing genes. These properties are maintained in transformed epidermal keratinocytes. However, with increasing malignant potential of the cells, the respective signaling pathway becomes independent from a differentiation stimulus and leads to constitutive activation of RARE-controlled genes.
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PMID:Retinoic acid signaling cascade in differentiating murine epidermal keratinocytes: alterations in papilloma- and carcinoma-derived cell lines. 932 36

Two genomic clones comprising the entire coding sequence of mouse SPRR1A and 1B genes were isolated and sequenced. Sequence analysis of the 1A and 1B genomic clones indicated that both genes contain two exons separated by an intron slightly larger than 1.1kb in size (1147 and 1152nt, respectively). This type of genomic structure is identical to the counterpart of human SPRR1 gene and other genes encoding for cornified envelope proteins. Primer extension analysis using 1A and 1B gene-specific primers indicates that 1A gene is expressed in squamous tissues such as skin and esophagus, whereas 1B gene is expressed in papilloma tumors but not in squamous tissues. The first 300nt of 5'-flanking region of the mouse SPRR 1A and 1B genes reveal an overall approximately 50% identity to the human counterpart. However, there is a high degree of identity (?75%) at the promoter region containing a TATA box and TRE/TRE-like motifs. Both TATA and TRE/TRE-like motifs are almost identical in sequence and positions to those found in the counterpart of human promoter. Using transient transfection for the analysis of promoter activity, we observed that both 1A and 1B 5'-flanking regions contain the promoter activity to direct the expression of the reporter gene, chloramphenicol acetyltransferase, in airway epithelial cells in a fashion similar to that observed in the human counterpart. These results indicate a conserved nature of genetic structure and regulation of SPRR1 gene expression between human and mouse.
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PMID:Structure and organization of the genes encoding mouse small proline-rich proteins, mSPRR1A and 1B. 993 36

Under ICH guidelines, short-term carcinogenicity assays such as the Tg.AC assay are allowed alternatives for one species in the 2-year rodent bioassay. The Tg.AC transgenic mouse, which carries the v-Ha-ras oncogene under control of the zeta-globin promoter, develops skin papillomas in response to dermal application of carcinogens and tumor promoters. The appropriate specificity of the Tg.AC model for testing pharmaceuticals has not been systematically evaluated. The selection of candidate test compounds among noncarcinogenic pharmaceuticals would be aided by a high-throughput in vitro prescreen correlative of activity in the in vivo Tg.AC assay. Here we describe the development of a prescreen based on correct response to 24 compounds tested previously in Tg.AC mice. The in vitro prescreens, chosen to reflect molecular pathways possibly involved in Tg.AC papilloma formation, consisted of a zeta-globin promoter-luciferase construct stably expressed in K562 cells (Zeta-Luc) and three of the stress-response element-chloramphenicol acetyltransferase (CAT) fusion constructs stably expressed in HepG2 cells that are part of the CAT-Tox (L)iver assay. The stress response elements chosen were the c-fos promoter, the gadd153 promoter, and p53 response element repeats. Of the four assays, the gadd153-CAT assay showed the strongest concordance with activity in the Tg.AC assay, correctly classifying 78% of Tg.AC positive and 83% of Tg.AC negative compounds. The correlation was further improved by adding the Zeta-Luc assay as a second-stage screen. These cell-based assays will be used in a novel approach to selecting candidate compounds that challenge the specificity of the Tg.AC assay toward pharmaceuticals.
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PMID:Evaluation of in vitro reporter gene induction assays for use in a rapid prescreen for compound selection to test specificity in the Tg.AC mouse short-term carcinogenicity assay. 1096 10


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