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)

Using a transient expression assay in Vero cells, we have shown that the protein product from gene 61 of varicella-zoster virus (VZV) can repress the function of the VZV encoded trans-activators on putative viral immediate-early, early, and late gene promoters. The repression is exerted at the transcriptional level and requires functional gene 61 protein. This trans-repressor is the herpes simplex type 1 ICP0 (a trans-activator) homolog, as defined by gene location, the sharing of a cysteine-rich putative zinc-binding finger in the amino-terminal region, and limited amino acid homology. Open reading frame 61 (ORF61)-mediated trans-repression appears to be specific for VZV-encoded trans-activators in that it has no effect on simian virus 40 and Rous sarcoma virus promoters. Moreover, it does not inhibit trans-activation of the human T-lymphotropic virus type I and human immunodeficiency virus long terminal repeats by tax and tat genes, respectively. We constructed plasmids with mutations in ORF61 and tested them for their ability to inhibit trans-activator (VZV genes 4 and 62)-mediated activation of the viral thymidine kinase promoter-chloramphenicol acetyltransferase construct. Mutants containing interruptions in ORF61 lost their trans-repressing ability, as demonstrated at both the protein and steady-state RNA levels. These results suggest that the ORF61 protein product can mediate down-regulation of VZV gene expression.
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PMID:Characterization of a potent varicella-zoster virus-encoded trans-repressor. 165 42

The chloramphenicol acetyltransferase-encoding reporter gene (cat) is used extensively in assessing the ability of transcriptional regulatory elements (TRE) to direct gene expression in eukaryotic cells. Two commonly utilized plasmids contain the cat coding sequences under the transcriptional control of the Rous sarcoma virus LTR (pRSVcat) or simian virus 40 early (SV40E) promoter (pSV2cat). In the present study, we have recloned the RSV-LTR and SV40E TRE into a pUC18 vector. Direct comparison of these TRE in different plasmid vectors, as well as reevaluation of their relative level of cat expression revealed: (1) a small but significant increases in SV40E-directed reporter gene expression was observed when the TRE was inserted into the pUC18 vector; and (2) a significant increase in SV40E-directed gene expression was realized by inclusion of the 69-bp 5' of the sequences present in pSV2cat. These distal sequences are required for maximal activity of the SV40 TRE in the cell lines tested.
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PMID:Importance of SV40 early 5' distal sequences in directing heterologous gene expression. 165 24

Rous sarcoma virus-based retroviral vectors were constructed to compare three different approaches for coexpressing two genes in individual infected cells. All vectors expressed the upstream gene (lacZ) from the Rous sarcoma virus long terminal repeat, while the downstream gene (the chloramphenicol acetyltransferase gene [cat] or v-src) was expressed in one of three ways: from a subgenomic mRNA generated by regulated splicing, from a strong internal promoter, or from the encephalomyocarditis virus internal ribosome entry site (IRES). Both biochemical and immunohistochemical assays of cultured cells showed that the encephalomyocarditis virus IRES provided the most efficient means for coexpressing two genes from a single provirus. Most importantly, most cells infected by a LacZ-IRES-CAT virus expressed both LacZ and CAT, whereas most cells infected by internal promoter or regulated splicing vectors expressed either LacZ or CAT but not both. In addition, viral titers were highest with IRES vectors. Presumably, use of the IRES avoids transcriptional controls and RNA processing steps that differentially affect expression of multiple genes from internal promoter and regulated splicing vectors. Finally, we injected a LacZ-IRES-v-Src virus into chicken embryos and then identified the progeny of infected cells with a histochemical stain for LacZ. LacZ-positive cells in both skin and mesenchyme displayed morphological abnormalities attributable to expression of v-src. Thus, IRES vectors can be used to coexpress a reporter gene and a bioactive gene in vivo.
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PMID:The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos. 165 18

Glucocorticoid receptors are ligand-dependent transcription factors that are subject to down-regulation by their cognate ligand; however, the mechanisms mediating this physiological response are not completely understood. Since analysis of the human glucocorticoid receptor (hGR) cDNA sequence revealed the presence of sequences with homology to both positive and negative glucocorticoid regulatory elements, we have examined the potential of hGR to bind to the hGR cDNA by Southwestern blot analysis. The data revealed that glucocorticoid receptors exhibited specific binding to their own cDNA. To determine whether this binding was of functional significance in the down-regulation of glucocorticoid receptors, we analyzed the effect of glucocorticoids on hGR protein levels from COS 1 cells transfected with an hGR cDNA expression vector. These transfected cells produced intact hGR that were capable of ligand-dependent regulation of a co-transfected glucocorticoid-responsive reporter gene. Glucocorticoid treatment of hGR-transfected cells resulted in down-regulation of hGR (assayed by both glucocorticoid binding capacity and hGR protein levels) within 24 h of steroid administration. To determine if the glucocorticoid-induced down-regulation of transfected hGR was compatible with effects at the levels of receptor gene expression and RNA stability, we examined hGR mRNA steady state levels. Reductions from 2- to 6-fold were observed in hGR mRNA levels following glucocorticoid treatment of transfected COS 1 cells. This down-regulation of transfected hGR mRNA could not be attributed to either the Rous sarcoma virus promoter, which drives hGR expression, or to other sequences present in the vector plasmid since transcription of a related plasmid containing a chloramphenicol acetyltransferase gene in place of the hGR cDNA was not regulated by glucocorticoids. Down-regulation of hGR mRNA by glucocorticoids in transfected cells occurred in a time- and dose-dependent manner that is consistent with a glucocorticoid receptor-mediated process. Glucocorticoid-induced down-regulation of hGR mRNa steady state levels was not observed in COS 1 cells transfected with cDNAs encoding mutant hGR (defective in either steroid or DNA binding), which indicates that functional steroid and DNA binding domains of the expressed hGR were required for down-regulation. Interestingly, treatment of transfected COS 1 cells with the glucocorticoid antagonist RU486 also resulted in down-regulation of transfected hGR mRNA. Deletion analysis revealed that the region of the hGR cDNA that was responsible in part for the observed down-regulation in response to glucocorticoid was contained within a 1-kilobase restriction fragment (from base pair +527 to +1526).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Human glucocorticoid receptor cDNA contains sequences sufficient for receptor down-regulation. 169 20

The macrolide FK-506, like the cyclic undecapeptide cyclosporin A (CsA), is a potent immunosuppressant that interferes with the transcriptional activation of several early-phase genes in T lymphocytes, including that for interleukin-2 (IL-2). We compared the effects of FK-506 and CsA on transcription from the 5' upstream activating sequences (UAS) of the human IL-2 gene and several cellular and viral UAS to define cis-acting sites which may be responsive to FK-506. The UAS surveyed included the human IL-2 receptor alpha-chain, human metallothionein II, simian virus 40 early, human cytomegalovirus immediate-early, adenovirus major late, and Rous sarcoma virus long terminal repeat UAS. In addition, we studied multimers of several defined promoter elements (NFIL-2A, NF-kappa B, or NF-AT1) which are found in the UAS of the human IL-2 gene and which have been reported to be responsive to CsA when linked to a minimal promoter element (TATA box and transcription start site). Each promoter-regulatory region was fused to the bacterial chloramphenicol acetyltransferase gene and used to transiently transfect Jurkat cells. Quantitative chloramphenicol acetyltransferase assay determinations indicated that the transcriptional activity of each UAS induced upon T-cell activation was (i) completely sensitive, (ii) partially sensitive, or (iii) resistant to inhibition by CsA and FK-506. The induced transcription driven by the IL-2 promoter elements NF-AT1 and NFIL-2A could be blocked completely by FK-506 or CsA. Gel mobility shift assays indicated that the binding activities of the factors specifically interacting with these sequences were detected in activated cells regardless of whether the cells were treated with FK-506 or CsA. The results suggest that FK-506 or CsA inhibits a transacting mechanism(s) without disrupting the binding activities of these transcription factors. The degree to which each UAS was resistant to FK-506 was consistent with the level of transcription induced by phorbol myristate acetate, while UAS which were sensitive to inhibition by FK-506 were dependent on the presence of both phorbol myristate acetate and ionomycin.
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PMID:The immunosuppressant FK-506 specifically inhibits mitogen-induced activation of the interleukin-2 promoter and the isolated enhancer elements NFIL-2A and NF-AT1. 171 1

The nucleotide sequence of the pol-env intergenic region of two isolates of caprine arthritis-encephalitis virus (CAEV) was determined. Two open reading frames (orfs) were identified, designated Q and S by homology with visna virus. CAEV orf S is a single exon encoding a deduced 87-amino acid gene product sharing 36 amino acid identities with the visna trans-acting transcriptional activator (Tat). Ten of these identities comprise a conserved CGCRLCNPGW sequence similar to a cysteine-rich domain essential for trans-activation by human immunodeficiency virus Tat. To determine if transcription promoted by the CAEV long terminal repeat (LTR) could be stimulated in CAEV-infected goat synovial membrane cells, a plasmid (pCAE-CAT) expressing bacterial chloramphenicol acetyltransferase (CAT) under control of the CAEV LTR was transfected into uninfected and infected cells. Sixfold enhancement of CAT activity was observed in infected cells using 100 ng of transfected plasmid. To determine if the pol-env region encodes a gene product which trans-activates the CAEV LTR, goat synovial membrane cells were cotransfected with pCAE-CAT and pRSV-1.9, a plasmid expressing the pol-env region under control of the Rous sarcoma virus LTR. Results indicated that the CAEV genome encodes a tat gene product attributable to orf S.
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PMID:Genetic structure of the pol-env region of the caprine arthritis-encephalitis lentivirus genome. 184 32

Herpes simplex virus type 1 (HSV-1) subgenomic sequences from 0.743 to 0.782 map units have been molecularly cloned as plasmid AT1 and shown to inhibit stable DNA-mediated gene transformation of Ltk- cells with the HSV-1 thymidine kinase (tk) gene. Here it is shown that AT1 also inhibits transient gene expression. Expression from the chloramphenicol acetyltransferase (CAT) gene under the control of either the HSV-1 tk gene or the Rous sarcoma virus (RSV) promoter was inhibited when cotransfected into Ltk- and CV-1 cells with equimolar amounts of AT1. AT1 was subcloned as three overlapping plasmids called AT1a, alpha 27 and AT1b. The alpha 27 plasmid encodes the HSV-1 immediate early gene, alpha 27; AT1a possesses sequences that specify an open reading frame in HSV-1 strain KOS used in these studies, although the significance of this open reading frame is unknown; AT1b possesses the sequences for UL55 and UL56, also genes for which no function has been reported. No single subclone or pair of subclones demonstrated significant inhibition of transient gene expression. Cotransfection of all three subclones did result in inhibition of RSV-CAT gene expression, suggesting that information from each subclone is necessary. One of the three subclones, alpha 27, contains the HSV-1 immediate early gene, alpha 27, so the possibility that other immediate early genes could substitute for alpha 27 was tested. Inhibition of RSV-CAT gene expression was also achieved by cotransfection of AT1a and AT1b with either an alpha 0- or alpha 4-containing plasmid, suggesting that the role of the alpha 27-containing plasmid can be replaced by other alpha genes with trans-regulating capability. Finally, AT1a and AT1b linker insertion mutants have been constructed and used to study the role these plasmids play in mediating inhibition. These results suggest that AT1 contains HSV-1 functions in addition to that of alpha 27 that interfere with gene expression.
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PMID:Inhibition of transient gene expression with plasmids encoding herpes simplex virus type 1 UL55 and alpha genes. 184 42

Liposome-mediated gene transfer is useful for DNA transfection into cells in culture. We wondered whether this method could be used to introduce new DNA into the intact lung. Fusion genes containing either the Rous sarcoma virus (RSV) promoter or the mouse mammary tumor virus (MMTV) promoter (which contains glucocorticoid response elements) were linked to the bacterial gene chloramphenicol acetyltransferase (CAT), an enzyme not present in mammalian cells. Plasmids containing the RSV-CAT fusion gene were mixed with cationic liposomes (Lipofectin; BRL, Inc., Grand Island, NY), and single doses were instilled into the cervical trachea of anesthetized rats. Control rats received either liposomes or plasmid. After 24, 48, and 72 h, lungs were perfused free of blood, homogenized, and analyzed for CAT enzyme activity. Liver and kidney tissue were also obtained. We found that rats given either intratracheal liposomes or plasmid had no detectable CAT activity. By contrast, 24 h after instillation of lipid:DNA complexes, lung CAT expression remained elevated for the next 48 h but was barely detectable in liver or kidney. In another group of rats, MMTV-CAT:liposome complexes were instilled intratracheally and then the rats were injected with either dexamethasone or saline. We found that the dexamethasone-treated rats had a 5- to 10-fold higher level of lung CAT expression at 24 and 48 h than the saline-treated controls had; liver and kidney CAT levels were negligible in both groups. Dexamethasone treatment did not increase RSV-CAT expression, indicating that the dexamethasone effect on MMTV-CAT expression was related to the presence of the MMTV promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Localization and induced expression of fusion genes in the rat lung. 184 84

In this communication we demonstrate that gene transfer methodology can be applied to study gene expression in intact retinal explant cultures. The appropriate enzyme activity is observed in extracts obtained after electroporation of embryonic day-10 chicken retina with plasmids containing the chloramphenicol acetyltransferase-encoding or beta-galactosidase-encoding reporter genes under transcriptional control by the Rous sarcoma virus long terminal repeat. Similar results are obtained using Ca.phosphate-mediated gene transfer. Moreover, it has been previously established that glucocorticoid hormones stimulate transcription of glutamine synthetase (Glns) mRNA in embryonic retina. We report here that, based on the results of gene transfer experiments with chimeric plasmids containing 5'-flanking DNA derived from the cloned chicken Glns-encoding gene (Glns), essential glucocorticoid response elements reside between approx. 1.3 kb and 2.5 kb upstream from the Glns transcription start point. These data show that transfection of explant cultures can provide a useful approach to the study of gene expression in complex systems.
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PMID:Glucocorticoid-inducible expression of a glutamine synthetase-CAT-encoding fusion plasmid after transfection of intact chicken retinal explant cultures. 197 78

Depression is often characterized by increased cortisol secretion caused by hyperactivity of the hypothalamic-pituitary-adrenal axis and by nonsuppression of cortisol secretion following dexamethasone administration. This hyperactivity of the hypothalamic-pituitary-adrenal axis could result from a reduced glucocorticoid receptor (GR) activity in neurons involved in its control. To investigate the effect of reduced neuronal GR levels, we have blocked cellular GR mRNA processing and/or translation by introduction of a complementary GR antisense RNA strand. Two cell lines were transfected with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under control of the mouse mammary tumor virus long terminal repeat (a glucocorticoid-inducible promoter). This gene construction permitted assay of the sensitivity of the cells to glucocorticoid hormones. Cells were also cotransfected with a plasmid containing 1,815 bp of GR cDNA inserted in the reverse orientation downstream from either a neurofilament gene promoter element or the Rous sarcoma virus promoter element. Northern (RNA) blot analysis demonstrated formation of GR antisense RNA strands. Measurement of the sensitivity of CAT activity to exogeneous dexamethasone showed that although dexamethasone increased CAT activity by as much as 13-fold in control incubations, expression of GR antisense RNA caused a 2- to 4-fold decrease in the CAT response to dexamethasone. Stable transfectants bearing the GR antisense gene fragment construction demonstrated a 50 to 70% decrease of functional GR levels compared with normal cells, as evidenced by a ligand-binding assay with the type II glucocorticoid receptor-specific ligand [3H]RU 28362. These results validate the use of antisense RNA to GR to decrease cellular response to glucocorticoids.
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PMID:Decreased glucocorticoid receptor activity following glucocorticoid receptor antisense RNA gene fragment transfection. 199 14


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