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)

Chimeric DNA expression vectors containing regulatory sequences proximal to the 5' end of coding sequences for mammalian genes provide valuable tools to study gene expression. Genes coding for easily measured products (reporter genes) can be used to study promoter strength and regulation of gene expression after transient expression of promoter-reporter constructs in mammalian cells. To determine the strength of a variety of mammalian and viral promoter-enhancer sequences in primary cultures of human mammary epithelial cells (HMEC), these sequences were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and transfected into HMEC using strontium phosphate. The long terminal repeat (LTR) of the endogenous murine leukemia virus AKR-623 was the most potent promoter of transient CAT expression in HMEC. A number of commonly available promoter sequences displayed a wide range of activities in these cells. The glucocorticoid responsive LTR promoter from the murine mammary tumor virus modulated expression of CAT and was sensitive to the concentration of dexamethasone in the growth media. In a similar fashion, the regulatory sequences from the murine metallothionein-1 gene retained responsiveness to zinc concentration in the growth media.
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PMID:Relative promoter activity in human mammary epithelial cells assayed by transient expression. 148 64

Activities of trout metallothionein-A promoter and mouse metallothionein-I promoter in fish cells and fry were examined using the chloramphenicol acetyltransferase (CAT) gene as a reporter. Transfection and transient CAT assays on a trout liver cell line indicated that both promoters were inducible by metals, including zinc. In vivo activities of both promoters were examined using microinjection system into medaka eggs. Some of the hatched fry were exposed to zinc and their CAT activities were assayed. Both promoters revealed considerable activities after exposure to zinc while only weak activities were detected in unexposed fry.
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PMID:Metal-inducible activities of metallothionein promoters in fish cells and fry. 162 32

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 TAR sequence of the 5' leader of HIV-1 long terminal repeat-directed mRNA was found to be able to bind to and to activate double-stranded RNA-dependent (2'-5')A synthetase. Binding of TAR to the purified synthetase in vitro was abolished by addition of HIV-1 Tat protein, which binds to this sequence with a high affinity. Inhibition of TAR-mediated activation of (2'-5')A synthetase by Tat was prevented in the presence of the Zn2+ and Cd2+ chelators o-phenanthroline and penicillamine, which did not impair TAR-synthetase interaction. Transient expression assays of bacterial chloramphenicol acetyltransferase (CAT) gene in HeLa cells revealed that the levels of both CAT mRNA and CAT protein decreased after treatment of the cells with interferon, if CAT gene was linked to HIV-1 TAR segment. Cotransfection of the cells with a tat sequence containing plasmid rendered CAT gene expression insensible to the action of interferon.
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PMID:Binding of Tat protein to TAR region of human immunodeficiency virus type 1 blocks TAR-mediated activation of (2'-5')oligoadenylate synthetase. 169 53

Egr-1 is an early growth response gene that encodes a protein with three zinc fingers and is involved in transcriptional regulation. In adult heart myocytes, in contrast to c-fos and c-myc, high levels of Egr-1 mRNA expression have been shown. Here we report that Egr-1 transactivates rat cardiac alpha-MHC gene expression. In serum-starved primary cultures of 18-day-old fetal rat heart myocytes, addition of serum evoked expression of both Egr-1 and alpha-MHC gene transcripts. Inclusion of 10 microM cycloheximide in these cultures for 48 h caused a greater increase in Egr-1 mRNA, whereas the expression of alpha-MHC transcripts was ablated. To examine the involvement of Egr-1 in alpha-MHC induction, we transfected primary cultures of cardiac myocytes with plasmids pCMVEgr-1 (Egr-1 expression vector) and pMP3.3CAT containing -2.9- to +0.42-kilobase sequences of the alpha-MHC gene fused to the coding region of the chloramphenicol acetyltransferase (CAT) gene. Cotransfection of pCMVEgr-1 stimulated expression of pMP3.3CAT 10-15-fold. Furthermore, pCMVEgr-1 also stimulated expression of the endogenous alpha-MHC gene in primary cultures of cardiac myocytes. Transactivation of pMP3.3CAT expression by pCMVEgr-1 was also observed by transfecting the myogenic cell line Sol 8, but not in L6E9 cells or in NIH3T3 fibroblasts. By creating progressive 5' deletions of the alpha-MHC gene, we found that the region extending between -1698 and -1283 base pairs is necessary for Egr-1-induced expression of the alpha-MHC/CAT construct. These results define a physiological target for the Egr-1 transcription factor and delineate a novel mechanism for regulation of the alpha-MHC gene.
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PMID:Egr-1, a serum-inducible zinc finger protein, regulates transcription of the rat cardiac alpha-myosin heavy chain gene. 207 71

cAMP regulates the expression of several genes by activation of a promoter consensus sequence which functions as a cAMP-response element. Evidence indicated that this is accomplished via cAMP dissociation of cAMP-dependent protein kinase into its regulatory (R) and catalytic (C) subunits. Our investigations of the role of these two subunits in gene expression provide direct and quantitative evidence that the C subunit is required for cAMP stimulation of the cAMP-response element in the vasoactive-intestinal-peptide gene in rat pheochromocytoma cells. After cotransfection of a metallothionein-regulated C-subunit expression vector (pCEV) and a vasoactive-intestinal-peptide--chloramphenicol acetyltransferase construct containing a cAMP-response element, we could demonstrate expression of transfected C-alpha-subunit mRNA (truncated size 1.7 kb) by Northern blot and a concentration-dependent C subunit stimulation of chloramphenicol acetyltransferase activity. Basal activity was stimulated 12- and 50-fold by pCEV (30 micrograms), in the absence and presence, respectively, of Zn2+. Metallothionein-regulated expression of C was demonstrated by results that showed a 2-4-fold increase in chloramphenicol acetyltransferase activity in the presence versus the absence of 90 microM Zn2+. In contrast, overexpression of the R-II beta regulatory subunit did not stimulate chloramphenicol acetyltransferase activity, and R-II beta transfected together with C (ratio 2:1 and 4:1) inhibited the stimulation by the C subunit 70% and 90% respectively. Our results indicate that transfection of cAMP-dependent protein kinase subunits results in functional expression of both C-alpha and R-II beta subunits. Expression of the C subunit mediated cAMP-regulated gene expression but this expression could be inhibited by cotransfected R-II beta subunit, indicating intracellular reconstitution of the inactive holoenzyme of cAMP-dependent protein kinase.
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PMID:Regulation of gene expression by transfected subunits of cAMP-dependent protein kinase. 215 96

Three nuclear factors, the Ah receptor, XF1, and XF2, bind sequence specifically to the Ah response elements or xenobiotic response elements (XREs) of the cytochrome P450IA1 (P450c) gene. The interactions of these factors with the Ah response element XRE1 were compared by three independent methods, methylation interference footprinting, orthophenanthroline-Cu+ footprinting, and mobility shift competition experiments, using a series of synthetic oligonucleotides with systematic alterations in the XRE core sequence. These studies established the following (i) all three factors interact sequence specifically with the core sequence of XRE1; (ii) the pattern of contacts made with this sequence by the Ah receptor are different from those made by XF1 and XF2; and (iii) although XF1 and XF2 can be distinguished by the mobility shift assay, the sequence specificities of their interactions with XRE1 are indistinguishable. Further characterization revealed the following additional differences among these three factors: (i) XF1 and XF2 could be extracted from nuclei under conditions quite different from those required for extraction of the Ah receptor; (ii) XF1 and XF2 were present in the nuclei of untreated cells and did not respond to polycyclic compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-napthoflavone, while nuclear Ah receptor was undetectable in untreated cells and rapidly increased in response to TCDD; (iii) inhibition of protein synthesis did not affect the TCDD-induced appearance of the Ah receptor but substantially decreased the constitutive activities of XF1 and XF2, suggesting that the Ah receptor must be present in untreated cells in an inactive form that can be rapidly activated by polycyclic compounds, while the constitutive expression of XF1 and XF2 depends on the continued synthesis of a relatively unstable protein; (iv) the receptor-deficient and nuclear translocation-defective mutants of the hepatoma cell line Hepa1, which are known to lack nuclear Ah receptor, expressed normal levels of XF1 and XF2, suggesting that the former factor is genetically distinct from the latter two; and (v) a divalent metal ion, probably Zn2+, is known to be an essential cofactor for the Ah receptor but was not required for the DNA-binding activities of XF1 and XF2. Together, these findings indicate that the Ah receptor is distinct from XF1 and XF2, while the latter two activities may be related. Because the DNA-binding domains of these three factors overlap substantially, their binding to XREs is probably mutually exclusive, which suggests that the interplay of these factors at Ah response elements may be important to the regulation of CYP1A1 gene transcription. The results of preliminary transfection experiments with constructs harboring XREs upstream of the chloramphenicol acetyltransferase gene driven by a minimal simian virus 40 promoter are presented that are consistent with this hypothesis.
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PMID:Multiple DNA-binding factors interact with overlapping specificities at the aryl hydrocarbon response element of the cytochrome P450IA1 gene. 217 7

The NGFI-A gene encodes a "zinc-finger" protein that is rapidly induced by nerve growth factor (NGF) in PC12 rat pheochromocytoma cells. The complete exon/intron organization and nucleotide sequence of the rat NGFI-A gene have been determined. The gene spans 3789 nucleotides (nt) and is interrupted by a single intron at nt 588. All three zinc-finger DNA-binding domains are contiguously coded for within the 3' exon; this is in contrast to the structure described by others for the Xenopus laevis transcription factor TFIIIA gene. To analyze the transcription of this gene, we have determined the transcription start site and nucleotide sequence of the 5' flanking region. Transfection of PC12 cells with a fragment from the 5' flanking region linked to the chloramphenicol acetyltransferase (CAT) gene revealed that it contains an element which imparts an NGF-inducible phenotype to the normally silent CAT gene. Several regions with homologies to recognizable sequence elements are present in this fragment, including a TATA box at nt -27, serum response elements at nt -84, -106, -370, and -408, a cAMP-responsive element at nt -140, and a transcription factor Sp1-binding site at nt -286. These results establish the genomic structure of this mammalian multifinger protein and demonstrate that an NGF-responsive element lies upstream of the NGFI-A gene.
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PMID:Structure of the NGFI-A gene and detection of upstream sequences responsible for its transcriptional induction by nerve growth factor. 249 4

The possible role of the catalytic subunit of the cAMP-dependent protein kinase in mediating the regulation of prolactin gene transcription has been investigated through the use of a synthetic gene encoding the heat-stable inhibitor of the cAMP-dependent protein kinase. To assess the effects of protein kinase inhibitor expression on cAMP induction of prolactin gene transcription, a marker gene containing the rat prolactin promoter and adjacent 5'-flanking sequences linked to the bacterial chloramphenicol acetyltransferase gene was cotransfected with a protein kinase inhibitor-expression vector. The results demonstrate that the protein kinase inhibitor-expression vector reduced both basal and cAMP-stimulated expression of the cotransfected prolactin-chloramphenicol acetyltransferase gene. A mutant protein kinase inhibitor-expression vector, coding for an inactive inhibitor protein, did not inhibit basal or cAMP-stimulated prolactin gene transcription. Furthermore, the protein kinase inhibitor-expression vector did not inhibit zinc induction of the metallothionein promoter. Analysis of protein kinase activity in transfected cells demonstrated that the protein kinase inhibitor expression vector reduced cAMP-dependent protein kinase activity but did not reduce protein kinase C activity. Nuclease protection experiments confirmed that the effects of the inhibitor vector involved changes in correctly initiated transcripts produced from the prolactin promoter. Surprisingly, the protein kinase inhibitor-expression vector reduced the effects of several different agents including epidermal growth factor, thyrotropin-releasing hormone, phorbol esters, and estrogen on prolactin gene expression to the same extent as it altered cAMP effects.
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PMID:A protein kinase inhibitor gene reduces both basal and multihormone-stimulated prolactin gene transcription. 253 42

The trout metallothionein (MT) genes consist of two members. We describe the structure of the first fish MT (tMT-B) gene which shows an overall resemblance but some remarkable differences with mammalian MT genes. The similarities included (i) tripartite structure of the gene, (ii) conservation of cysteine residues, and (iii) a TATAAA signal and two copies of metal-responsive elements (MREs). The differences consisted of (i) an AT-rich tMT-B promoter compared with highly GC-rich mammalian MT promoters and (ii) a lack of SP1-binding sites in the tMT-B promoter. Functional analysis of the tMT-B 5'-flanking region following fusion with the bacterial chloramphenicol acetyltransferase gene and its transfection into the rainbow trout hepatoma cell line revealed that sequences from positions -600 to +8 are sufficient for regulation by metals. Further deletion analyses of this fragment suggested that a minimum of 100 nucleotides upstream of the transcription initiation site are required for induction by cadmium and zinc. The tMT-B promoter was also functional in the human hepatoblastoma cell line, suggesting that an MT regulatory factor(s) is conserved in phylogenetically distant species like humans and fish.
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PMID:Structure of the rainbow trout metallothionein B gene and characterization of its metal-responsive region. 318 57


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