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)

We describe conditions under which exogenous DNA templates can be introduced for transient expression into primary murine T lymphocytes. T cells at various stages of development, including concanavalin A-activated splenic T cells, immature pre-T cells, and even small cortical thymocytes, could be successfully transfected. A variety of model DNA constructs were compared in which different viral promoter regions were used to drive expression of the chloramphenicol acetyltransferase (CAT) reporter gene. All showed enhanced expression in cells that had been acutely stimulated with the Ca2+ ionophore A23187 and phorbol ester as chemical proxies for T-cell receptor-mediated signals. In addition, splenocytes but not thymocytes required prior treatment with a mitogen and interleukin-2 in order to express these constructs, implying that even postmitotic thymocytes may be held in a quasiactivated state. A most striking result was the finding that the viral regulatory sequences in the Rous sarcoma virus long terminal repeat and the simian virus 40 early region were subject to sharply differential regulation, with a rank order that changed depending on the developmental stage of the T cells. The most immature thymic blasts and several lymphoma cell lines expressed the pRSV-Cat and pSV2-Cat constructs similarly, but cortical thymocytes exhibited a strong preference for pSV2-Cat. Splenic concanavalin A-stimulated blasts, on the other hand, slightly preferred pRSV-Cat, a tendency which became exaggerated in factor-dependent T-cell lines. The ratio of pRSV-Cat to pSV2-Cat expression varied according to cell type by as much as 500-fold. These results argue against a trivial linkage of promoter preference to cell cycle status but instead provide evidence that activation of T cells at distinct stages of differentiation results in the expression of different ensembles of nuclear regulatory proteins. In contrast to the simian virus 40 and Rous sarcoma virus promoter regions, the long terminal repeats of the retroviruses mink cell focus-forming virus and Akv were expressed well in all primary T-lineage cells. Thus, they represent excellent model promoters for engineering developmental stage-independent expression of exogenous genes in murine T cells.
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PMID:In vitro transfection of fresh thymocytes and T cells shows subset-specific expression of viral promoters. 131 65

Northern-blot analysis was used to demonstrate that an increase in extracellular glucose concentration increased the content of preproinsulin mRNA 2.3-fold in the beta-cell line HIT T15. A probe for the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase was used as a control. Mannoheptulose blocked this effect of glucose. A stimulatory effect on preproinsulin mRNA levels was also observed in response to mannose and to 4-methyl-2-oxopentanoate. However, galactose and arginine were ineffective. Glucagon, forskolin and dibutyryl cyclic AMP also elicited an increase in HIT-cell preproinsulin mRNA. The ability of the 5' upstream region of the preproinsulin gene to mediate the effect of glucose and other metabolites on transcription was studied by using a bacterial reporter gene technique. HIT cells were transfected with a plasmid, pOK1, containing the upstream region of the rat insulin-1 gene (-345 to +1) linked to chloramphenicol acetyltransferase (CAT). Co-transfection with a plasmid pRSV beta-gal containing beta-galactosidase driven by the Rous sarcoma virus promoter was used as a control for the efficiency of transfection; expression of CAT activity in transfected HIT cells was normalized by reference to expression of beta-galactosidase. Glucose caused a dose-dependent increase in expression of CAT activity, with a half-maximal effect at 5.5 mM and a maximum response of 4-fold. Mannoheptulose blocked this effect of glucose. Other metabolites (mannose, 4-methyl-2-oxopentanoate and leucine plus glutamine) were also able to increase insulin promoter-driven CAT expression, but galactose and arginine were ineffective. The stimulatory effect of glucose on CAT expression was not blocked by verapamil and was inhibited by increasing extracellular Ca2+ from 0.4 to 5 mM. Both dibutyryl cyclic AMP and forskolin caused an increase in insulin promoter-driven gene expression in the presence of 1 mM-glucose, but neither agent further increased the level of expression occurring in the presence of a maximally stimulating glucose concentration. The phorbol ester phorbol 12-myristate 13-acetate (PMA) also increased insulin promoter-driven CAT expression in the presence of 1 mM-, but not 11 mM-glucose. Staurosporine blocked the stimulatory effect not only of PMA but also of glucose and of dibutyryl cyclic AMP. We conclude that the 5' upstream region of the insulin gene contains sequences responsible for mediating the stimulatory effect of glucose on insulin-gene transcription.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Control of insulin gene expression by glucose. 132 37

Prolonged depolarization has been used as a model of adaptive changes in the expression of various proteins, such as ion channels and neurotransmitter biosynthetic enzymes, in response to increased trans-synaptic activity in the nervous system. In depolarized PC12 cells, tyrosine hydroxylase (TH) mRNA levels increased severalfold (Kilbourne, E. J., and Sabban, E. L. (1990) Mol. Brain Res. 8, 121-127). In this study, membrane depolarization caused an increase in the expression of the reporter gene chloramphenicol acetyltransferase (CAT), under transcriptional control of the 5' region of the rat TH gene. These results indicate that membrane depolarization leads to increased transcription of the TH gene. Protein kinase C inhibitors had no effect on the induction of TH mRNA by depolarization, as well as the increase in formation of CAT under control of the upstream region of the TH gene. The depolarization responsive element in the TH gene was mapped to the region containing the cAMP responsive element. This region of the TH gene also increased CAT activity in response to the calcium ionophore, ionomycin. Interestingly, combined treatment with cAMP analogs and membrane depolarization had a greater effect than either alone on TH mRNA levels, as well as on CAT activity in PC12 cells transfected with the plasmid containing the cAMP responsive element.
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PMID:Regulated expression of the tyrosine hydroxylase gene by membrane depolarization. Identification of the responsive element and possible second messengers. 134 5

The preproenkephalin A gene is a neurotransmitter gene whose expression can be modulated "trans-synaptically" by changes in neuronal activity. DNA sequences lying within 200 base pairs of this gene's transcription start site resemble consensus binding sites for several transcription factor families. In nonneuronal cell cultures, this promoter region is sufficient to mediate gene responses to depolarization, phorbol esters, adenylate cyclase, and calcium fluxes. To assess the role that these cis-acting elements could play in preproenkephalin expression and regulation in vivo, the expression of a construct containing this 200-base-pair region fused to the chloramphenicol acetyltransferase gene was examined in transgenic mice. This promoter confers modest expression in brain, adrenal, and small intestine, with substantially higher levels in testis. These elements confer trans-synaptic regulation in two well-studied models of trans-synaptic preproenkephalin upregulation but not in a third system, underscoring the specificity of the regulatory sequence elements implicated in the synaptic regulation of neuronal genes.
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PMID:Preproenkephalin promoter "cassette" confers brain expression and synaptic regulation in transgenic mice. 137 43

The promoter of the human gene encoding the stress-responsive protein polypeptide-binding protein/78 kDa glucose-regulated protein (BiP/GRP78) was isolated from Burkitt's lymphoma cells by PCR. This promoter DNA segment (termed BiP670) or one of its 5' deletion derivatives was fused to the bacterial chloramphenicol acetyltransferase gene and introduced into HeLa cells for transient expression. BiP670 retained transcriptional activity at both the basal and Ca2+ ionophore A23187-inducible levels. However, there was no significant increase in promoter activity following a 5 h induction with 7 microM-A23187, and less than 5-fold induction at 15 h. In contrast, the steady-state mRNA level was induced by 18-fold at 5 h. The in vivo transactivation assays with BiP670 5' deletion derivatives indicate that the putative A23187-inducible element is located within a 70 bp DNA segment (i.e. spanning -39 to -107 bp upstream of the transcriptional initiation site). Using an in vitro gel mobility shift assay, A23187-inducible nuclear factors were identified from HeLa cell extracts. DNA-binding competition experiments also suggest that the 70 bp DNA segment contains a potential sequence motif for the binding of the A23187-inducible nuclear factors.
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PMID:Cloning of a functional Burkitt's lymphoma polypeptide-binding protein/78 kDa glucose-regulated protein (BiP/GRP78) gene promoter by the polymerase chain reaction, and its interaction with inducible cellular factors. 138 10

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.
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PMID:The human prothrombin gene: transcriptional regulation in HepG2 cells. 146 33

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.
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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

The noncollagenous proteins (NCPs) that predominate the bone matrix have recently been the focus of intense investigation because of their potential influence on cell attachment, Ca2+ and hydroxyapatite binding, and the mineralization of bone tissue. With the advent of molecular biology, all of the major NCPs of bone have been cloned and their amino acid sequences completely determined. While each of the proteins has distinct structural properties, some proteins appear to be part of gene families. Examples include the small proteoglycans, decorin and biglycan, as well as the gamma carboxyglutamic acid proteins, such as matrix gla protein and osteocalcin (bone gla protein). Some of the NCPs that are clearly not members of any known gene family still share several common characteristics. One such example of this "convergent evolution" is bone sialoprotein and osteopontin. Both are highly posttranslationally modified glycoproteins that share the cell attachment amino acid sequence RGD (arginine-glycine-aspartic acid), which facilitates the attachment of bone cells in vitro, yet they are clearly not related genetically. Using cDNAs and antisera as probes, the precise temporal localization of NCP expression has been determined, and it has been shown that NCPs are produced in skeletal, and in most cases, nonskeletal tissue as well. This observation implies that the functions of the NCPs are not necessarily limited to bone tissue. Many of the promoters for these genes have been isolated and functional domains determined by a combination of chloramphenicol acetyltransferase assay, gel shift, and footprint analyses. The most extensively studied promoter in the NCP category is osteocalcin, whose sensitivity to 1,25-dihydroxycholecalciferol has been delineated in detail. Future studies on the individual and cooperative activities of the NCPs in bone are likely to involve site-directed mutagenesis of cloned DNA and a combination of in vitro and in vivo functional analyses.
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PMID:Structure, expression, and regulation of the major noncollagenous matrix proteins of bone. 149 20

Nuclear protein binding to the human interferon-gamma (IFN-gamma) promoter was investigated to determine the structural basis for the control of gene expression during T cell activation. DNase I footprinting of gel-shift complexes demonstrated that proteins bind to two downstream (-124 to -114 and -36 to -30) and one upstream (-534 to -486) element in the IFN-gamma gene promoter. Treatment of human peripheral blood lymphocytes or continuous T cell tumors with phorbol 12-myristate 13-acetate (PMA) plus phytohemagglutinin or calcium ionophore results in a pattern of response that is similar when using either the upstream or downstream elements. Upon induction of T cells, the lower mobility gel-shift band disappears. Yet the equivalent band which is also present in non-T cells is unperturbed after PMA + calcium ionophore treatment. The higher mobility band which is modified upon induction is restricted to the T cell lineage. Upstream and downstream elements share similar protein-binding motifs as indicated by the homology of footprinted sequences, the similarity of protein-binding patterns, and the ability of these elements to compete against each other in gel-shift protein-binding assays. Protein binding to the downstream elements appears to be interactive, since both sites are required for complex formation. When either of the two downstream elements is disrupted by site-directed mutagenesis, the higher mobility gel-shift band is diminished by an amount that is consistent with the reduction in reporter (chloramphenicol acetyltransferase) gene expression. Therefore, proteins in the ubiquitous gel-shift band appear to be associated with the inactive state of IFN-gamma, while the modified band is closely associated with the positive regulation of IFN-gamma gene expression.
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PMID:Characterization of nuclear protein binding to the interferon-gamma promoter in quiescent and activated human T cells. 151 29

Antigen triggering of the T-cell receptor results in an accumulation of activated GTP-bound p21ras protein. To assess the role of ras protein in T-cell activation we have cotransfected the murine thymoma line EL4 with a construct capable of expressing a constitutively active, oncogenic form of Ha-ras and a reporter construct containing the human interleukin-2 promoter fused upstream of the bacterial gene for chloramphenicol acetyltransferase. We show that the ras oncoprotein contributes to interleukin-2 promoter activation. Its pattern of synergism with a calcium ionophore or the lymphokine interleukin-1 indicates that it replaces a signal mediated by protein kinase C. Interleukin-2 promoter activity in the presence of ras oncoprotein was inhibited by H7, a potent inhibitor of protein kinase C, but not by HA1004, an inhibitor of cyclic nucleotide-dependent kinase, suggesting that protein kinase C mediates the ras effect. In addition, we show that in these cells, expression of activated ras results in activation of a synthetic promoter containing several copies of an NF kappa B binding site.
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PMID:Interleukin-2 promoter activation in T-cells expressing activated Ha-ras. 153 20


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