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)

Although tumor necrosis factor (TNF) is a major mediator of endotoxic shock, the normal function of TNF that has preserved this protein throughout mammalian evolution remains unknown. If the protein serves a role in normal development or homeostasis, it must be produced under physiologic conditions. To determine whether TNF secretion occurs in normal animals, and to define the tissue sources of the protein, we prepared a reporter construct in which the TNF coding sequence and introns are replaced by the chloramphenicol acetyltransferase (CAT) coding sequence. This construct was inserted into the murine genome, yielding 13 transgenic founders. Macrophages harvested from 4 of the transgenic lines expressed CAT activity after stimulation with Escherichia coli lipopolysaccharide in vitro. Each of these 4 transgenic lines also constitutively expressed CAT activity in the thymus but in no other tissue examined. Cultured thymocytes secrete TNF, as demonstrated both by cytotoxicity assays and by immunoprecipitation of radiolabeled thymic culture medium. CAT activity was associated with the thymic lymphocyte population and not with thymic macrophages or dendritic cells. CAT activity was present in thymic lymphocytes irrespective of CD4 or CD8 expression; T cells from the spleen, however, had no detectable CAT activity. The biosynthesis of TNF in the thymus of normal animals implies a role for this protein in the development or regulation of the immune response.
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PMID:Constitutive synthesis of tumor necrosis factor in the thymus. 159 85

The rat hemopexin (Hx) gene was isolated and studies of its transcriptional regulation initiated. For analysis by a transient expression assay, the sequence between -2400 and +21 and sequential 5' truncates were linked to the chloramphenicol acetyltransferase (CAT) gene. HepG2 cells transfected with these CAT constructs were treated with conditioned medium of lipopolysaccharide stimulated human monocytes, interleukin-1 (IL-1) or interleukin-6 (IL-6). The activities of putative regulatory regions joined to the SV40 promoter indicated that the flanking region of the rat Hx gene from -209 to -104 contains three functional regions designated proximal regulatory regions; PRR-I (-209 to -173), -II (-178 to -158) and -III (-154 to -104). We found that PRR-II contains a different class of IL-6 responsive element (RE) from that reported for the human Hx gene, and that PRR-I and PRR-III participate in the basal expression of rat Hx in HepG2 cells.
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PMID:Identification of an interleukin-6 responsive element and characterization of the proximal promoter region of the rat hemopexin gene. 159 80

Granulocyte colony-stimulating factor (G-CSF) plays an essential role in granulopoiesis during bacterial infection. Macrophages produce G-CSF in response to bacterial endotoxins such as lipopolysaccharide (LPS). To elucidate the mechanism of the induction of G-CSF gene in macrophages or macrophage-monocytes, we have examined regulatory cis elements in the promoter of mouse G-CSF gene. Analyses of linker-scanning and internal deletion mutants of the G-CSF promoter by the chloramphenicol acetyltransferase assay have indicated that at least three regulatory elements are indispensable for the LPS-induced expression of the G-CSF gene in macrophages. When one of the three elements was reiterated and placed upstream of the TATA box of the G-CSF promoter, it mediated inducibility as a tissue-specific and orientation-independent enhancer. Although this element contains a conserved NF-kappa B-like binding site, the gel retardation assay and DNA footprint analysis with nuclear extracts from macrophage cell lines demonstrated that nuclear proteins bind to the DNA sequence downstream of the NF-kappa B-like element, but not to the conserved element itself. The DNA sequence of the binding site was found to have some similarities to the LPS-responsive element which was recently identified in the promoter of the mouse class II major histocompatibility gene.
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PMID:Regulatory elements responsible for inducible expression of the granulocyte colony-stimulating factor gene in macrophages. 169 38

Mouse macrophage BAM3 cells produced colony-stimulating factors (CSFs) after stimulation with bacterial lipopolysaccharide (LPS). By assaying the CSF using various interleukin 3-dependent cell lines, it was shown that most of the CSFs produced by BAM3 cells were granulocyte CSF (G-CSF). The granulocyte-macrophage CSF (GM-CSF) gene was also expressed in BAM3 cells after stimulation with LPS. When BAM3 cells were fused with the mouse renal adenocarcinoma cell line RAG which does not produce G-CSF, two of four hybrid cell lines constitutively produced large quantities of G-CSF. About 300 bp of the promoter region of mouse G-CSF chromosomal gene was inserted upstream of the Escherichia coli chloramphenicol acetyltransferase gene, and introduced into BAM3, RAG and hybrid cells. The G-CSF promoter was activated by stimulation with LPS, in BAM3 cells, but was inert in RAG cells. On the other hand, there was significant constitutive CAT activity in the hybrid cells.
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PMID:Constitutive production of granulocyte colony-stimulating factor by hybrids of a SV40-transformed mouse macrophage and a renal adenocarcinoma cell line. 172 85

The ability of the promotor/enhancer region of the mouse ornithine decarboxylase gene to respond to various stimuli was studied. This region was subcloned into multiple fragments and these were inserted in front of the chloramphenicol acetyltransferase gene on an expression vector, pBLCAT3. These ODC/CAT constructs were transfected into a mouse macrophage-like cell line, RAW264. The transfected cells were stimulated by bacterial lipopolysaccharide, 8-bromo cAMP or both followed by analysis of chloramphenicol acetyltransferase activity. Optimal inducible chloramphenicol acetyltransferase expression was obtained when sequences from -90 to +12 (with respect to the transcriptional start site) were tested in cells treated with a combination of lipopolysaccharide and 8-bromo cAMP. A putative cyclic AMP response element located at -48 was altered by site-directed mutagenesis but these alterations did not diminish activity in response to stimulation with lipopolysaccharide and 8-bromo cAMP.
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PMID:Regulation of mouse ornithine decarboxylase gene expression in a macrophage-like cell line: synergistic induction by bacterial lipopolysaccharide and cAMP. 184 9

The genes of Yersinia enterocolitica serotype O:3 (YeO3) that determine the synthesis of the O-side-chain of the lipopolysaccharide, the rfb region, were cloned into plasmid pBR322. The O-side-chain of YeO3 was expressed by the clone both in Escherichia coli and Salmonella typhimurium indicating that the entire rfb region was included in the clone. It was shown by restriction mapping, deletion analysis and transposition mutagenesis that about 10.4 kilobase pairs of DNA was essential for the synthesis and expression of the O-side-chain. The correct assembly of the O-side-chain on the cell surface of the clone was confirmed by immunofluorescence microscopy and slide agglutination. Immunoblotting using monoclonal antibody specific for the O-side-chain of YeO3 revealed that the O-side-chain material synthesized by the clone in E. coli was similar to that of YeO3. The clone did not show the in vitro temperature variation in O-side-chain expression characteristic of YeO3. Instead analogous O-side-chain was produced both at 25 degrees C and at 37 degrees C. Using transposon Tn2507, which carries a promotorless chloramphenicol acetyltransferase (CAT) gene, transcriptional fusions with the target DNA were generated. When testing the ability of mutated clones to produce CAT, transcription was shown to occur in a uniform direction throughout the whole rfb region. In colony hybridizations, using the cloned insert as a probe, homologous DNA was detected only in pathogenic Y. enterocolitica serotypes.
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PMID:Expression cloning of Yersinia enterocolitica O:3 rfb gene cluster in Escherichia coli K12. 185 98

Previously we described a cell line OCI-LY3 derived from a patient with non-Hodgkin's lymphoma. The cell line produced interleukin-6 (IL-6) mRNA and protein and demonstrated an autocrine pattern of growth for IL-6. Southern blot analysis of the IL-6 gene did not reveal any rearrangement. To determine whether the production of IL-6 by OCI-LY3 was due to subtle changes in the promoter of IL-6 or due to the expression of trans-acting factors chloramphenicol acetyltransferase (CAT) reporter constructs containing from -1,180 to +13 to -112 to +13 of a normal IL-6 gene were electroporated into the cell line. When these constructs are transferred into unstimulated fibroblasts, no CAT activity is seen; however, CAT activity is induced when the cells are stimulated with either IL-1 alpha, lipopolysaccharide (LPS), or cyclic adenosine monophosphate (cAMP) analogues. When the cell line OCI-LY3 was transfected with these constructs, CAT activity was observed; it was not necessary to stimulate the cells with exogenous factors to observe this activity. No CAT activity was observed in a second lymphoma cell line, OCI-LY13.1, that does not produce IL-6. These results suggest that the constitutive production of IL-6 by the cell line OCI-LY3 is due to the presence of trans-acting factors that stimulate the expression of IL-6 and not due to a cis-acting mutation of the IL-6 promoter.
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PMID:Regulation of interleukin-6 expression in the lymphoma cell line OCI-LY3. 191 71

The promoter region of the interleukin-6 (IL-6) gene has a putative NF-kappa B-binding site. We found that a fragment of the IL-6 promoter containing the site specifically binds highly purified NF-kappa B protein and the NF-kappa B protein in nuclear extracts of phorbol ester-induced Jurkat cells. Mutations of the NF-kappa B site abolished complex formation with both purified NF-kappa B and the nuclear extract protein. Transient expression of chloramphenicol acetyltransferase (CAT) plasmids containing the IL-6 promoter revealed very little activity of the promoter in U-937 monocytic cells and in HeLa cells before stimulation. However, stimulation of U-937 and HeLa cells by inducers of NF-kappa B led to a dramatic increase in CAT activity. Mutations in the NF-kappa B-binding site abolished inducibility of IL-6 promoter-cat constructs in U-937 cells by lipopolysaccharide, tumor necrosis factor alpha, the double-stranded RNA poly(IC), or phytohemagglutinin and in HeLa cells by tumor necrosis factor alpha and drastically reduced but did not completely eliminate inducibility in HeLa cells stimulated by double-stranded RNA poly(IC) or phorbol 12-myristate 13-acetate. These results suggest that NF-kappa B is an important mediator for activation of the IL-6 gene by a variety of IL-6 inducers in both U-937 and HeLa cells and that alternative inducible enhancer elements contribute in a cell-specific manner to IL-6 gene induction. Because NF-kappa B is involved in the control of a variety of genes activated upon inflammation, NF-kappa B may play a central role in the inflammatory response to infection and tissue injury.
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PMID:Activation of interleukin-6 gene expression through the NF-kappa B transcription factor. 218 31

Analysis of immunoglobulin expression in mice transgenic for either a kappa light chain (driven by the kappa enhancer) or lambda light chain (driven by the IgH enhancer) revealed that the transgenic light chains are expressed by the majority of B lymphocytes in the neonatal mice. However, the proportion of B cells that express the transgenes at a detectable level decreases rapidly with age, with a concomitant increase in cells expressing rearrangements of one of the endogenous light chain loci. This appears to be the result of cellular selection. The down-regulation of transgene expression is not due to an irreversible mechanism as incubation of adult splenic lymphocytes with bacterial lipopolysaccharide leads to a rapid increase in the expression of the transgenic light chain on the B cell surface. In mice carrying the lambda transgene (but not in mice carrying the kappa transgene) the change with age in the pattern of transgene expression is accompanied by a shift towards B cells that do not express surface IgD. This shift towards IgM+/IgDlow B cells is also observed in mice transgenic for a chloramphenicol acetyltransferase gene linked to the IgH enhancer. This suggests that the down-regulation of IgD may either be due to the expression of a transgene that impairs B cell development or, alternatively, could be associated with the molecular events responsible for the down-regulation of IgH enhancer activity. The results also draw attention to the contribution of cellular selection in determining the pattern of expression of immunoglobulin transgenes and emphasize the importance of in vivo analysis of neonatal as well as adult transgenic mice.
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PMID:Cellular selection leads to age-dependent and reversible down-regulation of transgenic immunoglobulin light chain genes. 248 40

Using in vitro protein binding and in vivo functional studies, we have identified novel regulatory sequences near the 5' end of murine leukemia virus (MuLV) long terminal repeats (LTRs). These sequences are highly conserved in all MuLV LTRs as well as in feline leukemia virus and gibbon ape leukemia virus LTRs. In this upstream conserved region (UCR), gel retardation assays detected two overlapping but distinct binding sites (UCR-U and UCR-L) for nuclear proteins (UCRF-U and UCRF-L). Three lines of evidence suggest a negative regulatory role for the UCR in viral transcription: (i) an inverse correlation was found between MuLV transcripts and nuclear proteins binding the UCR in the spleens of five different mouse strains; (ii) in vivo treatment of NFS mice with lipopolysaccharide resulted in the induction of splenic viral transcripts and the concomitant disappearance of UCR-binding proteins; and (iii) in mouse L cells transfected with an MuLV LTR linked to the chloramphenicol acetyltransferase (CAT) gene, cotransfected UCR oligonucleotides increased CAT expression, presumably by competing for inhibitory trans-acting factors.
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PMID:Negative control region at the 5' end of murine leukemia virus long terminal repeats. 254 Apr 25


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