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)

Biotinylation of fusion proteins in E. coli was studied using a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit. As the biotinylation sequence, we examined two sequences: one was of amino acid residues [84-123] of 1.3S, a partial sequence containing a region from a conserved tetrapeptide (Ala-Met-Bct-Met) around the biotinyl lysine (Bct) to the carboxyl terminal; the other was of an almost entire sequence [18-123]. We constructed recombinant plasmids for fusion proteins of beta-galactosidase, of chloramphenicol acetyltransferase, and of alkaline phosphatase. We found the biotinylation in the [18-123] sequence fused to alkaline phosphatase.
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PMID:In vivo biotinylation of fusion proteins expressed in Escherichia coli with a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit. 136 26

A plasmid expression vector was constructed to direct the synthesis of foreign proteins in Escherichia coli as fusions with cyclomaltodextrin glucanotransferase (CGT) with cytoplasmic location (delta ssCGT). The ability of CGT to bind to covalently immobilized cyclodextrins was utilized in purifying fused target proteins. A large proportion of the cytoplasmically synthesized delta ssCGT formed inclusion bodies which adopted the active conformation at considerably high refolding concentration (67 microM delta ssCGT solution). By lowering the cultivation temperature the proportion of the soluble delta ssCGT was slightly increased. Intracellularly expressed delta ssCGT provides a potential affinity handle which forms easily refoldable inclusion bodies increasing the yield and stability, and possibly allows the expression of lethal target proteins. Interestingly, the interaction between one model fusion protein delta ssCGT-CAT (CAT, chloramphenicol acetyltransferase) and the E. coli heat shock protein GroEL was observed.
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PMID:Expression in E. coli and purification of intracellular proteins by fusion to cyclomaltodextrin glucanotransferase. 136 55

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

ELAM1 (endothelial leukocyte adhesion molecule 1, also known as E-selectin) is a highly tissue-specific adhesion molecule that is transiently and exclusively expressed on cytokine-induced endothelial cells. We have identified two proximal ELAM1 promoter elements and their DNA-binding factors that are, in addition to NF-kappa B, essential for ELAM1 transcription. Mutation of either element in promoter constructs carrying the first 383 nucleotides of the ELAM1 promoter markedly diminshed the expression of a fused chloramphenicol acetyltransferase reporter gene. Although multimers of either element failed to display enhancer activity on its own, fusion of the most upstream of these to the NF-kappa B element had a strong stimulatory effect. This site, ACATCAT, is recognized by a factor we have called NF-ELAM1. The site corresponds to NF-ELAM1's preferential binding sequence (A/T)CA(G/T)CA(G/T) as determined in a target definition assay. This element is identical to the T-cell delta A enhancer found in the T-cell receptor-alpha, -beta, and CD3 delta genes. Our results suggest that the delta A/NF-ELAM1 element can function as a modulator of NF-kappa B in endothelial cells both as well as a T-cell enhancer.
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PMID:A T-cell enhancer cooperates with NF-kappa B to yield cytokine induction of E-selectin gene transcription in endothelial cells. 138 98

The interactions of nuclear proteins from embryonal carcinoma cells (PCC3) with the long terminal repeats (LTRs) of murine intracisternal A particle (IAP) genes were studied. Two protein-DNA complexes were detected between PCC3 nuclear extract and IAP LTRs in a gel mobility shift assay. An additional complex was observed when enriched fractions from a heparin-agarose column were used as the source of proteins. Two regions within the LTR of IAP 81 were identified as the sites of protein interaction by DNase I protection. One region encompasses 43 nucleotides within the U3 region at the 5' end of LTR 81. The other covers a 78 base pair region lying within 100 nucleotides upstream from the transcription initiation site. Studies using constructs containing intact or deleted versions of the LTR fused to the bacterial chloramphenicol acetyltransferase gene indicated that the absence of the 5' 47 base pairs reduced the level of chloramphenicol acetyltransferase transcription to 20% of that driven by the entire LTR. Southwestern analysis of PCC3 nuclear extracts and column fractions revealed that a 28,000- and a 46,000-dalton protein were the major species that interact with the 5' end of IAP LTR 81.
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PMID:Nuclear protein binding to the 5' enhancer region of the intracisternal A particle long terminal repeat. 140 Apr 31

Basal expression of a chimeric gene (pMHO4CAT) consisting of approximately 7 kilobase pairs (kbp) of the 5'-flanking region of the mouse heme oxygenase-1 (HO-1) gene fused to the bacterial chloramphenicol acetyltransferase gene is 2- to 10-fold greater than that of an analogous construct containing only 1287 bp of the 5'-flanking region (pMHO1CAT) in transiently transfected cultured cells. The enhancer activity has been localized to a 268-base pair (bp) fragment positioned approximately 4 kilobase pairs upstream of the transcription initiation site. This fragment contains two high affinity protein binding sites, regions A and B, as determined by DNase I protection assays using nuclear protein extracts from rat C6 glioma cells. Both sites include core sequence elements, TGAGTCA (region A) and TGTGTCA (region B), that resemble the consensus binding site, TGA(G/C)TCA, of the Jun/Fos (AP-1) family of transcription factors. Purified, bacterially expressed AP-1 (c-Jun homodimer) specifically binds to both elements, exhibiting greater affinity for the region A motif. The expression of pMHO4CAT, but not of pMHO1CAT, is stimulated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), and the 268-bp enhancer fragment confers TPA inducibility and c-Jun/c-Fos transactivation to the heterologous SV40 promoter. These functions are mediated by the AP-1 binding sites as multiple copies of the region A motif also confer TPA induction and c-Jun/c-Fos transactivation upon a heterologous promoter.
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PMID:Distal AP-1 binding sites mediate basal level enhancement and TPA induction of the mouse heme oxygenase-1 gene. 140 Apr 99

Previous transfection experiments have shown that 162 base pairs (bp) of the 5' flanking sequence of the chicken alpha A-crystallin gene are required for promoter activity in primary chicken lens epithelial cells (PLE), while only 111 bp of the 5' flanking sequence are needed for activity of the mouse alpha A-crystallin promoter in transfected chicken PLE cells or in a SV40 T-antigen-transformed transfected mouse lens epithelial cell line (alpha TN4-1). The effect of site-directed mutations covering positions -111 to -34 of the mouse alpha A-crystallin promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) gene was compared in transfected chicken PLE cells and mouse alpha TN4-1 cells; selected mutations were also examined in a nontransformed rabbit lens epithelial cell line (N/N1003A). In general, the same mutations reduced promoter activity in the transfected lens cells from all three species, although differences were noted. The mutations severely affected regions -111/-106 and -69/-40 regions in all the transfected cells examined; by contrast, mutations at positions -105/-99 and -87/-70 had a somewhat greater effect in the chicken PLE than the mouse alpha TN4-1 cells, while mutations of the -93/-88 sequence reduced expression in the alpha TN4-1 but not the PLE cells. A partial cDNA with sequence similarity to alpha A-CRYPB1 of the mouse has been isolated from a chicken lens library; mouse alpha A-CRYBP1 is a putative transcription factor which binds to the -66/-55 sequence of the mouse alpha A-crystallin promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Conservation of mouse alpha A-crystallin promoter activity in chicken lens epithelial cells. 140 19

When preadipocytes differentiate into adipocytes, several differentiation-linked genes are activated. Lipoprotein lipase (LPL) is one of the first genes induced during this process. To investigate early events in adipocyte development, we have focused on the transcriptional activation of the LPL gene. For this purpose, we have cloned and fused different parts of intragenic and flanking sequences with a chloramphenicol acetyltransferase reporter gene. Transient transfection experiments and DNase I hypersensitivity assays indicate that several positive as well as negative elements contribute to transcriptional regulation of the LPL gene. When reporter gene constructs were stably introduced into preadipocytes, we were able to monitor and compare the activation patterns of different promoter deletion mutants at selected time points representing the process of adipocyte development. We could delimit two cis-regulatory elements important for gradual activation of the LPL gene during adipocyte development in vitro. These elements, LP-alpha (-702 to -666) and LP-beta (-468 to -430), contain a striking similarity to a consensus sequence known to bind the transcription factors HNF-3 and fork head. Results of gel mobility shift assays and DNase I and exonuclease III in vitro protection assays indicate that factors with DNA-binding properties similar to those of the HNF-3/fork head family of transcription factors are present in adipocytes and interact with LP-alpha and LP-beta. We also demonstrate that LP-alpha and LP-beta were both capable of conferring a differentiation-linked expression pattern to a heterolog promoter, thus mimicking the expression of the endogenous LPL gene during adipocyte differentiation. These findings indicate that interactions with LP-alpha and LP-beta could be a part of a differentiation switch governing induction of the LPL gene during adipocyte differentiation.
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PMID:Characterization of the human lipoprotein lipase (LPL) promoter: evidence of two cis-regulatory regions, LP-alpha and LP-beta, of importance for the differentiation-linked induction of the LPL gene during adipogenesis. 140 52

We have recently cloned a cDNA encoding a mutant form of PRL receptor (PRL-R) from Nb2 cells, a PRL-dependent T lymphocyte-derived cell line. This cDNA is identical to the long form of the rat PRL-R, except for a deletion of 594 base pairs in the cytoplasmic domain, resulting in a mature receptor protein of 393 amino acids. Although a segment containing three cytoplasmic regions of moderate to high amino acid sequence identity with members of the PRL/GH receptor family is missing in this receptor form, the region of highest (70%) identity is retained. In the following studies, a homologous functional assay was developed to test the activity of three forms of receptor with respect to their ability to transmit a lactogenic signal. In this system, CHO cells were transiently transfected with a construct containing 2300 base pairs of the 5'-flanking sequence of the rat beta-casein gene fused to the chloramphenicol acetyltransferase (CAT) gene and an expression vector containing the various forms of rat PRL-R cDNA. The transfected cells were grown in serum-free medium in the absence or presence of PRL. In cells transfected with the long form of the PRL-R and beta-casein/CAT construct, a 7.2- +/- 0.9-fold induction (n = 3) of CAT activity was seen when cells were cultured in the presence of 400 ng/ml PRL and 1 micrograms/ml hydrocortisone. This level of stimulation was similar to that observed for the ovine beta-lactoglobulin/CAT construct in which a 5.7- +/- 1.2-fold (n = 3) effect was found.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The Nb2 form of prolactin receptor is able to activate a milk protein gene promoter. 140 2


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