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)

Genomic clones containing 1.7 kilobases of the 5'-flanking region of the rat TSH receptor (TSHR) plus coding sequence from the ATG initiation codon [1 basepair (bp)] to the start of the first intron (170 bp) have been isolated and characterized. RNAase protection, primer extension, and cDNA sequences cloned by the anchored polymerase chain reaction identified multiple transcriptional start sites, the major ones clustered between -89 to -68 bp. This portion of the 5'-flanking region has neither a TATA nor a CCAAT box, is GC rich but has no GC box motif, and has features of promoters seen in "housekeeping" genes. Chimeras containing 1.7 kilobases (-1707 to -2 bp) of the 5'-flanking region, or deletions thereof, and the bacterial chloramphenicol acetyltransferase (CAT) gene expressed significant CAT activity when transfected into rat thyroid cell lines, FRTL-5 and FRT, but not BRL rat liver or HeLa cells. TSH decreased CAT activity in the FRTL-5 thyroid cells that had been stably transfected with the TSHR-CAT chimeric constructs. Negative regulation of promoter activity by TSH was duplicated by 10 microM forskolin in FRT thyroid cells, which express no TSHR mRNA. Deletion analyses indicated that a "minimal" region, exhibiting promoter activity, tissue specificity, and negative regulation by TSH, is located between -195 and -39 bp; this region is highly conserved in rat and human TSHR genes. Differential digestion of genomic DNA by MspI and HpaII revealed that the TSHR promoter is methylated in FRT, but not FRTL-5, cells; methylation of the promoter may be associated with loss of endogenous TSHR gene expression in FRT cells.
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PMID:Characterization of the 5'-flanking region of the rat thyrotropin receptor gene. 131 4

The genome of Sindbis virus encodes the polypeptides that are required for the replication and transcription of the virus RNA in infected cells. These polypeptides are translated as a polyprotein that is co- and post-translationally cleaved by an autoproteinase to give rise to four polypeptides designated nsP1, nsP2, nsP3 and nsP4. We have initiated a study of the functions of these proteins by expressing them in the Autographa californica baculovirus polyhedrin expression system. Spodoptera frugiperda cells infected with the recombinant baculovirus synthesized the four Sindbis polypeptides. We used a complementation assay which measures chloramphenicol acetyltransferase (CAT) activity to demonstrate that these proteins were biologically active. The infected cells were transfected with a Sindbis defective RNA that contains the CAT gene downstream of the promoter for the synthesis of the viral subgenomic RNA. CAT activity was found only in cells that had been infected with the recombinant baculovirus, not with wild type baculovirus, indicating that the required Sindbis nsP activities were present. Sindbis virions grew poorly in S. frugiperda cells and self-replicating Sindbis RNAs produced only very low levels of biological activity. Our results suggest that these cells are defective in their ability to replicate Sindbis RNAs and that the block is partially overcome when the Sindbis nsP mRNA is expressed under the control of the baculovirus DNA.
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PMID:Expression of the nonstructural proteins of Sindbis virus in insect cells by a baculovirus vector. 132 Jul 94

Expression from the promoter of the herpes simplex virus type 2 (HSV-2) large subunit of ribonucleotide reductase (ICP10) is stimulated by co-transfection with DNA that encodes the virion protein Vmw65 previously shown to activate in trans the transcription of all IE genes (Wymer et al., 1989). Specific cis response elements involved in ICP10 transcriptional regulation were studied by chloramphenicol acetyltransferase analysis with hybrid ICP10 promoter/CAT structural gene constructions containing wild type or site-directed mutations of the promoter sequences. The data indicate that Vmw65 activation requires an intact TAAT-GARAT motif while complex formation requires an intact Oct-1 element, and the AP-1 consensus elements in the ICP10 promoter are functional in vitro. Thus, expression from the wild type and GA-rich mutant constructions was enhanced 10-20-fold by co-transfection with DNA encoding Vmw65. The GARAT and POU homeobox (PHB) binding motifs were required for Vmw65 mediated activation but the mutant in the POU specific box (PSB) binding motif was activated at higher concentrations of Vmw65 DNA (1.0-3.0 micrograms). The PHB and PSB binding motifs were necessary for complex formation as determined by gel retardation analysis with in vitro synthesized OTF-1 and Vmw65 proteins. The GARAT and GA-rich elements were not required. CAT expression from pICP10-cat was enhanced by co-transfection with jun and fos encoding DNA, and the ICP10 promoter complexed with in vitro synthesized jun protein.
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PMID:Immediate early and functional AP-1 cis-response elements are involved in the transcriptional regulation of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10). 132 Jul 96

Transcriptional activation of human heat shock protein (HSP) genes by heat shock or other stresses is regulated by the activation of a heat shock factor (HSF). Activated HSF posttranslationally acquires DNA-binding ability. We previously reported that quercetin and some other flavonoids inhibited the induction of HSPs in HeLa and COLO 320DM cells, derived from a human colon cancer, at the level of mRNA accumulation. In this study, we examined the effects of quercetin on the induction of HSP70 promoter-regulated chloramphenicol acetyltransferase (CAT) activity and on the binding of HSF to the heat shock element (HSE) by a gel mobility shift assay with extracts of COLO 320DM cells. Quercetin inhibited heat-induced CAT activity in COS-7 and COLO 320DM cells which were transfected with plasmids bearing the CAT gene under the control of the promoter region of the human HSP70 gene. Treatment with quercetin inhibited the binding of HSF to the HSE in whole-cell extracts activated in vivo by heat shock and in cytoplasmic extracts activated in vitro by elevated temperature or by urea. The binding of HSF activated in vitro by Nonidet P-40 was not suppressed by the addition of quercetin. The formation of the HSF-HSE complex was not inhibited when quercetin was added only during the binding reaction of HSF to the HSE after in vitro heat activation. Quercetin thus interacts with HSF and inhibits the induction of HSPs after heat shock through inhibition of HSF activation.
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PMID:Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids. 132 38

The transgenic mouse strain CAT40 carries in its germ line one copy of a DNA construct consisting of the chloramphenicol acetyltransferase gene and the immunoglobulin heavy-chain enhancer. We show that transgene integration has resulted in a recessive lethal mutation that leads to death of homozygous CAT40 embryos shortly after implantation. The transgene has integrated adjacent to the 3' end of the gene coding for the beta subunit of the brain-specific Ca2+/calmodulin-dependent protein kinase II (Camk-2). The complete cDNA sequence of the Camk-2 gene and most of its exon/intron structure was determined. The deduced amino acid sequence is highly homologous to the previously described rat protein. The chromosomal location of the Camk-2 locus was mapped by interspecific backcross analysis to the proximal region of mouse chromosome 11. This region lacks previously identified recessive embryonic lethal mutations. During embryonic development, Camk-2-specific transcripts are first seen in the head section of 12.5-day-old embryos, and in adult mice the gene is expressed almost exclusively in the brain. Although transcription of the Camk-2 gene in heterozygous CAT40 mice is affected by transgene integration, it is unlikely that this gene is responsible for the mutant phenotype, since it is not expressed in blastocysts and the first transcripts during normal development are detected after the death of homozygous CAT40 embryos. Transgene integration is accompanied by a large deletion of cellular DNA; death is therefore most likely caused by the loss of a gene or genes that are important for early postimplantation development.
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PMID:Structure, expression, and chromosome location of the gene for the beta subunit of brain-specific Ca2+/calmodulin-dependent protein kinase II identified by transgene integration in an embryonic lethal mouse mutant. 132 43

Human phenylalanine hydroxylase (PAH) catalyzes the conversion of L-phenylalanine to L-tyrosine. Deficiency of this enzyme results in phenylketonuria, a common genetic disorder of amino acid metabolism that causes severe mental retardation. In primates, PAH is expressed specifically in the liver, while in rodents PAH activity is also present in kidney, although at a much lower level. A 9-kilobase genomic DNA fragment at the 5' end of the hPAH gene (hPAH) was fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The hPAH/CAT minigene was used to generate multiple transgenic mouse lines. In all expressing lines, CAT activity was detected predominantly in the liver and at much lower levels in the kidney. By immunohistochemical staining, CAT expression was localized to hepatocytes and renal epithelial cells, both of which also express the endogenous mouse PAH enzyme. Furthermore, both the transgene and the endogenous mouse PAH were activated at about the same stage of embryonic development in the mouse liver. These results suggest that the 9-kilobase DNA fragment flanking the 5' end of the human PAH gene contains all the necessary cis-acting elements to direct tissue- and developmental-specific expression in vivo.
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PMID:Tissue- and development-specific expression of the human phenylalanine hydroxylase/chloramphenicol acetyltransferase fusion gene in transgenic mice. 132 25

HeLa S3 cells, which have been fractionated into sequential and synchronous cell cycle phase-specific fractions, express c-fos at twice the basal levels in the earliest part of G1 phase. To determine whether this peak in c-fos synthesis has regulatory significance, a DNA construct was prepared which contained the human c-fos gene under the transcriptional control of the SV40 promoter complex. The pc-fos(human)-1 gene (9 kilobases) was inserted into the eukaryotic expression vector pSG5 (4.076 kilobases) at the EcoRI site. Electroporation with an exponentially decaying pulse was employed to cotransfect this construct into HeLa S3 cells along with the plasmids pRSVcat and the neomycin-resistance plasmid pF beta fos3' neo. The level of transient expression of each plasmid was determined. Transfection efficiency was determined as percentage fluorescent cells by measurement of immunofluorescence with a chloramphenicol acetyltransferase (CAT) antibody. Efficiency of transfection ranged up to approximately 5% of the cells. Transfected cells were selected on the basis of resistance to Geneticin (G418) at 400 micrograms/mL. CAT fluorescence and Geneticin resistance were employed to select permanently transformed cell lines. Compared with exponentially growing cells, successfully transfected cell lines expressed more than twice the level of c-fos mRNA as determined by dot-blot analysis and 16 times more of the 62-kilodalton c-fos protein as determined by Western blot analysis. As all cells in the population were not stable c-fos transfectants, this value is likely to be an underestimate of the overexpression level. In addition, expression was under the control of a strong serum induction insensitive promoter, unlike the native c-fos promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Growth of HeLa S3 cells cotransfected with plasmids containing a c-fos gene under the control of the SV40 promoter complex, pRSVcat, and G418 resistance. 132 4

DNA sequence analysis and 5'-end mapping of mRNA were used to identify and clone DNA fragments which contain the presumptive promoter (Pgl) and poly(A) site (An) of the bovine herpesvirus 1 (BHV1) gl glycoprotein. To confirm the presence of these regulatory regions in the above fragments, they were cloned together with a chloramphenicol acetyltransferase (CAT) reporter gene into pUC19. The recombinant plasmid formed, pEC3, was capable of inducing CAT activity when transfected into bovine cells demonstrating that the Pgl-CAT-An sequence constituted a functional CAT expression cassette. The cassette was excised from pEC3, transferred to a plasmid insertion vector (pIV3A) and subsequently inserted into the thymidine kinase (tk) gene of BHV1. Insertion in either orientation, relative to the tk gene, gave rise to BHV1 recombinants which expressed CAT activity in infected cells. Analysis of RNA from infected cells indicated that CAT transcripts were present in multiple species of RNA. This unexpected result was found to reflect temporal shifts in promoter and poly(A) site usage during infection. Although the poly(A) site which forms part of the expression cassette was used extensively early in infection, most CAT transcripts synthesized at late times read through this promoter-proximal site and terminated at the distal site normally used for tk mRNA synthesis.
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PMID:Bovine herpesvirus 1 as a live virus vector for expression of foreign genes. 132 99

P element mediated germ-line transformation was used to study the developmental specificity of Drosophila chorion gene regulatory sequences directing expression of the bacterial reporter genes for chloramphenicol acetyltransferase (CAT) and beta-galactosidase (lacZ). DNA fragments containing 5' flanking plus the entire 5' untranslated and the beginning of the coding region of either the s36 or the s15 chorion gene are able to confer on the reporter genes normal tissue as well as temporal specificity of expression, exclusively in the ovary of transformed female flies. However, if 5' untranslated and coding regions are omitted, normal ovarian expression is maintained but tissue specificity is relaxed: expression of the reporter gene is detected both in the ovary and in specific non-ovarian tissues of transformed females and males. The evidence suggests that the missing 5' untranslated and coding sequences may include negative elements that normally suppress expression in non-ovarian tissues, and that these putative elements are distinct from those that prevent premature expression in the ovarian follicles. The exact location of ectopic lacZ expression within the internal male genitalia depends on the constellation of 5' flanking chorion regulatory sequences included in the P element constructs. Ectopic expression of the CAT gene in the male genitalia under s15 promoter control can be abolished by mutating the hexamer TCACGT, a sequence previously shown to be essential for the normal expression of this chorion gene in the ovary.
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PMID:Chorion gene cis-regulatory DNA restricts tissue specificity of reporter gene expression in transformed Drosophila. 132 96

We tested the efficiency of several different cationic liposome formulations, complexed to one of two different chloramphenicol acetyltransferase (CAT) reporter plasmids, in transfecting freshly isolated, highly purified rat lung alveolar type II cells, alveolar macrophages, and three different human lung carcinoma cell lines, as well as NIH 3T3 cells, a rapidly dividing, transformed mouse fibroblast line. Our results demonstrated that several different cationic liposome formulations can mediate high-level CAT gene expression in all the cell types tested. Electron microscopic analysis confirmed that cationic liposome-DNA complexes are avidly bound and internalized by lung cells. The time course of expression of transfected genes in nontransformed cell types with low mitotic indices, such as type II cells, is poorly characterized. NIH 3T3 cells expressed maximal CAT activity by day 4 following transfection, with virtual disappearance of activity by day 11. Conversely, type II cells expressed maximal CAT activity between days 5 and 11, and CAT activity was still clearly present 35 days after transfection. Southern blot analysis of DNA isolated from transfected type II cells revealed that the CAT gene was largely present in an extrachromosomal form, rather than integrated into genomic DNA. These observations indicate that following cationic liposome-mediated transfection, rat alveolar type II cells (the majority of which do not divide in culture) can express transfected genes for prolonged periods, apparently mediated by expression of the transgene in an episomal form.
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PMID:Prolonged transgene expression in rodent lung cells. 132 13


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