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)

Treatment of cells with interferons induces various mRNAs and the corresponding proteins. We have described previously the isolation of a mouse cDNA clone (cDNA clone 202) which specifies an mRNA whose level is increased 20-fold in beta-interferon-treated Ehrlich ascites tumor cells. The increase is a consequence of an increased rate of transcription. The mRNA encodes a 56,000-dalton protein. We report here the isolation of a genomic clone including the 5' terminus of the 202 gene with the interferon-responsive region. Experiments involving primer extension and protection from cleavage by S1 nuclease revealed the existence of multiple 5' termini of 202 mRNAs in Ehrlich ascites tumor and Ltk- cells. Treatment with beta-interferon increased the level of these 202 mRNAs with different 5' termini nonuniformly. A 0.8-kilobase DNA segment from the 202 gene (including its 5' flanking region and its 5'-terminal exon) was ligated to the chloramphenicol acetyltransferase gene, and the resulting construct was transfected into mouse Ltk- cells. Treatment of these cells with beta-interferon increased the expression of the chloramphenicol acetyltransferase gene 5-10-fold. Within the first, untranslated exon of the 202 gene, we found a 29-nucleotide long sequence that is partially homologous to sequences which occur upstream from interferon-inducible human HLA and metallothionein IIA genes (Friedman, R. L., and Stark, G. R. (1985) Nature 314, 637-639).
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PMID:Interferons as gene activators. Cloning of the 5' terminus and the control segment of an interferon activated gene. 301 48

A human genomic clone (lambda hP-450mc-1), highly homologous to the rat cytochrome P-450c gene, was isolated and analyzed for the complete nucleotide sequence. The gene structure coincides with that of a recently reported human gene isolated from genomic DNA of a human breast carcinoma cell line, MCF-7 [Jaiswal, A.K., Gonzalez, F.J. & Nebert, D.W. (1985) Nucleic Acids Res. 13, 4503-4520] with notable exceptions in the first intron: a 320-base-pair fragment is inserted and a 650-base-pair fragment is deleted in the gene examined in the present study. The 320-base-pair insert appears to contain a moderately repetitive sequence (approx. 140 copies) in the human genome. The 650-base-pair fragment, present in intron 1 of the reported sequence, is dislocated in the lambda hP-450mc-1 to about 10(4) base pairs upstream from the putative transcription initiation site. The results of Southern blot analysis using human total DNA were compatible with the gene structure of lambda hP-450mc-1. A fusion gene, which was constructed by ligating the 5' flanking region of the gene to the structural gene for prokaryotic chloramphenicol acetyltransferase (CAT), inducibly expressed the CAT activity in mouse Hepa-1 cells in response to administered methylcholanthrene, indicating that the isolated human gene is indeed of methylcholanthrene inducibility.
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PMID:Structure and drug inducibility of the human cytochrome P-450c gene. 301 83

The human 4F2 cell surface antigen is a 120-kilodalton (kDa) disulfide-linked heterodimer which is composed of an 80- to 90-kDa glycosylated heavy chain (4F2HC) and a 35- to 40-kDa nonglycosylated light chain (4F2LC). 4F2 belongs to a family of inducible cell surface molecules which are involved in T-lymphocyte activation and growth. To better understand the molecular mechanism(s) that controls 4F2HC gene expression in both resting and activated T cells, a 4F2HC human genomic clone was isolated and structurally characterized. The 4F2HC gene spans 8 kilobases of chromosome 11 and is composed of nine exons. The 5' upstream region of the gene displays several properties which are characteristic of housekeeping genes. It is G+C rich and hypomethylated in peripheral blood lymphocyte DNA and contains multiple binding sites for the Sp1 transcription factor while lacking TATA or CCAAT sequences. This region of the gene also displays sequence homologies with several other inducible T-cell genes, including the interleukin-2, interleukin-2 receptor alpha chain, dihydrofolate reductase, thymidine kinase, and transferrin receptor genes. A 255-base-pair fragment of the 4F2HC gene which contains 154 base pairs of the 5' flanking sequence was able to efficiently promote expression of the bacterial chloramphenicol acetyltransferase gene in human Jurkat T cells, indicating that it contains promoter or enhancer (or both) sequences. Analyses of chromatin structure in resting and lectin-activated T cells revealed the presence of stable DNase I-hypersensitive sites within both the 5' flanking and intron 1 regions of the 4F2HC gene. Although the 4F2HC gene displayed many of the structural features characteristic of a constitutively expressed gene, lectin-mediated activation of resting peripheral blood T lymphocytes resulted in a dramatic increase in steady-state levels of 4F2HC mRNA.
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PMID:Isolation and structural characterization of the human 4F2 heavy-chain gene, an inducible gene involved in T-lymphocyte activation. 326 70

The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.
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PMID:Firefly luciferase gene: structure and expression in mammalian cells. 382 27

Three polypeptides are produced from the major immediate-early (IE) region of human cytomegalovirus by alternative splicing. The IE gene products regulate subsequent viral and cellular gene expression. We previously reported that cotransfection of a genomic clone of the major IE region stimulated transient expression of chloramphenicol acetyltransferase driven by the dihydrofolate reductase (DHFR) promoter and that an intact E2F site was required for the trans activation (M. Wade, T. F. Kowalik, M. Mudryj, E.-S. Huang, and J. C. Azizkhan, Mol. Cell. Biol. 12:4364-4374, 1992). With the availability of cDNA clones for the individual major IE proteins, we sought to determine which of these proteins exerted this effect and whether the IE protein(s) interacted with E2F. In this study, we use cotransfection to demonstrate that the 55- and 86-kDa major IE proteins from the IE2 region can each moderately trans activate the DHFR promoter and that the 72-kDa IE1 protein stimulates DHFR transcription to a much higher level. Furthermore, trans activation through the 72-kDa IE1 protein is in part E2F dependent, while activation by the 55- and 86-kDa IE proteins is E2F independent. We also demonstrate by in vitro pull-down assays that the 72-kDa IE1 protein can specifically interact with the DNA binding domain of E2F1 (amino acids 88 to 191) in the presence of nuclear extract. Moreover, antibodies to either E2F1 or IE72 will immunoprecipitate both E2F and IE72 from cells that stably express IE72, and antibody to E2F1 will immunoprecipitate IE72 from normal human fibroblast cells infected with human cytomegalovirus.
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PMID:Interaction of the 72-kilodalton human cytomegalovirus IE1 gene product with E2F1 coincides with E2F-dependent activation of dihydrofolate reductase transcription. 749 86

Platelet thromboxane receptors are acutely and reversibly upregulated after acute myocardial infarction. To determine if platelet thromboxane receptors are under transcriptional control, we isolated and characterized human genomic DNA clones containing the 5' flanking region of the thromboxane receptor gene. The exon-intron structure of the 5' portion of the thromboxane receptor gene was determined initially by comparing the nucleotide sequence of the 5' flanking genomic clone with that of a novel human uterine thromboxane receptor cDNA that extended the mRNA 141 bp further upstream than the previously identified human placental cDNA. A major transcription initiation site was located in three human tissues approximately 560 bp upstream from the translation initiation codon and 380 bp upstream from any previously identified transcription initiation site. The thromboxane receptor gene has neither a TATA nor a CAAT consensus site. Promoter function of the 5' flanking region of the thromboxane receptor gene was evaluated by transfection of thromboxane receptor gene promoter/chloramphenicol acetyltransferase (CAT) chimera plasmids into platelet-like K562 cells. Thromboxane receptor promoter activity, as assessed by CAT expression, was relatively weak but was significantly enhanced by phorbol ester treatment. Functional analysis of 5' deletion constructs in transfected K562 cells and gel mobility shift localized the major phorbol ester-responsive motifs in the thromboxane receptor gene promoter to a cluster of activator protein-2 (AP-2) binding consensus sites located approximately 1.8 kb 5' from the transcription initiation site. These studies are the first to determine the structure and organization of the 5' end of the thromboxane receptor gene and demonstrate that thromboxane receptor gene expression can be regulated by activation of protein kinase C via induction of an AP-2-like nuclear factor binding to upstream promoter elements. These findings strongly suggest that the mechanism for previously described upregulation of platelet thromboxane receptors after acute myocardial infarction is increased thromboxane receptor gene transcription in platelet-progenitor cells.
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PMID:Characterization of 5' end of human thromboxane receptor gene. Organizational analysis and mapping of protein kinase C--responsive elements regulating expression in platelets. 764 19

Neuron-specific enolase (NSE) occurs in mature neurons and paraneurons. We have isolated the genomic clone coding for rat NSE and clarified its gene structure. In order to analyze the regulatory sequence in the 5'-upstream region and introns, we carried out transient expression experiments of NSE genomic DNA fragments fused to chloramphenicol acetyltransferase (CAT) gene which were transfected into several cultured cells. The used cells were primary cultured rat neurons, PC12, neuroblastoma 35, neuroblastoma 103, C6, primary cultured rat glial cells and HeLa cells. The promoter sequence (190 bp) upstream to the transcription initiation site was important in the expression of CAT gene in these cells. From the experiments with external and internal deletion mutants of the fusion gene, the cis-acting regulatory region responsible for the enhanced expression of the CAT activity in the primary cultured neuron and PC12 cells was found to be localized at upstream 500 bp sequence of the intron 1 and 1.5 kbp upstream sequence of the transcription initiation site. In the upstream important sequences, there were the nearest sequences for AP-1 binding motif, AP-2 binding element, SP-1 binding sequence, cAMP response element, half site of glucocorticoid receptor (GRE) binding sequence, half site of thyroid hormor receptor (TR) or retinoic acid receptor (RAR) binding sequence and MTF-1 binding sequence. Furthermore, Octamer-6 binding motifs also were found. In the intron 1, 5' end upstream 50 bp and downstream 100 bp were the most important sequences. We found the nearest sequences for cAMP response element, E2F binding sequence, early growth response (EGR)-1 binding motif, half site of TCF-1 binding sequence and a neuron-specific element-like sequence in the intron 1.
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PMID:Upstream and intron regulatory regions for expression of the rat neuron-specific enolase gene. 770 74

A genomic clone (19 kb) harboring the intron-exon sequences and the promoter-regulatory region of the E1 beta gene of human pyruvate dehydrogenase complex was isolated by screening a placental genomic library. The nucleotide sequence of the promoter region (1245 bp) showed 18 differences (including mismatches, insertions, and deletions) as compared to the published sequence [Koike et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5594-5597]. The E1 beta promoter lacked a TATA box homology but contained initiator sequences (two) and Sp1 sites (three) which are frequently found in TATA-less promoters. The DNase I footprinting pattern of the promoter region with crude rat liver nuclear extracts showed at least seven regions of protein binding and nuclease protection (P1-P7). The DNase I protected regions contained consensus nucleotide sequences recognized by GATA-1, Sp1, IgNF-A, Lva, bicoid Q9, NF-kB, HNF-5, H4TF-1, WAP5, and ADH transription factors. Transient expression of chloramphenicol acetyltransferase (CAT) suggested the possible presence of negative elements located within the sequence from -2316 to -930, whereas deletion constructs containing -929 to +32 and -98 to +32 DNA sequences showed approximately 7- and 20-fold increases in CAT activity over the basal CAT activity. Additional studies indicated the presence of an orientation-dependent cis element (or elements) within the region from -282 to -397 that acts as an enhancer or a repressor upon a heterologous thymidine kinase promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the promoter regulatory region of the human pyruvate dehydrogenase beta gene. 782 76

CDK2 (cyclin-dependent kinase 2) is a serine/threonine kinase which is involved in regulating S-phase entry in higher eukaryotes. To investigate the transcriptional control of this gene, a 13-kb Xenopus laevis genomic clone containing the 5' flanking sequences was isolated. A 2.7-kb fragment containing the promoter region was sequenced and the transcription start point (tsp) was determined by primer extension. Several putative regulatory elements, such as the E2F-binding site, Y box and octamer-binding site, were localized in this region, but no TATA box was found. When fused to cat, a reporter gene encoding chloramphenicol acetyltransferase, the 5' flanking sequences were shown to function in oocytes and an enhancer activity was found in this region. During early embryogenesis, cdk2 promoter activity was tested and de novo transcription was detected at the mid-blastula transition.
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PMID:Cloning of the Xenopus laevis cdk2 promoter and functional analysis in oocytes and during early development. 782 9

A genomic clone encoding the hamster CYP1A1 gene was isolated from a hamster EMBL-3 genomic library and characterized. The CYP1A1 gene contained seven exons including the noncoding first exon as determined for CYP1A1 of other species. DNA sequence analysis up to -2307 bp of the CYP1A1 gene revealed the occurrence of five consensus xenobiotic responsive elements (XREs) and one basal transcription element (BTE) in addition to the canonical TATA box. For functional analysis, transfection experiments were performed in human hepatoma HepG2 cells with reporter gene constructs consisting of fragments with various lengths of the 5'-flanking region of the CYP1A1 gene and bacterial chloramphenicol acetyltransferase (CAT) gene. External deletion of the upstream region from the reporter gene resulted in a stepwise decrease of the CAT activity, suggesting that XREs were responsible for inducible expression of CYP1A1 gene by 3-methylcholanthrene (MC). A negative regulatory element (NRE) was also identified in the 5'-flanking region at -833 to -642. Removal of the NRE from the CYP1A1-CAT fusion gene resulted in about 3-fold increase of MC-inducible CAT activity. Using gel retardation assays with HepG2 nuclear extract, we demonstrated the presence of a specific protein which bound to the NRE fragment. Further competition analysis and methylation interference assays revealed that the nuclear protein bound to a 22-base fragment (from -688 to -709) of the NRE region, whose sequences were conserved among hamster, human, and rat CYP1A1 genes.
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PMID:Characterization of hamster CYP1A1 gene: inducible expression and negative regulation. 788 54


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