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)

Some human small cell lung carcinomas (SCLC) secrete proopiomelanocortin (POMC) derived peptides, but in contrast to the pituitary, glucocorticoids fail to inhibit this hormone production. We have previously described an in vitro model using human SCLC cell lines that express POMC and are resistant to glucocorticoids. We have now identified the glucocorticoid receptor (GR) in the SCLC cell line COR L24 using a whole cell ligand binding assay (Kd = 5.7 nM; Bmax = 11 fmol/million cells), while another cell line, DMS 79, lacked significant glucocorticoid binding. To analyze GR function both positive (GMCO) and negative (TRE)3-tkCAT), glucocorticoid-regulated reporter gene constructs were transfected into COR L24 cells. In the SCLC cell line, neither hydrocortisone nor dexamethasone (500-2,000 nM) significantly induced chloramphenicol acetyltransferase expression from GMCO; in addition, they did not suppress chloramphenicol acetyltransferase expression from (TRE)3-tkCAT. Similar results were obtained with two other POMC-expressing SCLC cell lines. Expression of wild type GR in COR L24 cells restored glucocorticoid signaling, with marked induction of GMCO reporter gene expression by dexamethasone (9,100 +/- 910%; n = 3), and an estimated EC50 of 10 nM. This failure of the GR explains the resistance of the POMC gene to glucocorticoid inhibition and may have implications for cell growth in SCLC.
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PMID:Human small cell lung cancer cell lines expressing the proopiomelanocortin gene have aberrant glucocorticoid receptor function. 816 65

We studied the effects of thyroid hormone (T3) on nuclear protein-DNA interactions by using dimethyl sulfate (DMS) and DNase I ligation-mediated PCR footprinting. We examined an endogenous gene the growth hormone (GH) gene, and a stably transfected plasmid containing the chicken lysozyme silencer (F2) T3 response element (TRE) gene, F2-TRE-TK-CAT, both in pituitary tumor (GC) cells. The 235-1 cell line, which expresses prolactin (PRL) and Pit-1, but not the T3 receptor (TR) or GH, was used as a control. DMS and DNase I footprinting identified protected G residues in the Pit-1, Sp1, and Zn-15 binding sites of the GH gene in GC, but not in 235-1, cells. There was no specific protection of the tripartite GH TRE at -180 bp against either DMS or DNase I in the absence or presence of T3 in either cell line. However, T3 increased protection of the Pit-1 and Sp1 binding sites against DMS in GC cells. In GC cells stably transfected with a plasmid containing F2-TRE-TK-CAT or TRalpha, chloramphenicol acetyltransferase expression was T3 inducible and DMS footprinting revealed both F2 TRE TR-binding half sites in a pattern suggesting the binding of TR homodimers before and during T3 exposure. We conclude that the GH gene is accessible to specific nuclear proteins in GC, but not in 235-1, cells and that T3 enhances this interaction, although there is no evidence of TR binding to the low-affinity rat GH TRE. The presence of TR binding to the high-affinity F2 TRE before and during T3 exposure suggests that reversible interaction of T3 with DNA-bound TRs, rather than transient T3-TR contact with TREs, determines the level of T3-stimulated transcriptional activation.
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PMID:In vivo genomic footprinting of thyroid hormone-responsive genes in pituitary tumor cell lines. 875 47

To identify the 5' sequences of the murine coagulation factor VII (fVII) gene that resulted in its efficient transcription, a variety of 5'-flanking sequences up to 7 kilobase pairs upstream of the translation ATG initiation codon were fused to the reporter gene, bacterial chloramphenicol acetyltransferase, and relative expression levels of this gene in mouse Hepa 1-6 cells were determined. It was found that the 5' region extending approximately 85 base pairs (bp) upstream of the transcriptional initiation site served as the minimal DNA region that provided full relative promoter activity for chloramphenicol acetyltransferase expression. This region of the gene also contains consensus sequences for liver-enriched transcription factors, C/EBP beta and HNF4, as well as for the ubiquitous protein factors, AP1, H4TF1, NF1, and Sp1. In vitro DNase I footprinting of the 200-bp proximal region of the promoter with a murine Hepa 1-6 cell nuclear extract revealed a clear footprint of a region corresponding to -80 to -28 bp of the murine fVII gene, suggesting that liver factors interact with this region of the DNA. Competitive gel shift and supershift assays with different synthetic oligonucleotide probes demonstrate that proteins contained in the nuclear extract, identified as C/EBP beta, H4TF1, and HNF4, bind to a region of the murine fVII DNA from 85 to 32 bp upstream of the transcription start site. Purified Sp1 also interacts with this region of the DNA at a site that substantially overlaps, but is not identical to, the H4TF1 binding locus. Binding of Sp1 to the mouse DNA was not observed with the nuclear extract as the source of the transcription factors, suggesting that Sp1 is likely displaced from its binding site by H4TF1 in the crude extract. In vivo dimethyl sulfate footprint analysis confirmed the existence of these sites and additionally revealed two other binding regions slightly upstream of the CCAAT/enhancer-binding protein (C/EBP) binding locus that are homologous to NF1 binding sequences. The data demonstrate that appropriate transcription factor binding sites exist in the proximal promoter region of the murine fVII gene that are consistent with its strong liver-based expression in a highly regulated manner.
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PMID:Characterization of transcriptional regulatory elements in the promoter region of the murine blood coagulation factor VII gene. 944 72

Bovine leukemia virus (BLV) is a member of the human T-cell leukemia virus (HTLV)/BLV group of retroviruses. These viruses regulate their own transcription by producing Tax, a protein which activates the virus promoter region, the long terminal repeat (LTR). To explore the molecular mechanisms involved in the transactivation, we identified protein binding elements by in vivo footprinting and analyzed their function by site- directed mutagenesis. We used in vivo dimethyl sulfate footprinting by ligation-mediated PCR to detect constitutive in vivo protein-DNA interactions in a BLV-producing cell line, Bat2Cl6. The U3 region and part of the R region of the LTR were footprinted. In addition to the cis-acting elements (three cyclic AMP-responsive elements [CREs] and two AP4 sites) reported by others to be important for Tax-mediated activation of the BLV LTR, we found footprints in regions flanking these elements and in the core promoter region. The importance of these sites for transcriptional activation was studied by site-directed mutagenesis followed by promoter function analysis of the mutants with a chloramphenicol acetyltransferase reporter system. Our data corroborate those of others showing that the CREs are necessary for transactivation of the LTR, and they identify two new functional sites not previously reported by others. We show that the middle region of the BLV U3 contains multiple dual-functioning cis-acting elements which act as either positive or negative regulatory elements depending on the cell type tested. This is the first report of a functional mapping of the cis-acting elements of a virus of the HTLV/BLV group.
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PMID:In vivo protein binding and functional analysis of cis-acting elements in the U3 region of the bovine leukemia virus long terminal repeat. 962 Oct 62

The murine Ki-ras promoter contains a unique polypurine--polypyrimidine [poly(R.Y)] sequence between -290 and -320 from the 3' boundary of exon phi. Previously we demonstrated triplex formation and transcription inhibition promoted by GT and AG oligonucleotides directed against this site [Alunni-Fabbroni et al. (1996) Biochemistry 35, 16361--16369]. In this work, we have investigated triplex formation and anti-gene activity of five 20-mer AG motif triplex-forming oligonucleotides specific for the Ki-ras poly(R.Y) target, derived from 5'-AGGGAGGGAGGAAGGGAGGG (20AG) by replacing an increasing number of phosphodiester linkages with phosphorothioate linkages (S(i)-20AG; i = 2, 3, 4, 5, 19). Electrophoretic mobility-shift experiments (EMSA) showed that four thioate oligonucleotides, S(i)-20AG (i = 2, 3, 4, 5), recognized the Ki-ras target and exhibited dissociation constants similar to that of 20AG: K(d) = 12 +/- 2 nM, while the all-thioate S(19)-20AG exhibited a K(d) of 128 +/- 15 nM. Moreover, the binding between the Ki-ras promoter and oligonucleotides S(i)-20AG (i = 2, 3, 4, 5, 19) was characterized by DMS/piperidine and DNase I footprinting experiments. We observed that the introduction in the phosphodiester oligonucleotide 20AG of sulfur atoms reduced its aggregation significantly and increased its nuclease resistance. Transient transfection experiments using preformed triplexes with a recombinant plasmid containing the reporter chloramphenicol acetyltransferase (CAT) gene under the control of Ki-ras promoter showed that oligonucleotides S(i)-20AG (i = 2, 3, 4, 5, 19) promote a strong inhibition of up to 75% of the CAT expression when compared with control Ki-ras unspecific oligonucleotides. Taken together, these data provide a guideline for designing triplex-forming effector molecules capable of controlling Ki-ras expression in vivo.
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PMID:Anti-gene effect in live cells of AG motif triplex-forming oligonucleotides containing an increasing number of phosphorothioate linkages. 1117 Apr 38