Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The rat gene encoding phenylethanolamine N-methyltransferase (PNMT) was cloned and a consensus sequence for a glucocorticoid response element (GRE) was found at -513 bp, 5' to the transcriptional start site. In order to define the function of this element, fusion genes containing the PNMT promoter and a chloramphenicol acetyltransferase (CAT) reporter gene were constructed. These constructs did not express after transfection into any of 7 continuous cell lines, none of which endogenously produce PNMT. A system for transfecting chromaffin cells in primary culture was therefore devised using constructs containing 200 bp of the proenkephalin (ENK) promoter, whose expression characteristics are well known. pENK beta GAL-1, containing the ENK promoter with a lac Z reporter, was introduced into these cells and beta-galactosidase activity was visualized in situ. Approximately 90% of cells transfected were chromaffin; transfection efficiency was 5%. High levels of CAT activity were measured in chromaffin cells transfected with pENKAT12, possessing a CAT reporter. In contrast to tumor cell lines, pENKAT12 induction in these cells by forskolin and phorbol esters did not require a phosphodiesterase inhibitor. In this chromaffin system, both basal and regulated expression of the PNMT fusion genes were detected. Dexamethasone (dex) induced expression of pPNMT3000 and pPNMT900, containing the putative GRE and 3000 bp or 863 bp of PNMT promoter sequence, 4- to 10-fold. Expression of pPNMT300 and pPNMT100, which lack the GRE and contain 273 bp or 99 bp of PNMT promoter sequence, was unaffected by dex. Addition of the PNMT region spanning -490 to -863 bp conferred full dex responsiveness to a thymidine kinase promoter. Deletion of the putative GRE sequence by site-directed mutagenesis abolished the dex response. These data identify the sequence at -513 bp in the rat PNMT gene as a functional, positively acting GRE. Primary cultures of bovine chromaffin cells provide a biologically relevant expression system for transcriptional studies of catecholamine genes and their related neuropeptides.
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PMID:Identification of a functional glucocorticoid response element in the phenylethanolamine N-methyltransferase promoter using fusion genes introduced into chromaffin cells in primary culture. 230 57

The human medulloblastoma TE 671 cell line has been evaluated as a model for studying expression of transiently transfected phenylethanolamine N-methyltransferase (PNMT) promoter-fusion gene constructs. Because TE 671 cells are one of few continuous lines exhibiting a neuronal phenotype, possess both nicotinic and muscarinic ligand binding properties, and express PNMT mRNA, they represent a likely candidate system for study of cholinergic-regulated PNMT gene expression. When transfected with constructs containing from 0.1 to 3 kb of the region 5' to the initiation site for PNMT gene transcription, TE 671 cells express at detectable levels only those constructs containing the -391 to -863 bp portion of the rat PNMT promoter fused to the chloramphenicol acetyltransferase (CAT) gene. Both general and selective cholinergic agents regulate expression of the -863 to -391 bp PNMT region in a TK promoter-CAT reporter construct. With this construct, carbachol stimulates CAT expression 1.5-fold in transfected TE 671 cells. The selective agonists nicotine and muscarine each stimulate expression of the upstream TK-CAT constructs 2.8- and 1.9-fold respectively, while antagonists for these receptors block induction. Because TE 671 cells respond to both nicotinic and muscarinic stimuli, this continuous line represents a useful model system for studying PNMT gene regulation by cholinergic stimuli.
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PMID:Regulation of PNMT gene promoter constructs transfected into the TE 671 human medulloblastoma cell line. 883 Mar 18