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)

Insulin has been shown to inhibit rat growth hormone (GH) gene transcription. The effects of insulin were therefore tested on the expression of a transfected human GH gene. A 2.6-kilobase EcoRI fragment of the human GH gene was propagated in pUC18 and transfected by calcium-phosphate shock into HeLa and GC cells, respectively. Transfected cells grown in serum-free medium for 72 h expressed human GH measured by specific radioimmunoassay, incorporation of [35S] methionine into newly synthesized GH, and the presence of the appropriately sized protected transcripts seen after RNase protection assay. Immunoprecipitation analysis showed that insulin (0.7-7 nM) suppressed both the basal as well as the hydrocortisone (100 nM)-stimulated expression of newly synthesized 22-kDa GH in a dose-dependent fashion. Insulin (7 nM) also suppressed the basal and hydrocortisone-stimulated GH mRNA transcripts in these cells. Control nontransfected cells did not express human GH. Cells transfected with the truncated pOGH gene and pTKGH gene failed to respond to insulin treatment, whereas the human GH promoter was able to confer insulin responsiveness to the chloramphenicol acetyltransferase reporter gene. cis-Acting regulatory sequences residing on the 497-base pair 5'-flanking region of the human GH gene therefore appear to be a requirement for human GH gene response to the insulin signal.
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PMID:Insulin regulates expression of the human growth hormone gene in transfected cells. 290 52

The glucagon gene is expressed specifically in the alpha cells of the pancreatic islets. We show here that 300 base pairs of the 5'-flanking region of the rat glucagon gene, linked to a chloramphenicol acetyltransferase reporter plasmid transfected into islet cell lines of different hormone-producing phenotypes, directs transcription only in glucagon-producing islet cells. Deletional and linker-scanning mutations and DNase I footprinting assays identify three transcriptional control elements within these 300 base pairs. Two of these elements (G2 and G3) independently display enhancerlike functions on both homologous and heterologous promoters in glucagon (alpha) cells, but only on heterologous promoters in insulin- (beta) and somatostatin- (delta) expressing cells, and not in non-islet cells. The proximal promoter element (G1), characterized by low intrinsic transcriptional activity, is critical for specific expression of the glucagon gene in alpha cells. However, nuclear extracts prepared from all three islet cell phenotypes give similar protection to the three control elements of the glucagon 5'-flanking sequence. We conclude that these phenotypically distinct islet cell lines all contain regulatory DNA-binding proteins interacting with the three control elements of the glucagon gene, but that factors interacting with the glucagon promoter result in transcriptional activation only in alpha cells, to restrict glucagon gene expression to these cells. These observations suggest that interactions of nuclear proteins with cis-control elements are involved in the programmed developmental expression of the islet polypeptide hormone genes.
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PMID:Alpha-cell-specific expression of the glucagon gene is conferred to the glucagon promoter element by the interactions of DNA-binding proteins. 306 72

Gene 33, a rat gene transcriptionally enhanced by glucocorticoids, insulin, or cyclic AMP, was isolated from a library of rat genomic DNA and characterized by sequence comparison to a full-length cDNA. The structural gene spans 13,500 bp encoding 2970 bp of exon sequences interrupted by three introns of about 9600, 101 and 811 bp, respectively. Exons (5' to 3') are 198, 194, 77 and 2501 bp in length; the first of these initiates at the transcriptional start point determined by S1 nuclease mapping. The 5'-flanking DNA contains several putative transcriptional control elements including TATA and CAAT boxes and a binding site for the Sp1 transcription factor in the usual locations proximal to the start point. Sequences resembling known glucocorticoid and cyclic AMP regulatory elements are also found upstream. A chimeric plasmid was constructed containing putative gene 33 regulatory elements fused to the Escherichia coli gene cat, encoding the enzyme chloramphenicol acetyltransferase, and transfected into cultured fibroblasts. Transient expression assays established that this gene 33 DNA is effective in promoting transcription.
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PMID:Structure of a multihormonally regulated rat gene. 322 31

We evaluated the mechanism of insulin and phorbol ester induction of the proto-oncogene c-fos in Chinese hamster ovary fibroblasts stably transformed with high levels of genes expressing normal or truncated human insulin receptors. Both insulin and the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) induced c-fos mRNA accumulation in cells expressing high numbers of normal human insulin receptors; PMA but not insulin was effective in the cells expressing the mutant receptor. Transient expression studies with plasmid constructions containing c-fos 5'-flanking sequences ligated to the bacterial chloramphenicol acetyltransferase gene indicated that sequences corresponding to the serum response element were required for induction of c-fos transcription by both insulin and PMA. The insulin-sensitive cells contained a nuclear factor, presumably a protein, which bound specifically to this sequence of the c-fos gene; the apparent affinity of this factor to the normal serum response element was not affected by prior treatment of the cells with insulin or PMA. This c-fos binding factor may prove to be important in the regulation of c-fos expression by insulin and activators of protein kinase C.
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PMID:Identification of c-fos sequences involved in induction by insulin and phorbol esters. 327 73

Glucagon, a peptide hormone which regulates hepatic carbohydrate metabolism, is processed from a larger precursor, proglucagon. The gene encoding proglucagon is expressed at high levels in the A cells of the pancreatic islets and the L cells of the intestine, indicating that specific factors present in these two phenotypically distinct cells direct cell-specific expression. To characterize the factors that mediate glucagon gene transcription, we analyzed the 5'-flanking region of the rat glucagon gene for the existence of cis-acting sequences that promote glucagon gene transcription. A series of fusion genes containing sequentially shortened 5'-flanking sequences of the rat glucagon gene were constructed and fused to the coding sequence of the reporter enzyme chloramphenicol acetyltransferase. Analyses of the transcription of these fusion genes after their transfection into choriocarcinoma cells, fibroblasts, and islet cell lines of different phenotypes indicate that cis-acting DNA elements promote glucagon gene transcription only in islet cell lines. Transcriptional activity was much higher in glucagon compared to insulin-producing islet cell lines with fusion genes containing 249 or more base pairs of glucagon 5'-flanking sequence. Deletion of DNA sequences upstream of -168 abolished the preferential expression in glucagon-producing cell lines, however glucagon-chloramphenicol acetyltransferase fusion genes containing 168 base pairs or more of 5'-flanking sequence remained transcriptionally active, but only in islet cell lines. Fusion genes containing 115 base pairs of glucagon gene 5'-flanking sequences were transcriptionally inactive. These studies indicate that cis-acting DNA sequences present in the 5'-flanking region of the rat glucagon gene mediate islet cell-specific gene transcription.
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PMID:Glucagon gene 5'-flanking sequences promote islet cell-specific gene transcription. 331 2

Two cis-acting elements, the enhancer and the promoter, independently contribute to the cell-specific expression of the rat insulin 1 gene. The activities of these elements are presumably mediated by trans-acting factors. We have performed intracellular competition experiments that suggest the presence of a negative factor(s) that represses the enhancer activity in cells that do not express the insulin gene. In these experiments fibroblast cells (COS-7) were transfected with two plasmids: a test plasmid containing the gene for chloramphenicol acetyltransferase under the control of the thymidine kinase promoter and the insulin enhancer; and a competitor plasmid containing insulin enhancer sequences and the simian virus 40 origin of replication to permit its replication in the recipient cells. The presence of the competitor plasmid led to a 5- to 6-fold increase in chloramphenicol acetyltransferase activity as compared with the activity detected when insulin enhancer was absent from either the competitor or the test plasmid. A 5-fold increase in chloramphenicol acetyltransferase activity was also seen when the rat amylase enhancer was present on the competitor plasmid; in contrast the simian virus 40 enhancer exerted no effect. Efficient derepression required additional sequences downstream from those essential for enhancer activity. We propose that the activity of the rat insulin 1 enhancer is modulated by a negative trans-acting factor(s) that is active in cells not expressing insulin but is overridden by the dominant positive trans-acting factor(s) present in insulin-producing cells.
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PMID:Regulation of rat insulin 1 gene expression: evidence for negative regulation in nonpancreatic cells. 351 53

A method of introducing actively expressed genes into intact mammals is described. DNA precipitated with calcium phosphate has been injected intraperitoneally into newborn rats. The injected genes have been taken up and expressed by the animal tissues. To examine the generality of the method we have injected newborn rats with the chloramphenicol acetyltransferase prokaryotic gene fused with various viral and cellular gene promoters and the gene for hepatitis B surface antigen, and we observed appearance of chloramphenicol acetyltransferase activity and hepatitis B surface antigen in liver and spleen. In addition, administration of genes coding for hormones (insulin or growth hormone) resulted in their expression.
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PMID:Direct introduction of genes into rats and expression of the genes. 354 Sep 43

DNA sequences containing the 5'-flanking regions of the insulin and chymotrypsin genes were linked to the coding sequence of the chloramphenicol acetyltransferase (CAT) gene. The insulin gene recombinant elicits preferential expression of CAT activity when introduced into cells producing insulin; similarly, the chymotrypsin gene recombinant elicits preferential expression in chymotrypsin-producing cells. Sequences located upstream of previously defined transcriptional control elements are essential for efficient expression in both cases.
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PMID:Cell-specific expression controlled by the 5'-flanking region of insulin and chymotrypsin genes. 635

Insulin increases expression of somatostatin-chloramphenicol acetyltransferase (CAT) constructs 10-fold and thymidine kinase-CAT constructs 5-fold in GH4 cells. These responses are similar to our previously reported data on insulin-increased prolactin-CAT expression. They are also observed in HeLa cells and are thus not cell type specific. The evidence suggests that the insulin responsiveness of these genes is mediated by an Ets-related transcription factor. First, linker-scanning mutations and/or deletions of the prolactin, somatostatin, and thymidine kinase promoters suggest that their insulin responsiveness is mediated by the sequence CGGA. This sequence is identical with the response element of the Ets-related transcription factors. Second, CGGA-containing sequences placed at -88 in the delta MTV-CAT reporter plasmid conferred insulin responsiveness to the mammary tumor virus promoter. Third, expression of the DNA-binding domain of c-Ets-2, which acts by blocking effects mediated by Ets-related transcription factors, inhibits the response of these promoters to insulin. Finally, the Ets-related proteins Sap and Elk-1 bind to the prolactin, somatostatin, and thymidine kinase insulin-response elements. An Ets-like element was found in all insulin-sensitive promoters examined and may serve a similar function in those promoters.
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PMID:A consensus insulin response element is activated by an Ets-related transcription factor. 749 46

In differentiated 3T3-F442A adipocytes, insulin stimulated rapid and transient phosphorylation of c-Jun. Insulin also stimulated phosphorylation of c-Fos and several Fos-related proteins (pp72, pp45, and pp39) as indicated by precipitation with anti-c-Fos antibody following exposure to denaturating conditions. Phosphorylation of c-Fos was stimulated by 7-fold by 60 min, while phosphorylation of Fos-related proteins reached maxima of 3.5-5.5-fold at 15 to 60 min. The increase in phosphorylated c-Fos was due to an increase in both c-Fos protein and the stoichiometry of c-Fos phosphorylation, and was not observed in c-fos (-/-) cells. Additionally, insulin stimulated phosphorylation of a protein with molecular mass of approximately 82 kDa on tyrosine residues by 2.5-fold within 30 min; this protein appeared to be immunologically related to c-Fos. These increases in the phosphorylation of AP-1 transcription factors correlated with a > 5-fold stimulation of expression of a 12-O-tetradecanoylphorbol-13-acetate-responsive element-chloramphenicol acetyltransferase reporter gene transiently transfected into 3T3-F442A cells. These results indicate that insulin stimulates the phosphorylation of AP-1 transcription factors and several Fos-related proteins on serine and tyrosine residues. This is associated with changes in AP-1-mediated gene expression in vivo, suggesting that AP-1 phosphorylation by insulin plays a role in insulin-regulated gene expression.
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PMID:Insulin stimulates phosphorylation of c-Jun, c-Fos, and Fos-related proteins in cultured adipocytes. 751 56


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