Abstract
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.
Highlights
The effects of hormones on transcription are mediated through response elements in the promoters of genes
Insulin Increases Expression from the Somatostatin and the Thymidine Kinase Promoters—The observation that the insulin and cAMP response elements of the prolactin gene were composed of overlapping sequences (8) suggested that other cAMP-responsive genes might be insulin responsive
Multiple lines of evidence presented here and previously (8) indicate that the sequence CGGA is a consensus response element for insulin effects in GH and HeLa cells
Summary
Materials—[32P]dCTP, 3000 Ci/mmol, and [14C]chloramphenicol, 50 mCi/mmol, were obtained from ICN Biochemical Corp. All other enzymes and linkers were obtained from either New England Biolabs or from Boehringer Mannheim and, unless otherwise indicated, were used under conditions recommended by the suppliers. Duplex poly(dI1⁄7dC) was obtained from Pharmacia Biotech Inc. Reagents used for gel electrophoresis were purchased from Fisher. Acetyl-CoA and silica gel plates for thin layer chromatography were obtained from Sigma. All other reagents were of the highest purity available and were obtained from Sigma, Behring Diagnostics, Bio-Rad, Eastman, Fisher, or Boehringer Mannheim.
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