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Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene. 总被引:29,自引:16,他引:13
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P G Quinn T W Wong M A Magnuson J B Shabb D K Granner 《Molecular and cellular biology》1988,8(8):3467-3475
Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been identified by analysis of a series of PEPCK promoter mutations in transfection experiments. Fusion genes containing wild-type and mutated PEPCK promoter sequences from -600 to +69 base pairs (bp) fused to the coding sequence for chloramphenicol acetyltransferase were studied. Internal deletion mutations that replaced specific bases with a 10-bp linker within the region from -129 bp to -18 bp of the PEPCK promoter were examined. In addition, wild-type and mutated DNA templates were used as probes in DNase I protection experiments to determine sites of protein-DNA interaction. The PEPCK promoter contains a binding site for nuclear factor 1-CAAT. Deletion of the 5' end of this binding site reduced the size of the DNase I footprint in this region but had no effect on promoter activity. In contrast, deletion or disruption of the 3' end of this binding site completely eliminated protein binding and reduced promoter activity by 50%. Deletion of core sequences of the cAMP regulatory element (CRE) resulted in loss of cAMP responsiveness and an 85% decrease in basal promoter activity, indicating that the CRE also functions as a basal stimulatory element. Mutation of the core sequence of the CRE resulted in loss of the DNase I footprint over the CRE. Internal deletions flanking the CRE showed no loss of induction by cAMP but did have reduced promoter activity. This delimits the CRE to an 18-bp region between nucleotides -100 and -82. Analysis of mutations that disrupted bases between the CRE and the initiation site identified a basal inhibitory element adjacent to a basal stimulatory element, both located just 3' of the CRE, as well as a basal stimulatory element coincident with the TATA consensus sequence centered at -27. These data demonstrate that several cis-acting elements are located within 130 nucleotides of the initiation site of the PEPCK gene and that the CRE is essential for both basal promoter activity and cAMP-regulated expression of this gene. 相似文献
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Expression of the glycoprotein hormone alpha-subunit gene in the placenta requires a functional cyclic AMP response element, whereas a different cis-acting element mediates pituitary-specific expression. 总被引:8,自引:2,他引:6
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J A Bokar R A Keri T A Farmerie R A Fenstermaker B Andersen D L Hamernik J Yun T Wagner J H Nilson 《Molecular and cellular biology》1989,9(11):5113-5122
The single-copy gene encoding the alpha subunit of glycoprotein hormones is expressed in the pituitaries of all mammals and in the placentas of only primates and horses. We have systematically analyzed the promoter-regulatory elements of the human and bovine alpha-subunit genes to elucidate the molecular mechanisms underlying their divergent patterns of tissue-specific expression. This analysis entailed the use of transient expression assays in a chorionic gonadotropin-secreting human choriocarcinoma cell line, protein-DNA binding assays, and expression of chimeric forms of human or bovine alpha subunit genes in transgenic mice. From the results, we conclude that placental expression of the human alpha-subunit gene requires a functional cyclic AMP response element (CRE) that is present as a tandem repeat in the promoter-regulatory region. In contrast, the promoter-regulatory region of the bovine alpha-subunit gene, as well as of the rat and mouse genes, was found to contain a single CRE homolog that differed from its human counterpart by a single nucleotide. This difference substantially reduced the binding affinity of the bovine CRE homolog for the nuclear protein that bound to the human alpha CRE and thereby rendered the bovine alpha-subunit promoter inactive in human choriocarcinoma cells. However, conversion of the bovine alpha CRE homolog to an authentic alpha CRE restored activity to the bovine alpha-subunit promoter in choriocarcinoma cells. Similarly, a human but not a bovine alpha transgene was expressed in placenta in transgenic mice. Thus, placenta-specific expression of the human alpha-subunit gene may be the consequence of the recent evolution of a functional CRE. Expression of the human alpha transgene in mouse placenta further suggests that evolution of placenta-specific trans-acting factors preceded the appearance of this element. Finally, in contrast to their divergent patterns of placental expression, both the human and bovine alpha-subunit transgenes were expressed in mouse pituitary, indicating differences in the composition of the enhancers required for pituitary- and placenta-specific expression. 相似文献
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The core promoter for human DNA polymerase beta contains discrete binding sites for mammalian nuclear proteins, as revealed by DNasel footprinting and gel mobility shift assays. Two sites correspond to sequences identical with the Sp1 factor binding element, and a third site includes an eight residue palindromic sequence, TGACGTCA, known as the CRE element of several cAMP responsive promoters; the 5 to 10 residues flanking this palindrome on each side have no apparent sequence homology with known elements in other promoters. Nuclear extract from a variety of tissues and cells were examined; these included rat liver and testes and cultured cells of human and hamster origin. The DNasel footprint is strong over and around the palindromic element for each of the extracts and is equivalent in size (approximately 22 residues); footprinting over the Sp1 binding sites is seen also. Two potential tissue-specific binding sites, present in liver but not in testes, were found corresponding to residues -13 to -10 and +33 to +48, respectively. Protein binding to the palindromic element was confirmed by an electrophoretic mobility shift assay with the core promoter as probe. Binding specificity of the 22 residue palindromic element, as revealed by oligonucleotide competition, is different from that of AP-1 binding element. Controlled proteolysis with trypsin was used to study structural properties of proteins forming the mobility shift bands. Following digestion with trypsin, most of the palindrome binding activity of each extract corresponded to a sharp, faster migrating band, potentially representing a DNA binding domain of the palindrome binding protein. 相似文献
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J H Nilson J A Bokar C M Clay T A Farmerie R A Fenstermaker D L Hamernik R A Keri 《Biology of reproduction》1991,44(2):231-237
Expression of the glycoprotein hormone alpha-subunit gene occurs in the pituitary of all mammals but in placenta of only primates and horses. In humans, two different elements, termed upstream regulatory element (URE) and cAMP response element (CRE), are required for placenta-specific expression of the alpha-subunit gene. The URE binds a protein unique to placenta whereas the CRE binds a ubiquitous protein. Comparative analysis of the promoter-regulatory region of the alpha-subunit gene from a number of mammals indicates that a functional URE has been retained and suggests the potential for placenta-specific expression. Indirect evidence also indicates that the URE-binding protein has been conserved, even in placenta from mammals that fail to express the alpha-subunit gene. Lack of expression of the alpha-subunit gene in placenta of rodents and cattle can be traced to a single nucleotide change that renders the CRE-like sequence of these genes incapable of binding the protein that confers responsiveness to cAMP. In contrast, although expression of the alpha-subunit gene occurs in horse placenta, the promoter-regulatory region lacks a functional CRE but appears to retain a functional URE. This suggests that either a different accessory element and cognate protein interacts with the horse URE to provide placenta-specific expression or that a completely different set of regulatory elements is required for placenta-specific expression in horses. 相似文献
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