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M Asano  M Nishizawa  S Nagata 《Gene》1991,107(2):241-246
At least three regulatory elements GPE1, GPE2 and GPE3 (G-CSF promoter elements) controlling the gene (G-CSF) encoding granulocyte colony-stimulating factor (G-CSF) are indispensable for the constitutive expression of the G-CSF gene in human CHU-2 cells and for its lipopolysaccharide(LPS)-inducible expression in macrophages. The enhancer activities of each regulatory element were examined with or without the SV40 enhancer element placed downstream from the reporter gene. A GPE1 tetramer mediated the constitutive expression in CHU-2 cells, and the LPS-inducible expression in macrophage cell lines, while the GPE2 element was active in CHU-2 and LPS-treated macrophage cell lines only in combination with the SV40 enhancer. A GPE3 tetramer had efficient enhancer activity in CHU-2 cells but not in macrophage cell lines without the SV40 enhancer. In combination with the SV40 enhancer, GPE3 worked as an LPS-inducible enhancer element in macrophage BAM3 cells. Gel retardation assay indicated that the CHU-2 and the macrophage cells contained nuclear factors which specifically bound to each GPE sequence.  相似文献   

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A genomic clone obtained from mouse liver DNA using a mouse cytokeratin EndoA cDNA probe revealed the complete sequence of the EndoA gene. The gene is divided into nine exons and the exon-intron pattern has been conserved compared to that of other type-II cytokeratin-encoding genes. The 5' upstream, 3' downstream and first and third introns contain potential regulatory sequences, including polyoma virus enhancer motifs (PEA1 and PEA3) and AP-1 elements. The 5' regions upstream of the EndoA, EndoB and Ck8 genes contain homologous sequences surrounding the TATA boxes. In addition, a CpG dinucleotide cluster region was located around the first exon. This CpG cluster region was found to be hypomethylated in endodermal PYS-2 cells, retinoic acid-treated F9 cells, and F9 embryonal carcinoma cells, but hypermethylated in BALB/C 3T3 fibroblast cells that do not express EndoA. These findings may provide a clue to understanding the molecular mechanisms of EndoA gene expression.  相似文献   

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We have examined the pattern of gene expression of mouse cytokeratin endo A and endo B during postimplantational development and in adult organs by Northern blot and in situ hybridization analyses. Both mRNAs localized in the ectoplacental cone, trophoblastic giant cells surrounding the parietal yolk sac, trophoblast cells in placenta, visceral yolk sac, and simple epithelium of the embryo during postimplantational development and in simple or transitional epithelial tissues in adult organs. These results indicate that endo A and endo B are coexpressed and may play some roles in these tissues.  相似文献   

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The cytokeratins, which form the intermediate filaments (IFs) characteristic of epithelial cells, are encoded by a large family of genes whose members are differentially expressed in patterns different in the various kinds of epithelia. To identify possible cis-regulatory DNA elements involved in the cell-type-specific expression of these genes, we examined, in transfection assays, 5' upstream sequence intercepts of a certain cytokeratin gene, i.e. that for bovine cytokeratin IV* (CKIV*), in combination with the coding portions of either the chloramphenicol acetyltransferase (CAT) gene or other cytokeratin genes. A 5' upstream region located between the cap-site and nucleotide -605 was found to enhance the specific expression of these reporter genes in bovine mammary gland-derived BMGE + H cells, which express the endogenous gene, but not in bovine kidney epithelium-derived MDBK cells which synthesize cytokeratins other than IV*. This epithelium-type-specific expression was also observed in heterologous combinations, e.g. in murine keratinocytes, but not in other murine cell lines such as 3T3 fibroblasts. When a fragment located between -180 and -605 was coupled to the HSV-TK promoter it stimulated the expression of the reporter gene in a cell-type-specific manner. The enhancer character of this 425 nucleotide long region is also demonstrated. Moreover, the CKIV* promoter/enhancer complex was able to direct the expression of epidermal cytokeratins characteristic for suprabasal differentiation, i.e. bovine cytokeratins Ia and VIb, in cells that normally do not express these genes. We show that the newly synthesized cytokeratins integrate into the pre-existing cytokeratin IF system of the transfected cells and that the forced expression of one of these cytokeratins does not induce the endogenous gene encoding its normal pair partner.  相似文献   

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Expression of the mouse cytokeratin EndoA gene is restricted in endodermal and epithelial cells, and is regulated by an enhancer that is located 1 kilobase (kb) 3' downstream from the gene. The enhancer consists of six direct repeats, of which each contains two predicted Ets binding sites (EBS1 and EBS2) containing GGAA as a core. Mutation analysis showed that EBS1 is essential for the enhancer activity and additional effects of EBS2, suggesting that some Ets-related proteins bind and activate the enhancer through EBS1. We also showed that Ets-2 mRNA is expressed in PYS-2 cells and that Ets-2 protein produced by E. coli interacts with EBS1 but not with EBS2. Using co-transfection assays, we showed that Ets-2 can trans-activate the enhancer in PYS-2 cells. Mutations that impair Ets-2 binding abolished the activity of the EndoA enhancer. The results obtained from the binding competition assay using an Ets-2 specific antibody, however, suggest that EBS1 binds to an Ets protein which is distinct from Ets-2. These data show that Ets-2 related protein binds and activates the EndoA enhancer in a sequence-specific fashion.  相似文献   

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IL-4 secreted by activated T cells is a pleiotropic cytokine affecting growth and differentiation of diverse cell types such as T cells, B cells, and mast cells. We investigated the upstream regulatory elements of the human IL-4 promoter. A novel T cell-specific negative regulatory element (NRE) composed of two protein-binding sites were mapped in the 5' flanking region of the IL-4 gene: -311CTCCCTTCT-303 (NRE-I) and -288CTTTTTGCTT-TGC-300 (NRE-II). A T cell-specific protein Neg-1 and a ubiquitous protein Neg-2 binding to NRE-I and NRE-II, respectively, were identified. Furthermore, a positive regulatory element was found 45 bp downstream of the NRE. The enhancer activity of the PRE was completely suppressed when the NRE was present. These data suggest that IL-4 promoter activity is normally down-regulated by an NRE via repression of the enhancer positive regulatory element. These data may have implications for the stringent control of IL-4 expression in T cells.  相似文献   

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Combinatorial control of a neuron-specific exon.   总被引:4,自引:1,他引:3       下载免费PDF全文
The mouse c-src gene contains a short neuron-specific exon, N1. N1 exon splicing is partly controlled by an intronic splicing enhancer sequence that activates splicing of a heterologous reporter exon in both neural and nonneural cells. Here we attempt to dissect all of the regulatory elements controlling the N1 exon and examine how these multiple elements work in combination. We show that the 3' splice site sequence upstream of exon N1 represses the activation of splicing by the downstream intronic enhancer. This repression is stronger in nonneural cells and these two regulatory sequences combine to make a reporter exon highly cell-type specific. Substitution of the 3' splice site of this test exon with sites from other exons indicates that activation by the enhancer is very dependent on the nature of the upstream 3' splice site. In addition, we identify a previously uncharacterized purine-rich sequence within exon N1 that cooperates with the downstream intronic enhancer to increase exon inclusion. Finally, different regulatory elements were tested in multiple cell lines of both neuronal and nonneuronal origin. The individual splicing regulatory sequences from the src gene vary widely in their activity between different cell lines. These results demonstrate how a simple cassette exon is controlled by a variety of regulatory elements that only in combination will produce the correct tissue specificity of splicing.  相似文献   

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