Cytoplasmic polyadenylation elements mediate masking and unmasking of cyclin B1 mRNA.
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During oocyte maturation, cyclin B1 mRNA is translationally activated by cytoplasmic polyadenylation. This process is dependent on cytoplasmic polyadenylation elements (CPEs) in the 3' untranslated region (UTR) of the mRNA. To determine whether a titratable factor might be involved in the initial translational repression (masking) of this mRNA, high levels of cyclin B1 3' UTR were injected into oocytes. While this treatment had no effect on the poly(A) tail length of endogenous cyclin B1 mRNA, it induced cyclin B1 synthesis. A mutational analysis revealed that the most efficient unmasking element in the cyclin 3' UTR was the CPE. However, other U-rich sequences that resemble the CPE in structure, but which do not bind the CPE-binding polyadenylation factor CPEB, failed to induce unmasking. When fused to the chloramphenical acetyl transferase (CAT) coding region, the cyclin B1 3' UTR inhibited CAT translation in injected oocytes. In addition, a synthetic 3' UTR containing multiple copies of the CPE also inhibited translation, and did so in a dose-dependent manner. Furthermore, efficient CPE-mediated masking required cap-dependent translation. During the normal course of progesterone-induced maturation, cytoplasmic polyadenylation was necessary for mRNA unmasking. A model to explain how cyclin B1 mRNA masking and unmasking could be regulated by the CPE is presented. (+info)
Estrogen responses in bovine fetal uterine cells involve pathways directed by both estrogen response element and activator protein-1.
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Objectives were to examine possible roles of estrogen receptor (ER) in development of the bovine uterine endometrium in the context of ER type, enhancer type, and ligand-independent activation. Expression vectors producing either ERalpha or ERbeta were introduced into fetal uterine cells from Day 110 to 120 of gestation (UBF120 cells) and into rat embryo fibroblasts (Rat-1 cells), neither of which express endogenous ER. Reporter constructs containing either an estrogen response element (ERE) or activator protein-1 (AP-1) response element were cotransfected. These reporters were also transfected into fetal uterine cells from Day 180 to 200 of gestation (UBF180 cells), which express ER. In UBF120 and Rat-1 cells transfected with either ERalpha or ERbeta, treatment with estradiol-17beta (E2) resulted in increased activity of an ERE reporter construct, but not an AP-1 element reporter construct. The antiestrogen ICI 182,780 (ICI) exhibited E2 antagonist activity with both ERalpha and ERbeta. Thus, all components were present for E2-dependent transcription from an ERE except ER; however, cells were not competent for E2-dependent transcription mediated through AP-1. In UBF180 cells, E2 treatment increased both ERE and AP-1 reporter activity. ICI exhibited E2 antagonist activity. Treatment with epidermal growth factor resulted in increased ERE reporter activity that was inhibited by ICI, indicative of ligand-independent activation of ER. These data suggest that multiple pathways for ER-mediated gene regulation occur in the developing fetal uterus and that nuclear components necessary for action of both ERalpha and ERbeta are present prior to expression of the receptor. (+info)
Liposomal lipid and plasmid DNA delivery to B16/BL6 tumors after intraperitoneal administration of cationic liposome DNA aggregates.
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The transfer of plasmid expression vectors to cells is essential for transfection after administration of lipid-based DNA formulations (lipoplexes). A murine i.p. B16/BL6 tumor model was used to characterize DNA delivery, liposomal lipid delivery, and gene transfer after regional (i.p.) administration of free plasmid DNA and DNA lipoplexes. DNA lipoplexes were prepared using cationic dioleoyldimethylammonium chloride/dioleoylphosphatidylethanolamine (50:50 mol ratio) liposomes mixed with plasmid DNA (1 microgram DNA/10 nmol lipid). The plasmid used contained the chloramphenicol acetyltransferase gene and chloramphenicol acetyltransferase expression (mU/g tumor) was measured to estimate transfection efficiency. Tumor-associated DNA and liposomal lipid levels were measured to estimate the efficiency of lipid-mediated DNA delivery to tumors. Plasmid DNA delivery was estimated using [3H]-labeled plasmid as a tracer, dot blot analysis, and/or Southern analysis. Liposomal lipid delivery was estimated using [14C]-dioleoylphosphatidylethanolamine as a liposomal lipid marker. Gene expression in the B16/BL6 tumors was highly variable, with values ranging from greater than 2,000 mU/g tumor to less than 100 mU/g tumor. There was a tendency to observe enhanced transfection in small (<250 mg) tumors. Approximately 18% of the injected dose of DNA was associated with these small tumors 2 h after i.p. administration. Southern analysis of extracted tumor DNA indicated that plasmid DNA associated with tumors was intact 24 h after administration. DNA and associated liposomal lipid are efficiently bound to tumors after regional administration; however, it is unclear whether delivery is sufficient to abet internalization and appropriate subcellular localization of the expression vector. (+info)
Brachyury regulatory region active in embryonal carcinoma P19 cells.
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Brachyury (T) is involved in mesoderm induction during early mouse development. We analyzed the region regulating expression of T in embryonal carcinoma P19 cells, which differentiate into mesoderm derivatives in vitro. Transfection of plasmids encoding reporter genes under the control of the 5'-flanking region showed positive regulatory elements between -298 and -129 bp are responsible for driving T expression in mesodermal cells. (+info)
The role of p38 mitogen-activated protein kinase in IL-1 beta transcription.
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Several reports have shown that bicyclic imidazoles, specific inhibitors of the p38 mitogen-activated protein kinase (MAPK), block cytokine synthesis at the translational level. In this study, we examined the role of p38 MAPK in the regulation of the IL-1beta cytokine gene in monocytic cell lines using the bicyclic imidazole SB203580. Addition of SB203580 30 min before stimulation of monocytes with LPS inhibited IL-1beta protein and steady state message in a dose-dependent manner in both RAW264.7 and J774 cell lines. The loss of IL-1beta message was due mainly to inhibition of transcription, since nuclear run-off analysis showed an approximately 80% decrease in specific IL-1 RNA synthesis. In contrast, SB203580 had no effect on the synthesis of TNF-alpha message. LPS-stimulated p38 MAPK activity in the RAW264.7 cells was blocked by SB203580, as measured by the inhibition of MAPKAP2 kinase activity, a downstream target of the p38 MAPK. CCAATT/enhancer binding protein (C/EBP)/NFIL-6-driven chloramphenicol acetyltransferase (CAT) reporter activity was sensitive to SB203580, indicating that C/EBP/NFIL-6 transcription factor(s) are also targets of p38 MAPK. In contrast, transfected CAT constructs containing NF-kappaB elements were only partially inhibited (approximately 35%) at the highest concentration of SB203580 after LPS stimulation. As measured by EMSA, LPS-stimulated NF-kappaB activation was not affected by SB203580. Overall, the results demonstrate, for the first time, a role for p38 MAPK in IL-1beta transcription by acting through C/EBP/NFIL-6 transcription factors. (+info)
Biosynthesis of osteogenic growth peptide via alternative translational initiation at AUG85 of histone H4 mRNA.
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The osteogenic growth peptide (OGP) is an extracellular mitogen identical to the histone H4 (H4) COOH-terminal residues 90-103, which regulates osteogenesis and hematopoiesis. By Northern analysis, OGP mRNA is indistinguishable from H4 mRNA. Indeed, cells transfected with a construct encoding [His102]H4 secreted the corresponding [His13]OGP. These results suggest production of OGP from H4 genes. Cells transfected with H4-chloramphenicol acetyltransferase (CAT) fusion genes expressed both "long" and "short" CAT proteins. The short CAT was retained following an ATG --> TTG mutation of the H4 ATG initiation codon, but not following mutation of the in-frame internal ATG85 codon, which, unlike ATG1, resides within a perfect context for translational initiation. These results suggest that a PreOGP is translated starting at AUG85. The translational initiation at AUG85 could be inhibited by optimizing the nucleotide sequence surrounding ATG1 to maximally support upstream translational initiation, thus implicating leaky ribosomal scanning in usage of the internal AUG. Conversion of the predicted PreOGP to OGP was shown in a cell lysate system using synthetic [His102]H4-(85-103) as substrate. Together, our results demonstrate that H4 gene expression diverges at the translational level into the simultaneous parallel production of both H4, a nuclear structural protein, and OGP, an extracellular regulatory peptide. (+info)
Bacteriocin release protein triggers dimerization of outer membrane phospholipase A in vivo.
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Bacteriocin release protein is known to activate outer membrane phospholipase A (OMPLA), which results in the release of colicin from Escherichia coli. In vivo chemical cross-linking experiments revealed that the activation coincides with dimerization of OMPLA. Permeabilization of the cell envelope and dimerization were characterized by a lag time of 2 h. (+info)
Multiple elements influence transcriptional regulation from the human testis-specific PGK2 promoter in transgenic mice.
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The PGK2 gene is expressed in a strictly tissue-specific manner in meiotic spermatocytes and postmeiotic spermatids during spermatogenesis in eutherian mammals. Previous results indicate that this is regulated at the transcriptional level by core promoter sequences that bind ubiquitous transcription factors and by sequences in a 40-base pair (bp) upstream enhancer region (E1/E4) that bind tissue-specific transcription factors. Transgenic mice carrying different PGK2 promoter sequences linked to the chloramphenicol acetyltransferase (CAT) reporter gene, one containing only the 40-bp E1/E4 enhancer sequence plus the core promoter and two containing 515 bp of PGK2 promoter but with either the E1/E4 enhancer region or the Sp1-binding site in the core promoter disrupted by in vitro mutagenesis, all showed levels of expression reduced to less than half that of the wild-type 515 PGK2/CAT transgene. These results indicate that multiple factor-binding regions normally regulate initiation of transcription from the PGK2 promoter. The single disruption of any one of these binding activities reduced, but did not abolish, transgene expression. This is consistent with an "enhanceosome"-like function in this promoter involving multiple bound activator proteins that interact in a combinatorial manner to synergistically promote testis-specific transcription. (+info)