Enterococcus hirae vacuolar ATPase is expressed in response to pH as well as sodium.
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The Enterococcus hirae ntp operon encodes both a vacuolar ATPase, which transports Na+ as well as Li+, and the KtrII K+ transporter. A plasmid, in which the chloramphenicol acetyltransferase gene (CAT) was placed downstream of the ntp promoter, was introduced into a mutant totally defective in Na+ extrusion. The CAT activity of this transformant was increased preferentially by addition of NaCl, but not by LiCl, in the media or by elevating the medium pH, correlating well with the increase in amounts of the ATPase subunits observed by Western blotting. The physiological significance of these responses of the ntp promoter is discussed. (+info)
Effect of CWG methylation on expression of plant genes.
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The presence of two DNA methyltransferases in Pisum raises the possibility that they serve different functions. In vitro methylation of CWG sequences in the strong cauliflower mosaic virus 35S promoter had no effect on reporter gene expression. In contrast, in vitro methylation of CWG sequences in the relatively weak, CG-deficient Phaseolus vulgaris rbcS2 promoter inhibited transcription. Expression of both constructs was strongly inhibited by extensive CG methylation. A search of published plant promoter sequences revealed that the CG content of promoters is very variable, with some promoters having typical CG islands. In contrast, the distribution of CWG sequences is more even with little evidence for CWG islands. (+info)
1.3 kilobases of the lung type I cell T1alpha gene promoter mimics endogenous gene expression patterns during development but lacks sequences to enhance expression in perinatal and adult lung.
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The T1alpha gene is one of few markers for the type I cell phenotype in the adult mammalian lung. Type I cells form a large, thin epithelial layer that facilitates gas exchange and transport of fluids between the air spaces and capillaries. The T1alpha gene has a complex pattern of developmental expression in lung and brain; in vitro studies indicate that expression is regulated in part by thyroid transcription factor 1, forkhead proteins, and Sp1/Sp3 proteins. To explore the mechanisms that confine T1alpha expression in intact adult animals to alveolar type I and choroid plexus epithelial cells, we generated mice bearing a 1.3-kb T1alpha promoter-chloramphenicol acetyltransferase (CAT) gene. In situ hybridization and RNase protection assays show that the 1.3-kb promoter confers a pattern of CAT expression that largely matches the endogenous T1alpha in embryos and mid-term fetuses in lung and central nervous system. However, the 1.3-kb promoter lacks elements important for perinatal up-regulation of T1alpha in the lung and maintenance of that expression in the adult lung and brain. The final adult pattern of T1alpha expression may be directed by elements outside the 1.3-kb fragment, perhaps those 5' to the 1.3-kb fragment as we show herein, or in 3' and intronic regions. Dev Dyn 1999;215:319-331. (+info)
Regulation of ATP citrate-lyase gene expression in hepatocytes and adipocytes in normal and genetically obese rats.
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Transcriptional regulation of ATP citrate-lyase (ACL, one of the lipogenic enzymes) gene by glucose/insulin, polyunsaturated fatty acid (PUFA), and leptin has been investigated in hepatocytes and adipocytes of obese Wistar fatty rats and their lean littermates. The sequence spanning nucleotides -64 to -41 of the ACL gene, which is responsive to glucose/insulin stimulation [Eur. J. Biochem. 247, 497-502, 1997], was linked to a reporter gene and transfected into rat hepatocytes or adipocytes. The chloramphenicol acetyltransferase (CAT) activities in the presence of glucose alone were similar in primary cultured cells from both obese and lean rats. In the presence of glucose/insulin, however, the CAT activities were markedly increased in the hepatocytes of lean rats, but were not significantly increased in those of obese rats. The stimulation by glucose/insulin was reduced in PUFA-treated cells of lean rats. The stimulation was also reduced in leptin-treated cells or ob gene expression vector-containing cells. However, PUFA- or leptin-treated cells from obese rats did not show a significant reduction in insulin stimulation. The same effects were observed at the endogenous mRNA and enzyme levels. Similar results were seen in adipocytes, although the stimulation and suppression levels were much smaller than in hepatocytes. The expression of endogenous insulin receptor in hepatocytes and adipocytes was reduced in the presence of leptin or PUFA. We previously found that insulin-binding capacities are also reduced in the presence of leptin or PUFA and are very low in obese rats in comparison with lean. Moreover, gel mobility shift assays using end-labeled ACL(-64/-41) revealed that nuclear factor(s) including Sp1 bind specifically to the sequence, and DNA-protein complex formation is reduced in the obese rats. Thus, the reductions in the insulin-stimulated ACL transcription may be ascribed in part to reductions in insulin binding to receptors and DNA-protein complex formation. (+info)
Tissue and site-specific methylation correlates with expression of the mouse lactoferrin gene.
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We have previously examined the regulatory region of the mouse lactoferrin gene and have identified sequences essential for basal and hormonally induced expression. In this study, we explore the relationship between the methylation state of the mouse lactoferrin gene promoter and its expression in selected mouse tissues. In a transient expression system, transcriptional activity was blocked after in vitro methylation of the regulatory region of the mouse lactoferrin gene. In addition, the in vivo methylation state of three promoter region sites was assessed using Southern blot analysis of DNA digested with methylation-insensitive and -sensitive restriction enzymes. The results showed that site -455, upstream of the mouse lactoferrin estrogen response module, was highly unmethylated in DNA from both hormone-treated and -untreated mouse lung, liver, and spleen tissues. Also, in both treated and untreated samples, the -54 site is uniquely highly unmethylated in liver DNA, while the -22 site is unmethylated in spleen DNA. Northern blot analysis showed lactoferrin expression in tissues that were unmethylated at a minimum of two sites. These results show that the alteration of the methylation status of the three sites are tissue-specific and are associated with constitutive expression of lactoferrin. (+info)
Tissue-specific regulation of the ecto-5'-nucleotidase promoter. Role of the camp response element site in mediating repression by the upstream regulatory region.
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We have isolated the 5' region of the ecto-5'-nucleotidase (low K(m) 5'-NT) gene and established that a 969-base pair (bp) fragment confers cell-specific expression of a CAT reporter gene that correlates with the expression of endogenous ecto-5'-NT mRNA and enzymatic activity. A 768-bp upstream negative regulatory region has been identified that conferred lymphocyte-specific negative regulation in a heterologous system with a 244-bp deoxycytidine kinase core promoter. DNase I footprinting identified several protected areas including Sp1, Sp1/AP-2, and cAMP response element (CRE) binding sites within the 201-bp core promoter region and Sp1, NRE-2a, TCF-1/LEF-1, and Sp1/NF-AT binding sites in the upstream regulatory region. Whereas the CRE site was essential in mediating the negative activity of the upstream regulatory region in Jurkat but not in HeLa cells, mutation of the Sp1/AP-2 site decreased promoter activity in both cell lines. Electrophoretic mobility shift assay analysis of proteins binding to the CRE site identified both ATF-1 and ATF-2 in Jurkat cells. Finally, phorbol 12-myristate 13-acetate increased the activity of both the core and the 969-bp promoter fragments, and this increase was abrogated by mutations at the CRE site. In summary, we have identified a tissue-specific regulatory region 5' of the ecto-5'-NT core promoter that requires the presence of a functional CRE site within the basal promoter for its suppressive activity. (+info)
The Epstein-Barr virus latency BamHI-Q promoter is positively regulated by STATs and Zta interference with JAK/STAT activation leads to loss of BamHI-Q promoter activity.
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In Epstein-Barr virus (EBV)-associated tumors in nonimmunocompromised patients, EBV gene expression is highly restricted. EBV-encoded nuclear antigen (EBNA)-1 is expressed, whereas the immunogenic and proliferative EBNAs are not. This pattern of EBNA expression is generated by usage of the BamHI-Q promoter (Qp). We have determined that the JAK/STAT pathway positively regulates Qp activity. In transient-transfection assays, a Qp-CAT reporter was activated by cotransfected JAK-1 and by treatment of cells with the cytokine IL-6. The ability of Qp to bind signal transducer and activator of transcription (STAT) proteins was directly demonstrated by electrophoretic mobility-shift assay, and mutation of potential STAT-binding sites reduced Qp responsiveness to Janus kinase (JAK)-1. Consistent with a role for STATs in Qp function, Qp using Burkitt's lymphoma Rael cells and cultured nasopharyngeal carcinoma (NPC) cells contained nuclear STAT protein. We investigated whether the inability to maintain EBV-positive NPC cell lines in culture was related to Qp activity. Passaging of the NPC cell line HK666 led to activation of expression of BZLF1, which encodes Zta and loss of Qp function. Transient expression assays linked Zta expression to the down-regulation of Qp. Cotransfection of Zta reduced Qp activity in reporter assays. This negative regulation required Zta DNA-binding activity. We provide evidence that Zta up-regulation of p53 leads to p53-mediated interference with JAK/STAT activation of Qp. The data imply that JAK/STAT signaling has a role in EBV-associated malignancies. (+info)
Differential expression of Myc1 and Myc2 isoforms in cells transformed by eIF4E: evidence for internal ribosome repositioning in the human c-myc 5'UTR.
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eIF4E is essential for translation initiation, but its overexpression causes malignant transformation. Recent work demonstrated that eIF4E/F participates in exposing and locating alternate translation start codons during scanning. Translation initiation of several important protooncogenes and growth-regulators, such as Myc and FGF-2, can start at CUG start codon(s) upstream of the normal open reading frame (ORF). The resulting amino-terminal extension alters the properties of these proteins and their intracellular distribution. In cells overexpressing eIF4E, c-myc is overexpressed and particularly the larger, CUG-initiated form (Myc1). Recent reports suggest that synthesis of Myc2, the normally expressed AUG-initiated form, is mediated by an IRES. To determine what role eIF4E might play in c-myc expression, the c-myc 5' untranslated region (UTR) was fused in-frame to CAT reporters, and several more derivative constructs were made. In vitro translation experiments (with and without eIF4E/F); expression in CHO cells transformed with eIF4E; and deletion/mutation analysis demonstrated that Myc1 is translated by a scanning mechanism, while Myc2 is translated by Internal Ribosome Repositioning. Moreover, the existence of a true IRES in the 5'UTR was contradicted by its failure to direct translation of a circular transcript, in contrast to hsp70. The c-myc 5'UTR also failed to engage in translation in the absence of functional eIF4F, after cleavage of the eIF4G component with CVB4 protease-2A. The Internal Repositioning Element (IRPE) in c-myc 5'UTR was delimited to nucleotides (nt) 394-440 from the P1 transcription start site. (+info)