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Deletion Analysis (deletion + analysis)
Selected AbstractsFunctional analysis of the basic helix-loop-helix transcription factor DEC1 in circadian regulationFEBS JOURNAL, Issue 22 2004Interaction with BMAL The basic helix-loop-helix transcription factor DEC1 is expressed in a circadian manner in the suprachiasmatic nucleus where it seems to play a role in regulating the mammalian circadian rhythm by suppressing the CLOCK/BMAL1-activated promoter. The interaction of DEC1 with BMAL1 has been suggested as one of the molecular mechanisms of the suppression [Honma, S., Kawamoto, T., Takagi, Y., Fujimoto, K., Sato, F., Noshiro, M., Kato, Y. & Honma, K. (2002) Nature419, 841,844]. Deletion analysis of DEC1 demonstrated that its N-terminal region, which includes the basic helix-loop-helix domain, was essential for both the suppressive activity and the interaction with BMAL1, as DEC1 lacking the basic region did not show any suppression or interaction. Furthermore, we found that Arg65 in the basic region, which is conserved among group B basic helix-loop-helix proteins, was responsible for the suppression, for the interaction with BMAL1 and for its binding to CACGTG E-boxes. However, substitution of His57 for Ala significantly reduced the E-box binding activity of DEC1, although it did not affect the interaction with BMAL1 or suppression of CLOCK/BMAL1-induced transcription. On the other hand, the basic region-deleted DEC1 acted in a dominant-negative manner for DEC1 activity, indicating that the basic region was not required for homodimer formation of DEC1. Moreover, mutant DEC1 also counteracted DEC2-mediated suppressive activity in a dominant-negative manner. The heterodimer formation of DEC1 and DEC2 was confirmed by pull-down assay. These findings suggest that the basic region of DEC1 participates in the transcriptional regulation through a protein,protein interaction with BMAL1 and DNA binding to the E-box. [source] Expression of immediate early gene pip92 during anisomycin-induced cell death is mediated by the JNK- and p38-dependent activation of Elk1FEBS JOURNAL, Issue 15 2000Kwang C. Chung We report here that immediate early gene pip92 is expressed during anisomycin-induced cell death in fibroblast NIH3T3 cells. To determine the mechanism by which this occurs and to identify downstream signaling pathways, we investigated the induction of the pip92 promoter. The activation of pip92 by anisomycin is mediated by the activation of MAP kinases, such as JNK and p38 kinase, but not ERK. Deletion analysis of the pip92 promoter indicated that pip92 activation occurs primarily within the region containing a serum response element (SRE). Further analysis of the SRE using a heterologous thymidine kinase promoter showed that both an Ets and CArG-like site are required for anisomycin-induced pip92 expression. Elk1, which binds to the Ets site, was phosphorylated by the JNK- and p38-dependent pathways and the phosphorylation of Elk1,GAL4 fusion proteins by these pathways was sufficient for the transactivation. Overall, this study suggested that different MAPK pathways are involved in the expression of immediate early gene pip92 by growth factors and environmental stresses. [source] Heregulin and forskolin-induced cyclin D3 expression in Schwann cells: Role of a CCAAT promoter element and CCAAT enhancer binding proteinGLIA, Issue 3 2004Luis Fuentealba Abstract Heregulin, a polypeptide growth factor, and forskolin, an adenylyl cyclase activator, synergistically stimulate expression of cyclin D3 and cell division in Schwann cells. Heregulin induces expression in Schwann cells of a luciferase reporter gene linked to the cyclin D3 promoter. Forskolin markedly augments reporter expression in the presence of heregulin. Deletion analysis identified several promoter sites that contribute to high-level reporter expression in heregulin- and forskolin-treated Schwann cells. A promoter fragment that contains 103 bp of 5,-flanking sequence produced significant reporter expression in heregulin- and forskolin-stimulated cells. Deletion of a consensus CCAAT site within this promoter fragment caused a nearly complete loss of reporter expression. Similar results were obtained when CCAAT site mutations were introduced into the promoter. Heregulin and forskolin increased steady-state levels of CCAAT/enhancer binding protein-, (C/EBP,) in Schwann cells. Mobility shift assays identified proteins in Schwann cell nuclear extracts that formed stable complexes with the cyclin D3 CCAAT promoter element and were disrupted by anti-C/EBP, antibody. Transfection of Schwann cells with C/EBP, cDNA increased cyclin D3 reporter expression. In contrast to these results, mutation of a cAMP response element in the cyclin D3 promoter had only a modest effect on heregulin- and forskolin-stimulated reporter expression. These findings demonstrate that C/EBP, plays a key role in the heregulin and cAMP-dependent regulation of cyclin D3 expression in Schwann cells. © 2003 Wiley-Liss, Inc. [source] Krüppel-Like Zinc Finger Protein Glis3 Promotes Osteoblast Differentiation by Regulating FGF18 Expression,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2007Ju Youn Beak Abstract The zinc finger protein Glis3 is highly expressed in human osteoblasts and acts synergistically with BMP2 and Shh in enhancing osteoblast differentiation in multipotent C3H10T1/2 cells. This induction of osteoblast differentiation is at least in part caused by the induction of FGF18 expression. This study supports a regulatory role for Glis3 in osteoblast differentiation. Introduction: Gli-similar 3 (Glis3) is closely related to members of the Gli subfamily of Krüppel-like zinc finger proteins, transcription factors that act downstream of sonic hedgehog (Shh). In this study, we analyzed the expression of Glis3 in human osteoblasts and mesenchymal stem cells (MSCs). Moreover, we examined the regulatory role of Glis3 in the differentiation of multipotent C3H10T1/2 cells into osteoblasts and adipocytes. Materials and Methods: Microarray analysis was performed to identify genes regulated by Glis3 in multipotent C3H10T1/2 cells. Reporter and electrophoretic mobility shift assays were performed to analyze the regulation of fibroblast growth factor 18 (FGF18) by Glis3. Results: Glis3 promotes osteoblast differentiation in C3H10T1/2 cells as indicated by the induction of alkaline phosphatase activity and increased expression of osteopontin, osteocalcin, and Runx2. In contrast, Glis3 expression inhibits adipocyte differentiation. Glis3 acts synergistically with BMP2 and Shh in inducing osteoblast differentiation. Deletion analysis indicated that the carboxyl-terminal activation function of Glis3 is needed for its stimulation of osteoblast differentiation. Glis3 is highly expressed in human osteoblasts and induced in MSCs during differentiation along the osteoblast lineage. Microarray analysis identified FGF18 as one of the genes induced by Glis3 in C3H10T1/2 cells. Promoter analysis and electrophoretic mobility shift assays indicated that a Glis3 binding site in the FGF18 promoter flanking region is important in its regulation by Glis3. Conclusions: Our study showed that Glis3 positively regulates differentiation of C3H10T1/2 cells into osteoblasts and inhibits adipocyte differentiation. Glis3 acts synergistically with BMP2 and Shh in inducing osteoblast differentiation. The promotion of osteoblast differentiation by Glis3 involves increased expression of FGF18, a positive regulator of osteogenesis. This, in conjunction with the induction of Glis3 expression during osteoblast differentiation in MSCs and its expression in osteoblasts, suggests that Glis3 is an important modulator of MSC differentiation. [source] Bone Morphogenetic Protein 2 Induces Cyclo-oxygenase 2 in Osteoblasts via a Cbfa1 Binding Site: Role in Effects of Bone Morphogenetic Protein 2 In Vitro and In VivoJOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2005Daichi Chikazu Abstract We tested the hypothesis that induction of cyclo-oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (BMP) 2 on bone. BMP-2 induced COX-2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP-2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX-2 promoter-luciferase reporter construct (Pluc) and in MC3T3-E1 cells transfected with Pluc. Deletion analysis identified the -300/-213-bp region of the COX-2 promoter as necessary for BMP-2 stimulation of luciferase activity. Mutation of core-binding factor activity 1 (muCbfa1) consensus sequence (5,-AACCACA-3,) at -267/-261 bp decreased BMP-2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonucleotide spanning the Cbfa1 site was inhibited or supershifted by specific antibodies to Cbfa1. In cultured osteoblasts from calvariae of COX-2 knockout (-/-) and wild-type (+/+) mice, the absence of COX-2 expression reduced the BMP-2 stimulation of both ALP activity and osteocalcin mRNA expression. In cultured marrow cells flushed from long bones, BMP-2 induced osteoclast formation in cells from COX-2+/+ mice but not in cells from COX-2,/, mice. In vivo, BMP-2 (10 ,g/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. Mineralization of pellets, measured by microcomputed tomography (,CT), was decreased by 78% in COX-2,/, mice compared with COX-2+/+ mice. We conclude that BMP-2 transcriptionally induces COX-2 in osteoblasts via a Cbfa1 binding site and that the BMP-2 induction of COX-2 can contribute to effects of BMP-2 on osteoblastic differentiation and osteoclast formation in vitro and to the BMP-2 stimulation of ectopic bone formation in vivo. [source] The correlation between alteration of p16 gene and clinical status in oral squamous cell carcinomaJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 9 2001Chung-Hung Tsai Abstract: The purpose of the study was to evaluate the presence of alteration of the tumor suppressor gene p16 and to correlate these changes with the clinical status of the patients in oral squamous cell carcinoma. Forty-eight oral squamous cell carcinomas were included in the analyses. Deletion analysis was performed by the polymerase chain reaction (PCR). Mutation analysis was restricted to exon 1 and exon 2 of the p16 gene, previously shown to have a high incidence of mutations. The sequences containing exon 1 and exon 2 were amplified by PCR and screened with a single-strand conformation polymorphism (SSCP) technique. Samples showing band shifts in SSCP were sequenced by PCR direct sequencing. Western blots were used to detect the protein expression of the p16 gene, and the results were evaluated with regard to their biological relevance in correlation with clinicopathological factors. Seven (14.6%) deletions were found; 5 (10.4%) mutations were discovered and located in different codons; 26 (54%) specimens had no p16 protein expression; in 11 specimens with p16 deletion or mutation, p16 protein could not be detected. One mutation was non-sense. The p16 gene alterations showed no relationship with location and clinical stage of cancer; however, a close relationship between p16 alterations and cancer metastasis to neck lymph node was found. The alteration rate gradually elevated from well to poorly differentiated grades. We perceive two results. First, the alterations of the p16 gene are common in oral squamous cell carcinoma. Second, the alterations of the p16 gene may attribute to the metastatic behavior or histological grade of cancer cells. [source] N-terminal residues regulate proteasomal degradation of AANATJOURNAL OF PINEAL RESEARCH, Issue 3 2010Zheping Huang Abstract:, Serotonin N -acetyltransferase (AANAT) catalyzes the conversion of serotonin to N -acetylserotonin, which is the immediate precursor for formation of melatonin. Although it is known that AANAT is degraded via the proteasomal proteolysis, detailed mechanisms are not defined. In this paper, we tested the in vivo role of proteasome inhibition on AANAT activity and melatonin release and examined the amino acid residues in AANAT that contribute to the proteasome degradation. We have shown that inhibition of proteasome activities in vivo in the intact pineal gland fails to prevent the light-induced suppression of melatonin secretion. Furthermore, in cell lines stably expressing AANAT, inhibition of proteasomal proteolysis, which resulted in a large accumulation of AANAT protein, similarly failed to increase AANAT enzyme activity proportional to the amount of proteins accumulated. Site-directed mutagenesis analysis of AANAT revealed that the AANAT degradation is independent of lysine and the two surface cysteine residues. Deletion analysis of N-terminus identified the second amino acid leucine (L2) as the key residue that contributes to the proteasomal proteolysis of AANAT protein. These results suggest that rat AANAT protein is degraded via the N-end rule pathway of proteasomal proteolysis and the leucine at the N-terminus appears to be the key residue recognized by N-end rule pathway. [source] Identification of a 251-bp Fragment of the PAI-1 Gene Promoter That Mediates the Ethanol-Induced Suppression of PAI-1 ExpressionALCOHOLISM, Issue 5 2001Hernan E. Grenett Background: Moderate alcohol consumption reduces the risk for coronary heart disease. This cardioprotection may be due to ethanol enhancement of fibrinolysis. Fibrinolysis involves the interaction of plasminogen activators (PAs) and the plasminogen activator inhibitor type-1 (PAI-1). Factor(s) that decrease endothelial cell (EC) PAI-1 expression increase fibrinolysis and may decrease the risk for cardiovascular disease. Methods: Five promoter deletion fragments were generated from a 1.1-kb PAI-1 promoter fragment and ligated to a luciferase reporter gene. Cultured human umbilical vein endothelial cells (HUVECs) were transiently transfected with these PAI-1 deletion constructs. A 251-base pair (bp) fragment of the PAI-1 promoter, positions ,800 to ,549, was cloned upstream of a heterologous promoter/enhancer. ECs luciferase activity was measured in the absence/presence of 20 mM ethanol. Electrophoresis mobility shift assays were performed with nuclear extracts from untreated and ethanol-treated ECs using this 251-bp fragment. Results: Deletion analysis showed a region between position ,800 and ,549 mediated ethanol repression of luciferase activity. This 251-bp promoter fragment also repressed the activity of a heterologous promoter/enhancer in the presence of ethanol. Using the labeled 251-bp fragment, nuclear extracts from ethanol-treated ECs contained two inducible bands and one enhanced band. Non-ethanol treated nuclear extracts also contained a band that was not observed in ethanol-treated samples. Competition using 100-fold molar excess of unlabeled probe abolished these four bands. Conclusions: Repression of PAI-I gene transcription in cultured HUVECs exposed to ethanol may involve the interaction of several transcription factors with binding sites localized between positions ,800 and ,549 of the PAI-1 gene promoter. [source] Analysis of a dextran-binding domain of the dextranase of Streptococcus mutansLETTERS IN APPLIED MICROBIOLOGY, Issue 3 2002H. Morisaki Aims: To examine the dextran-binding domain of the dextranase (Dex) of Streptococcus mutans. Methods and Results: Deletion mutants of the Dex gene of Strep. mutans were prepared by polymerase chain reaction and expressed in Escherichia coli cells. Binding of the truncated Dexs to dextran was measured with a Sephadex G-150 gel. Although the Dexs which lacked the N-terminal variable region lost enzyme activity, they still retained dextran-binding ability. In addition, further deletion into the conserved region from the N-terminal did not influence the dextran-binding ability. However, the Dex which carried a deletion in the C-terminus still possessed both enzyme activity and dextran-binding ability. Further deletion into the conserved region from the C-terminal resulted in complete disappearance of both enzyme and dextran-binding activities. Conclusions: Deletion analysis of the Dex gene of Strep. mutans showed that the C-terminal side (about 120 amino acid residues) of the conserved region of the Dex was essential for dextran-binding ability. Significance and Impact of the Study: The dextran-binding domain was present in a different area from the catalytic site in the conserved region of the Dex molecule. The amino acid sequence of the dextran-binding domain of the Dex differed from those of glucan-binding regions of other glucan-binding proteins reported. [source] Activation of enteropathogenic Escherichia coli (EPEC) LEE2 and LEE3 operons by LerMOLECULAR MICROBIOLOGY, Issue 4 2000Vanessa Sperandio Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing lesions (AE) on epithelial cells. The genes involved in the formation of the AE lesions are contained within a pathogenicity island named the locus of enterocyte effacement (LEE). The LEE comprises 41 open reading frames organized in five major operons: LEE1, LEE2, LEE3, LEE4 and tir. The first gene of the LEE1 operon encodes a transcription activator of the other LEE operons that is called the LEE-encoded regulator (Ler). The LEE2 and LEE3 operons are divergently transcribed with overlapping ,10 promoter regions, and gene fusion studies have shown that they are both activated by Ler. Deletion analysis, using lacZ reporter fusions, of the LEE2 and LEE3 promoters demonstrated that deletions extending closer to the LEE2 transcription start site than ,247 bp lead to loss of activation by Ler, whereas only 70 bp upstream of the LEE3 transcription start site is required for Ler-mediated activation. We have purified Ler as a His-tagged protein and used it to perform DNA-binding assays with LEE2 and LEE3. We observed that Ler bound to a DNA fragment containing the ,300 to +1 region of LEE2; however, it failed to bind to a DNA fragment containing the ,300 to +1 region of LEE3, suggesting that Ler activates both operons by only binding to the regulatory region upstream of LEE2. The Ler-activatable LEE3::lacZ fusions extended to what would be ,246 bp of the LEE2 operon. A lacZ fusion from the ,300 to +1 region of LEE3 failed to be activated by Ler, consistent with our hypothesis that Ler activates the expression of LEE2 and LEE3 by binding to a region located downstream of the LEE3 transcription start site. DNase I footprinting revealed that Ler protected a region of 121 bp upstream of LEE2. Purified Ler mutated in the coiled-coil domain was unable to activate transcription and to bind to the LEE2 regulatory region. These data indicate that Ler may bind as a multimer to LEE2 and activate both divergent operons by a novel mechanism potentially involving changes in the DNA structure. [source] Identification of regulatory elements involved in expression and induction by sucrose and UV-B light of the Arabidopsis thaliana COX5b-2 gene, encoding an isoform of cytochrome c oxidase subunit 5bPHYSIOLOGIA PLANTARUM, Issue 3 2009Raúl N. Comelli The promoter sequences required for expression of the Arabidopsis thaliana COX5b-2 gene, encoding an isoform of cytochrome c oxidase subunit 5b, were analyzed using plants transformed with deleted and mutagenized forms of the promoter fused to gus. A 1000-bp promoter fragment produces expression in root and shoot meristems, leaf and cotyledon tips, and anthers. Deletion analysis indicated the presence of positive and negative regulatory elements. A regulatory element located between ,660 and ,620 from the translation start site was identified as a G-box by mutagenic analysis. Mutation of the G-box, that is present within the coding region of the preceding gene in the genome, increases expression of COX5b-2 in cotyledon and leaf lamina and abolishes induction by ultraviolet-B (UV-B) light, which presumably acts through the removal of an inhibitory factor. Identified positive regulatory elements include a site II element (TGGGCC), a related element with the sequence TGGGTC and four initiator elements (YTCANTYY) that completely abolish expression when mutated in combination. Site II elements are also involved in the response to sucrose. The results imply that the COX5b-2 gene has retained expression characteristics presented by most respiratory chain component genes, but its expression mechanisms have diverged from those employed by COX5b-1, the other gene encoding cytochrome c oxidase subunit 5b in Arabidopsis. [source] The oligomerization of CynR in Escherichia coliPROTEIN SCIENCE, Issue 11 2009Gwendowlyn S. Knapp Abstract Deletion analysis and alanine-scanning based on a homology-based interaction model were used to identify determinants of oligomerization in the transcriptional regulator CynR, a member of the LysR-type transcriptional regulator (LTTR) family. Deletion analysis confirmed that the putative regulatory domain of CynR was essential for driving the oligomerization of , repressor-CynR fusion proteins. The interaction surface of a different LTTR and OxyR was mapped onto a multiple sequence alignment of the LTTR family. This mapping identified putative contacts in the CynR regulatory domain dimer interface, which were targeted for alanine-scanning mutagenesis. Oligomerization was assayed by the ability of mutant , repressor-CynR fusions to assemble in E. coli revealing interesting similarities and differences between OxyR and CynR. [source] Regulation of CCN2/Connective tissue growth factor expression in the nucleus pulposus of the intervertebral disc: Role of Smad and activator protein 1 signalingARTHRITIS & RHEUMATISM, Issue 7 2010Cassie M. Tran Objective To investigate transforming growth factor , (TGF,) regulation of connective tissue growth factor (CTGF) expression in cells of the nucleus pulposus of rats, mice, and humans. Methods Real-time reverse transcription,polymerase chain reaction and Western blot analyses were used to measure CTGF expression in the nucleus pulposus. Transfections were used to measure the effects of Smads 2, 3, and 7 and activator protein 1 (AP-1) on TGF,-mediated CTGF promoter activity. Results CTGF expression was lower in neonatal rat discs than in skeletally mature rat discs. An increase in CTGF expression and promoter activity was observed in rat nucleus pulposus cells after TGF, treatment. Deletion analysis indicated that promoter constructs lacking Smad and AP-1 motifs were unresponsive to treatment. Analysis showed that full-length Smad3 and the Smad3 MH-2 domain alone increased CTGF activity. Further evidence of Smad3 and AP-1 involvement was seen when DN-Smad3, SiRNA-Smad3, Smad7, and DN-AP-1 suppressed TGF,-mediated activation of the CTGF promoter. When either Smad3 or AP-1 sites were mutated, CTGF promoter induction by TGF, was suppressed. We also observed a decrease in the expression of CTGF in discs from Smad3-null mice as compared with those from wild-type mice. Analysis of human nucleus pulposus samples indicated a trend toward increasing CTGF and TGF, expression in the degenerated state. Conclusion TGF,, through Smad3 and AP-1, serves as a positive regulator of CTGF expression in the nucleus pulposus. We propose that CTGF is a part of the limited reparative response of the degenerated disc. [source] Promoter analysis of ventricular myosin heavy chain (vmhc) in zebrafish embryosDEVELOPMENTAL DYNAMICS, Issue 7 2009Daqing Jin Abstract In zebrafish, ventricular myosin heavy chain (vmhc) gene is initially expressed at the anterior lateral mesoderm and thereafter its expression is restricted to the cardiac ventricle. The transcriptional control mechanisms in regulating chamber-specific expression of myosin heavy chains are not well defined. We isolated and analyzed zebrafish vmhc upstream region to examine the spatial and temporal regulation of vmhc using transgenic and transient expression techniques. Promoter deletion analyses defined a basal promoter region sufficient to drive vmhc expression in the ventricle and an upstream fragment necessary for repressing ectopic vmhc expression in the atrium. The transcriptional mechanism that prevents vmhc expression in the atrium is mediated through Nkx2.5 binding elements (NKE). We have further discovered that paired-related homeobox transcriptional factor 2 (Prx2/S8)-like binding elements are required for promoting vmhc expression, and Prrx1b, a Prx-related homeobox protein, participates in the regulation of vmhc expression with other transcriptional factors. Developmental Dynamics 238:1760,1767, 2009. © 2009 Wiley-Liss, Inc. [source] Characterization and expression analysis of the aspartic protease gene family of Cynara cardunculus L.FEBS JOURNAL, Issue 10 2007Catarina Pimentel Cardosin A and cardosin B are two aspartic proteases mainly found in the pistils of cardoon Cynara cardunculus L., whose flowers are traditionally used in several Mediterranean countries in the manufacture of ewe's cheese. We have been characterizing cardosins at the biochemical, structural and molecular levels. In this study, we show that the cardoon aspartic proteases are encoded by a multigene family. The genes for cardosin A and cardosin B, as well as those for two new cardoon aspartic proteases, designated cardosin C and cardosin D, were characterized, and their expression in C. cardunculus L. was analyzed by RT-PCR. Together with cardosins, a partial clone of the cyprosin B gene was isolated, revealing that cardosin and cyprosin genes coexist in the genome of the same plant. As a first approach to understanding what dictates the flower-specific pattern of cardosin genes, the respective gene 5, regulatory sequences were fused with the reporter ,-glucuronidase and introduced into Arabidopsis thaliana. A subsequent deletion analysis of the promoter region of the cardosin A gene allowed the identification of a region of approximately 500 bp essential for gene expression in transgenic flowers. Additionally, the relevance of the leader intron of the cardosin A and B genes for gene expression was evaluated. Our data showed that the leader intron is essential for cardosin B gene expression in A. thaliana. In silico analysis revealed the presence of potential regulatory motifs that lay within the aforementioned regions and therefore might be important in the regulation of cardosin expression. [source] Human ATP-dependent RNA/DNA helicase hSuv3p interacts with the cofactor of survivin HBXIPFEBS JOURNAL, Issue 19 2005Michal Minczuk The human SUV3gene encodes an NTP-dependent DNA/RNA DExH box helicase predominantly localized in mitochondria. Its orthologue in yeast is a component of the mitochondrial degradosome complex involved in the mtRNA decay pathway. In contrast to this, the physiological function of human SUV3 remains to be elucidated. In this report we demonstrate that the hSuv3 protein interacts with HBXIP, previously identified as a cofactor of survivin in suppression of apoptosis and as a protein that binds the HBx protein encoded by the hepatitis B virus. Using deletion analysis we identified the region within the hSuv3 protein, which is responsible for binding to HBXIP. The HBXIP binding domain was found to be important for mitochondrial import and stability of the Suv3 protein in vivo. We discuss the possible involvement of the hSuv3p,HBXIP interaction in the survivin-dependent antiapoptotic pathway. [source] E2F1-mediated transcriptional inhibition of the plasminogen activator inhibitor type 1 geneFEBS JOURNAL, Issue 18 2001Magdalena Koziczak ,Gene expression of the plasminogen activation system is cell-cycle dependent. Previously, we showed that ectopic expression of E2F1 repressed the plasminogen activator inhibitor type 1 (PAI-1) promoter in a manner dependent on the presence of DNA-binding and transactivation domains of E2F1 but independent of binding to pocket-binding proteins, suggesting a novel mechanism for E2F-mediated negative gene regulation [Koziczak, M., Krek, W. & Nagamine, Y. (2000) Mol. Cell. Biol.20, 2014,2022]. However, it remains to be seen whether endogenous E2F can exert a similar effect. We report here that down-regulation of PAI-1 gene expression correlates with an increase in endogenous E2F activity. When cells were treated with a cdk2/4-specific inhibitor, which maintains E2F in an inactive state, the decline of serum-induced PAI-1 mRNA levels was suppressed. In mutant U2OS cells expressing a temperature-sensitive retinoblastoma protein (pRB), a shift to a permissive temperature induced PAI-1 mRNA expression. In U2OS cells stably expressing an E2F1-estrogen receptor chimeric protein that could be activated by tamoxifen, PAI-1 gene transcription was markedly reduced by tamoxifen even in the presence of cycloheximide. These results all indicate that endogenous E2F can directly repress the PAI-1 gene. DNase I hypersensitive-site analysis of the PAI-1 promoter suggested the involvement of conformation changes in chromatin structure of the PAI-1 promoter. 5, deletion analysis of the PAI-1 promoter showed that multiple sites were responsible for the E2F negative regulation, some of which were promoter dependent. Interestingly, one of these sites is a p53-binding element. [source] Expression of cardiotoxin-2 geneFEBS JOURNAL, Issue 6 2001Cloning, characterization, deletion analysis of the promoter This report is the first study of the regulation of expression of a toxin gene and it also demonstrates the novel finding that the cardiotoxin (CTX)-2 gene from Naja sputatrix is expressed in the venom gland as well as in other tissues in the snake, such as liver, heart and muscle. The venom gland produces a 500-bp (spliced) CTX-2 mRNA as the final transcript. However, the liver produces two types of CTX-2 mRNA, of which the unspliced transcript (1 kb) is predominant; the 500 bp spliced transcript is the minor species. This differential expression of the CTX gene has been attributed to the usage of alternative promoter consisting of independent TATA boxes and corresponding transcription initiation sites. Among the several transcription factors that have been identified by a search of the TFIID database, the participation of two glucocorticoid elements in the expression of the CTX gene has been demonstrated by promoter deletion analysis. Putative binding sites for SP-1, C/EBP, CACCC-binding factor and at least two unknown binding factors have also been identified by DNase I footprinting of the promoter. [source] Identification and characterization of a novel progesterone receptor-binding element in the mouse prostaglandin E receptor subtype EP2 geneGENES TO CELLS, Issue 9 2003Sohken Tsuchiya Background:, Gene expression of prostaglandin E receptor EP2 is induced in the luminal epithelium of the mouse uterus during peri-implantation period (day-5 of pseudopregnancy), suggesting the involvement of progesterone and its receptor (PR) in this expression. However it remains unclear whether PR affects EP2 gene expression through its binding. Results:, We investigated transcriptional regulation of EP2 gene expression with reporter gene analysis using HeLa cells with or without expression of the PR. The 5,-flanking region (,3260 to ,27, upstream of the translation initiation site) exhibited progesterone-induced promoter activation and basal promoter activity in the presence of PR. Using successive deletion analysis, we determined the six regulatory regions in the EP2 gene. Three regions were found to be involved in progesterone-induced promoter activation, whereas the other three regions were involved in basal promoter activity in the presence of PR. We identified a novel PR-binding sequence, 5,-G(G/A)CCGGA-3,, in the two basal promoter regions and Sp1- and Sp3-binding in the other basal promoter region. Conclusions:, We identified a novel PR-binding sequence, which may be involved in the regulation of basal promoter activity in the EP2 gene. [source] TCDD suppresses insulin-responsive glucose transporter (GLUT-4) gene expression through C/EBP nuclear transcription factors in 3T3-L1 adipocytesJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 2 2006Phillip Chin-Chen Liu Abstract TCDD is known to reduce significantly the level of the functionally active form of glucose transporter type 4 (GLUT4) in vivo in adipose tissue and muscles. To study the mechanistic basis of this phenomenon, we conducted transient transfection and DNA deletion analysis in 3T3-L1 cells using chloramphenicol acetyltransferase (CAT) reporter plasmids containing the GLUT4 promoter joined to the bacterial CAT. It was found that in transfected control samples, CAT activity was significantly higher in cells transfected with p469CAT and p273CAT than those with p78CAT, indicating that the region between ,78 and ,273 contained elements that play major roles in transactivation of this gene. Treatment with TCDD decreased CAT activity with p469CAT and p273CAT, but not with p78CAT, indicating the same region to contain the element(s) affected by TCDD. A gel-shift (EMSA) analysis result indicated that TCDD shows the profound effect only on the nuclear proteins binding to the [32P]-labeled probe containing C/EBP response element equivalent of the ,265 to ,242 stretch of the GLUT4 promoter. The results of supershift analysis showed that TCDD caused a decrease in the tier of C/EBP, and an increase in that of C/EBP, among the proteins bound to this C/EBP response element. We studied the effect of TCDD in cells overexpressing either C/EBP,, C/EBP,, or C/EBP, through transient transfection of p273CAT or p469CAT. The results clearly showed that the effect of TCDD to suppress the CAT activity of p273 or p469 disappeared in those cells overexpressing C/EBP, or C/EBP,. These results implicate the C/EBP proteins to be the main mediator of suppressive action of TCDD on GLUT4 gene expression in 3T3-L1 cells. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:79,87, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20120 [source] Transcriptional regulation of human excitatory amino acid transporter 1 (EAAT1): cloning of the EAAT1 promoter and characterization of its basal and inducible activity in human astrocytesJOURNAL OF NEUROCHEMISTRY, Issue 6 2003Seon-Young Kim Abstract Excitatory amino acid transporter 1 (EAAT1) is one of the two glial glutamate transporters that clear the extracellular glutamate generated during neuronal signal transmission. Here, we cloned and characterized a 2.1-kb promoter region of human EAAT1 and investigated its function in the transcriptional regulation of the EAAT1 gene in human primary astrocytes. The full-length promoter region lacked TATA and CCAAT boxes and an initiator element, it contained several potential transcription factor-binding sites and it exhibited promoter activity in primary astrocytes and in several types of transformed cells. Consecutive 5,-deletion analysis of the EAAT1 promoter indicated the presence of negative and positive regulatory regions and a putative core promoter between ,57 bp and +20 bp relative to the transcription start site (TSS). The core promoter contained a single GC-box in position ,52/,39 and one E-box near the TSS and the GC-box site that was responsible for 90% of the basal promoter activity as determined by mutational analysis. Electrophoretic mobility shift, supershift and competition assays demonstrated binding of stimulating proteins (Sp) 1 and 3 to the GC-box and upstream stimulating factor (USF) 1 to the E-box. Treatment of primary human astrocytes with cellular modulators 8-bromo cyclic AMP and epidermal growth factor increased EAAT1 promoter activity in transient transfection assays and increased cellular EAAT1 mRNA expression and glutamate uptake by astrocytes. Conversely, tumor necrosis factor-, reduced both EAAT promoter activity and cellular EAAT1 mRNA expression. These results enable studies of transcriptional regulation of EAAT1 gene at the promoter level. [source] Influence diagnostics and outlier tests for semiparametric mixed modelsJOURNAL OF THE ROYAL STATISTICAL SOCIETY: SERIES B (STATISTICAL METHODOLOGY), Issue 3 2002Wing-Kam Fung Summary. Semiparametric mixed models are useful in biometric and econometric applications, especially for longitudinal data. Maximum penalized likelihood estimators (MPLEs) have been shown to work well by Zhang and co-workers for both linear coefficients and nonparametric functions. This paper considers the role of influence diagnostics in the MPLE by extending the case deletion and subject deletion analysis of linear models to accommodate the inclusion of a nonparametric component. We focus on influence measures for the fixed effects and provide formulae that are analogous to those for simpler models and readily computable with the MPLE algorithm. We also establish an equivalence between the case or subject deletion model and a mean shift outlier model from which we derive tests for outliers. The influence diagnostics proposed are illustrated through a longitudinal hormone study on progesterone and a simulated example. [source] An amino-terminal domain of Enterococcus faecalis aggregation substance is required for aggregation, bacterial internalization by epithelial cells and binding to lipoteichoic acidMOLECULAR MICROBIOLOGY, Issue 4 2004Christopher M. Waters Summary Aggregation substance (AS), a plasmid-encoded surface protein of Enterococcus faecalis, plays important roles in virulence and antibiotic resistance transfer. Previous studies have suggested that AS-mediated aggregation of enterococcal cells could involve the binding of this protein to cell wall lipoteichoic acid (LTA). Here, a method to purify an undegraded form of Asc10, the AS of the plasmid pCF10, is described. Using this purified protein, direct binding of Asc10 to purified E. faecalis LTA was demonstrated. Equivalent binding of Asc10 to LTA purified from INY3000, an E. faecalis strain that is incapable of aggregation, was also observed. Surprisingly, mutations in a previously identified aggregation domain from amino acids 473 to 683 that abolished aggregation had no effect on LTA binding. In frame deletion analysis of Asc10 was used to identify a second aggregation domain located in the N-terminus of the protein from amino acids 156 to 358. A purified Asc10 mutant protein lacking this domain showed reduced LTA binding, while a purified N-terminal fragment from amino acids 44,331 had high LTA binding. Like the previously described aggregation domain, the newly identified Asc10(156,358) aggregation domain was also required for efficient internalization of E. faecalis into HT-29 enterocytes. Thus, Asc10 possess two distinct domains required for aggregation and eukaryotic cell internalization: an N-terminal domain that promotes binding to LTA and a second domain located near the middle of the protein. [source] Genetic analysis of the strong gyrase site (SGS) of bacteriophage Mu: localization of determinants required for promoting Mu replicationMOLECULAR MICROBIOLOGY, Issue 4 2000M. L. Pato The Mu strong gyrase site (SGS), located in the centre of the Mu genome, is required for efficient Mu replication, as it promotes synapsis of the prophage termini. Other gyrase sites tested, even very strong ones, were unable to substitute for the SGS in Mu replication. To determine the features required for its unique properties, a deletion analysis was performed on the SGS. For this analysis, we defined the 20 bp centred on the midpoint of the 4 bp staggered cleavage made by gyrase to be the ,core' and the flanking sequences to be the ,arms'. The deletion analysis showed that (i) ,,40 bp of the right arm is required, in addition to core sequences, for both efficient Mu replication and gyrase cleavage; and (ii) the left arm was not required for efficient Mu replication, although it was required for efficient gyrase cleavage. These observations implicated the right arm as the unique feature of the SGS. The second observation showed that strong gyrase cleavage and Mu replication could be dissociated and suggested that even weak gyrase sites, if supplied with the right arm of the SGS, could promote Mu replication. Hybrid sites were constructed with gyrase sites that could not support efficient Mu replication. The SGS right arm was used to replace one arm of the strong pSC101 gyrase site or the weaker pBR322 site. The pSC101 hybrid site allowed efficient Mu replication, whereas the pBR322 hybrid site allowed substantial, but reduced, replication. Hence, it appears that optimal Mu replication requires a central strong gyrase site with the properties imparted by the right arm sequences. Possible roles for the SGS right arm in Mu replication are addressed. [source] Autoinhibitory regulation of soluble adenylyl cyclaseMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2006James A. Chaloupka Abstract Soluble adenylyl cyclase is an evolutionarily conserved bicarbonate sensor that plays a crucial role in cAMP dependent processes that occur during mammalian fertilization. sAC protein is expressed at the highest levels in male germ cells, and is found to occur as one of two known isoforms: a truncated protein (sACt) that consists almost exclusively of the two conserved catalytic domains (C1 and C2), and a full-length form (sACfl) that contains an additional noncatalytic C-terminal region. Several studies suggested sACt was more active than sACfl. We now demonstrate that the specific activity of sACt is at least 10-fold higher than the specific activity of sACfl. Using deletion analysis and a novel genetic screen to identify activating mutations, we uncovered an autoinhibitory region just C-terminal to the C2 domain. Kinetic analysis of purified recombinant sAC revealed this autoinhibitory domain functions to lower the enzyme's Vmax without altering its affinity for substrate or regulation by any of the known modulators of sAC activity. Our results identify an additional regulatory mechanism specific to the sACfl isoform. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] Functional analysis of cauliflower mosaic virus 35S promoter: re-evaluation of the role of subdomains B5, B4 and B2 in promoter activityPLANT BIOTECHNOLOGY JOURNAL, Issue 6 2007Simran Bhullar Summary The cauliflower mosaic virus 35S (35S) promoter is used extensively for transgene expression in plants. The promoter has been delineated into different subdomains based on deletion analysis and gain-of-function studies. However, cis -elements important for promoter activity have been identified only in the domains B1 (as-2 element), A1 (as-1 element) and minimal promoter (TATA box). No cis -elements have been described in subdomains B2,B5, although these are reported to be important for the overall activity of the 35S promoter. We have re-evaluated the contribution of three of these subdomains, namely B5, B4 and B2, to 35S promoter activity by developing several modified promoters. The analysis of ,-glucuronidase gene expression driven by the modified promoters in different tissues of primary transgenic tobacco lines, as well as in seedlings of the T1 generation, revealed new facets about the functional organization of the 35S promoter. This study suggests that: (i) the 35S promoter truncated up to ,301 functions in a similar manner to the ,343 (full-length) 35S promoter; (ii) the Dof core and I-box core observed in the subdomain B4 are important for 35S promoter activity; and (iii) the subdomain B2 is essential for maintaining an appropriate distance between the proximal and distal regions of the 35S promoter. These observations will aid in the development of functional synthetic 35S promoters with decreased sequence homology. Such promoters can be used to drive multiple transgenes without evoking promoter homology-based gene silencing when attempting gene stacking. [source] A New De Novo Missense Mutation in Connexin 26 in a Sporadic Case of Nonsyndromic DeafnessTHE LARYNGOSCOPE, Issue 5 2007Paola Primignani PhD Abstract Objectives: Mutations in the GJB2 gene, encoding Connexin 26, can cause nonsyndromic recessive deafness or dominant hearing loss (HL) with or without keratoderma. The objective was to perform a molecular evaluation to establish the inherited pattern of deafness in the sporadic cases afferent to our center. Methods: The subject was a 2-year-old Italian girl with nonsyndromic early onset HL. We performed DNA sequencing of the GJB2 gene and deletion analysis of the GJB6 gene in all family members. Results: Direct sequencing of the gene showed a heterozygous C,G transition at nucleotide 172 resulting in a proline to alanine amino acid substitution at codon 58 (P58A). The analyses indicate that the P58A mutation appeared de novo in the proband with a possible dominant effect. Conclusions: This mutation occurs in the first extracellular domain (EC1), which seems to be very important for connexon-connexon interaction and for the control of voltage gating of the channel. The de novo occurrence of an EC1 mutation in a sporadic case of deafness is consistent with the assumption that P58A can cause dominant HL. [source] Fluorescence microscopy: a powerful technique to detect low GUS activity in vascular tissuesTHE PLANT JOURNAL, Issue 1 2003Philippe Rech Abstract We have previously shown that the Eucalyptus gunnii EgCAD2 promoter was preferentially expressed in vascular tissues in different transgenic plants (poplar, tobacco, Arabidopsis and grapevine). In order to delineate the cis elements governing this vascular expression pattern, promoter deletion analysis was performed allowing us to identify the proximal region [,340/,124] as essential for vascular cambium/xylem-specific expression. In plants transformed with the smallest promoter region [,124/+117], the GUS activity was difficult to detect using conventional bright field microscopy. To overcome this problem, we used fluorescence microscopy, enabling us to show that the [,124/+117] region contained cis -elements driving activity in phloem fibres but not in secondary xylem. The technical improvement of the histochemical detection of GUS activity using fluorescence microscopy enables accurate investigation of low GUS activity in phenol-rich tissues. [source] Crystallographic studies of a novel DNA-binding domain from the yeast transcriptional activator Ndt80ACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2002Sherwin P. Montano The Ndt80 protein is a transcriptional activator that plays a key role in the progression of the meiotic divisions in the yeast Saccharomyces cerevisiae. Ndt80 is strongly induced during the middle stages of the sporulation pathway and binds specifically to a promoter element called the MSE to activate transcription of genes required for the meiotic divisions. Here, the preliminary structural and functional studies to characterize the DNA-binding activity of this protein are reported. Through deletion analysis and limited proteolysis studies of Ndt80, a novel 32,kDa DNA-binding domain that is sufficient for DNA-binding in vitro has been defined. Crystals of the DNA-binding domain of Ndt80 in two distinct lattices have been obtained, for which diffraction data extend to 2.3,Å resolution. [source] | ||||||||||||||||||||||||||||