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Minimal Promoter (minimal + promoter)
Selected AbstractsTranscriptional regulation of ASK/Dbf4 in cutaneous melanoma is dependent on E2F1EXPERIMENTAL DERMATOLOGY, Issue 12 2008Sandeep Nambiar Background:, Melanoma is a complex genetic disease, the management of which will require an in-depth understanding of the biology underlying its initiation and progression. Recently, we have reported the differential regulation of a novel gene, namely ASK/Dbf4, in melanoma and suggested upregulation of ASK/Dbf4 as a novel molecular determinant with prognostic relevance that confers a proliferative advantage in cutaneous melanoma. As trans -acting factor binding is fundamental to understand the regulation of gene expression, this study focuses on characterization of the specific transcriptional regulation of ASK/Dbf4 in melanoma. Objective:, We investigated whether ASK/Dbf4 is a transcriptional target of the important cell cycle regulator E2F1 in melanoma. Results:, As evidenced by gel supershift assays on nuclear extracts from various melanoma cell lines (SK-MEL-28, MV3, M13, A375 and BLM), E2F1 bound to the ASK/Dbf4 minimal promoter (MP). In addition, cisplatin-mediated abrogation of E2F1 binding to the ASK/Dbf4 MP resulted in a transcriptional decrease in ASK/Dbf4. Further, the current study also demonstrated that ASK/Dbf4 regulation was refractory to UVB, a well-known risk factor for melanoma. Conclusions:, In summary, our study not only elucidated that ASK/Dbf4, a novel cell survival gene in melanoma was transcriptionally regulated by E2F1, but also that the induction of ASK/Dbf4 was refractory to UVB exposure suggesting that its upregulation was not an early event in melanomagenesis. [source] Nas transgenic mouse line allows visualization of Notch pathway activity in vivoGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 6 2006Céline Souilhol Abstract The Notch signaling pathway plays multiple and important roles in mammals. However, several aspects of its action, in particular, the precise mapping of its sites of activity, remain unclear. To address this issue, we generated a transgenic line carrying a construct consisting of a nls-lacZ reporter gene under the control of a minimal promoter and multiple RBP-J, binding sites. Here we show that this transgenic line, which we termed NAS (for Notch Activity Sensor), displays an expression profile that is consistent with current knowledge on Notch activity sites in mice, even though it may not report on all these sites. Moreover, we observe that NAS transgene expression is abolished in a RBP-J,-deficient background, indicating that it indeed requires Notch/RBP-J, signaling pathway activity. Thus, the NAS transgenic line constitutes a valuable and versatile tool to gain further insights into the complex and various functions of the Notch signaling pathway. genesis 44: 277,286, 2006. © 2006 Wiley-Liss, Inc. [source] Mapping of intrinsic bent DNA sites in the upstream region of DNA puff BhC4-1 amplified geneJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2001Adriana Fiorini Abstract We have identified bent DNA sites in the distal and proximal DNA puff BhC4-1 amplified gene promoter region of Bradysia hygida. The 2D modeling of the 3D DNA path and the ENDS ratio values calculated in this promoter region resulted in the identification of ten pronounced bent sites named BhC4B ,,9 to +,1. The 1847 bp fragment (,,3697 to ,,1850) in relation to the transcription start site shows multiple bending sites, BhC4B ,,9 to BhC4B ,,4, with periodicity ,300 bp. The analysis of the other identified bent region, starting at position ,,957, reveals that the BhC4B +,1 bent site colocalizes with the putative BhC4-1 minimal promoter. The sequence analysis of bent site BhC4B ,,4 shows a distribution of dA,dT at ,10 bp intervals between the middle of each tract, but intervals with more than one turn, ,20 bp, two helix turns, were detected in the other bent sites described here. The bent sites BhC4B ,,6 and BhC4B ,,4, contain two consensus sequences, with 60 bp each. The apparent molecular weight of fragments in the BhC4-1 promoter region were estimated in agarose gels and compared with the data obtained in polyacrylamide gels without and with ethidium bromide. The mobility reduction ratios (R -values) were determined, and a high R -value, 1.80, for a 1215 bp fragment in the distal promoter region and a 1.23 significant R -value for a 662 bp fragment in the proximal segment were found. To further analyze the predicted bent DNA sites in these fragments, the 2D trajectories of the 3D DNA path and other parameters, AT percentage, roll angle, ENDS ratio and ,G, were determined. The role of these bent sites in the BhC4-1 transcription regulation is discussed. © 2001 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] Identification of a 150 bp cis -acting element of the AtNRT2.1 promoter involved in the regulation of gene expression by the N and C status of the plantPLANT CELL & ENVIRONMENT, Issue 11 2007THOMAS GIRIN ABSTRACT The Arabidopsis thaliana AtNRT2.1 gene, which encodes a NO3 - transporter involved in high-affinity uptake by the roots, is a molecular target of several mechanisms responsible for the regulation of root NO3 - acquisition by the N status of the plant. All levels of AtNRT2.1 expression (promoter activity, transcript level, protein accumulation, transport activity) are coordinately up-regulated in the presence of NO3 - , and repressed by downstream N metabolites. Transgenic plants expressing the GUS reporter gene under the control of upstream sequences of AtNRT2.1 have been studied to identify elements targeted by these two regulatory mechanisms. A 150 bp sequence located upstream of the TATA box that is required for both stimulation by NO3 - and repression by N metabolites of the promoter has been identified. This sequence is able to confer these two regulations to a minimal promoter. Split-root experiments indicate that the stimulation of the chimaeric promoter by NO3 - occurs only at the local level, whereas its repression by N metabolites is mediated by a systemic signal spread to the whole plant. The activity of the cis -acting 150 bp element is also regulated by sucrose supply to the roots, suggesting a possible interaction between N and C signalling within this short region. Accordingly, multiple motifs potentially involved in regulations by N and/or C status are identified within this sequence by bioinformatic approaches. This is the first report of such a cis -acting element in higher plants. [source] Reversal of the silencing of tetracycline-controlled genes requires the coordinate action of distinctly acting transcription factorsTHE JOURNAL OF GENE MEDICINE, Issue 1 2005Renata Pankiewicz Abstract Background Regulation of genes transferred to eukaryotic organisms is often limited by the lack of consistent expression levels in all transduced cells, which may result in part from epigenetic gene silencing effects. This reduces the efficacy of ligand-controlled gene switches designed for somatic gene transfers such as gene therapy. Methods A doxycycline-controlled transgene was stably introduced in human cells, and clones were screened for epigenetic silencing of the transgene. Various regulatory proteins were targeted to the silent transgene, to identify those that would mediate regulation by doxycycline. Results A doxycycline-controlled minimal promoter was found to be prone to gene silencing, which prevents activation by a fusion of the bacterial TetR DNA-binding domain with the VP16 activator. DNA modification studies indicated that the silenced transgene adopts a poorly accessible chromatin structure. Several cellular transcriptional activators were found to restore an accessible DNA structure when targeted to the silent transgene, and they cooperated with Tet-VP16 to mediate regulation by doxycycline. Conclusions Reversal of the silencing of a tetracycline-regulated minimal promoter requires a chromatin-remodeling activity for subsequent promoter activation by the Tet-VP16 fusion protein. Thus, distinct regulatory elements may be combined to obtain long-term regulation and persistent expression of exogenous genes in eukaryotic cells. Copyright © 2004 John Wiley & Sons, Ltd. [source] Second-generation tetracycline-regulatable promoter: repositioned tet operator elements optimize transactivator synergy while shorter minimal promoter offers tight basal leakinessTHE JOURNAL OF GENE MEDICINE, Issue 7 2004Siamak Agha-Mohammadi Abstract Background The tetracycline-regulatable system is one of the most valuable tools for controlling gene expression. In its current form, however, the system is less than ideal for in vivo or gene therapy uses due to difficulties in set-up procedures, high basal leakiness, and unpredictable delivery and efficiency. Methods To address these issues, we have devised a second generation of tetracycline-regulated promoters (TREs). The second-generation TRE (SG-TRE) contains a shortened cytomegalovirus (CMV) minimal promoter together with eight tet operator sequences positioned in an optimized manner upstream of the TATA box. This construct displays far greater reduction in basal leakiness than maximal transgene expression. Conversely, maximal transgene expression is increased to a greater degree than basal leakiness by post-translational stabilization with bovine growth hormone poly A. Results In transient studies, the SG-TRE displays over 100 000-fold regulation efficiency in HeLa cells at 1:1 ratio of transactivator to reporter plasmid in the Tet-Off system. This novel promoter achieves a regulation efficiency 500- to 1000-fold higher than that of the original TRE (PhCMV*-1) in HeLa cells by displaying undetectable levels of basal leakiness without compromised maximal expression. In other cell lines, the SG-TRE proves to be more efficient than the original PhCMV*-1 in a cell-dependent manner. Furthermore, the SG-TRE preserves its enhanced regulation efficiency and its reduced basal leakiness in the context of a single positive feedback regulatory vector that presents ease of delivery of the system for use in vivo. Finally, in vivo, the biological function of granulocyte-macrophage colony stimulating factor is tightly regulated in the context of SG-TRE delivered via adeno-associated viruses. Copyright © 2004 John Wiley & Sons, Ltd. [source] Tachykinin expression in cartilage and function in human articular chondrocyte mechanotransductionARTHRITIS & RHEUMATISM, Issue 1 2003S. J. Millward-Sadler Objective To assess whether substance P and the corresponding neurokinin 1 (NK1) receptor are expressed in human articular cartilage, and whether these molecules have a role in chondrocyte mechanotransduction. Methods Transgenic studies, immunohistochemistry, Western blotting, and reverse transcriptase,polymerase chain reaction were used to assess the expression of the preprotachykinin (PPT) gene, substance P, and NK1 in developing mice, in adult human articular cartilage, and in human chondrocytes in culture. Chondrocytes obtained from PPT knockout mice and human articular chondrocytes were mechanically stimulated in the presence or absence of inhibitors of substance P signaling, and cell membrane potentials or relative levels of aggrecan messenger RNA (mRNA) were measured. Results Replacing a region of the PPT gene transcriptional site that contains a dominant repressor of the proximal promoter activity with the constitutive minimal promoter of the human ,-globin promoter allowed expression of a marker gene in areas of chondrogenesis during mouse development and in adult chondrocytes grown in culture. Adult human articular chondrocytes expressed endogenous PPT mRNA, substance P, and the corresponding NK1 receptor in vivo and in vitro. Blockade of substance P signaling by a chemical antagonist to the NK1 receptor inhibited chondrocyte responses to mechanical stimulation. Conclusion Substance P is expressed in human articular cartilage and is involved in chondrocyte mechanotransduction via the NK1 receptor in an autocrine and paracrine manner. This suggests that substance P and the NK1 receptor have roles in the maintenance of articular cartilage structure and function that were previously unrecognized. [source] | |||||||||||||