Results Significant hypomethylation of two CpG sites within IFI44L promoter, Site1 (Chr1: 79 085 222) and Site2 (Chr1: 79 085 250; cg06872964), was identified in patients with SLE compared with HCs, patients with RA and patients with pSS. In a comparison between patients with SLE and HCs included in the first validation cohort, Site1 methylation had a sensitivity of 93.6% and a specificity of 96.8% at a cut-off methylation level of 75.5% and Site2 methylation had a sensitivity of 94.1% and a specificity of 98.2% at a cut-off methylation level of 25.5%. The IFI44L promoter methylation marker was also validated in an European-derived cohort. In addition, the methylation levels of Site1 and Site2 within IFI44L promoter were significantly lower in patients with SLE with renal damage than those without renal damage. Patients with SLE showed significantly increased methylation levels of Site1 and Site2 during remission compared with active stage. ...
Gao, L., Smit, M. A., van den Oord, J. J., Goeman, J. J., Verdegaal, E. M. E., van der Burg, S. H., Stas, M., Beck, S., Gruis, N. A., Tensen, C. P., Willemze, R., Peeper, D. S. and van Doorn, R. (2013), Genome-wide promoter methylation analysis identifies epigenetic silencing of MAPK13 in primary cutaneous melanoma. Pigment Cell & Melanoma Research, 26: 542-554. doi: 10.1111/pcmr.12096 ...
The structure of the core promoter region (i.e., DNA sequences flanking the transcription start site and including the TATA, initiator, and downstream elements that interact with the general transcription machinery) of protein-coding genes has an important influence both on the efficiency of basal transcription and on the ability of the core promoter to respond to upstream promoter-bound activators in vivo and in vitro (reviewed in references33, 38, and 46). Although the general transcription machinery has been well characterized, little is known about the factors and mechanisms that control its activity in a core promoter-specific manner. Whereas various factors like E2F, YY1, TFII-I, and USF may regulate transcription not only through upstream promoter elements but also through interactions with the core promoter regions of certain genes (for reviews, see references38 and 50), several components of the basal transcription machinery have intrinsic and more general core promoter-selective ...
The RNA polymerase II (Pol II) core promoter is the strategic site of convergence of the signals that lead to the initiation of DNA transcription1-5, but the downstream core promoter in humans has been difficult to understand1-3. Here we analyse the human Pol II core promoter and use machine learning to generate predictive models for the downstream core promoter region (DPR) and the TATA box. We developed a method termed HARPE (high-throughput analysis of randomized promoter elements) to create hundreds of thousands of DPR (or TATA box) variants, each with known transcriptional strength. We then analysed the HARPE data by support vector regression (SVR) to provide comprehensive models for the sequence motifs, and found that the SVR-based approach is more effective than a consensus-based method for predicting transcriptional activity. These results show that the DPR is a functionally important core promoter element that is widely used in human promoters. Notably, there appears to be a duality between the
We have characterized the 5′ region of the human alpha 1(V) collagen gene (COL5A1). The transcriptional promoter is shown to have a number of features characteristic of the promoters of housekeeping and growth-control-related genes. It lacks obvious TATA and CAAT boxes, has multiple transcription start sites, has a high GC content, lies within a well-defined CpG island and has a number of consensus sites for the potential binding of transcription factor Sp1. This type of promoter structure, while unusual for a collagen gene, is consistent with the broad distribution of expression of COL5A1 and is reminiscent of the promoter structures of the genes encoding type VI collagen, which has a similarly broad distribution of expression. Stepwise deletion of COL5A1 5′ sequences, placed upstream of a heterologous reporter gene, yielded a gradual decrease in promoter activity, indicating that the COL5A1 promoter is composed of an array of cis-acting elements. A minimal promoter region contained ...
Our products have been quoted by publications of Association of CnB 5I/5D promoter gene polymorphism and serum calcineurin levels in early onset of coronary artery disease of south Indian cohort from Gene .
Our results establish that the extent of stable DNA wrapping in RPo depends on the sequence of the promoter and, in particular, on sequence determinants in the upstream region of the promoter (UP elements). The presence of αCTD and an intact α‐linker is required to maintain extensive stable DNA wrapping. Our results further indicate that the sequence of the upstream region of the promoter can affect DNA wrapping even in the absence of αCTD and thus even in the absence of αCTD-DNA interactions. For example, RPo prepared using ΔαCTDI/ΔαCTDII RNAP shows an apparent DNA compaction of 13±0.6 nm at lacUV5(UPfull) but only 4±0.8 nm at lacUV5(ICAP) (Fig 3E,F). We infer that the sequence of the upstream region of the promoter can affect compaction not only through effects on αCTD-DNA interaction but also through other effects. We suggest that these other effects involve intrinsic DNA curvature, noting that UP‐element subsites and UP elements are A/T‐rich sequences (Fig 1A; Ross et al, ...
Dual Luciferase Assay - posted in Molecular Cloning: I am planning to do a dual luciferase assay using the firefly and renilla luciferase vectors. Can someone suggest me a ratio for the co-reporter vectors to be added to the transfection mix. Regards Sankella
The present study describes a novel cell type-specific mechanism of transcriptional regulation of TSP-1 in vascular cells in response to glucose. We report here that unlike our recently identified short promoter region (−280/+66) responsible for the increased THBS1 transcription in ECs, a longer promoter fragment (−1270/+66) is required for THBS1 regulation in HASMCs, as was described for specialized pericytes and mesangial cells.27 Interestingly, glucose responsiveness in ECs was in fact inhibited by the distal fragment of the promoter,10 suggesting the presence of an inhibitory element in this region, which is not active in either VSMCs or mesangial cells.27 The longer promoter region, −1270/+66, responsible for the increased THBS1 transcription in HASMCs contained distinctly different binding elements, as identified by MatInspector, located in the distal end of the promoter. These binding elements had no similarity to those in the EC-specific THBS1 promoter fragment, −280/+66, ...
In this article, we review some of the expression systems that are available for Metabolic Control Analysis and Metabolic Engineering, and examine their advantages and disadvantages in different contexts. In a recent approach, artificial promoters for modulating gene expression in micro-organisms were constructed using synthetic degenerated oligonucleotides. From this work, a promoter library was obtained for Lactococcus lactis, containing numerous individual promoters and covering a wide range of promoter activities. Importantly, the range of promoter activities was covered in small steps of activity change. Promoter libraries generated by this approach allow for optimization of gene expression and for experimental control analysis in a wide range of biological systems by choosing from the promoter library promoters giving, e.g., 25%, 50%, 200%, and 400% of the normal expression level of the gene in question. If the relevant variable (e.g., the flux or yield) is then measured with each of these ...
No. The Promoter Selection Plate is not meant to be used to establish fundamental lentiviral transduction conditions, such as optimal cell density, in your cells of interest. The purpose of the Promoter Selection Plate is to visually assess relative promoter activity. Basic experimental conditions (such as cell density, with or without serum, Polybrene concentration) should be established prior to transduction of viral particles using the SMARTchoice Promoter Selection Plate. The Promoter Selection Plate contains duplicate wells of serially diluted lentiviral particles representing each promoter so that two separate conditions can be tested simultaneously. After identification of the optimal promoter for your cells of interest, additional transduction optimization should be performed to identify appropriate MOIs for optimal shRNA performance ...
An accurate identification of gene promoters remains an important challenge. Computational approaches for this problem rely on promoter sequence attributes that are believed to be critical for transcription initiation. Here we report a probabilistic model that captures two important properties of promoters, not used by previous methods, viz., the location preference and co-occurrence of promoter elements. Additionally, we found that many of the position-specific DNA elements are strongly linked with the function of the gene product. For instance, a highly conserved motif CCTTT at -1 position is strongly associated with protein synthesis, cellular and tissue development. Our comparative analysis of promoter classes reveals that the promoters devoid of CpG islands are more conserved and have fewer alternative transcription start sites. The discovered links between promoter elements and gene function allows us to infer genetic networks from promoter elements. The web server for the PSPA promoter ...
The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5 LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the
In this study, we indicated that the mouse β-chain promoter is functional only in PT18 mouse mast cell line, but not in monomacrophage and lymphoma cell lines. By using a series of 5′-deletion promoter constructs, the cell type-specific promoter was assigned within −69/+103, although a slight decrease in the promoter activity was observed by the deletion of −116/−70. This may suggest the presence of additional cis-enhancing elements recognizing the deleted region. Anyway, by EMSA using antitranscription factor Abs and various competitive oligonucleotides, GATA-1 protein was identified to bind the promoter region (−61/+3) of the β-chain where three possible GATA-1-binding (−53/−48, −46/−51, and −42/−47) and a single possible GATA-1-binding site (−31/−26) are present. Furthermore, reporter assay using PT18 cells (β-chain+, GATA-1+) and coexpression analysis using CV-1 cells (β-chain−, GATA-1−) indicated that those four GATA-1 sites are required for full ...
Fibulin-1 is a multifunctional extracellular protein involved in diverse biological processes including cardiovascular development, haemostasis and cancer. To investigate the transcriptional regulation of the gene encoding fibulin-1 we cloned and analysed about 4.0kb of the 5′-flanking regions of both the human and mouse fibulin-1 genes. The human and mouse fibulin-1 promoters share little sequence similarity except for a short region of approx. 150-170bp immediately upstream of the translation start site. The conserved region contains a TATA-like sequence (ATAATT) and multiple consensus binding sites for Sp1 and activator protein 2 (AP-2). That the short conserved region in each gene confers basal promoter activity is demonstrated by transient transfections of promoter deletion constructs for both the human and mouse genes into cells that express fibulin-1 constitutively. Co-transfections of promoter constructs with expression plasmids for Sp1, Sp3 and Sp4 into Drosophila SL2 cells indicate ...
The mdm2 gene is a target for transcriptional activation by the p53 tumor suppressor gene product. Previous work has revealed that the mouse mdm2 gene contains two promoters: one is located upstream to the gene and is active in the absence of p53, the other resides within the first intron and requires p53 for transcriptional activity. To determine whether this unique promoter activation pattern is biologically important, we investigated the structure and function of the corresponding region of the human mdm2 (hmdm2) gene. We report here that the hmdm2 gene also contains an intronic, p53-dependent promoter. The structural features of this promoter are highly conserved between mouse and man, as opposed to the lack of conservation of the first exon. This promoter is triggered in vivo in the presence of activated wild type p53, leading to the production of novel mRNA species. The intronic hmdm2 promoter contains two tandem p53 binding elements. Deletion analysis suggests that optimal promoter
IL-6 induces PAI-1 mRNA and protein accumulation.13,19 Although several transcription factor binding sites were identified in PAI-1 promoter, no classic inflammatory response element was found. Because promoter activity was increased by IL-6, the IL-6-responsive region was explored. The deletion and site mutation of the region from −239 to −210 bp decreased ,80% of the IL-6-inducible promoter activity, indicating that the region is critical for response. A computer-based database analysis indicated there is a putative C/EBP binding site (−226 to −212 bp). The promoters of most IL-6-inducible acute-phase protein genes have been characterized with C/EBP binding motifs.11 Using competition experiments, EMSA supershift analysis, and DNase I footprinting analysis, a C/EBP motif on PAI-1 promoter was verified, and 3 members of C/EBP family including α, β, and δ were involved in the DNA-protein complex formation. C/EBPβ was involved in the formation of 3 complexes because single-copy ...
Core promoter element analysis is performed in order to investigate the quality of the promoter collection. It exploits the fact that certain DNA motifs preferentially occur at characteristic distances from a TSS. For instance, the TATA-box occurs in a narrow region centered about 28 bp upstream of the TSS whereas the CCAAT-box occurs in a much wider area with a peak frequency at position −80. Based on these observations, we would expect a high-quality promoter collection to show high peaks for both sequence motifs. In addition, a narrow TATA-box peak at −28 would indicate precise TSS mapping. This analysis has been performed using OProf. Readers are encouraged to repeat this anlysis and perform others in order to check for the quality of the promoter list. TATA-box: this core promoter element is normally found 28 bp upstream the transcription start site. The following plot shows that EPDnew promoter collection has a more focused TATA-box distribution compared to Gramene annotation ...
Several mutants derived from transformed human B cell lines are defective in expressing major histocompatibility complex (MHC) class II genes. The failure to express a class II gene in at least one such mutant line has been mapped to the MHC class II X box, a conserved transcriptional element in the promoter region. A complementary DNA encoding a DNA-binding protein (human X box binding protein, hXBP-1) whose target is the human DR alpha X box and the 3 flanking region has now been cloned. This complementary DNA encoded a protein with structural similarities to the c-jun proto-oncogene product, and its target sequence was closely related to the palindromic target sequence of c-jun. Mutation of the hXBP-1 DNA target sequence decreased DR alpha promoter activity in vivo. These studies suggest that the hXBP-1 protein acts as a transcription factor in B cells. ...
REHOVOT, Israel and CUIABÁ, Brazil, Oct. 19, 2016-- Evogene Ltd., a leading biotechnology company for the improvement of crop productivity, and Instituto Mato-grossense do Algodão, a leading developer and marketer of cotton seeds, announced today a collaboration for the discovery and validation of novel genomic promoters to support IMAmts product...
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Binding of transcriptional activators to a promoter is a prerequisite process in transcriptional activation. It is well established that the efficiency of activator binding to a promoter is determined by the affinity of direct interactions between the DNA-binding domain of an activator and its specific target sequences. However, I describe here that activator binding to a promoter is augmented in vivo by the effects of two other determinants that have not been generally appreciated: (i) the number of activator binding sites present in a promoter and (ii) the potency of activation domains of activators. Multiple sites within a promoter can cooperatively recruit cognate factors regardless of whether they contain an effective activation domain. This cooperativity can result in the synergistic activation of transcription. The second effect is the enhancement of activator binding to a promoter by the presence of activation domains. In this case, activation domains are not simply tethered to the ...
Fig. 4. Analysis of the 5′ region of FEZ1 gene. A, primer extension analysis of the transcriptional start site of human FEZ1 gene. Poly(A)+ RNAs from human brain (Lane 1) and breast (Lane 2) were reverse-transcribed with a 5′-radiolabeled, gene-specific primer corresponding with the 1524-1499-bp upstream region from the first translatable methionine. Arrow, TSS, which is 69 bp upstream from the 3′-end of the primer. B, analysis of the human FEZ1 gene 5′ region. Bold bar at the top of the figure indicates the FEZ1 gene locus, in which exons are depicted in boxes. Cross-hatched boxes, deletion mutants used for the luciferase reporter assay. The 5′-end positions of the deleted promoter are indicated as nucleotide numbers from the TSS. C, luciferase reporter assay of the deleted FEZ1 promoter regions. Luciferase reporter plasmids were transfected into Fez1-positive 293 cells to identify the positive regulatory region for FEZ1 transcription. Left, 5′-end positions of the deleted promoter ...
A promoter is a region of DNA that facilitates the transcription of a particular gene. Promoters can be about 100-1000 [nucleotides] long.[1]. A promoter is on the template strand for the gene and near the gene in numbers of nucleotides (nts) along the DNA template strand. Usually, the promoter lies within the string of nucleotides between genes. Some promoters are called constitutive as they are active in all circumstances in the cell, while others are regulated becoming active in response to specific stimuli. These specific stimuli for a gene find a receptive portion within that genes promoter. In the case of genes that are used to produce proteins, the RNA polymerase II holoenzyme that actually performs the transcription from the template strand needs to find chemical cues for attachment to the DNA and where to begin transcription. Preceding this are chemical cues for which DNA strand is the template strand and in what direction transcription is to be performed. A promoter contains cues for ...
A promoter is a region of DNA that facilitates the transcription of a particular gene. Promoters can be about 100-1000 [nucleotides] long.[1]. A promoter is on the template strand for the gene and near the gene in numbers of nucleotides (nts) along the DNA template strand. Usually, the promoter lies within the string of nucleotides between genes. Some promoters are called constitutive as they are active in all circumstances in the cell, while others are regulated becoming active in response to specific stimuli. These specific stimuli for a gene find a receptive portion within that genes promoter. In the case of genes that are used to produce proteins, the RNA polymerase II holoenzyme that actually performs the transcription from the template strand needs to find chemical cues for attachment to the DNA and where to begin transcription. Preceding this are chemical cues for which DNA strand is the template strand and in what direction transcription is to be performed. A promoter contains cues for ...
This invention provides novel chimeric promoter/enhancers. The chimeric promoter/enhancers are particularly suitable for directing gene expression in mammalian cells.
The human aldolase A gene is transcribed from three different promoters, pN, pM, and pH, all of which are clustered within a small 1.6-kbp DNA domain. pM, which is highly specific to adult skeletal muscle, lies in between pN and pH, which are ubiquitous but particularly active in heart and skeletal muscle. A ubiquitous enhancer, located just upstream of pH start sites, is necessary for the activity of both pH and pN in transient transfection assays. Using transgenic mice, we studied the sequence controlling the muscle-specific promoter pM and the relations between the three promoters and the ubiquitous enhancer. A 4.3-kbp fragment containing the three promoters and the ubiquitous enhancer showed an expression pattern consistent with that known in humans. In addition, while pH was active in both fast and slow skeletal muscles, pM was active only in fast muscle. pM activity was unaltered by the deletion of a 1.8-kbp region containing the ubiquitous enhancer and the pH promoter, whereas pN remained ...
The functionality of a 3422-base pair promoter fragment from the mouse α1B-adrenergic receptor (α1BAR) gene was examined. This fragment, cloned from a mouse genomic library, was found to have significant sequence homology to the known human and rat α1BAR promoters. However, the consensus motif of several key cis-acting elements is not conserved among the rat, human, and mouse genes, suggesting species specificity. Confirming fidelity of the murine promoter, robust in vitro expression of a chloramphenicol acetyltransferase (CAT) reporter was detected in known α1BAR-expressing BC3H1, NB41A3, and DDT1MF-2 cells transiently transfected with a promoter-CAT construct. Conversely, minimal CAT expression was detected in known α1BAR-negative RAT-1 and R3T3 cells. These findings were extended by transfecting the same promoter-CAT construct into various primary cell types. In support of the hypothesis that α1ARs are differentially expressed in the smooth muscle of the vasculature, primary cultures of ...
Transcription initiation is an important step in the process of gene regulation in prokaryotes. Promoters are stretches of DNA sequence that are present in the upstream region of transcription start sites (TSSs), where RNA polymerase and other transcription factors bind to initiate transcription. Recent advancement in sequencing technologies has resulted in huge amount of raw data in the form of whole genome sequences. This sequence data has to be annotated, in order to identify coding, non-coding and regulatory regions. Computational tools are useful for a quick and fairly reliable annotation of many genome sequences. Promoter prediction is an important step in genome annotation process which is needed, not only for the validation of predicted genes, but also for the identification of novel genes, especially those coding for non-coding RNA, which are missed by gene prediction programs. DNA sequence dependent structural properties such as DNA duplex stability, bendability and intrinsic ...
Diversity in rates of gene expression is essential for basic cell functions and is controlled by a variety of intricate mechanisms. Revealing general mechanisms that control gene expression is important for understanding normal and pathological cell functions and for improving the design of expression systems. Here we analyzed the relationship between general features of genes and their contribution to expression levels. Genes were divided into four groups according to their core promoter type and their characteristics analyzed statistically. Surprisingly we found that small variations in the TATA box are linked to large differences in gene length. Genes containing canonical TATA are generally short whereas long genes are associated with either non-canonical TATA or TATA-less promoters. These differences in gene length are primarily determined by the size and number of introns. Generally, gene expression was found to be tightly correlated with the strength of the TATA-box. However significant reduction
Promoter Gene software free downloads. Promoter Gene shareware, freeware, demos: Gene Inspector by Textco BioSoftware Inc, Page Promoter by NetPromoter, Meta Tag Promoter by NetPromoter etc...
I am currently studying the regulation of several mammalian (mouse and human) promoters. Of obvious interest are known transcription factor binding sites. Since I wouldnt know an AP-1 site from an EcoRI site, I was hoping there might be a computer program on the net that would analyze a submitted sequence for consensus recognition sites. Does anyone out there have any advice? At this point, even a simple list of known consensus sites would be better than nothing. Thanks in advance, Benjamin S. Braun UCLA ...
RESULTS: A percentage of 36.17% controls and 38.6% patients were heterozygosis, considering Amplification-refractory mutation system (ARMS)-PCR assay while 23% and 22.85% were heterozygosis using Mismatch Amplification Mutation Assay (MAMA)-PCR. On the contrary, 1.3% and 1.4% were homozygosis A, while 75.7% and 75.75% presented homozygosis G, taking into account the MAMA-PCR results. The two assays were significantly different (P=0.0004 at χ2 Test), but MAMA-PCR showed a better performance for TNF-α -308 G/A gene polymorphism investigation ...
Potential binding sites of transcription factors are an example of biological event sequences where co-occurrence patterns and burstiness occur. We applied our techniques on 10 Mbp regions from human chromosomes 1-10 [11] (NCBI 36 assembly), where we identified potential binding sites as matches to known transcription factor binding motifs. The regions 30 - 40 Mbp were used for chromosomes 1-9, and 20 - 30 Mbp for chromosome 10, to avoid the centromere region. This dataset contains genome regions with different characteristics (e.g., C+G and gene densities), while being compact enough to be efficiently studied with several null models and window sizes. The motifs we consider are from the Jaspar collection [12] (Jaspar Core), all 138 motifs in the 2008 build. In these sequences we identified all matches for each Jaspar transcription factor (TF) matrix by the PoSSuMsearch program [13]. The threshold for a match was set with p ≤ 10-5, yielding approximately 30000 matches for each 10 Mbp sequence. ...
In article ,310FF66A.A0A at ulam.generes.ca,, kalch ,kalch at ulam.generes.ca, wrote: , Hi, , we are trying to map promoter elements from known DNA sequence. , However, we find that the DNA analysis programs we have in the lab , (MacVector and GeneWorks) are not suitable for this analysis. Could , someone please post (or respond to me directly) where I can send (or , download) a program that will search for promoter elements (e.g. , Sp1, ERE, AP1, AP2, etc)? , , Thanks, , , Michael MacVector does do this (Im not saying that it does it well, but it has the ability). I think that the newer versions ship with a transcription factor subsequence file that has over 600 sites bound by proteins in promoter regions. You just have to do a nucleic acid subsequence search. I ran a test against the junD promoter and it found all the sites that had been mapped by Shaul etal when they cloned it (except the TATA box. Im not sure why it didnt find that as one of the subsequences is exactly the same sequence ...
Generation of mutant mice. In the strategy of IMCT (Kobayashi et al., 1995), transgenic (Tg) mice are generated that express the human interleukin-2 receptor α-subunit (IL-2Rα) under the control of a cell type-specific promoter. These mice are then treated with a recombinant immunotoxin (IT), which is composed of the variable regions of the anti-IL-2Rα monoclonal antibody and a bacterial exotoxin fragment. The transgene construct contained a 10 kb DNA fragment encoding the 5′-flanking region of the mouse neuropsin (NP) gene (Hirata et al., 2001), the second intron of the rabbit β-globin gene (Kobayashi et al., 1992), the gene cassette encoding IL-2Rα fused to green fluorescent protein (IL-2Rα/GFP) (Watanabe et al., 1998), and the polyadenylation signals of the rabbit β-globin gene and simian virus 40 early gene (Kobayashi et al., 1992). The construct was microinjected into fertilized mouse eggs, which were then implanted into pseudopregnant females. Tg mice were identified by Southern ...
Promoters. Oriane Broustal BIO 535. Promoters. About promoters ( structure, function,…) Two types of human promoters based on CG content Bidirectional promoters in human genome. Questions:. What are the differences between eukaryotic and prokaryotic promoters? Slideshow 5386988 by ivana
Promoter elements play important roles in isoform and cell type-specific expression. We surveyed the epigenomic promoter landscape of gastric adenocarcinoma, analyzing 110 chromatin profiles (H3K4me3, H3K4me1, H3K27ac) of primary gastric cancers, gastric cancer lines, and nonmalignant gastric tissues. We identified nearly 2,000 promoter alterations (somatic promoters), many deregulated in various epithelial malignancies and mapping frequently to alternative promoters within the same gene, generating potential pro-oncogenic isoforms (RASA3). Somatic promoter-associated N-terminal peptides displaying relative depletion in tumors exhibited high-affinity MHC binding predictions and elicited potent T-cell responses in vitro, suggesting a mechanism for reducing tumor antigenicity. In multiple patient cohorts, gastric cancers with high somatic promoter usage also displayed reduced T-cell cytolytic marker expression. Somatic promoters are enriched in PRC2 occupancy, display sensitivity to EZH2 ...
Parts produced from this project: Bacterial-Mammalian/B-M (PyeaR-CArG) Hybrid Promoter -- Mammalian-Bacterial/M-B (CArG-PyeaR) Hybrid Promoter -- B-M + eCFP -- B-M + RFP -- M-B + eCFP -- M-B + RFP Our main project has resulted in the production of a hybrid bacterial and mammalian promoter optimised for induction by nitric oxide, nitrates and nitrites. We have ligated PyeaR, a known bacterial promoter and Part BBa_K216005 (Cambridge 2009) in the parts registry, with its mammalian counterpart, CArG. The resulting hybrid promoter has been synthesised in two orientations; PyeaR (bacterial, B) upstream of CArG (mammalian, M), nicknamed (B-M); and CArG upstream of PyeaR (M-B). These orientations were submitted to the parts registry as our first two biobricks. Each orientation of the promoter was ligated to enhanced cyan fluorescent protein (eCFP) and red fluorescent protein (RFP) to produce four new biobricks which have been submitted to the parts registry. These promoter + fluorescent protein ...
The hINV basal promoter, which encodes forty one nucleotides upstream of the transcription commence web site and no AP1 web sites, is not controlled by TAM67
Use this page to investigate EPDnew promoters for their chromatin status or motifs enrichment/distribution with our tools. If you want to restrict the analysis to a subset of promoters please use the promoter selection tool page. ...
The double truncation constructs, P3 and P4, are both lacking 449 nts immediately upstream the ATG translation start codon. The absence of this region does not seem to be critical for promoter functionality, since the activity was comparable to the full-length construct (Sps1) and other mutants (Sps2-Sps6) all of which include these initial 449 upstream nts at their 3 end. As concluded from P3, the removal of nts upstream 1167 from the 5 end did not reduce activity. Therefore a series of 5-truncations were made to narrow the critical promoter region. Up to construct Sps6 which includes nts 1-763 no significant reduction was observed whereas a dramatic loss near almost background levels was noticed for the shorter constructs Sps7 to Sps11. Since construct Sps5, containing 910 nts, exhibits full activity, this 5 end was considered critical and additional constructs were designed by successively truncating 3 nts (ds13-ds17). The four long constructs dS13 - dS16 showed fairly high promoter ...
I want to do a ChIP assay with an antibody against acetylated histone h3. I did a cDNA microarray before and now I want to check if the genes I found there are really regulated by histone acetylation. Thats why I want to do quantitative realtime PCR after the ChIP. The problem is (correct me if I am wrong) that I now have to design primer in the promoter region. And I have no idea how I can do this. Actually I do not even know how to search for the promoter region ...
PR is a classical estrogen-regulated gene (1, 2) and is frequently used as a surrogate marker for functional ERα activity. PR exists in 2 isoforms, PRA and PRB, which are the result of transcription from 2 alternative promoters, and initiation of translation at 2 different AUG codons (3). Structurally, PRB differs from PRA only in that the B receptor contains an additional 164 amino acids at the N-terminus of the protein (4). Despite structural similarities, PRA and PRB possess different functional activities. PRB has been found to be a stronger transcriptional activator than PRA, due in part to a third activation domain (AF-3) within the N-terminal 164 amino acids (5). On the other hand, PRA has been shown to act as a repressor which can inhibit other receptors, including ER and PRB (6). PRA and PRB regulate different sets of genes-of 94 progesterone-regulated genes, 65 were uniquely regulated by PRB, 4 uniquely by PRA, and only 25 by both (7). Moreover, the unliganded PR can regulate gene ...
Recent analysis of a Gal4 mutant (Gap71) carrying three point mutations (S22D, K23Q and K25F) in its DNA-binding domain (DBD), has demonstrated that it cannot occupy GAL promoters efficiently in cells and that it is not mono-ubiquitylated, suggesting a functional link between this modification and stable DNA binding in cells. The mechanistic underpinning of this phenotype is that this protein is hypersensitive to a newly discovered activity of the proteasomal ATPases--their ability to actively dissociate transcription factor-DNA complexes after direct interaction with the activation domain. In this paper, we examine the roles of each of the three point mutations contained in Gap71 individually. These experiments have revealed that serine 22 is a site of phosphorylation in the Gal4 DBD and that lysine 23 is essential for S22 phosphorylation, possibly acting as part of the kinase recognition site. Mutation of either residue blocks Gal4 DBD phosphorylation, its subsequent ubiquitylation and ...
As an anti-inflammatory mediator IL10 is beneficial in certain contexts and deleterious in others. As increased production of IL10 favours protection against inflammatory disease, whereas low production promotes elimination of foreign pathogens by the host, we investigated the possible influence of balancing selection at this locus. We began by resequencing 48 European and 48 African chromosomes across 2.2 kb of the IL10 promoter region, and compared this with four neighbouring gene regions: MK2, IL19, IL20 and IL24. Analysis of nucleotide diversity showed a positive Tajimas D-test for IL10 in Europeans, of borderline statistical significance (1.89, P=0.05). Analysis of F(st) values showed significant population divergence at MK2, IL19, IL20 and IL24 (P,0.01) but not at IL10. Taken together, these findings are consistent with the hypothesis that balancing selection has played a role in the evolution of polymorphisms in the IL10 promoter region.. ...
This interaction may be competed off with unlabelled oligo and supershifted making use of the YB one antibody. To further dissect YB one binding inside the 2a area we developed biotin labelled oligonucleotides during which the YB one responsive aspects had been mutated at 968, 940 or both web sites. Dropping either in the YREs resulted in significantly less YB 1 binding com pared with the wild form EGFR promoter sequence. These data verify that the 968 and 940 binding web sites are bona fide YREs. With each other these data demonstrate that YB 1 is ready to bind on the initial 1 kb with the EGFR promoter, and this leads to transactivation inside a phosphorylation dependent method. Offered on-line material 9 five R61 Figure five Y box binding protein 1 binds to distinct web pages inside the epidermal development aspect receptor promoter.. AVL292 Sequence of your EGFR2a oligonucleotide used in the gel shift assays. Highlighted sequences will be the possible YB 1 binding websites. The substitutions ...
Figure 1. Promoter activity varies across several human and rodent cell lines. Cells were plated at a density of 50,000 cells per well in a 24-well plate and transduced at MOI = 15 with SMARTvector Empty Vector Control Particles expressing TurboGFP. Promoter activity was assessed at 72 hours post-transduction by the fluorescence intensity of TurboGFP.. If you are uncertain of relative promoter activity in your cells, the Dharmacon SMARTchoice Promoter Selection Plate and the SMARTchoice Inducible Non-targeting Control 4-pack allow straightforward identification of the optimal promoter in your cells of interest.. ...
TY - JOUR. T1 - Patterns of gene promoter methylation in squamous cell cancer of the head and neck. AU - Hasegawa, Masayuki. AU - Nelson, Heather H.. AU - Peters, Edward. AU - Ringstrom, Elin. AU - Posner, Marshall. AU - Kelsey, Karl T.. N1 - Funding Information: Supported by: CA78609, ES08357, ES00002 and 1P01-DE12467-05.. PY - 2002/6/20. Y1 - 2002/6/20. N2 - Promoter methylation is an important pathway in transcriptional silencing of known and candidate tumor suppressor genes in Head and Neck Squamous Cell Carcinoma (HNSCC). In order to study the association of tumor suppressor gene promoter methylation in HNSCC with patient clinical characteristics, especially alcohol consumption and tobacco smoking, we examined promoter methylation of the p16INK4a, DAP-kinase, E-Cadherin, and RASSF1A genes using methylation-specific PCR (MSP) in 80 patients. The prevalence of p16INK4a, DAP-kinase, E-Cadherin, and RASSF1A promoter methylation was 26/80 (32.5%), 19/80 (23.8%), 29/80 (36.3%), 6/80 (7.5%) ...
TY - JOUR. T1 - ApoE -491A/T promoter polymorphism is not an independent risk factor, but associated with the ε4 allele in Hungarian Alzheimers dementia population. AU - Juhász, Anna. AU - Palotás, András. AU - Janka, Zoltán. AU - Rimanóczy, Ágnes. AU - Palotás, Miklós. AU - Bódi, Nikoletta. AU - Boda, Krisztina. AU - Zana, Marianna. AU - Vincze, Gábor. AU - Kálmán, János. PY - 2005/5/1. Y1 - 2005/5/1. N2 - Apolipoprotein E gene (Apoε) has three common alleles (ε2, ε3, and ε4), of which ε4 has been shown to be associated with an increased risk for Alzheimers disease (AD). Possible additional genetic factors, like the -491A variant of ApoE promoter may modify the development of AD, independently of the ApoE allele status. The objective of this study was to investigate whether A/T allelic polymorphism at site-491 of the ApoE promoter is associated with AD in a Hungarian population. The genomic DNA isolated from peripheral blood lymphocytes of 52 late-onset AD and 53 control ...
BTG3/ANA/APRO4 has been reported to be a tumor suppressor gene in some malignancies. It constitutes important negative regulatory mechanism for Src-mediated signaling, a negative regulator of the cell cycle and inhibits transcription factor E2F1. We report that BTG3 is downregulated in renal cancer and that the mechanism of inactivation is through promoter hypermethylation. Quantitative real-time polymerase chain reaction (PCR) showed that BTG3 was downregulated in cancer tissues and cells. Genistein and 5-aza-2-deoxycytidine (5Aza-C) induced BTG3 messenger RNA (mRNA) expression in A498, ACHN and HEK-293 renal cell carcinoma (RCC) cell lines. Bisulfite-modified PCR and DNA sequencing results showed complete methylation of BTG3 promoter in tumor samples and cancer cell lines. Genistein and 5Aza-C treatment significantly decreased promoter methylation, reactivating BTG3 expression. Chromatin immunoprecipitation assay revealed that genistein and 5Aza-C increased levels of acetylated histones 3, 4, ...
TY - JOUR. T1 - Assessment of gene promoter hypermethylation for detection of cervical neoplasia. AU - Wisman, G. Bea A.. AU - Nijhuis, Esther R.. AU - Hoque, Mohammad O.. AU - Reesink-Peters, Nathalie. AU - Koning, Alice J.. AU - Volders, Haukeline H.. AU - Buikema, Henk J.. AU - Boezen, H. Marike. AU - Hollema, Harry. AU - Schuuring, Ed. AU - Sidransky, David. AU - Van Der Zee, Ate G.J.. PY - 2006/10/15. Y1 - 2006/10/15. N2 - Current cervical cancer screening is based on morphological assessment of Pap smears and associated with significant false negative and false positive results. Previously, we have shown that detection of hypermethylated genes in cervical scrapings using quantitative methylation-specific PCR (QMSP) is a promising tool for identification of squamous cell cervical cancer. Aim of the present pilot-study was to evaluate presence of hypermethylated genes in cervical carcinogenesis, both in squamous cell as well as adenocarcinomas. Cervical scrapings were obtained from 30 ...
TY - JOUR. T1 - Exendin-4 as a stimulator of rat insulin I gene promoter activity via bZIP/CRE interactions sensitive to serine/threonine protein kinase inhibitor Ro 31-8220. AU - Chepurny, Oleg G.. AU - Hussain, Mehboob A.. AU - Holz, George G.. PY - 2002. Y1 - 2002. N2 - Signal transduction properties of exendin-4 (Ex-4) underlying its ability to stimulate rat insulin I gene promoter (RIP1) activity were assessed in the pancreatic β-cell line INS-1. Ex-4 acted via glucagon-like peptide-1 receptors to stimulate RIP1 in a glucose-dependent manner, as measured in cells transfected with a -410-bp RIP1-luciferase construct (RIP1-Luc). The action of Ex-4 was independent of cAMP and PKA because it was not blocked by cotransfection with dominant-negative Gαs, was unaffected by pretreatment with the membrane-permeant cAMP antagonist 8-Br-Rp-cAMPS, and remained apparent after treatment with PKA inhibitors H-89 or KT 5720. Similarly, cotransfection with a dominant-negative isoform of the type-2 ...
The vaccinia virus early transcription factor (VETF), in addition to the viral RNA polymerase, is required for efficient transcription of early genes in vitro. VETF is a heterodimeric protein that binds specifically to early gene promoters. In order to localize the VETF DNA binding domain, we have used photoreactive oligonucleotide probes with the sequence of the vaccinia virus growth factor promoter. The probes consisted of double-stranded oligonucleotides incorporating radiolabeled dAMP and 5-bromo-dUMP into sequences of the promoter known to contact VETF. Irradiation of a DNA probe having these nucleotides located upstream of the transcription start site in the presence of VETF resulted in the transfer of label to a polypeptide that comigrated with the small subunit of VETF. The label transfer reaction was shown to occur with the recombinant VETF small subunit in the absence of the large subunit. These results indicate that the small subunit comprises at least part of the VETF DNA binding ...
The physiological role of TFIIA was investigated by analyzing transcription in a yeast strain that contains a TATA-binding protein (TBP) mutant (N2-1) defective for interacting with TFIIA. In cells containing N2-1, transcription from a set of artificial his3 promoters dependent on different activators is generally reduced by a similar extent, indicating that TFIIA function is largely nonselective for activators. In addition, TATA element utilization, a core promoter function, is altered at his3 promoters dependent on weak activators. Genomic expression analysis reveals that 3% of the genes are preferentially affected by a factor of 4 or more. Chimeras of affected promoters indicate that the sensitivity to the TFIIA-TBP interaction can map either to the upstream or core promoter region. Unlike wild-type TBP or TFIIA, the N2-1 derivative does not activate transcription when artificially recruited to the promoter via a heterologous DNA binding domain, indicating that TFIIA is important for transcription
Differential usage of several transcription start sites in the human GnRH receptor gene was evident in human brain and pituitary. To locate the promoter responsible for a cluster of the 3′ CAP sites from -635 to -578 (relative to ATG) found in the pituitary, a proximal promoter element was identified at -677/-558 by 5′ and 3′ deletion mutant analysis. The promoter element drove a 13.1 ± 0.6-fold increase in reporter gene activity in an orientation-dependent manner in the mouse gonadotrope-derived αT3-1 cells. Within the core promoter element, two functional AT-rich Inr motifs, interacting with the same protein factor with different affinities, were identified. By Southwestern blot analysis and competitive gel mobility shift assays, multiple nuclear factors (36-150 kDa) were found to interact specifically with the core promoter element. Interestingly, these nuclear proteins also interacted with a previously identified distal promoter of the human GnRH receptor gene. Taken together, our ...
Lab Reagents Human IgG antibody Laboratories manufactures the single or dual luciferase assay reagents distributed by Genprice. The Single Or Dual Luciferase Assay reagent is RUO (Research Use Only) to test human serum or cell culture lab samples. To purchase these products, for the MSDS, Data Sheet, protocol, storage conditions/temperature or for the concentration, please contact luciferase assay. Other Single products are available in stock. Specificity: Single Category: Or Group: Dual Luciferase. Dual Luciferase information ...
title: RANTES gene promoter polymorphisms are associated with bronchial hyperresponsiveness in Korean children with asthma, doi: 10.1007/s00408-007-9049-3, category: Article
Succinoglycan (EPS I), the main acidic exopolysaccharide of Sinorhizobium meliloti, is required for the initiation and elongation of infection threads during nodulation of the host plant alfalfa. The gene products of the exoYFQ operon are involved in the first step of succinoglycan biosynthesis as well as in the polymerisation of subunits to the high-molecular-mass form of this exopolysaccharide. One promoter region that directs transcription of exoX and two promoter regions that drive transcription of exoY were mapped in the exoX-exoY intergenic region. The distal exoY promoter region containing three putative -10 promoter elements was active under standard growth conditions and was subject to ExoR-dependent regulation. Although this promoter region was stimulated in a phoB mutant, no PHO box-like sequences were found, suggesting an indirect regulatory effect of PhoB. The proximal promoter contains a PHO box-like sequence in the putative - 35 region and was affected by low and high phosphate ...
TY - JOUR. T1 - Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch. AU - Liu, Nan. AU - Hargreaves, Victoria V.. AU - Zhu, Qian. AU - Kurland, Jesse V.. AU - Hong, Jiyoung. AU - Kim, Woojin. AU - Sher, Falak. AU - Macias-Trevino, Claudio. AU - Rogers, Julia M.. AU - Kurita, Ryo. AU - Nakamura, Yukio. AU - Yuan, Guo Cheng. AU - Bauer, Daniel E.. AU - Xu, Jian. AU - Bulyk, Martha L.. AU - Orkin, Stuart H.. N1 - Funding Information: We thank Peter Skene and Steven Henikoff for advice on protocols for CUT&RUN, Birgit Knoechel for assistance with DNA sequencing, and Paul Bruno for improvements in protein purification. We appreciate the generosity of Merlin Crossley for sharing findings during the evolution of these studies. Fluorescence polarization experiments were performed at the Institute for Chemistry and Cell Biology-Longwood Screening Facility at HMS. Octet experiments were performed at the Center for Macromolecular Interactions in the Department of Biological ...
cytosol, nucleus, centromeric DNA binding, chromatin binding, DNA replication origin binding, RNA polymerase II core promoter proximal region sequence-specific DNA binding, sequence-specific DNA binding, transcriptional activator activity, RNA polymerase II core promoter proximal region sequence-specific binding, transcriptional repressor activity, RNA polymerase II core promoter proximal region sequence-specific binding, anatomical structure morphogenesis
A factor, present in transcriptionally active extracts prepared from purified vaccinia virus particles, binds to vaccinia early promoter sequences. The specificity of binding was demonstrated by electrophoretic mobility shift assays using the 5-terminal segments of two early genes and related and unrelated competitor DNA fragments. DNase I footprint analysis indicated that the factor formed a complex with promoter regions of both genes and protected sequences of 10-15 nucleotides centered 21-24 nucleotides upstream of the RNA start sites. The lack of protection of a late regulatory sequence and of an early promoter with transcriptionally inactivating single-nucleotide substitutions suggested that the protein is an early transcription factor. When subjected to glycerol gradient centrifugation, the DNA-binding factor was resolved from RNA polymerase and sedimented as a 7.5S species with an estimated molecular weight of 130,000. ...
Tumor‑specific promoter hypermethylation of large tumor suppressor, homolog 2 (LATS2), a tumor suppressor gene, has been investigated using methylation‑specific polymerase chain reaction (MSP) assays in different types of human cancer producing conflicting results. The aim of the present study was to evaluate the methylation status of the LATS2 promoter region using bisulfite sequencing with a next generation sequencer for breast cancer. In the 11 patients enrolled in the present study, the LATS2 promoter methylation index (MI) was uniformly high in tumor and normal tissues of the breast (median, 84.0 and 87.4%, respectively). The presence of LATS2 promoter hypermethylation was confirmed in isolated tumor cells and normal epithelial cells using the magnetic‑activated cell sorting method. In situ hybridization for LATS2 messenger RNA (mRNA) revealed that the mRNA expression of LATS2 was higher in normal epithelial cells, compared with tumor cells, however, it was not significantly ...
Transgenic techniques offer a valuable tool for determining gene functions. Although various promoters are available for use in gene overexpression, gene knockdown, and identification of transgenic individuals, there is nevertheless a lack of versatile promoters for such studies, and this dearth acts as a bottleneck, especially with regard to nonmodel organisms. Here, we succeeded in identifying a novel strong and ubiquitous promoter/enhancer in the silkworm. We identified a unique silkworm strain whose reporter gene showed strong and ubiquitous expression during the establishment of enhancer trap strains. In this strain, the transposon was inserted into the 5′UTR of hsp90, a housekeeping gene that is abundantly expressed in a range of tissues. To determine whether the promoter/enhancer of hsp90 could be used to induce strong gene expression, a 2.9-kb upstream genomic fragment of hsp90 was isolated (hsp90P2.9k), and its transcriptional activation activity was examined. Strikingly, hsp90P2.9k ...
Dual luciferase assay - proper controls? - posted in Cell Biology: Hey everyone, I am trying to perform a dual luciferase reporter assay in a cell line with promoters established from another cell line. cDNA and protein of the gene are present in both cell lines. Problem: In the new cell line, the luciferase activity for my promoters of interest is below pGL3, whereas TKpGL3 is as highly active as in the other cell line. Renilla activity was equal in all probes. So I am looking for appr...
In plant genetic engineering, the identification of gene promoters leading to particular expression patterns is crucial for the development of new genetically modified plant generations. This research was conducted in order to isolate and characterize several new promoters from cassava (Manihot esculenta Crantz) elongation factor 1 alpha (EF1A) gene family. Three promoters MeEF1A3, MeEF1A4 and MeEF1A5 were successfully isolated. Sequence analyses showed that all of the promoters contain three conserved putative cis-acting elements which are located upstream of the transcription start site. These elements are included a TEF1, a TELO and TATA boxes. In addition, all of the promoters also have the 5′UTR intron but with a different lengths. These promoters were constructed translationally with gusAreporter gene (promoter::gusA fusion) in pBI-121 binary vector to build a new binary vector using Overlap Extension PCR Cloning (OEPC) technique. Transient expression assay that was done by using ...
The tRNA(m5U54)methyltransferase, whose structural gene is designated trmA, catalyzes the formation of 5-methyluridine in position 54 of all tRNA species in Escherichia coli. The synthesis of this enzyme has previously been shown to be both growth rate dependent and stringently regulated, suggesting regulatory features similar to those of rRNA. We have determined the complete nucleotide sequence of the trmA operon in E. coli and the sequence of the trmA promoter region in Salmonella typhimurium and also analyzed the transcriptional regulation of the gene. The trmA and the btuB (encoding the vitamin B12 outer membrane receptor protein) promoters are divergent promoters separated by 102 bp between the transcriptional start sites. The trmA promoters of both E. coli and S. typhimurium share promoter elements with the rRNA P1 promoter. The sequence downstream from the -10 region of the trmA promoter is homologous to the discriminatory region found in stringently regulated promoters. Next to and ...
Background:Tumour-released DNA in blood represents a promising biomarker for cancer detection. Although epigenetic alterations such as aberrant promoter methylation represent an appealing perspective, the discordance existing between frequencies of alterations found in DNA extracted from tumour tissue and cell-free DNA (cfDNA) has challenged their practical clinical application. With the aim to explain this bias of agreement, we investigated whether protocadherin 10 (PCDH10) promoter methylation in tissue was associated with methylation pattern in matched cfDNA isolated from plasma of patients with colorectal cancer (CRC), and whether the strength of concordance may depend on levels of cfDNA, integrity index, as well as on different clinical-pathological features.Methods:A quantitative methylation-specific PCR was used to analyse a selected CpG site in the PCDH10 promoter of 67 tumour tissues, paired normal mucosae, and matched plasma samples. The cfDNA integrity index and cfDNA concentration ...
OBJECTIVE: Inactivating mutations in glucokinase (GCK) cause mild fasting hyperglycemia. Identification of a GCK mutation has implications for treatment and prognosis; therefore, it is important to identify these individuals. A significant number of patients have a phenotype suggesting a defect in glucokinase but no abnormality of GCK. We hypothesized that the GCK beta-cell promoter region, which currently is not routinely screened, could contain pathogenic mutations; therefore, we sequenced this region in 60 such probands. RESEARCH DESIGN AND METHODS: The beta-cell GCK promoter was sequenced in patient DNA. The effect of the identified novel mutation on GCK promoter activity was assessed using a luciferase reporter gene expression system. Electrophoretic mobility shift assays (EMSAs) were used to determine the impact of the mutation on Sp1 binding. RESULTS: A novel -71G|C mutation was identified in a nonconserved region of the human promoter sequence in six apparently unrelated probands. Family testing
Incubation of 3T3-L1 preadipocytes with isobutylmethylxanthine (IBMX), dexamethasone, and insulin, alone or in combination, demonstrated that IBMX, which increased cAMP-response element-binding protein (CREB) phosphorylation, was the predominant regulator of Pde3b expression. Real time PCR and immunoblotting indicated that in 3T3-L1 preadipocytes, IBMX-stimulated induction of Pde3b mRNA and protein was markedly inhibited by dominant-negative CREB proteins. By transfecting preadipocytes, differentiating preadipocytes, and HEK293A cells with luciferase reporter vectors containing different fragments of the 5- flanking region of the Pde3b gene, we identified a distal promoter that contained canonical cis-acting cAMP-response elements (CRE) and a proximal, GC-rich promoter region, which contained atypical CRE. Mutation of the CRE sequences dramatically reduced distal promoter activity; H89 inhibited IBMX-stimulated CREB phosphorylation and proximal and distal promoter activities. Distal promoter ...
Conventional wisdom holds that, owing to the dominance of features such as chromatin level control, the expression of a gene cannot be readily predicted from knowledge of promoter architecture. This is reflected, for example, in a weak or absent correlation between promoter divergence and expression divergence between paralogs. However, an inability to predict may reflect an inability to accurately measure or employment of the wrong parameters. Here we address this issue through integration of two exceptional resources: ENCODE data on transcription factor binding and the FANTOM5 high-resolution expression atlas. Consistent with the notion that in eukaryotes most transcription factors are activating, the number of transcription factors binding a promoter is a strong predictor of expression breadth. In addition, evolutionarily young duplicates have fewer transcription factor binders and narrower expression. Nonetheless, we find several binders and cooperative sets that are disproportionately associated
Background: Conventional wisdom holds that, owing to the dominance of features such as chromatin level control, the expression of a gene cannot be readily predicted from knowledge of promoter architecture. This is reflected, for example, in a weak or absent correlation between promoter divergence and expression divergence between paralogs. However, an inability to predict may reflect an inability to accurately measure or employment of the wrong parameters. Here we address this issue through integration of two exceptional resources: ENCODE data on transcription factor binding and the FANTOM5 high-resolution expression atlas. Results: Consistent with the notion that in eukaryotes most transcription factors are activating, the number of transcription factors binding a promoter is a strong predictor of expression breadth. In addition, evolutionarily young duplicates have fewer transcription factor binders and narrower expression. Nonetheless, we find several binders and cooperative sets that are ...
The family of repeats (FR) is a major upstream enhancer of the Epstein-Barr virus (EBV) latent C promoter (Cp) that controls transcription of six different latent nuclear proteins following interaction with the EBV nuclear protein EBNA1. Here, it was shown that Cp could also be activated by octamer-binding factor (Oct) proteins. Physical binding to the FR by the cellular transcription factors Oct-1 and Oct-2 was demonstrated by using an electrophoretic mobility-shift assay. Furthermore, Oct-1 in combination with co-regulator Bob.1, or Oct-2 alone, could drive transcription of a heterologous thymidine kinase promoter linked to the FR in both B cells and epithelial cells. Cp controlled by the FR was also activated by binding of Oct-2 to the FR. This may have direct implications for B cell-specific regulation of Cp.
In this large cohort, we found TERT promoter mutations to be common, particularly in FTC and BRAF mutation-positive PTC, and associated with aggressive clinicopathological characteristics.
The upstream regions of the human CYP11A and bovine CYP11B genes [have] a distal promoter in each gene. The distal promoters are located at −1.8 to −1.5 kb in the upstream region of the CYP11A gene and −1.5 to −1.1 kb in the upstream region of the CYP11B gene.[11] Using cloned chicken βA-globin genes, either individually or within the natural chromosomal locus, enhancer-dependent transcription is achieved in vitro at a distance of 2 kb with developmentally staged erythroid extracts. This occurs by promoter derepression and is critically dependent upon DNA topology. In the presence of the enhancer, genes must exist in a supercoiled conformation to be actively transcribed, whereas relaxed or linear templates are inactive. Distal protein-protein interactions in vitro may be favored on supercoiled DNA because of topological constraints.[12] Distal promoter regions may be a relatively small number of nucleotides, fairly close to the TSS such as (-253 to -54)[13] or several regions of ...
The Pem gene encodes an atypical homeodomain protein, distantly related to Prd/Pax family members, that we demonstrate is regulated in a complex transcriptional and post-transcriptional manner. We show that the rat Pem genomic structure includes three 5-untranslated (5-UT) exons and four coding exons, three of which encode the homeodomain. Several alternatively spliced transcripts were identified, including one that skips an internal coding exon, enabling this mRNA to express a novel form of the Pem protein. Other alternatively spliced mRNAs were characterized that possess different 5-UT regions, including a muscle-specific transcript. The different 5-UT termini present in Pem transcripts conferred different levels of translatability in vitro. Two promoters containing multiple transcription initiation sites were identified: a distal promoter (Pd) in the first 5-UT exon and a proximal promoter (Pp) located in the intron upstream of the first coding exon. The Pd was active in placenta, ovary, tumor
Results In vivo, the Saa3-promoter reporter activity was strongly upregulated at 1 and 2 days after the first and second SCW challenge. The Saa3-promoter activities during acute inflammation correlated with Tc uptake measurements but were more sensitive and able to respond to the ongoing synovitis in the chronic phase of SCW arthritis. Molecular stratification defined two inflammatory SF subtypes, unrelated to disease classification. Relative Saa3-promoter responses to interleukin 1β, tumour necrosis factor α and TLR4 agonist were significantly increased in OA/RA SF with a high compared to a low inflammatory profile subtype. Serum stimulation of the Saa3-promoter reporter cell-line could distinguish between healthy and RA patients.. ...
Naively, promoter strength is a function of 3 variables: holoenzyme affinity for promoter, equilibrium between closed and open complex and efficiency/frequency of promoter escape (this includes all events between opening of DNA helix and clearance of the promoter - in particular initiating synthesis of the first phosphodiester bond, idling/stuttering leading to the generation of nonproductive RNA oligos and finally clearance of the promoter). In the absence of activation/repression, the -40 to +15 region controls all 3 of these variables (-35,-10 define binding, -10-+4 define DNA opening, +1-+20 define promoter escape) - I think this is the absolute minimum we should use to define a promoter. More realistically, we should probably include back to -100 and forward to +20 as the vast majority of both repressors and activators act in this region (see a sampling of ~200 coli promoters below). ...
ERα and ERβ exert differential effects on the transcriptional activity of the human PAI-1 promoter in transient transfection studies using cultured endothelial cells. Our experiments demonstrate that ERα increases the promoter activity of PAI-1 promoter fragments in response to estrogen. ERα interacts with at least 1 ERE located at position −422 in the proximal PAI-1 promoter to elicit this estrogen-dependent activation of the PAI-1 promoter. In contrast, ERβ fails to induce, and in fact may suppress, the activity of PAI-1 promoter constructs tested in BAECs through an estrogen-independent mechanism. Furthermore, ERβ exerts a dominant-negative effect on ERα to blunt the estrogen-dependent activation of the PAI-1 promoter in BAECs. Based on these findings, we speculate that the balance of ERα and ERβ in vascular tissue modulates vascular PAI-1 production via estrogen-dependent and estrogen-independent mechanisms.. The observations reported here are consistent with recent studies that ...
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 ...
The His-1 gene is developmentally expressed in the murine choroid plexus but is silenced in the adult brain. To test the hypothesis that the gene contains cis-acting elements that contribute to this repression, we have analyzed segments of the proximal promoter for negative regulatory sequences by transient transfection analysis. The activity of the proximal promoter was moderately influenced by positively and negatively acting sequences located from 2335 to 2168 and 2617 to 2335, respectively. A strong His-1-positive regulatory element (HPRE, 118 to 129) was essential for maximal promoter activity and could also enhance the activity of the heterologous SV40 promoter in an orientation-dependent manner. The HPRE contains homology to the neuronal restrictive silencer element (NRSE) but interacted with nuclear proteins that were distinct from the NRSE-binding factor (NRSF). By contrast, a potent negative regulatory sequence (HNRE) was identified in the first exon that repressed either the His-1 or ...
Oncogenic activation of ras results in changes in the transcription of several genes leading to uncontrolled cell growth. In this paper, we demonstrate that transformation of fibroblast cells by the ras oncogene leads to transcriptional repression of the smooth muscle α-actin promoter. Transient transfection analysis of plasmids containing the 5′ upstream region of the human α-actin gene fused to human growth hormone or bacterial chloramphenicol acetyltransferase coding sequences into Rat-2 and ras-transformed Rat-2 (HO6) cells indicates that α-actin promoter is repressed in ras-transformed cells. In addition, stable rat fibroblast cell lines expressing human growth hormone or β-galactosidase under the control of α-actin promoter exhibit repressed reporter gene activity following transformation by the ras oncogene. α-Actin promoter-driven β-galactosidase activity is derepressed in revertants of ras-transformed stable cell lines. This revertant cell line expresses elevated levels of ras ...
Promoter Methylation Status of E-Cadherin, hMLH1, and p16 Genes in Nonneoplastic Gastric Epithelia: Silencing of tumor suppressor and tumor-related genes by hyp
One of the most intriguing aspects of Airn‐dependent gene silencing is the ability of Airn transcription to silence neighbouring genes in cis, although its own promoter is unaffected. In these studies, a mouse PGK promoter is able to silence Igf2r in differentiated ES cells, without being itself affected. Currently, two models have been suggested to explain the silencing activity of the Airn ncRNA. The RNA‐directed targeting model is based on parallels to X chromosome inactivation and proposes a function for the Airn ncRNA itself. This model proposes that the Airn ncRNA coats the silenced region and recruits effector proteins that induce widespread repressive epigenetic modifications (Pauler et al, 2007). Intuitively, this model implies a special ability of the Airn promoter to resist silencing as the induced epigenetic changes on the paternal allele silence Igf2r, Slc22a2 and Slc22a3, but not the Airn promoter. Our findings argue against this model, as the PGK promoter in the APD‐PGK ...
RNA polymerase II core promoter proximal region sequence-specific DNA binding, transcriptional activator activity, RNA polymerase II core promoter proximal region sequence-specific binding
Purpose. Damage to the corneal epithelium results in the massive secretion of fibronectin (FN) shortly after injury and induces the expression of its integrin receptor α5β1. The authors reported previously that FN induces α5 expression in human corneal epithelial cells and rabbit corneal epithelial cells by altering the binding of the transcription factor (TF) Sp1 to a regulatory element from the α5 promoter that it is also flanked by binding sites for the TFs NFI and AP-1. Here, they assessed the function of NFI and AP-1 on α5 gene expression and evaluated the contribution of FN to their overall regulatory influence. Methods. TF binding to the α5 promoter was evaluated in vitro by electrophoretic mobility shift assays and in vivo by ligation-mediated PCR or chromatin immunoprecipitation. TFs expression was monitored by Western blot, whereas their influence was assessed by transfection and RNAi analyses. Results. Coexpression of Sp1, NFI, and AP-1 was demonstrated in all cell types, and ...
TY - JOUR. T1 - Enhanced expression and HIV- 1 inhition of chimeric tRNALys3-Ribozymes under dual U6 snRNA and tRNA promoters. AU - Chang, Zongli. AU - Westaway, Shawn. AU - Li, Shirley. AU - Zaia, John A.. AU - Rossi, John J.. AU - Scherer, Lisa J.. PY - 2002/10/1. Y1 - 2002/10/1. N2 - We previously demonstrated that chimeric tRNALys3-ribozymes targeting the primer binding site of HIV produced virions with reduced infectivity. To further enhance the anti-HIV efficiency of these ribozymes by increasing their level of transcription, we designed several tRNALys3 promoter variants and compared their expression levels from the internal tRNALys3 promoters and also from an exogenous human U6 snRNA promoter. The dual U6/tRNA promoter constructs gave rise to much higher levels of expression than constructs that used only an internal tRNA promoter. The most abundant expression is produced when a U6 promoter drives a chimeric tRNALys3-ribozyme containing a mutation in the tRNA B box. As detected by ...
Bidirectional promoters are short (,1 kbp) intergenic regions of DNA between the 5 ends of the genes in a bidirectional gene pair.[14] A bidirectional gene pair refers to two adjacent genes coded on opposite strands, with their 5 ends oriented toward one another.[15] The two genes are often functionally related, and modification of their shared promoter region allows them to be co-regulated and thus co-expressed.[16] Bidirectional promoters are a common feature of mammalian genomes.[17] About 11% of human genes are bidirectionally paired.[14]. Bidirectionally paired genes in the Gene Ontology database shared at least one database-assigned functional category with their partners 47% of the time.[18] Microarray analysis has shown bidirectionally paired genes to be co-expressed to a higher degree than random genes or neighboring unidirectional genes.[14] Although co-expression does not necessarily indicate co-regulation, methylation of bidirectional promoter regions has been shown to ...
Cavalcanti, Alexandre B.; Berwanger, Otavio; Suzumura, Erica A.; Amato, Marcelo B. P.; Tallo, Fernando S.; Rezende, Ederlon A. C.; Telles, Jose M. M.; Romano, Edson; Guimaraes, Helio P.; Regenga, Marisa M.; Takahashi, Luzia N.; Teixeira, Cassiano; Oliveira, Roselaine P.; Carvalho, Vitor O.; Diaz-Quijano, Fredi A.; Carvalho, Carlos R. R.; Kodama, Alessandra A.; Ribeiro, Gisele F. M.; Abreu, Matheus O.; Oliveira, Ivonaldo M.; Guyatt, Gordon; Ferguson, Niall; Walter, Stephen; Vasconcelos, Marcia O. M.; Segundo, Valerio J.; Ferraz, Iris L.; Silva, Rosicley S.; Oliveira Filho, Wilson de; Silva, Nelson B.; Heirel, Debora C. B.; Takatani, Rodrigo R.; Sousa Neto, Jefferson A.; Neto, Jeronimo C. B.; Almeida, Samara D.; Chamy, Gauco; Goncalves Neto, Graciliano J. L.; Dias, Alysson P.; Silva, Rozangela R.; Tavares, Roberta C.; Souza, Marcia L. V. D.; Decio, Janaina C.; Lima, Cyntia M. L. S.; Ferreira Neto, Fleury; Oliveira, Katia R.; Dias, Polyana P. L. C.; Brandao, Andre L. S. B.; Ramos, Joroastro E.; ...
Title: Regulation of MAO-A and MAO-B Gene Expression. VOLUME: 11 ISSUE: 15. Author(s):J. C. Shih and K. Chen. Affiliation:Department of Cell andNeurobiology, Keck School of Medicine, University of SouthernCalifornia, 1985 Zonal Ave. Los Angeles, California, 90033, USA. Keywords:mao a, mao b, gene regulation, promoter, sp1, protein kinase c, mapkinase, egr-1. Abstract: MAO A and B genes are made of 15 exons with identical exon-intron organization. They are located on X-chromosome organized in opposite direction, tail to tail with 24kb apart. Both promoters are GC-rich and regulated by transcription factor Sp1. However, they have distinctly different features. MAO B gene, but not MAO A gene, has TATA box. MAO B promoter contains two clusters of overlapping Sp1 sites, the CACCC repressor element. Transcription factors Sp1 and Sp4 can activate MAO B promoter activity through the proximal cluster of Sp1 sites and its activation can be repressed by the over-expression of Sp3 and a related family ...
Hepatocytes (Heps) and sinusoidal endothelial cells (SECs) perform different roles in normal and pathological liver functions through the differential expression of fibronectin (FN) polypeptides. Nonetheless, the molecular basis underlying cell-type specific FN expression remains unknown. Using liver cell isolation techniques followed by short-term primary culture and transient transfection, here, we compare the transcriptional regulation of the FN promoter in Heps and SEC in conditions that closely resemble in vivo physiology. Transfection experiments allowed us to reveal cell-type specific regulatory elements operating through the proximal regions of the FN promoter. To investigate this further, we examined the occupation patterns of key elements of the FN promoter such as the -170 CRE and -150 CCAAT sites. Transcriptional activity of mutagenised promoter constructs confirmed that in Heps, these two sites behave as a composite element critical for normal promoter activity. In addition, ...
The PhoP binding pattern within the resA promoter is similar to other PhoP-regulated promoters(Liu, 1997; Liu and Hulett, 1997; 1998; Liu et al., 1998a, b), in that (i) both PhoP and PhoP∼P bind to these promoters; (ii) PhoP∼P extends the binding site further upstream and/or downstream, and has a higher binding affinity; and (iii) the core PhoP-binding region (region bound by PhoP and PhoP∼P) is located from about −22 to −60 in all these promoters(Liu and Hulett, 1998), including resA. A feature unique to the resA promoter among PhoP-regulated promoters is that it contains only two of the consensus PhoP binding repeats in the core binding region (Fig. 6), instead of at least four as seen in other Pho promoters studied. Ongoing expression and footprinting analyses of a site-specific mutagenized PhoP regulated promoter (S. Eder and F. M. Hulett, unpublished) indicate that PhoP binds to 6 bp repeated TT(A/T/C)ACA sequences separated by 4-5 bp, and that PhoP binding to one set of consensus ...
Read Cloning of a wheat puroindoline gene promoter by IPCR and analysis of promoter regions required for tissue-specific expression in transgenic riceseeds, Plant Molecular Biology on DeepDyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips.
Generation of the Mouse FasL Promoter Reporter Constructs and Motifs-Directed Mutagenesis. Mouse FasL promoter cloned in pGL3 vector was a gift from Dr. Timothy L. Ratiff (University of Iowa, Iowa City, IA). The cloning of FasL promoter and generation of luciferase reporter construct in pGL3 vector have been described previously (Crist et al., 2003).. To determine the role of NF-κB in regulation of FasL promoter in the presence of TCDD, we generated three FasL promoter constructs with or without NF-κB motifs. We designated these constructs FasL231, FasL191, and FasL324 based on the size of the respective PCR-amplified fragments. The primers used to generate various fragments of mouse FasL promoter are shown in Table 1. For directional cloning, all upstream primers included SacI restriction site and downstream primers included XhoI restriction site.. The amplified PCR fragments FasL231, FasL191, and FasL324 were first cloned into the polylinker sites of Topo PCR2.1 T/A vector and then sequenced ...
We present a system by which diverse transcriptional reporter elements, downstream of different signal transduction pathways, elicit cellular fluorescence in an agonist-dependent and agonist-selective fashion in live cells and embryos. By employing the constitutively active Rosa26 gene and analyzing its upstream promoter sequences, we made successive deletions with the intention of preserving the inherent openness of the locus and its constitutive activity, while diminishing core promoter function sufficiently to provide a low background of transcription in the absence of signal pathway activity. The −228 deletion endpoint satisfied these criteria, whereas the −60 deletion retained high constitutive activity. Fortunately, we discovered that insertion of all signaling elements studied here at −60 resulted in a marked diminution of basal activity. Not unexpectedly, the different −228 and −60 constructs provided slightly different results in terms of basal and induced activity with ...