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Transcriptional Start Site (transcriptional + start_site)
Selected AbstractsRequirement for ,B1-crystallin promoter of Xenopus laevis in embryonic lens development and lens regenerationDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2005Nobuhiko Mizuno Regulation of the lens-specific ,B1-crystallin promoter in Xenopus laevis was investigated using transgenic larvae and tadpoles. Comparison of the promoter sequence with that of chicken ,B1-crystallin gene indicates significant sequence similarity over a span of several hundred base pairs starting from the transcriptional start site. Remarkably, PL-1 and PL-2 sequences identified in the chicken promoter as essential binding sites of MAF, Pax6 and Prox1 transcription factors were conserved. Mutations of X (Xenopus) PL-1 and XPL-2 sequences eliminated the promoter activity, indicating a conserved mechanism regulating ,B1-crystallin promoter among vertebrate species. A stepwise deletion of the promoter sequence starting from 2800 bp indicated that the proximal 260 bp directly upstream of the transcription initiation site is sufficient for eliciting lens-specific expression, but the 150 bp promoter sequence is inactive despite it containing the XPL-1 and XPL-2 sequences, suggesting the presence of an additional and essential regulatory sequence located between ,150 and ,260 bp. Activity of the ,B1-crystallin promoter during lens regeneration from cornea was examined using transgenic tadpoles and found to have the same dependence on promoter regions as in embryonic lens development, indicating that gene regulation is largely shared by the two lens-generating processes. [source] A green light-absorbing phycoerythrin is present in the high-light-adapted marine cyanobacterium Prochlorococcus sp.ENVIRONMENTAL MICROBIOLOGY, Issue 10 2005Summary In the high-light-adapted unicellular marine cyanobacterium Prochlorococcus sp. MED4 the cpeB gene is the only gene coding for a structural phycobiliprotein. The absence of any other phycoerythrin gene in the fully sequenced genome of this organism, the previous inability to detect a gene product, and the mutation of two out of four cysteine residues, normally involved in binding chromophores, suggested that MED4- cpeB might not code for a functional protein. Here, transcription of MED4- cpeB at a low level was detected and the transcriptional start site was mapped. Enrichment of the protein identified phycoerythrobilin as its sole chromophore in vivo, which was confirmed by chromophorylation assays in vitro using the recombinant protein. Phycourobilin is the major chromophore in low-light-adapted Prochlorococcus ecotypes such as strain SS120. Therefore, spectrally tuned phycoerythrins are a characteristic feature of distinct Prochlorococcus ecotypes. Further in vitro mutagenesis experiments replacing one or both cysteines C61R/C82S by arginine or serine, respectively, revealed that only Cys82 is required for chromophore binding. Thus, an unusual green light-absorbing phycoerythrin evolved in the high-light-adapted ecotypes of Prochlorococcus, which potentially serves as a photoreceptor. [source] LIN54 is an essential core subunit of the DREAM/LINC complex that binds to the cdc2 promoter in a sequence-specific mannerFEBS JOURNAL, Issue 19 2009Fabienne Schmit Recently, the conserved human LINC/DREAM complex has been described as an important regulator of cell cycle genes. LINC consists of a core module that dynamically associates with E2F transcription factors, p130 and the B-MYB transcription factor in a cell cycle-dependent manner. In this study, we analyzed the evolutionary conserved LIN54 subunit of LINC. We found that LIN54 is required for cell cycle progression. Protein interaction studies demonstrated that a predicted helix,coil,helix motif is required for the interaction of LIN54 with p130 and B-MYB. In addition, we found that the cysteine-rich CXC domain of LIN54 is a novel DNA-binding domain that binds to the cdc2 promoter in a sequence-specific manner. We identified two binding sites for LIN54 in the cdc2 promoter, one of which overlaps with the cell cycle homology region at the transcriptional start site. Gel shift assays suggested that, in quiescent cells, the binding of LIN54 at the cell cycle homology region is stabilized by the binding of E2F4 to the adjacent cell cycle-dependent element. Our data demonstrate that LIN54 is an important and integral subunit of LINC. Structured digital abstract ,,MINT-7239362: LIN54 (uniprotkb:Q6MZP7) physically interacts (MI:0915) with p130 (uniprotkb:Q08999) by anti tag coimmunoprecipitation (MI:0007) ,,MINT-7239376: LIN54 (uniprotkb:Q6MZP7) physically interacts (MI:0915) with B-Myb (uniprotkb:P10244) by anti tag coimmunoprecipitation (MI:0007) [source] A human phospholamban promoter polymorphism in dilated cardiomyopathy alters transcriptional regulation by glucocorticoids,HUMAN MUTATION, Issue 5 2008Kobra Haghighi Abstract Depressed calcium handling by the sarcoplasmic reticulum (SR) Ca-ATPase and its regulator phospholamban (PLN) is a key characteristic of human and experimental heart failure. Accumulating evidence indicates that increases in the relative levels of PLN to Ca-ATPase in failing hearts and resulting inhibition of Ca sequestration during diastole, impairs contractility. Here, we identified a genetic variant in the PLN promoter region, which increases its expression and may serve as a genetic modifier in dilated cardiomyopathy (DCM). The variant AF177763.1:g.203A>C (at position ,36,bp relative to the PLN transcriptional start site) was found only in the heterozygous form in 1 out of 296 normal subjects and in 22 out of 381 cardiomyopathy patients (heart failure at age of 18,44 years, ejection fraction=22±9%). In vitro analysis, using luciferase as a reporter gene in rat neonatal cardiomyocytes, indicated that the PLN-variant increased activity by 24% compared to the wild type. Furthermore, the g.203A>C substitution altered the specific sequence of the steroid receptor for the glucocorticoid nuclear receptor (GR)/transcription factor in the PLN promoter, resulting in enhanced binding to the mutated DNA site. These findings suggest that the g.203A>C genetic variant in the human PLN promoter may contribute to depressed contractility and accelerate functional deterioration in heart failure. Hum Mutat 29(5), 640,647, 2008. © 2008 Wiley-Liss, Inc. [source] Acholeplasma laidlawii up-regulates granulysin gene expression via transcription factor activator protein-1 in a human monocytic cell line, THP-1IMMUNOLOGY, Issue 3 2001Yutaka Kida Summary An antimicrobial protein granulysin is constitutively expressed in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. However, little is known about the precise regulatory mechanisms underlying granulysin gene expression. In this study, we examined the regulatory mechanisms underlying granulysin gene expression using a human monocytic cell line, THP-1, treated with Acholeplasma laidlawii. The level of granulysin mRNA expression in THP-1 cells was significantly augmented in response to stimulation with A. laidlawii. The transfection of reporter gene constructs into THP-1 cells indicated that DNA sequences between residues ,329 and ,239, relative to the transcriptional start site of the granulysin gene, are responsible for mediating gene induction. In addition, mutagenesis of a putative activator protein-1 (AP-1)-binding site between residues ,277 and ,271 in the granulysin promoter resulted in the reduction of granulysin promoter activity. Electrophoretic mobility shift assays (EMSA) demonstrated that nuclear extract prepared from A. laidlawii- treated THP-1 cells can generate specific binding to DNA oligonucleotides encompassing the AP-1-binding site, whereas unstimulated nuclear extract from the cells failed to do so. Furthermore, competition and supershift assays confirmed that A. laidlawii can induce the activation of AP-1. These results indicate that AP-1 dominantly participates in the regulation of inducible granulysin gene expression in THP-1 cells. Therefore, the finding of inducible granulysin gene expression by A. laidlawii suggests that inducible granulysin in macrophages may function as a protective weapon when microbial invasion occurs. [source] The expression of recombinant genes in Escherichia coli can be strongly stimulated at the transcript production level by mutating the DNA-region corresponding to the 5,-untranslated part of mRNAMICROBIAL BIOTECHNOLOGY, Issue 3 2009Laila Berg Summary Secondary structures and the short Shine,Dalgarno sequence in the 5,-untranslated region of bacterial mRNAs (UTR) are known to affect gene expression at the level of translation. Here we report the use of random combinatorial DNA sequence libraries to study UTR function, applying the strong, ,32/,38 -dependent, and positively regulated Pm promoter as a model. All mutations in the libraries are located at least 8 bp downstream of the transcriptional start site. The libraries were screened using the ampicillin-resistance gene (bla) as reporter, allowing easy identification of UTR mutants that display high levels of expression (up to 20-fold increase relative to the wild-type at the protein level). Studies of the two UTR mutants identified by a modified screening procedure showed that their expression is stimulated to a similar extent at both the transcript and protein product levels. For one such mutant a model analysis of the transcription kinetics showed significant evidence of a difference in the transcription rate (about 18-fold higher than the wild type), while there was no evidence of a difference in transcript stability. The two UTR sequences also stimulated expression from a constitutive ,70 -dependent promoter (P1/Panti-tet), demonstrating that the UTR at the DNA or RNA level has a hitherto unrecognized role in transcription. [source] EthR, a repressor of the TetR/CamR family implicated in ethionamide resistance in mycobacteria, octamerizes cooperatively on its operatorMOLECULAR MICROBIOLOGY, Issue 1 2004Jean Engohang-Ndong Summary Ethionamide (ETH) is an important second-line antitubercular drug used for the treatment of patients infected with multidrug-resistant Mycobacterium tuberculosis. Although ETH is a structural analogue of isoniazid, only little cross-resistance to these two drugs is observed among clinical isolates. Both isoniazid and ETH are pro-drugs that need to be activated by mycobacterial enzymes to exert their antimicrobial activity. We have recently identified two M. tuberculosis genes, Rv3854c (ethA) and Rv3855 (ethR), involved in resistance to ETH. ethA encodes a protein that belongs to the Flavin-containing monooxygenase family catalysing the activation of ETH. We show here that ethR, which encodes a repressor belonging to the TetR/CamR family of transcriptional regulators, negatively regulates the expression of ethA. By the insertion of the ethA promoter region upstream of the lacZ reporter gene, overexpression of ethR in trans was found to cause a strong inhibition of ethA expression, independently of the presence of ETH in the culture media. Electrophoretic mobility shift assays indicated that EthR interacts directly with the ethA promoter region. This interaction was confirmed by DNA footprinting analysis, which, in addition, identified the EthR-binding region. Unlike other TetR/CamR members, which typically bind 15 bp operators, EthR recognises an unusually long 55 bp region suggesting multimerization of the repressor on its operator. Identification by primer-extension of the ethA transcriptional start site indicated that it is located within the EthR-binding region. Taken together, bacterial two-hybrid experiments and gel filtration assays suggested a dimerization of EthR in the absence of its operator. In contrast, surface plasmon resonance analyses showed that eight EthR molecules bind cooperatively to the 55 bp operator, which represents a novel repression mechanism for a TetR/CamR member. [source] Identification and Characterization of a DNase Hypersensitive Region of the Human Tyrosinase GenePIGMENT CELL & MELANOMA RESEARCH, Issue 6 2003James P. Fryer Mutations of the tyrosinase gene produce oculocutaneous albinism type 1 (OCA1). Most affected individuals are compound heterozygotes with different maternal and paternal mutations, but a substantial number of presumed tyrosinase alleles in these individuals have no identifiable mutation in the coding or proximal promoter region of the gene. This suggests that mutations in other regions of the gene, such as regulatory regions that are removed from the direct proximity of the coding sequence, may account for these currently unidentifiable mutations. The mouse tyrosinase gene has a distal enhancer or locus control region (LCR) that provides position-independent stimulation of gene expression, and a homologous regulatory region (HR) of the human gene could be the site of some of these mutations. We report a region 9 kb upstream of the human tyrosinase transcriptional start site that may be involved in regulation of this gene. Analysis of this region shows DNase I hypersensitivity in a cell lineage-specific pattern, a pattern indicative of regulatory regions of a gene. This region also has significant enhancer function when reporter vectors containing it are transfected into either human or mouse melanocyte cell lines, and elimination of specific sequences with homology to the mouse core enhancer in this region extinguishes the enhancer function. We believe that this region of homology contains sequences critical in the regulation of the human tyrosinase gene and is a candidate for the location of OCA1 mutations. [source] | ||||||||||