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Regulatory Regions (regulatory + regions)
Selected AbstractsFunctional analysis of synaptotagmin gene regulatory regions in two distantly related ascidian speciesDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 7 2008Jun Matsumoto We have studied the structure and function of a promoter region of the Halocynthia synaptotagmin (Hr-Syt) gene, which is abundantly expressed in neuronal cells. Our previous analysis suggested that the expression of Hr-Syt is regulated by at least one epidermal and two neuronal regulatory regions. In this study, the regulatory regions of Hr-Syt promoter were further characterized by using two species of ascidians, Halocynthia roretzi and Ciona intestinalis embryos. A putative GATA transcription factor binding site in the epidermal regulatory region has ectodermal enhancer activity in the Halocynthia embryo. Neuronal expression of Hr-Syt was regulated by multiple redundant enhancer regions. Among these enhancer regions, a 200-bp (,2900/,2700) region drove the reporter expression in neurons in both species of ascidian. Although the synaptotagmin promoter sequences did not show overall similarity between Hr-Syt and Ciona synaptotagmin (Ci-Syt), 5,-upsteream two short sequences of Ci-Syt have similarity to the ,2766/,2732 region of the Hr-Syt promoter. The homeodomain binding sites in this region are required for the neuronal enhancer activity. These results suggest that GATA and homeodomain transcription factors regulate the expression of synaptotagmin. [source] Endothelium-specific Cre recombinase activity in flk-1-Cre transgenic miceDEVELOPMENTAL DYNAMICS, Issue 2 2004Alexander H. Licht Abstract The use of the Cre-loxP recombination system allows the conditional inactivation of genes in mice. The availability of transgenic mice in which the Cre recombinase expression is highly cell type specific is a prerequisite to successfully use this system. We previously have characterized regulatory regions of the mouse flk-1 gene sufficient for endothelial cell-specific expression of the LacZ reporter gene in transgenic mice. These regions were fused to the Cre recombinase gene, and transgenic mouse lines were generated. In the resulting flk-1-Cre transgenic mice, specificity of Cre activity was determined by cross-breeding with the reporter mouse lines Rosa26R or CAG-CAT-LacZ. We examined double-transgenic mice at different stages of embryonic development (E9.5,E16.5) and organs of adult animals by LacZ staining. Strong endothelium-specific staining of most vascular beds was observed in embryos older than E11.5 in one or E13.5 in a second line. In addition, the neovasculature of experimental BFS-1 tumors expressed the transgene. These lines will be valuable for the conditional inactivation of floxed target genes in endothelial cells of the embryonic vascular system. Developmental Dynamics 229:312,318, 2004. © 2004 Wiley-Liss, Inc. [source] Analysis of regulatory elements of E-cadherin with reporter gene constructs in transgenic mouse embryosDEVELOPMENTAL DYNAMICS, Issue 2 2003Marc P. Stemmler Abstract Proper regulation of E-cadherin,mediated cell adhesion is important during early embryonic development and in organogenesis. In mice, E-cadherin is expressed from the fertilized egg onward and becomes down-regulated during gastrulation in mesoderm and its derivatives, but its expression is maintained in all epithelia. E-cadherin promoter analyses led to the identification of binding sites for two transcriptional repressors, Snail and SIP1, which are able to mediate down-regulation in vitro, but little is known about the regulatory elements that govern E-cadherin transcriptional activity in vivo. Here, we compared the developmentally regulated expression of a series of lacZ -reporter transgenes fused to different sequences of the murine E-cadherin gene between ,6 kb, including the promoter, and +16 kb, covering one third of intron 2. Four different segments with distinct regulatory properties were identified. The promoter fragment from +0.1 to ,1.5 kb remains inactive in most cases but occasionally induces ectopic expression in mesodermal tissues, although it contains binding sites for the repressors Snail and SIP1. This promoter fragment also lacks positive elements needed for the activation of transcription in ectoderm and endoderm. Sequences from ,1.5 to ,6 kb harbor regulatory elements for brain-specific expression and, in addition, insulator or silencer elements, because they are consistently inactive in the mesoderm. Only if sequences from +0.1 to +11 kb are combined with the promoter fragments is E-cadherin,specific transgene expression observed in endoderm and certain epithelia. Sequences between +11 and +16 kb contain cis -active elements that generally enhance transcription. Our analyses show that E-cadherin expression is governed by a complex interplay of multiple regulatory regions dispersed throughout large parts of the locus. Developmental Dynamics 227:238,245, 2003. © 2003 Wiley-Liss, Inc. [source] Further genetic evidence implicates the vasopressin system in childhood-onset mood disordersEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2009Emma L. Dempster Abstract Studies in both animals and humans advocate a role for the vasopressin (AVP) system in the aetiology of depressive symptoms. Attention has particularly focused on the role of AVP in the overactivation of the hypothalamic-pituitary-adrenal (HPA)-axis in mood disorders. Elevated AVP plasma levels have been found in mood disorder patients, which are often positively correlated with the severity of symptoms. We recently reported an association between childhood-onset mood disorders (COMD) and polymorphisms in the receptor responsible for the AVP-mediated activation of the HPA-axis (AVPR1B). As genetic variation in the vasopressinergic system could provide a mechanism to explain the endocrine alterations observed in mood disorders, we investigated other genes in this system. The gene encoding AVP is the strongest candidate, particularly as genetic variation in this gene in rodents is associated with anxiety-related behaviours. Six single-nucleotide polymorphisms (SNPs) were genotyped across the AVP gene in a sample comprised of 586 Hungarian nuclear families ascertained through affected probands with a diagnosis of COMD. In addition, AVP coding and putative regulatory regions were screened for mutations using denaturing high-performance liquid chromatography. One SNP, 3, to the AVP, gene reached significance (P = 0.03), as did the overtransmission of a five-marker haplotype with a frequency of 22% (P = 0.0001). The subsequent mutation screen failed to identify any putative functional polymorphisms. The outcome of this study, combined with our previous association between COMD and AVPR1B, implicates genetic variation in vasopressinergic genes in mediating vulnerability to COMD. [source] RESURRECTING THE ROLE OF TRANSCRIPTION FACTOR CHANGE IN DEVELOPMENTAL EVOLUTIONEVOLUTION, Issue 9 2008Vincent J. Lynch A long-standing question in evolutionary and developmental biology concerns the relative contribution of cis- regulatory and protein changes to developmental evolution. Central to this argument is which mutations generate evolutionarily relevant phenotypic variation? A review of the growing body of evolutionary and developmental literature supports the notion that many developmentally relevant differences occur in the cis -regulatory regions of protein-coding genes, generally to the exclusion of changes in the protein-coding region of genes. However, accumulating experimental evidence demonstrates that many of the arguments against a role for proteins in the evolution of gene regulation, and the developmental evolution in general, are no longer supported and there is an increasing number of cases in which transcription factor protein changes have been demonstrated in evolution. Here, we review the evidence that cis- regulatory evolution is an important driver of phenotypic evolution and provide examples of protein-mediated developmental evolution. Finally, we present an argument that the evolution of proteins may play a more substantial, but thus far underestimated, role in developmental evolution. [source] THE LOCUS OF EVOLUTION: EVO DEVO AND THE GENETICS OF ADAPTATIONEVOLUTION, Issue 5 2007Hopi E. Hoekstra An important tenet of evolutionary developmental biology ("evo devo") is that adaptive mutations affecting morphology are more likely to occur in the cis -regulatory regions than in the protein-coding regions of genes. This argument rests on two claims: (1) the modular nature of cis -regulatory elements largely frees them from deleterious pleiotropic effects, and (2) a growing body of empirical evidence appears to support the predominant role of gene regulatory change in adaptation, especially morphological adaptation. Here we discuss and critique these assertions. We first show that there is no theoretical or empirical basis for the evo devo contention that adaptations involving morphology evolve by genetic mechanisms different from those involving physiology and other traits. In addition, some forms of protein evolution can avoid the negative consequences of pleiotropy, most notably via gene duplication. In light of evo devo claims, we then examine the substantial data on the genetic basis of adaptation from both genome-wide surveys and single-locus studies. Genomic studies lend little support to the cis -regulatory theory: many of these have detected adaptation in protein-coding regions, including transcription factors, whereas few have examined regulatory regions. Turning to single-locus studies, we note that the most widely cited examples of adaptive cis -regulatory mutations focus on trait loss rather than gain, and none have yet pinpointed an evolved regulatory site. In contrast, there are many studies that have both identified structural mutations and functionally verified their contribution to adaptation and speciation. Neither the theoretical arguments nor the data from nature, then, support the claim for a predominance of cis -regulatory mutations in evolution. Although this claim may be true, it is at best premature. Adaptation and speciation probably proceed through a combination of cis -regulatory and structural mutations, with a substantial contribution of the latter. [source] Identification and characterization of a novel progesterone receptor-binding element in the mouse prostaglandin E receptor subtype EP2 geneGENES TO CELLS, Issue 9 2003Sohken Tsuchiya Background:, Gene expression of prostaglandin E receptor EP2 is induced in the luminal epithelium of the mouse uterus during peri-implantation period (day-5 of pseudopregnancy), suggesting the involvement of progesterone and its receptor (PR) in this expression. However it remains unclear whether PR affects EP2 gene expression through its binding. Results:, We investigated transcriptional regulation of EP2 gene expression with reporter gene analysis using HeLa cells with or without expression of the PR. The 5,-flanking region (,3260 to ,27, upstream of the translation initiation site) exhibited progesterone-induced promoter activation and basal promoter activity in the presence of PR. Using successive deletion analysis, we determined the six regulatory regions in the EP2 gene. Three regions were found to be involved in progesterone-induced promoter activation, whereas the other three regions were involved in basal promoter activity in the presence of PR. We identified a novel PR-binding sequence, 5,-G(G/A)CCGGA-3,, in the two basal promoter regions and Sp1- and Sp3-binding in the other basal promoter region. Conclusions:, We identified a novel PR-binding sequence, which may be involved in the regulation of basal promoter activity in the EP2 gene. [source] Hypomethylation of PRAME is responsible for its aberrant overexpression in human malignanciesGENES, CHROMOSOMES AND CANCER, Issue 9 2007Tino Schenk The preferentially expressed antigen of melanoma (PRAME) is expressed at high levels in large fractions of human malignancies, e.g., acute myeloid leukemia. Therefore, PRAME is an important marker for diagnosis of various malignant diseases and a relevant parameter for monitoring minimal residual disease. It is supposed to be involved in tumorigenic processes. Because of these important aspects we investigated its transcriptional regulation in detail. Most relevant was a detailed DNA methylation analysis of the PRAME 5, region by genomic sequencing in correlation with PRAME expression in various human patient samples and cell lines. In combination with DNA-truncation/transfection experiments with respect to DNA methylation, we show that changes in the methylation pattern in defined parts of the regulatory regions of PRAME are sufficient for its upregulation in cells usually not expressing the gene. © 2007 Wiley-Liss, Inc. [source] Nodal-related gene Xnr5 is amplified in the Xenopus genomeGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 7 2006Shuji Takahashi Abstract In Xenopus, six nodal-related genes (Xnrs) have been identified to date. We found numerous tandem duplications of Xnr5 in the Xenopus laevis and Xenopus tropicalis genomes that involve highly conserved copies of coding and regulatory regions. The duplicated versions of Xnr5 were expressed in both the superficial and deep layer of dorsal endoderm and in the deep layer of ventral endoderm, where the initial inducers of mesendoderm formation would be expected to be localized. Overexpression of secreted inhibitors of Xnrs led to a substantially enhanced transcription of the duplicated Xnr5 genes and Xnr6 in embryos. Therefore, Xnr5 and Xnr6 have a novel feedback loop to inhibit transcription of Xnr5 and Xnr6. These results suggest that the initialization of a strong Xnr5 and Xnr6 signal is enabled by the rapid transcription from multiple genes. The novel feedback loop may negatively regulate transcription of Xnr5s and Xnr6 to limit overproduction of these potent inducers, with the Xnr5/Xnr6 signal then activating positive (Xnrs) and negative (Xlefty) loops, which regulate the range of mesodermal tissues produced. genesis 44:309,321, 2006. [source] Genetic variability in the mitochondrial serine protease HTRA2 contributes to risk for Parkinson disease,HUMAN MUTATION, Issue 6 2008Veerle Bogaerts Abstract In one genetic study, the high temperature requirement A2 (HTRA2) mitochondrial protein has been associated with increased risk for sporadic Parkinson disease (PD). One missense mutation, p.Gly399Ser, in its C-terminal PDZ domain (from the initial letters of the postsynaptic density 95, PSD-95; discs large; and zonula occludens-1, ZO-1 proteins [Kennedy, 1995]) resulted in defective protease activation, and induced mitochondrial dysfunction when overexpressed in stably transfected cells. Here we examined the contribution of genetic variability in HTRA2 to PD risk in an extended series of 266 Belgian PD patients and 273 control individuals. Mutation analysis identified a novel p.Arg404Trp mutation within the PDZ domain predicted to freeze HTRA2 in an inactive form. Moreover, we identified six patient-specific variants in 5, and 3, regulatory regions that might affect HTRA2 expression as supported by data of luciferase reporter gene analyses. Our study confirms a role of the HTRA2 mitochondrial protein in PD susceptibility through mutations in its functional PDZ domain. In addition, it extends the HTRA2 mutation spectrum to functional variants possibly affecting transcriptional activity. The latter underpins a previously unrecognized role for altered HTRA2 expression as a risk factor relevant to parkinsonian neurodegeneration. Hum Mutat 29(6), 832,840, 2008. © 2008 Wiley-Liss, Inc. [source] Functional polymorphisms in dopamine and serotonin pathway genes,HUMAN MUTATION, Issue 1 2006Ursula M. D'Souza Abstract There is mounting evidence on the functional significance of single nucleotide and simple repeat sequence polymorphisms in both the coding and regulatory regions of genes in the monoamine neurotransmitter pathways. Many of these gene variants have been associated with human behavioral disorders and traits, and thus have important clinical relevance. This review summarizes the literature on the published functional studies from a molecular, cellular, and neurobiological perspective, and notes their possible behavioral consequences. Functional studies have adopted a variety of strategies. Pharmacological studies have focused on the effects of gene variation at the protein level in terms of binding to ligands or drugs. Other key investigations have determined effects on gene expression at the level of transcription in mammalian cell cultures, lymphoblasts, and/or human postmortem brain tissue. This has enabled the comparison of in vitro and in vivo data, and furthermore provides an improved perceptive of their respective advantages. Additionally, molecular biological approaches have identified transcription factors (DNA-binding proteins) that interact with the motifs within the polymorphisms themselves. Various neuroimaging studies have further determined the relationship of genotype with protein availability in the brain, and thus have contributed to our understanding of the in vivo functional significance of gene variants. Finally, there is growing evidence from both human and animal studies on the interaction of functional polymorphisms with the environment in determining a behavioral outcome. Taken together, these findings have contributed to a greater understanding of the plausible molecular mechanisms that underpin the functional significance of polymorphisms in monoamine neurotransmitter pathway genes, and how they may influence behavioral phenotypes. Hum Mutat 27(1), 1,13, 2006. © 2005 Wiley-Liss, Inc. [source] Defining a role for Sonic hedgehog pathway activation in desmoplastic medulloblastoma by identifying GLI1 target genesINTERNATIONAL JOURNAL OF CANCER, Issue 1 2009Joon Won Yoon Abstract A subgroup of medulloblastomas shows constitutive activation of the Sonic hedgehog pathway with expression of GLI1. We identified the subset of GLI1 transforming target genes specifically expressed in medulloblastomas by comparing GLI1 targets in RK3E cells transformed by GLI1 with the gene expression profile of Sonic hedgehog signature medulloblastomas. We identified 1,823 genes whose expression was altered more than 2-fold in 2 independent RK3E + GLI1 cell lines. We identified 25 whose expression was altered similarly in medulloblastomas expressing GLI1. We identified potential GLI binding elements in the regulatory regions of 10 of these genes, confirmed that GLI1 binds the regulatory regions and activates transcription of select genes, and showed that GLI1 directly represses transcription of Krox-20. We identified upregulation of CXCR4, a chemokine receptor that plays roles in the proliferation and migration of granule cell neuron precursors during development, supporting the concept that reinitiation of developmental programs may contribute to medulloblastoma tumorigenesis. In addition, the targets suggest a pathway through which GLI1 may ultimately affect medulloblastoma cell proliferation, survival and genomic stability by converging on p53, SGK1, MGMT and NTRK2. We identify a p53 mutation in RK3E + GLI1 cells, suggesting that p53 mutations may sometimes shift the balance toward dysregulated tumor cell survival. © 2008 Wiley-Liss, Inc. [source] Regulation of Dlx gene expression in the zebrafish pharyngeal arches: from conserved enhancer sequences to conserved activityJOURNAL OF APPLIED ICHTHYOLOGY, Issue 2 2010R. B. MacDonald Summary The Dlx genes play an important role in the development of the pharyngeal arches and the structures derived from these tissues, including the craniofacial skeleton. They are typically expressed in a nested pattern along the proximo-distal axis of the branchial arches in mice. This pattern is known as the "Dlx code" and has been proposed to be responsible for an early regional patterning of branchial arches in mammals. A number of cis - regulatory elements (CREs) have been identified within the Dlx loci, which target reporter expression to the developing pharyngeal arches of the mouse. Most of these CREs are located in the intergenic regions between the two genes constituting a Dlx bigene cluster. Therefore, the regionalized dlx expression in the branchial arches could be the result of the shared activities of these regulatory regions. Here we analyze previously published and new results showing these CREs are highly conserved in both their sequence and their activity in the pharyngeal arches of two distantly related vertebrates, the zebrafish and the mouse. A better understanding of Dlx gene regulation of the Dlx genes and of the genetic cascades in which they are involved can lead to clues explaining variations in morphology of the craniofacial regions of vertebrates. [source] CREB-dependent Nur77 induction following depolarization in PC12 cells and neurons is modulated by MEF2 transcription factorsJOURNAL OF NEUROCHEMISTRY, Issue 4 2010Brian Yee Hong Lam J. Neurochem. (2010) 112, 1065,1073. Abstract Expression of the nuclear orphan receptor gene Nur77 in neuronal cells is induced by activity-dependent increases in intracellular Ca2+ ions. Ca2+ responsiveness of the Nur77 gene has been attributed to two distinct DNA regulatory regions that recruit the transcription factors cAMP response element binding protein (CREB) and myocyte enhancer factor-2 (MEF2). Here we used dominant interfering and constitutively active mutants of CREB and MEF2 proteins to assess their relative contribution to depolarization-induced Nur77 expression in undifferentiated PC12 cells and hippocampal neurons. We show that while CREB is necessary for Ca2+ -activated Nur77 expression MEF2 functions to modulate CREB-dependent Nur77 expression by acting as a repressor in quiescent cells. [source] Transcriptional regulation of human excitatory amino acid transporter 1 (EAAT1): cloning of the EAAT1 promoter and characterization of its basal and inducible activity in human astrocytesJOURNAL OF NEUROCHEMISTRY, Issue 6 2003Seon-Young Kim Abstract Excitatory amino acid transporter 1 (EAAT1) is one of the two glial glutamate transporters that clear the extracellular glutamate generated during neuronal signal transmission. Here, we cloned and characterized a 2.1-kb promoter region of human EAAT1 and investigated its function in the transcriptional regulation of the EAAT1 gene in human primary astrocytes. The full-length promoter region lacked TATA and CCAAT boxes and an initiator element, it contained several potential transcription factor-binding sites and it exhibited promoter activity in primary astrocytes and in several types of transformed cells. Consecutive 5,-deletion analysis of the EAAT1 promoter indicated the presence of negative and positive regulatory regions and a putative core promoter between ,57 bp and +20 bp relative to the transcription start site (TSS). The core promoter contained a single GC-box in position ,52/,39 and one E-box near the TSS and the GC-box site that was responsible for 90% of the basal promoter activity as determined by mutational analysis. Electrophoretic mobility shift, supershift and competition assays demonstrated binding of stimulating proteins (Sp) 1 and 3 to the GC-box and upstream stimulating factor (USF) 1 to the E-box. Treatment of primary human astrocytes with cellular modulators 8-bromo cyclic AMP and epidermal growth factor increased EAAT1 promoter activity in transient transfection assays and increased cellular EAAT1 mRNA expression and glutamate uptake by astrocytes. Conversely, tumor necrosis factor-, reduced both EAAT promoter activity and cellular EAAT1 mRNA expression. These results enable studies of transcriptional regulation of EAAT1 gene at the promoter level. [source] Mutational analysis of HOXD13 and HOXA13 genes in the triphalangeal thumb,brachyectrodactyly syndromeJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2002A. Pérez-Cabrera Abstract The triphalangeal thumb-brachyectrodactyly syndrome is a very rare autosomal dominant disorder of unknown etiology characterized by an unusual pattern of limb malformations: triphalangeal thumbs and brachyectrodactyly in the hands, and ectrodactyly in the feet. In a previous report, we described the clinical and radiographical features of three related subjects with the disease and suggest that due to the unusual combination of limb defects and to its phenotypic similarity with the limb malformative pattern induced by disrupting the Hoxd13 gene in mouse, the triphalangeal thumb-brachyectrodactyly syndrome might be caused by mutations in a HOX gene. After sequencing the entire coding region of HOXD13 and the highly conserved homeodomain encoding region of HOXA13, we do not detect any deleterious mutation in any of the patients excluding that alterations at these sequences are responsible for the disease. Mutations in regulatory regions of these genes or in other genes involved in limb development might be responsible for the disease. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] CtsR controls class III heat shock gene expression in the human pathogen Listeria monocytogenesMOLECULAR MICROBIOLOGY, Issue 4 2000Shamila Nair Stress proteins play an important role in virulence, yet little is known about the regulation of stress response in pathogens. In the facultative intracellular pathogen Listeria monocytogenes, the Clp ATPases, including ClpC, ClpP and ClpE, are required for stress survival and intracellular growth. The first gene of the clpC operon of L. monocytogenes encodes a homologue of the Bacillus subtilis CtsR repressor of stress response genes. An L. monocytogenes ctsR -deleted mutant displayed enhanced survival under stress conditions (growth in the presence of 2% NaCl or at 42°C), but its level of virulence in the mouse was not affected. The virulence of a wild-type strain constitutively expressing CtsR is significantly attenuated, presumably because of repression of the stress response. Regulation of the L. monocytogenes clpC, clpP and clpE genes was investigated using transcriptional fusions in B. subtilis as a host. The L. monocytogenes ctsR gene was placed under the control of an inducible promoter, and regulation by CtsR and heat shock was demonstrated in vivo in B. subtilis. The purified CtsR protein of L. monocytogenes binds specifically to the clpC, clpP and clpE regulatory regions, and the extent of the CtsR binding sites was defined by DNase I footprinting. Our results demonstrate that this human pathogen possesses a CtsR regulon controlling class III heat shock genes, strikingly similar to that of the saprophyte B. subtilis. This is the first description of a stress response regulatory gene in a pathogen. [source] Characterisation of aurone biosynthesis in Antirrhinum majusPHYSIOLOGIA PLANTARUM, Issue 4 2006Kevin M. Davies Aurones are bright yellow flavonoids produced in petals of a limited range of plant species, including Antirrhinum majus. The biosynthesis of aurones is thought to occur by the action of aureusidin synthase (AUS), and possibly aureusidin 7- O -glucosyltransferase (A7GT). The temporal and spatial occurrence of AUS and A7GT transcript was examined in wild-type A. majus and two mutant lines; sulfurea, which has increased aurone production in petals, and violacea, which has reduced aurone production. AUS and A7GT transcript abundance was similar in all three lines, increasing during flower development coincident with yellow coloration. The spatial pattern of AUS occurrence was also similar in all three lines, being spatially restricted to the inner epidermis of the face and throat of the lower petal. A new recessive line (CFR1011) with greatly reduced aurone production in all parts of the petal was identified by ethylmethanesulfonate mutagenesis of the homozygous recessive sulfurea line. Transcript abundance for AUS was not changed in the CFR1011 line compared with the wild-type line, and neither were any point mutations detected in the coding sequences for AUS or A7GT. Thus, the sulfurea, violacea and CFR1011 mutations do not seem to control aurone production through a change in transcript abundance of the predicted biosynthetic genes AUS or A7GT. To examine AUS gene regulation further, the putative AUS gene promoter region was isolated and compared with other A. majus flavonoid gene promoters. A number of conserved potential regulatory regions were identified, in particular a consensus site for the MYB-type transcription factors. [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] Right atrial stretch alters fore- and hind-brain expression of c-fos and inhibits the rapid onset of salt appetiteTHE JOURNAL OF PHYSIOLOGY, Issue 15 2008Juliana Irani Fratucci De Gobbi The inflation of an intravascular balloon positioned at the superior vena cava and right atrial junction (SVC-RAJ) reduces sodium or water intake induced by various experimental procedures (e.g. sodium depletion; hypovolaemia). In the present study we investigated if the stretch induced by a balloon at this site inhibits a rapid onset salt appetite, and if this procedure modifies the pattern of immunohistochemical labelling for Fos protein (Fos-ir) in the brain. Male Sprague,Dawley rats with SVC-RAJ balloons received a combined treatment of furosemide (Furo; 10 mg (kg bw),1) plus a low dose of the angiotensin-converting enzyme inhibitor captopril (Cap; 5 mg (kg bw),1). Balloon inflation greatly decreased the intake of 0.3 m NaCl for as long as the balloon was inflated. Balloon inflation over a 3 h period following Furo,Cap treatment decreased Fos-ir in the organum vasculosum of the lamina terminalis and the subfornical organ and increased Fos-ir in the lateral parabrachial nucleus and caudal ventrolateral medulla. The effect of balloon inflation was specific for sodium intake because it did not affect the drinking of diluted sweetened condensed milk. Balloon inflation and deflation also did not acutely change mean arterial pressure. These results suggest that activity in forebrain circumventricular organs and in hindbrain putative body fluid/cardiovascular regulatory regions is affected by loading low pressure mechanoreceptors at the SVC-RAJ, a manipulation that also attenuates salt appetite. [source] Sequence variation in ,-methylacyl-CoA racemase and risk of early-onset and familial prostate cancerTHE PROSTATE, Issue 14 2007Albert M. Levin Abstract BACKGROUND Expression of the ,-methylacyl-CoA racemase (AMACR) gene has been established as a sensitive and specific biomarker for the diagnosis of prostate cancer. An initial study has also suggested that the risk of familial (but not sporadic) prostate cancer may be associated with germline variation in the AMACR gene. METHODS In a study of brothers discordant for the diagnosis of prostate cancer (including 449 affected and 394 unaffected men) from 332 familial and early-onset prostate cancer families, we used conditional logistic regression and family-based association tests to investigate the association between prostate cancer and five single nucleotide polymorphisms (SNPs) tagging common haplotype variation within the coding and regulatory regions of AMACR. RESULTS The strongest evidence for prostate cancer association was for SNP rs3195676, with an estimated odds ratio of 0.58 (95% confidence interval,=,0.38,0.90; P,=,0.01 for a recessive model). This non-synonymous SNP (nsSNP) results in a methionine-to-valine substitution at codon 9 (M9V) in exon 2 of the AMACR gene. Three additional nsSNPs showed suggestive evidence for prostate cancer association (P,,,0.10). CONCLUSIONS Our results confirm an initial report of association between the AMACR gene and the risk of familial prostate cancer. These findings emphasize the value of studying early-onset and familial prostate cancer when attempting to identify genetic variation associated with prostate cancer. Prostate 67: 1507,1513, 2007. © 2007 Wiley-Liss, Inc. [source] Common promoter deletion is associated with 3.9-fold differential transcription of ovine CCR5 and reduced proviral level of ovine progressive pneumonia virusANIMAL GENETICS, Issue 5 2009S. N. White Summary Chemokine (C-C motif) Receptor 5 (CCR5) is a chemokine receptor that regulates immune cell recruitment in inflammation and serves as a coreceptor for human immunodeficiency virus (HIV). A human CCR5 coding deletion (termed delta-32) results in strong resistance to HIV infection, and sequence variants in CCR5 regulatory regions have been implicated in delayed progression to acquired immune deficiency syndrome. Both ovine progressive pneumonia virus (OPPV), also known as maedi-visna, and HIV are macrophage-tropic lentiviruses, have similar genomic structures, and cause lifelong persistent host infection, suggesting CCR5 may have a role in regulating OPPV provirus levels. Therefore, the ovine CCR5 genomic sequence was determined, and sequence variants were obtained from the open reading frame and surrounding regulatory sites. One CCR5 variant contained a 4-base deletion within a binding site for octamer transcription factors in the promoter region. A test for differential transcription from each allele in heterozygous animals showed a 3.9-fold transcription difference (P < 0.0001). OPPV proviral levels were also measured in 351 naturally exposed Rambouillet, Polypay and Columbia sheep. Deletion homozygotes showed reduced OPPV proviral levels among these animals (P < 0.01). The association of this CCR5 promoter deletion with OPPV levels will need to be validated in additional populations before the deletion can be recommended for widespread use in marker-assisted selection. However, because of the large impact on transcription and because CCR5 has roles in inflammation, recruitment of effector cells, and cell-mediated immunity, this deletion may play a role in the control of infections of many diverse pathogens of sheep. [source] Identifying Putative Promoter Regions of Hermansky-Pudlak Syndrome Genes by Means of Phylogenetic FootprintingANNALS OF HUMAN GENETICS, Issue 4 2009Horia Stanescu Summary HPS is an autosomal recessive disorder characterized by oculocutaneous albinism and prolonged bleeding. Eight human genes are described resulting in the HPS subtypes 1,8. Certain HPS proteins combine to form Biogenesis of Lysosome-related Organelles Complexes (BLOCs), thought to function in the formation of intracellular vesicles such as melanosomes, platelet dense bodies, and lytic granules. Specifically, BLOC-2 contains the HPS3, HPS5 and HPS6 proteins. We used phylogenetic footprinting to identify conserved regions in the upstream sequences of HPS3, HPS5 and HPS6. These conserved regions were verified to have in vitro transcription activation activity using luciferase reporter assays. Transcription factor binding site analyses of the regions identified 52 putative sites shared by all three genes. When analysis was limited to the conserved footprints, seven binding sites were found shared among all three genes: Pax-5, AIRE, CACD, ZF5, Zic1, E2F and Churchill. The HPS3 conserved upstream region was sequenced in four patients with decreased fibroblast HPS3 RNA levels and only one HPS3 mutation in the coding exons and surrounding exon/intron boundaries; no mutation was found. These findings illustrate the power of phylogenetic footprinting for identifying potential regulatory regions in non-coding sequences and define the first putative promoter elements for any HPS genes. [source] Overexpression of the growth arrest and DNA damage,induced 45, gene contributes to autoimmunity by promoting DNA demethylation in lupus T cellsARTHRITIS & RHEUMATISM, Issue 5 2010Yaping Li Objective Demethylation of CD11a and CD70 regulatory regions in CD4+ T cells contributes to the development of autoreactivity and overstimulation of autoantibodies. Because growth arrest and DNA damage,induced 45, (GADD45,) reduces epigenetic silencing of genes by removing methylation marks, this study examined whether the gadd45A gene could contribute to autoimmunity by promoting DNA demethylation in T cells from patients with systemic lupus erythematosus (SLE). Methods Levels of GADD45,, CD11a, and CD70 messenger RNA (mRNA) and protein were detected by real-time reverse transcription,polymerase chain reaction and Western blotting or flow cytometry. Global DNA methylation was evaluated using Methylamp global DNA methylation quantification kits. Detection of CD4+ T cell proliferation and autologous B cell IgG antibodies was performed using commercially available kits. CD11a and CD70 promoter methylation was determined with bisulfite sequencing. Results Elevated gadd45A mRNA expression and global DNA hypomethylation were observed in CD4+ T cells from SLE patients. The levels of gadd45A mRNA were inversely proportional to the levels of DNA methylation. Positive correlations were found between gadd45A and CD11a/CD70 mRNA levels. Expression of gadd45A mRNA was increased in CD4+ T cells following ultraviolet B irradiation, and this was accompanied by increased levels of CD11a and CD70 mRNA. Moreover, increased expression of gadd45A, CD11a, and CD70 mRNA was accompanied by increased autoreactivity and excessive B cell stimulation in gadd45A -transfected CD4+ T cells. CD11a promoter methylation was also significantly reduced in transfected cells. Transfection of gadd45A small interfering RNA inhibited the autoreactivity of SLE CD4+ T cells and led to significant increases in the methylation levels of the CD11a and CD70 promoter regions. Conclusion These findings indicate that gadd45A may contribute to lupus-like autoimmunity by promoting DNA demethylation in SLE CD4+ T cells. [source] Nonoverlapping Clusters: Approximate Distribution and Application to Molecular BiologyBIOMETRICS, Issue 2 2001Xiaoping Su Summary. An approach is developed for the screening of genomic sequence data to identify gene regulatory regions. This approach is based on deciding if putative transcription factor binding sites are clustered together to a greater extent than one would expect by chance. Given n events occurring on an interval of width L (L base pairs), an r:w cluster is defined as r+ 1 consecutive events all contained within a window of length wL. Accurate and easily computable approximations are derived for the distribution of the number of nonoverlapping r:w clusters under the model that the positions of the n events have a uniform distribution. Simulations demonstrate that these approximations have greater accuracy than existing methods. The approximation is applied to detect erythroid-specific regulatory regions in genomic DNA sequences, first in an artificial case where r is specified a priori and then as part of an exploratory approach. [source] The combinations of TNF,,308 and IL-6 ,174 or IL-10 ,1082 genes polymorphisms suggest an association with susceptibility to sporadic late-onset Alzheimer's diseaseACTA NEUROLOGICA SCANDINAVICA, Issue 6 2009P. Vural Objective,,, Single nucleotide polymorphisms in the regulatory regions of the cytokine genes for tumor necrosis factor , (TNF,), interleukin (IL)-6 and IL-10 have been suggested to influence the risk of Alzheimer's disease (AD) with conflicting results. Aim,,, To investigate the TNF,,308, IL-6 ,174 and IL-10 ,1082 gene polymorphisms as susceptibility factors for AD. Methods,,, We analyzed genotype and allele distributions of these polymorphisms in 101 sporadic AD patients and 138 healthy controls. Results,,, Heterozygotes (AG) or combined genotype (AG+AA) for IL-10 ,1082 were associated with approximately two-fold increase in the risk of AD. Carriers of A alleles of both TNF,,308 and IL-10 ,1082 had 6.5 times higher risk for AD in comparison with non-carriers. Concomitant presence of both mutant TNF,,308 A and IL-6 ,174 C alleles raised three-fold the AD risk, whereas there was no notable risk for AD afflicted by IL-6 ,174 polymorphism alone. Conclusion,,, Our results suggest that TNF, and IL-10 promoter polymorphism might be a risk factor for AD. The combined effects of TNF,,308, IL-6 ,174 and IL-10 ,1082 variant alleles may be more decisive to induce functional differences and modify the risk for AD. [source] 2161: Development of a next-generation sequencing platform for retinal dystrophies, with LCA and RP as proof of conceptACTA OPHTHALMOLOGICA, Issue 2010F COPPIETERS Purpose Retinal dystrophies represent an emerging group of hereditary disorders that lead to degeneration of the photoreceptors and/or the retinal pigment epithelium, resulting in irreversible blindness. They are genetically complex, with over 200 disease loci identified so far. Current genetic screening consists of microarray analysis (Asper Ophthalmics) for the most recurrent mutations, and subsequent Sanger sequencing. However, the high cost and low throughput of the latter technology limits testing to only the most recurrent genes. This project aims to develop a high throughput and cost-effective platform for screening of all known disease genes for Leber Congenital Amaurosis (LCA) and retinitis pigmentosa (RP), using the next-generation sequencing (NGS) technology. Methods A NGS panel will be developed for all 16 and 47 known LCA and RP genes, respectively, including coding and untranslated regions, regulatory regions and microRNA binding sites. The protocol will consist of the following steps: 1) high throughput primerdesign and qPCR, 2) ligation, 3) shearing and 4) sequencing on the Illumina Genome Analyser IIx (GAIIx). This innovative protocol overcomes the need for short amplicons in order to render short-read sequences by the GAIIx. This sequencing instrument was chosen because of its high capacity, low cost per base and the absence of interpretation problems at homopolymeric regions. Analysis of the variants will be performed using in-house developed and commercial software, which ranks all variants according to their pathogenic potential. Conclusion Using the proposed protocol, comprehensive screening for all known disease genes for LCA and RP will be available for the first time. [source] | ||||||||||||||||||||||||||||