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Regulatory Elements (regulatory + element)
Kinds of Regulatory Elements Terms modified by Regulatory Elements Selected AbstractsSp1 and Sp3 are involved in up-regulation of human deoxyribonuclease II transcription during differentiation of HL-60 cellsFEBS JOURNAL, Issue 8 2003San-Fang Chou Expression of DNase II in macrophages is potentially crucially important in the removal of unwanted DNA. We have previously shown that DNase II expression is up-regulated at the transcriptional level during the phorbol 12-myristate-13-acetate (PMA)-induced differentiation of HL-60 and THP-1 cells. In this study, we investigated the cis -regulatory elements and transcription factors involved in this process in HL-60 cells. cis -Regulatory elements in the DNase II promoter were located by 5, deletion and site-directed mutagenesis of promoter-luciferase constructs and transient transfection of HL-60 cells. Furthermore, the binding proteins were identified by electrophoretic mobility shift assay (EMSA) in the presence of specific antibodies. In the DNase II promoter, 249 base pairs upstream of the transcription start site were essential for maximal promoter activity in both untreated and PMA-treated HL-60 cells and, within this region, three Sp1 and Sp3 binding sites were identified as essential for transcriptional regulation and PMA induction. Western blot analysis showed that PMA treatment resulted in increased levels of Sp1 and Sp3 proteins. Furthermore, cotransfection analysis in Drosophila SL2 cells showed that Sp1 was more potent than Sp3 in activating the DNase II promoter. We therefore conclude that Sp1 and/or Sp3 are involved in the up-regulation of DNase II expression during the differentiation of HL-60 cells. [source] How to make a zone of polarizing activity: Insights into limb development via the abnormality preaxial polydactylyDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2007Robert E. Hill Early in vertebrate limb development, a program initiates that polarizes the limb along the antero-posterior axis. The mesenchyme at the posterior margin is ultimately responsible for the asymmetry due to a region called the zone of polarizing activity (ZPA). The ZPA produces and secretes the molecule SHH, which coordinates the patterning of the resulting digits. Preaxial polydactyly (PPD) is a commonly occurring limb abnormality; investigating the genetic basis of this defect has provided insights into our understanding of digit patterning. PPD disrupts limb asymmetry by producing an ectopic ZPA at the opposite margin of the limb bud. Mutations in the long-range, limb-specific regulatory element of the Shh gene are responsible for the defect. Genetic analysis of this limb abnormality provides an important approach in understanding the mechanisms that control digit patterning. [source] Hoxb3 vagal neural crest-specific enhancer element for controlling enteric nervous system developmentDEVELOPMENTAL DYNAMICS, Issue 2 2005Kwok Keung Chan Abstract The neural and glial cells of the intrinsic ganglia of the enteric nervous system (ENS) are derived from the hindbrain neural crest at the vagal level. The Hoxb3 gene is expressed in the vagal neural crest and in the enteric ganglia of the developing gut during embryogenesis. We have identified a cis -acting enhancer element b3IIIa in the Hoxb3 gene locus. In this study, by transgenic mice analysis, we examined the tissue specificity of the b3IIIa enhancer element using the lacZ reporter gene, with emphasis on the vagal neural crest cells and their derivatives in the developing gut. We found that the b3IIIa-lacZ transgene marks only the vagal region and not the trunk or sacral region. Using cellular markers, we showed that the b3IIIa-lacZ transgene was expressed in a subset of enteric neuroblasts during early development of the gut, and the expression was maintained in differentiated neurons of the myenteric plexus at later stages. The specificity of the b3IIIa enhancer in directing gene expression in the developing ENS was further supported by genetic analysis using the Dom mutant, a spontaneous mouse model of Hirschsprung's disease characterized by the absence of enteric ganglia in the distal gut. The colonization of lacZ -expressing cells in the large intestine was incomplete in all the Dom/b3IIIa-lacZ hybrid mutants we examined. To our knowledge, this is the only vagal neural crest-specific genetic regulatory element identified to date. This element could be used for a variety of genetic manipulations and in establishing transgenic mouse models for studying the development of the ENS. Developmental Dynamics 233:473,483, 2005. © 2005 Wiley-Liss, Inc. [source] Discordance between intramuscular triglyceride and insulin sensitivity in skeletal muscle of Zucker diabetic rats after treatment with fenofibrate and rosiglitazoneDIABETES OBESITY & METABOLISM, Issue 5 2007K. J. Nadeau Aim:, Intramyocellular triglyceride (IMTG) correlates with insulin resistance, but there is no clear causal relationship. Insulin resistance and associated hyperinsulinaemia may increase IMTG, via the insulin-regulated transcription factor, sterol regulatory element,binding protein 1 (SREBP-1). PPAR agonists may also affect IMTG via changes in insulin sensitivity, SREBP-1 or other factors. Methods:, We examined skeletal muscle IMTG and SREBP-1 expression, and metabolic parameters in Zucker diabetic fatty rats (ZDF) after 25 weeks of PPAR-, or PPAR-, administration. Results:, Compared with Zucker lean rats (ZL), untreated ZDF had significantly higher weights, serum glucose, insulin, free fatty acids, total cholesterol and triglycerides. IMTG and SREBP-1c messenger RNA (mRNA) were also higher in untreated ZDF; both were decreased by fenofibrate (FF). Rosiglitazone (Rosi), despite marked improvement in glycaemia, hyperinsulinaemia and hyperlipidaemia, failed to affect SREBP-1 expression, and increased body weight and IMTG. Rosi/FF combination caused less weight gain and no IMTG increase, despite metabolic effects similar to Rosi alone. Conclusions:, IMTG and SREBP-1c mRNA are high in the ZDF. FF and Rosi both improved insulin sensitivity but had opposite effects on IMTG. Thus, there was a clear discordance between insulin sensitivity and IMTG with PPAR agonists, indicating that IMTG and insulin sensitivity do not share a simple relationship. [source] Molecular cloning of the Matrix Gla Protein gene from Xenopus laevisFEBS JOURNAL, Issue 7 2002Functional analysis of the promoter identifies a calcium sensitive region required for basal activity To analyze the regulation of Matrix Gla Protein (MGP) gene expression in Xenopus laevis, we cloned the xMGP gene and its 5, region, determined their molecular organization, and characterized the transcriptional properties of the core promoter. The Xenopus MGP (xMGP) gene is organized into five exons, one more as its mammalian counterparts. The first two exons in the Xenopus gene encode the DNA sequence that corresponds to the first exon in mammals whereas the last three exons show homologous organization in the Xenopus MGP gene and in the mammalian orthologs. We characterized the transcriptional regulation of the xMGP gene in transient transfections using Xenopus A6 cells. In our assay system the identified promoter was shown to be transcriptionally active, resulting in a 12-fold induction of reporter gene expression. Deletional analysis of the 5, end of the xMGP promoter revealed a minimal activating element in the sequence from ,70 to ,36 bp. Synthetic reporter constructs containing three copies of the defined regulatory element delivered 400-fold superactivation, demonstrating its potential for the recruitment of transcriptional activators. In gel mobility shift assays we demonstrate binding of X. laevis nuclear factors to an extended regulatory element from ,180 to ,36, the specificity of the interaction was proven in competition experiments using different fragments of the xMGP promoter. By this approach the major site of factor binding was demonstrated to be included in the minimal activating promoter fragment from ,70 to ,36 bp. In addition, in transient transfection experiments we could show that this element mediates calcium dependent transcription and increasing concentrations of extracellular calcium lead to a significant dose dependent activation of reporter gene expression. [source] Phylogenetic reconstruction of Gram-positive organisms based on comparative sequence analysis of molecular chaperones from the ruminal microorganism Ruminococcus flavefaciens FD-1FEMS MICROBIOLOGY LETTERS, Issue 1 2003Dionysios A. Antonopoulos Abstract Primers designed on the basis of nucleotide sequences conserved in DnaK and GroEL from Gram-positive organisms were used to PCR amplify internal regions of the cognate genes from the anaerobic ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1. Genome walking was then utilized to elucidate the remainder of the sequences in addition to upstream and downstream regions. The full sequence of the gene encoding the GroES protein (groES) was found directly upstream from groEL. The deduced amino acid sequence of the groEL gene showed the highest homology with the amino acid sequence of the Clostridium thermocellum GroEL protein (72% amino acid identity). Similarly, translation of the groES nucleotide sequence showed highest homology to the C. thermocellum GroES protein (61% amino acid identity). Analysis of the upstream region of this chaperonin operon revealed a CIRCE regulatory element 45 bp upstream from the putative start of the groES ORF. The deduced amino acid sequence of the putative dnaK gene showed the highest homology with the amino acid sequence of the Clostridium acetobutylicum DnaK protein (68% amino acid identity). Phylogenetic analyses based on the translated sequences reiterate this relationship between R. flavefaciens and the Clostridia. However, when the nucleotide sequences of Gram-positive organisms are analyzed, a different topology occurs of the relationship between high- and low-G+C Gram-positive organisms to the 16S rRNA interpretation. [source] A new Minos vector for eye-specific expression of white+ marker in Ceratitis capitata and in distantly related dipteran speciesINSECT MOLECULAR BIOLOGY, Issue 3 2006M. Salvemini Abstract The genetic transformation of insects by transposable elements is based on the use of selectable genetic markers required to identify transgenic individuals. Conserved regulatory sequences can be used to develop single constructs capable of adequate expression of a marker, across a range of different species. We present evidence that the Drosophila GBS regulatory element (Glass-binding site), derived from the Rh1 rhodopsin gene, is able to drive in vivo eye-specific expression of a Ccwhite+ transgene in the Mediterranean fruitfly Ceratitis capitata. The Ceratitis lineage diverged from that of Drosophila,120 Myr ago. As the GBS regulatory sequence seems to be partially conserved in the more distantly related dipteran species Anopheles gambiae (250 Myr), we propose that the GBS may be widely useful for driving eye-specific expression in a wide range of dipteran species. [source] Characterization of genomic DNA encoding cecropins from an Aedes albopictus mosquito cell lineINSECT MOLECULAR BIOLOGY, Issue 1 2002D. Sun Abstract We used cDNA probes from Aedes albopictus mosquito cecropins AalCecA, B, and C to obtain genomic DNA copies and flanking DNA. Two gene copies (AalCecA1 and A2, AalCecB1 and B2, AalCecC1 and C2) encoding each of the three mature cecropin peptides were recovered. All these genes had a similar organization, into two exons interrupted by a single short intron. AalCecA1 and AalCecA2 encode mature protein products that differ by one amino acid residue, while AalCecB1 and AalCecB2, AalCecC1 and AalCecC2 encode identical mature cecropin peptides, respectively. The AalCecB and C gene pairs each share a common intergenic region of approximately 1 kb, with the two coding regions transcribed in opposite directions. With the exception of small insertions/deletions, the intergenic spacer region was highly conserved between the B1/C1 and B2/C2 clones. In transfected cells, 0.8 kb of upstream sequence was sufficient for inducible expression of AalCecA1. Within this region, a 28 bp sequence at positions ,192 to ,165 upstream of the transcription initiation site was found to contain a potential regulatory element. In electrophoretic mobility shift assays, synthetic double-stranded DNA containing this 28 bp sequence retarded protein in cytoplasmic and nuclear extracts from C7-10 cells. [source] Basal and stimulated lactate fluxes in primary cultures of astrocytes are differentially controlled by distinct proteinsJOURNAL OF NEUROCHEMISTRY, Issue 3 2008Fumihiko Maekawa Abstract Lactate release by astrocytes is postulated to be of importance for neuroenergetics but its regulation is poorly understood. Basigin, a chaperone protein for specific monocarboxylate transporters (MCTs), represents a putatively important regulatory element for lactate fluxes. Indeed, basigin knockdown by RNA interference in primary cultures of astrocytes partially reduced both proton-driven lactate influx and efflux. But more strikingly, enhancement of lactate efflux induced by glutamate was prevented while the effect of sodium azide was significantly reduced by treatment of cultured astrocytes with anti-basigin small interfering RNA. Enhancement of glucose utilization was unaffected under the same conditions. Basal lactate uptake and release were significantly reduced by MCT1 knockdown, even more so than with basigin knockdown, whereas glutamate-driven or sodium azide-induced enhancement of lactate release was not inhibited by either MCT1, 2, or 4 small interfering RNAs. In conclusion, MCT1 plays a pivotal role in the control of basal proton-driven lactate flux in astrocytes while basigin is only partly involved, most likely via its interaction with MCT1. In contrast, basigin appears to critically regulate the enhancement of lactate release caused by glutamate (or sodium azide) but via an effect on another unidentified transporter at least present in astrocytes in vitro. [source] Significance of consensus CYC-binding sites found in the promoters of both ChCYC and ChRAD genes in Chirita heterotricha (Gesneriaceae)JOURNAL OF SYSTEMATICS EVOLUTION, Issue 4 2010Xia YANG Abstract,CYC -like genes are widely conserved in controlling floral dorsoventral asymmetry (zygomorphy) through persistent expression in corresponding domains in core eudicots. To understand how CYC -like gene expression is maintained during flower development, we selected Chirita heterotricha as a material and isolated the promoter sequences of the ChCYC1C and ChCYC1D genes, homologs of CYC, by inverse polymerase chain reaction. Further promoter analyses led to the identification of a putative cis -regulatory element in each promoter matching the consensus DNA binding site for Antirrhinum CYC protein: GGCCCCTC at ,165 for ChCYC1C, and GGCCCCCC at ,163 for ChCYC1D. This indicates that both the ChCYC1C and ChCYC1D genes have probably evolved autoregulatory loops to sustain their expression in developing flowers. We also isolated the coding and promoter sequences of the ChRAD gene, a homolog of Antirrhinum RAD. Promoter analysis showed that the ChRAD gene promoter also contained a putative CYC-binding site (GGCCCAC at ,134). Therefore, ChRAD is likely a direct target of the ChCYC1 genes, which is similar to Antirrhinum RAD. These results imply that the establishment of floral zygomorphy in Chirita may have been achieved by the evolution of an autoregulatory loop for CYC -like genes, which was probably accompanied by simultaneous co-option of the RAD -like gene into their regulatory network. [source] Rho A participates in the regulation of phosphatidylserine-dependent procoagulant activity at the surface of megakaryocytic cellsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 4 2004C. Kunzelmann Summary. Once exposed at the external surface of activated platelets or apoptotic cells, phosphatidylserine, an anionic phospholipid mostly sequestered in the inner leaflet of the plasma membrane, plays essential roles in hemostasis and phagocytosis. The mechanism governing the migration of the phosphatidylserine to the exoplasmic leaflet is not yet fully understood. We have proposed that store-operated calcium entry (SOCE) constitutes a key step of this process. ERK pathway is among the elements modulating SOCE and phosphatidylserine externalization in megakaryocytic HEL cells. Here, we investigated the role of small GTPase Rho A, which may interact with the ERK pathway. Specific inhibitors of Rho A (exoenzyme C3 and toxin B) reduced both SOCE and phosphatidylserine-dependent procoagulant activity. Simultaneous inhibition of Rho A and extracellular signal-regulated kinase (ERK) pathways did not elicit further reduction with respect to each individual one. Rho A can regulate SOCE and phosphatidylserine exposure through the reorganization of actin cytoskeleton, but not through ROCK pathway. Hence, Rho A is another regulatory element for the completion of SOCE-induced phosphatidylserine transmembrane redistribution in HEL cells. [source] Study of the effects of interferon a on several human hepatoma cell lines: analysis of the signalling pathway of the cytokine and of its effects on apoptosis and cell proliferationLIVER INTERNATIONAL, Issue 2 2004A. Legrand Background: Interferon , (IFN,), currently used for the treatment of chronic viral hepatitis, is also known to prevent the development of hepatocellular carcinoma (HCC), the mechanism of this action being still debatable. Aims: To study thoroughly in human hepatoma cell lines (HHL) , Hep3B, HepG2, HuH7, SKHep1, and Chang-Liver , submitted to rhIFN,, the signalling pathway of IFN,, the binding activity of the cytokine on specific gamma-activated sequence (GAS) and interferon-stimulated regulatory element (ISRE) nuclear sequences, and its effects on apoptosis and cell proliferation. Methods: The behaviour of signal transducer and activator of transcription (STAT)1, STAT2, p48IRF9 and the binding of nuclear proteins were investigated by immunoblot and electro-mobility shift assay. Expression of some IFN,-dependent proteins , p21/WAF1, inducible nitric oxide synthase, IRF1 and 2 , were studied by immunoblot. Apoptosis and the cell cycle were studied by morphological and biochemical methods. Results: Transduction of INF, was unaltered, although there were some variations in the different HHL. Nuclear protein binding to GAS or ISRE showed that ISRE was mainly involved. Apoptosis did not occur. The cell cycle was slightly modified in HuH7. Three GAS- and/or ISRE-dependent proteins increased, suggesting that IFN, may have some biological effects on HHL. Conclusions: The IFN, signalling pathway is functional in several HHL, but the cytokine has no apoptotic effect and a moderate anti-proliferative effect. This suggests that the preventive role of IFN, on HCC cannot be explained by an apoptotic and/or an anti-proliferative effect, but possibly by its action on several specific nuclear sequences that protect liver cells from transformation. [source] Regulation of bacterial gene expression by the NTP substrates of transcription initiationMOLECULAR MICROBIOLOGY, Issue 1 2008Charles L. Turnbough Summary Many mechanisms of gene regulation in bacteria do not employ repressor or activator proteins. One class of these mechanisms includes those in which the key regulatory element is the control of transcription initiation by the availability of NTP substrates. In this commentary, several distinct examples of initiating NTP-mediated gene regulation are discussed, including a mechanism reported by Krásnýet al. in this issue of Molecular Microbiology. These researchers show that during the stringent response induced by amino acid starvation of Bacillus subtilis, increases in the intracellular level of ATP permit upregulation of promoters with +1A start sites, while concurrent decreases in the intracellular level of GTP cause downregulation of promoters with +1G start sites. This regulation is restricted to stringently controlled promoters. [source] Identification of an ospC operator critical for immune evasion of Borrelia burgdorferiMOLECULAR MICROBIOLOGY, Issue 1 2007Qilong Xu Summary Timely expression of the outer surface protein C (OspC) is crucial for the pathogenic strategy of the Lyme disease spirochete Borrelia burgdorferi. The pathogen abundantly expresses OspC during initial infection when the antigen is required, but downregulates when its presence poses a threat to the spirochetes once the anti-OspC humoral response has developed. Here, we show that a large palindromic sequence immediately upstream of the ospC promoter is essential for the repression of ospC expression during murine infection and for the ability of B. burgdorferi to evade specific OspC humoral immunity. Deletion of the sequence completely diminished the ability of B. burgdorferi to avoid clearance by transferred OspC antibody in SCID mice. B. burgdorferi lacking the regulatory element was able to initiate infection but unable to persist in immunocompetent mice. Taken together, the regulatory element immediately upstream of the ospC promoter serves as an operator that may interact with an unidentified repressor(s) to negatively regulate ospC expression and is essential for the immune evasion of B. burgdorferi. [source] Microarray analysis of chitin elicitation in Arabidopsis thalianaMOLECULAR PLANT PATHOLOGY, Issue 5 2002Katrina M. Ramonell Summary Chitin oligomers, released from fungal cell walls by endochitinase, induce defence and related cellular responses in many plants. However, little is known about chitin responses in the model plant Arabidopsis. We describe here a large-scale characterization of gene expression patterns in Arabidopsis in response to chitin treatment using an Arabidopsis microarray consisting of 2375 EST clones representing putative defence-related and regulatory genes. Transcript levels for 71 ESTs, representing 61 genes, were altered three-fold or more in chitin-treated seedlings relative to control seedlings. A number of transcripts exhibited altered accumulation as early as 10 min after exposure to chitin, representing some of the earliest changes in gene expression observed in chitin-treated plants. Included among the 61 genes were those that have been reported to be elicited by various pathogen-related stimuli in other plants. Additional genes, including genes of unknown function, were also identified, broadening our understanding of chitin-elicited responses. Among transcripts with enhanced accumulation, one cluster was enriched in genes with both the W-box promoter element and a novel regulatory element. In addition, a number of transcripts had decreased abundance, encoding several proteins involved in cell wall strengthening and wall deposition. The chalcone synthase promoter element was identified in the upstream regions of these genes, suggesting that pathogen signals may suppress the expression of some genes. These data indicate that Arabidopsis should be an excellent model to elucidate the mechanisms of chitin elicitation in plant defence. [source] Differential regulation of the Oct-3/4 gene in cell culture model systems that parallel different stages of mammalian developmentMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 8 2008Sunil Kumar Mallanna Abstract Oct-3/4 is an essential transcription factor that regulates stem cell fate during embryogenesis. Previous reports have shown that the Oct-3/4 gene utilizes different enhancers to regulate its expression as development proceeds. However, the cis -elements contributing to the differential activity of these enhancers require further study. Here, we investigated the function of the HMG/POU cassette and LRH-1 site present in the distal enhancer (DE) and the proximal enhancer, respectively. F9 and P19 EC cells were the focus of this study because their differential utilization of Oct-3/4 enhancers parallels the use of these enhancers during different stages of development. We determined that the LRH-1 site functions as a positive and a negative cis -regulatory element in P19 and F9 EC cells, respectively. Furthermore, we determined that the HMG/POU cassette in the DE strongly activates the Oct-3/4 promoter in F9 cells, but is a much weaker positive regulatory element in P19 cells. Given that HMG/POU cassettes play key roles in the regulation of at least seven essential genes, the Oct-3/4 HMG/POU cassette was examined more closely by focusing on Sox2, which can bind to HMG/POU cassettes. Although chromatin immunoprecipitation demonstrated that Sox2 binds to the Oct-3/4 gene equally well in both EC cell lines, tethering Sox2 to the region of the HMG/POU cassette only activated the Oct-3/4 promoter in F9 EC cells. These and other findings suggest that the differential activity of the HMG/POU cassette of the Oct-3/4 gene in EC cells is due to differential action of Sox2 and its associated co-factors. Mol. Reprod. Dev. 75: 1247,1257, 2008. © 2008 Wiley-Liss, Inc. [source] Stage-specific regulatory element of mouse Sry gene,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2003Kou Yokouchi Abstract Sry expression is essential for initiating male sex differentiation, and the expression occurs only during a restricted period in the developing gonad. It is thought that Sry is part of a pathway of genes that regulate sex determination. Although the interactions of several genes with Sry expression have been suggested, the exact cascade of gene expression regulating Sry transcription is entirely obscure because there is no available cell line expressing Sry and reflecting an in vivo condition. The present study was carried out to investigate the cis -acting element of the mouse Sry that responds stage specifically to its expression, in part, using transgenic mice expressing GFP on the Y chromosome. Ten DNA fragments were generated by digesting the 5, upstream region (positions 5491,8039; 2,549 bp) of mouse Sry with appropriate restriction enzymes. In an electrophoretic mobility assay with these fragments, the region from position 5491 to position 5799 (309 bp) was identified as forming specific protein,DNA complexes with nuclear extracts from 11.5 days post coitus (dpc) gonads, but not from 12.5 and 13.5-dpc gonads. This region also formed specific protein,DNA complexes with the nuclear extracts from adult testicular germ cells that generate only a circular form from Sry. This stage-specific responsive region was narrowed down to positions 5559,5616 by DNase I footprinting analysis. The assay of DNase I hypersensitive (HS) using the nuclear lysates from the 11.5-dpc urogenital ridges demonstrated that the novel HS site was located in the proximity of position 5600. This region DNase I HS was also detected at the same position when the lysates from adult testicular germ cells were applied. The results indicate that the present HS site may be involved in the transcriptional regulation of the linear and/or circular molecule transcripts from mouse Sry gene. Mol. Reprod. Dev. 64: 389,396, 2003. © 2003 Wiley-Liss, Inc. [source] Identification of regulatory elements involved in expression and induction by sucrose and UV-B light of the Arabidopsis thaliana COX5b-2 gene, encoding an isoform of cytochrome c oxidase subunit 5bPHYSIOLOGIA PLANTARUM, Issue 3 2009Raúl N. Comelli The promoter sequences required for expression of the Arabidopsis thaliana COX5b-2 gene, encoding an isoform of cytochrome c oxidase subunit 5b, were analyzed using plants transformed with deleted and mutagenized forms of the promoter fused to gus. A 1000-bp promoter fragment produces expression in root and shoot meristems, leaf and cotyledon tips, and anthers. Deletion analysis indicated the presence of positive and negative regulatory elements. A regulatory element located between ,660 and ,620 from the translation start site was identified as a G-box by mutagenic analysis. Mutation of the G-box, that is present within the coding region of the preceding gene in the genome, increases expression of COX5b-2 in cotyledon and leaf lamina and abolishes induction by ultraviolet-B (UV-B) light, which presumably acts through the removal of an inhibitory factor. Identified positive regulatory elements include a site II element (TGGGCC), a related element with the sequence TGGGTC and four initiator elements (YTCANTYY) that completely abolish expression when mutated in combination. Site II elements are also involved in the response to sucrose. The results imply that the COX5b-2 gene has retained expression characteristics presented by most respiratory chain component genes, but its expression mechanisms have diverged from those employed by COX5b-1, the other gene encoding cytochrome c oxidase subunit 5b in Arabidopsis. [source] Genetics of human iris colour and patternsPIGMENT CELL & MELANOMA RESEARCH, Issue 5 2009Richard A. Sturm Summary The presence of melanin pigment within the iris is responsible for the visual impression of human eye colouration with complex patterns also evident in this tissue, including Fuchs' crypts, nevi, Wolfflin nodules and contraction furrows. The genetic basis underlying the determination and inheritance of these traits has been the subject of debate and research from the very beginning of quantitative trait studies in humans. Although segregation of blue-brown eye colour has been described using a simple Mendelian dominant-recessive gene model this is too simplistic, and a new molecular genetic perspective is needed to fully understand the biological complexities of this process as a polygenic trait. Nevertheless, it has been estimated that 74% of the variance in human eye colour can be explained by one interval on chromosome 15 that contains the OCA2 gene. Fine mapping of this region has identified a single base change rs12913832 T/C within intron 86 of the upstream HERC2 locus that explains almost all of this association with blue-brown eye colour. A model is presented whereby this SNP, serving as a target site for the SWI/SNF family member HLTF, acts as part of a highly evolutionary conserved regulatory element required for OCA2 gene activation through chromatin remodelling. Major candidate genes possibly effecting iris patterns are also discussed, including MITF and PAX6. [source] Mechanisms of imprint dysregulation,AMERICAN JOURNAL OF MEDICAL GENETICS, Issue 3 2010Bernhard Horsthemke Abstract Genomic imprinting is an epigenetic process by which the male and the female germ line confer specific marks (imprints) onto certain gene regions, so that one allele of an imprinted gene is active and the other allele is silent. Genomic imprints are erased in primordial germ cells, newly established during later stages of germ cell development, and stably inherited through somatic cell divisions during postzygotic development. Defects in imprint erasure, establishment, or maintenance result in a paternal chromosome carrying a maternal imprint or in a maternal chromosome carrying a paternal imprint. A wrong imprint leads to activation of an allele that should be silent or silencing of an allele that should be active. Since the dosage of imprinted genes is very important for development and growth, imprinting defects lead to specific diseases. Imprinting defects can occur spontaneously without any DNA sequence change (primary imprinting defect) or as the result of a mutation in a cis -regulatory element or a trans -acting factor (secondary imprinting defect). The distinction between primary and secondary imprinting defects is important for assessing the recurrence risk in affected families. © 2010 Wiley-Liss, Inc. [source] Identification of the Tctex-1 regulatory element that directs expression to neural stem/progenitor cells in developing and adult brainTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 16 2010Yun-Yu Tseng Abstract Previous studies showed that Tctex-1 immunoreactivity is selectively enriched in the germinal zones of adult brain. In this report we identify a regulatory region of the Tctex-1 gene that is capable of directing transgenic expression of green fluorescent protein (GFP) reporter that recapitulates the spatial and temporal expression pattern of endogenous Tctex-1. This construct specifically targeted expression to the nestin+/Pax6+/GLAST+ radial glial cells and Tbr2+ intermediate progenitors when the reporter construct was delivered to developing mouse neocortex via in utero electroporation. Characterization of mice transgenically expressing GFP under the same regulatory element showed that the GFP expression is faithful to endogenous Tctex-1 at the subgranular zone (SGZ) of dentate gyrus, ventricular/subventricular zone of lateral ventricles, and ependymal layer of 3rd ventricle of adult brains. Immunolocalization and bromodeoxyuridine incorporation studies of adult SGZ in four independent mouse lines showed that Tctex-1:GFP reporter selectively marks nestin+/GFAP+/Sox2+ neural stem-like cells in two mouse lines (4 and 13). In two other mouse lines (17 and 18), Tctex-1:GFP is selectively expressed in Type-2 and Type-3 transient amplifying progenitors and a small subset of young neuronal progeny. The P/E-Tctex-1 reporter mouse studies independently confirmed the specific enrichment of Tctex-1 at adult SGZ stem/progenitor cells. Furthermore, these studies supported the notion that an analogous transcriptional program may be used to regulate neurogenesis in embryonic cerebral cortex and adult hippocampus. Finally, the genomic sequences and the reporter mouse lines described here provide useful experimental tools to advance adult neural stem cell research. J. Comp. Neurol. 518:3327,3342, 2010. © 2010 Wiley-Liss, Inc. [source] Reconstituting retroviral (ReCon) vectors facilitating delivery of cytotoxic genes in cancer gene therapy approachesTHE JOURNAL OF GENE MEDICINE, Issue 2 2008Eva Maria Brandtner Abstract Background We have previously described the generation of reconstituting retroviral (ReCon) vectors designed for cancer gene therapy using cytotoxic gene products. The unique vector structure with a promoter physically separated from the transgene allows generation of stable virus producer cells irrespective of the toxic gene. The mechanism of synthesis of DNA from retroviral RNA dictates that infection leads to the reconstitution of functional expression cassettes in the target cell. Methods To improve vector titres, a cytomegalovirus enhancer was inserted upstream of the 5,-long-terminal repeat (LTR); the Woodchuck hepatitis virus post-transcriptional regulatory element and an elongated attachment site upstream of the 3,-LTR were included. In addition, a bacterial origin of replication was deleted and a functional internal polyadenylation signal mutated. Transcriptional targeting was attempted by introducing mammary tissue-specific promoters such as the U3 region of mouse mammary tumour virus or the promoter of the whey acidic protein encoding gene. All modifications were analysed in detail with respect to virus production and infectivity. Finally, the vector was armed with the ,-holin encoding gene and transduced cells were analysed for cytotoxic effects. Results Distinct modifications of the vector resulted in a titre improvement of more than 560-fold. Compatibility of the optimized vector with targeted cellular promoters was demonstrated. When equipped with the cytotoxic gene, stable producer cells could be successfully established and high titre virus infection resulted in rigorous target cell killing. Conclusions The ReCon vector in its optimized form is an attractive tool for cancer gene therapy approaches. Copyright © 2007 John Wiley & Sons, Ltd. [source] Enhancing rAAV vector expression in the lungTHE JOURNAL OF GENE MEDICINE, Issue 7 2005Isabel Virella-Lowell Abstract Despite favorable DNA transfer efficiency, gene expression from recombinant adeno-associated virus (rAAV2) vectors in the lung has been variable in the context of cystic fibrosis (CF) gene therapy. This is due, in part, to the large size of the CF transmembrane regulator (CFTR)-coding sequence which necessitates the use of compact endogenous promoter elements versus stronger exogenous promoters. We evaluated the possibility that gene expression from rAAV could be improved by using AAV capsid serotypes with greater tropism for the apical surface of airway cells (i.e. rAAV5 or rAAV1) and/or using strong promoters such as the cytomegalovirus (CMV) enhancer/chicken beta-actin hybrid (C,) promoter. The relative activity of the CMV immediate-early (CMVie) promoter, the C, promoter, and the C, promoter with a downstream woodchuck hepatitis virus post-transcriptional regulatory element (wpre) were assessed in vitro and in vivo in C57\Bl6 mice using human alpha-1 antitrypsin (hAAT) as a secreted reporter. In vivo, the C,-AAT-wpre group achieved maximum serum levels of 1.5 mg/ml of hAAT. AAV capsid serotypes were then compared in vivo utilizing the transcriptionally optimized CB-wpre cassette in rAAV serotype 1, 2 or 5 capsids (rAAV1, rAAV2, and rAAV5), utilizing luciferase as a reporter to compare expression over a wide dynamic range. The pulmonary luciferase levels at 8 weeks were similar in rAAV5 and rAAV1 groups (2.9 × 106 relative light units (RLU)/g tissue and 2.7 × 106 RLU/g tissue, respectively), both of which were much higher than rAAV2. Although the advantage of rAAV5 over rAAV2 in the lung has already been described, the availability of another serotype (rAAV1) capable of efficient gene transfer in the lung could be useful. Copyright © 2005 John Wiley & Sons, Ltd. [source] FUSCA3 from barley unveils a common transcriptional regulation of seed-specific genes between cereals and ArabidopsisTHE PLANT JOURNAL, Issue 6 2008Miguel Ángel Moreno-Risueno Summary Accumulation of storage compounds in the embryo and endosperm of developing seeds is a highly regulated process that allows seedling growth upon germination until photosynthetic capacity is acquired. A critical regulatory element in the promoters of seed storage protein (SSP) genes from dicotyledonous species is the RY box, a target of B3-type transcription factors. However, the functionality of this motif in the transcriptional regulation of SSP genes from cereals has not been fully established. We report here the identification and molecular characterization of barley FUSCA3, a B3-type transcription factor as yet uncharacterized in monocotyledonous plants. Our results show that both the barley and Arabidopsis FUS3 genes maintain a conserved functionality for the regulation of SSP genes and anthocyanin biosynthesis in these two distantly related phylogenetic groups. Complementation of the loss-of-function mutant fus3 in Arabidopsis by the barley HvFus3 gene resulted in restored transcription from the At2S3 gene promoter and normal accumulation of anthocyanins in the seed. In barley, HvFUS3 participates in transcriptional activation of the endosperm-specific genes Hor2 and Itr1. HvFUS3, which specifically binds to RY boxes in EMSA experiments, trans -activates Hor2 and Itr1 promoters containing intact RY boxes in transient expression assays in developing endosperms. Mutations in the RY boxes abolished the HvFUS3-mediated trans -activation. HvFus3 transcripts accumulate in the endosperm and in the embryo of developing seeds, peaking at mid maturation phase. Remarkably, HvFUS3 interacts with the Opaque2-like bZIP factor BLZ2 in yeast, and this interaction is essential for full trans -activation of the seed-specific genes in planta. [source] Analysis of two translocation breakpoints and identification of a negative regulatory element in patients with Rieger's syndromeBIRTH DEFECTS RESEARCH, Issue 2 2004Dimitri G. Trembath Abstract BACKGROUND Rieger's syndrome is an autosomal dominant disorder characterized by eye, tooth, and umbilical anomalies. A gene responsible for Rieger's syndrome, PITX2, has previously been cloned using two patients with balanced translocations, t(4;16) and t(4;11), with breakpoints that lie near the gene, but which do not interrupt it. METHODS We sequenced both breakpoint regions on chromosome 4 and screened this area for novel genes. Fluorescence in situ hybridization (FISH) was used to determine if PITX2 was still present on the 4:16 chromosome. Both the chromosome 16 and chromosome 11 breakpoints were cloned and sequenced using panhandle polymerase chain reaction (PHPCR). Transient transfection studies were performed to compare effects on a reporter gene between native chromosome 4 sequence and chromosome 11 sequence. RESULTS The region surrounding PITX2 on chromosome 4 is rich in repetitive elements, but no novel genes were identified. FISH demonstrated that PITX2 was intact on the 4:16 translocation chromosome. The PHPCR experiments demonstrated that the translocated regions of chromosomes 16 and 11 were repeat-rich, and transfection studies revealed a slight enhancer effect with the chromosome 4 sequence, and a strong silencer effect when the chromosome 11 sequence was present. CONCLUSIONS Given the lack of any novel genes near either breakpoint, changes in potential regulatory elements may be the best model to explain the loss of PITX2 expression in these patients and hence the Rieger's syndrome phenotype. Birth Defects Research (Part A), 2004. © 2004 Wiley-Liss, Inc. [source] Antisense modulation of the coding or regulatory sequence of the folate receptor (folate binding protein-1) in mouse embryos leads to neural tube defects,BIRTH DEFECTS RESEARCH, Issue 7 2003Deborah K. Hansen Abstract BACKGROUND Although folic acid decreases the incidence of neural tube defects (NTDs) in humans, the mechanism for this protection is unknown. We have employed antisense technology to alter expression of the gene for the folate receptor (folate binding protein-1 [Folbp1]) in mouse embryos cultured in vitro. METHODS Embryos were explanted on day 8 of gestation and cultured for 44 hr. Several oligodeoxyribonucleotides designed to modulate the coding region or a regulatory sequence in the 5,-untranslated region of Folbp1 were microinjected into the amniotic sac of embryos at the beginning of the culture period. RESULTS Two different antisense sequences to the 5, and 3, coding region in Folbp1 produced concentration-dependent increases in the number of embryos with NTDs. Coinjection of 5-methyltetrahydrofolate with these sequences decreased the frequency of abnormal embryos. A semi-quantitative RT-PCR technique used to measure the amount of Folbp1 mRNA in treated and control embryos confirmed that the mRNA level was decreased by treatment with the antisense sequences. An antisense oligodeoxyribonucleotide to a 17 base cis regulatory element also generated a concentration-dependent increase in the frequency of embryos with NTDs, and a decrease in the level of Folbp1 mRNA. CONCLUSIONS These results demonstrate that alterations in expression of Folbp1 by perturbing either the coding sequence or a critical regulatory cis -element can play a role in NTDs. Birth Defects Research (Part A) 67475,487, 2003. © 2003 Wiley-Liss, Inc. [source] The effect of NQO1 polymorphism on the inflammatory response in cardiopulmonary bypassCELL BIOCHEMISTRY AND FUNCTION, Issue 4 2008C. Selim Isbir Abstract Cardiopulmonary bypass (CPB) has been associated with systemic inflammatory response syndrome (SIRS). Endothelial dysfunction related to non-laminar flow during CPB is known to play a key role in this complex pathology. Antioxidant response element (ARE) dependent NAD(P)H:quinone oxidoreductase 1 (NQO1) promoter is a regulatory element involved in the anti-inflammatory mechanism in vasculature exposed to non-laminar flow. Mutation of the NQO1 could represent a novel anti-inflammatory effect in CPB. The goal of this study was to demonstrate whether genetic variants of NQO1 affect cytokine release after CPB. Eighteen patients who underwent standard coronary artery bypass grafting (CABG) operation were included in the study. Genotyping for NQO1 was performed. Serum Interleukin-6 (IL-6) levels were measured before induction, during CPB after declamping the aorta, and 24,h after operation. Clinical data were collected respectively. Seven patients were NQO1 T carriers and 11 patients were NQO1 T non-carriers. During CPB, IL-6 concentrations were increased in NQO1 T carriers compared to T non-carriers (p,=,0.038). Although ventilation times and blood loss were higher in T carriers these were not statistically significant. Patients with NQO1 T carriers showed significantly higher IL-6 levels during CPB. Non-laminar flow during CPB may diminish the transcriptional activation of the NQO1 in T carriers. Preoperative determination of this novel anti-inflammatory mechanism could be useful to improve operative outcome in CPB. Copyright © 2007 John Wiley & Sons, Ltd. [source] Manipulating gene activity in Wnt1-expressing precursors of neural epithelial and neural crest cellsDEVELOPMENTAL DYNAMICS, Issue 1 2010Wei Hsu Abstract Targeted gene disruption or expression often encounters lethality. Conditional approaches, permitting manipulation at desired stages, are required to overcome this problem in order to analyze gene function in later developmental processes. Wnt1 has been shown to be expressed in neural crest precursors at the dorsal midline region. However, its expression was not detected in emigrated neural crest cells, the descendants of Wnt1-expressing precursors. We have developed mouse transgenic systems to manipulate gene activity in the Wnt1-expressing precursors and their derivatives by integrating the tetracycline-dependent activation and Cre-mediated recombination methods. A new Wnt1-rtTA strain, carrying rtTA under control of Wnt1 regulatory elements, has been created for gene manipulation in a spatiotemporal-specific fashion. Together with our previously developed Wnt1-Cre;R26STOPrtTA model, these systems permit conditional gene expression and ablation in pre-migratory and/or post-migratory neural crest cells. This study demonstrated the versatility of our mouse models to achieve gene manipulation in early neural development. Developmental Dynamics 239:338,345, 2010. © 2009 Wiley-Liss, Inc. [source] HOXA13 directly regulates EphA6 and EphA7 expression in the genital tubercle vascular endotheliaDEVELOPMENTAL DYNAMICS, Issue 4 2007Carley A. Shaut Abstract Hypospadias, a common defect affecting the growth and closure of the external genitalia, is often accompanied by gross enlargements of the genital tubercle (GT) vasculature. Because Hoxa13 homozygous mutant mice also exhibit hypospadias and GT vessel expansion, we examined whether genes playing a role in angiogenesis exhibit reduced expression in the GT. From this analysis, reductions in EphA6 and EphA7 were detected. Characterization of EphA6 and EphA7 expression in the GT confirmed colocalization with HOXA13 in the GT vascular endothelia. Analysis of the EphA6 and EphA7 promoter regions revealed a series of highly conserved cis -regulatory elements bound by HOXA13 with high affinity. GT chromatin immunoprecipitation confirmed that HOXA13 binds these gene-regulatory elements in vivo. In vitro, HOXA13 activates gene expression through the EphA6 and EphA7 gene-regulatory elements. Together these findings indicate that HOXA13 directly regulates EphA6 and EphA7 in the developing GT and identifies the GT vascular endothelia as a novel site for HOXA13-dependent expression of EphA6 and EphA7. Developmental Dynamics 236:951,960, 2007. © 2007 Wiley-Liss, Inc. [source] Complementation of melanocyte development in SOX10 mutant neural crest using lineage-directed gene transferDEVELOPMENTAL DYNAMICS, Issue 1 2004Ling Hou Abstract An in vitro gene complementation approach has been developed to dissect gene function and regulation in neural crest (NC) development and disease. The approach uses the avian RCAS virus to express genes in NC cells derived from transgenic mice expressing the RCAS receptor TVA, under the control of defined promoter elements. Constructs for creating TVA transgenic mice were developed using site-specific recombination GATEWAY (GW), compatible vectors that can also be used to facilitate analysis of genomic fragments for transcriptional regulatory elements. By using these GW vectors to facilitate cloning, transgenic mouse lines were generated that express TVA in SOX10-expressing NC stem cells under the control of the Pax3 promoter. The Pax3-tv-a transgene was bred onto a Sox10 -deficient background, and the feasibility of complementing genetic NC defects was demonstrated by infecting the Pax3-tv-a cells with an RCAS- Sox10 expression virus, thereby rescuing melanocyte development of Sox10 -deficient NC cells. This system will be useful for assessing genetic hierarchies in NC development. Developmental Dynamics 229:54,62, 2004. © 2003 Wiley-Liss, Inc. [source] | |||||||||||||||||||||||||||||||||||||