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Sample records for globin gene promoters

  1. The human β-globin gene contains multiple regulatory regions: identification of one promoter and two downstream enhancers.

    NARCIS (Netherlands)

    M. Antoniou (Michael); E. de Boer (Ernie); G. Habets; F.G. Grosveld (Frank)

    1988-01-01

    textabstractWe have introduced into murine erythroleukaemia (MEL) cells several series of deletion mutants of hybrid genes between the human beta-globin gene and the murine H-2Kb gene. S1 nuclease and transcriptional run-off analysis showed that the human beta-globin gene contains at least three

  2. Transcriptional promiscuity of the human /alpha/-globin gene

    Energy Technology Data Exchange (ETDEWEB)

    Whitelaw, E.; Hogben, P.; Hanscombe, O.; Proudfoot, N.J.

    1989-01-01

    The human /alpha/-globin gene displays the unusual property of transcriptional promiscuity: that is, it functions in the absence of an enhancer when transfected into nonerythroid cell lines. It is also unusual in that its promoter region lies in a hypomethylated HpaII tiny fragment (HTF) island containing multiple copies of the consensus sequence for the SP1-binding site. The authors have investigated whether there is a relationship between these two observations. First, they investigated the mouse /alpha/-globin gene since it does not lie in an HTF island. They have demonstrated that it was not transcriptionally promiscuous. Second, they studied the transcriptional activity of the human /alpha/-globin gene in the absence of the GC-rich region containing putative SP1-binding sites and found a small (two- to threefold) but consistent positive effect of this region on transcriptional activity in both nonerythroid and erythroid cell lines. However, this effect did not account for the promiscuous nature of the human /alpha/-globin gene. They found that in a nonreplicating system, the human //a/-globin gene, like that of the mouse, required a simian virus 40 enhancer in order to be transcriptionally active in nonerythroid and erythroid cell lines. Since they only observed enhancer independence of the human /alpha/-globin gene in a high-copy-number replicating system, they suggest that competition for trans-acting factors could explain these results. Finally, the authors' experiments with the erythroid cell line Putko suggest that there are no tissue-specific enhancers within 1 kilobase 5' of the human /alpha/-globin cap site or within the gene itself.

  3. The globin gene family of the cephalochordate amphioxus: implications for chordate globin evolution

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    Marden Michael C

    2010-11-01

    Full Text Available Abstract Background The lancelet amphioxus (Cephalochordata is a close relative of vertebrates and thus may enhance our understanding of vertebrate gene and genome evolution. In this context, the globins are one of the best studied models for gene family evolution. Previous biochemical studies have demonstrated the presence of an intracellular globin in notochord tissue and myotome of amphioxus, but the corresponding gene has not yet been identified. Genomic resources of Branchiostoma floridae now facilitate the identification, experimental confirmation and molecular evolutionary analysis of its globin gene repertoire. Results We show that B. floridae harbors at least fifteen paralogous globin genes, all of which reveal evidence of gene expression. The protein sequences of twelve globins display the conserved characteristics of a functional globin fold. In phylogenetic analyses, the amphioxus globin BflGb4 forms a common clade with vertebrate neuroglobins, indicating the presence of this nerve globin in cephalochordates. Orthology is corroborated by conserved syntenic linkage of BflGb4 and flanking genes. The kinetics of ligand binding of recombinantly expressed BflGb4 reveals that this globin is hexacoordinated with a high oxygen association rate, thus strongly resembling vertebrate neuroglobin. In addition, possible amphioxus orthologs of the vertebrate globin X lineage and of the myoglobin/cytoglobin/hemoglobin lineage can be identified, including one gene as a candidate for being expressed in notochord tissue. Genomic analyses identify conserved synteny between amphioxus globin-containing regions and the vertebrate β-globin locus, possibly arguing against a late transpositional origin of the β-globin cluster in vertebrates. Some amphioxus globin gene structures exhibit minisatellite-like tandem duplications of intron-exon boundaries ("mirages", which may serve to explain the creation of novel intron positions within the globin genes

  4. Human γ-globin genes silenced independently of other genes in the β-globin locus.

    NARCIS (Netherlands)

    N.O. Dillon (Niall); F.G. Grosveld (Frank)

    1991-01-01

    textabstractErythropoiesis during human development is characterized by switches in expression of beta-like globin genes during the transition from the embryonic through fetal to adult stages. Activation and high-level expression of the genes is directed by the locus control region (LCR), located 5'

  5. Fate of a redundant gamma-globin gene in the atelid clade of New World monkeys: implications concerning fetal globin gene expression.

    Science.gov (United States)

    Meireles, C M; Schneider, M P; Sampaio, M I; Schneider, H; Slightom, J L; Chiu, C H; Neiswanger, K; Gumucio, D L; Czelusniak, J; Goodman, M

    1995-01-01

    Conclusive evidence was provided that gamma 1, the upstream of the two linked simian gamma-globin loci (5'-gamma 1-gamma 2-3'), is a pseudogene in a major group of New World monkeys. Sequence analysis of PCR-amplified genomic fragments of predicted sizes revealed that all extant genera of the platyrrhine family Atelidae [Lagothrix (woolly monkeys), Brachyteles (woolly spider monkeys), Ateles (spider monkeys), and Alouatta (howler monkeys)] share a large deletion that removed most of exon 2, all of intron 2 and exon 3, and much of the 3' flanking sequence of gamma 1. The fact that two functional gamma-globin genes were not present in early ancestors of the Atelidae (and that gamma 1 was the dispensible gene) suggests that for much or even all of their evolution, platyrrhines have had gamma 2 as the primary fetally expressed gamma-globin gene, in contrast to catarrhines (e.g., humans and chimpanzees) that have gamma 1 as the primary fetally expressed gamma-globin gene. Results from promoter sequences further suggest that all three platyrrhine families (Atelidae, Cebidae, and Pitheciidae) have gamma 2 rather than gamma 1 as their primary fetally expressed gamma-globin gene. The implications of this suggestion were explored in terms of how gene redundancy, regulatory mutations, and distance of each gamma-globin gene from the locus control region were possibly involved in the acquisition and maintenance of fetal, rather than embryonic, expression. Images Fig. 2 PMID:7535927

  6. Eos negatively regulates human γ-globin gene transcription during erythroid differentiation.

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    Hai-Chuan Yu

    Full Text Available BACKGROUND: Human globin gene expression is precisely regulated by a complicated network of transcription factors and chromatin modifying activities during development and erythropoiesis. Eos (Ikaros family zinc finger 4, IKZF4, a member of the zinc finger transcription factor Ikaros family, plays a pivotal role as a repressor of gene expression. The aim of this study was to examine the role of Eos in globin gene regulation. METHODOLOGY/PRINCIPAL FINDINGS: Western blot and quantitative real-time PCR detected a gradual decrease in Eos expression during erythroid differentiation of hemin-induced K562 cells and Epo-induced CD34+ hematopoietic stem/progenitor cells (HPCs. DNA transfection and lentivirus-mediated gene transfer demonstrated that the enforced expression of Eos significantly represses the expression of γ-globin, but not other globin genes, in K562 cells and CD34+ HPCs. Consistent with a direct role of Eos in globin gene regulation, chromatin immunoprecipitaion and dual-luciferase reporter assays identified three discrete sites located in the DNase I hypersensitivity site 3 (HS3 of the β-globin locus control region (LCR, the promoter regions of the Gγ- and Aγ- globin genes, as functional binding sites of Eos protein. A chromosome conformation capture (3C assay indicated that Eos may repress the interaction between the LCR and the γ-globin gene promoter. In addition, erythroid differentiation was inhibited by enforced expression of Eos in K562 cells and CD34+ HPCs. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that Eos plays an important role in the transcriptional regulation of the γ-globin gene during erythroid differentiation.

  7. Importance of globin gene order for correct developmental expression.

    NARCIS (Netherlands)

    O. Hanscombe (Olivia); D. Whyatt (David); P.J. Fraser (Peter); N. Yannoutsos (Nikos); D.R. Greaves (David); N.O. Dillon (Niall); F.G. Grosveld (Frank)

    1991-01-01

    textabstractWe have used transgenic mice to study the influence of position of the human globin genes relative to the locus control region (LCR) on their expression pattern during development. The LCR, which is located 5' of the globin gene cluster, is normally required for the activation of all the

  8. Molecular Characterization and Expression of α-Globin and β-Globin Genes in the Euryhaline Flounder (Platichthys flesus

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    Weiqun Lu

    2011-01-01

    Full Text Available In order to understand the possible role of globin genes in fish salinity adaptation, we report the molecular characterization and expression of all four subunits of haemoglobin, and their response to salinity challenge in flounder. The entire open reading frames of α1-globin and α2-globin genes were 432 and 435 bp long, respectively, whereas the β1-globin and β2-globin genes were both 447 bp. Although the head kidney (pronephros is the predicted major site of haematopoiesis, real-time PCR revealed that expression of α-globin and β-globin in kidney (mesonephros was 1.5 times higher than in head kidney. Notably, the α1-globin and β1-globin mRNA expression was higher than α2-globin and β2-globin in kidney. Expression levels of all four globin subunits were higher in freshwater- (FW- than in seawater- (SW-adapted fish kidney. If globins do play a role in salinity adaptation, this is likely to be more important in combating the hemodilution faced by fish in FW than the dehydration and salt loading which occur in SW.

  9. The role of EKLF in human beta-globin gene competition.

    Science.gov (United States)

    Wijgerde, M; Gribnau, J; Trimborn, T; Nuez, B; Philipsen, S; Grosveld, F; Fraser, P

    1996-11-15

    We have investigated the role of erythroid Kruppel-like factor (EKLF) in expression of the human beta-globin genes in compound EKLF knockout/human beta-locus transgenic mice. EKLF affects only the adult mouse beta-globin genes in homozygous knockout mice; heterozygous mice are unaffected. Here we show that EKLF knockout mice express the human epsilon and gamma-globin genes normally in embryonic red cells. However, fetal liver erythropoiesis, which is marked by a period of gamma- and beta-gene competition in which the genes are alternately transcribed, exhibits an altered ratio of gamma- to beta-gene transcription. EKLF heterozygous fetal livers display a decrease in the number of transcriptionally active beta genes with a reciprocal increase in the number of transcriptionally active gamma genes. beta-Gene transcription is absent in homozygous knockout fetuses with coincident changes in chromatin structure at the beta promoter. There is a further increase in the number of transcriptionally active gamma genes and accompanying gamma gene promoter chromatin alterations. These results indicate that EKLF plays a major role in gamma- and beta-gene competition and suggest that EKLF is important in stabilizing the interaction between the Locus Control Region and the beta-globin gene. In addition, these findings provide further evidence that developmental modulation of globin gene expression within individual cells is accomplished by altering the frequency and/or duration of transcriptional periods of a gene rather than changing the rate of transcription.

  10. Effect Alpha Globlin Gene Deletion And Gamma Globin Gene -158 ...

    African Journals Online (AJOL)

    ... have been unable to find a molecular basis for the benign clinical course in all our patients. Other genetic or acquired factors must be hypothesized which ameliorate the clinical condition. Keywords: β- thalassemia, Xmn1 polymorphism, α-globin gene deletion. Egyptian Journal of Biochemistry and Molecular Biology Vol.

  11. Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals

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    Warren Wesley C

    2008-07-01

    Full Text Available Abstract Background Vertebrate alpha (α- and beta (β-globin gene families exemplify the way in which genomes evolve to produce functional complexity. From tandem duplication of a single globin locus, the α- and β-globin clusters expanded, and then were separated onto different chromosomes. The previous finding of a fossil β-globin gene (ω in the marsupial α-cluster, however, suggested that duplication of the α-β cluster onto two chromosomes, followed by lineage-specific gene loss and duplication, produced paralogous α- and β-globin clusters in birds and mammals. Here we analyse genomic data from an egg-laying monotreme mammal, the platypus (Ornithorhynchus anatinus, to explore haemoglobin evolution at the stem of the mammalian radiation. Results The platypus α-globin cluster (chromosome 21 contains embryonic and adult α- globin genes, a β-like ω-globin gene, and the GBY globin gene with homology to cytoglobin, arranged as 5'-ζ-ζ'-αD-α3-α2-α1-ω-GBY-3'. The platypus β-globin cluster (chromosome 2 contains single embryonic and adult globin genes arranged as 5'-ε-β-3'. Surprisingly, all of these globin genes were expressed in some adult tissues. Comparison of flanking sequences revealed that all jawed vertebrate α-globin clusters are flanked by MPG-C16orf35 and LUC7L, whereas all bird and mammal β-globin clusters are embedded in olfactory genes. Thus, the mammalian α- and β-globin clusters are orthologous to the bird α- and β-globin clusters respectively. Conclusion We propose that α- and β-globin clusters evolved from an ancient MPG-C16orf35-α-β-GBY-LUC7L arrangement 410 million years ago. A copy of the original β (represented by ω in marsupials and monotremes was inserted into an array of olfactory genes before the amniote radiation (>315 million years ago, then duplicated and diverged to form orthologous clusters of β-globin genes with different expression profiles in different lineages.

  12. Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals.

    Science.gov (United States)

    Patel, Vidushi S; Cooper, Steven J B; Deakin, Janine E; Fulton, Bob; Graves, Tina; Warren, Wesley C; Wilson, Richard K; Graves, Jennifer A M

    2008-07-25

    Vertebrate alpha (alpha)- and beta (beta)-globin gene families exemplify the way in which genomes evolve to produce functional complexity. From tandem duplication of a single globin locus, the alpha- and beta-globin clusters expanded, and then were separated onto different chromosomes. The previous finding of a fossil beta-globin gene (omega) in the marsupial alpha-cluster, however, suggested that duplication of the alpha-beta cluster onto two chromosomes, followed by lineage-specific gene loss and duplication, produced paralogous alpha- and beta-globin clusters in birds and mammals. Here we analyse genomic data from an egg-laying monotreme mammal, the platypus (Ornithorhynchus anatinus), to explore haemoglobin evolution at the stem of the mammalian radiation. The platypus alpha-globin cluster (chromosome 21) contains embryonic and adult alpha- globin genes, a beta-like omega-globin gene, and the GBY globin gene with homology to cytoglobin, arranged as 5'-zeta-zeta'-alphaD-alpha3-alpha2-alpha1-omega-GBY-3'. The platypus beta-globin cluster (chromosome 2) contains single embryonic and adult globin genes arranged as 5'-epsilon-beta-3'. Surprisingly, all of these globin genes were expressed in some adult tissues. Comparison of flanking sequences revealed that all jawed vertebrate alpha-globin clusters are flanked by MPG-C16orf35 and LUC7L, whereas all bird and mammal beta-globin clusters are embedded in olfactory genes. Thus, the mammalian alpha- and beta-globin clusters are orthologous to the bird alpha- and beta-globin clusters respectively. We propose that alpha- and beta-globin clusters evolved from an ancient MPG-C16orf35-alpha-beta-GBY-LUC7L arrangement 410 million years ago. A copy of the original beta (represented by omega in marsupials and monotremes) was inserted into an array of olfactory genes before the amniote radiation (>315 million years ago), then duplicated and diverged to form orthologous clusters of beta-globin genes with different expression

  13. Synergistic effect of two β globin gene cluster mutations leading to the hereditary persistence of fetal hemoglobin (HPFH) phenotype.

    Science.gov (United States)

    Hariharan, Priya; Sawant, Madhavi; Gorivale, Manju; Manchanda, Ruma; Colah, Roshan; Ghosh, K; Nadkarni, Anita

    2017-10-01

    Co-inheritance of gamma and beta globin gene mutations in a compound heterozygous state is rare but of clinical interest as it provides an important data on understanding the HbF expression. Hematological analysis was carried out (Sysmex KX-21). F-cells were enumerated using flow cytometry. Beta globin gene was analysed by CRDB technique and by DNA sequencing. Gamma globin promoter region was sequenced and expression studies were carried out using real time Taqman assay. We report a family, where two inherited defects of the β globin gene cluster segregate. The proband and her sibling were compound heterozygotes for a novel G γ promoter mutation and the 619 bp deletion a common Indian β thalassemia mutation. Molecular characterization revealed that the father (HbA 2 5.1%, HbF 5.4%), proband (HbA 2 3.6%, HbF 31.7%) and her brother (HbA 2 3.9%, HbF 23.6%) were heterozygous for the 619 bp deletion. The mother (HbA 2 2.1%, HbF 3.4%) had a normal β globin gene. As both the children showed high HbF levels, the γ globin gene work up was carried out. The G γ-globin gene promoter analysis revealed that the mother and the two children were heterozygous for a 5 bp deletion -ATAAG (-533 to -529) that resides in the GATA binding site. These findings suggest that the 5 bp deletion in the G γ globin promoter has a functional role in silencing the γ-globin gene expression in adults by disrupting GATA-1 binding and the associated repressor complex and results in the up-regulation of gamma globin gene expression. When co-inherited with β -thalassemia trait it leads to a phenotype of HPFH.

  14. DNA sequences required for regulated expresson of the β-globin genes in murine erythroleukaemia cells.

    NARCIS (Netherlands)

    S. Wright; E. de Boer (Ernie); A. Rosenthal; R.A. Flavell (Richard); F.G. Grosveld (Frank)

    1984-01-01

    textabstractWe have introduced into murine erythroleukaemia (MEL) cells a series of human globin gene cosmids and two sets of hybrid genes constructed from the human beta-globin gene and the human gamma-globin or murine H-2Kbm1 genes. S1-nuclease analysis of the mRNA products from these genes before

  15. Repeated evolution of chimeric fusion genes in the β-globin gene family of laurasiatherian mammals.

    Science.gov (United States)

    Gaudry, Michael J; Storz, Jay F; Butts, Gary Tyler; Campbell, Kevin L; Hoffmann, Federico G

    2014-05-09

    The evolutionary fate of chimeric fusion genes may be strongly influenced by their recombinational mode of origin and the nature of functional divergence between the parental genes. In the β-globin gene family of placental mammals, the two postnatally expressed δ- and β-globin genes (HBD and HBB, respectively) have a propensity for recombinational exchange via gene conversion and unequal crossing-over. In the latter case, there are good reasons to expect differences in retention rates for the reciprocal HBB/HBD and HBD/HBB fusion genes due to thalassemia pathologies associated with the HBD/HBB "Lepore" deletion mutant in humans. Here, we report a comparative genomic analysis of the mammalian β-globin gene cluster, which revealed that chimeric HBB/HBD fusion genes originated independently in four separate lineages of laurasiatherian mammals: Eulipotyphlans (shrews, moles, and hedgehogs), carnivores, microchiropteran bats, and cetaceans. In cases where an independently derived "anti-Lepore" duplication mutant has become fixed, the parental HBD and/or HBB genes have typically been inactivated or deleted, so that the newly created HBB/HBD fusion gene is primarily responsible for synthesizing the β-type subunits of adult and fetal hemoglobin (Hb). Contrary to conventional wisdom that the HBD gene is a vestigial relict that is typically inactivated or expressed at negligible levels, we show that HBD-like genes often encode a substantial fraction (20-100%) of β-chain Hbs in laurasiatherian taxa. Our results indicate that the ascendancy or resuscitation of genes with HBD-like coding sequence requires the secondary acquisition of HBB-like promoter sequence via unequal crossing-over or interparalog gene conversion. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  16. In vivo protein-DNA interactions at the β-globin gene locus

    International Nuclear Information System (INIS)

    Tohru Ikuta; Yuet Wai Kan

    1991-01-01

    The authors have investigated in vivo protein-DNA interactions in the β-globin gene locus by dimethyl sulfate (DMS) footprinting in K562 cells, which express var-epsilon- and γ-globin but not β-globin. In the locus control region, hypersensitive site 2 (HS-2) exhibited footprints in several putative protein binding motifs. HS-3 was not footprinted. The β promoter was also not footprinted, while extensive footprints were observed in the promoter of the active γ-globin gene. No footprints were seen in the A γ and β3' enhancers. With several motifs, additional protein interactions and alterations in binding patterns occurred with hemin induction. In HeLa cells, some footprints were observed in some of the motifs in HS-2, compatible with the finding that HS-2 has some enhancer function in HeLa cells, albeit much weaker than its activity in K562 cells. No footprint was seen in B lymphocytes. In vivo footprinting is a useful method for studying relevant protein-DNA interactions in erythroid cells

  17. Expression of triplicated and quadruplicated alpha globin genes in sheep.

    Science.gov (United States)

    Vestri, R; Pieragostini, E; Yang, F; di Gregorio, P; Rando, A; Masina, P

    1991-01-01

    In the sheep alpha alpha alpha globin gene haplotype, the three genes display from the 5' to the 3' end the percentage efficiencies of about 30:14:6, as indicated by the amounts of the three types of alpha chain produced in the alpha alpha alpha/alpha alpha alpha homozygotes. The 3' gene in the alpha alpha alpha alpha haplotype appears to have an efficiency around 1%, as suggested by analysis of one quadruple alpha homozygote. Moreover, the total outputs of the alpha alpha alpha as well as of the alpha alpha alpha alpha haplotypes do not substantially differ from that of the common alpha alpha haplotype.

  18. The HS2 enhancer of the beta-globin locus control region initiates synthesis of non-coding, polyadenylated RNAs independent of a cis-linked globin promoter.

    Science.gov (United States)

    Ling, Jianhua; Baibakov, Boris; Pi, Wenhu; Emerson, Beverly M; Tuan, Dorothy

    2005-07-29

    The HS2 enhancer in the beta-globin locus control region (LCR) regulates transcription of the globin genes 10-50 kb away. Earlier studies show that a transcription mechanism initiated by the HS2 enhancer through the intervening DNA in the direction of the cis-linked promoter and gene mediates long-range enhancer function. Here, we further analyzed the enhancer-initiated RNAs and their mode of transcription from the HS2 enhancer in the endogenous genome of erythroid K562 cells, in plasmids integrated into K562 cells and in purified DNA used as template in in vitro transcription reactions. We found that the HS2 enhancer was able to initiate transcription autonomously in the absence of a cis-linked globin promoter. The enhancer-initiated, intergenic RNAs were different from the mRNA synthesized at the promoter in several aspects. The enhancer RNAs were synthesized not from a defined site but from multiple sites both within and as far as 1 kb downstream of the enhancer. The enhancer RNAs did not appear to contain a normal cap structure at the 5' ends. They were polyadenylated at multiple sites within 3 kb downstream of their initiation sites and were therefore shorter than 3 kb in lengths. The enhancer RNAs remained in discrete spots within the nucleus and were not processed into mRNA or translated into proteins. These particular features of enhancer-initiated transcription indicate that the transcriptional complex assembled by the enhancer was different from the basal transcription complex assembled at the promoter. The results suggest that in synthesizing non-coding, intergenic RNAs, the enhancer-assembled transcription complex could track through the intervening DNA to reach the basal promoter complex and activate efficient mRNA synthesis from the promoter.

  19. Molecular nature of alpha-globin genes in the Saudi population

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    J. Francis Borgio

    2015-11-01

    Full Text Available Alpha-thalassemia (α-thal is a disorder caused by the deletion of single or double α-globin genes, and/or point mutations in the α-globin genes. There are 2 common types of α-globin genes; HBA2 and HBA1. Recently, it has been discovered that the HBA2 gene is replaced by a unique HBA12 gene convert in 5.7% of the Saudi population. The α-globin genes have been emerging as a molecular target for the treatment of β-thalassemia (β-thal. Hence, it is essential to understand the molecular nature of α-globin genes to treat the most prevalent hemoglobin disorders, such as sickle cell disease, α-thal, and β-thal prevalent in the Kingdom of Saudi Arabia. Thirty-two different α-globin genotypes have been observed in the Saudi population. This review outlines the classification of the α-globin genes on the basis of their molecular nature and complex combinations of α-globin genes, and their variants predominant in Saudis.

  20. Variations in the β-Globin genes of Sickle Cell Anaemia Patients in ...

    African Journals Online (AJOL)

    2016-07-28

    Jul 28, 2016 ... Department Ahmadu Bello University (ABUTH) Zaria, Kaduna State, Nigeria. Methods and Material: The buccal swab specimens were collected and β-globin gene DNA sequencing was done. The sequences obtained were compared with a. Genbank Reference β-globin gene (NC_000011.9) using Basic ...

  1. The role of EKLF in human β-globin gene competition.

    NARCIS (Netherlands)

    M.G.J.M. Wijgerde (Mark); J.H. Gribnau (Joost); T. Trimborn (Tolleiv); B. Nuez (Beatriz); J.N.J. Philipsen (Sjaak); F.G. Grosveld (Frank); P.J. Fraser (Peter)

    1996-01-01

    textabstractWe have investigated the role of erythroid Kruppel-like factor (EKLF) in expression of the human beta-globin genes in compound EKLF knockout/human beta-locus transgenic mice. EKLF affects only the adult mouse beta-globin genes in homozygous knockout mice; heterozygous mice are

  2. Activation of Fetal γ-globin Gene Expression via Direct Protein Delivery of Synthetic Zinc-finger DNA-Binding Domains

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    Mir A Hossain

    2016-01-01

    Full Text Available Reactivation of γ-globin expression has been shown to ameliorate disease phenotypes associated with mutations in the adult β-globin gene, including sickle cell disease. Specific mutations in the promoter of the γ-globin genes are known to prevent repression of the genes in the adult and thus lead to hereditary persistence of fetal hemoglobin. One such hereditary persistence of fetal hemoglobin is associated with a sequence located 567 bp upstream of the Gγ-globin gene which assembles a GATA-containing repressor complex. We generated two synthetic zinc-finger DNA-binding domains (ZF-DBDs targeting this sequence. The -567Gγ ZF-DBDs associated with high affinity and specificity with the target site in the γ-globin gene promoter. We delivered the -567Gγ ZF-DBDs directly to primary erythroid cells. Exposure of these cells to the recombinant -567Gγ ZF-DBDs led to increased expression of the γ-globin gene. Direct protein delivery of ZF-DBDs that compete with transcription regulatory proteins will have broad implications for modulating gene expression in analytical or therapeutic settings.

  3. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids

    DEFF Research Database (Denmark)

    Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S

    2008-01-01

    Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian...... DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent...... cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor...

  4. A tissue and developmental specific enhancer is located downstream from the human β-globin gene.

    NARCIS (Netherlands)

    G. Kollias (George); J. Hurst; E. de Boer (Ernie); F.G. Grosveld (Frank)

    1987-01-01

    textabstractThe human P-globin gene is part of a multigene family and is expressed specifically in adult human erythroid tissue (for review, 1). When the human P-globin is introduced into fertilized mouse eggs, it is first activated in foetal liver and remains expressed in adult erythroid tissues

  5. Rapid and Sensitive Assessment of Globin Chains for Gene and Cell Therapy of Hemoglobinopathies

    NARCIS (Netherlands)

    Loucari, C.C. (Constantinos C.); Patsali, P. (Petros); T.B. van Dijk (Thamar); Stephanou, C. (Coralea); Papasavva, P. (Panayiota); Zanti, M. (Maria); Kurita, R. (Ryo); Nakamura, Y. (Yukio); S. Christou (Soteroula); Sitarou, M. (Maria); J.N.J. Philipsen (Sjaak); C.W. Lederer (Carsten); M. Kleanthous (Marina)

    2018-01-01

    textabstractThe β-hemoglobinopathies sickle cell anemia and β-thalassemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains because it indicates the level of anemia, likely toxicity of excess or aberrant globins, and therapeutic potential of

  6. Position-independent high level expression of the human β-globin gene in transgenic mice.

    NARCIS (Netherlands)

    F.G. Grosveld (Frank); G. Blom van Assendelft (Greet); D.R. Greaves (David); G. Kollias (George)

    1987-01-01

    textabstractWe have constructed a "minilocus" that contains the 5' and 3' flanking regions of the human beta-globin locus and the beta-globin gene. These regions are characterized by erythroid-specific DNAase I-superhypersensitive sites and are normally located approximately 50 kb 5' and 20 kb 3' of

  7. Geographical distribution of β-globin gene mutations in Syria.

    Science.gov (United States)

    Murad, Hossam; Moasses, Faten; Dabboul, Amir; Mukhalalaty, Yasser; Bakoor, Ahmad Omar; Al-Achkar, Walid; Jarjour, Rami A

    2018-04-11

    Objectives β-Thalassemia disease is caused by mutations in the β-globin gene. This is considered as one of the common genetic disorders in Syria. The aim of this study was to identify the geographical distribution of the β-thalassemia mutations in Syria. Methods β-Globin gene mutations were characterized in 636 affected patients and 94 unrelated carriers using the amplification refractory mutations system-polymerase chain reaction technique and DNA sequencing. Results The study has revealed the presence of 38 β-globin gene mutations responsible for β-thalassemia in Syria. Important differences in regional distribution were observed. IVS-I.110 [G > A] (22.2%), IVS-I.1 [G > A] (17.8%), Cd 39 [C > T] (8.2%), IVS-II.1 [G > A] (7.6%), IVS-I.6 [T > C] (7.1%), Cd 8 [-AA] (6%), Cd 5 [-CT] (5.6%) and IVS-I.5 [G > C] (4.1%) were the eight predominant mutations found in our study. The coastal region had higher relative frequencies (37.9 and 22%) than other regions. A clear drift in the distribution of the third common Cd 39 [C > T] mutation in the northeast region (34.8%) to the northwest region (2.5%) was noted, while the IVS-I.5 [G > C] mutation has the highest prevalence in north regions. The IVS-I.6 [T > C] mutation had a distinct frequency in the middle region. Ten mutations -86 [C > G], -31 [A > G], -29 [A > G], 5'UTR; +22 [G > A], CAP + 1 [A > C], Codon 5/6 [-TG], IVS-I (-3) or codon 29 [C > T], IVS-I.2 [T > A], IVS-I.128 [T > G] and IVS-II.705 [T > G] were found in Syria for the first time. Conclusions These data will significantly facilitate the population screening, genetic counseling and prenatal diagnosis in Syrian population.

  8. Ruminant globin gene structures suggest an evolutionary role for Alu-type repeats.

    OpenAIRE

    Schimenti, J C; Duncan, C H

    1984-01-01

    Bovine fetal and adult globin genes were cloned and subjected to DNA sequence analysis. Both of these genes contained insertions of Alu-type repetitive DNA within their introns. Comparison of cow and goat beta-type globin genes indicates that intragenic DNA insertions played a role in their evolution. These data support the theory that Alu-type repeats maintain genetic diversity by inhibiting gene conversion.

  9. Regulated expression of human A γ-, β-, and hybrid γ β-globin genes in transgenic mice: manipulation of the developmental expression patterns.

    NARCIS (Netherlands)

    G. Kollias (George); N. Wrighton; J. Hurst; F.G. Grosveld (Frank)

    1986-01-01

    textabstractWe have introduced the human fetal gamma- and adult beta-globin genes into the germ line of mice. Analysis of the resulting transgenic mice shows that the human gamma-globin gene is expressed like an embryonic mouse globin gene; the human beta-globin gene is expressed (as previously

  10. Rapid and Sensitive Assessment of Globin Chains for Gene and Cell Therapy of Hemoglobinopathies

    Science.gov (United States)

    Loucari, Constantinos C.; Patsali, Petros; van Dijk, Thamar B.; Stephanou, Coralea; Papasavva, Panayiota; Zanti, Maria; Kurita, Ryo; Nakamura, Yukio; Christou, Soteroulla; Sitarou, Maria; Philipsen, Sjaak; Lederer, Carsten W.; Kleanthous, Marina

    2018-01-01

    The β-hemoglobinopathies sickle cell anemia and β-thalassemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains because it indicates the level of anemia, likely toxicity of excess or aberrant globins, and therapeutic potential of induced or exogenous β-like globins. Reversed-phase high-performance liquid chromatography (HPLC) allows versatile and inexpensive globin quantification, but commonly applied protocols suffer from long run times, high sample requirements, or inability to separate murine from human β-globin chains. The latter point is problematic for in vivo studies with gene-addition vectors in murine disease models and mouse/human chimeras. This study demonstrates HPLC-based measurements of globin expression (1) after differentiation of the commonly applied human umbilical cord blood–derived erythroid progenitor-2 cell line, (2) in erythroid progeny of CD34+ cells for the analysis of clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of the globin regulator BCL11A, and (3) of transgenic mice holding the human β-globin locus. At run times of 8 min for separation of murine and human β-globin chains as well as of human γ-globin chains, and with routine measurement of globin-chain ratios for 12 nL of blood (tested for down to 0.75 nL) or of 300,000 in vitro differentiated cells, the methods presented here and any variant-specific adaptations thereof will greatly facilitate evaluation of novel therapy applications for β-hemoglobinopathies. PMID:29325430

  11. Nucleotide sequence of the goat embryonic alpha globin gene (zeta) and linkage and evolutionary analysis of the complete alpha globin cluster.

    Science.gov (United States)

    Wernke, S M; Lingrel, J B

    1986-12-05

    In previous studies we identified and sequenced clones containing two adult alpha globin genes of the goat. Additional studies have revealed the presence of an embryonic alpha globin gene termed zeta. Sequence analysis of the gene shows that it is the largest mammalian or avian globin gene cloned to date. Its unusual size is mainly due to a 14 base-pair tandem repeat sequence in its first intron. A similar sequence is also found in the first intron of the human zeta gene. The goat zeta coding sequence differs greatly from that of the adult alpha, particularly at amino acid position 38, where it codes for the amino acid replacement of Gln for Thr. This change may confer a higher intrinsic O2 affinity on the zeta globin protein, ensuring a sufficient O2 supply for the developing goat embryo. The cloning and sequencing of this gene completes the alpha globin locus of the goat, composed of three genes in the following order 5'-zeta-I alpha-II alpha-3'. Evolutionary comparisons of the goat alpha locus with other amphibian, avian and mammalian loci reveal several interesting features. Statistical analysis confirms the hypothesis that the embryonic alpha gene is much older (400 million years) than the embryonic beta gene (200 million years), and that it is descended from a primordial gene, whose present-day counterpart is the Xenopus larval alpha globin gene. Our results also suggest that after the divergence of the avian line, the alpha A gene converted the alpha D gene during the evolution of the pre-mammalian line. The alpha D globin gene remains unconverted in the avian line, potentially because of insertion/deletion sequences that may prevent any gene conversion event. The divergence rates of specific globin genes have been analyzed and found to form an essentially straight line, in agreement with the neutralist view of evolution.

  12. Effect of ATRX and G-Quadruplex Formation by the VNTR Sequence on α-Globin Gene Expression.

    Science.gov (United States)

    Li, Yue; Syed, Junetha; Suzuki, Yuki; Asamitsu, Sefan; Shioda, Norifumi; Wada, Takahito; Sugiyama, Hiroshi

    2016-05-17

    ATR-X (α-thalassemia/mental retardation X-linked) syndrome is caused by mutations in chromatin remodeler ATRX. ATRX can bind the variable number of tandem repeats (VNTR) sequence in the promoter region of the α-globin gene cluster. The VNTR sequence, which contains the potential G-quadruplex-forming sequence CGC(GGGGCGGGG)n , is involved in the downregulation of α-globin expression. We investigated G-quadruplex and i-motif formation in single-stranded DNA and long double-stranded DNA. The promoter region without the VNTR sequence showed approximately twofold higher luciferase activity than the promoter region harboring the VNTR sequence. G-quadruplex stabilizers hemin and TMPyP4 reduced the luciferase activity, whereas expression of ATRX led to a recovery in reporter activity. Our results demonstrate that stable G-quadruplex formation by the VNTR sequence downregulates the expression of α-globin genes and that ATRX might bind to and resolve the G-quadruplex. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Rearrangements of the beta-globin gene cluster in apparently typical betaS haplotypes.

    Science.gov (United States)

    Zago, M A; Silva, W A; Gualandro, S; Yokomizu, I K; Araujo, A G; Tavela, M H; Gerard, N; Krishnamoorthy, R; Elion, J

    2001-02-01

    The majority of the chromosomes with the betaS gene have one of the five common haplotypes, designated as Benin, Bantu, Senegal, Cameroon, and Arab-Indian haplotypes. However, 5-10% of the chromosomes have less common haplotypes, usually referred to as atypical haplotypes. We have demonstrated that most atypical haplotypes are generated by recombinations. The present study was carried out in order to explore whether recombination also occurs in chromosomes with the common (or typical) haplotypes. We screened the HS-2 region of the beta-globin gene locus control region (LCR) in 244 sickle cell patients who had typical restriction fragment length polymorphism (RFLP)-defined haplotypes of the betaS-gene cluster. For 14 cases in which the expected and the observed LCR repeat-sequence sizes were discrepant, the analysis was extended to other unexplored polymorphic markers of the bS-globin gene cluster, i.e.: pre-Ggamma framework, pre-Ggamma 6-bp deletion, HS-2 LCR (AT)xR(AT)y and pre-beta(AT)xTy repeats, and the intragenic beta-globin gene framework. In all 14 cases (15 chromosomes) in which the LCR repeat-sequence sizes were discrepant, a recombination involving a typical 3' segment of the betaS globin gene cluster was demonstrated. In most of the cases, the recombination site was located between the beta-globin gene and the betaLCR. Nine cases involving recombination were detected among 156 Brazilian HbS homozygotes and five among 88 African patients homozygotes for the Benin haplotype. INTERPRETATION AND CONCLUSIONS. Thus, 3.1% of apparently typical haplotypes linked to the sickle cell gene involve recombinations similar to those that generate the atypical haplotypes, a finding that reinforces the picture of the beta-globin gene cluster as highly dynamic.

  14. Chicken globin gene transcription is cell lineage specific during the time of the switch

    International Nuclear Information System (INIS)

    Lois, R.; Martinson, H.G.

    1989-01-01

    Posttranscriptional silencing of embryonic globin gene expression occurs during hemoglobin switching in chickens. Here the authors use Percoll density gradients to fractionate the red blood cells of 5-9 day embryos in order to determine the cellular source and the timing of this posttranscriptional process. By means of nuclear run-on transcription in vitro they show that it is within mature primitive cells that production of embryonic globin mRNA is terminated posttranscriptionally. In contrast, young definitive cells produce little (or no) embryonic globin mRNA because of regulation at the transcriptional level. Thus the lineage specificity of embryonic and adult globin gene expression is determined transcriptionally, and the posttranscriptional process described by Landes et al. is a property of the senescing primitive cells, not a mechanism operative in the hemoglobin switch. This conclusion is supported by [ 3 H]leucine incorporation experiments on Percoll-fractionated cells which reveal no posttranscriptional silencing of the embryonic genes during the early stages of the switch. In the course of these studies they have noticed a strong transcriptional pause near the second exon of the globin genes which is induced by 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) and which resembles a natural pause near that position

  15. NF-Y recruits both transcription activator and repressor to modulate tissue- and developmental stage-specific expression of human γ-globin gene.

    Directory of Open Access Journals (Sweden)

    Xingguo Zhu

    Full Text Available The human embryonic, fetal and adult β-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal γ-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal γ-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal γ-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed γ-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of γ-globin gene.

  16. Distinctive patterns of evolution of the δ-globin gene (HBD in primates.

    Directory of Open Access Journals (Sweden)

    Ana Moleirinho

    Full Text Available In most vertebrates, hemoglobin (Hb is a heterotetramer composed of two dissimilar globin chains, which change during development according to the patterns of expression of α- and β-globin family members. In placental mammals, the β-globin cluster includes three early-expressed genes, ε(HBE-γ(HBG-ψβ(HBBP1, and the late expressed genes, δ (HBD and β (HBB. While HBB encodes the major adult β-globin chain, HBD is weakly expressed or totally silent. Paradoxically, in human populations HBD shows high levels of conservation typical of genes under strong evolutionary constraints, possibly due to a regulatory role in the fetal-to-adult switch unique of Anthropoid primates. In this study, we have performed a comprehensive phylogenetic and comparative analysis of the two adult β-like globin genes in a set of diverse mammalian taxa, focusing on the evolution and functional divergence of HBD in primates. Our analysis revealed that anthropoids are an exception to a general pattern of concerted evolution in placental mammals, showing a high level of sequence conservation at HBD, less frequent and shorter gene conversion events. Moreover, this lineage is unique in the retention of a functional GATA-1 motif, known to be involved in the control of the developmental expression of the β-like globin genes. We further show that not only the mode but also the rate of evolution of the δ-globin gene in higher primates are strictly associated with the fetal/adult β-cluster developmental switch. To gain further insight into the possible functional constraints that have been shaping the evolutionary history of HBD in primates, we calculated dN/dS (ω ratios under alternative models of gene evolution. Although our results indicate that HBD might have experienced different selective pressures throughout primate evolution, as shown by different ω values between apes and Old World Monkeys + New World Monkeys (0.06 versus 0.43, respectively, these estimates

  17. The entire β-globin gene cluster is deleted in a form of τδβ-thalassemia.

    NARCIS (Netherlands)

    E.R. Fearon; H.H.Jr. Kazazian; P.G. Waber (Pamela); J.I. Lee (Joseph); S.E. Antonarakis; S.H. Orkin (Stuart); E.F. Vanin; P.S. Henthorn; F.G. Grosveld (Frank); A.F. Scott; G.R. Buchanan

    1983-01-01

    textabstractWe have used restriction endonuclease mapping to study a deletion involving the beta-globin gene cluster in a Mexican-American family with gamma delta beta-thalassemia. Analysis of DNA polymorphisms demonstrated deletion of the beta-globin gene from the affected chromosome. Using a DNA

  18. Dominant control region of the human β- like globin gene cluster

    NARCIS (Netherlands)

    Blom van Assendelft, Margaretha van

    1989-01-01

    The structure and regulation of the human β -like globin gene cluster has been studied extensively. Genetic disorders connected with this gene cluster are responsible for human diseases associated with high levels of morbidity and mortality, such as β-thalassaemia and sickle cell anaemia. The work

  19. TBP binding and the rate of transcription initiation from the human β-globin gene.

    NARCIS (Netherlands)

    M. Antoniou (Michael); E. Spanopoulou; F.G. Grosveld (Frank); E. de Boer (Ernie)

    1995-01-01

    textabstractDNA-protein interaction studies in vitro revealed several factors binding over the TATA box and the region of transcription initiation (cap) site of the human beta-globin promoter; TATA binding protein TBP at -30, Sp1 at -19, GATA-1 at -12 and +5, YY1 at -9 and a novel factor C1 over the

  20. Retroviral-mediated transfer and expression of human β-globin genes in cultured murine and human erythroid cells

    International Nuclear Information System (INIS)

    Weber-Benarous, A.; Cone, R.D.; London, I.M.; Mulligan, R.C.

    1988-01-01

    The authors cloned human β-globin DNA sequences from a genomic library prepared from DNA isolated from the human leukemia cell line K562 and have used the retroviral vector pZip-NeoSV(X)1 to introduce a 3.0-kilobase segment encompassing the globin gene into mouse erythroleukemia cells. Whereas the endogenous K562 β-globin gene is repressed in K562 cells, when introduced into mouse erythroleukemia cells by retroviral-mediated gene transfer, the β-globin gene from K562 cells was transcribed and induced 5-20-fold after treatment of the cells with dimethyl sulfoxide. The transcripts were correctly initiated, and expression and regulation of the K562 gene were identical to the expression of a normal human β-globin gene transferred into mouse erythroleukemia cells in the same way. They have also introduced the normal human β-globin gene into K562 cells using the same retrovirus vector. SP6 analysis of the RNA isolated from the transduced cells showed that the normal β-globin gene was transcribed at a moderately high level, before or after treatment with hemin. Based on these data, they suggest that the lack of expression of the endogenous β-globin gene in K562 cells does not result from an alteration in the gene itself and may not result from a lack of factor(s) necessary for β-lobin gene transcription. Retroviral-mediated transfer of the human β-globin gene may, however, uniquely influence expression of the gene K562 cells

  1. Gene conversion of two functional goat alpha-globin genes preserves only minimal flanking sequences.

    Science.gov (United States)

    Schon, E A; Wernke, S M; Lingrel, J B

    1982-06-25

    We have determined the complete nucleotide sequence of the nonallelic adult goat I alpha- and II alpha-globin genes and, as is the case with the duplicated human alpha-to each other. Such high homology (99%) has most likely been preserved via a gene conversion mechanism. The conversion unit in goats is only about 9000 base pairs in length, and contained within this short region are all the known signals required for accurate and efficient transcription, with the CCAAT box adjacent to the 5'-boundary of the conversion unit and the poly(A) addition site adjacent to the 3' end. This conversion unit is also flanked by a 23-base-pair direct repeat "boundary sequence," vestiges of which are also observable in the human and mouse alpha-globin genes and pseudogenes. These direct repeats imply that a transposition-like event may have been responsible for the insertion of an ancestral alpha-like sequence into a new chromosomal locus, and that this insertion event and subsequent gene duplication may have predated the mammalian radiation.

  2. Molecular evolution of globin genes in Gymnotiform electric fishes: relation to hypoxia tolerance.

    Science.gov (United States)

    Tian, Ran; Losilla, Mauricio; Lu, Ying; Yang, Guang; Zakon, Harold

    2017-02-13

    Nocturnally active gymnotiform weakly electric fish generate electric signals for communication and navigation, which can be energetically taxing. These fish mainly inhabit the Amazon basin, where some species prefer well-oxygenated waters and others live in oxygen-poor, stagnant habitats. The latter species show morphological, physiological, and behavioral adaptations for hypoxia-tolerance. However, there have been no studies of hypoxia tolerance on the molecular level. Globins are classic respiratory proteins. They function principally in oxygen-binding and -delivery in various tissues and organs. Here, we investigate the molecular evolution of alpha and beta hemoglobins, myoglobin, and neuroglobin in 12 gymnotiforms compared with other teleost fish. The present study identified positively selected sites (PSS) on hemoglobin (Hb) and myoglobin (Mb) genes using different maximum likelihood (ML) methods; some PSS fall in structurally important protein regions. This evidence for the positive selection of globin genes suggests that the adaptive evolution of these genes has helped to enhance the capacity for oxygen storage and transport. Interestingly, a substitution of a Cys at a key site in the obligate air-breathing electric eel (Electrophorus electricus) is predicted to enhance oxygen storage of Mb and contribute to NO delivery during hypoxia. A parallel Cys substitution was also noted in an air-breathing African electric fish (Gymnarchus niloticus). Moreover, the expected pattern under normoxic conditions of high expression of myoglobin in heart and neuroglobin in the brain in two hypoxia-tolerant species suggests that the main effect of selection on these globin genes is on their sequence rather than their basal expression patterns. Results indicate a clear signature of positive selection in the globin genes of most hypoxia-tolerant gymnotiform fishes, which are obligate or facultative air breathers. These findings highlight the critical role of globin genes in

  3. Activation of the alpha-globin gene expression correlates with dramatic upregulation of nearby non-globin genes and changes in local and large-scale chromatin spatial structure.

    Science.gov (United States)

    Ulianov, Sergey V; Galitsyna, Aleksandra A; Flyamer, Ilya M; Golov, Arkadiy K; Khrameeva, Ekaterina E; Imakaev, Maxim V; Abdennur, Nezar A; Gelfand, Mikhail S; Gavrilov, Alexey A; Razin, Sergey V

    2017-07-11

    In homeotherms, the alpha-globin gene clusters are located within permanently open genome regions enriched in housekeeping genes. Terminal erythroid differentiation results in dramatic upregulation of alpha-globin genes making their expression comparable to the rRNA transcriptional output. Little is known about the influence of the erythroid-specific alpha-globin gene transcription outburst on adjacent, widely expressed genes and large-scale chromatin organization. Here, we have analyzed the total transcription output, the overall chromatin contact profile, and CTCF binding within the 2.7 Mb segment of chicken chromosome 14 harboring the alpha-globin gene cluster in cultured lymphoid cells and cultured erythroid cells before and after induction of terminal erythroid differentiation. We found that, similarly to mammalian genome, the chicken genomes is organized in TADs and compartments. Full activation of the alpha-globin gene transcription in differentiated erythroid cells is correlated with upregulation of several adjacent housekeeping genes and the emergence of abundant intergenic transcription. An extended chromosome region encompassing the alpha-globin cluster becomes significantly decompacted in differentiated erythroid cells, and depleted in CTCF binding and CTCF-anchored chromatin loops, while the sub-TAD harboring alpha-globin gene cluster and the upstream major regulatory element (MRE) becomes highly enriched with chromatin interactions as compared to lymphoid and proliferating erythroid cells. The alpha-globin gene domain and the neighboring loci reside within the A-like chromatin compartment in both lymphoid and erythroid cells and become further segregated from the upstream gene desert upon terminal erythroid differentiation. Our findings demonstrate that the effects of tissue-specific transcription activation are not restricted to the host genomic locus but affect the overall chromatin structure and transcriptional output of the encompassing

  4. Dual role for the methyltransferase G9a in the maintenance of beta-globin gene transcription in adult erythroid cells.

    Science.gov (United States)

    Chaturvedi, Chandra-Prakash; Hosey, Alison M; Palii, Carmen; Perez-Iratxeta, Carolina; Nakatani, Yoshihiro; Ranish, Jeffrey A; Dilworth, F Jeffrey; Brand, Marjorie

    2009-10-27

    Using a proteomics screen, we have identified the methyltransferase G9a as an interacting partner of the hematopoietic activator NF-E2. We show that G9a is recruited to the beta-globin locus in a NF-E2-dependent manner and spreads over the entire locus. While G9a is often regarded as a corepressor, knocking down this protein in differentiating adult erythroid cells leads to repression of the adult beta(maj) globin gene and aberrant reactivation of the embryonic beta-like globin gene E(y). While in adult cells G9a maintains E(y) in a repressed state via dimethylation of histone H3 at lysines 9 and 27, it activates beta(maj) transcription in a methyltransferase-independent manner. Interestingly, the demethylase UTX is recruited to the beta(maj) (but not the E(y)) promoter where it antagonizes G9a-dependent H3K27 dimethylation. Collectively, these results reveal a dual role for G9a in maintaining proper expression (both repression and activation) of the beta-globin genes in differentiating adult erythroid cells.

  5. Association of Xmn I Polymorphism and Hemoglobin E Haplotypes on Postnatal Gamma Globin Gene Expression in Homozygous Hemoglobin E

    Directory of Open Access Journals (Sweden)

    Supachai Ekwattanakit

    2012-01-01

    Full Text Available Background and Objectives. To explore the role of cis-regulatory sequences within the β globin gene cluster at chromosome 11 on human γ globin gene expression related to Hb E allele, we analyze baseline hematological data and Hb F values together with β globin haplotypes in homozygous Hb E. Patients and Methods. 80 individuals with molecularly confirmed homozygous Hb E were analyzed for the β globin haplotypes and Xmn I polymorphism using PCR-RFLPs. 74 individuals with complete laboratory data were further studied for association analyses. Results. Eight different β globin haplotypes were found linked to Hb E alleles; three major haplotypes were (a (III, (b (V, and (c (IV accounting for 94% of Hb E chromosomes. A new haplotype (Th-1 was identified and most likely converted from the major ones. The majority of individuals had Hb F < 5%; only 10.8% of homozygous Hb E had high Hb F (average 10.5%, range 5.8–14.3%. No association was found on a specific haplotype or Xmn I in these individuals with high Hb F, measured by alkaline denaturation. Conclusion. The cis-regulation of γ globin gene expression might not be apparent under a milder condition with lesser globin imbalance such as homozygous Hb E.

  6. Beta-globin gene linked DNA haplotypes and frameworks in three South-East Asian populations.

    Science.gov (United States)

    Hundrieser, J; Sanguansermsri, T; Papp, T; Laig, M; Flatz, G

    1988-09-01

    DNA haplotypes and frameworks (numbers in parenthesis) linked to the beta-globin gene were determined by restriction fragment analysis using eight restriction endonucleases on 86 (97) chromosomes bearing the normal beta-globin gene (HBB*A) and 108 (118) chromosomes bearing HBB*E in subjects homozygous for HBB*A or HBB*E from three South-East Asian populations with high HBB*E frequencies (northern Thailand, north-eastern Thailand and Cambodia). A systematic nomenclature for beta-globin gene-linked haplotype characterized by six polymorphic sites is introduced. In all populations, HBB*A occurred preferentially (greater than 80%) in linkage with the haplotype 41 (+----+) and all three frameworks described by Antonarakis et al. (1982). In contrast, almost 80% of the HBB*E genes occurred with the haplotype 27 (-+- ). In northern and north-eastern Thailand, HBB*E was present almost exclusively in frame-work 2; HBB*E in framework 3 (Asian) was limited to the Khmer population of Cambodia, and the frequency of HBB*E-linked framework 3 increased from the west to the east in this country.

  7. Characteristic beta-globin gene cluster haplotypes of Evenkis and Oroqens in north China.

    Science.gov (United States)

    Shimizu, Koji; Marubayashi, Azusa; Tokimasa, Kozue; Harihara, Shinji; Omoto, Keiichi; Imanishi, Tadashi; Hao, Luping; Jin, Feng

    2004-10-01

    Haplotype frequencies of the beta-globin gene cluster were estimated for 114 Evenkis and 81 Oroqens from northeast China, and their characteristics were compared with those in Japanese, Koreans, and three Colombian Amerindian groups of South America (Wayuu, Kamsa, and Inga tribes). A major 5' subhaplotype (5' to the delta-globin gene) was + - - - - in Evenkis, whereas + - - - -, - + + - +, and - + - + + were the major subhaplotypes in Oroqens. One possible candidate for an ancestral 5' subhaplotype, - - - - -, was found in one Evenki (0.5%) and three Oroqen chromosomes (2.0%). They were observed as heterozygous forms for + ---- and -----. Major haplotypes were +-----+, + -----+-, and + - - - - + + in Evenkis, whereas they were +-----+,-++-+-+, +----+-, and -+-++-+ in Oroqens. The lowest Nei's genetic distance values of Evenkis or Oroqens based on the 5' subhaplotype frequency distributions were observed in relation to the Wayuu or Koreans, respectively, but those of Evenkis and Oroqens based on the haplotype frequency distributions were found in relation to Koreans.

  8. Restriction site polymorphisms in the pig beta-globin gene cluster.

    Science.gov (United States)

    Rando, A; Masina, P

    1985-01-01

    A restriction fragment length polymorphism was detected in pig DNA digested with Hind III restriction endonuclease and probed with rabbit beta 1-globin gene. Eight different phenotypes were observed and for six of them family data demonstrated that they are determined by three alleles. As this polymorphism is not found with four other restriction endonucleases (Bam HI, Eco RI, Kpn I, and Pst I), single point mutations are proposed to explain the observed differences.

  9. Regulation of the β-globin gene family expression, useful in the search for new therapeutic targets for hemoglobinopathies

    Directory of Open Access Journals (Sweden)

    Karen G. Scheps

    2016-12-01

    Full Text Available Different hemoglobin isoforms are expressed during the embryonic, fetal and postnatal stages. They are formed by combination of polypeptide chains synthesized from the α- and β-globin gene clusters. Based on the fact that the presence of high hemoglobin F levels is beneficial in both sickle cell disease and severe thalassemic syndromes, a revision of the regulation of the β-globin cluster expression is proposed, especially regarding the genes encoding the y-globin chains (HBG1 and HBG2. In this review we describe the current knowledge about transcription factors and epigenetic regulators involved in the switches of the β-globin cluster. It is expected that the consolidation of knowledge in this field will allow finding new therapeutic targets for the treatment of hemoglobinopathies.

  10. EFFECT OF CIS ACTING POTENTIAL REGULATORS IN THE ß GLOBIN GENE CLUSTER ON THE PRODUCTION OF HBF IN THALASSEMIA PATIENTS

    Directory of Open Access Journals (Sweden)

    Anita Nadkarni

    2013-02-01

    Full Text Available The clinical presentation of   b-thalassemia intermedia phenotypes are influenced by many factors .The persistence of fetal hemoglobin and  several polymorphisms located in the promoters of  g- and b-globin genes are some of them .The aim of this study was to evaluate the combined effect of  the -158Gg (CàT polymorphism and of the (ATx(Ty configuration, as well as their eventual association with elevated levels of HbF  in  b-thalassemia carriers, b-thalassemia Intermedia , b-thalassemia major and normal controls of Indian origin. The -158 Gg T allele was found to be associated with increased levels of HbF in b-thalassemia carriers, and not in wild-type subjects. In the homozygous group the -158 Gg T allele was significantly higher in the thalassemia intermedia group (66% as against the thalassemia major group (21%. The (AT9(T5 allele did not show any association with raised HbF levels. However 24% of milder cases showed presence of this allele. This study suggests that two regions of the b globin cluster, whether in cis or in trans to each other, can interact to enhance HbF expression when a b thalassemic determinant is present in heterozigosity and help in amelioration of the severity of the disease in homozygotes.

  11. Total alpha-globin gene cluster deletion has high frequency in Filipinos

    Energy Technology Data Exchange (ETDEWEB)

    Hunt, J.A.; Haruyama, A.Z.; Chu, B.M. [Kapiolani Medical Center, Honolulu, HI (United States)] [and others

    1994-09-01

    Most {alpha}-thalassemias [Thal] are due to large deletions. In Southeast Asians, the (--{sup SEA}) double {alpha}-globin gene deletion is common, 3 (--{sup Tot}) total {alpha}-globin cluster deletions are known: Filipino (--{sup Fil}), Thai (--{sup Thai}), and Chinese (--{sup Chin}). In a Hawaii Thal project, provisional diagnosis of {alpha}-Thal-1 heterozygotes was based on microcytosis, normal isoelectric focusing, and no iron deficiency. One in 10 unselected Filipinos was an {alpha}-Thal-1 heterozygote, 2/3 of these had a (--{sup Tot}) deletion: a {var_sigma}-cDNA probe consistently showed fainter intensity of the constant 5.5 kb {var_sigma}{sub 2} BamHI band, with no heterzygosity for {var_sigma}-globin region polymorphisms; {alpha}-cDNA or {var_sigma}-cDNA probes showed no BamHI or BglII bands diagnostic of the (--{sup SEA}) deletion; bands for the (-{alpha}) {alpha}-Thal-2 single {alpha}-globin deletions were only seen in Hb H cases. A reliable monoclonal anti-{var_sigma}-peptide antibody test for the (--{sup SEA}) deletion was always negative in (--{sup Tot}) samples. Southern digests with the Lo probe, a gift from D. Higgs of Oxford Univ., confirmed that 49 of 50 (--{sup Tot}) chromosomes in Filipinos were (--{sup Fil}). Of 20 {alpha}-Thal-1 hydrops born to Filipinos, 11 were (--{sup Fil}/--{sup SEA}) compound heterozygotes; 9 were (--{sup SEA}/--{sup SEA}) homozygotes, but none was a (--{sup Fil}/--{sup Fil}).

  12. Triplication of alpha-globin genes is responsible for unusual alpha 113Leu/alpha 113His-globin chain ratios in sheep.

    Science.gov (United States)

    Vestri, R; Masina, P; Rando, A; Testa, A; Di Gregorio, P

    1987-10-01

    By investigations at the DNA and protein level, it has been shown that in sheep a previously detected, presumed quantitative allele of the II alpha 113His gene, displaying a reduced efficiency (called the II alpha 113His decreases gene), is carried by a chromosome bearing three alpha-globin loci. In particular, five sheep having an alpha 113Leu/alpha 113His-chain ratio of about 13:1 (13:1 phenotype) possessed the -I alpha 113Leu-II alpha 113Leu-/-I alpha 113Leu-II alpha 113Leu-III alpha 113His decreases genotype. One sheep showing a alpha 113Leu/alpha 113His-chain ratio of about 3:1 (3:1 phenotype) had the -I alpha 113Leu-II alpha 113His-/-I alpha 113Leu-II alpha 113Leu-III alpha 113His decreases genotype, while one sheep having a chain ratio of about 6:1 (6:1 phenotype) carried the -I alpha 113Leu-II alpha 113Leu-II alpha 113His decreases-/-I alpha 113Leu-II alpha 113Leu-III alpha 113His decreases genotype. Nineteen sheep, displaying the common phenotypes, all possessed the alpha alpha/alpha alpha gene arrangement. Furthermore, the possible location of the gene with reduced efficiency and the expression of the three genes in the triple alpha-globin loci chromosome are discussed.

  13. Two Bg1II RFLPs of the human. alpha. -globin gene cluster in the American sickle cell population

    Energy Technology Data Exchange (ETDEWEB)

    Embury, S.H.; Blachman, T.; Kroop, G.L.; Suzuki, J.K.; Boyle, M. (Univ. of California and Northern California Comprehensive Sickle Cell Center, San Fransicso (USA))

    1989-11-11

    Human {alpha}-globin cDNA cloned into plasmid pMB9(JW101) was used as a hybridization probe for assessing the {alpha}-globin genotypes of 2271 Americans with sickle cell anemia. The normal duplicated human {alpha}-globin genes, {alpha}2 and {alpha}1, residue on separate Bg1 II fragments, each of which is cleaved by Hin dIII. Both {alpha} loci reside on a single 14 kb Bam HI fragment. The authors performed single Bg1 II and BAM HI digests to detect {alpha}-globin gene deletions in 2271 subjects enrolled in the National Cooperative Study of Sickel Cell Disease (NCSSCD). In addition to gene deletions and duplications, two Bg1 II RFLP were found. The human {alpha}-globin genes reside on the short arm of chromosome 16. The {alpha}2-specific RFLP occurs in linkage dysequilibrium and the mother of one subject with the {alpha}1-specific RFLP had this RFLP, suggesting their Mendelian inheritance.

  14. the characterization of exon-1 mutation(s) of beta globin gene in beta thalassemia

    International Nuclear Information System (INIS)

    Abass, M.M.E.

    2004-01-01

    β-thalassemia constitutes one of the most serious health problems worldwide, it is the most common chronic hemolytic anemia in egypt. the aim of this work is to study the mutations of exon-1 of β-globin gene in β-thalassaemic children in sharkia governorate. the present study was included 25 healthy children and 50 patients diagnosed as β-thalassemia. this work showed that the thalassaemic patients had significantly decrease in Hb conc . than the control group (p 2 showed a significant increase as compared with the control group

  15. In silico mutation analysis of human beta globin gene in sickle cell disease patients

    OpenAIRE

    Hira Mubeen; Rubab Zahra Naqvi; Ammara Masood; Muhammad Waseem Shoaib; Shahid Raza

    2016-01-01

    Background: Sickle cell disease is an inherited blood disorder that affects red blood cells. People with sickle cell conditions make a different form of hemoglobin a called hemoglobin S. Sickle cell conditions are inherited from parents in much the same way as blood type, hair color and texture, eye color and other physical traits. Sickle cell disease occurs due to a single mutation on the b-globin gene, namely, a substitution of glutamic acid for valine at position 6 of the b chain. Several ...

  16. S1 nuclease analysis of α-globin gene expression in preleukemic patients with acquired hemoglobin H disease after transfer to mouse erythroleukemia cells

    International Nuclear Information System (INIS)

    Helder, J.; Deisseroth, A.

    1987-01-01

    The loss of α-globin gene transcriptional activity rarely occurs as an acquired abnormality during the evolution of myeloproliferative disease or preleukemia. To test whether the mutation responsible for the loss of α-globin gene expression (hemoglobin H disease) in these patients is linked with the α-globin genes on chromosome 16, the authors transferred chromosome 16 from preleukemic patients with acquired hemoglobin H disease to mouse erythroleukemia cells and measured the transcriptional activity of the human α-globin genes. After transfer to mouse erythroleukemia cells, the expression of human α-globin genes from the peripheral blood or marrow cells of preleukemic patients with acquired hemoglobin H disease was similar to that of human α-globin genes transferred to mouse erythroleukemia cells from normal donors. These data showed that factor(s) in the mouse erythroleukemia cell can genetically complement the α-globin gene defect in these preleukemia patients with acquired hemoglobin H disease and suggest that altered expression of a gene in trans to the α-globin gene may be responsible for the acquisition of hemoglobin H disease in these patients

  17. Generation of a high-titer retroviral vector capable of expressing high levels of the human β-globin gene

    NARCIS (Netherlands)

    M. Sadelain (Michel); C.H.J. Wang (Jason); M. Antoniou (Michael); F.G. Grosveld (Frank); R.C. Mulligan

    1995-01-01

    textabstractRetrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and

  18. Same β-globin gene mutation is present on nine different β-thalassemia chromosomes in a Sardinian population

    International Nuclear Information System (INIS)

    Pirastu, M.; Galanello, R.; Doherty, M.A.; Tuveri, T.; Cao, A.; Kan, Y.W.

    1987-01-01

    The predominant β-thalassemia in Sardinia is the β 0 type in which no β-globin chains are synthesized in the homozygous state. The authors determined the β-thalassemia mutations in this population by the oligonucleotide-probe method and defined the chromosome haplotypes on which the mutation resides. The same β/sup 39(CAG→TAG)/ nonsense mutation was found on nine different chromosome haplotypes. Although this mutation may have arisen more than once, the multiple haplotypes could also be generated by crossing over and gene conversion events. These findings underscore the frequency of mutational events in the β-globin gene region

  19. Gamma-interferon alters globin gene expression in neonatal and adult erythroid cells

    International Nuclear Information System (INIS)

    Miller, B.A.; Perrine, S.P.; Antognetti, G.; Perlmutter, D.H.; Emerson, S.G.; Sieff, C.; Faller, D.V.

    1987-01-01

    The effect of gamma-interferon on fetal hemoglobin synthesis by purified cord blood, fetal liver, and adult bone marrow erythroid progenitors was studied with a radioligand assay to measure hemoglobin production by BFU-E-derived erythroblasts. Coculture with recombinant gamma-interferon resulted in a significant and dose-dependent decrease in fetal hemoglobin production by neonatal and adult, but not fetal, BFU-E-derived erythroblasts. Accumulation of fetal hemoglobin by cord blood BFU-E-derived erythroblasts decreased up to 38.1% of control cultures (erythropoietin only). Synthesis of both G gamma/A gamma globin was decreased, since the G gamma/A gamma ratio was unchanged. Picograms fetal hemoglobin per cell was decreased by gamma-interferon addition, but picograms total hemoglobin was unchanged, demonstrating that a reciprocal increase in beta-globin production occurred in cultures treated with gamma-interferon. No toxic effect of gamma-interferon on colony growth was noted. The addition of gamma-interferon to cultures resulted in a decrease in the percentage of HbF produced by adult BFU-E-derived cells to 45.6% of control. Fetal hemoglobin production by cord blood, fetal liver, and adult bone marrow erythroid progenitors, was not significantly affected by the addition of recombinant GM-CSF, recombinant interleukin 1 (IL-1), recombinant IL-2, or recombinant alpha-interferon. Although fetal progenitor cells appear unable to alter their fetal hemoglobin program in response to any of the growth factors added here, the interaction of neonatal and adult erythroid progenitors with gamma-interferon results in an altered expression of globin genes

  20. Screening for mutations in human alpha-globin genes by nonradioactive single-strand conformation polymorphism

    Directory of Open Access Journals (Sweden)

    Jorge S.B.

    2003-01-01

    Full Text Available Point mutations and small insertions or deletions in the human alpha-globin genes may produce alpha-chain structural variants and alpha-thalassemia. Mutations can be detected either by direct DNA sequencing or by screening methods, which select the mutated exon for sequencing. Although small (about 1 kb, 3 exons and 2 introns, the alpha-globin genes are duplicate (alpha2 and alpha1 and highy G-C rich, which makes them difficult to denature, reducing sequencing efficiency and causing frequent artifacts. We modified some conditions for PCR and electrophoresis in order to detect mutations in these genes employing nonradioactive single-strand conformation polymorphism (SSCP. Primers previously described by other authors for radioactive SSCP and phast-SSCP plus denaturing gradient gel electrophoresis were here combined and the resultant fragments (6 new besides 6 original per alpha-gene submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions including two electrophoretic apparatus (PhastSystem(TM and GenePhor(TM, Amersham Biosciences, different polyacrylamide gel concentrations, run temperatures and denaturing agents, and entire and restriction enzyme cut fragments. One hundred percent of sensitivity was achieved with four of the new fragments formed, using the PhastSystem(TM and 20% gels at 15ºC, without the need of restriction enzymes. This nonradioactive PCR-SSCP approach showed to be simple, rapid and sensitive, reducing the costs involved in frequent sequencing repetitions and increasing the reliability of the results. It can be especially useful for laboratories which do not have an automated sequencer.

  1. Total beta-globin gene deletion has high frequency in Filipinos

    Energy Technology Data Exchange (ETDEWEB)

    Patrick, N.; Miyakawa, F.; Hunt, J.A. [Univ. of Hawaii, Honolulu, HI (United States)] [and others

    1994-09-01

    The distribution of {beta}-thalassemia [{beta}{sup Th}] mutations is unique to each ethnic group. Most mutations affect one or a few bases; large deletions have been rare. Among families screened in Hawaii, [{beta}{sup Th}] heterozygotes were diagnosed by microcytosis, absence of abnormal hemoglobins on isoelectric focusing, and raised Hb A{sub 2} by chromatography. Gene frequency for {beta}{sup Th} was 0.02 in Filipinos. In Filipinos, polymerase chain reaction [PCR] with denaturing gradient gel electrophoresis for {beta}{sup Th} mutations detected a mutation in only 6 of 42 {beta}{sup Th} heterozygotes; an IVS2-666 C/T polymorphism showed non-heterozygosity in 37 and heterozygosity in only 5 of these {beta}{sup Th} heterozygotes. One {beta}{sup Th}/{beta}{sup Th} major patient and his mother had no mutation detected by allele-specific oligomer hybridization; PCR failed to amplify any DNA from his {beta}-globin gene. After a total {beta}-globin gene deletion [{beta}{sup Del}] was found in a Filipino family in Ontario, specific PCR amplification for {beta}{sup Del} detected this in 43 of 53 {beta}{sup Th} Filipino samples tested; the above {beta}{sup Th}/{beta}{sup Th} patient was a ({beta}{sup Del}/{beta}{sup Del}) homozygote. The {beta}{sup Del} may account for over 60% of all {beta}{sup Th} alleles in Filipinos; this is the highest proportion of a deletion {beta}{sup Th} mutation reported from any population. Most but not all {beta}{sup Del} heterozygotes had high Hb F [5.13 {plus_minus} 3.94 mean {plus_minus} 1 s.d.] compared to the codon 41/42 four base deletion common in Chinese [2.30 {plus_minus} 0.86], or to {beta}{sup Th} heterozygotes with normal {alpha}-globin genes [2.23 {plus_minus} 0.80].

  2. Role of the duplicated CCAAT box region in γ-globin gene regulation and hereditary persistence of fetal haemoglobin.

    NARCIS (Netherlands)

    A. Ronchi (Antonella); M. Berry (Meera); S. Raguz (Selina); A.M.A. Imam (Ali); N. Yannoutsos (Nikos); S. Ottolenghi (Sergio); F.G. Grosveld (Frank); N.O. Dillon (Niall)

    1996-01-01

    textabstractHereditary persistence of fetal haemoglobin (HPFH) is a clinically important condition in which a change in the developmental specificity of the gamma-globin genes results in varying levels of expression of fetal haemoglobin in the adult. The condition is benign and can significantly

  3. AB089. Prevalence of the most common β-globin gene mutations in Filipino β-thalassemia patients

    Science.gov (United States)

    Silao, Catherine Lynn T.; Fabella, Terence Diane F.; Naranjo, Maria Liza T.; Padilla, Carmencita D.; Yuson, Ernesto D. J.

    2017-01-01

    Background Beta (β)-thalassemia is an autosomal recessive disorder characterized by reduced or absent β-globin chain synthesis resulting in decreased hemoglobin in red blood cells (RBC) and consequently, microcytic, hypochromic anemia with varying degrees of severity. The disease is a result of mutations in the human β-globin (HBB) gene. This genetic disorder is considered an emerging global health burden, especially in Southeast Asia, as 50% of world carriers of β-thalassemia, corresponding to 40 million people, are found in this region alone. Thus, prevalence of HBB gene mutations should be identified in our population. This study aimed to determine the prevalence of common β-globin gene mutations in Filipino β-thalassemia patients. Methods Fifteen [15] patients underwent DNA extraction and Beta globin strip assay mutational analysis. Results The HbE (20%) was the most common allele detected using the strip assay method. Interestingly, a suspected unnamed deletion was detected in 13.33% of patients tested. A high number of normal genotype was also detected which composed majority (60%) of the patients. Conclusions The result of the study suggests the limitation of strip assay method in the detection of Filipino HBB gene mutations. Further analysis of the samples tested via the sequencing method is recommended.

  4. Characterization of a large deletion in the {beta}-globin gene cluster in a newborn with hemoglobin FE

    Energy Technology Data Exchange (ETDEWEB)

    Louie, E.; Dietz, L.; Shafer, F. [Children`s Hosptial, Oakland, CA (United States)] [and others

    1994-09-01

    A sample on a newborn with hemoglobin FE screen results was obtained to investigate whether E/E or B/{beta}{degrees} thalassemia was present using polymerase chain reaction (PCR) methodology. The newborn appeared homozygous for the hemoglobin E mutation in our initial study, but the parents` genotypes did not support this diagnosis. The father is homozygous for the absence of the hemoglobin E mutation (non E/non E) and the mother is heterozygous (E/non E) for this mutation. The limitation of PCR analysis is an assumption that the amplification of the two {beta}-globin alleles is equivalent. A large deletion on one {beta}-globin gene, which would produce E/{beta}{degrees} thalassemia, would be missed if it included part or the entire region subjected to amplification. The family results were consistent with either non-paternity, sample mix-up or such a deletion of the {beta}-globin gene in the father and child. To rule out the possibility of non-paternity, two polymorphic loci (HLA on chromosome 6 and a VNTR system of chromosome 17) that are outside of the {beta}-globin gene were analyzed and show that inheritance is consistent and the likelihood of a sample mix-up is then reduced. We therefore believe there is a gene deletion in this family. At the present time, analyses of the RFLPs that are 5{prime} of the {beta}-globin gene cluster show that the polymorphisms most distal from the 5{prime} {beta}-globin gene are not being inherited as expected. These results support our interpretation that a deletion exists in the father and was inherited by the child. The father`s clinical picture of possible HPFH (the father has 12% hemoglobin F) also supports the interpretation of a deletion in this family. Deletions of the {beta}-globin gene within this ethnic group are rare. Currently, Southern blots on the family are being probed to determine the extent of the putative deletion.

  5. Changes in body temperature pattern in vertebrates do not influence the codon usages of alpha-globin genes.

    Science.gov (United States)

    Hamada, Kazuo; Horiike, Tokumasa; Kanaya, Shigehiko; Nakamura, Hiroshi; Ota, Hidetoshi; Yatogo, Takayuki; Okada, Kazuhisa; Nakamura, Hiroshi; Shinozawa, Takao

    2002-06-01

    Codon usages are known to vary among vertebrates chiefly due to variations in isochore structure. Under the assumption that marked differences exist in isochore structure between warm-blooded and cold-blooded animals, the variations among vertebrates were previously attributed to an adaptation to homeothermy. However, based on data from a turtle species and a crocodile (Archosauromorpha), it was recently proposed that the common ancestors of mammals, birds and extent reptiles already had the "warm-blooded" isochore structure. We determined the nucleotide sequences of alpha-globin genes from two species of heterotherms, cuckoo (Cuculus canorus) and bat (Pipistrellus abramus), and three species of snakes (Lepidosauromorpha), Naja kaouthia from a tropical terrestrial habitat, Elaphe climacophora from a temperate terrestrial habitat, and Hydrophis melanocephalus from a tropical marine habitat. Our purposes were to assess the influence of differential body temperature patterns on codon usage and GC content at the third position of a codon (GC3), and to test the hypothesis concerning the phylogenetic position at which GC contents had increased in vertebrates. The results of principal component analysis (PCA) using the present data and data for other taxa from GenBank indicate that the primary difference in codon usage in globin genes among amniotes and other vertebrates lies in GC3. The codon usages (and GC3) in alpha-globin genes from two heterotherms and three snakes are similar to those in alpha-globin genes from warm-blooded vertebrates. These results refute the influence of body temperature pattern upon codon usages (and GC3) in alpha-globin genes, and support the hypothesis that the increase in GC content in the genome occurred in the common ancestor of amniotes.

  6. Polymorphic restriction sites in the horse beta-globin gene cluster.

    Science.gov (United States)

    Rando, A; Di Gregorio, P; Masina, P

    1986-01-01

    Horse DNA samples digested with PstI and probed with the rabbit beta 1 globin gene show three phenotypes determined by one fragment of variable length (about 5.1 or 3.3 kb). Family data demonstrate that these fragments segregate as Mendelian alleles. The frequencies of the two alleles are 0.66 for the 3.3-kb fragment and 0.34 for the 5.1-kb one. Another polymorphism has been detected with BamHI. Again three phenotypes determined by two alleles (fragments of 7.5 and 3.8 kb) have been observed. Allelic frequencies of the 7.5- and 3.8-kb fragments are 0.24 and 0.76 respectively. The two polymorphic sites are non-randomly associated.

  7. Restriction fragment length polymorphism in the 3' flanking region of the rabbit beta 1-globin gene.

    Science.gov (United States)

    Masina, P; Rando, A; Cocozza, S

    1984-10-01

    By Southern blot analysis, a restriction fragment length polymorphism in the 3' flanking region of the rabbit beta 1-globin gene was detected. Two alleles, characterized by 9.7- and 12.4-kb BamHI fragments and by 15.3- and 18.0-kb HindIII fragments, have been detected in a small population of White New Zealand rabbits. The long allele is the most frequent (about 70%). The simultaneous changes in the restriction patterns of the two endonucleases and the constant distance between BamHI and HindIII sites in short and long fragments suggest the possibility that the two alleles arise from a rearrangement phenomenon involving a DNA segment 2.7 kb long. In addition, the presence of the two alleles in individuals genetically unrelated to the White New Zealand breed suggests that this polymorphism is widespread.

  8. Genetic relationships among native americans based on beta-globin gene cluster haplotype frequencies

    Directory of Open Access Journals (Sweden)

    Rita de Cassia Mousinho-Ribeiro

    2003-01-01

    Full Text Available The distribution of b-globin gene haplotypes was studied in 209 Amerindians from eight tribes of the Brazilian Amazon: Asurini from Xingú, Awá-Guajá, Parakanã, Urubú-Kaapór, Zoé, Kayapó (Xikrin from the Bacajá village, Katuena, and Tiriyó. Nine different haplotypes were found, two of which (n. 11 and 13 had not been previously identified in Brazilian indigenous populations. Haplotype 2 (+ - - - - was the most common in all groups studied, with frequencies varying from 70% to 100%, followed by haplotype 6 (- + + - +, with frequencies between 7% and 18%. The frequency distribution of the b-globin gene haplotypes in the eighteen Brazilian Amerindian populations studied to date is characterized by a reduced number of haplotypes (average of 3.5 and low levels of heterozygosity and intrapopulational differentiation, with a single clearly predominant haplotype in most tribes (haplotype 2. The Parakanã, Urubú-Kaapór, Tiriyó and Xavante tribes constitute exceptions, presenting at least four haplotypes with relatively high frequencies. The closest genetic relationships were observed between the Brazilian and the Colombian Amerindians (Wayuu, Kamsa and Inga, and, to a lesser extent, with the Huichol of Mexico. North-American Amerindians are more differentiated and clearly separated from all other tribes, except the Xavante, from Brazil, and the Mapuche, from Argentina. A restricted pool of ancestral haplotypes may explain the low diversity observed among most present-day Brazilian and Colombian Amerindian groups, while interethnic admixture could be the most important factor to explain the high number of haplotypes and high levels of diversity observed in some South-American and most North-American tribes.

  9. Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β-Globin Gene with the IVSI-6 Thalassemia Mutation

    Science.gov (United States)

    Mancini, Irene; Lampronti, Ilaria; Salvatori, Francesca; Fabbri, Enrica; Zuccato, Cristina; Cosenza, Lucia C.; Montagner, Giulia; Borgatti, Monica; Altruda, Fiorella; Fagoonee, Sharmila; Carandina, Gianni; Aiello, Vincenzo; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2015-01-01

    Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin mu α-globin2/hu β-globin2 and, more importantly, (d) the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia. PMID:26097845

  10. A review on the origin and spread of deleterious mutants of the beta-globin gene in Indian populations.

    Science.gov (United States)

    Das, S K; Talukder, G

    2001-01-01

    Deleterious mutations of the human beta-globin gene are responsible for beta-thalassaemia and other haemoglobinopathies, which are the most common genetic diseases in Indian populations. A highly heterogeneous distribution of those mutations is observed in India and certain mutations are restricted to some extent to particular groups only. The reasons behind the geographical clustering and origin of the mutations in India is a highly debated issue and the evidence is conflicting. Our present article aims at tracing the origin of the deleterious beta-globin mutation and evaluates the role of different evolutionary forces responsible for the spread and present distribution of those mutations in Indian populations, using data from molecular biology and statistical methods. Mutations are generated essentially randomly, but "hot-spot" sites for mutation are reported for the beta-globin gene cluster, indicating sequence dependency of mutation. A single origin of a deleterious beta-globin mutation, followed by recombination (in a hot spot region) and/or interallelic gene conversion (within beta-globin gene) through time is the most plausible hypothesis to explain the association of those mutations with multiple haplotype backgrounds and frameworks. It is suggested that India is the place of origin of HbE and HbD mutations and that they dispersed to other parts of the would by migration. HbS mutants present in Indian populations are not of Middle East origin but rather a fresh mutation is the probable explanation for the prevalence among tribal groups. beta-thalassaemia represents a heterogeneous group of mutant alleles in India. Five common and twelve rare mutations have been reported in variable frequencies among different Indian populations. Gene flow of those mutant alleles from different populations of the world by political, military and commercial interactions possibly accounts for the heterogenous nature of beta-thalassaemia among Indians. A multiple allelic

  11. A 21 Nucleotide Duplication on the α1- and α2-Globin Genes Involves a Variety of Hypochromic Microcytic Anemias, From Mild to Hb H Disease.

    Science.gov (United States)

    Farashi, Samaneh; Faramarzi Garous, Negin; Zeinali, Fatemeh; Vakili, Shadi; Ashki, Mehri; Imanian, Hashem; Najmabadi, Hossein; Azarkeivan, Azita; Tamaddoni, Ahmad

    2015-01-01

    α-Thalassemia (α-thal) is a common genetic disorder in Iran and many parts of the world. Genetic defects in the α-globin gene cluster can result in α-thal that may develop into a clinical phenotype varying from almost asymptomatic to a lethal hemolytic anemia. Loss of one functional α gene, indicated as heterozygous α(+)-thal, shows minor hematological abnormalities. Homozygosity for α(+)- or heterozygosity for α(0)-thal have more severe hematological abnormalities due to a markedly reduced α chain output. At the molecular level, the absence of three α-globin genes resulting from the compound heterozygous state for α(0)- and α(+)-thal, lead to Hb H disease. Here we present a 21 nucleotide (nt) duplication consisting of six amino acids and 3 bp of intronic sequence at the exon-intron boundary, in both the α-globin genes, detected by direct DNA sequencing. This duplication was identified in three patients originating from two different Iranian ethnic groups and one Arab during more than 12 years. The clinical presentation of these individuals varies widely from a mild asymptomatic anemia (heterozygote in α1-globin gene) to a severely anemic state, diagnosed as an Hb H individual requiring blood transfusion (duplication on the α2-globin gene in combination with the - -(MED) double α-globin gene deletion). The third individual, who was homozygous for this nt duplication on the α1-globin gene, showed severe hypochromic microcytic anemia and splenomegaly. In the last decade, numerous α-globin mutations have demonstrated the necessity of prenatal diagnosis (PND) for α-thal, and this study has contributed another mutation as important enough that needs to be considered.

  12. Self-catalytic DNA depurination underlies human β-globin gene mutations at codon 6 that cause anemias and thalassemias.

    Science.gov (United States)

    Alvarez-Dominguez, Juan R; Amosova, Olga; Fresco, Jacques R

    2013-04-19

    The human β-globin gene contains an 18-nucleotide coding strand sequence centered at codon 6 and capable of forming a stem-loop structure that can self-catalyze depurination of the 5'G residue of that codon. The resultant apurinic lesion is subject to error-prone repair, consistent with the occurrence about this codon of mutations responsible for 6 anemias and β-thalassemias and additional substitutions without clinical consequences. The 4-residue loop of this stem-loop-forming sequence shows the highest incidence of mutation across the gene. The loop and first stem base pair-forming residues appeared early in the mammalian clade. The other stem-forming segments evolved more recently among primates, thereby conferring self-depurination capacity at codon 6. These observations indicate a conserved molecular mechanism leading to β-globin variants underlying phenotypic diversity and disease.

  13. The Locus Control Region Is Necessary for Gene Expression in the Human β-Globin Locus but Not the Maintenance of an Open Chromatin Structure in Erythroid Cells

    Science.gov (United States)

    Reik, Andreas; Telling, Agnes; Zitnik, Galynn; Cimbora, Daniel; Epner, Elliot; Groudine, Mark

    1998-01-01

    Studies in many systems have led to the model that the human β-globin locus control region (LCR) regulates the transcription, chromatin structure, and replication properties of the β-globin locus. However the precise mechanisms of this regulation are unknown. We have developed strategies to use homologous recombination in a tissue culture system to examine how the LCR regulates the locus in its natural chromosomal environment. Our results show that when the functional components of the LCR, as defined by transfection and transgenic studies, are deleted from the endogenous β-globin locus in an erythroid background, transcription of all β-globin genes is abolished in every cell. However, formation of the remaining hypersensitive site(s) of the LCR and the presence of a DNase I-sensitive structure of the β-globin locus are not affected by the deletion. In contrast, deletion of 5′HS5 of the LCR, which has been suggested to serve as an insulator, has only a minor effect on β-globin transcription and does not influence the chromatin structure of the locus. These results show that the LCR as currently defined is not necessary to keep the locus in an “open” conformation in erythroid cells and that even in an erythroid environment an open locus is not sufficient to permit transcription of the β-like globin genes. PMID:9742116

  14. The spectrum of beta-globin gene mutations in children with beta-thalassaemia major from Kota Kinabalu, Sabah, Malaysia.

    Science.gov (United States)

    Thong, M K; Soo, T L

    2005-07-01

    Beta-thalassaemia major is one of the commonest genetic disorders in South East Asia. The strategy for the community control of beta-thalassaemia major requires the characterisation of the spectrum of beta-globin gene mutations in any multi-ethnic population. There is only a single report of mutation analyses of the beta-globin gene in an isolated Kadazandusun community in Kota Belud, Sabah, Malaysia, which showed the presence of a common 45 kb deletion. To confirm the observation that this large deletion is the commonest beta-globin gene mutation among the Kadazandusun and other indigenous populations in Sabah, Malaysia, we performed polymerase chain reaction (PCR) analysis of the beta-globin gene in ten children with beta-thalassaemia major attending the Thalassaemia Centre, Queen Elizabeth Hospital, the major paediatric referral centre in Kota Kinabalu, Sabah. The 45 kb deletion was confirmed to be the commonest mutation found in the Kadazandusun, Bajau and Murut populations, whereby it was detected in 19 out of the 20 (95 percent) alleles analysed. The other mutation was due to an IVS-1 position 1 G > T mutation. This finding confirmed the deletion in the homozygous state was associated with a severe phenotype. The reason for the predominance of this mutation in Kota Kinabalu is most likely to be due to founder effects and possibly intermarriages between the various ethnic groups. Prenatal diagnosis using PCR for this common mutation is feasible in this community. Medical workers and scientists at molecular diagnostic centres serving large South East Asian populations should incorporate a diagnostic strategy for this deletion in the appropriate population. Future studies on these indigenous ethnic groups in other areas and other groups in Sabah are required.

  15. Comparative analysis of the ATRX promoter and 5' regulatory region reveals conserved regulatory elements which are linked to roles in neurodevelopment, alpha-globin regulation and testicular function

    Directory of Open Access Journals (Sweden)

    Argentaro Anthony

    2011-06-01

    Full Text Available Abstract Background ATRX is a tightly-regulated multifunctional protein with crucial roles in mammalian development. Mutations in the ATRX gene cause ATR-X syndrome, an X-linked recessive developmental disorder resulting in severe mental retardation and mild alpha-thalassemia with facial, skeletal and genital abnormalities. Although ubiquitously expressed the clinical features of the syndrome indicate that ATRX is not likely to be a global regulator of gene expression but involved in regulating specific target genes. The regulation of ATRX expression is not well understood and this is reflected by the current lack of identified upstream regulators. The availability of genomic data from a range of species and the very highly conserved 5' regulatory regions of the ATRX gene has allowed us to investigate putative transcription factor binding sites (TFBSs in evolutionarily conserved regions of the mammalian ATRX promoter. Results We identified 12 highly conserved TFBSs of key gene regulators involved in biologically relevant processes such as neural and testis development and alpha-globin regulation. Conclusions Our results reveal potentially important regulatory elements in the ATRX gene which may lead to the identification of upstream regulators of ATRX and aid in the understanding of the molecular mechanisms that underlie ATR-X syndrome.

  16. Mutation spectrum of β-globin gene in thalassemia patients at Hasan Sadikin Hospital - West Java Indonesia.

    Science.gov (United States)

    Maskoen, Ani Melani; Rahayu, Nurul S; Reniarti, Lelani; Susanah, Susi; Laksono, Bremmy; Fauziah, Prima Nanda; Zada, Almira; Hidayat, Dadang S

    2017-12-30

    Thalassemia is the most common hereditary haemolytic anemia in Southeast Asia, in which Indonesia is among countries that are at a high risk for thalassemia. It has been reported that mutation in the beta-globin gene is responsible in severe Thalassemia. However, the spectrum of beta-globin gene mutations in Indonesian population varies in different regions . Thus, this study aimed to identify the most prevalent mutation of Thalassemia patients from the Hasan Sadikin Hospital, Bandung, using this as a reference hospital for Thalassemia in West Java. The three most prevalent mutations of beta globin (IVS1nt5, Cd26 (HbE), and IVS1nt1), were conducted in the beginning of this study. Mutations of 291 samples were detected by PCR-RFLP in the Molecular Genetic Laboratory, Faculty of Medicine Universitas Padjadjaran, Bandung. The prevalence of the beta globin gene mutation types were 47.4% IVS1nt5 homozygote, 9.9% compound heterozygote IVS1nt5/HbE, 5.4% compound heterozygote IVS1nt5/IVS1nt1, 1.4% compound heterozygote HbE/IVS1nt1, 1% HbE homozygote, 14.4% Compound heterzygote IVS1nt5/… (no paired mutation), 2.06% compound heterozygote HbE/… (no paired mutation), 1.3% compound heterozygote IVS1nt1/… (no paired mutation), and 7 samples were unidentified. The thalassemia mutation IVS1nt5 homozygote is the most common mutation found in Thalassemia patients at Hasan Sadikin Hospital, Bandung. The samples with unidentified results might carry mutations other than the three that are observed in the present study.

  17. Two α1-Globin Gene Point Mutations Causing Severe Hb H Disease.

    Science.gov (United States)

    Jiang, Hua; Huang, Lv-Yin; Zhen, Li; Jiang, Fan; Li, Dong-Zhi

    Hb H disease is generally a moderate form of α-thalassemia (α-thal) that rarely requires regular blood transfusions. In this study, two Chinese families with members carrying transfusion-dependent Hb H disease were investigated for rare mutations on the α-globin genes (HBA1, HBA2). In one family, Hb Zürich-Albisrieden [α59(E8)Gly→Arg; HBA1: c.178G>C] in combination with the Southeast Asian (- - SEA ) deletion was the defect responsible for the severe phenotype. In another family, a novel hemoglobin (Hb) variant named Hb Sichuan (HBA1: c.393_394insT), causes α-thal and a severe phenotype when associated with the - - SEA deletion. As these two HBA1 mutations can present as continuous blood transfusion-dependent α-thal, it is important to take this point into account for detecting the carriers, especially in couples in which one partner is already a known α 0 -thal carrier.

  18. Genetic heterogeneity of the β-globin gene in various geographic populations of Yunnan in southwestern China.

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    Jie Zhang

    Full Text Available The aim of this study was to investigate the geographic distribution of β-globin gene mutations in different ethnic groups in Yunnan province.From 2004 to 2014, 1,441 subjects with hemoglobin disorders, identified by PCR-reverse dot blot and DNA sequencing, were studied according to ethnicity and geographic origin. Haplotypes were examined among 41 unrelated thalassemia chromosomes.Eighteen β-thalassemia mutations and seven hemoglobin variants were identified for 1,616 alleles in 22 different ethnic groups from all 16 prefecture-level divisions of Yunnan. The prevalence of β-thalassemia was heterogeneous and regionally specific. CD 41-42 (-TCTT was the most prevalent mutation in the populations of northeastern Yunnan. CD 17 (A>T was the most common mutation in the populations of southeastern Yunnan, especially for the Zhuang minority, whereas Hb E (CD 26, G>A was the most prevalent mutation in populations of southwestern Yunnan, especially for the Dai minority. Among the seven types of haplotypes identified, CD 17 (A>T was mainly linked to haplotype VII (+ - - - - - + and IVS-II-654 (C>T was only linked to haplotype I (+ - - - - + +.Our data underline the heterogeneity of β-globin gene mutations in Yunnan. This distribution of β-globin mutations in the geographic regions and ethnic populations provided a detailed ethnic basis and evolutionary view of humans in southern China, which will be beneficial for genetic counseling and prevention strategies.

  19. The sequence and phylogenesis of the ?-globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon) and Cyprus mouflon (Ovis aries ophion).

    Science.gov (United States)

    Pirastru, Monica; Multineddu, Chiara; Mereu, Paolo; Sannai, Mara; El Sherbini, El Said; Hadjisterkotis, Eleftherios; Nàhlik, Andràs; Franceschi, Paul; Manca, Laura; Masala, Bruno

    2009-09-01

    In order to investigate the polymorphism of ?-globin chain of hemoglobin amongst caprines, the linked (I)? and (II)? globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon), and Cyprus mouflon (Ovis aries ophion) were completely sequenced, including the 5? and 3? untranslated regions. European and Cyprus mouflons, which do not show polymorphic ? globin chains, had almost identical ? globin genes, whereas Barbary sheep exhibit two different chains encoded by two nonallelic genes. Four different ? genes were observed and sequenced in goat, validating previous observations of the existence of allelic and nonallelic polymorphism. As in other vertebrates, interchromosomal gene conversion appears to be responsible for such polymorphism. Evaluation of nucleotide sequences at the level of molecular evolution of the (I)?-globin gene family in the caprine taxa suggests a closer relationship between the genus Ammotragus and Capra. Molecular clock estimates suggest sheep-mouflon, goat-aoudad, and ancestor-caprine divergences of 2.8, 5.7, and 7.1 MYBP, respectively.

  20. Role of novel and rare nucleotide substitutions of the β-globin gene

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    Margherita Vinciguerra

    2012-11-01

    Full Text Available The Laboratory for Molecular Prenatal Diagnosis of Hemoglobinopathies at the Villa Sofia-Cervello Hospital in Palermo, Italy, carries out an intensive screening program aimed at identifying the healthy carriers of thalassemia and, consequently, the couples at risk of bearing an affected fetus. The diagnostic process is basically divided into two phases: i hematologic and hemoglobin data; ii molecular analysis of globin genes and, when possible, a genetic study of the family. Since 2003, we have been performing DNA sequence analysis on those cases in which classical molecular methods failed to give a complete diagnostic response, particularly in phenotypes with borderline values of HbA2 with mild or absent microcytosis. During ten years of screening activities (from 2003 to 2012, twenty- seven unknown or rare nucleotide changes of the β-globin gene have been identified; hematologic and hemoglobin data have been carefully evaluated and, wherever possible, we have conducted a family study to evaluate whether a phenotypic expression could be associated to these nucleotide changes. Because of the limited numbers of cases for each mutation, the significance of these nucleotide substitutions has still not been fully clarified, and this raises a number of questions that need to be answered when carrying out appropriate genetic counseling for couples presumed to be at risk. 意大利巴勒莫Villa Sofia-Cervello医院血红蛋白病分子产前诊断实验室进行密集的筛选程序,旨在识别健康的地中海贫血携带者和有怀上地中海贫血胎儿风险的夫妇。 诊断过程基本上分为两个阶段:1)血液及血红蛋白数据;2)珠蛋白基因分子分析以及家族遗传研究(如有可能)。 自2003年以来,我们已对这类病例进行DNA序列分析:传统的分子方法无法给出完整的诊断响应,尤其是有轻微小红细胞症或缺乏小红细胞症的HbA2临界值表型。

  1. Beta-globin gene cluster haplotypes in the Mapuche Indians of Argentina

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    Letícia Kaufman

    1998-12-01

    Full Text Available Haplotypes derived from five polymorphic restriction sites in the beta-globin gene cluster were investigated in 86 chromosomes from the Argentinian Mapuche. These results were integrated with those previously obtained for ten Brazilian Indian tribes. Eight haplotypes were identified, the most frequent being 2 (57% and 6 (27%. The presence of haplotype 3 in 2% of the Mapuche chromosomes is probably an evidence of admixture with individuals of African ancestry. Due to the high number of haplotypes observed, heterozygosity as measured by the Gini-Simpson index was higher in the Mapuche than in Brazilian Indians. The haplotypic distribution in the Mapuche was also significantly different from those of all Brazilian tribes investigated. This heterogeneity could be at least partially explained by admixture with non-Indian populations.Haplótipos derivados de cinco sítios de restrição polimórficos presentes no agrupamento da globina beta foram investigados em 86 cromossomos da população mapuche da Argentina. Esses resultados foram analisados em conjunto com os previamente obtidos para dez tribos indígenas brasileiras. Oito haplótipos foram identificados, dos quais os mais freqüentes foram o 2 (57% e o 6 (27%. A presença do haplótipo 3 em 2% dos cromossomos dos Mapuches é uma evidência de mistura com indivíduos de ancestralidade africana. Devido ao alto número de haplótipos, a heterozigosidade medida pelo índice Gini-Simpson é mais alta nos Mapuches do que nos índios brasileiros. A distribuição haplotípica nos Mapuches é também significativamente diferente da observada nas tribos brasileiras. Essa heterogeneidade poderia ser parcialmente explicada pela mistura com populações não-indígenas.

  2. Nuclear topography of beta-like globin gene cluster in IL-3-stimulated human leukemic K-562 cells

    Czech Academy of Sciences Publication Activity Database

    Galiová-Šustáčková, Gabriela; Bártová, Eva; Kozubek, Stanislav

    2004-01-01

    Roč. 33, č. 1 (2004), s. 4-14 ISSN 1079-9796 R&D Projects: GA ČR GA301/01/0186; GA AV ČR KSK5052113; GA AV ČR IAA5004306; GA ČR GA202/04/0907; GA MŠk ME 565 Institutional research plan: CEZ:AV0Z5004920 Keywords : beta-like globin gene cluster * K-562 cells * nuclear topography Subject RIV: BO - Biophysics Impact factor: 2.549, year: 2004

  3. An unusual DNA sequence observed in the. gamma. globin gene loci of two members of a Chinese family

    Energy Technology Data Exchange (ETDEWEB)

    Ryan, Q.C.

    1989-01-01

    There are two nonallelic human {gamma} globin genes located on the short arm of chromosome No. 11 in the order 5{prime}-{sup G}{sub {gamma}}-{sup A}{sub {gamma}}-3{prime}. Various modifications of the two {gamma} genes have been reported and include: deletions, triplications, quadruplications and recently a quintuplication. These are generally created by one or more unequal crossovers in the {gamma} globin gene regions on adjacent chromosomes. During the course of looking for a {gamma}{sup {degree}} thalassemia, which might be due to a crossover of looking for a {gamma} genes, two cases were found in the family W. Bgl II mapping studies showed a 5 kb deletion at the {gamma} gene loci in these individuals. The Bgl II fragment from the {gamma} gene loci of R.W. was cloned into the phage vector QR1. Phage mapping showed that two out of the three Pst I sites within the Bgl II fragment were missing which suggested that the crossover might have occurred within the {gamma} gene, possibly within the {gamma}IVS II region. Sequence analysis of the cloned fragment revealed an unusual sequence which had no sequence homology with the {gamma} gene region except for a small 264 bp region near the 3{prime} end. The orientation of the 264 bp fragment is inverted relative to homologous sequences in the {sup G}{sub {gamma}} and {sup A}{sub {gamma}} IVS II. The unusual sequence was computer analyzed for homology with every DNA sequence file in the EMBL database and GenBank and did not show any significant homologies to all the available DNA sequences except for the 264 bp {gamma}IVS II homology.

  4. alpha-Globin genes: thalassemic and structural alterations in a Brazilian population

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    M.R.S.C. Wenning

    2000-09-01

    Full Text Available Seven unrelated patients with hemoglobin (Hb H disease and 27 individuals with alpha-chain structural alterations were studied to identify the alpha-globin gene mutations present in the population of Southeast Brazil. The -alpha3.7, --MED and -(alpha20.5 deletions were investigated by PCR, whereas non-deletional alpha-thalassemia (alphaHphalpha, alphaNcoIalpha, aaNcoI, alphaIcalpha and alphaTSaudialpha was screened with restriction enzymes and by nested PCR. Structural alterations were identified by direct DNA sequencing. Of the seven patients with Hb H disease, all of Italian descent, two had the -(alpha20.5/-alpha3.7 genotype, one had the --MED/-alpha3.7 genotype, one had the --MED/alphaHphalpha genotype and three showed interaction of the -alpha3.7 deletion with an unusual, unidentified form of non-deletional alpha-thalassemia [-alpha3.7/(aaT]. Among the 27 patients with structural alterations, 15 (of Italian descent had Hb Hasharon (alpha47Asp->His associated with the -alpha3.7 deletion, 4 (of Italian descent were heterozygous for Hb J-Rovigo (alpha53Ala->Asp, 4 (3 Blacks and 1 Caucasian were heterozygous for Hb Stanleyville-II (alpha78Asn->Lys associated with the alpha+-thalassemia, 1 (Black was heterozygous for Hb G-Pest (alpha74Asp->Asn, 1 (Caucasian was heterozygous for Hb Kurosaki (alpha7Lys->Glu, 1 (Caucasian was heterozygous for Hb Westmead (alpha122His->Gln, and 1 (Caucasian was the carrier of a novel silent variant (Hb Campinas, alpha26Ala->Val. Most of the mutations found reflected the Mediterranean and African origins of the population. Hbs G-Pest and Kurosaki, very rare, and Hb Westmead, common in southern China, were initially described in individuals of ethnic origin differing from those of the carriers reported in the present study and are the first cases to be reported in the Brazilian population.

  5. A Dual Reporter Mouse Model of the Human β-Globin Locus: Applications and Limitations

    NARCIS (Netherlands)

    P. Papadopoulos (Petros); L. Gutiérrez (Laura); R. van der Linden (Reinier); J. Kong-a-San (John); A. Maas (Alex); D.D. Drabek (Dubravka); G.P. Patrinos (George); J.N.J. Philipsen (Sjaak); F.G. Grosveld (Frank)

    2012-01-01

    textabstractThe human β-globin locus contains the β-like globin genes (i.e. fetal γ-globin and adult β-globin), which heterotetramerize with α-globin subunits to form fetal or adult hemoglobin. Thalassemia is one of the commonest inherited disorders in the world, which results in quantitative

  6. Effect ALPHA Globalin Gene Deletion and GAMMA Globin Gene -158 (C/T) Polymorphism in BETA- Thalassaemic Patients

    International Nuclear Information System (INIS)

    EL Serafi, T.I.; Ismail, E.F.; Mahmoud, M.A.; Mohamed, M.A.; Ghattas, M.H.; Badran, D.I.; El Serafi, I.T.; Mohamed, H.S.

    2008-01-01

    The beta-thalassemias (β- thalassemias) are among the most common autosomal recessive disorders. They have a remarkably high frequency in the Mediterranean region and represent one of the most common genetic diseases in Egypt. In this study, the spectrum of P- thalassemia mutations and genotype-to-phenotype correlations were defined in 32 β- thalassaemic patients (β- thalassemias major and intermedia) with varying disease severity in two cities of the Suez Canal region. Ten different mutations were identified and the most frequent ones were: Isi-6 (T-C) (37.5%), IVSI-110 (G-A) (34.4%) and both IVSI-1 (G-A), IVSII-745 (C-G) and -102 (C-G) (12.5% each). There was a wide spectrum of phenotypic severity in all patients. We studied the Xmnl polymorphism (C/T) in γ- globin gene position -158 of P- thalassemia as a modulating factor of the disease severity. Presence of the polymorphism was found in two patients and this was not sufficient to explain the diversity of the phenotype encountered. Co-inheritance of alpha thalassaemia as a modulating factor was not evident in our patients. In conclusion, we have been unable to find a molecular basis for the benign clinical course in all our patients. Other genetic or acquired factors must be hypothesized which ameliorate the clinical condition.

  7. Beta-globin gene cluster haplotypes and alpha-thalassemia in sickle cell disease patients from Trinidad.

    Science.gov (United States)

    Jones-Lecointe, Altheia; Smith, Erskine; Romana, Marc; Gilbert, Marie-Georges; Charles, Waveney P; Saint-Martin, Christian; Kéclard, Lisiane

    2008-01-01

    In this study, we have determined the frequency of beta(S) haplotypes in 163 sickle cell disease patients from Trinidad. The alpha(3.7) globin gene deletion status was also studied with an observed gene frequency of 0.17. Among the 283 beta(S) chromosomes analyzed, the Benin haplotype was the most prevalent (61.8%) followed by Bantu (17.3%), Senegal (8.5%), Cameroon (3.5%), and Arab-Indian (3.2%), while 5.7% of them were atypical. This beta(S) haplotypes distribution differed from those previously described in other Caribbean islands (Jamaica, Guadeloupe, and Cuba), in agreement with the known involvement of the major colonial powers (Spain, France, and Great Britain) in the slave trade in Trinidad and documented an Indian origin of the beta(S) gene.

  8. Detecting deletions, insertions, and single nucleotide substitutions in cloned β-globin genes and new polymorphic nucleotide substitutions in β-globin genes in a Japanese population using ribonuclease cleavage at mismatches in RNA: DNA duplexes

    International Nuclear Information System (INIS)

    Hiyama, Keiko; Kodaira, Mieko; Satoh, Chiyoko.

    1990-08-01

    The applicability of ribonuclease (RNase) cleavage at mismatches in RNA:DNA duplexes (the RNase cleavage method) for determining nucleotide variant rates was examined in a Japanese population. DNA segments of various lengths obtained from four different regions of one normal and three thalassemic cloned human β-globin genes were inserted into transcription vectors. Sense and antisense RNA probes uniformly labeled with 32 P were prepared. When RNA probes of 771 nucleotides (nt) or less were hybridized with cloned DNAs and the resulting duplexes were treated with a mixture of RNases A and T1, the length of products agreed with theoretical values. Twelve possible mismatches were examined. Since both sense and antisense probes were used, uncleavable mismatches such as G:T and G:G which were made from one combination of RNA and DNA strands could be converted to the cleavable C:A and C:C mismatches, respectively, by using the opposite combination. Deletions and insertions of one (G), four(TTCT), five (ATTTT), and 10 (ATTTTATTTT) nt were easily detected. A polymorphic substitution of T to C at position 666 of the second intervening sequence (IVS2-666) of the β-globin gene was detected using genomic DNAs from cell lines established from the peripheral B lymphocytes of 59 unrelated Japanese from Hiroshima or those amplified by polymerase chain reaction (PCR). The frequency of the gene with C at the IVS2-666 (allele C) was 0.48 and that of the gene with T (allene T) was 0.52. Two new polymorphic substitutions of C to A and A to T were detected at nucleotide positions 1789 and 1945 from the capping site, respectively, using genomic DNAs amplified by PCR. We conclude that it would be feasible to use the RNase cleavage method combined with PCR for large-scale screening of variation in chromosomal DNA. (J.P.N.)

  9. Nuclear localization and histone acetylation: a pathway for chromatin opening and transcriptional activation of the human β-globin locus

    Science.gov (United States)

    Schübeler, Dirk; Francastel, Claire; Cimbora, Daniel M.; Reik, Andreas; Martin, David I.K.; Groudine, Mark

    2000-01-01

    We have investigated the mechanism, structural correlates, and cis-acting elements involved in chromatin opening and gene activation, using the human β-globin locus as a model. Full transcriptional activity of the human β-globin locus requires the locus control region (LCR), composed of a series of nuclease hypersensitive sites located upstream of this globin gene cluster. Our previous analysis of naturally occurring and targeted LCR deletions revealed that chromatin opening and transcriptional activity in the endogenous β-globin locus are dissociable and dependent on distinct cis-acting elements. We now report that general histone H3/H4 acetylation and relocation of the locus away from centromeric heterochromatin in the interphase nucleus are correlated and do not require the LCR. In contrast, LCR-dependent promoter activation is associated with localized histone H3 hyperacetylation at the LCR and the transcribed β-globin-promoter and gene. On the basis of these results, we suggest a multistep model for gene activation; localization away from centromeric heterochromatin is required to achieve general hyperacetylation and an open chromatin structure of the locus, whereas a mechanism involving LCR/promoter histone H3 hyperacetylation is required for high-level transcription of the β-globin genes. PMID:10783166

  10. Lentiviral Transfer of γ-Globin with Fusion Gene NUP98-HOXA10HD Expands Hematopoietic Stem Cells and Ameliorates Murine β-Thalassemia.

    Science.gov (United States)

    Zhao, Hui Fen; Abraham, Allistair; Kim, Yoon-Sang; Wang, Yong-Dong; Pestina, Tamara; Zhan, Jun; Humphries, Keith; Nienhuis, Arthur W; Persons, Derek A

    2017-03-01

    Recently, an engineered Homeobox-nucleoporin fusion gene, NUP98-HOXA10HD or NA10HD, was reported to expand and maintain murine hematopoietic stem cells (HSCs). We postulated that NA10HD would increase the number of human γ-globin-expressing cells to therapeutic levels. We developed a double gene lentiviral vector encoding both human γ-globin and NA10HD, which was used to transduce human peripheral blood CD34 + cells and increased engraftment 2- to 2.5-fold at 15 weeks post-transplantation in immunodeficient mice. In β-thalassemic mice transplanted with β-thalassemic HSCs transduced with the γ-globin/NA10HD vector, the number of fetal hemoglobin (HbF)-expressing cells was significantly increased after 3 months, leading to resolution of the anemia. Furthermore, the increases in HbF were maintained at 6 months and persisted after secondary transplantation. In addition, NA10HD enrichment of transduced HSCs led to HbF increases without affecting homeostasis of the white blood cell lineages. Our results suggest that NA10HD increases the number of γ-globin-transduced HSCs that engraft, leading to an elevated number of fetal hemoglobin-containing red cells. These effects of NA10HD provide an improved platform for testing of the therapeutic efficacy of novel globin vectors and provide further impetus to develop safe and effective methods for selective expansion of genetically modified cells. Copyright © 2017. Published by Elsevier Inc.

  11. Chromosome 11-linked determinant controls fetal globin expression and the fetal-to-adult globin switch

    International Nuclear Information System (INIS)

    Melis, M.; Demopulos, G.; Najfeld, V.; Zhang, J.W.; Brice, M.; Papayannopoulou, T.; Stamatoyannopoulos, G.

    1987-01-01

    Hybrids formed by fusing mouse erythroleukemia (MEL) cells with human fetal erythroid cells produce human fetal globin, but they switch to adult globin production as culture time advances. To obtain information on the chromosomal assignment of the elements that control γ-to-β switching, the authors analyzed the chromosomal composition of hybrids producing exclusively or predominantly human fetal globin and hybrids producing only adult human globin. No human chromosome was consistently present in hybrids expressing fetal globin and consistently absent in hybrids expressing adult globin. Subcloning experiments demonstrated identical chromosomal compositions in subclones displaying the fetal globin program and those that had switched to expression of the adult globin program. These data indicate that retention of only one human chromosome -- i.e., chromosome 11 -- is sufficient for expression of human fetal globin and the subsequent γ-to-β switch. The results suggest that the γ-to-β switch is controlled either cis to the β-globin locus of by a trans-acting mechanism, the genes of which reside on human chromosome 11

  12. Hemoglobin variants, hematological parameters and beta-globin gene cluster haplotypes in an isolated Amerindian group from the Orinoco River Delta.

    Science.gov (United States)

    Arends, Anabel; Chacín, Marycarmen; Bravo-Urquiola, Martha; Tibisay, Arends De O; Alvarez, Maritza; Castillo, Omar; Guevara, Jose M

    2008-01-01

    Several previous studies reported that the Venezuelan Warao Indians presented unusual genetic characteristics. The present study checked previous reports of a high frequency of hereditary persistence of fetal hemoglobin (HPFH) and examined other hematological traits. Standard hematology, electrophoresis on cellulose acetate, fetal hemoglobin alkali denaturation, gamma-globin chain, DNA amplification and sequencing, and denaturing gradient gel electrophoresis determinations were performed in 269 individuals living in two localities of the Orinoco River Delta. Two beta(s) genes, in apparently non-related individuals, were found. HPFH, detected in this same population of Warao Indians 25 years ago, was present in heterozygous form in five individuals from a large kindred, with hemoglobin F levels ranging from 3.7% to 8%, and with a pancellular distribution. The HPFH mutation was of the deletional type. beta-globin gene haplotypes were determined by direct counting (through family studies) in 150 chromosomes; 26% of the 150 examined cluster presented haplotype 2, 22% haplotype 6, and 13% a new, Warao haplotype. Haplotype 3, of probable African origin, was also found with a frequency of 5%. The presence of the HPFH mutation was confirmed, and the new beta-globin gene haplotype together with the presence of other rare variants indicates that the Warao are very distinctive in relation to other Native Americans. Evidence was also found of a slight admixture from Africa-derived subjects (Layrisse et al. 1988).

  13. Dissection of the beta-globin replication-initiation region reveals specific requirements for replicator elements during gene amplification.

    Directory of Open Access Journals (Sweden)

    Naoya Okada

    Full Text Available Gene amplification plays a pivotal role in malignant transformation of human cells. A plasmid with both a mammalian replication-initiation region (IR/origin/replicator and a nuclear matrix-attachment region (MAR is spontaneously amplified in transfected cells by a mechanism that involves amplification at the extrachromosomal site, followed by amplification at the chromosomal arm, ultimately generating a long homogeneously staining region (HSR. Several observations suggest that replication initiation from IR sequences might mediate amplification. To test this idea, we previously dissected c-myc and DHFR IRs to identify the minimum sequence required to support amplification. In this study, we applied an improved analysis that discriminates between two amplification steps to the ß-globin RepP IR, which contains separate elements already known to be essential for initiation on the chromosome arm. The IR sequence was required at least for the extrachromosomal amplification step. In addition to the vector-encoded MAR, amplification also required an AT-rich region and a MAR-like element, consistent with the results regarding replicator activity on the chromosome. However, amplification did not require the AG-rich tract necessary for replicator activity, but instead required a novel sequence containing another AG-rich tract. The differential sequence requirement might be a consequence of extrachromosomal replication.

  14. Frequency of Gγ-globin promoter -158 (C>T) XmnI polymorphism in patients with homozygous/compound heterozygous beta thalassaemia.

    Science.gov (United States)

    Ali, Nadir; Ayyub, Muhammad; Khan, Saleem Ahmed; Ahmed, Suhaib; Abbas, Kazim; Malik, Hamid Saeed; Tashfeen, Sunila

    2015-03-01

    Response to hydroxyurea therapy in homozygous or compound heterozygous beta thalassaemia (BT) has been reported as more favourable in the presence of XmnI polymorphism. The prevalence of XmnI polymorphism may vary with BT phenotypes and genotypes, and differs geographically in distribution. Prevalence of XmnI polymorphism is not known in northern Pakistan. To determine the frequency of Gγ-globin promoter -158 (C>T) XmnI polymorphism (XmnI polymorphism) in patients with homozygous or compound heterozygous beta thalassaemia. Polymerase chain reaction (PCR) for common beta thalassaemia mutations and Gγ-globin promoter -158 (C>T) XmnI polymorphism was performed on 107 blood samples of transfusion dependent beta thalassaemia (BT) patients in Pakistan. One hundred samples of unrelated BT traits and 94 samples of healthy subjects as controls were also analysed for BT mutations and XmnI polymorphism. Out of 301 DNA samples, XmnI polymorphism was detected in 71(24%); in normal controls, XmnI polymorphism was detected in 34/94 (36%) subjects; while in homozygous/compound heterozygous BT, it was detected in 14/107(13%) patients (Fisher's exact test, p=.0002). In heterozygous BT group, XmnI polymorphism was detected in 23/100 subjects (Fisher's exact test, p=.03 with normal controls, and p=.049 with homozygous/compound heterozygous BT). The most common BT genotype was Frame Shift (Fr) 8-9/Fr 8-9, and none of the patients with this genotype had XmnI polymorphism. The second most common genotype was IVSI-5/IVSI-5; 4/26 (15%). Cases with this genotype had XmnI polymorphism. XmnI polymorphism in homozygous/compound heterozygous BT group is 13%. The most common genotype associated with XmnI polymorphism was IVSI-5/IVSI-5. Copyright © 2015 King Faisal Specialist Hospital & Research Centre. Published by Elsevier B.V. All rights reserved.

  15. Differences in the number of embryonic and pseudo-beta-globin genes between HbA and HbB sheep.

    Science.gov (United States)

    Rando, A; Di Gregorio, P; Masina, P

    1989-02-01

    DNA samples obtained from 8 goats, 1 moufflon, and 84 sheep with HbA, HbAB, and HbB belonging to different breeds were digested with BamHI, EcoRI, HindIII and PstI and probed with the 5' end of the goat epsilon IV- and psi beta Z-globin genes. Sheep homozygous for HbA show a different restriction pattern than sheep homozygous fo HbB with each of these endonucleases. The main difference is that HbB sheep lack the epsilon II and psi beta X genes. These results, in addition to those previously obtained using a probe specific for beta-globin genes, suggest that HbB sheep probably lack the preadult four-gene set. The DNAs from moufflon and sheep homozygous for HbA show indistinguishable restriction patterns. Furthermore, a number of restriction fragment length polymorphisms (RFLPs) are detected in the epsilon IV and psi beta Z DNA regions, and one HindIII RFLP in the epsilon VI DNA region.

  16. molecular analysis of intron-1 mutation in β-Globin gene of β-Thalassemia

    International Nuclear Information System (INIS)

    Kamel, S.A.L.M.

    2004-01-01

    β-thalassemia is considered the most common genetic disorder worldwide, it occurs in a particularly high frequency in abroad belt extending from the mediterranean basin through the middle east, and abundance in egypt. the thalassemias are a group of genetic (inherited) blood disorders that share in common one feature, the defective production of hemoglobin. there are many different disorders with defective hemoglobin synthesis and, hence, many types of thalassemia. about 3% of the world's population (180 million people) carry β-thalassemia genes.the present study was carried out in the biological application department of nuclear research center, atomic energy authority and microbiology department and hematology unit of pediatrics department, faculty of medicine, Zagazig University

  17. Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS Are Active In Vivo in Anemic Baboons and Transgenic Mice.

    Directory of Open Access Journals (Sweden)

    Michael S Boosalis

    Full Text Available High-level fetal (γ globin expression ameliorates clinical severity of the beta (β hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies.

  18. Characterization of Hb Calvino (HBB: c.406G > A): a new silent β-globin gene variant found in coexistence with α-thalassemia in a family of African origin.

    Science.gov (United States)

    Marsella, Maria; Salvagno, Gianluca; Dolcini, Bernadetta; Ferlini, Alessandra; Ravani, Anna; Harteveld, Cornelis L; Giordano, Piero C; Borgna-Pignatti, Caterina

    2014-01-01

    We report a new silent β-globin gene variant found in a family from Angola living in the north eastern Italian city of Ferrara. The probands, two young sisters, presented with hematological parameters compatible with a β-thalassemia (β-thal) minor but with normal Hb A₂ levels and normal hemoglobin (Hb) separation on high performance liquid chromatography (HPLC). Molecular analyses revealed a homozygosity for the common -α(3.7) (rightward) deletion and heterozygosity for a novel transition (GCT > ACT) at codon 135 of the β-globin gene, leading to an Ala → Thr single amino acid substitution that was inherited from the healthy father.

  19. Hb Dartmouth (HBA2: c.200T>C): An α2-Globin Gene Associated with Hb H Disease in One Homozygous Patient.

    Science.gov (United States)

    Farashi, Samaneh; Faramarzi Garous, Negin; Ashki, Mehri; Vakili, Shadi; Zeinali, Fatemah; Imanian, Hashem; Azarkeivan, Azita; Najmabadi, Hossein

    2015-01-01

    Hb H (β4) disease is caused by deletion or inactivation of three out of four α-globin genes. A high incidence of Hb H disease has been reported all over the world. There is a wide spectrum of phenotypic presentations, from clinically asymptomatic to having significant hepatosplenomegaly and requiring occasional or even regular blood transfusions, even more severe anemia, Hb Bart's (γ4) hydrops fetalis syndrome that can cause death in the affected fetuses late in gestation. We here present a case who was diagnosed with Hb H disease that represents a new genotype for this hereditary disorder. Hb Dartmouth is a variant caused by a missense mutation at codon 66 of the α2-globin gene (HBA2: c.200T>C), resulting in the substitution of leucine by proline. We here emphasize the importance of this point mutation involving Hb H disease and also the necessity for prenatal diagnosis (PND) for those who carry this point mutation in the heterozygous state.

  20. A New Intergenic α-Globin Deletion (α-αΔ125) Found in a Kabyle Population.

    Science.gov (United States)

    Singh, Amrathlal Rabbind; Lacan, Philippe; Cadet, Estelle; Bignet, Patricia; Dumesnil, Cécile; Vannier, Jean-Pierre; Joly, Philippe; Rochette, Jacques

    2016-01-01

    We have identified a deletion of 125 bp (α-α(Δ125)) (NG_000006.1: g.37040_37164del) in the α-globin gene cluster in a Kabyle population. A combination of singlex and multiplex polymerase chain reaction (PCR)-based assays have been used to identify the molecular defect. Sequencing of the abnormal PCR amplification product revealed a novel α1-globin promoter deletion. The endpoints of the deletion were characterized by sequencing the deletion junctions of the mutated allele. The observed deletion was located 378 bp upstream of the α1-globin gene transcription initiation site and leaves the α2 gene intact. In some patients, the α-α(Δ125) deletion was shown to segregate with Hb S (HBB: c.20A>T) and/or Hb C (HBB: c.19G>A) or a β-thalassemic allele. The α-α(Δ125) deletion has no discernible effect on red cell indices when inherited with no other abnormal globin genes. The family study demonstrated that the deletion is heritable. This is the only example of an intergenic α2-α1 non coding DNA deletion, leaving the α2-globin gene and the α1 coding part intact.

  1. Sox6 directly silences epsilon globin expression in definitive erythropoiesis.

    Directory of Open Access Journals (Sweden)

    2006-02-01

    Full Text Available Sox6 is a member of the Sox transcription factor family that is defined by the conserved high mobility group (HMG DNA binding domain, first described in the testis determining gene, Sry. Previous studies have suggested that Sox6 plays a role in the development of the central nervous system, cartilage, and muscle. In the Sox6-deficient mouse, p100H, epsilony globin is persistently expressed, and increased numbers of nucleated red cells are present in the fetal circulation. Transfection assays in GM979 (erythroleukemic cells define a 36-base pair region of the epsilony proximal promoter that is critical for Sox6 mediated repression. Electrophoretic mobility shift assay (EMSA and chromatin immunoprecipitation (ChIP assays demonstrate that Sox6 acts as a repressor by directly binding to the epsilony promoter. The normal expression of Sox6 in wild-type fetal liver and the ectopic expression of epsilony in p100H homozygous fetal liver demonstrate that Sox6 functions in definitive erythropoiesis. The present study shows that Sox6 is required for silencing of epsilony globin in definitive erythropoiesis and suggests a role for Sox6 in erythroid cell maturation. Thus, Sox6 regulation of epsilony globin might provide a novel therapeutical target in the treatment of hemoglobinopathies such as sickle cell anemia and thalassemia.

  2. Targeted correction of a thalassemia-associated beta-globin mutation induced by pseudo-complementary peptide nucleic acids

    DEFF Research Database (Denmark)

    Lonkar, Pallavi; Kim, Ki-Hyun; Kuan, Jean Y

    2009-01-01

    Beta-thalassemia is a genetic disorder caused by mutations in the beta-globin gene. Triplex-forming oligonucleotides and triplex-forming peptide nucleic acids (PNAs) have been shown to stimulate recombination in mammalian cells via site-specific binding and creation of altered helical structures...... hindrance, pcPNAs are unable to form pcPNA-pcPNA duplexes but can bind to complementary DNA sequences via double duplex-invasion complexes. We demonstrate here that pcPNAs, when co-transfected with donor DNA fragments, can promote single base pair modification at the start of the second intron of the beta......-globin gene. This was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta globin fusion gene. We also demonstrate that pcPNAs are effective in stimulating recombination in human fibroblast cells in a manner dependent on the nucleotide excision repair...

  3. Long-Term Engraftment and Fetal Globin Induction upon BCL11A Gene Editing in Bone-Marrow-Derived CD34+ Hematopoietic Stem and Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Kai-Hsin Chang

    2017-03-01

    Full Text Available To develop an effective and sustainable cell therapy for sickle cell disease (SCD, we investigated the feasibility of targeted disruption of the BCL11A gene, either within exon 2 or at the GATAA motif in the intronic erythroid-specific enhancer, using zinc finger nucleases in human bone marrow (BM CD34+ hematopoietic stem and progenitor cells (HSPCs. Both targeting strategies upregulated fetal globin expression in erythroid cells to levels predicted to inhibit hemoglobin S polymerization. However, complete inactivation of BCL11A resulting from bi-allelic frameshift mutations in BCL11A exon 2 adversely affected erythroid enucleation. In contrast, bi-allelic disruption of the GATAA motif in the erythroid enhancer of BCL11A did not negatively impact enucleation. Furthermore, BCL11A exon 2-edited BM-CD34+ cells demonstrated a significantly reduced engraftment potential in immunodeficient mice. Such an adverse effect on HSPC function was not observed upon BCL11A erythroid-enhancer GATAA motif editing, because enhancer-edited CD34+ cells achieved robust long-term engraftment and gave rise to erythroid cells with elevated levels of fetal globin expression when chimeric BM was cultured ex vivo. Altogether, our results support further clinical development of the BCL11A erythroid-specific enhancer editing in BM-CD34+ HSPCs as an autologous stem cell therapy in SCD patients.

  4. Co-inheritance of the rare β hemoglobin variants Hb Yaounde, Hb Görwihl and Hb City of Hope with other alterations in globin genes: impact in genetic counseling.

    Science.gov (United States)

    Vinciguerra, Margherita; Passarello, Cristina; Leto, Filippo; Cassarà, Filippo; Cannata, Monica; Maggio, Aurelio; Giambona, Antonino

    2015-04-01

    Nearly 1183 different molecular defects of the globin genes leading to hemoglobin variants have been identified (http://globin.bx.psu.edu) over the past decades. The purpose of this study was to report three cases, never described in the literature, of co-inheritance of three β hemoglobin variants with other alterations in globin genes and to evaluate the clinical significance to conduct an appropriate genetic counseling. We report the molecular study performed in three probands and their families, sampling during the screening program conducted at the Laboratory for Molecular Prenatal Diagnosis of Hemoglobinopathies at Villa Sofia-Cervello Hospital in Palermo, Italy. This work allowed us to describe the co-inheritance of three rare β hemoglobin variants with other alterations in globin genes: the β hemoglobin variant Hb Yaounde [β134(H12)Val>Ala], found for the first time in combination with ααα(anti3.7) arrangement, and the β hemoglobin variants Hb Görwihl [β5(A2)Pro>Ala] and Hb City of Hope [β69(E13)Gly>Ser], found both in association with β(0) -thalassemia. The present work emphasizes the importance of a careful evaluation of the hematological data, especially in cases of atypical hematological parameters, to carry out an adequate and complete molecular study and to formulate an appropriate genetic counseling for couples at risk. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Production of β-globin and adult hemoglobin following G418 treatment of erythroid precursor cells from homozygous β039 thalassemia patients

    Science.gov (United States)

    Salvatori, Francesca; Breveglieri, Giulia; Zuccato, Cristina; Finotti, Alessia; Bianchi, Nicoletta; Borgatti, Monica; Feriotto, Giordana; Destro, Federica; Canella, Alessandro; Brognara, Eleonora; Lampronti, Ilaria; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2013-01-01

    In several types of thalassemia (including β039-thalassemia), stop codon mutations lead to premature translation termination and to mRNA destabilization through nonsense-mediated decay. Drugs (for instance aminoglycosides) can be designed to suppress premature termination, inducing a ribosomal readthrough. These findings have introduced new hopes for the development of a pharmacologic approach to the cure of this disease. However, the effects of aminoglycosides on globin mRNA carrying β-thalassemia stop mutations have not yet been investigated. In this study, we have used a lentiviral construct containing the β039- thalassemia globin gene under control of the β-globin promoter and a LCR cassette. We demonstrated by fluorescence-activated cell sorting (FACS) analysis the production of β-globin by K562 cell clones expressing the β039-thalassemia globin gene and treated with G418. More importantly, after FACS and high-performance liquid chromatography (HPLC) analyses, erythroid precursor cells from β039-thalassemia patients were demonstrated to be able to produce β-globin and adult hemoglobin after treatment with G418. This study strongly suggests that ribosomal readthrough should be considered a strategy for developing experimental strategies for the treatment of β0-thalassemia caused by stop codon mutations. PMID:19810011

  6. Wide diversity in structure and expression profiles among members of the Caenorhabditis elegans globin protein family

    Directory of Open Access Journals (Sweden)

    Vinogradov Serge

    2007-10-01

    Full Text Available Abstract Background The emergence of high throughput genome sequencing facilities and powerful high performance bioinformatic tools has highlighted hitherto unexpected wide occurrence of globins in the three kingdoms of life. In silico analysis of the genome of C. elegans identified 33 putative globin genes. It remains a mystery why this tiny animal might need so many globins. As an inroad to understanding this complexity we initiated a structural and functional analysis of the globin family in C. elegans. Results All 33 C. elegans putative globin genes are transcribed. The translated sequences have the essential signatures of single domain bona fide globins, or they contain a distinct globin domain that is part of a larger protein. All globin domains can be aligned so as to fit the globin fold, but internal interhelical and N- and C-terminal extensions and a variety of amino acid substitutions generate much structural diversity among the globins of C. elegans. Likewise, the encoding genes lack a conserved pattern of intron insertion positioning. We analyze the expression profiles of the globins during the progression of the life cycle, and we find that distinct subsets of globins are induced, or repressed, in wild-type dauers and in daf-2(e1370/insulin-receptor mutant adults, although these animals share several physiological features including resistance to elevated temperature, oxidative stress and hypoxic death. Several globin genes are upregulated following oxygen deprivation and we find that HIF-1 and DAF-2 each are required for this response. Our data indicate that the DAF-2 regulated transcription factor DAF-16/FOXO positively modulates hif-1 transcription under anoxia but opposes expression of the HIF-1 responsive globin genes itself. In contrast, the canonical globin of C. elegans, ZK637.13, is not responsive to anoxia. Reduced DAF-2 signaling leads to enhanced transcription of this globin and DAF-16 is required for this effect

  7. Synthetic promoter libraries- tuning of gene expression

    DEFF Research Database (Denmark)

    Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal

    2006-01-01

    The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene...... be met by using promoter libraries. This approach generally consists of inserting a library of promoters in front of the gene to be studied, whereby the individual promoters might deviate either in their spacer sequences or bear slight deviations from the consensus sequence of a vegetative promoter. Here......, we describe the two different methods for obtaining promoter libraries and compare their applicability....

  8. Interaction between Hb E and Hb Yala (HBB:c.129delT); a novel frameshift beta globin gene mutation, resulting in Hemoglobin E/β0 thalassemia.

    Science.gov (United States)

    Ekwattanakit, Supachai; Riolueang, Suchada; Viprakasit, Vip

    2018-03-01

    There are more than 200 known mutations found in patients with β-thalassemia, a possibility to identify an unknown or novel mutation becomes less possible. Here, we report a novel mutation in a patient from Thailand who presented with chronic hemolytic anemia. A comprehensive hematology and DNA analysis was applied in the index patient and her mother. Hematological and hemoglobin analyses were consistent with the clinical diagnosis of Hb E/β 0 -thalassemia. However, we could find only Hb E heterozygous mutation using our common polymerase chain reaction-based mutation detection of the β-globin genes. Furthermore, the molecular analysis demonstrated a novel T-deletion at codon 42 of the second exon of the β-globin gene which we named 'Hb Yala' according to the origin of this index family. This mutation was assumed to generate a truncated β-globin chain terminating at codon 60 with possible unstable variant leading to a 'null' or β 0 -thalassemia. However, the clinical phenotype was surprisingly mild and no other ameliorating genetic factors, including co-inheritance of α-thalassemia and high propensity of Hb F by Xmn I polymorphism, were found. This report has provided evidence that genotype-phenotype correlation in thalassemia syndromes is highly complex and a correct clinical severity classification of thalassemia should be mainly based on clinical evaluation.

  9. Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda.

    Science.gov (United States)

    Bracke, An; Hoogewijs, David; Dewilde, Sylvia

    2018-02-15

    Globins are among the best investigated proteins in biological and medical sciences and represent a prime tool for the study of the evolution of genes and the structure-function relationship of proteins. Here, we explore the recombinant expression of globins in three different expression systems: Escherichia coli, Pichia pastoris and the baculovirus infected Spodoptera frugiperda. We expressed two different human globin types in these three expression systems: I) the well-characterized neuroglobin and II) the uncharacterized, circular permutated globin domain of the large chimeric globin androglobin. It is clear from the literature that E.coli is the most used expression system for expression and purification of recombinant globins. However, the major disadvantage of E. coli is the formation of insoluble aggregates. We experienced that, for more complex multi-domain globins, like the chimeric globin androglobin, it is recommended to switch to a higher eukaryotic expression system. Copyright © 2017. Published by Elsevier Inc.

  10. A dominant control region from the human β-globin locus conferring integration site-independent gene expression.

    NARCIS (Netherlands)

    D. Talbot; P. Collis; M. Antoniou (Michael); M. Vidal; F.G. Grosveld (Frank); D.R. Greaves (David)

    1989-01-01

    textabstractThe regulatory elements that determine the expression pattern of a number of eukaryotic genes expressed specifically in certain tissues have been defined and studied in detail. In general, however, the expression conferred by these elements on genes reintroduced into the genomes of cell

  11. Clinical presentation and molecular identification of four uncommon alpha globin variants in Thailand. Initiation codon mutation of α2-globin Gene (HBA2:c.1delA), donor splice site mutation of α1-globin gene (IVSI-1, HBA1:c.95 + 1G>A), hemoglobin Queens Park/Chao Pra Ya (HBA1:c.98T>A) and hemoglobin Westmead (HBA2:c.369C>G).

    Science.gov (United States)

    Viprakasit, Vip; Ekwattanakit, Supachai; Chalaow, Nipon; Riolueang, Suchada; Wijit, Sirirat; Tanyut, Porntep; Chat-Uthai, Nunthawut; Tachavanich, Kalaya

    2014-01-01

    Alpha thalassemia is the most common genetic disease in the world with the prevalence of carriers ranging from 5-50% in several populations. Coinheritance of two defective α-globin genes usually gives rise to a symptomatic condition, hemoglobin (Hb) H disease. Previously, it has been suggested from several studies in different populations that nondeletional Hb H disease (--/α(T)α or --/αα(T)) is generally more severe than the deletional type (--/-α). In this report, we describe four rare nondeletional α-thalassemia mutations in Thai individuals, including initiation codon mutation (HBA2:c.1delA), donor splice site mutation (IVSI-1, HBA1:c.95 + 1G>A), Hb Queens Park (HBA1:c.98T>A) [α32(B13)Met>Lys], and Hb Westmead (HBA2:c.369C>G) [α122(H5)His>Gln]. Interactions of the first three mutations with the α(0)-thalassemia resulted in nondeletional Hb H disease; however, their clinical presentations were rather mild and some were detected accidentally. This suggests that a genotype-phenotype correlation of α-thalassemia syndrome might be more heterogeneous and so the type of mutation does not simply imply the prediction of the resulting phenotype. Our data will be of use in future genetic counseling of such conditions that are increasingly identified thanks to the improvement of molecular analysis in routine laboratories. © 2013 S. Karger AG, Basel.

  12. Application of Single Strand Conformational Polymorphism (PCR-SSCP) in Identification of Some Beta-Globin Gene Mutations in A Group of Egyptian Beta-Thalassemia Patients and Carriers

    International Nuclear Information System (INIS)

    Somaya, E.T.; Soliman, M.D

    2010-01-01

    The present study investigated whether the single-strand conformational polymorphism (SSCP) method could be employed to identify (rather than simply detect) four of the most common beta-globin gene mutations in the Egyptian population: IVS-I-110, IVS-I-6, the IVS-I-1, and Codon 39. Using DNA from 90 beta-thalassemia patients and carriers, by PCR the appropriate 238-bp region of the human beta-globin gene was amplified, the reaction products (Single-stranded DNA) were analyzed by none denaturing polyacrylamide gel electrophoresis, and the bands visualized by silver staining. Single-stranded DNA (ssDNA) fragments showed reproducible pattern of bands that were characteristic of the mutations present. With the use of control samples containing six of the 10 possible combinations of the four beta-globin gene mutations under study, we were able to predict the mutations present in 23 out of 90 (26.4%) of the patients studied. These predictions were confirmed independently by the amplification refractory mutation system (ARMS) method. It is concluded that this non-radioactive PCR-SSCP method can be used to reliably identify mutations in beta-thalassemia patients, provided that suitable controls are available. However, usefulness of this method for determining the genotype of beta-thalassaemic individuals is obviously limited by the great number of controls required. Moreover, the ability to detect mutations by SSCP is in general lower compared to other methods, ARMS, DGGE or DHPLC, which are reported to detect 49.5% to 73% of the mutations present. The SSCP method is nevertheless much easier to employ than other methods and is especially successful for beta-thalassemia carriers. This method would thus be particularly useful for an initial screening of target groups (prenatal diagnosis)

  13. Structural analysis of the 5' flanking region of the β-globin gene in African sickle cell anemia patients: Further evidence for three origins of the sickle cell mutation in Africa

    International Nuclear Information System (INIS)

    Chebloune, Y.; Pagnier, J.; Trabuchet, G.; Faure, C.; Verdier, G.; Labie, D.; Nigon, V.

    1988-01-01

    Haplotype analysis of the β-globin gene cluster shows two regions of DNA characterized by nonrandom association of restriction site polymorphisms. These regions are separated by a variable segment containing the repeated sequences (ATTTT) n and (AT) x T y , which might be involved in recombinational events. Studies of haplotypes linked to the sickle cell gene in Africa provide strong argument for three origins of the mutation: Benin, Senegal, and the Central African Republic. The structure of the variable segment in the three African populations was studied by S1 nuclease mapping of genomic DNA, which allows a comparison of several samples. A 1080-base-pair DNA segment was sequenced for one sample from each population. S1 nuclease mapping confirmed the homogeneity of each population with regard to both (ATTTT) n and (AT) x T y repeats. The authors found three additional structures for (AT) x T y correlating with the geographic origin of the patients. Ten other nucleotide positions, 5' and 3' to the (AT) x T y copies, were found to be variable when compared to homologous sequences from human and monkey DNAs. These results allow us to propose an evolutionary scheme for the polymorphisms in the 5' flanking region of the β-globin gene. The results strongly support the hypothesis of three origins for the sickle mutation in Africa

  14. Erythroid Krüppel-like factor (EKLF) is active in primitive and definitive erythroid cells and is required for the function of 5'HS3 of the beta-globin locus control region.

    Science.gov (United States)

    Tewari, R; Gillemans, N; Wijgerde, M; Nuez, B; von Lindern, M; Grosveld, F; Philipsen, S

    1998-04-15

    Disruption of the gene for transcription factor EKLF (erythroid Krüppel-like factor) results in fatal anaemia caused by severely reduced expression of the adult beta-globin gene, while other erythroid-specific genes, including the embryonic epsilon- and fetal gamma-globin genes, are expressed normally. Thus, EKLF is thought to be a stage-specific factor acting through the CACC box in the beta-gene promoter, even though it is already present in embryonic red cells. Here, we show that a beta-globin gene linked directly to the locus control region (LCR) is expressed at embryonic stages, and that this is only modestly reduced in EKLF-/- embryos. Thus, embryonic beta-globin expression is not intrinsically dependent on EKLF. To investigate whether EKLF functions in the locus control region, we analysed the expression of LCR-driven lacZ reporters. This shows that EKLF is not required for reporter activation by the complete LCR. However, embryonic expression of reporters driven by 5'HS3 of the LCR requires EKLF. This suggests that EKLF interacts directly with the CACC motifs in 5'HS3 and demonstrates that EKLF is also a transcriptional activator in embryonic erythropoiesis. Finally, we show that overexpression of EKLF results in an earlier switch from gamma- to beta-globin expression. Adult mice with the EKLF transgene have reduced platelet counts, suggesting that EKLF levels affect the balance between the megakaryocytic and erythroid lineages. Interestingly, the EKLF transgene rescues the lethal phenotype of EKLF null mice, setting the stage for future studies aimed at the analysis of the EKLF protein and its role in beta-globin gene activation.

  15. Frequency and origin of haplotypes associated with the beta-globin gene cluster in individuals with trait and sickle cell anemia in the Atlantic and Pacific coastal regions of Colombia

    Directory of Open Access Journals (Sweden)

    Cristian Fong

    2013-01-01

    Full Text Available Sickle cell anemia is a genetic disease with high prevalence in people of African descent. There are five typical haplotypes associated with this disease and the haplotypes associated with the beta-globin gene cluster have been used to establish the origin of African-descendant people in America. In this work, we determined the frequency and the origin of haplotypes associated with hemoglobin S in a sample of individuals with sickle cell anemia (HbSS and sickle cell hemoglobin trait (HbAS in coastal regions of Colombia. Blood samples from 71 HbAS and 79 HbSS individuals were obtained. Haplotypes were determined based on the presence of variable restriction sites within the β-globin gene cluster. On the Pacific coast of Colombia the most frequent haplotype was Benin, while on the Atlantic coast Bantu was marginally higher than Benin. Eight atypical haplotypes were observed on both coasts, being more diverse in the Atlantic than in the Pacific region. These results suggest a differential settlement of the coasts, dependent on where slaves were brought from, either from the Gulf of Guinea or from Angola, where the haplotype distributions are similar. Atypical haplotypes probably originated from point mutations that lost or gained a restriction site and/or by recombination events.

  16. Determination of the spectrum of beta-thalassemia genes in Spain by use of dot-blot analysis of amplified beta-globin DNA.

    OpenAIRE

    Amselem, S; Nunes, V; Vidaud, M; Estivill, X; Wong, C; d'Auriol, L; Vidaud, D; Galibert, F; Baiget, M; Goossens, M

    1988-01-01

    We have delineated the molecular lesions causing beta-thalassemia in Spain, a country that has witnessed the passage of different Mediterranean populations over the centuries, in order to evaluate the extent of heterogeneity of these mutations and to make possible simplified prenatal diagnosis of the disorder in that country. The use of the polymerase chain-reaction (PCR) technique to preferentially amplify beta-globin DNA sequences that contain the most frequent beta-thalassemia mutations in...

  17. Molecular phylogeny of ateline new world monkeys (Platyrrhini, atelinae) based on gamma-globin gene sequences: evidence that brachyteles is the sister group of lagothrix.

    Science.gov (United States)

    Meireles, C M; Czelusniak, J; Schneider, M P; Muniz, J A; Brigido, M C; Ferreira, H S; Goodman, M

    1999-06-01

    Nucleotide sequences, each spanning approximately 7 kb of the contiguous gamma1 and gamma2 globin genomic loci, were determined for seven species representing all extant genera (Ateles, Lagothrix, Brachyteles, and Alouatta) of the New World monkey subfamily Atelinae. After aligning these seven ateline sequences with outgroup sequences from several other primate (non-ateline) genera, they were analyzed by maximum parsimony, maximum likelihood, and neighbor-joining algorithms. All three analyzes estimated the same phylogenetic relationships: [Alouatta [Ateles (Brachyteles, Lagothrix)

  18. The GATA1-HS2 enhancer allows persistent and position-independent expression of a β-globin transgene.

    Directory of Open Access Journals (Sweden)

    Annarita Miccio

    Full Text Available Gene therapy of genetic diseases requires persistent and position-independent expression of a therapeutic transgene. Transcriptional enhancers binding chromatin-remodeling and modifying complexes may play a role in shielding transgenes from repressive chromatin effects. We tested the activity of the HS2 enhancer of the GATA1 gene in protecting the expression of a β-globin minigene delivered by a lentiviral vector in hematopoietic stem/progenitor cells. Gene expression from proviruses carrying GATA1-HS2 in both LTRs was persistent and resistant to silencing at most integration sites in the in vivo progeny of human hematopoietic progenitors and murine long-term repopulating stem cells. The GATA1-HS2-modified vector allowed correction of murine β-thalassemia at low copy number without inducing clonal selection of erythroblastic progenitors. Chromatin immunoprecipitation studies showed that GATA1 and the CBP acetyltransferase bind to GATA1-HS2, significantly increasing CBP-specific histone acetylations at the LTRs and β-globin promoter. Recruitment of CBP by the LTRs thus establishes an open chromatin domain encompassing the entire provirus, and increases the therapeutic efficacy of β-globin gene transfer by reducing expression variegation and epigenetic silencing.

  19. Co-inheritance of novel ATRX gene mutation and globin (α & β) gene mutations in transfusion dependent beta-thalassemia patients.

    Science.gov (United States)

    Al-Nafie, Awatif N; Borgio, J Francis; AbdulAzeez, Sayed; Al-Suliman, Ahmed M; Qaw, Fuad S; Naserullah, Zaki A; Al-Jarrash, Sana; Al-Madan, Mohammed S; Al-Ali, Rudaynah A; AlKhalifah, Mohammed A; Al-Muhanna, Fahad; Steinberg, Martin H; Al-Ali, Amein K

    2015-06-01

    α-Thalassemia X-linked mental retardation syndrome is a rare inherited intellectual disability disorder due to mutations in the ATRX gene. In our previous study of the prevalence of β-thalassemia mutations in the Eastern Province of Saudi Arabia, we confirmed the widespread coinheritance of α-thalassemia mutation. Some of these subjects have a family history of mental retardation, the cause of which is unknown. Therefore, we investigated the presence or absence of mutations in the ATRX gene in these patients. Three exons of the ATRX gene and their flanking regions were directly sequenced. Only four female transfusion dependent β-thalassemia patients were found to be carriers of a novel mutation in the ATRX gene. Two of the ATRX gene mutations, c.623delA and c.848T>C were present in patients homozygous for IVS I-5(G→C) and homozygous for Cd39(C → T) β-thalassemia mutation, respectively. While the other two that were located in the intronic region (flanking regions), were present in patients homozygous for Cd39(C → T) β-thalassemia mutation. The two subjects with the mutations in the coding region had family members with mental retardation, which suggests that the novel frame shift mutation and the missense mutation at coding region of ATRX gene are involved in ATRX syndrome. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Genome scan identifies a locus affecting gamma-globin expression in human beta-cluster YAC transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Lin, S.D.; Cooper, P.; Fung, J.; Weier, H.U.G.; Rubin, E.M.

    2000-03-01

    Genetic factors affecting post-natal g-globin expression - a major modifier of the severity of both b-thalassemia and sickle cell anemia, have been difficult to study. This is especially so in mice, an organism lacking a globin gene with an expression pattern equivalent to that of human g-globin. To model the human b-cluster in mice, with the goal of screening for loci affecting human g-globin expression in vivo, we introduced a human b-globin cluster YAC transgene into the genome of FVB mice . The b-cluster contained a Greek hereditary persistence of fetal hemoglobin (HPFH) g allele resulting in postnatal expression of human g-globin in transgenic mice. The level of human g-globin for various F1 hybrids derived from crosses between the FVB transgenics and other inbred mouse strains was assessed. The g-globin level of the C3HeB/FVB transgenic mice was noted to be significantly elevated. To map genes affecting postnatal g-globin expression, a 20 centiMorgan (cM) genome scan of a C3HeB/F VB transgenics [prime] FVB backcross was performed, followed by high-resolution marker analysis of promising loci. From this analysis we mapped a locus within a 2.2 cM interval of mouse chromosome 1 at a LOD score of 4.2 that contributes 10.4% of variation in g-globin expression level. Combining transgenic modeling of the human b-globin gene cluster with quantitative trait analysis, we have identified and mapped a murine locus that impacts on human g-globin expression in vivo.

  1. Genome scan identifies a locus affecting gamma-globin level in human beta-cluster YAC transgenic mice.

    Science.gov (United States)

    Lin, S D; Cooper, P; Fung, J; Weier, H U; Rubin, E M

    2000-11-01

    Genetic factors affecting postnatal gamma-globin expression--a major modifier of the severity of both beta-thalassemia and sickle cell anemia--have been difficult to study. This is especially so in mice, an organism lacking a globin gene with an expression pattern equivalent to that of human gamma-globin. To model the human beta-cluster in mice, with the goal of screening for loci affecting human gamma-globin expression in vivo, we introduced a human beta-globin cluster YAC transgene into the genome of FVB/N mice. The beta-cluster contained a Greek hereditary persistence of fetal hemoglobin (HPFH) gamma allele, resulting in postnatal expression of human gamma-globin in transgenic mice. The level of human gamma-globin for various F1 hybrids derived from crosses between the FVB/N transgenics and other inbred mouse strains was assessed. The gamma-globin level of the (C3HeB/FeJ x FVB/N)F1 transgenic mice was noted to be significantly elevated. To map genes affecting postnatal y-globin expression, we performed a 20-centiMorgan (cM) genome scan of a (C3HeB/FeJ x FVB/N)F1 transgenics x FVB/N backcross, followed by high-resolution marker analysis of promising loci. From this analysis we mapped a locus within an 18-cM interval of mouse Chromosome (Chr) 1 (LOD = 4.3) that contributes 10.9% of variation in gamma-globin level. Combining transgenic modeling of the human beta-globin gene cluster with quantitative trait analysis, we have identified and mapped a murine locus that impacts on human gamma-globin level in vivo.

  2. Monomethylfumarate induces γ-globin expression and fetal hemoglobin production in cultured human retinal pigment epithelial (RPE) and erythroid cells, and in intact retina.

    Science.gov (United States)

    Promsote, Wanwisa; Makala, Levi; Li, Biaoru; Smith, Sylvia B; Singh, Nagendra; Ganapathy, Vadivel; Pace, Betty S; Martin, Pamela M

    2014-05-13

    Sickle retinopathy (SR) is a major cause of vision loss in sickle cell disease (SCD). There are no strategies to prevent SR and treatments are extremely limited. The present study evaluated (1) the retinal pigment epithelial (RPE) cell as a hemoglobin producer and novel cellular target for fetal hemoglobin (HbF) induction, and (2) monomethylfumarate (MMF) as an HbF-inducing therapy and abrogator of oxidative stress and inflammation in SCD retina. Human globin gene expression was evaluated by RT-quantitative (q)PCR in the human RPE cell line ARPE-19 and in primary RPE cells isolated from Townes humanized SCD mice. γ-Globin promoter activity was monitored in KU812 stable dual luciferase reporter expressing cells treated with 0 to 1000 μM dimethylfumarate, MMF, or hydroxyurea (HU; positive control) by dual luciferase assay. Reverse transcriptase-qPCR, fluorescence-activated cell sorting (FACS), immunofluorescence, and Western blot techniques were used to evaluate γ-globin expression and HbF production in primary human erythroid progenitors, ARPE-19, and normal hemoglobin producing (HbAA) and homozygous β(s) mutation (HbSS) RPE that were treated similarly, and in MMF-injected (1000 μM) HbAA and HbSS retinas. Dihydroethidium labeling and nuclear factor (erythroid-derived 2)-like 2 (Nrf2), IL-1β, and VEGF expression were also analyzed. Retinal pigment epithelial cells express globin genes and synthesize adult and fetal hemoglobin MMF stimulated γ-globin expression and HbF production in cultured RPE and erythroid cells, and in HbSS mouse retina where it also reduced oxidative stress and inflammation. The production of hemoglobin by RPE suggests the potential involvement of this cell type in the etiology of SR. Monomethylfumarate influences multiple parameters consistent with improved retinal health in SCD and may therefore be of therapeutic potential in SR treatment. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  3. Characterization of histone H3K27 modifications in the β-globin locus

    International Nuclear Information System (INIS)

    Kim, Yea Woon; Kim, AeRi

    2011-01-01

    Research highlights: → The β-globin locus control region is hyperacetylated and monomethylated at histone H3K27. → Highly transcribed globin genes are marked by H3K27ac, but H3K27me2 is remarkable at silent globin genes in erythroid K562 cells. → Association of PRC2 subunits is comparable with H3K27me3 pattern. → Modifications of histone H3K27 are established in an enhancer-dependent manner. -- Abstract: Histone H3K27 is acetylated or methylated in the environment of nuclear chromatin. Here, to characterize the modification pattern of H3K27 in locus control region (LCR) and to understand the correlation of various H3K27 modifications with transcriptional activity of genes, we analyzed the human β-globin locus using the ChIP assay. The LCR of the human β-globin locus was enriched by H3K27ac and H3K27me1 in erythroid K562 cells. The highly transcribed globin genes were hyperacetylated at H3K27, but the repressed globin genes were highly dimethylated at this lysine in these cells. However, in non-erythroid 293FT cells, the β-globin locus was marked by a high level of H3K27me3. EZH2 and SUZ12, subunits of polycomb repressive complex 2, were comparably detected with the H3K27me3 pattern in K562 and 293FT cells. In addition, H3K27ac, H3K27me1 and H3K27me3 were established in an enhancer-dependent manner in a model minichromosomal locus containing an enhancer and its target gene. Taken together, these results show that H3K27 modifications have distinctive correlations with the chromatin state or transcription level of genes and are influenced by an enhancer.

  4. Deletion of a region that is a candidate for the difference between the deletion forms of hereditary persistence of fetal hemoglobin and deltabeta-thalassemia affects beta- but not gamma-globin gene expression.

    NARCIS (Netherlands)

    R. Calzolari (Roberta); T. McMorrow (Tara); N. Yannoutsos (Nikos); A. Langeveld (An); F.G. Grosveld (Frank)

    1999-01-01

    textabstractThe analysis of a number of cases of beta-globin thalassemia and hereditary persistence of fetal hemoglobin (HPFH) due to large deletions in the beta-globin locus has led to the identification of several DNA elements that have been implicated in the switch

  5. Análise dos haplótipos do gene da betaS-globina no Ceará Analysis of betaS-globin gene haplotypes in Ceará, Brazil

    Directory of Open Access Journals (Sweden)

    Gentil Claudino de Galiza Neto

    2005-10-01

    Full Text Available No presente trabalho abordam-se vários aspectos relacionados à natureza molecular da anemia falciforme (AF, desordem hematológica de caráter hereditário. A descoberta do polimorfismo do DNA no grupamento do gene betaS, originando diferentes haplótipos da doença, permitiu ampliar o conhecimento em torno da heterogeneidade clínica observada nos pacientes falcêmicos nas mais diversas regiões do mundo. Analisaram-se os diferentes haplótipos e seus parâmetros hematológicos, presentes em um grupo de 22 pacientes naturais e procedentes do estado do Ceará. A distribuição das freqüências dos haplótipos encontrados foi de 55,9% para Benin; 41,2% para República Centro-Africana (CAR; e de 2,9% para o haplótipo Senegal. Esses dados, em comparação com os demais estudos realizados no Brasil, mostram associação entre os seus valores para um alfa de 5% (p The present work deals with numerous aspects related to the molecular nature of sickle cell anemia. The discovery of the DNA polymorphism in the betas-globin gene cluster, gave origin to different haplotypes of the disease, making possible to enlarge the knowledge about the clinical heterogenity observed on the patients with sickle cell disease, in the various regions of the world. The different haplotypes and its hematological parameters were analysed in a group of 22 patients born in the State of Ceará, northeast of Brazil. The distribution found in the haplotypes frequency was of 55.9% for the Benin, of 41.2% for the CAR, and of 2.9% for Senegal haplotype. The data, compared to the others works done in Brazil, show relations among their values to alpha of 5% (p < 0,05. The results make possible a full understanding of the pathophisiology of the illness and of its clinical complexity in our State, as well as it allows a better knowledge of the sickle cell disease in our country.

  6. Search for antisense copies of beta-globin mRNA in anemic mouse spleen

    Directory of Open Access Journals (Sweden)

    Taylor John M

    2001-03-01

    Full Text Available Abstract Background Previous studies by Volloch and coworkers have reported that during the expression of high levels of β-globin mRNA in the spleen of anemic mice, they could also detect small but significant levels of an antisense (AS globin RNA species, which they postulated might have somehow arisen by RNA-directed RNA synthesis. For two reasons we undertook to confirm and possibly extend these studies. First, previous studies in our lab have focussed on what is an unequivocal example of host RNA-directed RNA polymerase activity on the RNA genome of human hepatitis delta virus. Second, if AS globin species do exist they could in turn form double-stranded RNA species which might induce post-transcriptional gene silencing, a phenomenon somehow provoked in eukaryotic cells by AS RNA sequences. Results We reexamined critical aspects of the previous globin studies. We used intraperitoneal injections of phenylhydrazine to induce anemia in mice, as demonstrated by the appearance and ultimate disappearance of splenomegaly. While a 30-fold increase in globin mRNA was detected in the spleen, the relative amount of putative AS RNA could be no more than 0.004%. Conclusions Contrary to earlier reports, induction of a major increase in globin transcripts in the mouse spleen was not associated with a detectable level of antisense RNA to globin mRNA.

  7. Variation in RNA-Seq transcriptome profiles of peripheral whole blood from healthy individuals with and without globin depletion.

    Directory of Open Access Journals (Sweden)

    Heesun Shin

    Full Text Available BACKGROUND: The molecular profile of circulating blood can reflect physiological and pathological events occurring in other tissues and organs of the body and delivers a comprehensive view of the status of the immune system. Blood has been useful in studying the pathobiology of many diseases. It is accessible and easily collected making it ideally suited to the development of diagnostic biomarker tests. The blood transcriptome has a high complement of globin RNA that could potentially saturate next-generation sequencing platforms, masking lower abundance transcripts. Methods to deplete globin mRNA are available, but their effect has not been comprehensively studied in peripheral whole blood RNA-Seq data. In this study we aimed to assess technical variability associated with globin depletion in addition to assessing general technical variability in RNA-Seq from whole blood derived samples. RESULTS: We compared technical and biological replicates having undergone globin depletion or not and found that the experimental globin depletion protocol employed removed approximately 80% of globin transcripts, improved the correlation of technical replicates, allowed for reliable detection of thousands of additional transcripts and generally increased transcript abundance measures. Differential expression analysis revealed thousands of genes significantly up-regulated as a result of globin depletion. In addition, globin depletion resulted in the down-regulation of genes involved in both iron and zinc metal ion bonding. CONCLUSIONS: Globin depletion appears to meaningfully improve the quality of peripheral whole blood RNA-Seq data, and may improve our ability to detect true biological variation. Some concerns remain, however. Key amongst them the significant reduction in RNA yields following globin depletion. More generally, our investigation of technical and biological variation with and without globin depletion finds that high-throughput sequencing by RNA

  8. Variation in RNA-Seq transcriptome profiles of peripheral whole blood from healthy individuals with and without globin depletion.

    Science.gov (United States)

    Shin, Heesun; Shannon, Casey P; Fishbane, Nick; Ruan, Jian; Zhou, Mi; Balshaw, Robert; Wilson-McManus, Janet E; Ng, Raymond T; McManus, Bruce M; Tebbutt, Scott J

    2014-01-01

    The molecular profile of circulating blood can reflect physiological and pathological events occurring in other tissues and organs of the body and delivers a comprehensive view of the status of the immune system. Blood has been useful in studying the pathobiology of many diseases. It is accessible and easily collected making it ideally suited to the development of diagnostic biomarker tests. The blood transcriptome has a high complement of globin RNA that could potentially saturate next-generation sequencing platforms, masking lower abundance transcripts. Methods to deplete globin mRNA are available, but their effect has not been comprehensively studied in peripheral whole blood RNA-Seq data. In this study we aimed to assess technical variability associated with globin depletion in addition to assessing general technical variability in RNA-Seq from whole blood derived samples. We compared technical and biological replicates having undergone globin depletion or not and found that the experimental globin depletion protocol employed removed approximately 80% of globin transcripts, improved the correlation of technical replicates, allowed for reliable detection of thousands of additional transcripts and generally increased transcript abundance measures. Differential expression analysis revealed thousands of genes significantly up-regulated as a result of globin depletion. In addition, globin depletion resulted in the down-regulation of genes involved in both iron and zinc metal ion bonding. Globin depletion appears to meaningfully improve the quality of peripheral whole blood RNA-Seq data, and may improve our ability to detect true biological variation. Some concerns remain, however. Key amongst them the significant reduction in RNA yields following globin depletion. More generally, our investigation of technical and biological variation with and without globin depletion finds that high-throughput sequencing by RNA-Seq is highly reproducible within a large dynamic

  9. Genetics Home Reference: methemoglobinemia, beta-globin type

    Science.gov (United States)

    ... Home Health Conditions Methemoglobinemia, beta-globin type Methemoglobinemia, beta-globin type Printable PDF Open All Close All ... to view the expand/collapse boxes. Description Methemoglobinemia, beta-globin type is a condition that affects the ...

  10. Genetic origin of Behçet's disease population in Denizli, Turkey; population genetics data analysis; historical demography and geographical perspectives based on β-globin gene cluster haplotype variation.

    Science.gov (United States)

    Ozturk, O; Arikan, S; Bahadir, A; Atalay, A; Atalay, E O

    2017-01-01

    In our study, we aimed to investigate the possible genetic drift, relationships, expansion and historical origin based on haplotype frequencies of the β-globin gene cluster of normal and Behçet's disease (BD) population in Denizli, Turkey. We examined blood DNA samples obtained from our DNA bank. The association of population genetic parameters such as haplotypes, diversity, differentiation, Hardy-Weinberg equilibrium and demographic analysis for two populations was performed by Arlequin ver. 3.5. Our results show that both populations have high similarity in genetic parameters in terms of development and expansion based on haplotype diversity through the history. We found that historical levels of gene flow were significantly higher between the two populations. According to historical population, growth parameter of τ values for normal and BD populations dated approximately 42 000 to 38 000 ybp, respectively. In conclusion, historically, two populations show similar genetic parameters and unimodal growth distribution. Our results are consistent with the view that the BD may have occurred in area, independent from Silk Road.

  11. A membrane-bound vertebrate globin.

    Directory of Open Access Journals (Sweden)

    Miriam Blank

    Full Text Available The family of vertebrate globins includes hemoglobin, myoglobin, and other O(2-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and membrane localization of globin X. To the best of our knowledge, this is the first time that a vertebrate globin has been identified as component of the cell membrane. Globin X has a hexacoordinate binding scheme and displays cooperative O(2 binding with a variable affinity (P(50∼1.3-12.5 torr, depending on buffer conditions. A respiratory function of globin X is unlikely, but analogous to some prokaryotic membrane-globins it may either protect the lipids in cell membrane from oxidation or may act as a redox-sensing or signaling protein.

  12. Isolating Barley ( Hordeum vulgare L.) B1 Hordein Gene Promoter ...

    African Journals Online (AJOL)

    Promoters play the most important role in determining the temporal and spatial expression pattern and transcript level of a gene. Some strong constitutive promoters, such as cauliflower mosaic virus 35s promoter, are widely used in plant genetic engineering research. However, the expression levels of the foreign genes in ...

  13. A novel in vivo transcription assay demonstrates the presence of globin-inducing trans-acting factors in uninduced Murine Erythroleukemia cells.

    NARCIS (Netherlands)

    N. Wrighton; F.G. Grosveld (Frank)

    1988-01-01

    textabstractWe report the development of a novel in vivo transcription assay for trans-acting factors regulating the human gamma- and beta-globin genes. A cDNA coding for the human tissue-type plasminogen activator (t-PA) was inserted into the globin genes. Simian virus 40 small T-antigen splice and

  14. Reconstructing Dynamic Promoter Activity Profiles from Reporter Gene Data

    DEFF Research Database (Denmark)

    Kannan, Soumya; Sams, Thomas; Maury, Jérôme

    2018-01-01

    Accurate characterization of promoter activity is important when designing expression systems for systems biology and metabolic engineering applications. Promoters that respond to changes in the environment enable the dynamic control of gene expression without the necessity of inducer compounds, ...

  15. Correlation between RAGE gene promoter methylation and diabetic retinal inflammation.

    Science.gov (United States)

    Kan, Shifeng; Wu, Jing; Sun, Chengxi; Hao, Jing; Wu, Zhen

    2018-01-01

    The methylation status of the receptor for advanced glycation end products (RAGE) gene promoter in peripheral blood mononuclear cells (PBMCs) of type 2 diabetic retinopathy (DR) patients was evaluated to investigate the correlation between RAGE gene promoter methylation and diabetic retinal inflammation. Eighty patients admitted and diagnosed as type 2 DR in Qilu Hospital, Shandong University during the period from October, 2013 to October, 2015 were enrolled in this study. They were the observation group and 40 healthy subjects were enrolled in the control group. PBMCs were collected from patients using density gradient centrifugation, and the methylation status of RAGE gene promoters was detected using methylation-specific PCP (MSP). Interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) levels of in the serum were measured using enzyme-linked immunosorbent assay (ELISA). PBMCs in patients with positive RAGE gene promoter methylation were isolated and cultured and RAGE gene promoter methylation was inhibited using the demethylating agent, 5'-aza-2'-deoxycytidine (5-aza-dC). The methylation status of RAGE gene promoters in PBMCs was detected via MSP. IL-1β, IL-6 and TNF-α levels in the supernatant of PBMC culture solution were evaluated using ELISA. MSP results showed that there were 26 cases (32.50%) of RAGE gene promoter methylation in PBMCs in DR patients. RAGE gene promoters were methylated in all normal healthy subjects. IL-1β, IL-6 and TNF-α levels in serum for positive RAGE gene promoter methylation group were significantly lower than those in negative RAGE gene promoter methylation group (pRAGE gene promoter methylation of PBMCs in patients with positive RAGE gene promoter methylation. The inhibition of methylation in RAGE gene promoter increased the levels of IL-1β, IL-6 and TNF-α in supernatant of culture solution. In conclusion, RAGE gene promoter hypomethylation was detected in DR patients, indicating that RAGE gene promoter

  16. Erythroid Krüppel-like factor (EKLF) is active in primitive and definitive erythroid cells and is required for the function of 5'HS3 of the beta-globin locus control region

    NARCIS (Netherlands)

    Tewari, R.; Gillemans, N.; Wijgerde, M.; Nuez, B.; von Lindern, M.; Grosveld, F.; Philipsen, S.

    1998-01-01

    Disruption of the gene for transcription factor EKLF (erythroid Krüppel-like factor) results in fatal anaemia caused by severely reduced expression of the adult beta-globin gene, while other erythroid-specific genes, including the embryonic epsilon- and fetal gamma-globin genes, are expressed

  17. A randomized Phase I/II Trial of HQK-1001, an oral fetal globin gene inducer, in β–thalassaemia intermedia and HbE/β–thalassaemia

    Science.gov (United States)

    Fucharoen, Suthat; Inati, Adlette; Siritanaratku, Noppadol; Thein, Swee Lay; Wargin, William C.; Koussa, Suzanne; Taher, Ali; Chaneim, Nattawara; Boosalis, Michael; Berenson, Ronald; Perrine, Susan P.

    2014-01-01

    β–thalassemia intermedia syndromes (BTI) cause hemolytic anemia, ineffective erythropoiesis, and widespread complications. Higher fetal globin expression within genotypes reduces globin imbalance and ameliorates anemia. Sodium 2,2 dimethylbutyrate (HQK-1001), an orally bioavailable short-chain fatty acid derivative, induces γ-globin expression experimentally and is well-tolerated in normal subjects. Accordingly, a randomized, blinded, placebo-controlled, Phase I/II trial was performed in 21 adult BTI patients (14 with HbE/β0 thalassemia and 7 with β+/β0 thalassemia intermedia, to determine effective doses for fetal globin induction, safety, and tolerability. HQK-1001 or placebo were administered once daily for 8 weeks at four dose levels (10, 20, 30, or 40 mg/kg/day), and subjects were monitored for laboratory and clinical events. Pharmacokinetic profiles demonstrated a t1/2 of 10–12 hours. Adverse events with HQK-1001 treatment were not significantly different from placebo treatment. Median HbF increased with the 20 mg/kg treatment doses above baseline levels by 6.6% and 0.44 g/dL (p <0.01) in 8/9 subjects; total hemoglobin (Hgb) increased by a mean of 1.1 gm/dL in 4/9 subjects. These findings identify a safe oral therapeutic which induces fetal globin in BTI. Further investigation of HQK-1001 with longer dosing to definitively evaluate its hematologic potential appears warranted. PMID:23530969

  18. Precise regulation of gene expression dynamics favors complex promoter architectures.

    Directory of Open Access Journals (Sweden)

    Dirk Müller

    2009-01-01

    Full Text Available Promoters process signals through recruitment of transcription factors and RNA polymerase, and dynamic changes in promoter activity constitute a major noise source in gene expression. However, it is barely understood how complex promoter architectures determine key features of promoter dynamics. Here, we employ prototypical promoters of yeast ribosomal protein genes as well as simplified versions thereof to analyze the relations among promoter design, complexity, and function. These promoters combine the action of a general regulatory factor with that of specific transcription factors, a common motif of many eukaryotic promoters. By comprehensively analyzing stationary and dynamic promoter properties, this model-based approach enables us to pinpoint the structural characteristics underlying the observed behavior. Functional tradeoffs impose constraints on the promoter architecture of ribosomal protein genes. We find that a stable scaffold in the natural design results in low transcriptional noise and strong co-regulation of target genes in the presence of gene silencing. This configuration also exhibits superior shut-off properties, and it can serve as a tunable switch in living cells. Model validation with independent experimental data suggests that the models are sufficiently realistic. When combined, our results offer a mechanistic explanation for why specific factors are associated with low protein noise in vivo. Many of these findings hold for a broad range of model parameters and likely apply to other eukaryotic promoters of similar structure.

  19. Human fetal globin DNA sequences suggest novel conversion event.

    OpenAIRE

    Stoeckert, C J; Collins, F S; Weissman, S M

    1984-01-01

    DNA sequencing studies of two recently cloned human A gamma globin alleles has revealed a number of base differences which are clustered in the large intron (IVS-2). One allele has a previously undescribed IVS-2 sequence. Most of the allelic differences can be explained as resulting from a gene conversion event involving G gamma as a donor. A novel feature of this event is that three G gamma-like regions occur interspersed among unconverted areas of the A gamma gene. We propose that an altern...

  20. Modification of human beta-globin locus PAC clones by homologous recombination in Escherichia coli

    NARCIS (Netherlands)

    G.P. Patrinos (George); M. de Krom (Mariken); S. Bottardi; R.J. Janssens; E. Katsantoni (Eleni); A.W. Wai; D.J. Sherratt; F.G. Grosveld (Frank); A.M.A. Imam (Ali)

    2000-01-01

    textabstractWe report here modifications of human beta-globin PAC clones by homologous recombination in Escherichia coli DH10B, utilising a plasmid temperature sensitive for replication, the recA gene and a wild-type copy of the rpsL gene which allows for an efficient selection for

  1. Reconstructing Dynamic Promoter Activity Profiles from Reporter Gene Data.

    Science.gov (United States)

    Kannan, Soumya; Sams, Thomas; Maury, Jérôme; Workman, Christopher T

    2018-03-16

    Accurate characterization of promoter activity is important when designing expression systems for systems biology and metabolic engineering applications. Promoters that respond to changes in the environment enable the dynamic control of gene expression without the necessity of inducer compounds, for example. However, the dynamic nature of these processes poses challenges for estimating promoter activity. Most experimental approaches utilize reporter gene expression to estimate promoter activity. Typically the reporter gene encodes a fluorescent protein that is used to infer a constant promoter activity despite the fact that the observed output may be dynamic and is a number of steps away from the transcription process. In fact, some promoters that are often thought of as constitutive can show changes in activity when growth conditions change. For these reasons, we have developed a system of ordinary differential equations for estimating dynamic promoter activity for promoters that change their activity in response to the environment that is robust to noise and changes in growth rate. Our approach, inference of dynamic promoter activity (PromAct), improves on existing methods by more accurately inferring known promoter activity profiles. This method is also capable of estimating the correct scale of promoter activity and can be applied to quantitative data sets to estimate quantitative rates.

  2. Activator-mediated Recruitment of the MLL2 Methyltransferase Complex to the β-globin Locus

    Science.gov (United States)

    Demers, Celina; Chaturvedi, Chandra-Prakash; Ranish, Jeffrey A.; Juban, Gaetan; Lai, Patrick; Morle, Francois; Aebersold, Ruedi; Dilworth, F. Jeffrey; Groudine, Mark; Brand, Marjorie

    2007-01-01

    Summary MLL-containing complexes methylate histone H3 at lysine 4 (H3K4) and have been implicated in the regulation of transcription. However, it is unclear how MLL complexes are targeted to specific gene loci. Here, we show that the MLL2 complex associates with the hematopoietic activator NF-E2 in erythroid cells and is important for H3K4 trimethylation and maximal levels of transcription at the β-globin locus. Furthermore, recruitment of the MLL2 complex to the β-globin locus is dependent upon NF-E2 and coincides spatio-temporally with NF-E2 binding during erythroid differentiation. Thus a DNA-bound activator is important initially for guiding MLL2 to a particular genomic location. Interestingly, while the MLL2-associated subunit Ash2L is restricted to the β-globin locus control region 38 kb upstream of the βmaj-globin gene, the MLL2 protein spreads across the β-globin locus, suggesting a previously undefined mechanism by which an activator influences transcription and H3K4 trimethylation at a distance. PMID:17707229

  3. Activator-mediated recruitment of the MLL2 methyltransferase complex to the beta-globin locus.

    Science.gov (United States)

    Demers, Celina; Chaturvedi, Chandra-Prakash; Ranish, Jeffrey A; Juban, Gaetan; Lai, Patrick; Morle, Francois; Aebersold, Ruedi; Dilworth, F Jeffrey; Groudine, Mark; Brand, Marjorie

    2007-08-17

    MLL-containing complexes methylate histone H3 at lysine 4 (H3K4) and have been implicated in the regulation of transcription. However, it is unclear how MLL complexes are targeted to specific gene loci. Here, we show that the MLL2 complex associates with the hematopoietic activator NF-E2 in erythroid cells and is important for H3K4 trimethylation and maximal levels of transcription at the beta-globin locus. Furthermore, recruitment of the MLL2 complex to the beta-globin locus is dependent upon NF-E2 and coincides spatio-temporally with NF-E2 binding during erythroid differentiation. Thus, a DNA-bound activator is important initially for guiding MLL2 to a particular genomic location. Interestingly, while the MLL2-associated subunit ASH2L is restricted to the beta-globin locus control region 38 kb upstream of the beta(maj)-globin gene, the MLL2 protein spreads across the beta-globin locus, suggesting a previously undefined mechanism by which an activator influences transcription and H3K4 trimethylation at a distance.

  4. Variation in the number of alpha-globin loci in sheep.

    Science.gov (United States)

    Rando, A; Ramunno, L; Masina, P

    1986-03-01

    Southern blot analysis was used to compare sheep and goat restriction-endonuclease maps of the DNA region containing the alpha-globin genes. The identical digestion patterns observed in both species with three endonucleases (BamHI, BstEII, and PstI) show that in sheep a single chromosome normally bears two nonallelic alpha-globin genes positioned at the same distance as in goat. Variant digestion patterns with enzymes that cleave outside (BamHI and HindIII) and within (EcoRI) the alpha-globin loci allowed us to infer that chromosomes with different numbers of alpha-globin loci are also present in sheep. In particular, in the 60 sheep considered, four individuals were heterozygous (alpha alpha/alpha alpha alpha) and one was homozygous (alpha alpha alpha/alpha alpha alpha) for chromosomes with three loci and one individual was heterozygous for a chromosome with four loci (alpha alpha/alpha alpha alpha alpha). This variation in the number of copies of alpha-globin loci can be explained by means of unequal crossovers.

  5. Hereditary Persistence of Fetal Hemoglobin Caused by Single Nucleotide Promoter Mutations in Sickle Cell Trait and Hb SC Disease.

    Science.gov (United States)

    Akinbami, Anthony O; Campbell, Andrew D; Han, Zeqiu J; Luo, Hong-Yuan; Chui, David H K; Steinberg, Martin H

    2016-01-01

    Hereditary persistence of fetal hemoglobin (HPFH) can be caused by point mutations in the γ-globin gene promoters. We report three rare cases: a child compound heterozygous for Hb S (HBB: c.20A > T) and HPFH with a novel point mutation in the (A)γ-globin gene promoter who had 42.0% Hb S, 17.0% Hb A and 38.0% Hb F; a man with Hb SC (HBB: c.19G > A) disease and a point mutation in the (G)γ-globin gene promoter who had 54.0% Hb S, 18.0% Hb C and 25.0% Hb F; a child heterozygous for Hb S and HPFH due to mutations in both the (A)γ- and (G)γ-globin gene promoters in cis [(G)γ(A)γ(β(+)) HPFH], with 67.0% Hb A, 6.5% Hb S and 25.0% Hb F.

  6. Splice-site mutations cause Rrp6-mediated nuclear retention of the unspliced RNAs and transcriptional down-regulation of the splicing-defective genes.

    Directory of Open Access Journals (Sweden)

    Andrea B Eberle

    Full Text Available BACKGROUND: Eukaryotic cells have developed surveillance mechanisms to prevent the expression of aberrant transcripts. An early surveillance checkpoint acts at the transcription site and prevents the release of mRNAs that carry processing defects. The exosome subunit Rrp6 is required for this checkpoint in Saccharomyces cerevisiae, but it is not known whether Rrp6 also plays a role in mRNA surveillance in higher eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an in vivo system to study nuclear mRNA surveillance in Drosophila melanogaster. We have produced S2 cells that express a human beta-globin gene with mutated splice sites in intron 2 (mut beta-globin. The transcripts encoded by the mut beta-globin gene are normally spliced at intron 1 but retain intron 2. The levels of the mut beta-globin transcripts are much lower than those of wild type (wt ss-globin mRNAs transcribed from the same promoter. We have compared the expression of the mut and wt beta-globin genes to investigate the mechanisms that down-regulate the production of defective mRNAs. Both wt and mut beta-globin transcripts are processed at the 3', but the mut beta-globin transcripts are less efficiently cleaved than the wt transcripts. Moreover, the mut beta-globin transcripts are less efficiently released from the transcription site, as shown by FISH, and this defect is restored by depletion of Rrp6 by RNAi. Furthermore, transcription of the mut beta-globin gene is significantly impaired as revealed by ChIP experiments that measure the association of the RNA polymerase II with the transcribed genes. We have also shown that the mut beta-globin gene shows reduced levels of H3K4me3. CONCLUSIONS/SIGNIFICANCE: Our results show that there are at least two surveillance responses that operate cotranscriptionally in insect cells and probably in all metazoans. One response requires Rrp6 and results in the inefficient release of defective mRNAs from the transcription site. The

  7. β-Thalassemia trait association with autoimmune diseases: β-globin locus proximity to the immunity genes or role of hemorphins?

    Science.gov (United States)

    Altinoz, Meric A; Gedikoglu, Gunduz; Deniz, Gunnur

    2012-04-01

    Thalassemia major continues to be a significant health problem for Mediterranean, Afro-Arabic countries, India and South Easth Asia. It was generally assumed that the β-thalassemia heterozygotes do not bear significant medical risks except a mild microcytic anemia. Nonetheless, increasing number of reports associate β-thalassemia trait with autoimmune conditions, nephritis, diabetes, arthritis, fibromyalgia and asthma. Available sparse data indicate reduced incidence of systemic lupus erythematosus (SLE) in β-thalassemia heterozygotes; yet, if two conditions coexist, the SLE manifestations occur much severer. These associations make sense when considering that the hemoglobin β-chain locus at 11p15.5 resides in close proximity to eight genes with profound roles in immune regulation: STIM1, CD151, TC21/RRAS2, SIGIRR/TOLL/IL1R8, pp52/LSP1 (lymphocyte specific protein), TRIM21, toll interacting protein (TOLLIP) and SLEN3. β-Thalassemia trait accompaniment to autoimmune disease may be the result of haplotypal associations between the close proximity genes. An alternative explanation to thalassemia heterozygosity: autoimmune disease association may be the changed concentrations of hemorphins. Hemorphins are endogenous opioid peptides derived via proteolytical cleavage of hemoglobin. They are shown to bind diverse opioid receptors and act anti-inflammatory. Their reduced expression in thalassemia heterozygosity may explain a proinflammatory stage and autoimmunity vulnerability.

  8. Inferring gene expression from ribosomal promoter sequences, a crowdsourcing approach.

    Science.gov (United States)

    Meyer, Pablo; Siwo, Geoffrey; Zeevi, Danny; Sharon, Eilon; Norel, Raquel; Segal, Eran; Stolovitzky, Gustavo

    2013-11-01

    The Gene Promoter Expression Prediction challenge consisted of predicting gene expression from promoter sequences in a previously unknown experimentally generated data set. The challenge was presented to the community in the framework of the sixth Dialogue for Reverse Engineering Assessments and Methods (DREAM6), a community effort to evaluate the status of systems biology modeling methodologies. Nucleotide-specific promoter activity was obtained by measuring fluorescence from promoter sequences fused upstream of a gene for yellow fluorescence protein and inserted in the same genomic site of yeast Saccharomyces cerevisiae. Twenty-one teams submitted results predicting the expression levels of 53 different promoters from yeast ribosomal protein genes. Analysis of participant predictions shows that accurate values for low-expressed and mutated promoters were difficult to obtain, although in the latter case, only when the mutation induced a large change in promoter activity compared to the wild-type sequence. As in previous DREAM challenges, we found that aggregation of participant predictions provided robust results, but did not fare better than the three best algorithms. Finally, this study not only provides a benchmark for the assessment of methods predicting activity of a specific set of promoters from their sequence, but it also shows that the top performing algorithm, which used machine-learning approaches, can be improved by the addition of biological features such as transcription factor binding sites.

  9. DNA methylation of PTEN gene promoter region is not correlated ...

    African Journals Online (AJOL)

    PTEN promoter hypermethylation has been found to be involved in many kinds of cancers. Up to date, no report about the relationships between methylation of PTEN promoter region and bladder cancer has been found. To investigate the methylation pattern of PTEN gene transcriptional regulation region (TRR), ...

  10. Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells

    Science.gov (United States)

    Aimola, Idowu A.; Inuwa, Hajiya M.; Nok, Andrew J.; Mamman, Aisha I.; Bieker, James J.

    2017-01-01

    Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24 h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ9 desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ9-desaturase inhibited cells 48 h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer PMID:26879870

  11. Comparative analysis of ADS gene promoter in seven Artemisia ...

    Indian Academy of Sciences (India)

    2014-12-23

    Dec 23, 2014 ... tent in Artemisia annua by enhancing the expression of genes in artemisinin biosynthetic pathway. Biologia 64, 319–323. Kiran K., Ansari S. A., Srivastava R., Lodhi N., Chaturvedi C. P.,. Sawant S. V. et al. 2006 The TATA-Box sequence in the basal promoter contributes to determining light-dependent gene.

  12. Bradykinin stimulation of nitric oxide production is not sufficient for gamma-globin induction

    Directory of Open Access Journals (Sweden)

    Čokić Vladan P.

    2014-01-01

    Full Text Available Introduction. Hydroxycarbamide, used in therapy of hemoglobinopathies, enhances nitric oxide (NO production both in primary human umbilical vein endothelial cells (HUVECs and human bone marrow endothelial cell line (TrHBMEC. Moreover, NO increases γ-globin and fetal hemoglobin levels in human erythroid progenitors. Objective. In order to find out whether simple physiologic stimulation of NO production by components of hematopoietic microenvironment can increase γ-globin gene expression, the effects of NO-inducer bradykinin were examined in endothelial cells. Methods. The study was performed in co-cultures of human erythroid progenitors, TrHBMEC and HUVECs by ozone-based chemiluminescent determination of NO and real-time quantitative RT-PCR. Results. In accordance with previous reports, the endogenous factor bradykinin increased endothelial cell production of NO in a dose- and time-dependent manner (0.1-0.6 μM up to 30 minutes. This induction of NO in HUVECs and TrHBMEC by bradykinin was blocked by competitive inhibitors of NO synthase (NOS, demonstrating NOS-dependence. It has been shown that bradykinin significantly reduced endothelial NOS (eNOS mRNA level and eNOS/Я-actin ratio in HUVEC (by twofold. In addition, bradykinin failed to increase γ-globin mRNA expression in erythroid progenitors only, as well as in co-culture studies of erythroid progenitors with TrHBMEC and HUVEC after 24 hours of treatment. Furthermore, bradykinin did not induce γ/β globin ratio in erythroid progenitors in co-cultures with HUVEC. Conclusion. Bradykinin mediated eNOS activation leads to short time and low NO production in endothelial cells, insufficient to induce γ-globin gene expression. These results emphasized the significance of elevated and extended NO production in augmentation of γ-globin gene expression. [Projekat Ministarstva nauke Republike Srbije, br. 175053

  13. Firefly luciferase gene contains a cryptic promoter

    Czech Academy of Sciences Publication Activity Database

    Vopálenský, V.; Mašek, T.; Horváth, Ondřej; Vicenová, B.; Mokrejš, M.; Pospíšek, M.

    2008-01-01

    Roč. 14, č. 9 (2008), s. 1720-1729 ISSN 1355-8382 Grant - others:GAČR(CZ) GA204/03/1487; GAČR(CZ) GA301/07/0607; Mšk(CZ) LC06066 Program:LC Institutional research plan: CEZ:AV0Z50520514 Keywords : luciferase * cryptic promoter * hepatitis C virus Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.018, year: 2008

  14. Cross-Sectional Study for the Detection of Mutations in the Beta-Globin Gene Among Patients with Hemoglobinopathies in the Bengali Population.

    Science.gov (United States)

    Panja, Amrita; Chowdhury, Prosanto; Chakraborty, Sharmistha; Ghosh, Tapan Kumar; Basu, Anupam

    2017-01-01

    Thalassemia is a common autosomal recessive blood disorder, which is most prevalent in South East Asian and Mediterranean populations. It is considered as a major health burden in the Indian population. The aims of the present study were to investigate the common, as well as uncommon, mutations responsible for thalassemia in the Bengali population. The Bengali state was divided into four sampling zones. Mutation detection was done using Sanger sequencing of the HBB gene. A total of 14 different mutations were observed, including rare mutations IVS1-130(G>C), IVS1-129(A>C), -90(T>C), CD16(-C), -30(T>C), CD15(-T), and a novel mutation CD53(C>T). The frequencies of IVS1-5(G>C) and CD26(G>A) mutations were higher than other mutations. There were also some silent polymorphisms found in the studied group, CD3(T>C), CD10(C>A), IVSII-16(G>C), IVSII-74(T>G), -42(C>G). The present study is the first attempt to screen for β-thalassemia-causing mutations by direct sequencing in different districts of West Bengal. The information obtained from the present study may be helpful for thalassemia management and prenatal mutation detection.

  15. Mechanosensitive promoter region in the human HB-GAM gene

    DEFF Research Database (Denmark)

    Liedert, Astrid; Kassem, Moustapha; Claes, Lutz

    2009-01-01

    expression through specific transcription factor binding sites in the promoter region of mechanosensitive genes. In the present study, we demonstrate that the expression of HB-GAM, which is known to have stimulating effects on osteogenic differentiation, is rapidly induced by mechanical loading in hMSC-TERT4...... cells. Analysis of the human HB-GAM gene upstream regulatory region with luciferase reporter gene assays revealed that the upregulation of HB-GAM expression occurred at the transcriptional level and was mainly dependent on the HB-GAM promoter region most upstream containing three potential AP-1 binding...

  16. Highly efficient expression of interleukin-2 under the control of rabbit β-globin intron II gene enhances protective immune responses of porcine reproductive and respiratory syndrome (PRRS DNA vaccine in pigs.

    Directory of Open Access Journals (Sweden)

    Yijun Du

    Full Text Available Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV had caused catastrophic losses in swine industry in China. The current inactivated vaccine provided only limited protection, and the attenuated live vaccine could protect piglets against the HP-PRRSV but there was a possibility that the attenuated virus returned to high virulence. In this study, the eukaryotic expression vector pVAX1© was modified under the control of rabbit β-globin intron II gene and the modified vector pMVAX1© was constructed. Porcine interleukin-2 (IL-2 and GP3-GP5 fusion protein of HP-PRRSV strain SD-JN were highly expressed by pMVAX1©. Mice inoculated with pMVAX1©-GP35 developed significantly higher PRRSV-specific antibody responses and T cell proliferation than those vaccinated with pVAX1©-GP35. pMVAX1©-GP35 was selected as PRRS DNA vaccine candidate and co-administrated with pVAX1©-IL-2 or pMVAX1©-IL-2 in pigs. pMVAX1©-IL-2+pMVAX1©-GP35 could provide enhanced PRRSV-specific antibody responses, T cell proliferation, Th1-type and Th2-type cytokine responses and CTL responses than pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35. Following homologous challenge with HP-PRRSV strain SD-JN, similar with attenuated PRRS vaccine group, pigs inoculated with pMVAX1©-IL-2+pMVAX1©-GP35 showed no clinical signs, almost no lung lesions and no viremia, as compared to those in pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35 groups. It indicated that pMVAX1©-IL-2 effectively increases humoral and cell mediated immune responses of pMVAX1©-GP35. Co-administration of pMVAX1©-IL-2 and pMVAX1©-GP35 might be attractive candidate vaccines for preventing HP-PRRSV infections.

  17. Highly efficient expression of interleukin-2 under the control of rabbit β-globin intron II gene enhances protective immune responses of porcine reproductive and respiratory syndrome (PRRS) DNA vaccine in pigs.

    Science.gov (United States)

    Du, Yijun; Lu, Yu; Wang, Xinglong; Qi, Jing; Liu, Jiyu; Hu, Yue; Li, Feng; Wu, Jiaqiang; Guo, Lihui; Liu, Junzhen; Tao, Haiying; Sun, Wenbo; Chen, Lei; Cong, Xiaoyan; Ren, Sufang; Shi, Jianli; Li, Jun; Wang, Jinbao; Huang, Baohua; Wan, Renzhong

    2014-01-01

    Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) had caused catastrophic losses in swine industry in China. The current inactivated vaccine provided only limited protection, and the attenuated live vaccine could protect piglets against the HP-PRRSV but there was a possibility that the attenuated virus returned to high virulence. In this study, the eukaryotic expression vector pVAX1© was modified under the control of rabbit β-globin intron II gene and the modified vector pMVAX1© was constructed. Porcine interleukin-2 (IL-2) and GP3-GP5 fusion protein of HP-PRRSV strain SD-JN were highly expressed by pMVAX1©. Mice inoculated with pMVAX1©-GP35 developed significantly higher PRRSV-specific antibody responses and T cell proliferation than those vaccinated with pVAX1©-GP35. pMVAX1©-GP35 was selected as PRRS DNA vaccine candidate and co-administrated with pVAX1©-IL-2 or pMVAX1©-IL-2 in pigs. pMVAX1©-IL-2+pMVAX1©-GP35 could provide enhanced PRRSV-specific antibody responses, T cell proliferation, Th1-type and Th2-type cytokine responses and CTL responses than pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35. Following homologous challenge with HP-PRRSV strain SD-JN, similar with attenuated PRRS vaccine group, pigs inoculated with pMVAX1©-IL-2+pMVAX1©-GP35 showed no clinical signs, almost no lung lesions and no viremia, as compared to those in pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35 groups. It indicated that pMVAX1©-IL-2 effectively increases humoral and cell mediated immune responses of pMVAX1©-GP35. Co-administration of pMVAX1©-IL-2 and pMVAX1©-GP35 might be attractive candidate vaccines for preventing HP-PRRSV infections.

  18. Evolution of Drosophila ribosomal protein gene core promoters.

    Science.gov (United States)

    Ma, Xiaotu; Zhang, Kangyu; Li, Xiaoman

    2009-03-01

    The coordinated expression of ribosomal protein genes (RPGs) has been well documented in many species. Previous analyses of RPG promoters focus only on Fungi and mammals. Recognizing this gap and using a comparative genomics approach, we utilize a motif-finding algorithm that incorporates cross-species conservation to identify several significant motifs in Drosophila RPG promoters. As a result, significant differences of the enriched motifs in RPG promoter are found among Drosophila, Fungi, and mammals, demonstrating the evolutionary dynamics of the ribosomal gene regulatory network. We also report a motif present in similar numbers of RPGs among Drosophila species which does not appear to be conserved at the individual RPG gene level. A module-wise stabilizing selection theory is proposed to explain this observation. Overall, our results provide significant insight into the fast-evolving nature of transcriptional regulation in the RPG module.

  19. Interleukin-18 gene promoter polymorphisms and recurrent spontaneous abortion.

    Science.gov (United States)

    Naeimi, Sirous; Ghiam, Alireza Fotouhi; Mojtahedi, Zahra; Dehaghani, Alamtaj Samsami; Amani, Dawar; Ghaderi, Abbas

    2006-01-01

    IL-18 is a multifunctional cytokine capable of inducing either Th1 or Th2 polarization depending on the immunologic milieu. IL-18 is detected at the materno-fetal interface very soon in early pregnancy. Two polymorphisms in the promoter region of the IL-18 gene at positions of -607 and -137 appear to have functional impacts. This study attempts to evaluate the frequency of these two polymorphisms in the IL-18 gene promoter in patients with recurrent spontaneous abortion (RSA) and normal pregnant women. One hundred and two RSA patients and 103 healthy pregnant women were enrolled in this study. Single nucleotide polymorphisms of the IL-18 gene at positions -607 (C/A) and -137 (G/C) were analyzed by the sequence-specific PCR method. There was no significant association between the allele, genotype, and haplotype frequencies of the two single nucleotide polymorphisms (SNPs) in the IL-18 gene promoter and RSA. The results of this study showed that IL-18 gene promoter polymorphisms at positions -607 and -137 did not confer susceptibility to RSA in southern Iranian patients.

  20. A novel BDNF gene promoter directs expression to skeletal muscle

    Directory of Open Access Journals (Sweden)

    Heinrich Gerhard

    2003-06-01

    Full Text Available Abstract Background Cell-specific expression of the gene that encodes brain-derived neurotrophic factor (BDNF is required for the normal development of peripheral sensory neurons and efficient synaptic transmission in the mature central and peripheral nervous system. The control of BDNF gene expression involves multiple tissue and cell-specific promoters that are differentially regulated. The molecular mechanisms that are responsible for tissue and cell-specific expression of these promoters are still incompletely understood. Results The cloning and analysis of three additional zebrafish (Danio rerio BDNF gene exons and two associated promoters, is reported. Among them are two exons that generate a novel tripartite mature transcript. The exons were located on the transcription unit, whose overall organization was determined by cloning, Southern blot hybridization and sequence analysis, and compared with the pufferfish (Fugu rubripes and mammalian BDNF loci, revealing a conserved but more compact organization. Structural and functional analysis of the exons, their adjacent promoters and 5' flanks, showed that they are expressed cell-specifically. The promoter associated with the 5' exon of the tripartite transcript is GC-rich, TATA-less and the 5' flank adjacent to it contains multiple Sp1, Mef2, and AP1 elements. A fusion gene containing the promoter and 1.5 KB of 5' flank is directed exclusively to skeletal muscle of transiently transfected embryos. The second promoter, whose associated 5' exon contains a 25-nucleotide segment of identity with a mammalian BDNF gene exon, was transiently expressed in yolk of the early embryo. RT-PCR analysis of total RNA from whole juvenile fish and adult female skeletal muscle revealed tissue-specific expression of the 5' exons but the novel exon could not be detected even after two rounds of nested PCR. Conclusion The zebrafish BDNF gene is as complex as the mammalian gene yet much more compact. Its exons are

  1. Structural and functional properties of designed globins

    Indian Academy of Sciences (India)

    Administrator

    De novo protein design is now thought to be an essential approach to elucidate the principles of protein ... We have proposed a new computational method to find the amino acid sequence that fits to a given protein .... Furthermore, the extinction coefficients of these peaks are also similar to those of natural globins and are ...

  2. Comparative analysis of ADS gene promoter in seven Artemisia ...

    Indian Academy of Sciences (India)

    Artemisinin is the most effective antimalarial drug that is derived from Artemisia annua. Amorpha-4,11-diene synthase (ADS) controls the first committed step in artemisinin biosynthesis. The ADS gene expression is regulated by transcription factors which bind to the cis-acting elements on the ADS promoter and are probably ...

  3. Interleukin-10 gene promoter polymorphism as a potential host ...

    African Journals Online (AJOL)

    10) gene have been associated with altered levels of circulating IL-10, a Th2 cytokine that plays a key role in the pathogenesis of TB. We analyzed the frequencies of IL-10 promoter polymorphisms in 82 TB patients and 99 healthy Pakistani ...

  4. Interleukin 10 gene promoter polymorphism and risk of diffuse large ...

    African Journals Online (AJOL)

    Purpose: Given the importance of understanding the genetic variations involved in the pathogenesis of non-Hodgkin's lymphoma (NHL), this work was designed to study the impact of IL-10 (1082 G/A; rs1800896 and 819 C/T; rs1800871) gene promoter polymorphism on susceptibility of Egyptians to diffuse large B cell ...

  5. Isolating Barley (Hordeum vulgare L.) B1 Hordein Gene Promoter ...

    African Journals Online (AJOL)

    Yomi

    2012-04-10

    Apr 10, 2012 ... Isolating Barley (Hordeum vulgare L.) B1 Hordein Gene. Promoter and Using Sequencing Analaysis. For The Identification of Conserved Regulatory. Elements By Bioinformatic Tools. Kobra Nalbandi1, Bahram Baghban Kohnehrouz2*, Khalil Alami Saeed1 and Ashraf. Gholizadeh3. 1Ramin Agricultural ...

  6. Gene promoter hypermethylation in leukoplakia of the oral mucosa

    Directory of Open Access Journals (Sweden)

    Mingli Liu

    2010-07-01

    Full Text Available Mingli Liu1, Lei Feng2, Ximing Tang3, Shanchun Guo41Department of Physics, Tufts University School of Medicine, Boston, Massachussetts; 2Department of Thoracic/Head and Neck Medical Oncology, 3Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, Texas; 4Sylvester Cancer Center, University of Miami School of Medicine, Florida, USAAbstract: To examine whether aberrant DNA methylation in the promoter region might occur earlier in tumorigenesis, particularly in premalignant lesions, we examined biopsies from 111 participants in a chemoprevention trial aimed at reversal of oral leukoplakia, using methylation-specific polymerase chain reaction for the promoter regions of the tumor suppressor gene CDKN2A (p16, the putative metastasis suppressor gene for death-associated protein kinase (DAP-K, the DNA repair gene O6-methyguanine-DNA-methyltransferase (MGMT, and the detoxification gene glutathione S-transferase p1(GSTP1. p16 promoter hypermethylation was detected in 21 of 82 (25.6%, DAP-K hypermethylation in 28 of 87 (32.2%, and MGMT hypermethylation in 32 of 106 (30.2% oral leukoplakia lesions analyzed. No aberrant methylation was found at the GSTP1 gene in 110 lesions examined. Among 68 biopsies analyzed for all three genes (p16, DAP-K, MGMT, 17 biopsies were detected with an abnormal methylation pattern at only one gene, 15 at two genes, and 8 at all three genes. Among clinical characteristics and their correlation with methylation, only alcohol consumption was correlated with DAP-K methylation (P = 0.027, while MGMT methylation was more frequent in females (P = 0.003 and nonsmokers (P = 0.0005. A significant correlation was found between p16 and DAP-K hypermethylation; p16 promoter was methylated in 14 (56% of 25 lesions with DAP-K methylation, and only 5 (11.1% of 45 DAP-K methylation-negative lesions (P = 0.0001. DAP-K aberrant methylation was also significantly correlated with MGMT methylation (16 of 31 in MGMT methylation

  7. Halophytes: Potential Resources for Salt Stress Tolerance Genes and Promoters.

    Science.gov (United States)

    Mishra, Avinash; Tanna, Bhakti

    2017-01-01

    Halophytes have demonstrated their capability to thrive under extremely saline conditions and thus considered as one of the best germplasm for saline agriculture. Salinity is a worldwide problem, and the salt-affected areas are increasing day-by-day because of scanty rainfall, poor irrigation system, salt ingression, water contamination, and other environmental factors. The salinity stress tolerance mechanism is a very complex phenomenon, and some pathways are coordinately linked for imparting salinity tolerance. Though a number of salt responsive genes have been reported from the halophytes, there is always a quest for promising stress-responsive genes that can modulate plant physiology according to the salt stress. Halophytes such as Aeluropus, Mesembryanthemum, Suaeda, Atriplex, Thellungiella, Cakile , and Salicornia serve as a potential candidate for the salt-responsive genes and promoters. Several known genes like antiporters ( NHX, SOS, HKT, VTPase ), ion channels (Cl - , Ca 2+ , aquaporins), antioxidant encoding genes ( APX, CAT, GST, BADH, SOD ) and some novel genes such as USP, SDR1, SRP etc. were isolated from halophytes and explored for developing stress tolerance in the crop plants (glycophytes). It is evidenced that stress triggers salt sensors that lead to the activation of stress tolerance mechanisms which involve multiple signaling proteins, up- or down-regulation of several genes, and finally the distinctive or collective effects of stress-responsive genes. In this review, halophytes are discussed as an excellent platform for salt responsive genes which can be utilized for developing salinity tolerance in crop plants through genetic engineering.

  8. Halophytes: Potential Resources for Salt Stress Tolerance Genes and Promoters

    Directory of Open Access Journals (Sweden)

    Avinash Mishra

    2017-05-01

    Full Text Available Halophytes have demonstrated their capability to thrive under extremely saline conditions and thus considered as one of the best germplasm for saline agriculture. Salinity is a worldwide problem, and the salt-affected areas are increasing day-by-day because of scanty rainfall, poor irrigation system, salt ingression, water contamination, and other environmental factors. The salinity stress tolerance mechanism is a very complex phenomenon, and some pathways are coordinately linked for imparting salinity tolerance. Though a number of salt responsive genes have been reported from the halophytes, there is always a quest for promising stress-responsive genes that can modulate plant physiology according to the salt stress. Halophytes such as Aeluropus, Mesembryanthemum, Suaeda, Atriplex, Thellungiella, Cakile, and Salicornia serve as a potential candidate for the salt-responsive genes and promoters. Several known genes like antiporters (NHX, SOS, HKT, VTPase, ion channels (Cl−, Ca2+, aquaporins, antioxidant encoding genes (APX, CAT, GST, BADH, SOD and some novel genes such as USP, SDR1, SRP etc. were isolated from halophytes and explored for developing stress tolerance in the crop plants (glycophytes. It is evidenced that stress triggers salt sensors that lead to the activation of stress tolerance mechanisms which involve multiple signaling proteins, up- or down-regulation of several genes, and finally the distinctive or collective effects of stress-responsive genes. In this review, halophytes are discussed as an excellent platform for salt responsive genes which can be utilized for developing salinity tolerance in crop plants through genetic engineering.

  9. Conservation of the primary structure, organization, and function of the human and mouse β-globin locus-activating regions

    International Nuclear Information System (INIS)

    Moon, A.M.; Ley, T.J.

    1990-01-01

    DNA sequences located in a region 6-18 kilobases (kb) upstream from the human ε-globin gene are known as the locus-activating region (LAR) or dominant control region. This region is thought to play a key role in chromatin organization of the β-like globin gene cluster during erythroid development. Since the human β-globin LAR is functional in mice, the authors reasoned that critical LAR sequence elements might be conserved between mice and humans. They therefore cloned murine genomic sequences homologous to one portion of the human LAR. They found that this murine DNA fragment (mouse LAR site II) and sequences homologous to human LAR sites I and III are located upstream from the mouse β-like globin gene cluster and determined that their locations relative to the cluster are similar to that of their human counterparts. The homologous site II sequences are 70% identical between mice and humans over a stretch of ∼800 base pairs. These results suggest that primary structural elements endash and the spatial organization of these elements endash are important for function of the β-globin LAR

  10. Specific expression and promoter analysis of the albumin gene promoter of the duck (Anas platyrhynchos domesticus).

    Science.gov (United States)

    Chen, Y; Yu, Q; Huang, Z; Wang, B; Xu, Q; Lan, L; Chang, G; Zhang, Y; Chen, G

    2017-02-01

    1. Albumin (ALB) is a serum protein most highly expressed in liver and regarded as an effective indicator for liver pathologies. The objectives of this study were to determine the expression of duck ALB gene (duALB) in various non-hepatic tissues and identify the potential cis-regulatory elements in the promoter. 2. A model was established to assess duALB promoter activity in different cell lines by construction of a duALB promoter-driven GFP (Green Fluorescent Protein)-expressing vector, which exhibited high expression activity in liver-derived cells and lower expression in other cells. Through the firefly luciferase reporter gene driven by a series of constructs carrying progressive deletions, the core transcriptional regulatory region within the duALB promoter was identified. Mutations in candidate-binding sites were made by site-directed mutagenesis. 3. The core transcriptional regulatory region was located in the -190/-51 bp region. This region contains three potential transcription factor-binding sites, one each for hepatocyte nuclear factor (HNF-3β) (-158/-149), CCAAT/Enhancer-binding protein element (C/EBPα) (-119/-107) and nuclear factor-1 (HNF-1) (-67/-57). Site-directed mutagenesis of HNF-1 and C/EBPα-binding sites resulted in a significant reduction in duALB promoter activity. Two potential cis-regulatory elements (C/EBPα and HNF-1) were responsible for its transcriptional activity in liver-derived cells. 4. These findings contribute to the further understanding of the fundamental mechanisms of ALB gene regulation and the use of tissue-specific gene promoters to regulate tissue-specific expression of exogenous genes in vivo.

  11. Promoter DNA hypermethylation and gene repression in undifferentiated Arabidopsis cells.

    Directory of Open Access Journals (Sweden)

    María Berdasco

    Full Text Available Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.

  12. The replication timing program of the Chinese hamster β-globin locus is established coincident with its repositioning near peripheral heterochromatin in early G1 phase

    Science.gov (United States)

    Li, Feng; Chen, Jianhua; Izumi, Masako; Butler, Mark C.; Keezer, Susan M.; Gilbert, David M.

    2001-01-01

    We have examined the dynamics of nuclear repositioning and the establishment of a replication timing program for the actively transcribed dihydrofolate reductase (DHFR) locus and the silent β-globin gene locus in Chinese hamster ovary cells. The DHFR locus was internally localized and replicated early, whereas the β-globin locus was localized adjacent to the nuclear periphery and replicated during the middle of S phase, coincident with replication of peripheral heterochromatin. Nuclei were prepared from cells synchronized at various times during early G1 phase and stimulated to enter S phase by introduction into Xenopus egg extracts, and the timing of DHFR and β-globin replication was evaluated in vitro. With nuclei isolated 1 h after mitosis, neither locus was preferentially replicated before the other. However, with nuclei isolated 2 or 3 h after mitosis, there was a strong preference for replication of DHFR before β-globin. Measurements of the distance of DHFR and β-globin to the nuclear periphery revealed that the repositioning of the β-globin locus adjacent to peripheral heterochromatin also took place between 1 and 2 h after mitosis. These results suggest that the CHO β-globin locus acquires the replication timing program of peripheral heterochromatin upon association with the peripheral subnuclear compartment during early G1 phase. PMID:11470818

  13. The replication timing program of the Chinese hamster ?-globin locus is established coincident with its repositioning near peripheral heterochromatin in early G1 phase

    OpenAIRE

    Li, Feng; Chen, Jianhua; Izumi, Masako; Butler, Mark C.; Keezer, Susan M.; Gilbert, David M.

    2001-01-01

    We have examined the dynamics of nuclear repositioning and the establishment of a replication timing program for the actively transcribed dihydrofolate reductase (DHFR) locus and the silent ?-globin gene locus in Chinese hamster ovary cells. The DHFR locus was internally localized and replicated early, whereas the ?-globin locus was localized adjacent to the nuclear periphery and replicated during the middle of S phase, coincident with replication of peripheral heterochromatin. Nuclei were pr...

  14. The altered promoter methylation of oxytocin receptor gene in autism.

    Science.gov (United States)

    Elagoz Yuksel, Mine; Yuceturk, Betul; Karatas, Omer Faruk; Ozen, Mustafa; Dogangun, Burak

    Autism spectrum disorder (ASD) is one of the lifelong existing disorders. Abnormal methylation status of gene promoters of oxytonergic system has been implicated as among the etiologic factors of ASDs. We, therefore, investigated the methylation frequency of oxytocin receptor gene (OXTR) promoter from peripheral blood samples of children with autistic features. Our sample includes 66 children in total (22-94 months); 27 children with ASDs according to the DSM-IV-TR and the Childhood Autism Rating Scale (CARS) and 39 children who do not have any autistic like symptoms as the healthy control group. We investigated the DNA methylation status of OXTR promoter by methylation specific enzymatic digestion of genomic DNA and polymerase chain reaction. A significant relationship has been found between ASDs and healthy controls for the reduction of methylation frequency of the regions MT1 and MT3 of OXTR. We could not find any association in the methylation frequency of MT2 and MT4 regions of OXTR. Although our findings indicate high frequency of OXTR promoter hypomethylation in ASDs, there is need for independent replication of the results for a bigger sample set. We expect that future studies with the inclusion of larger, more homogeneous samples will attempt to disentangle the causes of ASDs.

  15. Polymorphisms in the leptin gene promoter in Brazilian beef herds.

    Science.gov (United States)

    Guimarães, R C; Azevedo, J S N; Corrêa, S C; Campelo, J E G; Barbosa, E M; Gonçalves, E C; Silva Filho, E

    2016-12-02

    Brazil is the world's largest producer of beef cattle; however, the quality of its herds needs to be improved. The use of molecular markers as auxiliary tools in selecting animals for reproduction with high pattern for beef production would significantly improve the quality of the final beef product in Brazil. The leptin gene has been demonstrated to be an excellent candidate gene for bovine breeding. The objective of this study was to sequence and compare the leptin gene promoter of Brazil's important cattle breeds in order to identify polymorphisms in it. Blood samples of the Nellore, Guzerat, Tabapuã, and Senepol breeds were collected for genomic DNA extraction. The genomic DNA was used as a template for polymerase chain reaction (PCR) to amplify a 1575-bp fragment, which in turn was sequenced, aligned, and compared between animals of different breeds. Twenty-three single nucleotide polymorphic sites, including transitions and transversions, were detected at positions -1457, -1452, -1446, -1397, -1392, -1361, -1238, -963,-901, -578, -516, -483, -478, -470, -432, -430, -292, -282, -272, -211, -202, -170, and -147. Additionally, two insertion sites at positions -680 and -416 and two deletion sites at positions -1255 and -1059 were detected. As the promoter region of the leptin gene has been demonstrated to vary among breeds, these variations must be tested for their use as potential molecular markers for artificial selection of animals for enhanced beef production in different systems of bovine production in Brazil.

  16. Aberrant gene promoter methylation associated with sporadic multiple colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Victoria Gonzalo

    Full Text Available BACKGROUND: Colorectal cancer (CRC multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect. METHODOLOGY/PRINCIPAL FINDINGS: We examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2, RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008 and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047 as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006. Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17, SFRP1 (r = 0.83, 0.06, HPP1 (r = 0.64, p = 0.17, 3OST2 (r = 0.83, p = 0.06 and GATA4 (r = 0.6, p = 0.24. Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant

  17. Diversity of [beta]-globin mutations in Israeli ethnic groups reflects recent historic events

    Energy Technology Data Exchange (ETDEWEB)

    Filon, D.; Oron, V.; Krichevski, S.; Shaag, A.; Goldfarb, A.; Aker, M.; Rachmilewitz, E.A.; Rund, D.; Oppenheim, A. (Hebrew Univ. Hadassah-Medical School, Jerusalem (Israel)) (and others)

    1994-05-01

    The authors characterized nearly 500 [beta]-thalassemia genes from the Israeli population representing a variety of ethnic subgroups. They found 28 different mutations in the [beta]-globin gene, including three mutations ([beta][sup S], [beta][sup C], and [beta][sup O-Arab]) causing hemoglobinopathies. Marked genetic heterogeneity was observed in both the Arab (20 mutations) and Jewish (17 mutations) populations. On the other hand, two ethnic isolates - Druze and Samaritans - had a single mutation each. Fifteen of the [beta]-thalassemia alleles are Mediterranean in type, 5 originated in Kurdistan, 2 are of Indian origin, and 2 sporadic alleles came from Europe. Only one mutant allele-nonsense codon 37-appears to be indigenous to Israel. While human habitation in Israel dates back to early prehistory, the present-day spectrum of [beta]-globin mutations can be largely explained by migration events that occurred in the past millennium. 26 refs., 2 figs., 3 tabs.

  18. Promoter Methylation Analysis of IDH Genes in Human Gliomas.

    Science.gov (United States)

    Flanagan, Simon; Lee, Maggie; Li, Cheryl C Y; Suter, Catherine M; Buckland, Michael E

    2012-01-01

    Mutations in isocitrate dehydrogenase (IDH)-1 or -2 are found in the majority of WHO grade II and III astrocytomas and oligodendrogliomas, and secondary glioblastomas. Almost all described mutations are heterozygous missense mutations affecting a conserved arginine residue in the substrate binding site of IDH1 (R132) or IDH2 (R172). But the exact mechanism of IDH mutations in neoplasia is not understood. It has been proposed that IDH mutations impart a "toxic gain-of-function" to the mutant protein, however a dominant-negative effect of mutant IDH has also been described, implying that IDH may function as a tumor suppressor gene. As most, if not all, tumor suppressor genes are inactivated by epigenetic silencing, in a wide variety of tumors, we asked if IDH1 or IDH2 carry the epigenetic signature of a tumor suppressor by assessing cytosine methylation at their promoters. Methylation was quantified in 68 human brain tumors, including both IDH-mutant and IDH wildtype, by bisulfite pyrosequencing. In all tumors examined, CpG methylation levels were less than 8%. Our data demonstrate that inactivation of IDH function through promoter hypermethylation is not common in human gliomas and other brain tumors. These findings do not support a tumor suppressor role for IDH genes in human gliomas.

  19. Promoter methylation analysis of IDH genes in human gliomas

    Directory of Open Access Journals (Sweden)

    Simon eFlanagan

    2012-12-01

    Full Text Available Mutations in isocitrate dehydrogenase (IDH -1 or -2 are found in the majority of WHO grade II and III astrocytomas and oligodendrogliomas, and secondary glioblastomas. Almost all described mutations are heterozygous missense mutations affecting a conserved arginine residue in the substrate binding site of IDH1 (R132 or IDH2 (R172. But the exact mechanism of IDH mutations in neoplasia is not understood. It has been proposed that IDH mutations impart a ‘toxic gain of function’ to the mutant protein, however a dominant-negative effect of mutant IDH has also been described, implying that IDH may function as a tumour suppressor gene. As most, if not all, tumour suppressor genes are inactivated by epigenetic silencing, in a wide variety of tumours, we asked if IDH1 or IDH2 carry the epigenetic signature of a tumour suppressor by assessing cytosine methylation at their promoters. Methylation was quantified in 68 human brain tumours, including both IDH-mutant and IDH wildtype, by bisulfite pyrosequencing. In all tumours examined, CpG methylation levels were less than 8%. Our data demonstrate that inactivation of IDH function through promoter hypermethylation is not common in human gliomas and other brain tumours. These findings do not support a tumour suppressor role for IDH genes in human gliomas.

  20. Promoter Methylation Analysis of IDH Genes in Human Gliomas

    International Nuclear Information System (INIS)

    Flanagan, Simon; Lee, Maggie; Li, Cheryl C. Y.; Suter, Catherine M.; Buckland, Michael E.

    2012-01-01

    Mutations in isocitrate dehydrogenase (IDH)-1 or -2 are found in the majority of WHO grade II and III astrocytomas and oligodendrogliomas, and secondary glioblastomas. Almost all described mutations are heterozygous missense mutations affecting a conserved arginine residue in the substrate binding site of IDH1 (R132) or IDH2 (R172). But the exact mechanism of IDH mutations in neoplasia is not understood. It has been proposed that IDH mutations impart a “toxic gain-of-function” to the mutant protein, however a dominant-negative effect of mutant IDH has also been described, implying that IDH may function as a tumor suppressor gene. As most, if not all, tumor suppressor genes are inactivated by epigenetic silencing, in a wide variety of tumors, we asked if IDH1 or IDH2 carry the epigenetic signature of a tumor suppressor by assessing cytosine methylation at their promoters. Methylation was quantified in 68 human brain tumors, including both IDH-mutant and IDH wildtype, by bisulfite pyrosequencing. In all tumors examined, CpG methylation levels were less than 8%. Our data demonstrate that inactivation of IDH function through promoter hypermethylation is not common in human gliomas and other brain tumors. These findings do not support a tumor suppressor role for IDH genes in human gliomas.

  1. Senataxin Mutation Reveals How R-Loops Promote Transcription by Blocking DNA Methylation at Gene Promoters.

    Science.gov (United States)

    Grunseich, Christopher; Wang, Isabel X; Watts, Jason A; Burdick, Joshua T; Guber, Robert D; Zhu, Zhengwei; Bruzel, Alan; Lanman, Tyler; Chen, Kelian; Schindler, Alice B; Edwards, Nancy; Ray-Chaudhury, Abhik; Yao, Jianhua; Lehky, Tanya; Piszczek, Grzegorz; Crain, Barbara; Fischbeck, Kenneth H; Cheung, Vivian G

    2018-02-01

    R-loops are three-stranded nucleic acid structures found abundantly and yet often viewed as by-products of transcription. Studying cells from patients with a motor neuron disease (amyotrophic lateral sclerosis 4 [ALS4]) caused by a mutation in senataxin, we uncovered how R-loops promote transcription. In ALS4 patients, the senataxin mutation depletes R-loops with a consequent effect on gene expression. With fewer R-loops in ALS4 cells, the expression of BAMBI, a negative regulator of transforming growth factor β (TGF-β), is reduced; that then leads to the activation of the TGF-β pathway. We uncovered that genome-wide R-loops influence promoter methylation of over 1,200 human genes. DNA methyl-transferase 1 favors binding to double-stranded DNA over R-loops. Thus, in forming R-loops, nascent RNA blocks DNA methylation and promotes further transcription. Hence, our results show that nucleic acid structures, in addition to sequences, influence the binding and activity of regulatory proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Human β-globin locus control region: Analysis of the 5' DNase I hypersensitive site HS 2 in transgenic mice

    International Nuclear Information System (INIS)

    Caterina, J.J.; Ryan, T.M.; Pawlik, K.M.; Townes, T.M.; Brinster, R.L.; Behringer, R.R.; Palmiter, R.D.

    1991-01-01

    The human β-globin locus control region (LCR) is essential for high-level expression of human var-epsilon-, γ-, and β-globin genes. Developmentally stable DNase I hypersensitive sites (designated HS) mark sequences within this region that are important for LCR activity. A 1.9-kilobase (kb) fragment containing the 5' HS 2 site enhances human β-globin gene expression 100-fold in transgenic mice and also confers position-independent expression. To further define important sequences within this region, deletion mutations of the 1.9-kb fragment were introduced upstream of the human β-globin gene, and the constructs were tested for activity in transgenic mice. Although enhancer activity was gradually lost with deletion of both 5' and 3' sequences, a 373-base-pair (BP) fragment retained the ability to confer relative position-independent expression. Three prominent DNase I footprints were observed in this region with extracts from the human erythroleukemia cell line K-562, one of which contained duplicated binding sites for transcription factor AP-1 (activator protein 1). When the 1.9-kb fragment containing an 19-bp deletion of the AP-1 binding sites was tested in transgenic mice, enhancer activity decreased 20-fold but position-independent expression was retained

  3. A five' splice-region G → C mutation in exon 1 of the human β-globin gene inhibits pre-mRNA splicing: A mechanism for β+-thalassemia

    International Nuclear Information System (INIS)

    Vidaud, M.; Vidaud, D.; Amselem, S.; Rosa, J.; Goossens, M.; Gattoni, R.; Stevenin, J.; Chibani, J.

    1989-01-01

    The authors have characterized a Mediterranean β-thalassemia allele containing a sequence change at codon 30 that alters both β-globin pre-mRNA splicing and the structure of the homoglobin product. Presumably, this G → C transversion at position -1 of intron 1 reduces severely the utilization of the normal 5' splice site since the level of the Arg → Thr mutant hemoglobin (designated hemoglobin Kairouan) found in the erythrocytes of the patient is very low (2% of total hemoglobin). Since no natural mutations of the guanine located at position -1 of the CAG/GTAAGT consensus sequence had been isolated previously. They investigated the role of this nucleotide in the constitution of an active 5' splice site by studying the splicing of the pre-mRNA in cell-free extracts. They demonstrate that correct splicing of the mutant pre-mRNA is 98% inhibited. Their results provide further insights into the mechanisms of pre-mRNA maturation by revealing that the last residue of the exon plays a role at least equivalent to that of the intron residue at position +5

  4. Long-Distance Control of Origin Choice and Replication Timing in the Human β-Globin Locus Are Independent of the Locus Control Region

    Science.gov (United States)

    Cimbora, Daniel M.; Schübeler, Dirk; Reik, Andreas; Hamilton, Joan; Francastel, Claire; Epner, Elliot M.; Groudine, Mark

    2000-01-01

    DNA replication in the human β-globin locus is subject to long-distance regulation. In murine and human erythroid cells, the human locus replicates in early S phase from a bidirectional origin located near the β-globin gene. This Hispanic thalassemia deletion removes regulatory sequences located over 52 kb from the origin, resulting in replication of the locus from a different origin, a shift in replication timing to late S phase, adoption of a closed chromatin conformation, and silencing of globin gene expression in murine erythroid cells. The sequences deleted include nuclease-hypersensitive sites 2 to 5 (5′HS2-5) of the locus control region (LCR) plus an additional 27-kb upstream region. We tested a targeted deletion of 5′HS2-5 in the normal chromosomal context of the human β-globin locus to determine the role of these elements in replication origin choice and replication timing. We demonstrate that the 5′HS2-5-deleted locus initiates replication at the appropriate origin and with normal timing in murine erythroid cells, and therefore we conclude that 5′HS2-5 in the classically defined LCR do not control replication in the human β-globin locus. Recent studies also show that targeted deletion of 5′HS2-5 results in a locus that lacks globin gene expression yet retains an open chromatin conformation. Thus, the replication timing of the locus is closely correlated with nuclease sensitivity but not globin gene expression. PMID:10891496

  5. Investigating alpha-globin structural variants: a retrospective review of 135,000 Brazilian individuals

    Directory of Open Access Journals (Sweden)

    Elza Miyuki Kimura

    2015-04-01

    Full Text Available Background: Brazil has a multiethnic population with a high diversity of hemoglobinopathies. While screenings for beta-globin mutations are far more common, alterations affecting alpha-globin genes are usually more silent and less well known. The aim of this study was to describe the results of a screening program for alpha-globin gene mutations in a representative sample of the Southeastern Brazilian population. Methods: A total of 135,000 individuals, including patients with clinical suspicion of hemoglobinopathies and their family members, randomly chosen individuals submitted to blood tests and blood donors who were abnormal hemoglobin carriers were analyzed. The variants were screened by alkaline and acid electrophoreses, isoelectric focusing and cation-exchange high performance liquid chromatography (HPLC and the abnormal chains were investigated by reverse-phase high performance liquid chromatography (RP-HPLC. Mutations were identified by molecular analyses, and the oxygen affinity, heme-heme cooperativity and Bohr effect of the variants were evaluated by functional tests. Results: Four new and 22 rare variants were detected in 98 families. Some of these variants were found in co-inheritance with other hemoglobinopathies. Of the rare hemoglobins, Hasharon, Stanleyville II and J-Rovigo were the most common, the first two being S-like and associated with alpha-thalassemia. Conclusion: The variability of alpha-globin alterations reflects the high degree of racial miscegenation and an intense internal migratory flow between different Brazilian regions. This diversity highlights the importance of programs for diagnosing hemoglobinopathies and preventing combinations that may lead to important clinical manifestations in multiethnic populations.

  6. Globins Scavenge Sulfur Trioxide Anion Radical*

    Science.gov (United States)

    Gardner, Paul R.; Gardner, Daniel P.; Gardner, Alexander P.

    2015-01-01

    Ferrous myoglobin was oxidized by sulfur trioxide anion radical (STAR) during the free radical chain oxidation of sulfite. Oxidation was inhibited by the STAR scavenger GSH and by the heme ligand CO. Bimolecular rate constants for the reaction of STAR with several ferrous globins and biomolecules were determined by kinetic competition. Reaction rate constants for myoglobin, hemoglobin, neuroglobin, and flavohemoglobin are large at 38, 120, 2,600, and ≥ 7,500 × 106 m−1 s−1, respectively, and correlate with redox potentials. Measured rate constants for O2, GSH, ascorbate, and NAD(P)H are also large at ∼100, 10, 130, and 30 × 106 m−1 s−1, respectively, but nevertheless allow for favorable competition by globins and a capacity for STAR scavenging in vivo. Saccharomyces cerevisiae lacking sulfite oxidase and deleted of flavohemoglobin showed an O2-dependent growth impairment with nonfermentable substrates that was exacerbated by sulfide, a precursor to mitochondrial sulfite formation. Higher O2 exposures inactivated the superoxide-sensitive mitochondrial aconitase in cells, and hypoxia elicited both aconitase and NADP+-isocitrate dehydrogenase activity losses. Roles for STAR-derived peroxysulfate radical, superoxide radical, and sulfo-NAD(P) in the mechanism of STAR toxicity and flavohemoglobin protection in yeast are suggested. PMID:26381408

  7. Novel "Superspreader" Bacteriophages Promote Horizontal Gene Transfer by Transformation.

    Science.gov (United States)

    Keen, Eric C; Bliskovsky, Valery V; Malagon, Francisco; Baker, James D; Prince, Jeffrey S; Klaus, James S; Adhya, Sankar L

    2017-01-17

    Bacteriophages infect an estimated 10 23 to 10 25 bacterial cells each second, many of which carry physiologically relevant plasmids (e.g., those encoding antibiotic resistance). However, even though phage-plasmid interactions occur on a massive scale and have potentially significant evolutionary, ecological, and biomedical implications, plasmid fate upon phage infection and lysis has not been investigated to date. Here we show that a subset of the natural lytic phage population, which we dub "superspreaders," releases substantial amounts of intact, transformable plasmid DNA upon lysis, thereby promoting horizontal gene transfer by transformation. Two novel Escherichia coli phage superspreaders, SUSP1 and SUSP2, liberated four evolutionarily distinct plasmids with equal efficiency, including two close relatives of prominent antibiotic resistance vectors in natural environments. SUSP2 also mediated the extensive lateral transfer of antibiotic resistance in unbiased communities of soil bacteria from Maryland and Wyoming. Furthermore, the addition of SUSP2 to cocultures of kanamycin-resistant E. coli and kanamycin-sensitive Bacillus sp. bacteria resulted in roughly 1,000-fold more kanamycin-resistant Bacillus sp. bacteria than arose in phage-free controls. Unlike many other lytic phages, neither SUSP1 nor SUSP2 encodes homologs to known hydrolytic endonucleases, suggesting a simple potential mechanism underlying the superspreading phenotype. Consistent with this model, the deletion of endonuclease IV and the nucleoid-disrupting protein ndd from coliphage T4, a phage known to extensively degrade chromosomal DNA, significantly increased its ability to promote plasmid transformation. Taken together, our results suggest that phage superspreaders may play key roles in microbial evolution and ecology but should be avoided in phage therapy and other medical applications. Bacteriophages (phages), viruses that infect bacteria, are the planet's most numerous biological

  8. A strong promoter of a non-cry gene directs expression of the cry1Ac gene in Bacillus thuringiensis.

    Science.gov (United States)

    Zhang, Xin; Gao, Tantan; Peng, Qi; Song, Lai; Zhang, Jie; Chai, Yunrong; Sun, Dongmei; Song, Fuping

    2018-04-01

    Bacillus thuringiensis bacteria show insecticidal activities that rely upon the production of insecticidal crystal proteins, which are encoded by cry or cyt genes and can target a variety of insect pests. It has been shown that cry1Ac is the only cry gene in B. thuringiensis subsp. kurstaki HD73 (B. thuringiensis HD73) and its expression is controlled by both σ E and σ K . Here, we report a novel σ E -dependent strong promoter of a non-cry gene (HD73_5014), which can direct strong cry1Ac gene expression in B. thuringiensis HD73. We constructed an E. coli-B. thuringiensis shuttle vector (pHT315-P 5014 -1Ac) for cry1Ac gene expression, using the HD73_5014 gene promoter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis showed that expression of the cry1Ac gene directed by the HD73_5014 gene promoter was at the same level as that directed by the previously known strongest cry promoter, P cry8E . However, this strain did not form typical bipyramidal crystals in mother cells, as observed by transmission electron microscopy and atomic force microscope. The strain with Cry1Ac protein expression under the control of the HD73_5014 gene promoter (P 5014 -cry1Ac) showed insecticidal activity against Plutella xylostella similar to that under the control of the orf1cry8E gene promoter (P cry8E -cry1Ac). Collectively, these results suggest that the HD73_5014 gene promoter, as a non-cry gene promoter, would be an efficient transcriptional element for cry gene expression. These data also show the possibility for improving Cry production by searching for transcriptional elements in not only cry genes, but also non-cry genes.

  9. IL6 gene promoter polymorphisms and type 2 diabetes

    DEFF Research Database (Denmark)

    Huth, Cornelia; Heid, Iris M; Vollmert, Caren

    2006-01-01

    Several lines of evidence indicate a causal role of the cytokine interleukin (IL)-6 in the development of type 2 diabetes in humans. Two common polymorphisms in the promoter of the IL-6 encoding gene IL6, -174G>C (rs1800795) and -573G>C (rs1800796), have been investigated for association with type...... 2 diabetes in numerous studies but with results that have been largely equivocal. To clarify the relationship between the two IL6 variants and type 2 diabetes, we analyzed individual data on >20,000 participants from 21 published and unpublished studies. Collected data represent eight different...... countries, making this the largest association analysis for type 2 diabetes reported to date. The GC and CC genotypes of IL6 -174G>C were associated with a decreased risk of type 2 diabetes (odds ratio 0.91, P = 0.037), corresponding to a risk modification of nearly 9%. No evidence for association was found...

  10. Promoter methylation and gene polymorphism are two independent events in regulation of GSTP1 gene expression.

    Science.gov (United States)

    Bhat, Aaliya; Masood, A; Wani, K A; Bhat, Younus Ahmad; Nissar, Bushra; Khan, Nuzhat Shaheen; Ganai, B A

    2017-04-01

    Breast carcinogenesis is a multistep process, involving both genetic and epigenetic modification process of genes, involved in diverse pathways ranging from DNA repair to metabolic processes. This study was undertaken to assess the role of promoter methylation of GSTP1 gene, a member of glutathione-S-transferase family of enzymes, in relation to its expression, polymorphism, and clinicopathological parameters. Tissue samples were taken from breast cancer patients and paired with their normal adjacent tissues. A total of 51 subjects were studied, in which the frequency of promoter methylation in cancerous tissue was 37.25% as against 11% in the normal tissues ( p ≤ 0.001). The hypermethylated status of the gene was significantly associated with the loss of the protein expression ( r = -0.449, p = 0.001, odds ratio = 7.42, 95% confidence interval = 2.05-26.92). Furthermore, when compared with the clinical parameters, the significant association was found between the promoter hypermethylation and lymph node metastasis ( p ≤ 0.001), tumor stage ( p = 0.039), tumor grade ( p = 0.028), estrogen receptor status ( p = 0.018), and progesterone receptor status ( p = 0.046). Our study is the first of its kind in Kashmiri population, which indicates that GSTP1 shows aberrant methylation pattern in the breast cancer with the consequent loss in the protein expression. Furthermore, it also shows that the gene polymorphism (Ile105Val) at codon 105 is not related to the promoter methylation and two are the independent events in breast cancer development.

  11. Heterologous gene expression driven by carbonic anhydrase gene promoter in Dunaliella salina

    Science.gov (United States)

    Yurong, Chai; Yumin, Lu; Tianyun, Wang; Weihong, Hou; Lexun, Xue

    2006-12-01

    Dunaliella salina, a halotolerant unicellular green alga without a rigid cell wall, can live in salinities ranging from 0.05 to 5 mol/L NaCl. These features of D. salina make it an ideal host for the production of antibodies, oral vaccine, and commercially valuable polypeptides. To produce high level of heterologous proteins from D. salina, highly efficient promoters are required to drive expression of target genes under controlled condition. In the present study, we cloned a 5' franking region of 1.4 kb from the carbonic anhydrase ( CAH) gene of D. salina by genomic walking and PCR. The fragment was ligated to the pMD18-T vector and characterized. Sequence analysis indicated that this region contained conserved motifs, including a TATA- like box and CAAT-box. Tandem (GT)n repeats that had a potential role of transcriptional control, were also found in this region. The transcription start site (TSS) of the CAH gene was determined by 5' RACE and nested PCR method. Transformation assays showed that the 1.4 kb fragment was able to drive expression of the selectable bar (bialaphos resistance) gene when the fusion was transformed into D. salina by biolistics. Northern blotting hybridizations showed that the bar transcript was most abundant in cells grown in 2 mol/L NaCl, and less abundant in 0.5 mol/L NaCl, indicating that expression of the bar gene was induced at high salinity. These results suggest the potential use of the CAH gene promoter to induce the expression of heterologous genes in D. salina under varied salt condition.

  12. Characterization and Comparative Immunoreactivity of Antibody to Newt (T. cristatus) Globins

    OpenAIRE

    Kowalski, Louis A.; Walsh, Anne W.; Merrow, Martha; Paulekus, Wayne; Mackin, William; Grasso, Joseph A.

    1984-01-01

    1. Rabbit antisera to newt (T. cristatus) globin were produced by repeated injections of globin and antiglobin antibodies purified by chromatography on globin-Sepharose 4B. 2. Ouchterlony and SDS PAGE analysis indicated that the material eluted from the affinity column was rabbit IgG. 3. The antiglobin antibodies tested by immunodiffusion and ELISA cross-reacted with native hemoglobin and globin from T. cristatus and to varying extents with globins of N. viridescens, R. pipiens and X. laevis,...

  13. Genome-wide analysis of regions similar to promoters of histone genes

    KAUST Repository

    Chowdhary, Rajesh

    2010-05-28

    Background: The purpose of this study is to: i) develop a computational model of promoters of human histone-encoding genes (shortly histone genes), an important class of genes that participate in various critical cellular processes, ii) use the model so developed to identify regions across the human genome that have similar structure as promoters of histone genes; such regions could represent potential genomic regulatory regions, e.g. promoters, of genes that may be coregulated with histone genes, and iii/ identify in this way genes that have high likelihood of being coregulated with the histone genes.Results: We successfully developed a histone promoter model using a comprehensive collection of histone genes. Based on leave-one-out cross-validation test, the model produced good prediction accuracy (94.1% sensitivity, 92.6% specificity, and 92.8% positive predictive value). We used this model to predict across the genome a number of genes that shared similar promoter structures with the histone gene promoters. We thus hypothesize that these predicted genes could be coregulated with histone genes. This hypothesis matches well with the available gene expression, gene ontology, and pathways data. Jointly with promoters of the above-mentioned genes, we found a large number of intergenic regions with similar structure as histone promoters.Conclusions: This study represents one of the most comprehensive computational analyses conducted thus far on a genome-wide scale of promoters of human histone genes. Our analysis suggests a number of other human genes that share a high similarity of promoter structure with the histone genes and thus are highly likely to be coregulated, and consequently coexpressed, with the histone genes. We also found that there are a large number of intergenic regions across the genome with their structures similar to promoters of histone genes. These regions may be promoters of yet unidentified genes, or may represent remote control regions that

  14. [The methylation of ZHX2 gene promoter enhances AFP gene expression in hepatocellular carcinoma].

    Science.gov (United States)

    Lv, Zili; DU, Yangjun; Wen, Jianming

    2013-07-01

    To investigate the relationship between Zinc-fingers and homeoboxes 2 (ZHX2) promoter methylation and alpha-fetoprotein (AFP) gene expression, and analyze the mechanism of AFP gene expression. HepG2 cell line was cultured with 0.5, 1.0 or 5.0 μmol/L of 5-aza-deoxycytidine (5-Aza-Dc). RT-PCR and Western blotting were used to detect the expressions of ZHX2 and AFP in HepG2 cell line. Methylation-specific PCR was used to detect ZHX2 promoter methylation in 38 hepatocellular carcinoma tissues. The HepG2 cell line showed a low level of ZHX2 mRNA, negative expression of ZHX2 protein, but high expression of AFP at both mRNA and protein levels. After the HepG2 cells were treated with 1.0 or 5.0 μmol/L 5-Aza-Dc for 6 d, the expression of ZHX2 mRNA and protein increased and the expression of AFP mRNA and protein decreased. Among 38 hepatocellular carcinoma tissues, ZHX2 promoter methylation was found in 16 hepatocellular carcinoma tissues with AFP>25 ng/mL in serum. No methylation of ZHX2 promoter was found in 8 hepatocellular carcinoma tissues with AFPexpression.

  15. Cooperative binding of transcription factors promotes bimodal gene expression response.

    Directory of Open Access Journals (Sweden)

    Pablo S Gutierrez

    Full Text Available In the present work we extend and analyze the scope of our recently proposed stochastic model for transcriptional regulation, which considers an arbitrarily complex cis-regulatory system using only elementary reactions. Previously, we determined the role of cooperativity on the intrinsic fluctuations of gene expression for activating transcriptional switches, by means of master equation formalism and computer simulation. This model allowed us to distinguish between two cooperative binding mechanisms and, even though the mean expression levels were not affected differently by the acting mechanism, we showed that the associated fluctuations were different. In the present generalized model we include other regulatory functions in addition to those associated to an activator switch. Namely, we introduce repressive regulatory functions and two theoretical mechanisms that account for the biphasic response that some cis-regulatory systems show to the transcription factor concentration. We have also extended our previous master equation formalism in order to include protein production by stochastic translation of mRNA. Furthermore, we examine the graded/binary scenarios in the context of the interaction energy between transcription factors. In this sense, this is the first report to show that the cooperative binding of transcription factors to DNA promotes the "all-or-none" phenomenon observed in eukaryotic systems. In addition, we confirm that gene expression fluctuation levels associated with one of two cooperative binding mechanism never exceed the fluctuation levels of the other.

  16. Expression of RNA-interference/antisense transgenes by the cognate promoters of target genes is a better gene-silencing strategy to study gene functions in rice.

    Science.gov (United States)

    Li, Jing; Jiang, Dagang; Zhou, Hai; Li, Feng; Yang, Jiawei; Hong, Laifa; Fu, Xiao; Li, Zhibin; Liu, Zhenlan; Li, Jianming; Zhuang, Chuxiong

    2011-03-03

    Antisense and RNA interference (RNAi)-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.

  17. Human glucagon gene promoter sequences regulating tissue-specific versus nutrient-regulated gene expression.

    Science.gov (United States)

    Nian, Min; Gu, Jun; Irwin, David M; Drucker, Daniel J

    2002-01-01

    The glucagon-like peptides (GLPs) are synthesized and secreted in a nutrient-dependent manner in rodents; however, the factors regulating human GLP-1 and GLP-2 biosynthesis remain unclear. To understand how nutrients regulate human proglucagon gene expression, we studied the expression of a human proglucagon promoter-growth hormone (GH) transgene in 1.6 human glucagon-GH transgenic mice. Fasting-refeeding significantly decreased and increased the levels of circulating mouse insulin and transgene-derived hGH (P fasting vs. refeeding) and decreased and upregulated, respectively, the levels of endogenous mouse proglucagon RNA in the ileum but not in the jejunum or colon. High-fiber feeding significantly increased the levels of glucose-stimulated circulating hGH and upregulated levels of mouse intestinal proglucagon gene expression in the jejunum, ileum, and colon (P fasting-refeeding nor a high-fiber diet upregulated the expression of the human proglucagon promoter-hGH transgene. These findings demonstrate that human proglucagon gene regulatory sequences specifying tissue-specific expression in gut endocrine cells are not sufficient for recognition of energy-derived signals regulating murine glucagon gene expression in enteroendocrine cells in vivo.

  18. HOX Gene Promoter Prediction and Inter-genomic Comparison: an Evo-Devo Study

    OpenAIRE

    Endriga, Maria A; de la Paz, Victoria Karenina R; Sazon, Jezreel Marie G; Co, Elisa L; Deocaris, Custer C

    2010-01-01

    Homeobox genes direct the anterior-posterior axis of the body plan in eukaryotic organisms. Promoter regions upstream of the Hox genes jumpstart the transcription process. CpG islands found within the promoter regions can cause silencing of these promoters. The locations of the promoter regions and the CpG islands of Homeo sapiens sapiens (human), Pan troglodytes (chimpanzee), Mus musculus (mouse), and Rattus norvegicus (brown rat) are compared and related to the possib...

  19. Genome-wide prediction of transcriptional regulatory elements of human promoters using gene expression and promoter analysis data

    Directory of Open Access Journals (Sweden)

    Kim Seon-Young

    2006-07-01

    Full Text Available Abstract Background A complete understanding of the regulatory mechanisms of gene expression is the next important issue of genomics. Many bioinformaticians have developed methods and algorithms for predicting transcriptional regulatory mechanisms from sequence, gene expression, and binding data. However, most of these studies involved the use of yeast which has much simpler regulatory networks than human and has many genome wide binding data and gene expression data under diverse conditions. Studies of genome wide transcriptional networks of human genomes currently lag behind those of yeast. Results We report herein a new method that combines gene expression data analysis with promoter analysis to infer transcriptional regulatory elements of human genes. The Z scores from the application of gene set analysis with gene sets of transcription factor binding sites (TFBSs were successfully used to represent the activity of TFBSs in a given microarray data set. A significant correlation between the Z scores of gene sets of TFBSs and individual genes across multiple conditions permitted successful identification of many known human transcriptional regulatory elements of genes as well as the prediction of numerous putative TFBSs of many genes which will constitute a good starting point for further experiments. Using Z scores of gene sets of TFBSs produced better predictions than the use of mRNA levels of a transcription factor itself, suggesting that the Z scores of gene sets of TFBSs better represent diverse mechanisms for changing the activity of transcription factors in the cell. In addition, cis-regulatory modules, combinations of co-acting TFBSs, were readily identified by our analysis. Conclusion By a strategic combination of gene set level analysis of gene expression data sets and promoter analysis, we were able to identify and predict many transcriptional regulatory elements of human genes. We conclude that this approach will aid in decoding

  20. Development of chimeric gene promoters responsive to hypoxia and ionizing radiation

    International Nuclear Information System (INIS)

    Zheng Aiqing; Yu Jinming

    2004-01-01

    The authors describe two systems that make use of gene-directed enzyme prodrug therapy, regulated by radiation or hypoxic-responsive promoters. The use of treatment-, condition- or tumor-specific promoters to control gene-directed enzyme prodrug therapy is one such method for targeting gene expression to the tumor. The development of such strategies that achieve tumor targeted expression of genes via selective promoters will enable improved specificity and targeting thereby addressing one of the major limitations of cancer gene therapy

  1. ABERRANT METHYLATION OF THE PROMOTER OF APC, CDH13 AND MGMT GENES IN COLORECTAL CANCER PATIENTS

    Directory of Open Access Journals (Sweden)

    O. I. Kit

    2016-01-01

    Full Text Available Aberrant methylation of gene promoter regions is the main epigenetic change characterizing colorectal cancer. Methylation levels of 42 CpG-sites of promoter regions of the MGMT, APC and CDH13 genes in colorectal cancer were studied in comparison with methylation levels of the adjacent normal tissue in 25 patients. Pyrosequencing showed an increase in methylation levels of promoter regions of the MGMT, APC and CDH13 genes in tumor samples by 3 to 5 times. These tumor samples were screened for activating SNP-mutations in the KRAS (40 %, NRAS (0 % and BRAF (0 % oncogenes. SNP-mutations in the KRAS gene were accompanied by hypermethylation of one or more promoters of the studied genes. Association of this epigenetic index with tumor metastasis was proved. The data on an increase in methylation of the promoter regions of oncosupressor genes can be used as sensitive prognostic markers of progression and metastasis of colorectal cancer.

  2. Viral promoters can initiate expression of toxin genes introduced into Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jacob Daniela

    2005-06-01

    Full Text Available Abstract Background The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. Results We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. Conclusion According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.

  3. Archaeal promoter architecture and mechanism of gene activation

    DEFF Research Database (Denmark)

    Peng, Nan; Ao, Xiang; Liang, Yun Xiang

    2011-01-01

    element named ara box directing arabinose-inducible expression and the basal promoter element TATA, serving as the binding site for the TATA-binding protein. Strikingly, these promoters possess a modular structure that allows an essentially inactive basal promoter to be strongly activated. The invoked...

  4. DNA entropy reveals a significant difference in complexity between housekeeping and tissue specific gene promoters.

    Science.gov (United States)

    Thomas, David; Finan, Chris; Newport, Melanie J; Jones, Susan

    2015-10-01

    The complexity of DNA can be quantified using estimates of entropy. Variation in DNA complexity is expected between the promoters of genes with different transcriptional mechanisms; namely housekeeping (HK) and tissue specific (TS). The former are transcribed constitutively to maintain general cellular functions, and the latter are transcribed in restricted tissue and cells types for specific molecular events. It is known that promoter features in the human genome are related to tissue specificity, but this has been difficult to quantify on a genomic scale. If entropy effectively quantifies DNA complexity, calculating the entropies of HK and TS gene promoters as profiles may reveal significant differences. Entropy profiles were calculated for a total dataset of 12,003 human gene promoters and for 501 housekeeping (HK) and 587 tissue specific (TS) human gene promoters. The mean profiles show the TS promoters have a significantly lower entropy (pentropy distributions for the 3 datasets show that promoter entropies could be used to identify novel HK genes. Functional features comprise DNA sequence patterns that are non-random and hence they have lower entropies. The lower entropy of TS gene promoters can be explained by a higher density of positive and negative regulatory elements, required for genes with complex spatial and temporary expression. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Positional bias of general and tissue-specific regulatory motifs in mouse gene promoters

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    Farré Domènec

    2007-12-01

    Full Text Available Abstract Background The arrangement of regulatory motifs in gene promoters, or promoter architecture, is the result of mutation and selection processes that have operated over many millions of years. In mammals, tissue-specific transcriptional regulation is related to the presence of specific protein-interacting DNA motifs in gene promoters. However, little is known about the relative location and spacing of these motifs. To fill this gap, we have performed a systematic search for motifs that show significant bias at specific promoter locations in a large collection of housekeeping and tissue-specific genes. Results We observe that promoters driving housekeeping gene expression are enriched in particular motifs with strong positional bias, such as YY1, which are of little relevance in promoters driving tissue-specific expression. We also identify a large number of motifs that show positional bias in genes expressed in a highly tissue-specific manner. They include well-known tissue-specific motifs, such as HNF1 and HNF4 motifs in liver, kidney and small intestine, or RFX motifs in testis, as well as many potentially novel regulatory motifs. Based on this analysis, we provide predictions for 559 tissue-specific motifs in mouse gene promoters. Conclusion The study shows that motif positional bias is an important feature of mammalian proximal promoters and that it affects both general and tissue-specific motifs. Motif positional constraints define very distinct promoter architectures depending on breadth of expression and type of tissue.

  6. Tumor Restrictive Suicide Gene Therapy for Glioma Controlled by the FOS Promoter.

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    Jianqing Pan

    Full Text Available Effective suicide gene delivery and expression are crucial to achieving successful effects in gene therapy. An ideal tumor-specific promoter expresses therapeutic genes in tumor cells with minimal normal tissue expression. We compared the activity of the FOS (FBJ murine osteosarcoma viral oncogene homolog promoter with five alternative tumor-specific promoters in glioma cells and non-malignant astrocytes. The FOS promoter caused significantly higher transcriptional activity in glioma cell lines than all alternative promoters with the exception of CMV. The FOS promoter showed 13.9%, 32.4%, and 70.8% of the transcriptional activity of CMV in three glioma cell lines (U87, U251, and U373. Importantly, however, the FOS promoter showed only 1.6% of the transcriptional activity of CMV in normal astrocytes. We also tested the biologic activity of recombinant adenovirus containing the suicide gene herpes simplex virus thymidine kinase (HSV-tk driven by the FOS promoter, including selective killing efficacy in vitro and tumor inhibition rate in vivo. Adenoviral-mediated delivery of the HSV-tk gene controlled by the FOS promoter conferred a cytotoxic effect on human glioma cells in vitro and in vivo. This study suggests that use of the FOS-tk adenovirus system is a promising strategy for glioma-specific gene therapy but still much left for improvement.

  7. Gene promoter evolution targets the center of the human protein interaction network.

    Directory of Open Access Journals (Sweden)

    Jordi Planas

    Full Text Available Assessing the contribution of promoters and coding sequences to gene evolution is an important step toward discovering the major genetic determinants of human evolution. Many specific examples have revealed the evolutionary importance of cis-regulatory regions. However, the relative contribution of regulatory and coding regions to the evolutionary process and whether systemic factors differentially influence their evolution remains unclear. To address these questions, we carried out an analysis at the genome scale to identify signatures of positive selection in human proximal promoters. Next, we examined whether genes with positively selected promoters (Prom+ genes show systemic differences with respect to a set of genes with positively selected protein-coding regions (Cod+ genes. We found that the number of genes in each set was not significantly different (8.1% and 8.5%, respectively. Furthermore, a functional analysis showed that, in both cases, positive selection affects almost all biological processes and only a few genes of each group are located in enriched categories, indicating that promoters and coding regions are not evolutionarily specialized with respect to gene function. On the other hand, we show that the topology of the human protein network has a different influence on the molecular evolution of proximal promoters and coding regions. Notably, Prom+ genes have an unexpectedly high centrality when compared with a reference distribution (P=0.008, for Eigenvalue centrality. Moreover, the frequency of Prom+ genes increases from the periphery to the center of the protein network (P=0.02, for the logistic regression coefficient. This means that gene centrality does not constrain the evolution of proximal promoters, unlike the case with coding regions, and further indicates that the evolution of proximal promoters is more efficient in the center of the protein network than in the periphery. These results show that proximal promoters

  8. Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene.

    Science.gov (United States)

    Nuez, B; Michalovich, D; Bygrave, A; Ploemacher, R; Grosveld, F

    1995-05-25

    Erythroid Krüppel-like factor (EKLF) was originally isolated from erythroid cell RNA by differential screening and shown to be erythroid-specific, although a low level of EKLF was found in mast cell lines. EKLF contains three zinc-fingers homologous to those found in the Krüppel family of transcription factors. Because it binds the sequence CCACACCCT, EKLF may affect erythroid development as a result of its ability to bind to the CAC box in the promoter of the beta-globin gene. Mutation of this element leads to reduced beta-globin expression and it appears to mediate the effect of the globin locus control region on the promoter. Here we inactivate the EKLF gene through insertion of a lacZ reporter gene by homologous recombination in embryonic stem (ES) cells. Heterozygous EKLF+/- mice show that the reporter gene is expressed in a developmentally specific manner in all types of erythroblasts in the fetal liver and adult bone marrow. Homozygous EKLF-/- mice appear normal during the embryonic stage of haematopoiesis in the yolk sac, but develop a fatal anaemia during early fetal life when haematopoiesis has switched to the fetal liver. Enucleated erythrocytes are formed but these do not contain the proper amount of haemoglobin. We conclude that the transcription factor EKLF is essential for the final steps of definitive erythropoiesis in fetal liver.

  9. Expression of a Fungal Cellulase Gene by b-glucanase Promoter of Streptococcus bovis

    OpenAIRE

    EKİNCİ, Mehmet Sait

    2014-01-01

    The promoter region of the §-glucanase gene was identified using a transcriptional fusion between the upstream region of the Streptococcus bovis b-glucanase gene and the celA gene. Using the transcriptional and protein localisation signals of the S. bovis b-glucanase gene, an in-frame tarnslational fusion between the end of the b-glucanase signal sequence and the ATG of the Neocallimastix patriciarum celA gene was constructed. The b-glucanase promoter-celA fusion was expressed in both E....

  10. A Leader Intron of a Soybean Elongation Factor 1A (eEF1A) Gene Interacts with Proximal Promoter Elements to Regulate Gene Expression in Synthetic Promoters.

    Science.gov (United States)

    Zhang, Ning; McHale, Leah K; Finer, John J

    2016-01-01

    Introns, especially the first intron in the 5' untranslated region (5'UTR), can significantly impact gene expression via intron-mediated enhancement (IME). In this study, we demonstrate the leader intron of a soybean elongation factor 1A (eEF1A) gene (GmScreamM8) was essential for the high activity of the native promoter. Furthermore, the interaction of the GmScreamM8 leader intron with regulatory element sequences from several soybean eEF1A promoters was studied using synthetic promoters, which consisted of element tetramers upstream of a core promoter used to regulate a green fluorescent protein (gfp) reporter gene. Element tetramers, placed upstream of a GmScreamM8 core promoter, showed very high activity using both transient expression in lima bean cotyledons and stable expression in soybean hairy roots, only if the native leader intron was included, suggesting an interaction between intronic sequences and promoter elements. Partial deletions of the leader intron showed that a 222 bp intronic sequence significantly contributed to very high levels of GFP expression. Generation of synthetic intron variants with a monomeric or trimeric repeat of the 222 bp intronic sequence, yielded almost two-fold higher expression compared to the original intron, while partial deletion of the 222 bp intronic repeated sequence significantly decreased gene expression, indicating that this intronic sequence was essential for the intron-element interaction enhancement.

  11. Investigation of Interleukin-17 gene polymorphism on DAP-Kinase gene promoter methylation in Patients with Breast Cancer

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    S Naeimi

    2016-02-01

    Full Text Available Background & Aim: Breast cancer is the most common malignancy in women . Studies have shown that increased in methylation of CpG islands (CpG island hyper methylation, CIHM, is one of the important mechanisms in gene down regulation. DAP-Kinase protein plays an important role in the process of Apoptosis. Interleukin-17 is an proinflammatory cytokine and inflammation,is one of the factors  that affect on gene methylation . the purpose of this study was to evaluate the polymorphism of the IL-17 gene promoter methylation Dap-kinase and its relationship to breast cancer. Methods: In this case - control study, A total of 40 Women with Breast cancer and 40 healthy women in Iran were examined.DNA was extracted by saluting out method and Single nucleotide Polymorphisms of the IL-17 gene were analyzed by the PCR-RFLP method and To study gene promoter methylation Dap-kinase, MSPCR method was used.data were compared in both groups by using Pearson’s chi-square and Hardy-weinberg equilibrium test. RESULTS: Results confirm the fact that, there is a relationship between DAP-kinase gene promoter methylation and breast cancer disease So that the promoter of this gene in patients than in healthy individuals was much more methylated( p0.05 Conclusion: Due to the fact, that promoter genes methylation is one of the mechanisms of epigenetic genes silencing, it seems that DAP-kinase gene promoter methylation increases is associated with the risk of breast cancer in women.

  12. Effectiveness of B-actin promoter on driving target gene expression in common carp transgenesis

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    Andi Aliah Hidayani

    2011-01-01

    Full Text Available Promoter in transgene construct plays an important role on regulating of transgene expression level in transgenic fish. In fish transgenesis, researcher convinced that use all-fish gene construct is safety and prospective. This study was performed to compare effectiveness b-actin promoter, - the promoter which has ubiquitous, constitutive, housekeeping characteristics, from common carp (homologous and from tilapia and medaka b-actin promoters (heterologous in driving of green fluorescent protein (GFP expression as a model of target gene on common carp transgenesis. These gene constructs were separately microinjected into cytoplasm of 60 one-cell-stage common carp embryos. The results suggested that 70% survival rate at embryo stage and 45% hatching rate values showed that the microinjection was performed successfully. Percentage of embryos expressing GFP gene were slightly higher when injected using common carp and medaka promoters than those of using tilapia promoter. Percentage of larvae expressing GFP using common carp promoter was similar with medaka promoter. Furthermore, GFP expression using common carp b-actin promoter could be detected at one-week-old larvae, while GFP expressing using medaka b-actin promoter was lasted at 2-day-old larvae. The results demonstrated that homologous promoter more effective in driving of a target gene expression than that of heterologous promoter.  Key words: homologous promoter, GFP, transgenesis, common carp   ABSTRAK Promoter dalam konstruksi transgen berperan penting dalam pengaturan tingkat ekspresi transgen pada ikan transgenik. Dalam transgenesis ikan, peneliti meyakini bahwa penggunaan konstruksi gen "all-fish" adalah aman dan prospektif.  Penelitian ini dilakukan untuk membandingkan efektivitas promoter β-aktin, - promoter yang memiliki ciri ubiquitous, constitutive, dan housekeeping, dari ikan dari ikan mas (homolog dan ikan nila dan ikan medaka (heterolog dalam mengendalikan ekspresi gen GFP

  13. 5'HS5 of the human beta-globin locus control region is dispensable for the formation of the beta-globin active chromatin hub.

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    Ping Kei Chan

    Full Text Available Hypersensitive site 5 (5'HS5 of the beta-globin Locus Control Region functions as a developmental stage-specific border in erythroid cells. Here, we have analyzed the role of 5'HS5 in the three dimensional organization of the beta-gene locus using the Chromatin Conformation Capture (3C technique. The results show that when 5'HS5 is deleted from the locus, both remote and internal regulatory elements are still able to interact with each other in a three-dimensional configuration termed the Active Chromatin Hub. Thus, the absence of 5'HS5 does not have an appreciable effect on the three dimensional organization of the beta-globin locus. This rules out models in which 5'HS5 nucleates interactions with remote and/or internal regulatory elements. We also determined the binding of CTCF, the only defined insulator protein in mammalian cells, to 5'HS5 by using chromatin immunoprecipitation (ChIP assays. We detect low levels of CTCF binding to 5'HS5 in primitive erythroid cells, in which it functions as a border element. Surprisingly, we also observe binding levels of CTCF to 5'HS5 in definitive erythroid cells. Thus, binding of CTCF to 5'HS5 per se does not render it a functional border element. This is consistent with the previous data suggesting that CTCF has dual functionality.

  14. The progress of tumor gene-radiotherapy induced by Egr-1 promoter

    International Nuclear Information System (INIS)

    Guo Rui; Li Biao

    2010-01-01

    The promoter of early growth response gene-1 (Egr-1) is a cis-acting element of Egr-1, and its activity is regulated by inducers such as ionizing radiation, free radical. In designated gene-radiotherapy system, radiation combined with therapeutic gene (such as tumor necrosis factor-α gene, suicide gene) can spatially and temporally regulate therapeutic gene expression in the irradiated field, produced a marked effect, while little systemic toxicities were observed. The combination of radiotherapy and gene therapy is promising in tumor therapy. (authors)

  15. Identification and functional analysis of an alternative promoter of human intersectin 1 gene

    Directory of Open Access Journals (Sweden)

    Rynditch A. V.

    2010-04-01

    Full Text Available Aim. Intersectin 1 (ITSN1 gene encodes an evolutionarily conserved adaptor protein that functions in clathrin-mediated endocytosis, cell signalling, apoptosis and cytoskeleton rearrangements. Its expression is characterized by multiple alternative splicing. Alternative promoter usage is an additional way to create diversity and flexibility in the regulation of gene expression. The aim of this study was to identify possible alternative promoters of ITSN1 gene. Methods. In silico prediction, 5' RACE, RT-PCR and reporter gene expression assay were used for identification and functional characterization of alternative promoter region. Results. We detected an alternative promoter of human ITSN1 gene which is located in intron 5 and generates 5' truncated transcripts containing in-frame ATG codon with strong Kozak sequence and could encode an N-terminally truncated isoforms lacking first EH domain. The region located 246–190 bp upstream of exon 6 is required for alternative promoter activity. ITSN1 transcripts generated from an alternative promoter were detected in human kidney, liver, lung and brain tissues. However, the level of their expression was significantly lower than that of major ITSN1 isoforms. Conclusion. The results obtained suggest that alternative promoter region located in intron 5 of ITSN1 gene functions as a weak promoter. Further experiments are required to clarify the role of 5' truncated ITSN1 transcripts.

  16. MDM2 negatively regulates the human telomerase RNA gene promoter

    Directory of Open Access Journals (Sweden)

    Keith W Nicol

    2005-01-01

    Full Text Available Abstract Background We have previously demonstrated that NF-Y and Sp1 interact with the human telomerase RNA (hTR promoter and play a central role in its regulation. We have also shown that pRB activates the hTR promoter, but the mechanism of pRb directed activation is unknown. It has recently been reported that pRB induces Sp1 activity by relieving inhibition mediated by mdm2. The aim was to investigate possible roles for mdm2 in hTR promoter regulation. Methods Chromatin immunoprecipitation was used to determine binding of mdm2 to the hTR promoter. Transfection and luciferase assays were used to investigate mdm2 repression of the promoter activity and interaction with known transcriptional modulators. Results Here we show using chromatin immunoprecipitation that mdm2 specifically binds the hTR promoter in vivo. Transient co-transfection experiments using an hTR promoter luciferase reporter construct show that hTR promoter activity is inhibited by over-expression of mdm2 in 5637 bladder carcinoma cells (p53 and pRB negative, low mdm2. Titration of mdm2 was able to antagonise activation of hTR promoter activity mediated by pRB or Sp1 over-expression, although in the presence of pRB, mdm2 could not repress promoter activity below basal levels. Using an Sp1 binding site mutation construct we showed that mdm2 repression did not absolutely require Sp1 binding sites in the hTR promoter, suggesting the possibility of pRB/Sp1 independent mechanisms of repression. Finally, we show that NF-Y mediated transactivation of the hTR promoter was also suppressed by mdm2 in a dose-dependent manner. Conclusions These studies suggest that mdm2 may inhibit the hTR promoter by multiple mechanisms. Mdm2 may directly repress activation by both pRB and Sp1, or activation by NF-Y. Furthermore, the ability of mdm2 to interact and interfere with components of the general transcription machinery might partly explain the general repressive effect seen here. Elucidation of

  17. Analysis of promoter regions of co-expressed genes identified by microarray analysis

    Directory of Open Access Journals (Sweden)

    Höglund Mattias

    2006-08-01

    Full Text Available Abstract Background The use of global gene expression profiling to identify sets of genes with similar expression patterns is rapidly becoming a widespread approach for understanding biological processes. A logical and systematic approach to study co-expressed genes is to analyze their promoter sequences to identify transcription factors that may be involved in establishing specific profiles and that may be experimentally investigated. Results We introduce promoter clustering i.e. grouping of promoters with respect to their high scoring motif content, and show that this approach greatly enhances the identification of common and significant transcription factor binding sites (TFBS in co-expressed genes. We apply this method to two different dataset, one consisting of micro array data from 108 leukemias (AMLs and a second from a time series experiment, and show that biologically relevant promoter patterns may be obtained using phylogenetic foot-printing methodology. In addition, we also found that 15% of the analyzed promoter regions contained transcription factors start sites for additional genes transcribed in the opposite direction. Conclusion Promoter clustering based on global promoter features greatly improve the identification of shared TFBS in co-expressed genes. We believe that the outlined approach may be a useful first step to identify transcription factors that contribute to specific features of gene expression profiles.

  18. Selection of Arabidopsis mutants overexpressing genes driven by the promoter of an auxin-inducible glutathione S-transferase gene

    NARCIS (Netherlands)

    Kop, D.A.M. van der; Schuyer, M.; Pinas, J.E.; Zaal, B.J. van der; Hooykaas, P.J.J.

    1999-01-01

    Transgenic arabidopsis plants were isolated that contained a T-DNA construct in which the promoter of an auxin-inducible glutathione S-transferase (GST) gene from tobacco was fused to the kanamycin resistance (nptII) as well as to the β-glucuronidase (gusA) reporter gene. Subsequently, seeds were

  19. γδβ-thalassaemias 1 and 2 are the result of a 100 kpb deletion in the human β-globin cluster.

    NARCIS (Netherlands)

    R. Taramelli; D. Kioussis; E. Vanin; K. Bartram; J. Groffen; J. Hurst; F.G. Grosveld (Frank)

    1986-01-01

    textabstractThe DNA spanning two large deletions in the human beta-globin gene cluster (gamma beta-thalassaemia 1 and 2) has been cloned by cosmid cloning and chromosomal walking. The entire region was mapped and analyzed for the presence of repetitive sequences. The results show that the affected

  20. A novel method for the determination of basal gene expression of tissue-specific promoters: an analysis of prostate-specific promoters.

    NARCIS (Netherlands)

    Poel, H.G. van der; McCadden, J.; Verhaegh, G.W.C.T.; Kruszewski, M.; Ferrer, F.; Schalken, J.A.; Carducci, M.; Rodriguez, R.

    2001-01-01

    Because the toxicity of suicide gene therapeutics is directly related to basal promoter activity, we developed an assay to test for promoter "leakiness" using a diphtheria toxin mutant. Sequences of 15 prostate-specific gene promoter constructs were cloned in an expression plasmid (pBK; Stratagene,

  1. Enhancer-core-promoter specificity separates developmental and housekeeping gene regulation.

    Science.gov (United States)

    Zabidi, Muhammad A; Arnold, Cosmas D; Schernhuber, Katharina; Pagani, Michaela; Rath, Martina; Frank, Olga; Stark, Alexander

    2015-02-26

    Gene transcription in animals involves the assembly of RNA polymerase II at core promoters and its cell-type-specific activation by enhancers that can be located more distally. However, how ubiquitous expression of housekeeping genes is achieved has been less clear. In particular, it is unknown whether ubiquitously active enhancers exist and how developmental and housekeeping gene regulation is separated. An attractive hypothesis is that different core promoters might exhibit an intrinsic specificity to certain enhancers. This is conceivable, as various core promoter sequence elements are differentially distributed between genes of different functions, including elements that are predominantly found at either developmentally regulated or at housekeeping genes. Here we show that thousands of enhancers in Drosophila melanogaster S2 and ovarian somatic cells (OSCs) exhibit a marked specificity to one of two core promoters--one derived from a ubiquitously expressed ribosomal protein gene and another from a developmentally regulated transcription factor--and confirm the existence of these two classes for five additional core promoters from genes with diverse functions. Housekeeping enhancers are active across the two cell types, while developmental enhancers exhibit strong cell-type specificity. Both enhancer classes differ in their genomic distribution, the functions of neighbouring genes, and the core promoter elements of these neighbouring genes. In addition, we identify two transcription factors--Dref and Trl--that bind and activate housekeeping versus developmental enhancers, respectively. Our results provide evidence for a sequence-encoded enhancer-core-promoter specificity that separates developmental and housekeeping gene regulatory programs for thousands of enhancers and their target genes across the entire genome.

  2. Characterization and Comparative Immunoreactivity of Antibody to Newt (T. cristatus) Globins

    NARCIS (Netherlands)

    Kowalski, Louis A.; Walsh, Anne W.; Merrow, Martha; Paulekus, Wayne; Mackin, William; Grasso, Joseph A.

    1984-01-01

    1. Rabbit antisera to newt (T. cristatus) globin were produced by repeated injections of globin and antiglobin antibodies purified by chromatography on globin-Sepharose 4B. 2. Ouchterlony and SDS PAGE analysis indicated that the material eluted from the affinity column was rabbit IgG. 3. The

  3. Nup98 promotes antiviral gene expression to restrict RNA viral infection in Drosophila.

    Science.gov (United States)

    Panda, Debasis; Pascual-Garcia, Pau; Dunagin, Margaret; Tudor, Matthew; Hopkins, Kaycie C; Xu, Jie; Gold, Beth; Raj, Arjun; Capelson, Maya; Cherry, Sara

    2014-09-16

    In response to infection, the innate immune system rapidly activates an elaborate and tightly orchestrated gene expression program to induce critical antimicrobial genes. While many key players in this program have been identified in disparate biological systems, it is clear that there are additional uncharacterized mechanisms at play. Our previous studies revealed that a rapidly-induced antiviral gene expression program is active against disparate human arthropod-borne viruses in Drosophila. Moreover, one-half of this program is regulated at the level of transcriptional pausing. Here we found that Nup98, a virus-induced gene, was antiviral against a panel of viruses both in cells and adult flies since its depletion significantly enhanced viral infection. Mechanistically, we found that Nup98 promotes antiviral gene expression in Drosophila at the level of transcription. Expression profiling revealed that the virus-induced activation of 36 genes was abrogated upon loss of Nup98; and we found that a subset of these Nup98-dependent genes were antiviral. These Nup98-dependent virus-induced genes are Cdk9-dependent and translation-independent suggesting that these are rapidly induced primary response genes. Biochemically, we demonstrate that Nup98 is directly bound to the promoters of virus-induced genes, and that it promotes occupancy of the initiating form of RNA polymerase II at these promoters, which are rapidly induced on viral infection to restrict human arboviruses in insects.

  4. Promoter-sharing by different genes in human genome – CPNE1 and RBM12 gene pair as an example

    Directory of Open Access Journals (Sweden)

    Yiu Siu-Ming

    2008-10-01

    Full Text Available Abstract Background Regulation of gene expression plays important role in cellular functions. Co-regulation of different genes may indicate functional connection or even physical interaction between gene products. Thus analysis on genomic structures that may affect gene expression regulation could shed light on the functions of genes. Results In a whole genome analysis of alternative splicing events, we found that two distinct genes, copine I (CPNE1 and RNA binding motif protein 12 (RBM12, share the most 5' exons and therefore the promoter region in human. Further analysis identified many gene pairs in human genome that share the same promoters and 5' exons but have totally different coding sequences. Analysis of genomic and expressed sequences, either cDNAs or expressed sequence tags (ESTs for CPNE1 and RBM12, confirmed the conservation of this phenomenon during evolutionary courses. The co-expression of the two genes initiated from the same promoter is confirmed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR in different tissues in both human and mouse. High degrees of sequence conservation among multiple species in the 5'UTR region common to CPNE1 and RBM12 were also identified. Conclusion Promoter and 5'UTR sharing between CPNE1 and RBM12 is observed in human, mouse and zebrafish. Conservation of this genomic structure in evolutionary courses indicates potential functional interaction between the two genes. More than 20 other gene pairs in human genome were found to have the similar genomic structure in a genome-wide analysis, and it may represent a unique pattern of genomic arrangement that may affect expression regulation of the corresponding genes.

  5. A Forward Genetic Screening for Prostate Cancer Progression Genes

    Science.gov (United States)

    2012-10-01

    manifestation of phenotype, it is not desirable to sacrifice valuable experimental animals during the course of the screen. It would be ideal to have a...transposon (Figure 1A, PB[mut]) to induce ectopic gene expression in multiple genomic contexts. The CMV early enhancer/ chicken b-actin promoter was cloned...PBase plasmid. Two copies of the chicken b-globin HS4 core enhancer from the plasmid pNI-CD were also cloned upstream of the Actin promoter. PBaseER was

  6. Brain alpha- and beta-globin expression after intracerebral hemorrhage

    OpenAIRE

    He, Yangdong; Hua, Ya; Lee, Jin-Yul; Liu, Wenquan; Keep, Richard F; Wang, Michael M.; Xi, Guohua

    2010-01-01

    Our recent study has demonstrated that hemoglobin (Hb) is present in cerebral neurons and neuronal Hb is inducible after cerebral ischemia. In the present study, we examined the effects of intracerebral hemorrhage (ICH) on the mRNA levels of the α-globin (HbA) and the β-globin (HbB) components of Hb and Hb protein in the brain in vivo and in vitro. In vivo, male Sprague-Dawley rats received either a needle insertion (sham) or an infusion of autologous whole blood into the basal ganglia and we...

  7. REVIEW ARTICLE One gene, many phenotypes

    African Journals Online (AJOL)

    salah

    One gene, many phenotypes. A- Modifier causing less severe (re- duced) phenotype of Beta thalassemia. The severity of anemia in beta thalas- semia reflects the degree of globin chain imbalance. The excess of alpha globin chain precipitates in red cell pre- cursors leading to ineffective erythro- poiesis. This imbalance can ...

  8. A novel binary T-vector with the GFP reporter gene for promoter characterization.

    Directory of Open Access Journals (Sweden)

    Shu-Ye Jiang

    Full Text Available Several strategies have been developed to clone PCR fragments into desired vectors. However, most of commercially available T-vectors are not binary vectors and cannot be directly used for Agrobacterium-mediated plant genetic transformation. In this study, a novel binary T-vector was constructed by integrating two AhdI restriction sites into the backbone vector pCAMBIA 1300. The T-vector also contains a GFP reporter gene and thus, can be used to analyze promoter activity by monitoring the reporter gene. On the other hand, identification and characterization of various promoters not only benefit the functional annotation of their genes but also provide alternative candidates to be used to drive interesting genes for plant genetic improvement by transgenesis. More than 1,000 putative pollen-specific rice genes have been identified in a genome-wide level. Among them, 67 highly expressed genes were further characterized. One of the pollen-specific genes LOC_Os10g35930 was further surveyed in its expression patterns with more details by quantitative real-time reverse-transcription PCR (qRT-PCR analysis. Finally, its promoter activity was further investigated by analyzing transgenic rice plants carrying the promoter::GFP cassette, which was constructed from the newly developed T-vector. The reporter GFP gene expression in these transgenic plants showed that the promoter was active only in mature but not in germinated pollens.

  9. Transcriptional analysis of heterologous gene expression using the endogenous sD promoter from Bacillus halodurans

    CSIR Research Space (South Africa)

    Crampton, Michael C

    2010-07-01

    Full Text Available This presentation focused on the transcriptional analysis of heterologous gene expression using the endogenous sD promoter from Bacillus halodurans. It concludes to a successful implementation of a high throughput mRNA sandwich hybridisation...

  10. Directed evolution of promoters and tandem gene arrays for customizing RNA synthesis rates and regulation.

    Science.gov (United States)

    Tyo, Keith E J; Nevoigt, Elke; Stephanopoulos, Gregory

    2011-01-01

    Manipulating RNA synthesis rates is a primary method the cell uses to adjust its physiological state. Therefore to design synthetic genetic networks and circuits, precise control of RNA synthesis rates is of the utmost importance. Often, however, a native promoter does not exist that has the precise characteristics required for a given application. Here, we describe two methods to change the rates and regulation of RNA synthesis in cells to create RNA synthesis of a desired specification. First, error-prone PCR is discussed for diversifying the properties of native promoters, that is, changing the rate of synthesis in constitutive promoters and the induction properties for an inducible promoter. Specifically, we describe techniques for generating diversified promoter libraries of the constitutive promoters P(L)tetO-1 in Escherichia coli and TEF1 in Saccharomyces cerevisiae as well as the inducible, oxygen-repressed promoter DAN1 in S. cerevisiae. Beyond generating promoter libraries, we discuss techniques to quantify the parameters of each new promoter. Promoter characteristics for each promoter in hand, the designer can then pick and choose the promoters needed for the specific genetic circuit described in silico. Second, Chemically Induced Chromosomal Evolution (CIChE) is presented as an alternative method to finely adjust RNA synthesis rates in E. coli by variation of gene cassette copy numbers in tandem gene arrays. Both techniques result in precisely defined RNA synthesis and should be of great utility in synthetic biology. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Minimal enhancer elements of the leghemoglobin lba and lbc3 gene promoters from Glycine max L. have different properties

    DEFF Research Database (Denmark)

    She, Q; Lauridsen, P; Stougaard, J

    1993-01-01

    The characteristics of the soybean leghemoglobin lba gene promoter were analyzed and important promoter elements from the lba and lbc3 promoters were compared using transgenic Lotus corniculatus plants. A 5' deletion analysis of the lba promoter delimited two cis-acting elements controlling expre...... function. This may reflect the differential expression of the two lb genes of Glycine max L....

  12. Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae, Fabaceae and Poaceae

    Directory of Open Access Journals (Sweden)

    Fauteux François

    2009-10-01

    Full Text Available Abstract Background Accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate DNA sequences bound by several interacting factors. Enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conserved transcriptional regulatory mechanisms. This provides a basis for the computational identification of cis-regulatory motifs. Results We have used a discriminative seeding DNA motif discovery algorithm for an in-depth analysis of 54 seed storage protein (SSP gene promoters from three plant families, namely Brassicaceae (mustards, Fabaceae (legumes and Poaceae (grasses using backgrounds based on complete sets of promoters from a representative species in each family, namely Arabidopsis (Arabidopsis thaliana (L. Heynh., soybean (Glycine max (L. Merr. and rice (Oryza sativa L. respectively. We have identified three conserved motifs (two RY-like and one ACGT-like in Brassicaceae and Fabaceae SSP gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. Fabaceae SSP gene promoter sequences are also enriched in a novel, seed-specific E2Fb-like motif. Conserved motifs identified in Poaceae SSP gene promoters include a GCN4-like motif, two prolamin-box-like motifs and an Skn-1-like motif. Evidence of the presence of a variant of the TATA-box is found in the SSP gene promoters from the three plant families. Motifs discovered in SSP gene promoters were used to score whole-genome sets of promoters from Arabidopsis, soybean and rice. The highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. Conclusion Seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination

  13. Identification, isolation and evaluation of a constitutive sucrose phosphate synthase gene promoter from tomato

    International Nuclear Information System (INIS)

    Naqvi, R.Z.; Mubeen, H.; Maqsood, A.; Khatoon, A.

    2017-01-01

    Sucrose phosphate synthase (SPS) is one of the abundantly expressed genes in plants. The promoters of SPS gene was identified, analyzed and retrieved from high throughput genomic sequence (HTGS) database. The cis-acting regulatory elements and transcription start sites of promoter were identified through different bioinformatics tools. The SPS promoter was isolated from Solanum lycopersicum and was initially cloned in TA vector (pTZ57R/T). Later on this promoter was transferred to a plant expression binary vector, pGR1 (pGRSPS) that was used for the transient GUS expression studies in various tissues of Nicotiana tabacum. SPS promoter was also cloned in plant stable expression vector pGA482 (pGASPS) and was transformed in Nicotiana tabacum through Agrobacterium-mediated transformation method. The histochemical GUS expression analysis of both transient and stable transgenic plants for this promoter indicated its functional importance in regulating gene expression in a constitutive manner. It was concluded that SPS promoter is constitutively expressed with a strength equivalent to CaMV 2X35S promoter. The promoter isolated through these studies may be effectively substituted in plant genetic engineering with other constitutive promoter for transgene expression in economically important agricultural crops. (author)

  14. Prediction of DNA methylation in the promoter of gene suppressor tumor.

    Science.gov (United States)

    Saif, Imane; Kasmi, Yassine; Allali, Karam; Ennaji, Moulay Mustapha

    2018-04-20

    The epigenetics methylation of cytosine is the most common epigenetic form in DNA sequences. It is highly concentrated in the promoter regions of the genes, leading to an inactivation of tumor suppressors regardless of their initial function. In this work, we aim to identify the highly methylated regions; the cytosine-phosphate-guanine (CpG) island located on the promoters and/or the first exon gene known for their key roles in the cell cycle, hence the need to study gene-gene interactions. The Frommer and hidden Markov model algorithms are used as computational methods to identify CpG islands with specificity and sensitivity up to 76% and 80%, respectively. The results obtained show, on the one hand, that the genes studied are suspected of developing hypermethylation in the promoter region of the gene involved in the case of a cancer. We then showed that the relative richness in CG results from a high level of methylation. On the other hand, we observe that the gene-gene interaction exhibits co-expression between the chosen genes. This let us to conclude that the hidden Markov model algorithm predicts more specific and valuable information about the hypermethylation in gene as a preventive and diagnostics tools for the personalized medicine; as that the tumor-suppresser-genes have relative co-expression and complementary relations which the hypermethylation affect in the samples studied in our work. Copyright © 2018. Published by Elsevier B.V.

  15. Fanconi anemia core complex gene promoters harbor conserved transcription regulatory elements.

    Directory of Open Access Journals (Sweden)

    Daniel Meier

    Full Text Available The Fanconi anemia (FA gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS. In the 5' region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3' regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs, and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters.

  16. Promoter polymorphisms in genes involved in porcine myogenesis influence their transcriptional activity.

    Science.gov (United States)

    Bongiorni, Silvia; Tilesi, Francesca; Bicorgna, Silvia; Iacoponi, Francesca; Willems, Daniela; Gargani, Maria; D'Andrea, MariaSilvia; Pilla, Fabio; Valentini, Alessio

    2014-11-07

    Success of meat production and selection for improvement of meat quality is among the primary aims in animal production. Meat quality traits are economically important in swine; however, the underlying genetic nature is very complex. Therefore, an improved pork production strongly depends on identifying and studying how genetic variations contribute to modulate gene expression. Promoters are key regions in gene modulation as they harbour several binding motifs to transcription regulatory factors. Therefore, polymorphisms in these regions are likely to deeply affect RNA levels and consequently protein synthesis. In this study, we report the identification of single nucleotide polymorphisms (SNPs) in promoter regions of candidate genes involved in development, cellular differentiation and muscle growth in Sus scrofa. We identified SNPs in the promoter regions of genes belonging to the Myogenic Regulatory Factors (MRF) gene family (the Myogenic Differentiation gene, MYOD1) and to Growth and Differentiation Factors (GDF) gene family (Myostatin gene, MSTN, GDF8), in Casertana and Large White breeds. The purpose of this study was to investigate if polymorphisms in the promoters could affect the transcriptional activity of these genes. With this aim, we evaluated in vitro the functional activity of the luciferase reporter gene luc2 activity, driven by two constructs carrying different promoter haplotypes. We tested the effects of the G302A (U12574) transition on the promoter efficiency in MYOD1 gene. We ascertained a difference in transcription efficiency for the two variants. A stronger activity of the A-carrying construct is more evident in C2C12. The luciferase expression driven by the MYOD1-A allelic variant displayed a 3.8-fold increased transcriptional activity. We investigated the activity of two haplotype variants (AY527152) in the promoter of GDF8 gene. The haploptype-1 (A435-A447-A879) up-regulated the expression of the reporter gene by a two-fold increase, and

  17. Approach of combined cancer gene therapy and radiation: response of promoters to ionizing radiation

    International Nuclear Information System (INIS)

    Anstett, A.

    2005-09-01

    Gene therapy is an emerging cancer treatment modality. We are interested in developing a radiation-inducible gene therapy system to sensitize the tumor vasculature to the effects of ionizing radiation (IR) treatment. An expression system based on irradiation-inducible promoters will drive the expression of anti-tumor genes in the tumor vasculature. Solid tumors are dependent on angio genesis, a process in which new blood vessels are formed from the pre-existing vasculature. Vascular endothelial cells are un transformed and genetically stable, thus avoiding the problem of resistance to the treatments. Vascular endothelial cells may therefore represent a suitable target for this therapeutic gene therapy strategy.The identification of IR-inducible promoters native to endothelial cells was performed by gene expression profiling using cDNA micro array technology. We describe the genes modified by clinically relevant doses of IR. The extension to high doses aimed at studying the effects of total radiation delivery to the tumor. The radio-inductiveness of the genes selected for promoter study was confirmed by RT-PCR. Analysis of the activity of promoters in response to IR was also assessed in a reporter plasmid. We found that authentic promoters cloned onto a plasmid are not suitable for cancer gene therapy due to their low induction after IR. In contrast, synthetic promoters containing repeated sequence-specific binding sites for IR-activated transcription factors such as NF-κB are potential candidates for gene therapy. The activity of five tandemly repeated TGGGGACTTTCCGC elements for NF-κB binding in a luciferase reporter was increased in a dose-dependent manner. Interestingly, the response to fractionated low doses was improved in comparison to the total single dose. Thus, we put present evidence that a synthetic promoter for NF-κB specific binding may have application in the radio-therapeutic treatment of cancer. (author)

  18. Type 1 plaminogen activator inhibitor gene: Functional analysis and glucocorticoid regulation of its promoter

    Energy Technology Data Exchange (ETDEWEB)

    Van Zonneveld, A.J.; Curriden, S.A.; Loskutoff, D.J. (Scripps Clinic and Research Foundation, La Jolla, CA (USA))

    1988-08-01

    Plasminogen activator inhibitor type 1 is an important component of the fibrinolytic system and its biosynthesis is subject to complex regulation. To study this regulation at the level of transcription, the authors have identified and sequenced the promoter of the human plasminogen activator inhibitor type 1 gene. Nuclease protection experiments were performed by using endothelial cell mRNA and the transcription initiation (cap) site was established. Sequence analysis of the 5{prime} flanking region of the gene revealed a perfect TATA box at position {minus}28 to position {minus}23, the conserved distance from the cap site. Comparative functional studies with the firefly luciferase gene as a reporter gene showed that fragments derived from this 5{prime} flanking region exhibited high promoter activity when transfected into bovine aortic endothelial cells and mouse Ltk{sup {minus}} fibroblasts but were inactive when introduced into HeLa cells. These studies indicate that the fragments contain the plasminogen activator inhibitor type 1 promoter and that it is expressed in a tissue-specific manner. Although the fragments were also silent in rat FTO2B hepatoma cells, their promoter activity could be induced up to 40-fold with the synthetic glucocorticoid dexamethasone. Promoter deletion mapping experiments and studies involving the fusion of promoter fragments to a heterologous gene indicated that dexamethasone induction is mediated by a glucocorticoid responsive element with enhancer-like properties located within the region between nucleotides {minus}305 and +75 of the plasminogen activator inhibitor type 1 gene.

  19. Aberrant gene promoter methylation in sputum from individuals exposed to smoky coal emissions

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Y.; Lan, Q.; Shen, M.; Jin, J.; Mumford, J.; Ren, D.X.; Keohavong, P. [University of Pittsburgh, Pittsburgh, PA (United States). Dept. of Environment and Occupational Health

    2008-07-15

    Recent studies suggested the potential for aberrant gene promoter methylation in sputum as a predictive marker for lung cancer. Here, the promoter methylation of p16, MGMT, RASSF1A and DAPK genes was investigated in sputum of individuals exposed to smoky coal emissions in Xuan Wei, China, where the lung cancer rate is more than 6 times the Chinese national average. Sputum DNA of 107 noncancer individuals and 58 lung cancer patients was screened for promoter methylation using methylation-specific PCR. Promoter methylation of the p16 gene was detected in about half (51.4% (551107)) of sputum DNA from noncancer individuals, a frequency higher than that observed for the RASSF1A (29.9%), MGMT (17.8%) and DAPK (15.9%) genes. Furthermore, the p16 gene was affected by promoter methylation at a frequency even higher among the lung cancer group, compared with the noncancer group (70.7% (41/58) versus 51.7% (55/107), p=0.017). Individuals exposed to smoky coal emissions in this region harbored frequent promoter methylation of these genes in their sputum and some of such alterations may be involved in lung tumor development.

  20. Functional analysis of a novel human serotonin transporter gene promoter in immortalized raphe cells

    DEFF Research Database (Denmark)

    Mortensen, O V; Thomassen, M; Larsen, M B

    1999-01-01

    were found to possess the additional 379 bp fragment. The integrity of the promoter was furthermore confirmed by genomic Southern blotting. The promoter activity was analyzed by reporter gene assays in neuronal and non-neuronal serotonergic cell lines. In immortalized serotonergic raphe neurons, RN46A...

  1. Comparative analysis of ADS gene promoter in seven Artemisia ...

    Indian Academy of Sciences (India)

    2014-12-23

    Dec 23, 2014 ... ness, salicylic acid and abscisic acid responsiveness, induc- tion upon fungal elicitation, endosperm expression, MeJA- responsiveness, low-temperature responsiveness, elicitation, wounding and pathogen responsiveness were also found in. ADS promoters of all or some of the seven Artemisia species.

  2. Oxygen Association-Dissociation and Stability Analysis on Mouse Hemoglobins with Mutant α- and β-Globins

    Science.gov (United States)

    D'Surney, S. J.; Popp, R. A.

    1992-01-01

    Oxygen association-dissociation and hemoglobin stability analysis were performed on mouse hemoglobins with amino acid substitutions in an α-globin (α89, His to Leu) and a β-globin (β59, Lys to Ile). The variant α-globin, designated chain 5(m) in the Hba(g2) haplotype, had a high oxygen affinity and was stable. The variant β-globin, (β(s2)) of the Hbb(s2) haplotype, also had an elevated oxygen affinity and in addition was moderately unstable in 19% isopropanol. Hemoglobins from the expected nine (Hba(g2)/Hba(g2);Hbb(s)/Hbb(s) X Hba(a)/Hba(a);Hbb(s2)/Hbb(s2)) F(2) genotypes can be grouped into five classes of P(50) values characterized by strict additivity and dependency on mutant globin gene dosage; physiologically, both globin variants gave indistinguishable effects on oxygen affinity. The hemoglobin of normal mice (Hba(a)/Hba(a);Hbb(s)/Hbb(s)) had a P(50) = 40 mm Hg and the hemoglobin of Hba(g2)/Hba(g2);Hbb(s2)/Hbb(s2) F(2) mice had a P(50) = 25 mm Hg (human P(50) = 26 mm Hg). Peripheral blood from Hba(g2)/Hba(g2);Hbb(s)/Hbb(s), Hba(a)/Hba(a);Hbb(s2)/Hbb(s2) and Hba(g2)/Hba(g2);Hbb(s2)/Hbb(s2) mice exhibited normal hematological values except for a slightly higher hematocrit for Hba(g2)/Hba(g2);Hbb(s)/Hbb(s) and Hba(g2)/Hba(g2);Hbb(s2)/Hbb(s2) mice, slightly elevated red cell counts for mice of the three mutant genotypes, and significantly lower values for the mean corpuscular volume and mean corpuscular hemoglobin for Hba(g2)/Hba(g2);Hbb(s2)/Hbb(s2) mice. PMID:1427042

  3. [Cloning of ID4 gene expression regulation promoter and subcloning of recombinant ID4 promoter luciferase reporter].

    Science.gov (United States)

    Li, Wei; Huang, Yu; Yang, Bo; Chi, Xiao-Hua; Liu, Li-Hong; Zhang, Feng; Yan, Jiang-Wei; Lu, Xue-Chun

    2010-04-01

    The present study was aimed to clone ID4 gene promoter and upstream regulatory region, and to construct a series of recombinant promoter-luciferase reporter for exploring the mechanism of ID4 gene expression regulation. the upstream 5' flanking sequence of 2242 bp from transcriptional start site (TSS) and downstream 5' non-coding region of 212 bp on ID4 gene were searched out and downloaded from human genome databank of NCBI using whole length of ID4 gene cDNA as a probe; On-line promoter analysis softwares, including TESS and Genomax, were employed to analyze the characteristics of ID4 gene promoter and upstream regulatory elements. Then, based on the analytic results, PCR primers were designed and synthesized. Segmental amplification method was adopted to obtain two fragments of 1829 bp and 784 bp. The two fragments were inserted into the plasmid pGEM-T, transformed into TOP10 competent E. coli., and positive recombinants were screened respectively. Subsequently, restriction enzymes KpnI/NheI and KpnI/EcoRI were used to digest the above-mentioned two plasmids pGEM-T and pGL3, and ligation was completed by T4 DNA ligase. After transformation to TOP10 competent E. coli. and screening of positive colonies, the basic recombinant ID4 gene promoter-pGL3 was successfully constructed. KpnI/NheI double digestion and sequencing showed that the target fragment was 2 459 bp and consistent with the corresponding sequence of GenBank; Using the 2459 bp fragment as a template, 5 pairs of primers with identical 3' terminus and different 5' terminus were designed and synthesized for half-nest PCR amplification. 5 fragments with an interval of approximate 400 bp each other, i.e. 2112 bp, 1703 bp, 1290 bp, 784 bp and 496 bp, were produced and inserted into pGEM-T after recovery and purification for transformation to TOP10 competent E. coli. and screening of positive colonies. After that, KpnI/NheI was used to digest the above-mentioned five pGEM-T recombinant plasmids and pGL3 basic

  4. The host factor polyhedrin promoter binding protein (PPBP) is involved in transcription from the baculovirus polyhedrin gene promoter.

    Science.gov (United States)

    Ghosh, S; Jain, A; Mukherjee, B; Habib, S; Hasnain, S E

    1998-09-01

    Hypertranscription and temporal expression from the Autographa californica nuclear polyhedrosis (AcNPV) baculovirus polyhedrin promoter involves an alpha-amanitin-resistant RNA polymerase and requires a trans-acting viral factor(s). We previously reported that a 30-kDa host factor, polyhedrin promoter binding protein (PPBP), binds with unusual affinity, specificity, and stability to the transcriptionally important motif AATAAATAAGTATT within the polyhedrin (polh) initiator promoter and also displays coding strand-specific single-stranded DNA (ssDNA)-binding activity (S. Burma, B. Mukherjee, A. Jain, S. Habib, and S. E. Hasnain, J. Biol. Chem. 269:2750-2757, 1994; B. Mukherjee, S. Burma, and S. E. Hasnain, J. Biol. Chem. 270:4405-4411, 1995). We now present evidence which indicates that an additional factor(s) is involved in stabilizing PPBP-duplex promoter and PPBP-ssDNA interactions. TBP (TATA box binding protein) present in Spodoptera frugiperda (Sf9) cells is characteristically distinct from PPBP and does not interact directly with the polh promoter. Replacement of PPBP cognate sequences within the polh promoter with random nucleotides abolished PPBP binding in vitro and also failed to express the luciferase reporter gene in vivo. Phosphocellulose fractions of total nuclear extract from virus-infected cells which support in vitro transcription from the polh promoter contain PPBP activity. When PPBP was sequestered by the presence of oligonucleotides containing PPBP cognate sequence motifs, in vitro transcription of a C-free reporter cassette was affected but was restored by the exogenous addition of nuclear extract containing PPBP. When PPBP was mopped out in vivo by a plasmid carrying PPBP cognate sequence present in trans, polh promoter-driven expression of the luciferase reporter was abolished, demonstrating that binding of PPBP to the polh promoter is essential for transcription.

  5. Cloning and analysis of plant fatty acid desaturase 7 gene promoter ...

    African Journals Online (AJOL)

    In order to investigate the regulation mode of Brassica napus FAD7 gene in response of thermal stress, we measured the protein levels of BnFAD7 in plant at low and high temperature, and then analyzed promoter activity of 5'-flanking regions of BnFAD7 by transient gene expression in B. napus protoplasts at different ...

  6. Cloning and expression of Icc1 Laccase gene promoter in Aspergillus niger

    International Nuclear Information System (INIS)

    Marqueda-Galvez, A. P.; Loera Carrol, O.; Xaconostle cazares, B.; Tellez-Jurado, A.; Arana-Cuenca, A.

    2009-01-01

    The white rot fungus Trametes sp. I-62 is a strain with laccase activity and a great potential for biotechnological applications given its ability to detoxify distillery effluents. The Icc1, Icc2 and Icc3 laccase genes of this basidiomycetes have been cloned and sequenced. The promoter region of Icc1 laccase gene contains a putative site for xenobiotics (XRE). (Author)

  7. The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei

    NARCIS (Netherlands)

    Zomerdijk, J. C.; Ouellette, M.; ten Asbroek, A. L.; Kieft, R.; Bommer, A. M.; Clayton, C. E.; Borst, P.

    1990-01-01

    The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site

  8. Interleukin 10 gene promoter polymorphism and risk of diffuse large B cell lymphoma (DLBCL

    Directory of Open Access Journals (Sweden)

    Roba M. Talaat

    2014-01-01

    Conclusions: Taken together, our findings demonstrated that IL-10 promoter gene polymorphism (−1082 and −819 may not have an influence on the clinical outcome of DLBCL, especially in terms of overall secretion level. Further investigations of other cytokine gene polymorphisms will lead to a better understanding of the disease’s biological background.

  9. Computational design and application of endogenous promoters for transcriptionally targeted gene therapy for rheumatoid arthritis.

    NARCIS (Netherlands)

    Geurts, J.; Joosten, L.A.B.; Takahashi, N.; Arntz, O.J.; Gluck, A.; Bennink, M.B.; Berg, W.B. van den; Loo, F.A.J. van de

    2009-01-01

    The promoter regions of genes that are differentially regulated in the synovial membrane during the course of rheumatoid arthritis (RA) represent attractive candidates for application in transcriptionally targeted gene therapy. In this study, we applied an unbiased computational approach to define

  10. Altered gene-expression profile in rat plasma and promoted body ...

    African Journals Online (AJOL)

    ... among which five GO annotations and four KEGG pathways were annotated. Findings indicate that EE during pregnancy could positively promote the body and nervous system development of offspring, involving the evidence for altered gene expression profile. Keywords: Environmental enrichment, rats, gene expression ...

  11. Cloning and expression of Icc1 Laccase gene promoter in Aspergillus niger

    Energy Technology Data Exchange (ETDEWEB)

    Marqueda-Galvez, A. P.; Loera Carrol, O.; Xaconostle cazares, B.; Tellez-Jurado, A.; Arana-Cuenca, A.

    2009-07-01

    The white rot fungus Trametes sp. I-62 is a strain with laccase activity and a great potential for biotechnological applications given its ability to detoxify distillery effluents. The Icc1, Icc2 and Icc3 laccase genes of this basidiomycetes have been cloned and sequenced. The promoter region of Icc1 kaccase gene contains a putative site for xenobiotics (XRE). (Author)

  12. Spectrin interactions with globin chains in the presence of ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    We have shown the differential interactions of the erythroid skeletal protein spectrin with the globin subunits of adult haemoglobin ... high molecular weight complex; Kd, binding dissociation constant; Mg/ATP, ATP in presence of 10-fold molar excess of MgCl2; PMB, ... mercuribenzoic acid sodium salt (PMB), sodium azide,.

  13. Inducible Promoter Systems for Gene Perturbation Experiments in Arabidopsis.

    Science.gov (United States)

    Thomson, Bennett; Graciet, Emmanuelle; Wellmer, Frank

    2017-01-01

    Assessing molecular changes that occur through altering a gene's activity is often hampered by difficulties that arise due to the typically static nature of the introduced perturbation. This is especially problematic when investigating molecular events at specific stages and/or in certain tissues or organs during Arabidopsis development. To circumvent these issues, we have employed chemically inducible artificial microRNAs (amiRNAs) for the specific knockdown of developmental regulators. For our own research, we have combined this gene perturbation approach with a floral induction system, which allows the simultaneous induction of a large number of flowers on the inflorescence of a single plant, and the ability to knock down a gene's activity at any given stage of development. To enable the plant community to avail of the full benefits of these systems, we describe, in this chapter, strategies for amiRNA-mediated gene perturbations and address some common problems that can be encountered when generating inducible amiRNA constructs, growing these plants, and collecting floral buds for analysis.

  14. A cII-dependent promoter is located within the Q gene of bacteriophage lambda.

    OpenAIRE

    Hoopes, B C; McClure, W R

    1985-01-01

    We have found a cII-dependent promoter, PaQ, within the Q gene of bacteriophage lambda. Transcription experiments and abortive initiation assays performed in vitro showed that the promoter strength and the cII affinity of PaQ were comparable to the other cII-dependent lambda promoters, PE and PI. The location and leftward direction of PaQ suggests a possible role in the delay of lambda late-gene expression by cII protein, a phenomenon that has been called cII-dependent inhibition. We have con...

  15. Characteristic differences between the promoters of intron-containing and intronless ribosomal protein genes in yeast

    Directory of Open Access Journals (Sweden)

    Vingron Martin

    2008-10-01

    Full Text Available Abstract Background More than two thirds of the highly expressed ribosomal protein (RP genes in Saccharomyces cerevisiae contain introns, which is in sharp contrast to the genome-wide five percent intron-containing genes. It is well established that introns carry regulatory sequences and that the transcription of RP genes is extensively and coordinately regulated. Here we test the hypotheses that introns are innately associated with heavily transcribed genes and that introns of RP genes contribute regulatory TF binding sequences. Moreover, we investigate whether promoter features are significantly different between intron-containing and intronless RP genes. Results We find that directly measured transcription rates tend to be lower for intron-containing compared to intronless RP genes. We do not observe any specifically enriched sequence motifs in the introns of RP genes other than those of the branch point and the two splice sites. Comparing the promoters of intron-containing and intronless RP genes, we detect differences in number and position of Rap1-binding and IFHL motifs. Moreover, the analysis of the length distribution and the folding free energies suggest that, at least in a sub-population of RP genes, the 5' untranslated sequences are optimized for regulatory function. Conclusion Our results argue against the direct involvement of introns in the regulation of transcription of highly expressed genes. Moreover, systematic differences in motif distributions suggest that RP transcription factors may act differently on intron-containing and intronless gene promoters. Thus, our findings contribute to the decoding of the RP promoter architecture and may fuel the discussion on the evolution of introns.

  16. Nup98 regulation of histone methylation promotes normal gene expression and may drive leukemogenesis.

    Science.gov (United States)

    Sump, Bethany; Brickner, Jason H

    2017-11-15

    Nuclear pore proteins (Nups) interact with chromosomes to regulate gene expression and chromatin structure. A new study by Franks and colleagues (pp. 2222-2234) provides new mechanistic insight into the molecular basis by which Nup98 promotes gene activation in normal hematopoietic cells and how that process is altered by translocations to cause excess expression of developmental genes in leukemia. © 2017 Sump and Brickner; Published by Cold Spring Harbor Laboratory Press.

  17. Different promoter affinities account for specificity in MYC-dependent gene regulation

    Science.gov (United States)

    Lorenzin, Francesca; Benary, Uwe; Baluapuri, Apoorva; Walz, Susanne; Jung, Lisa Anna; von Eyss, Björn; Kisker, Caroline; Wolf, Jana; Eilers, Martin; Wolf, Elmar

    2016-01-01

    Enhanced expression of the MYC transcription factor is observed in the majority of tumors. Two seemingly conflicting models have been proposed for its function: one proposes that MYC enhances expression of all genes, while the other model suggests gene-specific regulation. Here, we have explored the hypothesis that specific gene expression profiles arise since promoters differ in affinity for MYC and high-affinity promoters are fully occupied by physiological levels of MYC. We determined cellular MYC levels and used RNA- and ChIP-sequencing to correlate promoter occupancy with gene expression at different concentrations of MYC. Mathematical modeling showed that binding affinities for interactions of MYC with DNA and with core promoter-bound factors, such as WDR5, are sufficient to explain promoter occupancies observed in vivo. Importantly, promoter affinity stratifies different biological processes that are regulated by MYC, explaining why tumor-specific MYC levels induce specific gene expression programs and alter defined biological properties of cells. DOI: http://dx.doi.org/10.7554/eLife.15161.001 PMID:27460974

  18. Role of promoter element in c-mpl gene expression induced by TPO.

    Science.gov (United States)

    Sunohara, Masataka; Morikawa, Shigeru; Fuse, Akira; Sato, Iwao

    2013-01-01

    Thrombopoietin (TPO) and its receptor, c-Mpl, play the crucial role for the development of megakaryocyte and considered to regulate megakaryocytopoiesis. Previously we reported that TPO increased the c-mpl promoter activity determined by a transient expression system using a vector containing the luciferase gene as a reporter and the expression of the c-mpl gene is modulated by transcription through a protein kinase C (PKC)-dependent pathway in the megakaryoblastic cells. In this research, to elucidate the required elements in c-mpl promoter, the promoter activity of the deletion constructs and site-directed mutagenesis were measured by a transient transfection assay system. Destruction of -77GATA in c-mpl promoter decreased the activity by 22.8%. Our study elucidated that -77GATA involved in TPO-induced c-mpl gene expression in a human megakaryoblastic cell line, CMK.

  19. Tissue specific promoters improve the localization of radiation-inducible gene expression

    International Nuclear Information System (INIS)

    Hallahan, Dennis; Kataoka, Yasushi; Kuchibhotla, Jaya; Virudachalam, Subbu; Weichselbaum, Ralph

    1996-01-01

    Purpose: Site-specific activation of gene expression can be achieved by the use of a promoter that is induced by physical agents such as x-rays. The purpose of the present study was to determine whether site-specific activation of gene therapy can also be achieved within the vascular endothelium by use of radiation-inducible promoters. We studied induction of promoter-reporter gene constructs using previously identified radiation-promoters from c-jun, c-fos, Egr-1, ICAM-1, ELAM-1 after transfection into in the vascular endothelium. Methods: The following radiation-inducible genetic constructs were created: The ELAM-1 promoter fragment was cloned into pOGH to obtain the pE-sel(-587 +35)GH reporter construct. The ICAM-1 promoter fragment (-1162/+1) was cloned upstream of the CAT coding region of the pCAT-plasmid (Promega) after removal of the SV40 promoter by Bgl2/Stu1 digestion to create the pBS-CAT plasmid. The 132 to +170 bp segment of the 5' untranslated region of the c-jun promoter was cloned to the CAT reporter gene to create the -132/+170 cjun-CAT. The Egr-1 promoter fragment (-425/+75) was cloned upstream of the CAT coding region to create the pE425-CAT plasmid. Tandem repeats of the AP-1 binding site were cloned upstream of the CAT coding region (3 xTRE-CAT). Tandem repeats of the Egr binding site (EBS) were cloned upstream of the CAT coding region (EBS-CAT). Human vascular endothelial cells from both large vessel and small vessel origin (HUVEC and HMEC), as well as human tumor cell lines were transfected with plasmids -132/+170 cjun-CAT, pE425-CAT, 3 xTRE-CAT, EBS-CAT, pE-sel-GH and pBS-CAT by use of liposomes. Humor tumor cell lines included SQ20B (squamous), RIT3 (sarcoma), and HL525 (leukemia). Each plasmid was cotransfected with a plasmid containing a CMV promoter linked to the LacZ gene (1 μg). Transfected cells were treated with mock irradiation or x-rays. Cell extracts were assayed for reporter gene expression. Results: Radiation-induced gene

  20. Promoter polymorphism of transforming growth factor-beta1 gene and ulcerative colitis.

    Science.gov (United States)

    Tamizifar, B; Lankarani, K B; Naeimi, S; Rismankar Zadeh, M; Taghavi, A; Ghaderi, A

    2008-01-14

    To elucidate the possible difference in two promoter polymorphisms of the transforming growth factor-beta1 (TGF-beta1) gene (-800G > A, -509C > T) between ulcerative colitis (UC) patients and normal subjects. A total of 155 patients with established ulcerative colitis and 139 normal subjects were selected as controls. Two single nucleotide polymorphisms within the promoter region of TGF-beta1 gene (-509C > T and -800G > A) were genotyped using PCR-RFLP. There was a statistically significant difference in genotype and allele frequency distributions between UC patients and controls for the -800G > A polymorphism of the TGF-beta1 gene (P A of TGF-beta1 gene promoter between Iranian patients with UC and normal subjects.

  1. Tissue-specific and ubiquitous expression patterns from alternative promoters of human genes.

    Directory of Open Access Journals (Sweden)

    Edwin Jacox

    2010-08-01

    Full Text Available Transcriptome diversity provides the key to cellular identity. One important contribution to expression diversity is the use of alternative promoters, which creates mRNA isoforms by expanding the choice of transcription initiation sites of a gene. The proximity of the basal promoter to the transcription initiation site enables prediction of a promoter's location based on the gene annotations. We show that annotation of alternative promoters regulating expression of transcripts with distinct first exons enables a novel methodology to quantify expression levels and tissue specificity of mRNA isoforms.The use of distinct alternative first exons in 3,296 genes was examined using exon-microarray data from 11 human tissues. Comparing two transcripts from each gene we found that the activity of alternative promoters (i.e., P1 and P2 was not correlated through tissue specificity or level of expression. Furthermore neither P1 nor P2 conferred any bias for tissue-specific or ubiquitous expression. Genes associated with specific diseases produced transcripts whose limited expression patterns were consistent with the tissue affected in disease. Notably, genes that were historically designated as tissue-specific or housekeeping had alternative isoforms that showed differential expression. Furthermore, only a small number of alternative promoters showed expression exclusive to a single tissue indicating that "tissue preference" provides a better description of promoter activity than tissue specificity. When compared to gene expression data in public databases, as few as 22% of the genes had detailed information for more than one isoform, whereas the remainder collapsed the expression patterns from individual transcripts into one profile.We describe a computational pipeline that uses microarray data to assess the level of expression and breadth of tissue profiles for transcripts with distinct first exons regulated by alternative promoters. We conclude that

  2. Isolation and characterization of an ubiquitin extension protein gene (JcUEP) promoter from Jatropha curcas.

    Science.gov (United States)

    Tao, Yan-Bin; He, Liang-Liang; Niu, Long-Jian; Xu, Zeng-Fu

    2015-04-01

    The JcUEP promoter is active constitutively in the bio-fuel plant Jatropha curcas , and is an alternative to the widely used CaMV35S promoter for driving constitutive overexpression of transgenes in Jatropha. Well-characterized promoters are required for transgenic breeding of Jatropha curcas, a biofuel feedstock with great potential for production of bio-diesel and bio-jet fuel. In this study, an ubiquitin extension protein gene from Jatropha, designated JcUEP, was identified to be ubiquitously expressed. Thus, we isolated a 1.2 kb fragment of the 5' flanking region of JcUEP and evaluated its activity as a constitutive promoter in Arabidopsis and Jatropha using the β-glucuronidase (GUS) reporter gene. As expected, histochemical GUS assay showed that the JcUEP promoter was active in all Arabidopsis and Jatropha tissues tested. We also compared the activity of the JcUEP promoter with that of the cauliflower mosaic virus 35S (CaMV35S) promoter, a well-characterized constitutive promoter conferring strong transgene expression in dicot species, in various tissues of Jatropha. In a fluorometric GUS assay, the two promoters showed similar activities in stems, mature leaves and female flowers; while the CaMV35S promoter was more effective than the JcUEP promoter in other tissues, especially young leaves and inflorescences. In addition, the JcUEP promoter retained its activity under stress conditions in low temperature, high salt, dehydration and exogenous ABA treatments. These results suggest that the plant-derived JcUEP promoter could be an alternative to the CaMV35S promoter for driving constitutive overexpression of transgenes in Jatropha and other plants.

  3. A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Li Mingji

    2012-02-01

    Full Text Available Abstract Background For metabolic engineering, many rate-limiting steps may exist in the pathways of accumulating the target metabolites. Increasing copy number of the desired genes in these pathways is a general method to solve the problem, for example, the employment of the multi-copy plasmid-based expression system. However, this method may bring genetic instability, structural instability and metabolic burden to the host, while integrating of the desired gene into the chromosome may cause inadequate transcription or expression. In this study, we developed a strategy for obtaining gene overexpression by engineering promoter clusters consisted of multiple core-tac-promoters (MCPtacs in tandem. Results Through a uniquely designed in vitro assembling process, a series of promoter clusters were constructed. The transcription strength of these promoter clusters showed a stepwise enhancement with the increase of tandem repeats number until it reached the critical value of five. Application of the MCPtacs promoter clusters in polyhydroxybutyrate (PHB production proved that it was efficient. Integration of the phaCAB genes with the 5CPtacs promoter cluster resulted in an engineered E.coli that can accumulate 23.7% PHB of the cell dry weight in batch cultivation. Conclusions The transcription strength of the MCPtacs promoter cluster can be greatly improved by increasing the tandem repeats number of the core-tac-promoter. By integrating the desired gene together with the MCPtacs promoter cluster into the chromosome of E. coli, we can achieve high and stale overexpression with only a small size. This strategy has an application potential in many fields and can be extended to other bacteria.

  4. Correlation of MGMT promoter methylation status with gene and protein expression levels in glioblastoma

    Science.gov (United States)

    Uno, Miyuki; Oba-Shinjo, Sueli Mieko; Camargo, Anamaria Aranha; Moura, Ricardo Pereira; de Aguiar, Paulo Henrique; Cabrera, Hector Navarro; Begnami, Marcos; Rosemberg, Sérgio; Teixeira, Manoel Jacobsen; Marie, Suely Kazue Nagahashi

    2011-01-01

    OBJECTIVES: 1) To correlate the methylation status of the O6-methylguanine-DNA-methyltransferase (MGMT) promoter to its gene and protein expression levels in glioblastoma and 2) to determine the most reliable method for using MGMT to predict the response to adjuvant therapy in patients with glioblastoma. BACKGROUND: The MGMT gene is epigenetically silenced by promoter hypermethylation in gliomas, and this modification has emerged as a relevant predictor of therapeutic response. METHODS: Fifty-one cases of glioblastoma were analyzed for MGMT promoter methylation by methylation-specific PCR and pyrosequencing, gene expression by real time polymerase chain reaction, and protein expression by immunohistochemistry. RESULTS: MGMT promoter methylation was found in 43.1% of glioblastoma by methylation-specific PCR and 38.8% by pyrosequencing. A low level of MGMT gene expression was correlated with positive MGMT promoter methylation (p = 0.001). However, no correlation was found between promoter methylation and MGMT protein expression (p = 0.297). The mean survival time of glioblastoma patients submitted to adjuvant therapy was significantly higher among patients with MGMT promoter methylation (log rank = 0.025 by methylation-specific PCR and 0.004 by pyrosequencing), and methylation was an independent predictive factor that was associated with improved prognosis by multivariate analysis. DISCUSSION AND CONCLUSION: MGMT promoter methylation status was a more reliable predictor of susceptibility to adjuvant therapy and prognosis of glioblastoma than were MGMT protein or gene expression levels. Methylation-specific polymerase chain reaction and pyrosequencing methods were both sensitive methods for determining MGMT promoter methylation status using DNA extracted from frozen tissue. PMID:22012047

  5. Correlation of MGMT promoter methylation status with gene and protein expression levels in glioblastoma

    Directory of Open Access Journals (Sweden)

    Miyuki Uno

    2011-01-01

    Full Text Available OBJECTIVES: 1 To correlate the methylation status of the O6-methylguanine-DNA-methyltransferase (MGMT promoter to its gene and protein expression levels in glioblastoma and 2 to determine the most reliable method for using MGMT to predict the response to adjuvant therapy in patients with glioblastoma. BACKGROUND: The MGMT gene is epigenetically silenced by promoter hypermethylation in gliomas, and this modification has emerged as a relevant predictor of therapeutic response. METHODS: Fifty-one cases of glioblastoma were analyzed for MGMT promoter methylation by methylation-specific PCR and pyrosequencing, gene expression by real time polymerase chain reaction, and protein expression by immunohistochemistry. RESULTS: MGMT promoter methylation was found in 43.1% of glioblastoma by methylation-specific PCR and 38.8% by pyrosequencing. A low level of MGMT gene expression was correlated with positive MGMT promoter methylation (p = 0.001. However, no correlation was found between promoter methylation and MGMT protein expression (p = 0.297. The mean survival time of glioblastoma patients submitted to adjuvant therapy was significantly higher among patients with MGMT promoter methylation (log rank = 0.025 by methylation-specific PCR and 0.004 by pyrosequencing, and methylation was an independent predictive factor that was associated with improved prognosis by multivariate analysis. DISCUSSION AND CONCLUSION: MGMT promoter methylation status was a more reliable predictor of susceptibility to adjuvant therapy and prognosis of glioblastoma than were MGMT protein or gene expression levels. Methylation-specific polymerase chain reaction and pyrosequencing methods were both sensitive methods for determining MGMT promoter methylation status using DNA extracted from frozen tissue.

  6. Identification of learning and memory genes in canine; promoter investigation and determining the selective pressure.

    Science.gov (United States)

    Seifi Moroudi, Reihane; Masoudi, Ali Akbar; Vaez Torshizi, Rasoul; Zandi, Mohammad

    2014-12-01

    One of the important behaviors of dogs is trainability which is affected by learning and memory genes. These kinds of the genes have not yet been identified in dogs. In the current research, these genes were found in animal models by mining the biological data and scientific literatures. The proteins of these genes were obtained from the UniProt database in dogs and humans. Not all homologous proteins perform similar functions, thus comparison of these proteins was studied in terms of protein families, domains, biological processes, molecular functions, and cellular location of metabolic pathways in Interpro, KEGG, Quick Go and Psort databases. The results showed that some of these proteins have the same performance in the rat or mouse, dog, and human. It is anticipated that the protein of these genes may be effective in learning and memory in dogs. Then, the expression pattern of the recognized genes was investigated in the dog hippocampus using the existing information in the GEO profile. The results showed that BDNF, TAC1 and CCK genes are expressed in the dog hippocampus, therefore, these genes could be strong candidates associated with learning and memory in dogs. Subsequently, due to the importance of the promoter regions in gene function, this region was investigated in the above genes. Analysis of the promoter indicated that the HNF-4 site of BDNF gene and the transcription start site of CCK gene is exposed to methylation. Phylogenetic analysis of protein sequences of these genes showed high similarity in each of these three genes among the studied species. The dN/dS ratio for BDNF, TAC1 and CCK genes indicates a purifying selection during the evolution of the genes.

  7. Expression of the 90K immunostimulator gene is controlled by a promoter with unique features

    DEFF Research Database (Denmark)

    Brakebusch, C; Jallal, B; Fusco, O

    1997-01-01

    for promoter activity. This minimal promoter showed increased activity after stimulation with interferon-gamma or poly(I.C), a substance mimicking viral infection. Essential for both inductions was the integrity of an interferon regulatory factor element within this sequence, a potential binding site...... and was localized on chromosome 11, region E. RNase protection identified one major transcription start site (+1) and three minor ones (-3, +32, +34). The mouse 90K gene was found to have a TATA-less promoter of unusual structure. The 2. 3-kilobase pair 5'-flanking region exhibited strong promoter activity in NIH 3...

  8. Low-Dose Radiation Induces Genes Promoting Cell Survival

    International Nuclear Information System (INIS)

    Liu, Shu-Zheng; Chen, Dong; Mu, Ying

    1999-01-01

    Apoptosis is an important process controlling homeostasis of the body. It is influenced by stimuli constantly arising from the external and internal environment of the organism. It is well known that radiation could induce apoptosis of cells in vitro and in vivo. However, the dose-effect relationship of apoptosis extending to the low-dose range has scarcely been studied. Here, the molecular basis of the phenomenon is explored by examining the changes in expression of some of the proapoptotic and antiapoptotic genes

  9. Promoter sequence of 3-phosphoglycerate kinase gene 1 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

    2002-10-15

    The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 1 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

  10. Promoter sequence of 3-phosphoglycerate kinase gene 2 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

    2003-03-04

    The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 2 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

  11. Characterization of the sporophyte-preferential gene promoter from the red alga Porphyra yezoensis using transient gene expression.

    Science.gov (United States)

    Uji, Toshiki; Mizuta, Hiroyuki; Saga, Naotsune

    2013-04-01

    The life cycle of plants entails an alternation of generations, the diploid sporophyte and haploid gametophyte stages. There is little information about the characteristics of gene expression during each phase of marine macroalgae. Promoter analysis is a useful method for understanding transcriptional regulation; however, there is no report of promoter analyses in marine macroalgae. In this study, with the aim of elucidating the differences in the transcriptional regulatory mechanisms between the gametophyte and sporophyte stages in the marine red alga Porphyra yezoensis, we isolated the promoter from the sporophyte preferentially expressed gene PyKPA1, which encodes a sodium pump, and analyzed its promoter using a transient gene expression system with a synthetic β-glucuronidase (PyGUS) reporter. The deletion of -1432 to -768 relative to the transcription start site resulted in decreased GUS activity in sporophytes. In contrast, deletion from -767 to -527 increased GUS activity in gametophytes. Gain-of-function analyses showed that the -1432 to -760 region enhanced the GUS activity of a heterologous promoter in sporophytes, whereas the -767 to -510 region repressed it in gametophytes. Further mutation and gain-of-function analyses of the -767 to -510 region revealed that a 20-bp GC-rich sequence (-633 to -614) is responsible for the gametophyte-specific repressed expression. These results showed that the sporophyte-specific positive regulatory region and gametophyte-specific negative regulatory sequence play a crucial role in the preferential expression of PyKPA1 in P. yezoensis sporophytes.

  12. Regional differences in gene expression and promoter usage in aged human brains

    KAUST Repository

    Pardo, Luba M.

    2013-02-19

    To characterize the promoterome of caudate and putamen regions (striatum), frontal and temporal cortices, and hippocampi from aged human brains, we used high-throughput cap analysis of gene expression to profile the transcription start sites and to quantify the differences in gene expression across the 5 brain regions. We also analyzed the extent to which methylation influenced the observed expression profiles. We sequenced more than 71 million cap analysis of gene expression tags corresponding to 70,202 promoter regions and 16,888 genes. More than 7000 transcripts were differentially expressed, mainly because of differential alternative promoter usage. Unexpectedly, 7% of differentially expressed genes were neurodevelopmental transcription factors. Functional pathway analysis on the differentially expressed genes revealed an overrepresentation of several signaling pathways (e.g., fibroblast growth factor and wnt signaling) in hippocampus and striatum. We also found that although 73% of methylation signals mapped within genes, the influence of methylation on the expression profile was small. Our study underscores alternative promoter usage as an important mechanism for determining the regional differences in gene expression at old age.

  13. IFN-gamma increases the hGH gene promoter activity in rat GH3 cells.

    Science.gov (United States)

    Gong, Feng-Ying; Deng, Jie-Ying; Shi, Yi-Fan

    2003-01-01

    To study the effect(s) of interferon gamma (IFN-gamma) on the activity of human growth hormone (hGH) gene promoter in rat pituitary GH3 cells and the molecular mechanism underlying the effect(s). Cell transfection and luciferase reporter gene were used. IFN-gamma (10(2) and 10(3) U/ml) increased the activity of hGH in GH3 cells. The addition of the mitogen-activated protein kinase inhibitor PD98059 (40 micromol/l) to the cells blocked the stimulatory effect of IFN-gamma. Neither overexpression of Pit-1 nor inhibiting Pit-1 expression affected IFN-gamma induction of hGH promoter activity. To identify the DNA sequence that mediated the effect of IFN-gamma, four deletion constructs of hGH gene promoter were created. The stimulatory effect of IFN-gamma was abolished following deletion of the -250 to -132 fragment. IFN-gamma increases the activity of hGH gene promoter in rat pituitary GH3 cells. This stimulatory effect of IFN-gamma appears to require the intracellular mitogen-activated protein kinase-dependent signaling pathway. The effect of IFN-gamma requires the promoter sequence that spans the -250 to -132 fragment of the gene, but is unrelated to Pit-1 protein. Copyright 2003 S. Karger AG, Basel

  14. Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

    Energy Technology Data Exchange (ETDEWEB)

    Kurayoshi, Kenta [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan); Ozono, Eiko [Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary, University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ (United Kingdom); Iwanaga, Ritsuko; Bradford, Andrew P. [Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Anschutz Medical Campus, 12800 East 19th Avenue, Aurora, CO 80045 (United States); Komori, Hideyuki [Center for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109 (United States); Ohtani, Kiyoshi, E-mail: btm88939@kwansei.ac.jp [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan)

    2014-07-18

    Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter

  15. GAP promoter library for fine-tuning of gene expression in Pichia pastoris.

    Science.gov (United States)

    Qin, Xiulin; Qian, Jiangchao; Yao, Gaofeng; Zhuang, Yingping; Zhang, Siliang; Chu, Ju

    2011-06-01

    A library of engineered promoters of various strengths is a useful genetic tool that enables the fine-tuning and precise control of gene expression across a continuum of broad expression levels. The methylotrophic yeast Pichia pastoris is a well-established expression host with a large academic and industrial user base. To facilitate manipulation of gene expression spanning a wide dynamic range in P. pastoris, we created a functional promoter library through mutagenesis of the constitutive GAP promoter. Using yeast-enhanced green fluorescent protein (yEGFP) as the reporter, 33 mutants were chosen to form the functional promoter library. The 33 mutants spanned an activity range between ∼0.6% and 19.6-fold of the wild-type promoter activity with an almost linear fluorescence intensity distribution. After an extensive characterization of the library, the broader applicability of the results obtained with the yEGFP reporter was confirmed using two additional reporters (β-galactosidase and methionine adenosyltransferase [MAT]) at the transcription and enzyme activity levels. Furthermore, the utility of the promoter library was tested by investigating the influence of heterologous MAT gene expression levels on cell growth and S-adenosylmethionine (SAM) production. The extensive characterization of the promoter strength enabled identification of the optimal MAT activity (around 1.05 U/mg of protein) to obtain maximal volumetric SAM production. The promoter library permits precise control of gene expression and quantitative assessment that correlates gene expression level with physiologic parameters. Thus, it is a useful toolbox for both basic and applied research in P. pastoris.

  16. Functional characterization of genetic polymorphisms identified in the promoter region of the bovine PEPS gene.

    Science.gov (United States)

    Ju, Zhihua; Zheng, Xue; Huang, Jinming; Qi, Chao; Zhang, Yan; Li, Jianbin; Zhong, Jifeng; Wang, Changfa

    2012-06-01

    Peptidase S (PEPS) is a metallopeptidase that cleaves N-terminal residues from proteins and peptides. PEPS is used as a cell maintenance enzyme with critical roles in peptide turnover. The promoter region located upstream of the initiation site plays an important role in regulating gene expression. Polymorphism in the promoter region can alter gene expression and lead to biological changes. In the current study, polymorphisms in the promoter region of the PEPS gene were investigated. Polymerase chain reaction (PCR)-restriction fragment length polymorphism and DNA sequencing methods were used to screen sequence variations in the promoter region of DNA samples from 743 Chinese Holstein cattle. Two polymorphisms (g. -534 T>C and g. -2545 G>A) were identified and eight haplotypes were classified by haplotype analysis. The two genetic polymorphisms and haplotypes were associated with fat percentage and somatic cell score in Chinese Holstein cattle. The results of real-time PCR showed that cow kidneys exhibit the highest PEPS expression level. Moreover, bioinformatics analysis predicted that the single-nucleotide polymorphism g. -534 T>C is located in the core promoter region and in the transcription factor binding sites. The promoter activities of the polymorphism of -543 T>C were measured by luciferase assay in the human kidney epithelial cell line 293T. Transcriptional activity is significantly lower in cell lines transfected with the reporter construct containing 2.5 kb upstream fragments with -543 C than in those with wild-type -543 T. The results indicated that genetic variation at locus -543 influences PEPS promoter activity. The genetic variation in the promoter region of PEPS gene may regulate PEPS gene transcription and might have consequences at a regulatory level.

  17. [TEC promoter mediates P210(bcr/abl) gene expression in BaF3 cells].

    Science.gov (United States)

    Zhu, Yu-Feng; Wang, Yuan-Zhan; Meng, Fan-Yi

    2012-06-01

    P210(bcr/abl) transgene mouse is a good model to research the chronic myelogenous leukemia (CML), but the P210(bcr/abl) gene has a lethal effect on embryogenesis if driven by the constitutive promoter. So, the use of promoter which induces the special expression in hematopoietic tissue is the key to construct CML transgenic mice. This study was purposed to investigate the TEC promoter mediated P210(bcr/abl) gene expression in BaF3 cells. The CMVie promotes of IRES2-eGFP vector was replaced with the -364-+22 domain of TEC promoter cloned from mouse genome, and the P210(bcr/abl) gene was inserted into the EcoR I site of TEC-IRES2-eGFP vector. Then, the constructed vector was transfected into the BaF3 cells and 293 cells respectively. The expression levels of eGFP gene and P210(bcr/abl) gene in BaF3 and 293 cells were detected. The results showed that with fluorescent microscopy and flow cytometry, the eGFP gene was found to be expressed in the BaF3 cells, the expression rate was 7.10%, 23.35%, 64.61% at 6, 24, 72 h respectively after transfection, but the fluorescence was not seen in 293 cells. A 372 bp fragment of BCR/ABL mRNA was amplified by RT-PCR in BaF3 cells, but not in 293 cells. It is concluded that the -364-+22 domain of TEC promoter can mediate high-effective and specific expression of related genes in hematopoietic tissue, which can be used to construct P210(bcr/abl) transgene mice model.

  18. Culex tarsalis vitellogenin gene promoters investigated in silico and in vivo using transgenic Drosophila melanogaster.

    Science.gov (United States)

    Chen, Song; Rasgon, Jason L

    2014-01-01

    Genetic modification, or transgenesis, is a powerful technique to investigate the molecular interactions between vector-borne pathogens and their arthropod hosts, as well as a potential novel approach for vector-borne disease control. Transgenesis requires the use of specific regulatory regions, or promoters, to drive expression of genes of interest in desired target tissues. In mosquitoes, the vast majority of described promoters are from Anopheles and Aedes mosquitoes. Culex tarsalis is one of the most important vectors of arboviruses (including West Nile virus) in North America, yet it has not been the subject of molecular genetic study. In order to facilitate molecular genetic work in this important vector species, we isolated four fat body-specific promoter sequences located upstream of the Cx. tarsalis vitellogenin genes (Vg1a, Vg1b, Vg2a and Vg2b). Sequences were analyzed in silico to identify requisite cis-acting elements. The ability for promoter sequences to drive expression of green fluorescent protein (GFP) in vivo was investigated using transgenic Drosophila melanogaster. All four promoters were able to drive GFP expression but there was dramatic variation between promoters and between individual Drosophila lines, indicating significant position effects. The highest expression was observed in line Vg2bL3, which was >300-fold higher than the lowest line Vg1aL2. These new promoters will be useful for driving expression of genes of interest in transgenic Cx. tarsalis and perhaps other insects.

  19. Culex tarsalis vitellogenin gene promoters investigated in silico and in vivo using transgenic Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Song Chen

    Full Text Available Genetic modification, or transgenesis, is a powerful technique to investigate the molecular interactions between vector-borne pathogens and their arthropod hosts, as well as a potential novel approach for vector-borne disease control. Transgenesis requires the use of specific regulatory regions, or promoters, to drive expression of genes of interest in desired target tissues. In mosquitoes, the vast majority of described promoters are from Anopheles and Aedes mosquitoes.Culex tarsalis is one of the most important vectors of arboviruses (including West Nile virus in North America, yet it has not been the subject of molecular genetic study. In order to facilitate molecular genetic work in this important vector species, we isolated four fat body-specific promoter sequences located upstream of the Cx. tarsalis vitellogenin genes (Vg1a, Vg1b, Vg2a and Vg2b. Sequences were analyzed in silico to identify requisite cis-acting elements. The ability for promoter sequences to drive expression of green fluorescent protein (GFP in vivo was investigated using transgenic Drosophila melanogaster. All four promoters were able to drive GFP expression but there was dramatic variation between promoters and between individual Drosophila lines, indicating significant position effects. The highest expression was observed in line Vg2bL3, which was >300-fold higher than the lowest line Vg1aL2.These new promoters will be useful for driving expression of genes of interest in transgenic Cx. tarsalis and perhaps other insects.

  20. Characterization of promoter sequence of toll-like receptor genes in Vechur cattle

    Directory of Open Access Journals (Sweden)

    R. Lakshmi

    2016-06-01

    Full Text Available Aim: To analyze the promoter sequence of toll-like receptor (TLR genes in Vechur cattle, an indigenous breed of Kerala with the sequence of Bos taurus and access the differences that could be attributed to innate immune responses against bovine mastitis. Materials and Methods: Blood samples were collected from Jugular vein of Vechur cattle, maintained at Vechur cattle conservation center of Kerala Veterinary and Animal Sciences University, using an acid-citrate-dextrose anticoagulant. The genomic DNA was extracted, and polymerase chain reaction was carried out to amplify the promoter region of TLRs. The amplified product of TLR2, 4, and 9 promoter regions was sequenced by Sanger enzymatic DNA sequencing technique. Results: The sequence of promoter region of TLR2 of Vechur cattle with the B. taurus sequence present in GenBank showed 98% similarity and revealed variants for four sequence motifs. The sequence of the promoter region of TLR4 of Vechur cattle revealed 99% similarity with that of B. taurus sequence but not reveals significant variant in motifregions. However, two heterozygous loci were observed from the chromatogram. Promoter sequence of TLR9 gene also showed 99% similarity to B. taurus sequence and revealed variants for four sequence motifs. Conclusion: The results of this study indicate that significant variation in the promoter of TLR2 and 9 genes in Vechur cattle breed and may potentially link the influence the innate immunity response against mastitis diseases.

  1. Enhancer activity of Helitron in sericin-1 gene promoter from Bombyx mori.

    Science.gov (United States)

    Huang, Ke; Li, Chun-Feng; Wu, Jie; Wei, Jun-Hong; Zou, Yong; Han, Min-Jin; Zhou, Ze-Yang

    2016-06-01

    Sericin is a kind of water-soluble protein expressed specifically in the middle silk gland of Bombyx mori. When the sericin-1 gene promoter was cloned and a transgenic vector was constructed to express a foreign protein, a specific Helitron, Bmhel-8, was identified in the sericin-1 gene promoter sequence in some genotypes of Bombyx mori and Bombyx mandarina. Given that the Bmhel-8 Helitron transposon was present only in some genotypes, it could be the source of allelic variation in the sericin-1 promoter. The length of the sericin-1 promoter sequence is approximately 1063 or 643 bp. The larger size of the sequence or allele is ascribed to the presence of Bmhel-8. Silkworm genotypes can be homozygous for either the shorter or larger promoter sequence or heterozygous, containing both alleles. Bmhel-8 in the sericin-1 promoter exhibits enhancer activity, as demonstrated by a dual-luciferase reporter system in BmE cell lines. Furthermore, Bmhel-8 displays enhancer activity in a sericin-1 promoter-driven gene expression system but does not regulate the tissue-specific expression of sericin-1. © 2016 Institute of Zoology, Chinese Academy of Sciences.

  2. Octamer and heat shock elements regulate transcription from the AcMNPV polyhedrin gene promoter.

    Science.gov (United States)

    Kumar, M Senthil; Ramachandran, Aruna; Hasnain, Seyed E; Bashyam, Murali Dharan

    2009-01-01

    The baculovirus expression vector system exploits the polyhedrin (polh) promoter for high expression of foreign proteins in insect cells. The mechanism of basal and hyperactivated transcription from this promoter, however, remains poorly understood. We have analyzed the 4-kb upstream region of the polh promoter; deletion of two separate parts of the 4-kb upstream region, harboring the Oct binding site and the heat shock element, respectively, resulted in significant reduction of reporter gene expression regulated by the polh promoter. Insect cell host factors could bind to these elements in vitro. Moreover, these elements could activate polh transcription during viral infection when present upstream of a minimal polh promoter in transient expression reporter assays. Our results suggest the possible existence of transcription factors belonging to the POU and heat shock transcription factor family in Spodoptera frugiperda cells and support the hypothesis that host proteins may play a major role in activating transcription from the polh promoter.

  3. Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.

    Science.gov (United States)

    Montiel, Daniel; Kang, Hahk-Soo; Chang, Fang-Yuan; Charlop-Powers, Zachary; Brady, Sean F

    2015-07-21

    Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters.

  4. Identifying Growth Conditions for Nicotiana benthimiana Resulting in Predictable Gene Expression of Promoter-Gus Fusion

    Science.gov (United States)

    Sandoval, V.; Barton, K.; Longhurst, A.

    2012-12-01

    Revoluta (Rev) is a transcription factor that establishes leaf polarity inArabidopsis thaliana. Through previous work in Dr. Barton's Lab, it is known that Revoluta binds to the ZPR3 promoter, thus activating the ZPR3 gene product inArabidopsis thaliana. Using this knowledge, two separate DNA constructs were made, one carrying revgene and in the other, the ZPR3 promoter fussed with the GUS gene. When inoculated in Nicotiana benthimiana (tobacco), the pMDC32 plasmid produces the Rev protein. Rev binds to the ZPR3 promoter thereby activating the transcription of the GUS gene, which can only be expressed in the presence of Rev. When GUS protein comes in contact with X-Gluc it produce the blue stain seen (See Figure 1). In the past, variability has been seen of GUS expression on tobacco therefore we hypothesized that changing the growing conditions and leaf age might improve how well it's expressed.

  5. Identification of MGMT promoter methylation sites correlating with gene expression and IDH1 mutation in gliomas.

    Science.gov (United States)

    Zhang, Jie; Yang, Jian-Hui; Quan, Jia; Kang, Xing; Wang, Hui-Juan; Dai, Peng-Gao

    2016-10-01

    O 6 -methylguanine-DNA methyltransferase (MGMT) gene promoter methylation was reported to be an independent prognostic and predictive factor in glioma patients who received temozolomide treatment. However, the predictive value of MGMT methylation was recently questioned by several large clinical studies. The purpose of this study is to identify MGMT gene promoter CpG sites or region whose methylation were closely correlated with its gene expression to elucidate this contradictory clinical observations. The methylation status for all CpG dinucleotides in MGMT promoter and first exon region were determined in 42 Chinese glioma patients, which were then correlated with MGMT gene expression, IDH1 mutation, and tumor grade. In whole 87 CpG dinucleotides analyzed, three distinct CpG regions covering 28 CpG dinucleotides were significantly correlated with MGMT gene expression; 10 CpG dinucleotides were significantly correlated with glioma classification (p MGMT gene hypermethylation significantly co-existed, but not for MGMT gene expression. The validation cohort of gliomas treated with standard of care and comparison of the CpGs we identified with the current CpGs used in clinical setting will be very important for gliomas individual medicine in the future.

  6. GAL promoter-driven heterologous gene expression in Saccharomyces cerevisiae Δ strain at anaerobic alcoholic fermentation.

    Science.gov (United States)

    Ahn, Jungoh; Park, Kyung-Min; Lee, Hongweon; Son, Yeo-Jin; Choi, Eui-Sung

    2013-02-01

    The removal of Gal80 protein by gene disruption turned into efficient GAL promoter-driven heterologous gene expression under anaerobic alcoholic fermentation of Saccharomyces cerevisiae. Using lipase B from Candida antarctica as a reporter, the relative strength of GAL10 promoter (P(GAL10) ) in Δgal80 mutant that does not require galactose as an inducer was compared to those of ADH1, PDC1, and PGK promoters, which have been known to work well anaerobically in actively fermenting yeast cells under high glucose concentration. P(GAL10) in the Δgal80 mutant showed 0.8-fold (ADH1), fourfold (PDC1), and 50-fold (PGK) in promoter strength. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  7. The promoter of the glucoamylase-encoding gene of Aspergillus niger functions in Ustilago maydis

    Energy Technology Data Exchange (ETDEWEB)

    Smith, T.L. (Dept. of Agriculture, Madison, WI (United States) Univ. of Wisconsin, Madison (United States)); Gaskell, J.; Cullen, D. (Dept. of Agriculture, Madison, WI (United States)); Berka, R.M.; Yang, M.; Henner, D.J. (Genentech Inc., San Francisco, CA (United States))

    1990-01-01

    Promoter sequences from the Aspergillus niger glucoamylase-encoding gene (glaA) were linked to the bacterial hygromycin (Hy) phosphotransferase-encoding gene (hph) and this chimeric marker was used to select Hy-resistant (Hy[sup R]) Ustilago maydis transformants. This is an example of an Ascomycete promoter functioning in a Basidiomycete. Hy[sup R] transformants varied with respect to copy number of integrated vector, mitotic stability, and tolerance to Hy. Only 216 bp of glaA promoter sequence is required for expression in U. maydis but this promoter is not induced by starch as it is in Aspergillus spp. The transcription start points are the same in U. maydis and A. niger.

  8. Global mapping of DNA methylation in mouse promoters reveals epigenetic reprogramming of pluripotency genes.

    Science.gov (United States)

    Farthing, Cassandra R; Ficz, Gabriella; Ng, Ray Kit; Chan, Chun-Fung; Andrews, Simon; Dean, Wendy; Hemberger, Myriam; Reik, Wolf

    2008-06-27

    DNA methylation patterns are reprogrammed in primordial germ cells and in preimplantation embryos by demethylation and subsequent de novo methylation. It has been suggested that epigenetic reprogramming may be necessary for the embryonic genome to return to a pluripotent state. We have carried out a genome-wide promoter analysis of DNA methylation in mouse embryonic stem (ES) cells, embryonic germ (EG) cells, sperm, trophoblast stem (TS) cells, and primary embryonic fibroblasts (pMEFs). Global clustering analysis shows that methylation patterns of ES cells, EG cells, and sperm are surprisingly similar, suggesting that while the sperm is a highly specialized cell type, its promoter epigenome is already largely reprogrammed and resembles a pluripotent state. Comparisons between pluripotent tissues and pMEFs reveal that a number of pluripotency related genes, including Nanog, Lefty1 and Tdgf1, as well as the nucleosome remodeller Smarcd1, are hypomethylated in stem cells and hypermethylated in differentiated cells. Differences in promoter methylation are associated with significant differences in transcription levels in more than 60% of genes analysed. Our comparative approach to promoter methylation thus identifies gene candidates for the regulation of pluripotency and epigenetic reprogramming. While the sperm genome is, overall, similarly methylated to that of ES and EG cells, there are some key exceptions, including Nanog and Lefty1, that are highly methylated in sperm. Nanog promoter methylation is erased by active and passive demethylation after fertilisation before expression commences in the morula. In ES cells the normally active Nanog promoter is silenced when targeted by de novo methylation. Our study suggests that reprogramming of promoter methylation is one of the key determinants of the epigenetic regulation of pluripotency genes. Epigenetic reprogramming in the germline prior to fertilisation and the reprogramming of key pluripotency genes in the early

  9. Global mapping of DNA methylation in mouse promoters reveals epigenetic reprogramming of pluripotency genes.

    Directory of Open Access Journals (Sweden)

    Cassandra R Farthing

    2008-06-01

    Full Text Available DNA methylation patterns are reprogrammed in primordial germ cells and in preimplantation embryos by demethylation and subsequent de novo methylation. It has been suggested that epigenetic reprogramming may be necessary for the embryonic genome to return to a pluripotent state. We have carried out a genome-wide promoter analysis of DNA methylation in mouse embryonic stem (ES cells, embryonic germ (EG cells, sperm, trophoblast stem (TS cells, and primary embryonic fibroblasts (pMEFs. Global clustering analysis shows that methylation patterns of ES cells, EG cells, and sperm are surprisingly similar, suggesting that while the sperm is a highly specialized cell type, its promoter epigenome is already largely reprogrammed and resembles a pluripotent state. Comparisons between pluripotent tissues and pMEFs reveal that a number of pluripotency related genes, including Nanog, Lefty1 and Tdgf1, as well as the nucleosome remodeller Smarcd1, are hypomethylated in stem cells and hypermethylated in differentiated cells. Differences in promoter methylation are associated with significant differences in transcription levels in more than 60% of genes analysed. Our comparative approach to promoter methylation thus identifies gene candidates for the regulation of pluripotency and epigenetic reprogramming. While the sperm genome is, overall, similarly methylated to that of ES and EG cells, there are some key exceptions, including Nanog and Lefty1, that are highly methylated in sperm. Nanog promoter methylation is erased by active and passive demethylation after fertilisation before expression commences in the morula. In ES cells the normally active Nanog promoter is silenced when targeted by de novo methylation. Our study suggests that reprogramming of promoter methylation is one of the key determinants of the epigenetic regulation of pluripotency genes. Epigenetic reprogramming in the germline prior to fertilisation and the reprogramming of key pluripotency

  10. Relationship between promoter methylation & tissue expression of MGMT gene in ovarian cancer

    Directory of Open Access Journals (Sweden)

    V Shilpa

    2014-01-01

    Full Text Available Background & objectives: Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumour suppressor genes. O 6 -methyguanine-DNA methyltransferase (MGMT is a DNA repair gene that removes mutagenic and cytotoxic adducts from the O 6 -position of guanine induced by alkylating agents. MGMT promoter hypermethylation and reduced expression has been found in some primary human carcinomas. We studied DNA methylation of CpG islands of the MGMT gene and its relation with MGMT protein expression in human epithelial ovarian carcinoma. Methods: A total of 88 epithelial ovarian cancer (EOC tissue samples, 14 low malignant potential (LMP tumours and 20 benign ovarian tissue samples were analysed for MGMT promoter methylation by nested methylation-specific polymerase chain reaction (MSP after bisulphite modification of DNA. A subset of 64 EOC samples, 10 LMP and benign tumours and five normal ovarian tissue samples were analysed for protein expression by immunohistochemistry. Results: The methylation frequencies of the MGMT gene promoter were found to be 29.5, 28.6 and 20 per cent for EOC samples, LMP tumours and benign cases, respectively. Positive protein expression was observed in 93.8 per cent of EOC and 100 per cent in LMP, benign tumours and normal ovarian tissue samples. Promoter hypermethylation with loss of protein expression was seen only in one case of EOC. Interpretation & conclusions: Our results suggest that MGMT promoter hypermethylation does not always reflect gene expression.

  11. Relationship between promoter methylation & tissue expression of MGMT gene in ovarian cancer.

    Science.gov (United States)

    Shilpa, V; Bhagat, Rahul; Premalata, C S; Pallavi, V R; Ramesh, G; Krishnamoorthy, Lakshmi

    2014-11-01

    Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumour suppressor genes. O 6-methyguanine-DNA methyltransferase (MGMT) is a DNA repair gene that removes mutagenic and cytotoxic adducts from the O 6 -position of guanine induced by alkylating agents. MGMT promoter hypermethylation and reduced expression has been found in some primary human carcinomas. We studied DNA methylation of CpG islands of the MGMT gene and its relation with MGMT protein expression in human epithelial ovarian carcinoma. A total of 88 epithelial ovarian cancer (EOC) tissue samples, 14 low malignant potential (LMP) tumours and 20 benign ovarian tissue samples were analysed for MGMT promoter methylation by nested methylation-specific polymerase chain reaction (MSP) after bisulphite modification of DNA. A subset of 64 EOC samples, 10 LMP and benign tumours and five normal ovarian tissue samples were analysed for protein expression by immunohistochemistry. The methylation frequencies of the MGMT gene promoter were found to be 29.5, 28.6 and 20 per cent for EOC samples, LMP tumours and benign cases, respectively. Positive protein expression was observed in 93.8 per cent of EOC and 100 per cent in LMP, benign tumours and normal ovarian tissue samples. Promoter hypermethylation with loss of protein expression was seen only in one case of EOC. Our results suggest that MGMT promoter hypermethylation does not always reflect gene expression.

  12. Inhibition of SIRT1 reactivates silenced cancer genes without loss of promoter DNA hypermethylation.

    Directory of Open Access Journals (Sweden)

    Kevin Pruitt

    2006-03-01

    Full Text Available The class III histone deactylase (HDAC, SIRT1, has cancer relevance because it regulates lifespan in multiple organisms, down-regulates p53 function through deacetylation, and is linked to polycomb gene silencing in Drosophila. However, it has not been reported to mediate heterochromatin formation or heritable silencing for endogenous mammalian genes. Herein, we show that SIRT1 localizes to promoters of several aberrantly silenced tumor suppressor genes (TSGs in which 5' CpG islands are densely hypermethylated, but not to these same promoters in cell lines in which the promoters are not hypermethylated and the genes are expressed. Heretofore, only type I and II HDACs, through deactylation of lysines 9 and 14 of histone H3 (H3-K9 and H3-K14, respectively, had been tied to the above TSG silencing. However, inhibition of these enzymes alone fails to re-activate the genes unless DNA methylation is first inhibited. In contrast, inhibition of SIRT1 by pharmacologic, dominant negative, and siRNA (small interfering RNA-mediated inhibition in breast and colon cancer cells causes increased H4-K16 and H3-K9 acetylation at endogenous promoters and gene re-expression despite full retention of promoter DNA hypermethylation. Furthermore, SIRT1 inhibition affects key phenotypic aspects of cancer cells. We thus have identified a new component of epigenetic TSG silencing that may potentially link some epigenetic changes associated with aging with those found in cancer, and provide new directions for therapeutically targeting these important genes for re-expression.

  13. Association of polymorphisms of interleukin-18 gene promoter region with polycystic ovary syndrome in chinese population

    Directory of Open Access Journals (Sweden)

    Li Mei-zhi

    2010-10-01

    Full Text Available Abstract Background Recent research shows that polycystic ovary syndrome (PCOS may have an association with low-grade chronic inflammation, and that PCOS may induce an increase in serum interleukin-18 (IL-18 levels. Methods To investigate the polymorphisms of the IL-18 gene promoters with PCOS, two single nucleotide polymorphisms (SNPs in the promoter of the IL-18 gene (at positions -607C/A and -137G/C in 118 Chinese women with PCOS and 79 controls were evaluated using polymerase chain reaction (PCR. Results No significant differences were found in the genotype distribution, allele frequency and haplotype frequency between the PCOS and control groups. Further analysis demonstrated a relationship between IL-18 gene promoter polymorphisms and PCOS insulin resistance (IR. Regarding the -137 allele frequency, G and C allele frequencies were 93.5% and 6.5%, respectively, in the PCOS with IR patients; G and C allele frequencies were 85.4% and 14.6%, respectively, in PCOS patients without IR (chi2 = 3.601, P = 0.048. Conclusions The presence of a polymorphism in the IL-18 gene was found to have no correlation with the occurrence of PCOS. Carriage of the C allele at position -137 in the promoter of the IL-18 gene may play a protective role from the development of PCOS IR.

  14. Neuroglobin and cytoglobin: two new members of globin family

    Directory of Open Access Journals (Sweden)

    Priscilla Tosqui

    2011-01-01

    Full Text Available The globin family has long been defined by myoglobin and hemoglobin, proteins with the functions of oxygen storage and transportation, respectively. Recently, two new members of this family were discovered: neuroglobin present in neurons and retinal cells and cytoglobin found in various types of tissue. The increased expression of these proteins in hypoxic conditions first suggested a role in oxygen supply. However structural and functional differences, such as the hexacoordinated heme, a high autoxidation rate and different concentrations between different cellular types, have dismissed this hypothesis. The protective role of these globins has already been established. In vitro and in vivo studies have demonstrated increased survival of neurons under stress in the presence of neuroglobin and increased resistance to neurodegenerative diseases. However the mechanism remains unknown. Functions, including detoxification of nitric oxide, free radical scavenging and as an antioxidant and signaling of apoptosis, have also been suggested for neuroglobin and an antifibrotic function for cytoglobin.

  15. Oxygen binding properties of non-mammalian nerve globins

    DEFF Research Database (Denmark)

    Hundahl, Christian; Fago, Angela; Dewilde, Sylvia

    2006-01-01

    Oxygen-binding globins occur in the nervous systems of both invertebrates and vertebrates. While the function of invertebrate nerve haemoglobins as oxygen stores that extend neural excitability under hypoxia has been convincingly demonstrated, the physiological role of vertebrate neuroglobins...... is less well understood. Here we provide a detailed analysis of the oxygenation characteristics of nerve haemoglobins from an annelid (Aphrodite aculeata), a nemertean (Cerebratulus lacteus) and a bivalve (Spisula solidissima) and of neuroglobin from zebrafish (Danio rerio). The functional differences...... temperatures investigated and exhibited large enthalpies of oxygenation, the hexacoordinate globins showed reverse Bohr effects (at least at low temperature) and approximately twofold lower oxygenation enthalpies. Only S. solidissima nerve haemoglobin showed apparent cooperativity in oxygen binding, suggesting...

  16. A cell cycle-regulated histone H3 gene of alfalfa with an atypical promoter structure.

    Science.gov (United States)

    Robertson, A J; Kapros, T; Waterborg, J H

    1997-01-01

    The control of cell cycle expression of histone genes in plants is incompletely understood. A new histone H3 gene was cloned from alfalfa (Medicago sativa) that codes for the replication-dependent histone H3.1 variant protein. Despite lacking all promoter sequence motifs that have been associated with cell cycle-dependent histone gene expression in plants, northern analysis of synchronized cells clearly linked gene expression to DNA replication. TTAATNA was recognized as a new sequence element in the 3' untranslated regions of this and all other cell cycle-dependent histone H3 genes of dicotyledonous plants. It is not found in the replication-independent histone H3 genes.

  17. Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells

    Science.gov (United States)

    Ede, Christopher; Chen, Ximin; Lin, Meng-Yin; Chen, Yvonne Y.

    2016-01-01

    Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning two orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality. PMID:26883397

  18. Expression of the 90K immunostimulator gene is controlled by a promoter with unique features

    DEFF Research Database (Denmark)

    Brakebusch, C; Jallal, B; Fusco, O

    1997-01-01

    and was localized on chromosome 11, region E. RNase protection identified one major transcription start site (+1) and three minor ones (-3, +32, +34). The mouse 90K gene was found to have a TATA-less promoter of unusual structure. The 2. 3-kilobase pair 5'-flanking region exhibited strong promoter activity in NIH 3......T3 cells; however, it contained neither a TATA-box nor a SP1 site and was not GC-rich. No known initiator motif was found around the transcription start site. 5'- and 3'-deletions defined a minimal promoter of 51 base pairs (-66 --> -16), not including the start site, essential and sufficient...

  19. Novel strong tissue specific promoter for gene expression in human germ cells

    Directory of Open Access Journals (Sweden)

    Kuzmin Denis

    2010-08-01

    Full Text Available Abstract Background Tissue specific promoters may be utilized for a variety of applications, including programmed gene expression in cell types, tissues and organs of interest, for developing different cell culture models or for use in gene therapy. We report a novel, tissue-specific promoter that was identified and engineered from the native upstream regulatory region of the human gene NDUFV1 containing an endogenous retroviral sequence. Results Among seven established human cell lines and five primary cultures, this modified NDUFV1 upstream sequence (mNUS was active only in human undifferentiated germ-derived cells (lines Tera-1 and EP2102, where it demonstrated high promoter activity (~twice greater than that of the SV40 early promoter, and comparable to the routinely used cytomegaloviral promoter. To investigate the potential applicability of the mNUS promoter for biotechnological needs, a construct carrying a recombinant cytosine deaminase (RCD suicide gene under the control of mNUS was tested in cell lines of different tissue origin. High cytotoxic effect of RCD with a cell-death rate ~60% was observed only in germ-derived cells (Tera-1, whereas no effect was seen in a somatic, kidney-derived control cell line (HEK293. In further experiments, we tested mNUS-driven expression of a hyperactive Sleeping Beauty transposase (SB100X. The mNUS-SB100X construct mediated stable transgene insertions exclusively in germ-derived cells, thereby providing further evidence of tissue-specificity of the mNUS promoter. Conclusions We conclude that mNUS may be used as an efficient promoter for tissue-specific gene expression in human germ-derived cells in many applications. Our data also suggest that the 91 bp-long sequence located exactly upstream NDUFV1 transcriptional start site plays a crucial role in the activity of this gene promoter in vitro in the majority of tested cell types (10/12, and an important role - in the rest two cell lines.

  20. Recruitment of Histone Deacetylase 3 to the Interferon-A Gene Promoters Attenuates Interferon Expression

    Science.gov (United States)

    Génin, Pierre; Lin, Rongtuan; Hiscott, John; Civas, Ahmet

    2012-01-01

    Background Induction of Type I Interferon (IFN) genes constitutes an essential step leading to innate immune responses during virus infection. Sendai virus (SeV) infection of B lymphoid Namalwa cells transiently induces the transcriptional expression of multiple IFN-A genes. Although transcriptional activation of IFN-A genes has been extensively studied, the mechanism responsible for the attenuation of their expression remains to be determined. Principal Findings In this study, we demonstrate that virus infection of Namalwa cells induces transient recruitment of HDAC3 (histone deacetylase 3) to IFN-A promoters. Analysis of chromatin-protein association by Chip-QPCR demonstrated that recruitment of interferon regulatory factor (IRF)3 and IRF7, as well as TBP correlated with enhanced histone H3K9 and H3K14 acetylation, whereas recruitment of HDAC3 correlated with inhibition of histone H3K9/K14 acetylation, removal of IRF7 and TATA-binding protein (TBP) from IFN-A promoters and inhibition of virus-induced IFN-A gene transcription. Additionally, HDAC3 overexpression reduced, and HDAC3 depletion by siRNA enhanced IFN-A gene expression. Furthermore, activation of IRF7 enhanced histone H3K9/K14 acetylation and IFN-A gene expression, whereas activation of both IRF7 and IRF3 led to recruitment of HDAC3 to the IFN-A gene promoters, resulting in impaired histone H3K9 acetylation and attenuation of IFN-A gene transcription. Conclusion Altogether these data indicate that reversal of histone H3K9/K14 acetylation by HDAC3 is required for attenuation of IFN-A gene transcription during viral infection. PMID:22685561

  1. Hypermethylation of MGMT and DAPK gene promoters is associated with tumorigenesis and metastasis in oral squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Yong-Kie Wong

    2011-09-01

    Conclusions: Our study supports the hypothesis that hypermethylation of p16 gene promoters may indicate a high risk of oral cancer, and hypermethylation of the MGMT and DAPK genes may be a major indicator of early OSCC metastasis.

  2. Identification of a Gene Sharing a Promoter and Peroxisome Proliferator-Response Elements With Acyl-CoA Oxidase Gene

    Directory of Open Access Journals (Sweden)

    Mst. Hasina Akter

    2006-01-01

    Full Text Available Many mammalian genes are clustered on the genomes, and hence the genes in the same cluster can be regulated through a common regulatory element. We indeed showed previously that the perilipin/PEX11α gene pair is transactivated tissue-selectively by PPARγ and PPARα, respectively, through a common binding site. In the present study, we identified a gene, named GSPA, neighboring a canonical PPAR target, acyl-CoA oxidase (AOX gene. GSPA expression was induced by a peroxisome proliferator, Wy14,643, in the liver of wild-type mice, but not PPARα-null mice. GSPA and AOX share the promoter and two peroxisome proliferator-response elements. GSPA mRNA was also found in the heart and skeletal muscle, as well as 3T3-L1 cells. GSPA encodes a protein of 161 amino acids that is enriched in 3T3-L1 cells. Even other gene pairs might be regulated through common sequence elements, and conversely it would be interesting how each gene is aptly regulated in clusters.

  3. Stepwise colonization of the Andes by ruddy ducks and the evolution of novel β-globin variants.

    Science.gov (United States)

    Muñoz-Fuentes, V; Cortázar-Chinarro, M; Lozano-Jaramillo, M; McCracken, K G

    2013-03-01

    Andean uplift played a key role in Neotropical bird diversification, yet past dispersal and genetic adaptation to high-altitude environments remain little understood. Here we use multilocus population genetics to study population history and historical demographic processes in the ruddy duck (Oxyura jamaicensis), a stiff-tailed diving duck comprising three subspecies distributed from Canada to Tierra del Fuego and inhabiting wetlands from sea level to 4500 m in the Andes. We sequenced the mitochondrial DNA, four autosomal introns and three haemoglobin genes (α(A), α(D), β(A)) and used isolation-with-migration (IM) models to study gene flow between North America and South America, and between the tropical and southern Andes. Our analyses indicated that ruddy ducks dispersed first from North America to the tropical Andes, then from the tropical Andes to the southern Andes. While no nonsynonymous substitutions were found in either α globin gene, three amino acid substitutions were observed in the β(A) globin. Based on phylogenetic reconstruction and power analysis, the first β(A) substitution, found in all Andean individuals, was acquired when ruddy ducks dispersed from low altitude in North America to high altitude in the tropical Andes, whereas the two additional substitutions occurred more recently, when ruddy ducks dispersed from high altitude in the tropical Andes to low altitude in the southern Andes. This stepwise colonization pattern accompanied by polarized β(A) globin amino acid replacements suggest that ruddy ducks first acclimatized or adapted to the Andean highlands and then again to the lowlands. In addition, ruddy ducks colonized the Andean highlands via a less common route as compared to other waterbird species that colonized the Andes northwards from the southern cone of South America. © 2013 Blackwell Publishing Ltd.

  4. Gene Promoter Hypermethylation in Tumors and Plasma of Breast Cancer Patients

    Science.gov (United States)

    Shim, Young Ran; Choi, Joon Hyuk; Kim, Mi Jin; Gabrielson, Edward; Lee, Soo Jung; Hwang, Tae Yoon; Shin, Sei One

    2005-01-01

    Purpose To measure the hypermethylation of four genes in primary tumors and paired plasma samples to determine the feasibility of gene promoter hypermethylation markers for detecting breast cancer in the plasma. Materials and Methods DNA was extracted from the tumor tissues and peripheral blood plasma of 34 patients with invasive breast cancer, and the samples examined for aberrant hypermethylation in cyclin D2, retinoic acid receptor β (RARβ), twist and high in normal-1 (HIN-1) genes using methylation-specific PCR (MSP), and the results correlated with the clinicopathological parameters. Results Promoter hypermethylation was detected at high frequency in the primary tumors for cyclin D2 (53%), RARβ (56%), twist (41%) and HIN-1 (77%). Thirty-three of the 34 (97%) primary tumors displayed promoter hypermethylation in at least one of the genes examined. The corresponding plasma samples showed hyperme thylation of the same genes, although at lower frequencies (6% for cyclin D2, 16% for RARβ, 36% for twist, and 54% for HIN-1). Overall, 22 of the 33 (67%) primary tumors with hypermethylation of at least one of the four genes also had abnormally hypermethylated DNA in their matched plasma samples. No significant relationship was recognized between any of the clinical or pathological parameters (tumor size, axillary lymph node metastasis, stage, or Ki-67 labeling index) with the frequency of hypermethylated DNA in the primary tumor or plasma. Conclusion The detection of aberrant promoter hypermethylation of cancer-related genes in the plasma may be a useful tool for the detection of breast cancer. PMID:19956520

  5. Monoamine Oxidase a Promoter Gene Associated with Problem Behavior in Adults with Intellectual/Developmental Disabilities

    Science.gov (United States)

    May, Michael E.; Srour, Ali; Hedges, Lora K.; Lightfoot, David A.; Phillips, John A., III; Blakely, Randy D.; Kennedy, Craig H.

    2009-01-01

    A functional polymorphism in the promoter of the gene encoding monoamine oxidase A has been associated with problem behavior in various populations. We examined the association of MAOA alleles in adult males with intellectual/developmental disabilities with and without established histories of problem behavior. These data were compared with a…

  6. Understanding the Pathogenicity of Noncoding Mismatch Repair Gene Promoter Variants in Lynch Syndrome.

    Science.gov (United States)

    Liu, Qing; Thompson, Bryony A; Ward, Robyn L; Hesson, Luke B; Sloane, Mathew A

    2016-05-01

    Lynch syndrome is the most common familial cancer condition that mainly predisposes to tumors of the colon and endometrium. Cancer susceptibility is caused by the autosomal dominant inheritance of a loss-of-function mutation or epimutation in one of the DNA mismatch repair (MMR) genes. Cancer risk assessment is often possible with nonsynonymous coding region mutations, but in many cases patients present with DNA sequence changes within noncoding regions, including the promoters, of MMR genes. The pathogenic role of promoter variants, and hence clinical significance, is unclear and this hinders the clinical management of carriers. In this review, we provide an overview of the classification of MMR gene variants, outline the laboratory assays and online resources that can be used to assess the causality of promoter variants in Lynch syndrome, and highlight some of the practical challenges of demonstrating the pathogenicity of these variants. In conclusion, we propose a guide that could be integrated into the current InSiGHT classification scheme to help determine if a MMR gene promoter variant is pathogenic. © 2016 WILEY PERIODICALS, INC.

  7. Transcriptional regulation of teleost aicda genes. Pt 1 suppressors of promiscuous promoters

    Science.gov (United States)

    In order to better understand antibody affinity maturation in fishes we sought to identify gene regulatory elements that could drive expression of activated B-cell specific fluorescent reporter transgenes in zebrafish. Specifically the promoter and several non-coding regions of the channel catfish (...

  8. Characterization of endogenous promoters for control of recombinant gene expression in Acidithiobacillus ferrooxidans.

    Science.gov (United States)

    Kernan, Timothy; West, Alan C; Banta, Scott

    2017-11-01

    Acidithiobacillus ferrooxidans is an important iron- and sulfur-oxidizing acidophilic chemolithoautotroph that is used extensively in metal extraction and refining, and more recently in the bioproduction of chemicals. However, a lack of genetic tools has limited the further development of this organism for industrial bioprocesses. Using prior microarray studies that identified genes, which may express differentially in response to the availability of iron and sulfur, the cycA1 and tusA promoter sequences have been characterized for their ability to drive green fluorescent protein expression. The promoters exhibited opposite control behavior, where the cycA1 sequence was repressed and the tusA promoter was induced by the presence of sulfur in the growth medium. Sulfur was found to be the dominant signal. The sulfur IC 50 for cycA1 was 0.56 mM (18 mg/L), whereas the sulfur EC 50 of tusA was 2.5 mM (80 mg/L). Together these sequences provide two new tools to selectively induce or repress gene expression in A. ferrooxidans. Acidithiobacillus ferrooxidans is an important industrial organism; however, genetic tools for control of gene expression do not exist. Here, we report the identification of promoter sequences that allow for the development of control of gene expression for engineering this organism. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  9. Sumoylation of Rap1 mediates the recruitment of TFIID to promote transcription of ribosomal protein genes.

    Science.gov (United States)

    Chymkowitch, Pierre; Nguéa, Aurélie P; Aanes, Håvard; Koehler, Christian J; Thiede, Bernd; Lorenz, Susanne; Meza-Zepeda, Leonardo A; Klungland, Arne; Enserink, Jorrit M

    2015-06-01

    Transcription factors are abundant Sumo targets, yet the global distribution of Sumo along the chromatin and its physiological relevance in transcription are poorly understood. Using Saccharomyces cerevisiae, we determined the genome-wide localization of Sumo along the chromatin. We discovered that Sumo-enriched genes are almost exclusively involved in translation, such as tRNA genes and ribosomal protein genes (RPGs). Genome-wide expression analysis showed that Sumo positively regulates their transcription. We also discovered that the Sumo consensus motif at RPG promoters is identical to the DNA binding motif of the transcription factor Rap1. We demonstrate that Rap1 is a molecular target of Sumo and that sumoylation of Rap1 is important for cell viability. Furthermore, Rap1 sumoylation promotes recruitment of the basal transcription machinery, and sumoylation of Rap1 cooperates with the target of rapamycin kinase complex 1 (TORC1) pathway to promote RPG transcription. Strikingly, our data reveal that sumoylation of Rap1 functions in a homeostatic feedback loop that sustains RPG transcription during translational stress. Taken together, Sumo regulates the cellular translational capacity by promoting transcription of tRNA genes and RPGs. © 2015 Chymkowitch et al.; Published by Cold Spring Harbor Laboratory Press.

  10. A study of the frequency of methylation of gene promoter regions in ...

    Indian Academy of Sciences (India)

    2013-04-02

    Apr 2, 2013 ... 2Graduate Institute of Life Sciences, and 3School of Public Health, National Defense Medical Center, No.161 Sec. 6,. Minquan E. Rd. ... of gene promoter regions in colorectal cancer in the Taiwanese population. J. Genet. 92, 109–113] .... Clinical characteristics of 132 CRC patients. Number of patients (per ...

  11. Isolation and characterization of a copalyl diphosphate synthase gene promoter from Salvia miltiorrhiza

    Directory of Open Access Journals (Sweden)

    Piotr Szymczyk

    2016-09-01

    Full Text Available The promoter, 5' UTR, and 34-nt 5' fragments of protein encoding region of the Salvia miltiorrhiza copalyl diphosphate synthase gene were cloned and characterized. No tandem repeats, miRNA binding sites, or CpNpG islands were observed in the promoter, 5' UTR, or protein encoding fragments. The entire isolated promoter and 5' UTR is 2235 bp long and contains repetitions of many cis-active elements, recognized by homologous transcription factors, found in Arabidopsis thaliana and other plant species. A pyrimidine-rich fragment with only 6 non-pyrimidine bases was localized in the 33-nt stretch from nt 2185 to 2217 in the 5' UTR. The observed cis-active sequences are potential binding sites for trans-factors that could regulate spatio-temporal CPS gene expression in response to biotic and abiotic stress conditions. Obtained results are initially verified by in silico and co-expression studies based on A. thaliana microarray data. The quantitative RT-PCR analysis confirmed that the entire 2269-bp copalyl diphosphate synthase gene fragment has the promoter activity. Quantitative RT-PCR analysis was used to study changes in CPS promoter activity occurring in response to the application of four selected biotic and abiotic regulatory factors; auxin, gibberellin, salicylic acid, and high-salt concentration.

  12. Synthetic gene involving azobenzene-tethered T7 promoter for the photocontrol of gene expression by visible light.

    Science.gov (United States)

    Kamiya, Yukiko; Takagi, Toshiki; Ooi, Hideaki; Ito, Hiroshi; Liang, Xingguo; Asanuma, Hiroyuki

    2015-04-17

    In the present study, we demonstrate photoregulation of gene expression in a cell-free translation system from a T7 promoter containing two azobenzene derivatives at specific positions. As photoswitches, we prepared azobenzene-4'-carboxlyic acid (Azo) and 2,6-dimethylazobenzene-4'-carboxylic acid (DM-Azo), which were isomerized from trans to cis upon irradiation with UV light (λ azobenzene-4'-carobxylic acid (S-DM-Azo), which were cis-isomerized by irradiation with 400 nm visible light. Expression of green fluorescent protein from a promoter modified with S-Azo or S-DM-Azo could be induced by harmless visible light whereas that from a promoter modified with Azo or DM-Azo was induced only by UV light (340-360 nm). Thus, efficient photoregulation of green fluorescent protein production was achieved in a cell-free translation system with visible light without photodamage.

  13. Italian Mediterranean river buffalo CSN2 gene structure and promoter analysis

    Directory of Open Access Journals (Sweden)

    Gianfranco Cosenza

    2010-01-01

    Full Text Available The nucleotide sequence of the whole buffalo β-casein encoding gene (CSN2 plus 1,476 nt at the 5' flanking region and 51 nt at the 3' flanking region was determined. The gene is spread over 10.2 kb and consists of 9 exons varying in length from 24 (exon 5 to 498 bp (exon 7 and 8 introns from 92 bp (intron 5 to 22 59 bp (intron 1. Furthermore, highly conserved sequences, mainly located in the 5' flanking region, were found between this gene and the β-casein encoding genes of other species. The comparison between the obtained promoter and exonic regions and buffalo sequences present in EMBL evidenced different polymorphic sites. Finally, 5 interspersed repeated elements (4 in the bovine CSN2 gene were also identified at 3 different locations of the sequenced region: 5' untranscribed region, intron 1, and intron 4.

  14. Examination of the phosphoenolpyruvate carboxykinase gene promoter in patients with noninsulin-dependent diabetes mellitus

    Energy Technology Data Exchange (ETDEWEB)

    Ludwig, D.S. [Beth Israel Hospital, Boston, MA (United States)]|[Children`s Hospital, Boston, MA (United States); Vidal-Puig, A.; Moller, D.E. [Beth Israel Hospital, Boston, MA (United States)] [and others

    1996-02-01

    Expression of phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme in gluconeogenesis, is under dominant negative regulation by insulin. In this study, we sought to test the hypothesis that mutations in the PEPCK gene promoter may impair the ability of insulin to suppress hepatic glucose production, thereby contributing to both the insulin resistance and increased rate of gluconeogenesis characteristic of NIDDM. The proximal PEPCK promoter region in 117 patients with noninsulin-dependent diabetes mellitus and 20 obese Pima Indians was amplified by PCR and analyzed with single strand conformation of polymorphism techniques. In addition, limited direct DNA sequencing was performed on the insulin response sequence and flanking regions. No DNA sequence polymorphisms were found in any patient. This result suggests that mutations in cis-acting PEPCK gene regulatory elements do not constitute a common cause of noninsulin-dependent diabetes mellitus. The significance of genetic variation in promoter regions to human disease is discussed. 40 refs., 1 figs., 1 tab.

  15. Effect of promoter architecture on the cell-to-cell variability in gene expression.

    Directory of Open Access Journals (Sweden)

    Alvaro Sanchez

    2011-03-01

    Full Text Available According to recent experimental evidence, promoter architecture, defined by the number, strength and regulatory role of the operators that control transcription, plays a major role in determining the level of cell-to-cell variability in gene expression. These quantitative experiments call for a corresponding modeling effort that addresses the question of how changes in promoter architecture affect variability in gene expression in a systematic rather than case-by-case fashion. In this article we make such a systematic investigation, based on a microscopic model of gene regulation that incorporates stochastic effects. In particular, we show how operator strength and operator multiplicity affect this variability. We examine different modes of transcription factor binding to complex promoters (cooperative, independent, simultaneous and how each of these affects the level of variability in transcriptional output from cell-to-cell. We propose that direct comparison between in vivo single-cell experiments and theoretical predictions for the moments of the probability distribution of mRNA number per cell can be used to test kinetic models of gene regulation. The emphasis of the discussion is on prokaryotic gene regulation, but our analysis can be extended to eukaryotic cells as well.

  16. Methylation of the SLC6a2 gene promoter in major depression and panic disorder.

    Directory of Open Access Journals (Sweden)

    Richard Bayles

    Full Text Available Reduced function of the noradrenaline transporter (NET has been demonstrated in patients with major depressive disorder (MDD and panic disorder. Attempts to explain NET dysfunction in MDD and panic disorder by genetic variation in the NET gene SLC6a2 have been inconclusive. Transcriptional silencing of the SLC6a2 gene may be an alternative mechanism which can lead to NET dysfunction independent of DNA sequence. The objective of this study was to characterise the DNA methylation state of the SLC6a2 gene promoter in patients with MDD and panic disorder. SLC6a2 promoter methylation was also analysed before and after antidepressant treatment. This study was performed with DNA from blood, using bisulphite sequencing and EpiTYPER methylation analyses. Patients with MDD or panic disorder were not found to differ significantly from healthy controls in the pattern of methylation of the SLC6a2 gene promotor. While significant correlations between methylation levels at some CpG sites and physiological measures were identified, overall the variation in DNA methylation between patients was small, and the significance of this variation remains equivocal. No significant changes in SLC6a2 promoter methylation were observed in response to antidepressant treatment. Further in-depth analysis of alternative mechanisms of transcriptional regulation of the SLC6a2 gene in human health and disease would be of value.

  17. Promoter architecture and transcriptional regulation of Abf1-dependent ribosomal protein genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Fermi, Beatrice; Bosio, Maria Cristina; Dieci, Giorgio

    2016-07-27

    In Saccharomyces cerevisiae, ribosomal protein gene (RPG) promoters display binding sites for either Rap1 or Abf1 transcription factors. Unlike Rap1-associated promoters, the small cohort of Abf1-dependent RPGs (Abf1-RPGs) has not been extensively investigated. We show that RPL3, RPL4B, RPP1A, RPS22B and RPS28A/B share a common promoter architecture, with an Abf1 site upstream of a conserved element matching the sequence recognized by Fhl1, a transcription factor which together with Ifh1 orchestrates Rap1-associated RPG regulation. Abf1 and Fhl1 promoter association was confirmed by ChIP and/or gel retardation assays. Mutational analysis revealed a more severe requirement of Abf1 than Fhl1 binding sites for RPG transcription. In the case of RPS22B an unusual Tbf1 binding site promoted both RPS22B and intron-hosted SNR44 expression. Abf1-RPG down-regulation upon TOR pathway inhibition was much attenuated at defective mutant promoters unable to bind Abf1. TORC1 inactivation caused the expected reduction of Ifh1 occupancy at RPS22B and RPL3 promoters, but unexpectedly it entailed largely increased Abf1 association with Abf1-RPG promoters. We present evidence that Abf1 recruitment upon nutritional stress, also observed for representative ribosome biogenesis genes, favours RPG transcriptional rescue upon nutrient replenishment, thus pointing to nutrient-regulated Abf1 dynamics at promoters as a novel mechanism in ribosome biogenesis control. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Highly specific expression of luciferase gene in lungs of naive nude mice directed by prostate-specific antigen promoter

    International Nuclear Information System (INIS)

    Li Hongwei; Li Jinzhong; Helm, Gregory A.; Pan Dongfeng

    2005-01-01

    PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10 9 PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases

  19. The Histone Methyltransferase DOT1L Promotes Neuroblastoma by Regulating Gene Transcription.

    Science.gov (United States)

    Wong, Matthew; Tee, Andrew E L; Milazzo, Giorgio; Bell, Jessica L; Poulos, Rebecca C; Atmadibrata, Bernard; Sun, Yuting; Jing, Duohui; Ho, Nicholas; Ling, Dora; Liu, Pei Yan; Zhang, Xu Dong; Hüttelmaier, Stefan; Wong, Jason W H; Wang, Jenny; Polly, Patsie; Perini, Giovanni; Scarlett, Christopher J; Liu, Tao

    2017-05-01

    Myc oncoproteins exert tumorigenic effects by regulating expression of target oncogenes. Histone H3 lysine 79 (H3K79) methylation at Myc-responsive elements of target gene promoters is a strict prerequisite for Myc-induced transcriptional activation, and DOT1L is the only known histone methyltransferase that catalyzes H3K79 methylation. Here, we show that N-Myc upregulates DOT1L mRNA and protein expression by binding to the DOT1L gene promoter. shRNA-mediated depletion of DOT1L reduced mRNA and protein expression of N-Myc target genes ODC1 and E2F2 DOT1L bound to the Myc Box II domain of N-Myc protein, and knockdown of DOT1L reduced histone H3K79 methylation and N-Myc protein binding at the ODC1 and E2F2 gene promoters and reduced neuroblastoma cell proliferation. Treatment with the small-molecule DOT1L inhibitor SGC0946 reduced H3K79 methylation and proliferation of MYCN gene-amplified neuroblastoma cells. In mice xenografts of neuroblastoma cells stably expressing doxycycline-inducible DOT1L shRNA, ablating DOT1L expression with doxycycline significantly reduced ODC1 and E2F2 expression, reduced tumor progression, and improved overall survival. In addition, high levels of DOT1L gene expression in human neuroblastoma tissues correlated with high levels of MYCN, ODC1 , and E2F2 gene expression and independently correlated with poor patient survival. Taken together, our results identify DOT1L as a novel cofactor in N-Myc-mediated transcriptional activation of target genes and neuroblastoma oncogenesis. Furthermore, they characterize DOT1L inhibitors as novel anticancer agents against MYCN-amplified neuroblastoma. Cancer Res; 77(9); 2522-33. ©2017 AACR . ©2017 American Association for Cancer Research.

  20. CpG promoter methylation of the ALKBH3 alkylation repair gene in breast cancer.

    Science.gov (United States)

    Stefansson, Olafur Andri; Hermanowicz, Stefan; van der Horst, Jasper; Hilmarsdottir, Holmfridur; Staszczak, Zuzanna; Jonasson, Jon Gunnlaugur; Tryggvadottir, Laufey; Gudjonsson, Thorkell; Sigurdsson, Stefan

    2017-07-05

    DNA repair of alkylation damage is defective in various cancers. This occurs through somatically acquired inactivation of the MGMT gene in various cancer types, including breast cancers. In addition to MGMT, the two E. coli AlkB homologs ALKBH2 and ALKBH3 have also been linked to direct reversal of alkylation damage. However, it is currently unknown whether ALKBH2 or ALKBH3 are found inactivated in cancer. Methylome datasets (GSE52865, GSE20713, GSE69914), available through Omnibus, were used to determine whether ALKBH2 or ALKBH3 are found inactivated by CpG promoter methylation. TCGA dataset enabled us to then assess the impact of CpG promoter methylation on mRNA expression for both ALKBH2 and ALKBH3. DNA methylation analysis for the ALKBH3 promoter region was carried out by pyrosequencing (PyroMark Q24) in 265 primary breast tumours and 30 proximal normal breast tissue samples along with 8 breast-derived cell lines. ALKBH3 mRNA and protein expression were analysed in cell lines using RT-PCR and Western blotting, respectively. DNA alkylation damage assay was carried out in cell lines based on immunofluorescence and confocal imaging. Data on clinical parameters and survival outcomes in patients were obtained and assessed in relation to ALKBH3 promoter methylation. The ALKBH3 gene, but not ALKBH2, undergoes CpG promoter methylation and transcriptional silencing in breast cancer. We developed a quantitative alkylation DNA damage assay based on immunofluorescence and confocal imaging revealing higher levels of alkylation damage in association with epigenetic inactivation of the ALKBH3 gene (P = 0.029). In our cohort of 265 primary breast cancer, we found 72 cases showing aberrantly high CpG promoter methylation over the ALKBH3 promoter (27%; 72 out of 265). We further show that increasingly higher degree of ALKBH3 promoter methylation is associated with reduced breast-cancer specific survival times in patients. In this analysis, ALKBH3 promoter methylation at >20

  1. Single-nucleotide polymorphisms in the promoter region of the PARKIN gene and Parkinson's disease.

    Science.gov (United States)

    Mata, Ignacio F; Alvarez, Victoria; García-Moreira, Vanessa; Guisasola, Luis M; Ribacoba, René; Salvador, Carlos; Blázquez, Marta; Sarmiento, Rogelio González; Lahoz, Carlos H; Menes, Bernardino B; García, Eliecer Coto

    2002-08-30

    Mutations in the PARKIN gene have been identified in families with recessively inherited Parkinson disease (PD). Common DNA-polymorphisms at the PARKIN gene could contribute to the risk for PD in the general population. Here we searched for DNA-polymorphisms in the PARKIN promoter. We found two single nucleotide polymorphisms (-324 A/G and -797 A/G). In order to analyse the association of PD with these and two previously described polymorphisms (1281 G/A, Asp394Asn, and 601 G/A, Ser167Asn) we genotyped 105 patients and 150 healthy controls. Allele and genotype frequencies for the four polymorphisms did not differ between patients and controls, or between patients with an early-onset (40 years; n = 85). According to our data, the genetic variation at the PARKIN gene (including promoter polymorphisms) did not contribute to the risk of developing PD in the general population. Copyright 2002 Elsevier Science Ireland Ltd.

  2. Genetic variation in TGF-beta 1 gene promoter and risk of gestational trophoblastic disease.

    Science.gov (United States)

    Dehaghani, Alamtaj Samsami; Zamanpour, Tarlan; Naeimi, Sirous; Sameni, Safoura; Robati, Minoo; Ghaderi, Abbas

    2010-01-01

    To examine the relationship of transforming growth factor beta 1 (TGF-beta 1) gene polymorphisms at promoter positions -509 (C/T) and -800 (G/A) with the risk of gestational trophoblastic disease (GTD) as compared to normal controls Polymerase chain reaction-restriction fragment length polymorphism was performed on peripheral blood of 102 patients with GTD and 124 normal, healthy, pregnant women as the control group. In this study, TGF-beta 1 gene polymorphisms at positions -509 (C/T) and -800 (G/A) failed to correlate with GTD. Our findings suggest that promoter gene polymorphisms of TGF-beta 1 do not play major roles in GTD and may not be risk factors for this disease.

  3. Control of stochastic gene expression by host factors at the HIV promoter.

    Directory of Open Access Journals (Sweden)

    John C Burnett

    2009-01-01

    Full Text Available The HIV promoter within the viral long terminal repeat (LTR orchestrates many aspects of the viral life cycle, from the dynamics of viral gene expression and replication to the establishment of a latent state. In particular, after viral integration into the host genome, stochastic fluctuations in viral gene expression amplified by the Tat positive feedback loop can contribute to the formation of either a productive, transactivated state or an inactive state. In a significant fraction of cells harboring an integrated copy of the HIV-1 model provirus (LTR-GFP-IRES-Tat, this bimodal gene expression profile is dynamic, as cells spontaneously and continuously flip between active (Bright and inactive (Off expression modes. Furthermore, these switching dynamics may contribute to the establishment and maintenance of proviral latency, because after viral integration long delays in gene expression can occur before viral transactivation. The HIV-1 promoter contains cis-acting Sp1 and NF-kappaB elements that regulate gene expression via the recruitment of both activating and repressing complexes. We hypothesized that interplay in the recruitment of such positive and negative factors could modulate the stability of the Bright and Off modes and thereby alter the sensitivity of viral gene expression to stochastic fluctuations in the Tat feedback loop. Using model lentivirus variants with mutations introduced in the Sp1 and NF-kappaB elements, we employed flow cytometry, mRNA quantification, pharmacological perturbations, and chromatin immunoprecipitation to reveal significant functional differences in contributions of each site to viral gene regulation. Specifically, the Sp1 sites apparently stabilize both the Bright and the Off states, such that their mutation promotes noisy gene expression and reduction in the regulation of histone acetylation and deacetylation. Furthermore, the NF-kappaB sites exhibit distinct properties, with kappaB site I serving a

  4. Computational promoter analysis of mouse, rat and human antimicrobial peptide-coding genes

    Directory of Open Access Journals (Sweden)

    Kai Chikatoshi

    2006-12-01

    Full Text Available Abstract Background Mammalian antimicrobial peptides (AMPs are effectors of the innate immune response. A multitude of signals coming from pathways of mammalian pathogen/pattern recognition receptors and other proteins affect the expression of AMP-coding genes (AMPcgs. For many AMPcgs the promoter elements and transcription factors that control their tissue cell-specific expression have yet to be fully identified and characterized. Results Based upon the RIKEN full-length cDNA and public sequence data derived from human, mouse and rat, we identified 178 candidate AMP transcripts derived from 61 genes belonging to 29 AMP families. However, only for 31 mouse genes belonging to 22 AMP families we were able to determine true orthologous relationships with 30 human and 15 rat sequences. We screened the promoter regions of AMPcgs in the three species for motifs by an ab initio motif finding method and analyzed the derived promoter characteristics. Promoter models were developed for alpha-defensins, penk and zap AMP families. The results suggest a core set of transcription factors (TFs that regulate the transcription of AMPcg families in mouse, rat and human. The three most frequent core TFs groups include liver-, nervous system-specific and nuclear hormone receptors (NHRs. Out of 440 motifs analyzed, we found that three represent potentially novel TF-binding motifs enriched in promoters of AMPcgs, while the other four motifs appear to be species-specific. Conclusion Our large-scale computational analysis of promoters of 22 families of AMPcgs across three mammalian species suggests that their key transcriptional regulators are likely to be TFs of the liver-, nervous system-specific and NHR groups. The computationally inferred promoter elements and potential TF binding motifs provide a rich resource for targeted experimental validation of TF binding and signaling studies that aim at the regulation of mouse, rat or human AMPcgs.

  5. Analysis of Promoters of Arabidopsis thaliana Divergent Gene Pair SERAT3;2 and IDH-III Shows SERAT3;2 Promoter is Nested Within the IDH-III Promoter.

    Science.gov (United States)

    Raipuria, Ritesh Kumar; Kumar, Vajinder; Guruprasad, Kadur Narayan; Bhat, Shripad Ramachandra

    2017-07-01

    Intergenic regions of divergent gene pairs show bidirectional promoter activity but whether regulatory sequences for gene expression in opposite directions are shared is not established. In this study, promoters of divergently arranged gene pair At4g35640-At4g35650 (SERAT3;2-IDH-III) of Arabidopsis thaliana were analyzed to identify overlapping regulatory regions. Both genes showed the highest expression in flower buds and flowers. 5' RACE experiments extended the intergenic region from 161 bp shown in TAIR annotation to 512 bp. GUS analysis of transgenic A. thaliana plants carrying the 691 bp fragment (512 bp intergenic region plus 5' UTR of both the genes) linked to uidA gene revealed that SERAT3;2 promoter drives gene expression in the tapetum, whereas IDH-III promoter functions specifically in microspores/pollen. Serial 5' deletion of the 691 bp fragment showed SERAT3;2 promoter extends up to -355 position, whereas IDH-III promoter encompasses the 512 bp intergenic region. In transgenics, uidA transcript levels were lower than native SERAT3;2 and IDH-III transcripts indicating presence of additional cis regulatory elements beyond the 691 bp fragment. The present study demonstrated for the first time occurrence of a nested promoter in plants and identified a novel bidirectional promoter capable of driving gene expression in tapetum and microspores/pollen.

  6. Increased expression of alpha- and beta-globin mRNAs at the pituitary following exposure to estrogen during the critical period of neonatal sex differentiation in the rat

    DEFF Research Database (Denmark)

    Leffers, H; Navarro, V M; Nielsen, John E

    2006-01-01

    in the neuroendocrine system controlling development and function of the reproductive axis; the HP unit being highly sensitive to the organizing effects of endogenous and exogenous sex steroids. To gain knowledge on the molecular mode of action and potential biomarkers of exposure to estrogenic compounds at the HP unit...... by combination of RT-PCR analyses and in situ hybridization. Induction of alpha- and beta-globin mRNA expression at the pituitary by neonatal exposure to estrogen was demonstrated as dose-dependent and it was persistently detected up to puberty. In contrast, durable up-regulation of alpha- and beta-globin genes...

  7. The goat alphas1-casein gene: gene structure and promoter analysis.

    Science.gov (United States)

    Ramunno, Luigi; Cosenza, Gianfranco; Rando, Andrea; Illario, Rosa; Gallo, Daniela; Di Berardino, Dino; Masina, Piero

    2004-06-09

    The level of alphas1-casein in goat milk shows strong variations determined by at least 15 alleles associated with four different efficiencies of protein synthesis. The nucleotide sequence of the whole goat alphas1-casein-encoding gene (CSN1S1) plus 1973 nucleotides at the 5' flanking region and 610 nucleotides at the 3' flanking region was determined and aligned with its bovine counterpart. The gene is spread over 16.7 kb and consists of 19 exons varying in length from 24 bp (exons 5, 6, 7, 8, 10, 13 and 16) to 385 bp (exon 19) and 18 introns from 90 bp of intron 10 to 1685 bp of intron 2. Furthermore, highly conserved sequences, mainly located in the 5' flanking region, were found between this gene and other casein-encoding genes. Finally, seven interspersed repeated elements (10 in the bovine CSN1S1 gene) were also identified at four different locations of the sequenced region: 5' untranscribed region and introns 2, 8 and 11.

  8. A database of annotated promoters of genes associated with common respiratory and related diseases

    KAUST Repository

    Chowdhary, Rajesh

    2012-07-01

    Many genes have been implicated in the pathogenesis of common respiratory and related diseases (RRDs), yet the underlying mechanisms are largely unknown. Differential gene expression patterns in diseased and healthy individuals suggest that RRDs affect or are affected by modified transcription regulation programs. It is thus crucial to characterize implicated genes in terms of transcriptional regulation. For this purpose, we conducted a promoter analysis of genes associated with 11 common RRDs including allergic rhinitis, asthma, bronchiectasis, bronchiolitis, bronchitis, chronic obstructive pulmonary disease, cystic fibrosis, emphysema, eczema, psoriasis, and urticaria, many of which are thought to be genetically related. The objective of the present study was to obtain deeper insight into the transcriptional regulation of these disease-associated genes by annotating their promoter regions with transcription factors (TFs) and TF binding sites (TFBSs). We discovered many TFs that are significantly enriched in the target disease groups including associations that have been documented in the literature. We also identified a number of putative TFs/TFBSs that appear to be novel. The results of our analysis are provided in an online database that is freely accessible to researchers at http://www.respiratorygenomics.com. Promoter-associated TFBS information and related genomic features, such as histone modification sites, microsatellites, CpG islands, and SNPs, are graphically summarized in the database. Users can compare and contrast underlying mechanisms of specific RRDs relative to candidate genes, TFs, gene ontology terms, micro-RNAs, and biological pathways for the conduct of metaanalyses. This database represents a novel, useful resource for RRD researchers. Copyright © 2012 by the American Thoracic Society.

  9. Computational construction of 3D chromatin ensembles and prediction of functional interactions of alpha-globin locus from 5C data.

    Science.gov (United States)

    Gürsoy, Gamze; Xu, Yun; Kenter, Amy L; Liang, Jie

    2017-11-16

    Conformation capture technologies measure frequencies of interactions between chromatin regions. However, understanding gene-regulation require knowledge of detailed spatial structures of heterogeneous chromatin in cells. Here we describe the nC-SAC (n-Constrained-Self Avoiding Chromatin) method that transforms experimental interaction frequencies into 3D ensembles of chromatin chains. nC-SAC first distinguishes specific from non-specific interaction frequencies, then generates 3D chromatin ensembles using identified specific interactions as spatial constraints. Application to α-globin locus shows that these constraints (∼20%) drive the formation of ∼99% all experimentally captured interactions, in which ∼30% additional to the imposed constraints is found to be specific. Many novel specific spatial contacts not captured by experiments are also predicted. A subset, of which independent ChIA-PET data are available, is validated to be RNAPII-, CTCF-, and RAD21-mediated. Their positioning in the architectural context of imposed specific interactions from nC-SAC is highly important. Our results also suggest the presence of a many-body structural unit involving α-globin gene, its enhancers, and POL3RK gene for regulating the expression of α-globin in silent cells. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Prolonged treatment with DNMT inhibitors induces distinct effects in promoters and gene-bodies.

    Directory of Open Access Journals (Sweden)

    Yan-Fung Wong

    Full Text Available Treatment with the demethylating drugs 5-azacytidine (AZA and decitabine (DAC is now recognised as an effective therapy for patients with Myelodysplastic Syndromes (MDS, a range of disorders arising in clones of hematopoietic progenitor cells. A variety of cell models have been used to study the effect of these drugs on the methylation of promoter regions of tumour suppressor genes, with recent efforts focusing on the ability of these drugs to inhibit DNA methylation at low doses. However, it is still not clear how nano-molar drug treatment exerts its effects on the methylome. In this study, we have characterised changes in DNA methylation caused by prolonged low-dose treatment in a leukemic cell model (SKM-1, and present a genome-wide analysis of the effects of AZA and DAC. At nano-molar dosages, a one-month continuous treatment halved the total number of hypermethylated probes in leukemic cells and our analysis identified 803 candidate regions with significant demethylation after treatment. Demethylated regions were enriched in promoter sequences whereas gene-body CGIs were more resistant to the demethylation process. CGI methylation in promoters was strongly correlated with gene expression but this correlation was lost after treatment. Our results indicate that CGI demethylation occurs preferentially at promoters, but that it is not generally sufficient to modify expression patterns, and emphasises the roles of other means of maintaining cell state.

  11. Cloning and characterization of the human integrin β6 gene promoter.

    Directory of Open Access Journals (Sweden)

    Mingyan Xu

    Full Text Available The integrin β6 (ITGB6 gene, which encodes the limiting subunit of the integrin αvβ6 heterodimer, plays an important role in wound healing and carcinogenesis. The mechanism underlying ITGB6 regulation, including the identification of DNA elements and cognate transcription factors responsible for basic transcription of human ITGB6 gene, remains unknown. This report describes the cloning and characterization of the human ITGB6 promoter. Using 5'-RACE (rapid amplification of cDNA ends analysis, the transcriptional initiation site was identified. Promoter deletion analysis identified and functionally validated a TATA box located in the region -24 to -18 base pairs upstream of the ITGB6 promoter. The regulatory elements for transcription of the ITGB6 gene were predominantly located -289 to -150 from the ITGB6 promoter and contained putative binding sites for transcription factors such as STAT3 and C/EBPα. Using chromatin immunoprecipitation assays, this study has demonstrated, for the first time, that transcription factors STAT3 and C/EBPα are involved in the positive regulation of ITGB6 transcription in oral squamous cell carcinoma cells. These findings have important implications for unraveling the mechanism of abnormal ITGB6 activation in tissue remodeling and tumorigenesis.

  12. Development of radiation-inducible promoters for use in nitric oxide synthase gene therapy of cancer

    International Nuclear Information System (INIS)

    Hirst, D.G.; Worthington, J.; Adams, C.; Robson, T.; Scott, S.D.

    2003-01-01

    Full text: The free radical nitric oxide (NO) at nM concentrations performs multiple signaling roles that are essential for survival. These processes are regulated via the enzymes nNOS and eNOS, but another isoform, inducible nitric oxide synthase (iNOS) is capable of generating much higher concentrations (mM) over longer periods, resulting in the generation of very toxic species such as peroxynitrite. At high concentrations NO has many of the characteristics of an ideal anticancer molecule: it is cytotoxic (pro-apoptotic via peroxynitrite), it is a potent chemical radiosensitizer, it is anti-angiogenic and anti-metastatic. Thus, we see iNOS gene therapy as a strategy for targeting the generation of high concentrations of NO to tumours for therapeutic benefit. iNOS gene therapy should be used in combination with radiotherapy; so it is logical that the use of a radiation-inducible promoter should be part of the targeting strategy. We have tested several candidate promoters in vitro and in vivo. The WAF1 promoter has many of the properties desirable for therapeutic use including: rapid 3-4 fold induction at X-ray doses of 2 and 4Gy and no significant leakiness. WAF1 also has the advantage of being inducible by hypoxia and by the final product, NO. We have also tested the synthetic CArG promoter and demonstrated that, in addition to a high level of radiation inducibility, it is also inducible by NO. We have also been able to demonstrate potent radiosensitization (SER 2.0-2.5) in tumour cells in vitro and in vivo using iNOS gene transfer with constitutive or radiation-inducible promoters. We have also tested the use of iNOS gene therapy in combination with cisplatin and shown significant enhancement

  13. Characterization of the mouse cyclin D3 gene: Exon/intron organization and promoter activity

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhengyu; Zhang, Ying; Ravid, K. [Boston Univ. School of Medicine, Boston, MA (United States)] [and others

    1996-07-01

    The three D-type cyclins have been shown to be differentially expressed in a number of cell types, suggesting that they play distinct roles in cell cycle regulation in particular cell lineages. We have determined the complete nucleotide sequence (-1681 to +6582) of the mouse cyclin D3 gene, which encodes a G1 phase cyclin. The gene consists of five exons and four introns, varying in length from 422 to 2472 bp. Primer extension analysis revealed one major transcription initiation site at the position 107 bp 5{prime} upstream of the translation start. The promoter region lacks both canonical {open_quotes}TATA{close_quotes} and {open_quotes}CAAT{close_quotes} boxes. It contains, however, multiple transcription factor recognition by GATA, NF-{kappa}B, ATF, E2F, and TRE/AP1 transcription factors, E box binding myogenic factors, and the IL-6 induced-transcription factor, APRF. Promoter activity of the 1681-bp fragment upstream of the transcription initiation site was confirmed by linking it to a reporter gene and subjecting it to transient expression experiments in various cell types. Promoter activity was high in cell lines that expressed high levels of endogenous D3 mRNA, as indicated by Northern blot analysis, and was significantly reduced when the promoter was truncated to -122 bp. The characterization of the mouse cyclin D3 gene and insight into its promoter region will allow further studies defining the molecular events regulating the expression of this cyclin in proliferating and quiescent cells. 60 refs., 4 figs., 1 tab.

  14. A novel method for the determination of basal gene expression of tissue-specific promoters: an analysis of prostate-specific promoters.

    Science.gov (United States)

    van der Poel, H G; McCadden, J; Verhaegh, G W; Kruszewski, M; Ferrer, F; Schalken, J A; Carducci, M; Rodriguez, R

    2001-12-01

    Because the toxicity of suicide gene therapeutics is directly related to basal promoter activity, we developed an assay to test for promoter "leakiness" using a diphtheria toxin mutant. Sequences of 15 prostate-specific gene promoter constructs were cloned in an expression plasmid (pBK; Stratagene, La Jolla, CA) backbone driving expression of an attenuated mutant of diphtheria toxin A (tox176). Low expression levels of the DT-tox176 result in significant protein synthesis inhibition reflected by a decreased expression of the luciferase activity of a simultaneously transfected CMV luciferase construct. ID50 (dose of plasmid with 50% luciferase inhibition) was calculated for each promoter construct in different cell lines. Highest transactivational activity (ID50 CN65 (PSA promoter/enhancer) and PSE-hK2 (PSA enhancer and basal human kallikrein 2 promoter) in HEK293 and DLD cells indicating "leakiness" of these promoter constructs. Low basal promoter activity in nonprostate cell lines was found for the minimal PSA promoter, hK2, DD3, and OC promoters. The DT-tox176 assay can better predict basal promoter activity compared to less sensitive dual luciferase assay.

  15. VNTR internal structure mapping at the {alpha}-globin 3{prime}HVR locus reveals a hierachy of related lineages in oceania

    Energy Technology Data Exchange (ETDEWEB)

    Martinson, J.J.; Clegg, J.B.; Boyce, A.J. [Univ. of Oxford (United Kingdom)

    1994-09-01

    Analysis of the {alpha}-globin gene complex in Oceania has revealed many different rearrangements which remove one of the adult globin genes. Frequencies of these deletion chromosomes are elevated by malarial resistance conferred by the resulting {alpha}-thalassaemia. One particular deletion chromosome, designated -{alpha}{sup 3.7}III, is found at high levels in Melanesia and Polynesia: RFLP haplotype analysis shows that this deletion is always found on chromosomes bearing the IIIa haplotype and is likely to be the product of one single rearrangement event. A subset of the -{alpha}{sup 3.7}III chromosomes carries a more recent mutation which generates the haemoglobin variant HbJ{sup Tongariki}. We have characterized the allelic variation at the 3{prime}HVR VNTR locus located 6 kb from the globin genes in each of these groups of chromosomes. We have determined the internal structure of these alleles by RFLP mapping of PCR-amplified DNA: within each group, the allelic diversity results from the insertion and/or deletion of small {open_quotes}motifs{close_quotes} of up to 6 adjacent repeats. Mapping of 3{prime}HVR alleles associated with other haplotypes reveals that these are composed of repeat arrays that are substantially different to those derived from IIIa chromosomes, indicating that interchromosomal recombination between heterologous haplotypes does not account for any of the diversity seen to date. We have recently shown that allelic size variation at the two VNTR loci flanking the {alpha}-globin complex is very closely linked to the haplotypes known to be present at this locus. Here we show that, within a haplotype, VNTR alleles are very closely related to each other on the basis of internal structure and demonstrate that intrachromosomal mutation processes involving small numbers of tandem repeats are the main cause of variation at this locus.

  16. DNA demethylases target promoter transposable elements to positively regulate stress responsive genes in Arabidopsis.

    Science.gov (United States)

    Le, Tuan-Ngoc; Schumann, Ulrike; Smith, Neil A; Tiwari, Sameer; Au, Phil Chi Khang; Zhu, Qian-Hao; Taylor, Jennifer M; Kazan, Kemal; Llewellyn, Danny J; Zhang, Ren; Dennis, Elizabeth S; Wang, Ming-Bo

    2014-09-17

    DNA demethylases regulate DNA methylation levels in eukaryotes. Arabidopsis encodes four DNA demethylases, DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 (DML2), and DML3. While DME is involved in maternal specific gene expression during seed development, the biological function of the remaining DNA demethylases remains unclear. We show that ROS1, DML2, and DML3 play a role in fungal disease resistance in Arabidopsis. A triple DNA demethylase mutant, rdd (ros1 dml2 dml3), shows increased susceptibility to the fungal pathogen Fusarium oxysporum. We identify 348 genes differentially expressed in rdd relative to wild type, and a significant proportion of these genes are downregulated in rdd and have functions in stress response, suggesting that DNA demethylases maintain or positively regulate the expression of stress response genes required for F. oxysporum resistance. The rdd-downregulated stress response genes are enriched for short transposable element sequences in their promoters. Many of these transposable elements and their surrounding sequences show localized DNA methylation changes in rdd, and a general reduction in CHH methylation, suggesting that RNA-directed DNA methylation (RdDM), responsible for CHH methylation, may participate in DNA demethylase-mediated regulation of stress response genes. Many of the rdd-downregulated stress response genes are downregulated in the RdDM mutants nrpd1 and nrpe1, and the RdDM mutants nrpe1 and ago4 show enhanced susceptibility to F. oxysporum infection. Our results suggest that a primary function of DNA demethylases in plants is to regulate the expression of stress response genes by targeting promoter transposable element sequences.

  17. MGMT, GATA6, CD81, DR4, and CASP8 gene promoter methylation in glioblastoma

    Directory of Open Access Journals (Sweden)

    Skiriute Daina

    2012-06-01

    Full Text Available Abstract Background Methylation of promoter region is the major mechanism affecting gene expression in tumors. Recent methylome studies of brain tumors revealed a list of new epigenetically modified genes. Our aim was to study promoter methylation of newly identified epigenetically silenced genes together with already known epigenetic markers and evaluate its separate and concomitant role in glioblastoma genesis and patient outcome. Methods The methylation status of MGMT, CD81, GATA6, DR4, and CASP8 in 76 patients with primary glioblastomas was investigated. Methylation-specific PCR reaction was performed using bisulfite treated DNA. Evaluating glioblastoma patient survival time after operation, patient data and gene methylation effect on survival was estimated using survival analysis. Results The overwhelming majority (97.3% of tumors were methylated in at least one of five genes tested. In glioblastoma specimens gene methylation was observed as follows: MGMT in 51.3%, GATA6 in 68.4%, CD81 in 46.1%, DR4 in 41.3% and CASP8 in 56.8% of tumors. Methylation of MGMT was associated with younger patient age (p CASP8 with older (p MGMT methylation was significantly more frequent event in patient group who survived longer than 36 months after operation (p CASP8 was more frequent in patients who survived shorter than 36 months (p MGMT, GATA6 and CASP8 as independent predictors for glioblastoma patient outcome (p MGMT and GATA6 were independent predictors for patient survival in younger patients’ group, while there were no significant associations observed in older patients’ group when adjusted for therapy. Conclusions High methylation frequency of tested genes shows heterogeneity of glioblastoma epigenome and the importance of MGMT, GATA6 and CASP8 genes methylation in glioblastoma patient outcome.

  18. Predominant gain of promoter TATA box after gene duplication associated with stress responses.

    Science.gov (United States)

    Zou, Yangyun; Huang, Wei; Gu, Zhenglong; Gu, Xun

    2011-10-01

    TATA box, the core promoter element, exists in a broad range of eukaryotes, and the expression of TATA-containing genes usually responds to various environmental stresses. Hence, the evolution of TATA-box in duplicate genes may provide some clues for the interrelationship among environmental stress, expression differentiation, and duplicate gene preservation. In the present study, we observed that the TATA box is significantly overrepresented in duplicate genes compared with singletons in human, worm, Arabidopsis, and yeast genomes. We then conducted an extensive functional genomic analysis to investigate the evolution of TATA box along over 700 yeast gene family phylogenies. After reconstructing the ancestral TATA-box states (presence or absence), we found that significantly higher numbers of TATA box gain events than loss events had occurred after yeast gene duplications-the overall gain-loss ratio is about 3-4 to 1. Interestingly, these TATA-gain duplicate genes on average have experienced greater expression divergence from the ancestral expression states than their most closely related TATA-less duplicate partners, but only under environmental stress conditions (asymmetric evolution); indeed, under normal physiological conditions, they have similar expression divergence (symmetric evolution). Moreover, we showed that TATA-gain duplicates are enriched in stress-associated functional categories but that is not the case for TATA-ancestral duplicates (those inherited from their ancestors prior to duplication). Together, we conclude that after the gene duplication, gain of the TATA box in duplicate promoters may have played an important role in yeast duplicate preservation by accelerating expression divergence that may facilitate the adaptive evolution of the organism in response to environmental changes.

  19. Structure and promoter analysis of an ABA- and stress-regulated barley gene, HVA1.

    Science.gov (United States)

    Straub, P F; Shen, Q; Ho, T D

    1994-10-01

    A single-copy barley gene, HVA1, encoding a class 3 late embryogenesis-abundant protein, can be induced by either treatment with abscisic acid (ABA) or by stress conditions such as drought, cold, heat and salinity. We have isolated an HVA1 genomic clone containing about 400 bp of 5'-upstream sequence, a single 109 bp intron, and the full coding sequence. Linker scan mutagenesis and transient expression studies were used to test the function of four HVA1 promoter elements conserved in ABA-responsive genes. Mutations in two of these elements, the C box and the putative ABRE 1 (ABA-responsive element) containing an ACGT core, resulted in no significant change in transcription level or ABA induction. In contrast, mutations of the other two elements, putative ABRE 2 & 3 cause the level of transcription to drop to 10-20% of that obtained with the wild-type promoter indicating that the high level of expression of HVA1 is dependent on both pABRE 2 & 3. Interestingly, despite their low level of expression, the mutated promoters still gave more than 20-fold induction in response to ABA treatment. We suggest that the ABA induction of barley HVA1 gene is governed by a complex consisting of pABRE 2 & 3 working together to regulate the absolute level of expression, and either of these elements or a possible third element may regulate ABA inducibility. Phylogenetic analysis by parsimony indicates that the barley HVA1 and wheat pMA2005 sequences share a recent common ancester. These two genes are closely related to the carrot Dc3 and cotton D-7 genes with which they share a similar structural gene organization.

  20. Rat tenascin-R gene: structure, chromosome location and transcriptional activity of promoter and exon 1.

    Science.gov (United States)

    Leprini, A; Gherzi, R; Vecchi, E; Borsi, L; Zardi, L; Siri, A

    1998-01-01

    Tenascin-R is an extracellular matrix protein expressed exclusively in the central nervous system where it is thought to play a relevant role in regulating neurite outgrowth. We have i) cloned the cDNA of the rat tenascin-R 5' region; ii) defined its genomic organization, obtaining the sequence of two novel untranslated exons; iii) mapped the gene to rat chromosome 13q23 and suggested a previously unreported synteny between rat chromosome 13q23, human chromosome 1q24, and mouse chromosome 4E; and iv) sequenced and characterized the elements responsible for its neural cell-restricted transcription. We found that two discrete regions of the rat gene (the first in the proximal promoter, the second in the first exon) are independently able to activate to a high degree the transcription of a reporter gene in either human or rat neuroblastoma cell lines but not in other cell lines. Based on this observation, we re-evaluated the arrangement of transcriptionally active regions in the human tenascin-R gene we recently cloned and found that the human gene also contains an exon sequence able to initiate and sustain transcription independently of promoter sequences.

  1. The odd-skipped family of zinc finger genes promotes Drosophila leg segmentation.

    Science.gov (United States)

    Hao, Irene; Green, Ryan B; Dunaevsky, Olga; Lengyel, Judith A; Rauskolb, Cordelia

    2003-11-15

    Notch signaling controls formation of joints at leg segment borders and growth of the developing Drosophila leg. Here, we identify the odd-skipped gene family as a key group of genes that function downstream of the Notch receptor to promote morphological changes associated with joint formation during leg development. odd, sob, drm, and bowl are expressed in a segmental pattern in the developing leg, and their expression is regulated by Notch signaling. Ectopic expression of odd, sob, or drm can induce invaginations in the leg disc epithelium and morphological changes in the adult leg that are characteristic of endogenous invaginating joint cells. These effects are not due to an alteration in the expression of other genes of the developing joint. While odd or drm mutant clones do not affect leg segmentation, and thus appear to act redundantly, bowl mutant clones do perturb leg development. Specifically, bowl mutant clones result in a failure of joint formation from the distal tibia to tarsal segment 5, while more proximal clones cause melanotic protrusions from the leg cuticle. Together, these results indicate that the odd-skipped family of genes mediates Notch function during leg development by promoting a specific aspect of joint formation, an epithelial invagination. As the odd-skipped family genes are involved in regulating cellular morphogenesis during both embryonic segmentation and hindgut development, we suggest that they may be required in multiple developmental contexts to induce epithelial cellular changes.

  2. Developmental regulation of tandem promoters for the major outer membrane protein gene of Chlamydia trachomatis.

    Science.gov (United States)

    Stephens, R S; Wagar, E A; Edman, U

    1988-01-01

    Chlamydia trachomatis has a biphasic developmental cycle which is characterized by qualitative and quantitative changes in protein expression. The molecular mechanisms that mediate these changes are unknown. Evidence for transcriptional regulation of the chlamydial major outer membrane protein gene (omp1) was found by Northern hybridization of RNA isolated sequentially during the chlamydial developmental cycle. Early in the growth cycle a single transcript was detected, which was followed hours later in the cycle by an additional transcript. Mapping of the initiating nucleotide for each transcript suggested that this gene is regulated by differential transcription from tandem promoters. Images PMID:2448291

  3. Nonlinear Dynamics in Gene Regulation Promote Robustness and Evolvability of Gene Expression Levels.

    Directory of Open Access Journals (Sweden)

    Arno Steinacher

    Full Text Available Cellular phenotypes underpinned by regulatory networks need to respond to evolutionary pressures to allow adaptation, but at the same time be robust to perturbations. This creates a conflict in which mutations affecting regulatory networks must both generate variance but also be tolerated at the phenotype level. Here, we perform mathematical analyses and simulations of regulatory networks to better understand the potential trade-off between robustness and evolvability. Examining the phenotypic effects of mutations, we find an inverse correlation between robustness and evolvability that breaks only with nonlinearity in the network dynamics, through the creation of regions presenting sudden changes in phenotype with small changes in genotype. For genotypes embedding low levels of nonlinearity, robustness and evolvability correlate negatively and almost perfectly. By contrast, genotypes embedding nonlinear dynamics allow expression levels to be robust to small perturbations, while generating high diversity (evolvability under larger perturbations. Thus, nonlinearity breaks the robustness-evolvability trade-off in gene expression levels by allowing disparate responses to different mutations. Using analytical derivations of robustness and system sensitivity, we show that these findings extend to a large class of gene regulatory network architectures and also hold for experimentally observed parameter regimes. Further, the effect of nonlinearity on the robustness-evolvability trade-off is ensured as long as key parameters of the system display specific relations irrespective of their absolute values. We find that within this parameter regime genotypes display low and noisy expression levels. Examining the phenotypic effects of mutations, we find an inverse correlation between robustness and evolvability that breaks only with nonlinearity in the network dynamics. Our results provide a possible solution to the robustness-evolvability trade-off, suggest

  4. Nonlinear Dynamics in Gene Regulation Promote Robustness and Evolvability of Gene Expression Levels

    Science.gov (United States)

    Steinacher, Arno; Bates, Declan G.; Akman, Ozgur E.; Soyer, Orkun S.

    2016-01-01

    Cellular phenotypes underpinned by regulatory networks need to respond to evolutionary pressures to allow adaptation, but at the same time be robust to perturbations. This creates a conflict in which mutations affecting regulatory networks must both generate variance but also be tolerated at the phenotype level. Here, we perform mathematical analyses and simulations of regulatory networks to better understand the potential trade-off between robustness and evolvability. Examining the phenotypic effects of mutations, we find an inverse correlation between robustness and evolvability that breaks only with nonlinearity in the network dynamics, through the creation of regions presenting sudden changes in phenotype with small changes in genotype. For genotypes embedding low levels of nonlinearity, robustness and evolvability correlate negatively and almost perfectly. By contrast, genotypes embedding nonlinear dynamics allow expression levels to be robust to small perturbations, while generating high diversity (evolvability) under larger perturbations. Thus, nonlinearity breaks the robustness-evolvability trade-off in gene expression levels by allowing disparate responses to different mutations. Using analytical derivations of robustness and system sensitivity, we show that these findings extend to a large class of gene regulatory network architectures and also hold for experimentally observed parameter regimes. Further, the effect of nonlinearity on the robustness-evolvability trade-off is ensured as long as key parameters of the system display specific relations irrespective of their absolute values. We find that within this parameter regime genotypes display low and noisy expression levels. Examining the phenotypic effects of mutations, we find an inverse correlation between robustness and evolvability that breaks only with nonlinearity in the network dynamics. Our results provide a possible solution to the robustness-evolvability trade-off, suggest an explanation for

  5. Inducing gene expression by targeting promoter sequences using small activating RNAs

    Directory of Open Access Journals (Sweden)

    Ji Wang

    2015-02-01

    Full Text Available Vector-based systems comprised of exogenous nucleic acid sequences remain the standard for ectopic expression of a particular gene. Such systems offer robust overexpression, but have inherent drawbacks such the tedious process of construction, excluding sequences (e.g. introns and untranslated regions important for gene function and potential insertional mutagenesis of host genome associated with the use of viral vectors. We and others have recently reported that short double-stranded RNAs (dsRNAs can induce endogenous gene expression by targeting promoter sequences in a phenomenon referred to as RNA activation (RNAa and such dsRNAs are termed small activating RNAs (saRNAs. To date, RNAa has been successfully utilized to induce the expression of different genes such as tumor suppressor genes. Here, we describe a detailed protocol for target selection and dsRNA design with associated experiments to facilitate RNAa in cultured cells. This technique may be applied to selectively activate endogenous gene expression for studying gene function, interrogating molecular pathways and reprogramming cell fate.

  6. Identification and characterization of the promoter of human ATF5 gene.

    Science.gov (United States)

    Wei, Yuanyan; Ge, Yuqing; Zhou, Fengbiao; Chen, Hong; Cui, Chunhong; Liu, Dan; Yang, Zhiyuan; Wu, Guoqiang; Gu, Jianxin; Jiang, Jianhai

    2010-08-01

    ATF5, a member of ATF/CREB family of b-ZIP transcription factors, is highly expressed in a wide variety of neoplasms and regulates cell differentiation, cell survival and apoptosis. However, the mechanism of human ATF5 transcriptional regulation has not been clarified. Here, we identified the transcription start site of the ATF5 gene, cloned its 5'-flanking region and identified the region -105 to +3 relative to the transcription start site as that having promoter activity. This region contained potential binding sites for several transcription factors, including EBF1, Sp1 and E2F1. EBF1 transcription factor binds to the ATF5 promoter and regulates the ATF5 transcription in an EBF-binding site independent manner. Thus, our studies not only provided molecular basis of ATF5 transcriptional regulation, but also identified ATF5 as a target gene of EBF1 transcription factor.

  7. Finding Combination of Features from Promoter Regions for Ovarian Cancer-related Gene Group Classification

    KAUST Repository

    Olayan, Rawan S.

    2012-12-01

    In classification problems, it is always important to use the suitable combination of features that will be employed by classifiers. Generating the right combination of features usually results in good classifiers. In the situation when the problem is not well understood, data items are usually described by many features in the hope that some of these may be the relevant or most relevant ones. In this study, we focus on one such problem related to genes implicated in ovarian cancer (OC). We try to recognize two important OC-related gene groups: oncogenes, which support the development and progression of OC, and oncosuppressors, which oppose such tendencies. For this, we use the properties of promoters of these genes. We identified potential “regulatory features” that characterize OC-related oncogenes and oncosuppressors promoters. In our study, we used 211 oncogenes and 39 oncosuppressors. For these, we identified 538 characteristic sequence motifs from their promoters. Promoters are annotated by these motifs and derived feature vectors used to develop classification models. We made a comparison of a number of classification models in their ability to distinguish oncogenes from oncosuppressors. Based on 10-fold cross-validation, the resultant model was able to separate the two classes with sensitivity of 96% and specificity of 100% with the complete set of features. Moreover, we developed another recognition model where we attempted to distinguish oncogenes and oncosuppressors as one group from other OC-related genes. That model achieved accuracy of 82%. We believe that the results of this study will help in discovering other OC-related oncogenes and oncosuppressors not identified as yet.

  8. Remodeling epigenetic modifications at tumor suppressor gene promoters with bovine oocyte extract.

    Science.gov (United States)

    Wang, Zhenfei; Yue, Yongli; Han, Pengyong; Sa, Rula; Ren, Xiaolv; Wang, Jie; Bai, Haidong; Yu, Haiquan

    2013-09-01

    Epigenetic silencing of tumor suppressor genes by aberrant DNA methylation and histone modifications at their promoter regions plays an important role in the initiation and progression of cancer. The therapeutic effect of the widely used epigenetic drugs, including DNA methyltransferase inhibitors and histone deacetylase inhibitors, remains unsatisfactory. One important underlying factor in the ineffectiveness of these drugs is that their actions lack specificity. To investigate whether oocyte extract can be used for epigenetic re-programming of cancer cells, H460 human lung cancer cells were reversibly permeabilized and incubated with bovine oocyte extract. Bisulfite sequencing showed that bovine oocyte extract induced significant demethylation at hypermethylated promoter CpG islands of the tumor suppressor genes RUNX3 and CDH1; however, the DNA methylation levels of repetitive sequences were not affected. Chromatin immunoprecipitation showed that bovine oocyte extract significantly reduced transcriptionally repressive histone modifications and increased transcriptionally activating histone modifications at the promoter regions of RUNX3 and CDH1. Bovine oocyte extract reactivated the expression of RUNX3 and CDH1 at both the messenger RNA and the protein levels without up-regulating the transcription of pluripotency-associated genes. At the functional level, anchorage-independent proliferation, migration and invasion of H460 cells was strongly inhibited. These results demonstrate that bovine oocyte extract reactivates epigenetically silenced tumor suppressor genes by remodeling the epigenetic modifications at their promoter regions. Bovine oocyte extract may provide a useful tool for investigating epigenetic mechanisms in cancer and a valuable source for developing novel safe therapeutic approaches that target epigenetic alterations. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  9. Association between Osteopontin Promoter Gene Polymorphisms and Haplotypes with Risk of Diabetic Nephropathy

    Directory of Open Access Journals (Sweden)

    Balneek Singh Cheema

    2015-06-01

    Full Text Available Background: Osteopontin (OPN C-443T promoter polymorphism has been shown as a genetic risk factor for diabetic nephropathy (DN in type 2 diabetic patients (T2D. Methods: In the present study we investigated the association of three functional promoter gene polymorphisms C-443T, delG-156G, and G-66T and their haplotypes with the risk of DN and estimated Glomerular Filtration Rate (eGFR in Asian Indians T2D patients using Real time PCR based Taqman assay. A total of 1165 T2D patients, belonging to two independently ascertained Indian Asian cohorts, were genotyped for three OPN promoter polymorphisms C-443T (rs11730582, delG-156G (rs17524488 and G-66T (rs28357094. Results: -156G allele and GG genotypes (delG-156G and haplotypes G-C-G and T-C-G (G-66T, C-443T, delG-156G were associated with decreased risk of DN and higher eGFR. Haplotype G-T-delG and T-T-delG (G-66T, C-443T, delG-156G were identified as risk haplotypes, as shown by lower eGFR. Conclusion: This is the first study to report an association of OPN promoter gene polymorphisms; G-66T and delG-156G and their haplotypes with DN in T2D. Our results suggest an association between OPN promoter gene polymorphisms and their haplotypes with DN.

  10. Characterization of Saccharomyces cerevisiae promoters for heterologous gene expression in Kluyveromyces marxianus.

    Science.gov (United States)

    Lee, Ki-Sung; Kim, Jun-Seob; Heo, Paul; Yang, Tae-Jun; Sung, Young-Je; Cheon, Yuna; Koo, Hyun Min; Yu, Byung Jo; Seo, Jin-Ho; Jin, Yong-Su; Park, Jae Chan; Kweon, Dae-Hyuk

    2013-03-01

    Kluyveromyces marxianus is now considered one of the best choices of option for industrial applications of yeast because the strain is able to grow at high temperature, utilizes various carbon sources, and grows fast. However, the use of K. marxianus as a host for industrial applications is still limited. This limitation is largely due to a lack of knowledge on the characteristics of the promoters since the time and amount of protein expression is strongly dependent on the promoter employed. In this study, four well-known constitutive promoters (P(CYC), P(TEF), P(GPD), and P(ADH)) of Saccharomyces cerevisiae were characterized in K. marxianus in terms of protein expression level and their stochastic behavior. After constructing five URA3-auxotrophic K. marxianus strains and a plasmid vector, four cassettes each comprising one of the promoters--the gene for the green fluorescence protein (GFP)--CYC1 terminator (T(CYC)) were inserted into the vector. GFP expression under the control of each one of the promoters was analyzed by reverse transcription PCR, fluorescence microscopy, and flow cytometer. Using these combined methods, the promoter strength was determined to be in the order of P(GPD) > P(ADH) ∼ P(TEF) > P(CYC). All promoters except for the P(CYC) exhibited three distinctive populations, including non-expressing cells, weakly expressing cells, and strongly expressing cells. The relative ratios between populations were strongly dependent on the promoter and culture time. Forward scattering was independent of GFP fluorescence intensity, indicating that the different fluorescence intensities were not just due to different cell sizes derived from budding. It also excluded the possibility that the non-expressing cells resulted from plasmid loss because plasmid stability was maintained at almost 100 % over the culture time. The same cassettes, cloned into a single copy plasmid pRS416 and transformed into S. cerevisiae, showed only one population. When the

  11. Identification and annotation of conserved promoters and macrophage-expressed genes in the pig genome.

    Science.gov (United States)

    Robert, Christelle; Kapetanovic, Ronan; Beraldi, Dario; Watson, Mick; Archibald, Alan L; Hume, David A

    2015-11-18

    The FANTOM5 consortium used Cap Analysis of Gene Expression (CAGE) tag sequencing to produce a comprehensive atlas of promoters and enhancers within the human and mouse genomes. We reasoned that the mapping of these regulatory elements to the pig genome could provide useful annotation and evidence to support assignment of orthology. For human transcription start sites (TSS) associated with annotated human-mouse orthologs, 17% mapped to the pig genome but not to the mouse, 10% mapped only to the mouse, and 55% mapped to both pig and mouse. Around 17% did not map to either species. The mapping percentages were lower where there was not clear orthology relationship, but in every case, mapping to pig was greater than to mouse, and the degree of homology was also greater. Combined mapping of mouse and human CAGE-defined promoters identified at least one putative conserved TSS for >16,000 protein-coding genes. About 54% of the predicted locations of regulatory elements in the pig genome were supported by CAGE and/or RNA-Seq analysis from pig macrophages. Comparative mapping of promoters and enhancers from humans and mice can provide useful preliminary annotation of other animal genomes. The data also confirm extensive gain and loss of regulatory elements between species, and the likelihood that pigs provide a better model than mice for human gene regulation and function.

  12. Identification of the MUC2 Promoter as a Strong Promoter for Intestinal Gene Expression through Generation of Transgenic Quail Expressing GFP in Gut Epithelial Cells.

    Science.gov (United States)

    Woodfint, Rachel M; Chen, Paula R; Ahn, Jinsoo; Suh, Yeunsu; Hwang, Seongsoo; Lee, Sang Suk; Lee, Kichoon

    2017-01-19

    Identification of tissue- and stage-specific gene promoters is valuable for delineating the functional roles of specific genes in genetically engineered animals. Here, through the comparison of gene expression in different tissues by analysis of a microarray database, the intestinal specificity of mucin 2 ( MUC2 ) expression was identified in mice and humans, and further confirmed in chickens by RT-PCR (reverse transcription-PCR) analysis. An analysis of cis -acting elements in avian MUC2 gene promoters revealed conservation of binding sites, within a 2.9 kb proximal promoter region, for transcription factors such as caudal type homeobox 2 (CDX2), GATA binding protein 4 (GATA4), hepatocyte nuclear factor 4 α (HNF4A), and transcription factor 4 (TCF4) that are important for maintaining intestinal homeostasis and functional integrity. By generating transgenic quail, we demonstrated that the 2.9 kb chicken MUC2 promoter could drive green fluorescent protein (GFP) reporter expression exclusively in the small intestine, large intestine, and ceca. Fluorescence image analysis further revealed GFP expression in intestine epithelial cells. The GFP expression was barely detectable in the embryonic intestine, but increased during post-hatch development. The spatiotemporal expression pattern of the reporter gene confirmed that the 2.9 kb MUC2 promoter could retain the regulatory element to drive expression of target genes in intestinal tissues after hatching. This new transgene expression system, using the MUC2 promoter, will provide a new method of overexpressing target genes to study gene function in the avian intestine.

  13. Identification of the MUC2 Promoter as a Strong Promoter for Intestinal Gene Expression through Generation of Transgenic Quail Expressing GFP in Gut Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Rachel M. Woodfint

    2017-01-01

    Full Text Available Identification of tissue- and stage-specific gene promoters is valuable for delineating the functional roles of specific genes in genetically engineered animals. Here, through the comparison of gene expression in different tissues by analysis of a microarray database, the intestinal specificity of mucin 2 (MUC2 expression was identified in mice and humans, and further confirmed in chickens by RT-PCR (reverse transcription-PCR analysis. An analysis of cis-acting elements in avian MUC2 gene promoters revealed conservation of binding sites, within a 2.9 kb proximal promoter region, for transcription factors such as caudal type homeobox 2 (CDX2, GATA binding protein 4 (GATA4, hepatocyte nuclear factor 4 α (HNF4A, and transcription factor 4 (TCF4 that are important for maintaining intestinal homeostasis and functional integrity. By generating transgenic quail, we demonstrated that the 2.9 kb chicken MUC2 promoter could drive green fluorescent protein (GFP reporter expression exclusively in the small intestine, large intestine, and ceca. Fluorescence image analysis further revealed GFP expression in intestine epithelial cells. The GFP expression was barely detectable in the embryonic intestine, but increased during post-hatch development. The spatiotemporal expression pattern of the reporter gene confirmed that the 2.9 kb MUC2 promoter could retain the regulatory element to drive expression of target genes in intestinal tissues after hatching. This new transgene expression system, using the MUC2 promoter, will provide a new method of overexpressing target genes to study gene function in the avian intestine.

  14. Structure of 5' region of human tenascin-R gene and characterization of its promoter.

    Science.gov (United States)

    Gherzi, R; Leprini, A; Siri, A; Zardi, L

    1998-03-01

    The tenascin-R (TN-R) gene encodes a multidomain extracellular matrix protein belonging to the tenascin family, previously detected only in the central nervous system. In this report, we describe the structure of the 5' region of the human TN-R gene and characterize the activity of its promoter. We cloned two previously unreported nontranslated exons (exons 1 and 2, 539 and 101 bp in length, respectively) separated by a large (> or = 40-kb) intron. The intron between exons 2 and 3 (containing the ATG codon) is 122 kb in length. Tenascin-R transcripts in fetal, adult, and neoplastic human brain contain both exons 1 and 2, as demonstrated by S1 nuclease analysis and reverse transcriptase-polymerase chain reaction. The human TN-R promoter displays relatively unusual features in terms of sequence in that it lacks any TATA box, CAAT box, GC-rich regions, or initiator element. The promoter displays its activity only in cultured cells of neural and glial origin, not in transformed epithelial cells and melanoma cells. All the elements required for the full and cell-specific activity of the promoter are contained in the 57-bp sequence closest to the transcription startpoint.

  15. Promoter Hypermethylation of the EMP3 Gene in a Series of 229 Human Gliomas

    Directory of Open Access Journals (Sweden)

    Marta Mellai

    2013-01-01

    Full Text Available The epithelial membrane protein 3 (EMP3 is a candidate tumor suppressor gene in the critical region 19q13.3 for several solid tumors, including tumors of the nervous systems. The aim of this study was to investigate the EMP3 promoter hypermethylation status in a series of 229 astrocytic and oligodendroglial tumors and in 16 GBM cell lines. The analysis was performed by methylation-specific PCR and capillary electrophoresis. Furthermore, the EMP3 expression at protein level was evaluated by immunohistochemistry and Western blotting analysis. Associations of EMP3 hypermethylation with total 1p/19q codeletion, MGMT promoter hypermethylation, IDH1/IDH2 and TP53 mutations, and EGFR amplification were studied, as well as its prognostic significance. The EMP3 promoter hypermethylation has been found in 39.5% of gliomas. It prevailed in low-grade tumors, especially in gliomas with an oligodendroglial component, and in sGBMs upon pGBMs. In oligodendroglial tumors, it was strongly associated with both IDH1/IDH2 mutations and total 1p/19q codeletion and inversely with EGFR gene amplification. No association was found with MGMT hypermethylation and TP53 mutations. In the whole series, the EMP3 hypermethylation status correlated with 19q13.3 loss and lack of EMP3 expression at protein level. A favorable prognostic significance on overall survival of the EMP3 promoter hypermethylation was found in patients with oligodendroglial tumors.

  16. Genomic organization and promoter cloning of the human X11α gene APBA1.

    LENUS (Irish Health Repository)

    Chai, Ka-Ho

    2012-05-01

    X11α is a brain specific multi-modular protein that interacts with the Alzheimer\\'s disease amyloid precursor protein (APP). Aggregation of amyloid-β peptide (Aβ), an APP cleavage product, is believed to be central to the pathogenesis of Alzheimer\\'s disease. Recently, overexpression of X11α has been shown to reduce Aβ generation and to ameliorate memory deficit in a transgenic mouse model of Alzheimer\\'s disease. Therefore, manipulating the expression level of X11α may provide a novel route for the treatment of Alzheimer\\'s disease. Human X11α is encoded by the gene APBA1. As evidence suggests that X11α expression can be regulated at transcription level, we have determined the gene structure and cloned the promoter of APBA1. APBA1 spans over 244 kb on chromosome 9 and is composed of 13 exons and has multiple transcription start sites. A putative APBA1 promoter has been identified upstream of exon 1 and functional analysis revealed that this is highly active in neurons. By deletion analysis, the minimal promoter was found to be located between -224 and +14, a GC-rich region that contains a functional Sp3 binding site. In neurons, overexpression of Sp3 stimulates the APBA1 promoter while an Sp3 inhibitor suppresses the promoter activity. Moreover, inhibition of Sp3 reduces endogenous X11α expression and promotes the generation of Aβ. Our findings reveal that Sp3 play an essential role in APBA1 transcription.

  17. Promotion of growth by Coenzyme Q10 is linked to gene expression in C. elegans.

    Science.gov (United States)

    Fischer, Alexandra; Niklowitz, Petra; Menke, Thomas; Döring, Frank

    2014-10-03

    Coenzyme Q (CoQ, ubiquinone) is an essential component of the respiratory chain, a cofactor of pyrimidine biosynthesis and acts as an antioxidant in extra mitochondrial membranes. More recently CoQ has been identified as a modulator of apoptosis, inflammation and gene expression. CoQ deficient Caenorhabditis elegans clk-1 mutants show several phenotypes including a delayed postembryonic growth. Using wild type and two clk-1 mutants, here we established an experimental set-up to study the consequences of endogenous CoQ deficiency or exogenous CoQ supply on gene expression and growth. We found that a deficiency of endogenous CoQ synthesis down-regulates a cluster of genes that are important for growth (i.e., RNA polymerase II, eukaryotic initiation factor) and up-regulates oxidation reactions (i.e., cytochrome P450, superoxide dismutase) and protein interactions (i.e., F-Box proteins). Exogenous CoQ supply partially restores the expression of these genes as well as the growth retardation of CoQ deficient clk-1 mutants. On the other hand exogenous CoQ supply does not alter the expression of a further sub-set of genes. These genes are involved in metabolism (i.e., succinate dehydrogenase complex), cell signalling or synthesis of lectins. Thus, our work provides a comprehensive overview of genes which can be modulated in their expression by endogenous or exogenous CoQ. As growth retardation in CoQ deficiency is linked to the gene expression profile we suggest that CoQ promotes growth via gene expression. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Bicarbonate increases binding affinity of Vibrio cholerae ToxT to virulence gene promoters.

    Science.gov (United States)

    Thomson, Joshua J; Withey, Jeffrey H

    2014-11-01

    The major Vibrio cholerae virulence gene transcription activator, ToxT, is responsible for the production of the diarrhea-inducing cholera toxin (CT) and the major colonization factor, toxin coregulated pilus (TCP). In addition to the two primary virulence factors mentioned, ToxT is responsible for the activation of accessory virulence genes, such as aldA, tagA, acfA, acfD, tcpI, and tarAB. ToxT activity is negatively modulated by bile and unsaturated fatty acids found in the upper small intestine. Conversely, previous work identified another intestinal signal, bicarbonate, which enhances the ability of ToxT to activate production of CT and TCP. The work presented here further elucidates the mechanism for the enhancement of ToxT activity by bicarbonate. Bicarbonate was found to increase the activation of ToxT-dependent accessory virulence promoters in addition to those that produce CT and TCP. Bicarbonate is taken up into the V. cholerae cell, where it positively affects ToxT activity by increasing DNA binding affinity for the virulence gene promoters that ToxT activates regardless of toxbox configuration. The increase in ToxT binding affinity in the presence of bicarbonate explains the elevated level of virulence gene transcription. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  19. Structure of the gene for human β2-adrenergic receptor: expression and promoter characterization

    International Nuclear Information System (INIS)

    Emorine, L.J.; Marullo, S.; Delavier-Klutchko, C.; Kaveri, S.V.; Durieu-Trautmann, O.; Strosberg, A.D.

    1987-01-01

    The genomic gene coding for the human β 2 -adrenergic receptor (β 2 AR) from A431 epidermoid cells has been isolated. Transfection of the gene into eukaryotic cells restores a fully active receptor/GTP-binding protein/adenylate cyclase complex with β 2 AR properties. Southern blot analyses with β 2 AR-specific probes show that a single β 2 AR gene is common to various human tissues and that its flanking sequences are highly conserved among humans and between man and rabbit, mouse, and hamster. Functional significance of these regions is supported by the presence of a promoter region (including mRNA cap sites, two TATA boxes, a CAAT box, and three G + C-rich regions that resemble binding sites for transcription factor Sp1) 200-300 base pairs 5' to the translation initiation codon. In the 3' flanking region, sequences homologous to glucocorticoid-response elements might be responsible for the increased expression of the β 2 AR gene observed after treatment of the transfected cells with hydrocortisone. In addition, 5' to the promoter region, an open reading frame encodes a 251-residue polypeptide that displays striking homologies with protein kinases and other nucleotide-binding proteins

  20. Cloning and characterization of largemouth bass ( Micropterus salmoides) myostatin encoding gene and its promoter

    Science.gov (United States)

    Li, Shengjie; Bai, Junjie; Wang, Lin

    2008-08-01

    Myostatin or GDF-8, a member of the transforming growth factor-β (TGF-β) superfamily, has been demonstrated to be a negative regulator of skeletal muscle mass in mammals. In the present study, we obtained a 5.64 kb sequence of myostatin encoding gene and its promoter from largemouth bass ( Micropterus salmoides). The myostatin encoding gene consisted of three exons (488 bp, 371 bp and 1779 bp, respectively) and two introns (390 bp and 855 bp, respectively). The intron-exon boundaries were conservative in comparison with those of mammalian myostatin encoding genes, whereas the size of introns was smaller than that of mammals. Sequence analysis of 1.569 kb of the largemouth bass myostatin gene promoter region revealed that it contained two TATA boxes, one CAAT box and nine putative E-boxes. Putative muscle growth response elements for myocyte enhancer factor 2 (MEF2), serum response factor (SRF), activator protein 1 (AP1), etc., and muscle-specific Mt binding site (MTBF) were also detected. Some of the transcription factor binding sites were conserved among five teleost species. This information will be useful for studying the transcriptional regulation of myostatin in fish.

  1. Changes in Methylation Patterns of Kiss1 and Kiss1r Gene Promoters across Puberty

    Directory of Open Access Journals (Sweden)

    Amanda K. Wyatt

    2013-01-01

    Full Text Available The initiation of mammalian puberty is underpinned by an increase in Kisspeptin (Kiss1 signaling via its receptor (Kiss1r/GPR54 on gonadotropin-releasing hormone (GnRH neurons. Animals and humans with loss-of-function mutations in Kiss1 or Kiss1r fail to go through puberty. The timing of puberty is dependent on environmental factors, and malleability in puberty timing suggests a mechanism that can translate environmental signals into patterns of Kiss1/Kiss1r gene expression. Epigenetics is a powerful mechanism that can control gene expression in an environment-dependent manner. We investigated whether epigenetic DNA methylation is associated with gene expression changes at puberty. We used bisulfite-PCR-pyrosequencing to define the methylation in the promoters of Kiss1 and Kiss1r before and after puberty in female rats. Both Kiss1 and Kiss1r showed highly significant puberty-specific differential promoter methylation patterns. By identifying key differentially methylated residues associated with puberty, these findings will be important for further studies investigating the control of gene expression across the pubertal transition.

  2. Pattern of cavities in globins: the case of human hemoglobin.

    Science.gov (United States)

    Savino, Carmelinda; Miele, Adriana E; Draghi, Federica; Johnson, Kenneth A; Sciara, Giuliano; Brunori, Maurizio; Vallone, Beatrice

    2009-12-01

    Our aim is to shed light on the conservation of potential ligand docking sites that play an important role in ligand dynamics of globins by using the technique of filling internal cavities naturally present in hemoglobin and myoglobin with xenon atoms. In particular, we present the high resolution structures of the Xe-adduct of deoxygenated wild type human hemoglobin and a quadruple mutant (L(B10)Y and H(E7)Q in alpha and beta chains). For the sake of comparison we also determined under the same experimental conditions the xenon complex of wild type sperm whale myoglobin. The analysis revealed that the number and position of Xe binding cavities are different in the alpha and beta subunits, the latter being more similar to myoglobin. Notably, no proximal Xe docking site was detected in hemoglobin, at variance with myoglobin. The pattern of internal cavities accessibility and affinity for xenon suggests a different role for the dynamics of ligand migration in the two types of hemoglobin chains as compared to myoglobin. The number and position of hydrophobic cavities in hemoglobin are briefly discussed also in comparison with the data available for other members of the globin superfamily.

  3. Over-expression of Gene FaASR Promotes Strawberry Fruit Coloring

    Directory of Open Access Journals (Sweden)

    Liu Zhongjie

    2015-11-01

    Full Text Available Fruit development and ripening is a complicate process. Although much progress has been made on the ripenig process, the molecular mechamism of fruit development is not yet clear. In this study, we used ‘Sweet Charlie’ strawberry as test materials, based on cloning the strawberries ASR homologous gene, we carried out the bioinformatics and temporal expression analysis of FaASR, by manipulating ASR gene expression level in strawberry fruit, we tested the changes of physiological indicators, including sugar, ABA, pigments content, and fruit firmness, as well as phenotypic changes. In addition, we measured the expression changes of some anthocyanin-related gene, such as CHS and UFGT, by which we revealed the regulation mechanisms of ASR gene over strawberry fruit ripening. Strawberry ASR contained a typical domain of ABA/WDS that was related to fruit ripening and stress-resistance, and ASR gene over-expression could promote strawberry fruit coloring, endogenous ABA and sucrose accumulation, fruit softening, and induced the transcription levels of anthocyanin-related genes CHS and UFGT. The present study will further reveal the molecular mechanisms of information transmission in fruit development, and will also play an important foundation for future molecular improvement of strawberries breeding.

  4. Association of Interleukin-10 Gene Promoter Polymorphisms in Saudi Patients with Vitiligo

    Directory of Open Access Journals (Sweden)

    Abdullah Abanmi

    2008-01-01

    Full Text Available The promoter region of human Interleukin −10 gene is highly polymorphic and has been associated with numerous autoimmune diseases. Recent studies have linked vitiligo with defective autoimmune system. This study is aimed to explore a possible association between IL-10 gene polymorphism and vitiligo in Saudi population. This case control study consisted of 184 Saudi subjects including 83 vitiligo patients (40 males, 43 females mean age 27.85 ± 12.43 years and 101 matched controls. Genomic DNA was extracted from the blood samples of healthy controls and Vitiligo patients visiting out patient clinic of Department of Dermatology, Riyadh Armed Forces Hospital, using QIA ampR DNA mini kit (Qiagen CA, USA. Interleukin-10 gene was amplified by polymerase chain reaction (PCR using Arms primers to detect any polymorphism involved at positions −592, −819 and −1082.

  5. Multiplexed CRISPR/Cas9- and TAR-Mediated Promoter Engineering of Natural Product Biosynthetic Gene Clusters in Yeast.

    Science.gov (United States)

    Kang, Hahk-Soo; Charlop-Powers, Zachary; Brady, Sean F

    2016-09-16

    The use of DNA sequencing to guide the discovery of natural products has emerged as a new paradigm for revealing chemistries encoded in bacterial genomes. A major obstacle to implementing this approach to natural product discovery is the transcriptional silence of biosynthetic gene clusters under laboratory growth conditions. Here we describe an improved yeast-based promoter engineering platform (mCRISTAR) that combines CRISPR/Cas9 and TAR to enable single-marker multiplexed promoter engineering of large gene clusters. mCRISTAR highlights the first application of the CRISPR/Cas9 system to multiplexed promoter engineering of natural product biosynthetic gene clusters. In this method, CRISPR/Cas9 is used to induce DNA double-strand breaks in promoter regions of biosynthetic gene clusters, and the resulting operon fragments are reassembled by TAR using synthetic gene-cluster-specific promoter cassettes. mCRISTAR uses a CRISPR array to simplify the construction of a CRISPR plasmid for multiplex CRISPR and a single auxotrophic selection to improve the inefficiency of using a CRISPR array for multiplex gene cluster refactoring. mCRISTAR is a simple and generic method for multiplexed replacement of promoters in biosynthetic gene clusters that will facilitate the discovery of natural products from the rapidly growing collection of gene clusters found in microbial genome and metagenome sequencing projects.

  6. Identification of a novel first exon in the human dystrophin gene and of a new promoter located more than 500 kb upstream of the nearest known promoter

    Energy Technology Data Exchange (ETDEWEB)

    Yanagawa, H.; Nishio, H.; Takeshima, Y. [Kobe Univ. School of Medicine (Japan)] [and others

    1994-09-01

    The dystrophin gene, which is muted in patients with Duchenne and Becker muscular dystrophies, is the largest known human gene. Five alternative promoters have been characterized until now. Here we show that a novel dystrophin isoform with a different first exon can be produced through transcription initiation at a previously-unidentified alternative promoter. The case study presented is that of patient with Duchenne muscular dystrophy who had a deletion extending from 5{prime} end of the dystrophin gene to exon 2, including all promoters previously mapped in the 5{prime} part of the gene. Transcripts from lymphoblastoid cells were found to contain sequences corresponding to exon 3, indicating the presence of new promoter upstream of this exon. The nucleotide sequence of amplified cDNA corresponding to the 5{prime} end of the new transcript indicated that the 5{prime} end of exon 3 was extended by 9 codons, only the last (most 3{prime}) of which codes for methionine. The genomic nucleotide sequence upstream from the new exon, as determined using inverse polymerase chain reaction, revealed the presence of sequences similar to a TATA box, an octamer motif and an MEF-2 element. The identified promoter/exon did not map to intron 2, as might have been expected, but to a position more than 500 kb upstream of the most 5{prime} of the previously-identified promoters, thereby adding 500 kb to the dystrophin gene. The sequence of part of the new promoter region is very similar to that of certain medium reiteration frequency repetitive sequences. These findings may help us understand the molecular evolution of the dystrophin gene.

  7. Effect of TNF{alpha} on activities of different promoters of human apolipoprotein A-I gene

    Energy Technology Data Exchange (ETDEWEB)

    Orlov, Sergey V., E-mail: serge@iem.sp.ru [Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg (Russian Federation); Department of Embryology, St. Petersburg State University, 199034 St. Petersburg (Russian Federation); Mogilenko, Denis A. [Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg (Russian Federation); Department of Embryology, St. Petersburg State University, 199034 St. Petersburg (Russian Federation); Shavva, Vladimir S. [Department of Embryology, St. Petersburg State University, 199034 St. Petersburg (Russian Federation); Dizhe, Ella B.; Ignatovich, Irina A. [Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg (Russian Federation); Perevozchikov, Andrej P., E-mail: app@iem.sp.ru [Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg (Russian Federation); Department of Embryology, St. Petersburg State University, 199034 St. Petersburg (Russian Federation)

    2010-07-23

    Research highlights: {yields} TNF{alpha} stimulates the distal alternative promoter of human apoA-I gene. {yields} TNF{alpha} acts by weakening of promoter competition within apoA-I gene (promoter switching). {yields} MEK1/2 and nuclear receptors PPAR{alpha} and LXRs take part in apoA-I promoter switching. -- Abstract: Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoproteins. The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1{beta} and TNF{alpha}. Recently, two novel additional (alternative) promoters for human apoA-I gene have been identified. Nothing is known about the role of alternative promoters in TNF{alpha}-mediated downregulation of apoA-I gene. In this article we report for the first time about the different effects of TNF{alpha} on two alternative promoters of human apoA-I gene. Stimulation of HepG2 cells by TNF{alpha} leads to activation of the distal alternative apoA-I promoter and downregulation of the proximal alternative and the canonical apoA-I promoters. This effect is mediated by weakening of the promoter competition within human apoA-I 5'-regulatory region (apoA-I promoter switching) in the cells treated by TNF{alpha}. The MEK1/2-ERK1/2 cascade and nuclear receptors PPAR{alpha} and LXRs are important for TNF{alpha}-mediated apoA-I promoter switching.

  8. Promoter characterization and genomic organization of the human X11β gene APBA2.

    LENUS (Irish Health Repository)

    Hao, Yan

    2012-02-15

    Overexpression of neuronal adaptor protein X11β has been shown to decrease the production of amyloid-β, a toxic peptide deposited in Alzheimer\\'s disease brains. Therefore, manipulation of the X11β level may represent a potential therapeutic strategy for Alzheimer\\'s disease. As X11β expression can be regulated at the transcription level, we determined the genomic organization and the promoter of the human X11β gene, amyloid β A4 precursor protein-binding family A member 2 (APBA2). By RNA ligase-mediated rapid amplification of cDNA ends, a single APBA2 transcription start site and the complete sequence of exon 1 were identified. The APBA2 promoter was located upstream of exon 1 and was more active in neurons. The core promoter contains several CpG dinucleotides, and was strongly suppressed by DNA methylation. In addition, mutagenesis analysis revealed a putative Pax5-binding site within the promoter. Together, APBA2 contains a potent neuronal promoter whose activity may be regulated by DNA methylation and Pax5.

  9. Distinct promoters mediate the regulation of Ebf1 gene expression by interleukin-7 and Pax5.

    Science.gov (United States)

    Roessler, Stephanie; Györy, Ildiko; Imhof, Sascha; Spivakov, Mikhail; Williams, Ruth R; Busslinger, Meinrad; Fisher, Amanda G; Grosschedl, Rudolf

    2007-01-01

    Early differentiation of B lymphocytes requires the function of multiple transcription factors that regulate the specification and commitment of the lineage. Loss- and gain-of-function experiments have provided important insight into the transcriptional control of B lymphopoiesis, whereby E2A was suggested to act upstream of EBF1 and Pax5 downstream of EBF1. However, this simple hierarchy cannot account for all observations, and our understanding of a presumed regulatory network, in which transcription factors and signaling pathways operate, is limited. Here, we show that the expression of the Ebf1 gene involves two promoters that are differentially regulated and generate distinct protein isoforms. We find that interleukin-7 signaling, E2A, and EBF1 activate the distal Ebf1 promoter, whereas Pax5, together with Ets1 and Pu.1, regulates the stronger proximal promoter. In the absence of Pax5, the function of the proximal Ebf1 promoter and accumulation of EBF1 protein are impaired and the replication timing and subcellular localization of the Ebf1 locus are altered. Taken together, these data suggest that the regulation of Ebf1 via distinct promoters allows for the generation of several feedback loops and the coordination of multiple determinants of B lymphopoiesis in a regulatory network.

  10. Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription.

    Science.gov (United States)

    Knight, Britta; Kubik, Slawomir; Ghosh, Bhaswar; Bruzzone, Maria Jessica; Geertz, Marcel; Martin, Victoria; Dénervaud, Nicolas; Jacquet, Philippe; Ozkan, Burak; Rougemont, Jacques; Maerkl, Sebastian J; Naef, Félix; Shore, David

    2014-08-01

    In yeast, ribosome production is controlled transcriptionally by tight coregulation of the 138 ribosomal protein genes (RPGs). RPG promoters display limited sequence homology, and the molecular basis for their coregulation remains largely unknown. Here we identify two prevalent RPG promoter types, both characterized by upstream binding of the general transcription factor (TF) Rap1 followed by the RPG-specific Fhl1/Ifh1 pair, with one type also binding the HMG-B protein Hmo1. We show that the regulatory properties of the two promoter types are remarkably similar, suggesting that they are determined to a large extent by Rap1 and the Fhl1/Ifh1 pair. Rapid depletion experiments allowed us to define a hierarchy of TF binding in which Rap1 acts as a pioneer factor required for binding of all other TFs. We also uncovered unexpected features underlying recruitment of Fhl1, whose forkhead DNA-binding domain is not required for binding at most promoters, and Hmo1, whose binding is supported by repeated motifs. Finally, we describe unusually micrococcal nuclease (MNase)-sensitive nucleosomes at all RPG promoters, located between the canonical +1 and -1 nucleosomes, which coincide with sites of Fhl1/Ifh1 and Hmo1 binding. We speculate that these "fragile" nucleosomes play an important role in regulating RPG transcriptional output. © 2014 Knight et al.; Published by Cold Spring Harbor Laboratory Press.

  11. Association of a Human FABP1 Gene Promoter Region Polymorphism with Altered Serum Triglyceride Levels.

    Directory of Open Access Journals (Sweden)

    Xian-E Peng

    Full Text Available Liver fatty acid-binding protein (L-FABP, also known as fatty acid-binding protein 1 (FABP1, is a key regulator of hepatic lipid metabolism. Elevated FABP1 levels are associated with an increased risk of cardiovascular disease (CVD and metabolic syndromes. In this study, we examine the association of FABP1 gene promoter variants with serum FABP1 and lipid levels in a Chinese population. Four promoter single-nucleotide polymorphisms (SNPs of FABP1 gene were genotyped in a cross-sectional survey of healthy volunteers (n = 1,182 from Fuzhou city of China. Results showed that only the rs2919872 G>A variant was significantly associated with serum TG concentration(P = 0.032.Compared with the rs2919872 G allele, rs2919872 A allele contributed significantly to reduced serum TG concentration, and this allele dramatically decreased the FABP1 promoter activity(P < 0.05. The rs2919872 A allele carriers had considerably lower serum FABP1 levels than G allele carriers (P < 0.01. In the multivariable linear regression analysis, the rs2919872 A allele was negatively associated with serum FABP1 levels (β = -0.320, P = 0.003, while serum TG levels were positively associated with serum FABP1 levels (β = 0.487, P = 0.014. Our data suggest that compared with the rs2919872 G allele, the rs2919872 A allele reduces the transcriptional activity of FABP1 promoter, and thereby may link FABP1 gene variation to TG level in humans.

  12. COX-2 gene expression in colon cancer tissue related to regulating factors and promoter methylation status

    International Nuclear Information System (INIS)

    Asting, Annika Gustafsson; Carén, Helena; Andersson, Marianne; Lönnroth, Christina; Lagerstedt, Kristina; Lundholm, Kent

    2011-01-01

    Increased cyclooxygenase activity promotes progression of colorectal cancer, but the mechanisms behind COX-2 induction remain elusive. This study was therefore aimed to define external cell signaling and transcription factors relating to high COX-2 expression in colon cancer tissue. Tumor and normal colon tissue were collected at primary curative operation in 48 unselected patients. COX-2 expression in tumor and normal colon tissue was quantified including microarray analyses on tumor mRNA accounting for high and low tumor COX-2 expression. Cross hybridization was performed between tumor and normal colon tissue. Methylation status of up-stream COX-2 promoter region was evaluated. Tumors with high COX-2 expression displayed large differences in gene expression compared to normal colon. Numerous genes with altered expression appeared in tumors of high COX-2 expression compared to tumors of low COX-2. COX-2 expression in normal colon was increased in patients with tumors of high COX-2 compared to normal colon from patients with tumors of low COX-2. IL1β, IL6 and iNOS transcripts were up-regulated among external cell signaling factors; nine transcription factors (ATF3, C/EBP, c-Fos, Fos-B, JDP2, JunB, c-Maf, NF-κB, TCF4) showed increased expression and 5 (AP-2, CBP, Elk-1, p53, PEA3) were decreased in tumors with high COX-2. The promoter region of COX-2 gene did not show consistent methylation in tumor or normal colon tissue. Transcription and external cell signaling factors are altered as covariates to COX-2 expression in colon cancer tissue, but DNA methylation of the COX-2 promoter region was not a significant factor behind COX-2 expression in tumor and normal colon tissue

  13. Specific expression of bioluminescence reporter gene in cardiomyocyte regulated by tissue specific promoter

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Vu Hong; Tae, Seong Ho; Le, Nguyen Uyen Chi; Min, Jung Joon [Chonnam National University Medical School, Gwangju (Korea, Republic of)

    2007-07-01

    As the human heart is not capable of regenerating the great numbers of cardiac cells that are lost after myocardial infarction, impaired cardiac function is the inevitable result of ischemic disease. Recently, human embryonic stem cells (hESCs) have gained popularity as a potentially ideal cell candidate for tissue regeneration. In particular, hESCs are capable of cardiac lineage-specific differentiation and confer improvement of cardiac function following transplantation into animal models. Although such data are encouraging, the specific strategy for in vivo and non-invasive detection of differentiated cardiac lineage is still limited. Therefore, in the present study, we established the gene construction in which the optical reporter gene Firefly luciferase was controlled by Myosin Heavy Chain promoter for specific expressing in heart cells. The vector consisting of - MHC promoter and a firefly luciferase coding sequence flanked by full-length bovine growth hormone (BGH) 3'-polyadenylation sequence based on pcDNA3.1- vector backbone. To test the specific transcription of this promoter in g of MHC-Fluc or CMV-Flue (for control) plasmid DNA in myocardial tissue, 20 phosphate-buffered saline was directly injected into mouse myocardium through a midline sternotomy and liver. After 1 week of injection, MHC-Fluc expression was detected from heart region which was observed under cooled CCD camera of in vivo imaging system but not from liver. In control group injected with CMV-Flue, the bioluminescence was detected from all these organs. The expression of Flue under control of Myosin Heavy Chain promoter may become a suitable optical reporter gene for stem cell-derived cardiac lineage differentiation study.

  14. Performance- and safety-enhanced lentiviral vectors containing the human interferon-beta scaffold attachment region and the chicken beta-globin insulator.

    Science.gov (United States)

    Ramezani, Ali; Hawley, Teresa S; Hawley, Robert G

    2003-06-15

    Retroviral vectors are the most efficient means of stable gene delivery to hematopoietic stem cells (HSCs). However, transgene expression from retroviral vectors is frequently subject to the negative influence of chromosomal sequences flanking the site of integration. Toward the development of autonomous transgene expression cassettes, we inserted the human interferon-beta scaffold attachment region (IFN-SAR) and the chicken beta-globin 5' DNase I hypersensitive site 4 (5'HS4) insulator both separately and together into a series of self-inactivating (SIN) lentiviral vector backbones. Transduced cells of the human CD34+ hematopoietic progenitor line KG1a-pooled populations as well as individual clones harboring single integrants--were analyzed for reporter expression during culture periods of up to 4 months. Vectors carrying both the 5'HS4 insulator and the IFN-SAR consistently outperformed control vectors without inserts as well as vectors carrying either element alone. The performance of a set of vectors containing the murine stem cell virus long terminal repeat as an internal promoter was subsequently assessed during in vitro monocytic differentiation of transduced primary human CD34+ cord blood cells. Similar to what was observed in the KG1a hematopoietic progenitor cell model, optimal reporter expression in primary monocytes was obtained with the vector bearing both regulatory elements. These findings indicate that the 5'HS4/IFN-SAR combination is particularly effective at maintaining open chromatin domains permissive for high-level transgene expression at early and late stages of hematopoietic development, and thus could be of utility in HSC-directed retroviral vector-mediated gene transfer applications.

  15. [Morphological features of transgenic tobacco plants expressing the AINTEGUMENTA gene of rape under control of the Dahlia mosaic virus promoter].

    Science.gov (United States)

    Kuluev, B R; Kniazev, A V; Cheremis, A V; Vakhitov, V A

    2013-01-01

    Transgenic tobacco plants expressing the AINTEGUMENTA gene of rape under control of the 35S promoter and the promoter of dahlia mosaic virus were obtained. The transgenic plants were characterized by increase in the length of the leaves, flower sizes, stem height, and weight of seeds; at the same time, the degree of increase was greater in the case of use of the dahlia mosaic virus promoter as a regulator of transcription. Ectopic expression of the AINTEGUMENTA gene promoted prolongation of leaf growth, while sizes of epidermal cells of the leaves remained unchanged.

  16. Putative DNA G-quadruplex formation within the promoters of Plasmodium falciparum var genes

    Directory of Open Access Journals (Sweden)

    Rowe J

    2009-08-01

    Full Text Available Abstract Background Guanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can downregulate gene expression, possibly by blocking the transcriptional machinery. Here we have used a genome-wide bioinformatic approach to identify Putative G-Quadruplex Sequences (PQS in the Plasmodium falciparum genome, along with biophysical techniques to examine the physiological stability of P. falciparum PQS in vitro. Results We identified 63 PQS in the non-telomeric regions of the P. falciparum clone 3D7. Interestingly, 16 of these PQS occurred in the upstream region of a subset of the P. falciparum var genes (group B var genes. The var gene family encodes PfEMP1, the parasite's major variant antigen and adhesin expressed at the surface of infected erythrocytes, that plays a key role in malaria pathogenesis and immune evasion. The ability of the PQS found in the upstream regions of group B var genes (UpsB-Q to form stable G-quadruplex structures in vitro was confirmed using 1H NMR, circular dichroism, UV spectroscopy, and thermal denaturation experiments. Moreover, the synthetic compound BOQ1 that shows a higher affinity for DNA forming quadruplex rather than duplex structures was found to bind with high affinity to the UpsB-Q. Conclusion This is the first demonstration of non-telomeric PQS in the genome of P. falciparum that form stable G-quadruplexes under physiological conditions in vitro. These results allow the generation of a novel hypothesis that the G-quadruplex sequences in the upstream regions of var genes have the potential to play a role in the transcriptional control of this major virulence-associated multi-gene family.

  17. Opposite responses of rabbit and human globin mRNAs to translational inhibition by cap analogues

    International Nuclear Information System (INIS)

    Shakin, S.H.; Liebhaber, S.A.

    1987-01-01

    The translational efficiency of an mRNA may be determined at the step of translational initiation by the efficiency of its interaction with the cap binding protein complex. To further investigate the role of these interactions in translational control, the authors compare in vitro the relative sensitivities of rabbit and human α- and β-globin mRNAs to translational inhibition by cap analogues. They find that rabbit β-globin mRNA is more resistant to translational inhibition by cap analogues than rabbit α-globin mRNA, while in contrast, human β-globin mRNA is more sensitive to cap analogue inhibition than human α- and β-globin mRNAs is unexpected as direct in vivo and in vitro comparisons of polysome profiles reveal parallel translational handling of the α- and β-globin mRNAs from these two species. This discordance between the relative translational sensitivities of these mRNAs to cap analogues and their relative ribosome loading activities suggests that cap-dependent events may not be rate limiting in steady-state globin translation

  18. Beta globin messenger RNA content of bone marrow erythroblasts in heterozygous beta-thalassemia.

    Science.gov (United States)

    Benz, E J; Pritchard, J; Hillman, D; Glass, J; Forget, B G

    1984-01-01

    RNA from bone marrow erythroblasts and peripheral blood reticulocytes of patients with heterozygous beta-thalassemia was analyzed for relative content of alpha and beta globin messenger RNA by molecular hybrization. Erythroblasts from nonthalassemic patients exhibited approximately the same alpha and beta globin mRNA content (beta/alpha mRNA ratio = 0.8-1.0) as circulating reticulocytes (beta/alpha mRNA ratio = 0.74-1.2). The mRNA ratios corresponded well to levels of globin synthesis observed in bone marrow and peripheral blood. Erythroblasts from four patients with heterozygous beta-thalassemia also exhibited approximately the same beta/alpha mRNA ratios in bone marrow erythroblasts (0.34-0.59) as in reticulocytes (0.34-0.4): beta globin mRNA was clearly deficient in bone marrow erythroblasts. Globin biosynthesis by erythroblasts of beta-thalassemia heterozygotes was balanced despite the mRNA deficiency (beta/alpha = 0.9-1.0), suggesting that post-translational phenoma (eg, proteolysis of free globin chains), rather than instability of beta mRNA, accounts for the balanced globin chain synthesis frequently observed in bone marrow erythroblasts of patients with beta-thalassemia trait.

  19. Functional analysis of the genetic variability in the F7 gene promoter.

    Science.gov (United States)

    Sabater-Lleal, Maria; Chillón, Miguel; Howard, Tom E; Gil, Estel; Almasy, Laura; Blangero, John; Fontcuberta, Jordi; Soria, José Manuel

    2007-12-01

    The FVII level is considered a risk factor for cardiovascular disease. Some of the polymorphic differences in the promoter of the F7 gene have been associated with variations in FVII levels. However, linkage disequilibrium among those polymorphisms has made it difficult to pinpoint the true functional variants, so contradictory results have often appeared among various studies. We provide new findings of the effect of the polymorphisms in the promoter region of F7. In vitro transfection of 15 plasmids containing different combinations of F7 promoter polymorphisms was performed in HepG2 cells. We found that allelic variants -323ins10 and -122C strongly reduced promoter activity and that allelic variant -402A significantly increased promoter activity. We report the effect of a novel variant (-2989A) that significantly increases F7 expression levels. However, this novel allelic variant is in strong linkage disequilibrium with the -323ins10 variant in our Spanish population, which has a clear dominant effect over the -2989A variant and completely masks its effect. Our results have important implications for mapping genes affecting complex diseases using association studies. That is, they imply that true functional variants should be chosen to confirm the analyses and to ensure that the results can be reproduced in other populations. In addition, our results suggest that it would be informative to screen for the -2989A variant in other populations, since it may well be a risk factor for cardiovascular disease in populations where it does not appear with the decanucleotide insertion.

  20. Functional analysis of promoter variants in the microsomal triglyceride transfer protein (MTTP) gene.

    Science.gov (United States)

    Rubin, Diana; Schneider-Muntau, Alexandra; Klapper, Maja; Nitz, Inke; Helwig, Ulf; Fölsch, Ulrich R; Schrezenmeir, Jürgen; Döring, Frank

    2008-01-01

    The microsomal triglyceride transfer protein (MTTP) is required for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins from the intestine and liver. According to this function, polymorphic sites in the MTTP gene showed associations to low-density lipoprotein (LDL) cholesterol and related traits of the metabolic syndrome. Here we studied the functional impact of common MTTP promoter polymorphisms rs1800804:T>C (-164T>C), rs1800803:A>T (-400A>T), and rs1800591:G>T (-493G>T) using gene-reporter assays in intestinal Caco-2 and liver Huh-7 cells. Significant results were obtained in Huh-7 cells. The common MTTP promoter haplotype -164T/-400A/-493G showed about two-fold lower activity than the rare haplotype -164C/-400T/-493T. MTTP promoter mutant constructs -164T/-400A/-493T and -164T/-400T/-493T exhibited similar activity than the common haplotype. Activities of mutants -164C/-400A/-493G and -164C/-400A/-493T resembled the rare MTTP promoter haplotype. Electrophoretic mobility shift assays (EMSAs) revealed higher binding capacity of the transcriptional factor Sterol regulatory element binding protein1a (SREBP1a) to the -164T probe in comparison to the -164C probe. In conclusion, our study indicates that the polymorphism -164T>C mediates different activities of common MTTP promoter haplotypes via SREBP1a. This suggested that the already described SREBP-dependent modulation of MTTP expression by diet is more effective in -164T than in -164C carriers. (c) 2007 Wiley-Liss, Inc.

  1. Cathepsin L gene expression and promoter activation in rodent granulosa cells.

    Science.gov (United States)

    Sriraman, Venkataraman; Richards, JoAnne S

    2004-02-01

    The cysteine protease cathepsin L exhibits hormone-regulated expression during ovulation. In situ hybridization analyses of immature and pregnant mare serum gonadotropin-treated mouse and rat ovaries showed that cathepsin L expression in granulosa cells of small, growing follicles increased in periovulatory follicles after human chorionic gonadotropin stimulation. In the rat ovary, cathepsin L was also expressed in follicles with signs of atresia. To determine the molecular mechanisms that mediate the diverse regulation of this gene in granulosa cells, rat cathepsin L promoter-reporter constructs were analyzed by transient transfection assays in rat granulosa cells and EMSAs. A construct containing the transcriptional start site and -244 bp of upstream promoter sequence (-244/+33 bp) exhibited inducibility by forskolin, the phorbol ester phorbol myristate acetate, and an additive effect of both. Within this region, three functional specificity protein 1 (Sp1) sites, an overlapping early growth response protein-1 site, and a cAMP regulatory element-binding protein site were identified. Single or double mutants of the above-mentioned sites did not alter forskolin/phorbol myristate acetate inducibility of the promoter. Mutation of all three Sp1/specificity protein 3 (Sp3) sites, which also mutated the early growth response protein-1 site, reduced the promoter activation. Mutation of the cAMP regulatory element-binding protein site in the triple Sp1 mutant construct completely blocked the inducibility of the promoter. When these same constructs were transfected into MCF-7 human breast cancer cells or were cotransfected with an Sp1 expression vector in Drosophila SL2 cells, similar results were obtained. Collectively, the data document that three Sp1/specificity protein 3 binding GC-rich regions and a functional cAMP regulatory element constitute an important transcriptional regulatory complex for expression of the cathepsin L gene in rat granulosa cells.

  2. Comparisons of Ribosomal Protein Gene Promoters Indicate Superiority of Heterologous Regulatory Sequences for Expressing Transgenes in Phytophthora infestans.

    Science.gov (United States)

    Poidevin, Laetitia; Andreeva, Kalina; Khachatoorian, Careen; Judelson, Howard S

    2015-01-01

    Molecular genetics approaches in Phytophthora research can be hampered by the limited number of known constitutive promoters for expressing transgenes and the instability of transgene activity. We have therefore characterized genes encoding the cytoplasmic ribosomal proteins of Phytophthora and studied their suitability for expressing transgenes in P. infestans. Phytophthora spp. encode a standard complement of 79 cytoplasmic ribosomal proteins. Several genes are duplicated, and two appear to be pseudogenes. Half of the genes are expressed at similar levels during all stages of asexual development, and we discovered that the majority share a novel promoter motif named the PhRiboBox. This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis. Promoters from the three P. infestans genes encoding ribosomal proteins S9, L10, and L23 and their orthologs from P. capsici were tested for their ability to drive transgenes in stable transformants of P. infestans. Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters. With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression. Since cross-talk between native and transgene loci may trigger gene silencing, we encourage the use of heterologous promoters in transformation studies.

  3. Integration of molecular biology tools for identifying promoters and genes abundantly expressed in flowers of Oncidium Gower Ramsey

    Directory of Open Access Journals (Sweden)

    Tung Shu-Yun

    2011-04-01

    Full Text Available Abstract Background Orchids comprise one of the largest families of flowering plants and generate commercially important flowers. However, model plants, such as Arabidopsis thaliana do not contain all plant genes, and agronomic and horticulturally important genera and species must be individually studied. Results Several molecular biology tools were used to isolate flower-specific gene promoters from Oncidium 'Gower Ramsey' (Onc. GR. A cDNA library of reproductive tissues was used to construct a microarray in order to compare gene expression in flowers and leaves. Five genes were highly expressed in flower tissues, and the subcellular locations of the corresponding proteins were identified using lip transient transformation with fluorescent protein-fusion constructs. BAC clones of the 5 genes, together with 7 previously published flower- and reproductive growth-specific genes in Onc. GR, were identified for cloning of their promoter regions. Interestingly, 3 of the 5 novel flower-abundant genes were putative trypsin inhibitor (TI genes (OnTI1, OnTI2 and OnTI3, which were tandemly duplicated in the same BAC clone. Their promoters were identified using transient GUS reporter gene transformation and stable A. thaliana transformation analyses. Conclusions By combining cDNA microarray, BAC library, and bombardment assay techniques, we successfully identified flower-directed orchid genes and promoters.

  4. Interleukin 4 (IL-4) gene promoter polymorphisms in Rhombomys opimus, the main reservoir of zoonotic cutaneous leishmaniasis.

    Science.gov (United States)

    Bakhshi, H; Borhani, N; Mohebali, M; Khamesipour, A; Abai, M R; Hajjaran, H; Tajedin, L; Rassi, Y; Akhavan, A A; Mohtarami, F; Oshaghi, M A

    2014-01-01

    Great gerbils (Rhombomys opimus) are the most common gerbils in center to northeast of Iran as well as central Asia and serve as reservoirs for the zoonotic agents, including Leishmania major, the principal etiologic agent of zoonotic cutaneous leishmaniasis (ZCL). The outcome of L. major infection in gerbils is not uniform. Among several immune-related factors including cytokine genes, the polymorphism in interleukin 4 (IL-4) promoter gene showed a great impact on outcome and pathological symptoms of L. major infection at least in mouse model. In this study gerbils' IL-4 promoter gene polymorphism is assessed. Specific primers were designed to develop a PCR-based assay to amplify IL-4 promoter gene to possibly define IL-4 promoter gene polymorphism in great gerbil populations with a range of Leishmania infection and symptoms collected from different foci of the central, north and northeast regions of Iran. The results showed that the designed primers amplify 689bp of the promoter gene. Sequence analysis of the promoter gene revealed five polymorphic sites assembly six haplotypes among the gerbil populations. Further studies are needed to assess whether or not the five polymorphisms cause different outcome phenotypes following infection with L. major in great gerbils. The data might be used to characterize the immune responses of R. opimus against L. major infection. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Hypermethylation of gene promoters in peripheral blood leukocytes in humans long term after radiation exposure

    International Nuclear Information System (INIS)

    Kuzmina, Nina S.; Lapteva, Nellya Sh.; Rubanovich, Alexander V.

    2016-01-01

    Some human genes known to undergo age-related promoter hypermethylation. These epigenetic modifications are similar to those occurring in the course of certain diseases, e.g. some types of cancer, which in turn may also associate with age. Given external genotoxic factors may additionally contribute to hypermethylation, this study was designed to analyzes, using methylation-sensitive polymerase chain reaction (PCR), the CpG island hypermethylation in RASSF1A, CDKN2A (including p16/INK4A and p14/ARF) and GSTP1 promoters in peripheral blood leukocytes of individuals exposed to ionizing radiation long time ago. One hundred and twenty-four irradiated subjects (24–77 years old at sampling: 83 Chernobyl Nuclear Power Plant clean-up workers, 21 nuclear workers, 20 residents of territories with radioactive contamination) and 208 unirradiated volunteers (19–77 years old at sampling) were enrolled. In addition, 74 non-exposed offspring (2–51 years old at sampling) born to irradiated parents were examined. The frequency of individuals displaying promoter methylation of at least one gene in exposed group was significantly higher as compared to the control group (OR=5.44, 95% CI=2.62–11.76, p=3.9×10 −7 ). No significant difference was found between the frequency of subjects with the revealed promoter methylation in the group of offspring born to irradiated parents and in the control group. The increase in the number of methylated loci of RASSF1A and p14/ARF was associated with age (β=0.242; p=1.7×10 −5 ). In contrast, hypermethylation of p16/INK4A and GSTP1 genes correlated with the fact of radiation exposure only (β=0.290; p=1.7×10 −7 ). The latter finding demonstrates that methylation changes in blood leukocytes of healthy subjects exposed to radiation resemble those reported in human malignancies. Additional studies are required to identify the dose-response of epigenetic markers specifically associating with radiation-induced premature aging and/or with

  6. Cloning, expression promoter analysis of vasa gene in Japanese flounder (Paralichthys olivaceus).

    Science.gov (United States)

    Wu, Xiaomeng; Wang, Zhongkai; Jiang, Jiajun; Gao, Jinning; Wang, Jing; Zhou, Xiaosu; Zhang, Quanqi

    2014-01-01

    Vasa is a DEAD box helicase and has shown essential functions during gametogenesis and embryogenesis. In most species, research revealed a specific expression of vasa gene in the germ cells. Thus, vasa has become the candidate gene in identifying germ cells. In this study, the vasa gene was isolated from gonads of Japanese flounder (Paralichthys olivaceus). In the 11.4kb genomic sequence, 23 exons were identified besides 5' and 3' flanking regions. The promoter region contained several putative TF binding sites which may have the function of regulating vasa expression. Quantitative real-time PCR analysis showed that vasa gene expression was restricted to adult gonads, with a higher level in the ovary. Development expression profiling revealed a maternal deposit and constant embryonic expression at early stages, but the relative mRNA amount decreased after gastrula. Nine other PoVasa transcripts were detected and their expression in gonads and during early development was not all the same, implying potential different functions during gametogenesis or early embryonic development. These results together confirmed the feasibility of using vasa as a marker of germ cells and that vasa gene had an important role in spermatogenesis and oogenesis. Furthermore, our study laid the groundwork for identifying fish primordial germ cells (PGCs) and investigating germ cell biology. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Distinguishing the Transcription Regulation Patterns in Promoters of Human Genes with Different Function or Evolutionary Age

    KAUST Repository

    Alam, Tanvir

    2012-07-01

    Distinguishing transcription regulatory patterns of different gene groups is a common problem in various bioinformatics studies. In this work we developed a methodology to deal with such a problem based on machine learning techniques. We applied our method to two biologically important problems related to detecting a difference in transcription regulation of: a/ protein-coding and long non-coding RNAs (lncRNAs) in human, as well as b/ a difference between primate-specific and non-primate-specific long non-coding RNAs. Our method is capable to classify RNAs using various regulatory features of genes that transcribe into these RNAs, such as nucleotide frequencies, transcription factor binding sites, de novo sequence motifs, CpG islands, repetitive elements, histone modification marks, and others. Ten-fold cross-validation tests suggest that our model can distinguish protein-coding and non-coding RNAs with accuracy above 80%. Twenty-fold cross-validation tests suggest that our model can distinguish primate-specific from non-primate-specific promoters of lncRNAs with accuracy above 80%. Consequently, we can hypothesize that transcription of the groups of genes mentioned above are regulated by different mechanisms. Feature selection techniques allowed us to reduce the number of features significantly while keeping the accuracy around 80%. Consequently, we can conclude that selected features play significant role in transcription regulation of coding and non-coding genes, as well as primate-specific and non-primate-specific lncRNA genes.

  8. Aquilegia B gene homologs promote petaloidy of the sepals and maintenance of the C domain boundary

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    Bharti Sharma

    2017-11-01

    Full Text Available Abstract The model Aquilegia coerulea x “Origami” possesses several interesting floral features, including petaloid sepals that are morphologically distinct from the true petals and a broad domain containing many whorls of stamens. We undertook the current study in an effort to understand the former trait, but additionally uncovered data that inform on the latter. The Aquilegia B gene homolog AqPI is shown to contribute to the production of anthocyanin in the first whorl sepals, although it has no major role in their morphology. Surprisingly, knockdown of AqPI in Aquilegia coerulea x “Origami” also reveals a role for the B class genes in maintaining the expression of the C gene homolog AqAG1 in the outer whorls of stamens. These findings suggest that the transference of pollinator function to the first whorl sepals included a non-homeotic recruitment of the B class genes to promote aspects of petaloidy. They also confirm results in several other Ranunculales that have revealed an unexpected regulatory connection between the B and C class genes.

  9. ADAM33 gene silencing by promoter hypermethylation as a molecular marker in breast invasive lobular carcinoma

    Directory of Open Access Journals (Sweden)

    de Souza Emanuel M

    2009-03-01

    Full Text Available Abstract Background ADAM33 protein is a member of the family of transmembrane glycoproteins composed of multidomains. ADAM family members have different activities, such as proteolysis and adhesion, making them good candidates to mediate the extracellular matrix remodelling and changes in cellular adhesion that characterise certain pathologies and cancer development. It was reported that one family member, ADAM23, is down-regulated by promoter hypermethylation. This seems to correlate with tumour progression and metastasis in breast cancer. In this study, we explored the involvement of ADAM33, another ADAM family member, in breast cancer. Methods First, we analysed ADAM33 expression in breast tumour cell lines by RT-PCR and western blotting. We also used 5-aza-2'-deoxycytidine (5azadCR treatment and DNA bisulphite sequencing to study the promoter methylation of ADAM33 in breast tumour cell lines. We evaluated ADAM33 methylation in primary tumour samples by methylation specific PCR (MSP. Finally, ADAM33 promoter hypermethylation was correlated with clinicopathological data using the chi-square test and Fisher's exact test. Results The expression analysis of ADAM33 in breast tumour cell lines by RT-PCR revealed gene silencing in 65% of tumour cell lines. The corresponding lack of ADAM33 protein was confirmed by western blotting. We also used 5-aza-2'-deoxycytidine (5-aza-dCR demethylation and bisulphite sequencing methodologies to confirm that gene silencing is due to ADAM33 promoter hypermethylation. Using MSP, we detected ADAM33 promoter hypermethylation in 40% of primary breast tumour samples. The correlation between methylation pattern and patient's clinicopathological data was not significantly associated with histological grade; tumour stage (TNM; tumour size; ER, PR or ERBB2 status; lymph node status; metastasis or recurrence. Methylation frequency in invasive lobular carcinoma (ILC was 76.2% compared with 25.5% in invasive ductal carcinoma

  10. ADAM33 gene silencing by promoter hypermethylation as a molecular marker in breast invasive lobular carcinoma

    International Nuclear Information System (INIS)

    Seniski, Gerusa G; Zanata, Silvio M; Costa, Fabrício F; Klassen, Giseli; Camargo, Anamaria A; Ierardi, Daniela F; Ramos, Edneia AS; Grochoski, Mariana; Ribeiro, Enilze SF; Cavalli, Iglenir J; Pedrosa, Fabio O; Souza, Emanuel M de

    2009-01-01

    ADAM33 protein is a member of the family of transmembrane glycoproteins composed of multidomains. ADAM family members have different activities, such as proteolysis and adhesion, making them good candidates to mediate the extracellular matrix remodelling and changes in cellular adhesion that characterise certain pathologies and cancer development. It was reported that one family member, ADAM23, is down-regulated by promoter hypermethylation. This seems to correlate with tumour progression and metastasis in breast cancer. In this study, we explored the involvement of ADAM33, another ADAM family member, in breast cancer. First, we analysed ADAM33 expression in breast tumour cell lines by RT-PCR and western blotting. We also used 5-aza-2'-deoxycytidine (5azadCR) treatment and DNA bisulphite sequencing to study the promoter methylation of ADAM33 in breast tumour cell lines. We evaluated ADAM33 methylation in primary tumour samples by methylation specific PCR (MSP). Finally, ADAM33 promoter hypermethylation was correlated with clinicopathological data using the chi-square test and Fisher's exact test. The expression analysis of ADAM33 in breast tumour cell lines by RT-PCR revealed gene silencing in 65% of tumour cell lines. The corresponding lack of ADAM33 protein was confirmed by western blotting. We also used 5-aza-2'-deoxycytidine (5-aza-dCR) demethylation and bisulphite sequencing methodologies to confirm that gene silencing is due to ADAM33 promoter hypermethylation. Using MSP, we detected ADAM33 promoter hypermethylation in 40% of primary breast tumour samples. The correlation between methylation pattern and patient's clinicopathological data was not significantly associated with histological grade; tumour stage (TNM); tumour size; ER, PR or ERBB2 status; lymph node status; metastasis or recurrence. Methylation frequency in invasive lobular carcinoma (ILC) was 76.2% compared with 25.5% in invasive ductal carcinoma (IDC), and this difference was

  11. Association of the Resistin Gene Promoter Region Polymorphism with Kawasaki Disease in Chinese Children

    Directory of Open Access Journals (Sweden)

    Ruixi Liu

    2012-01-01

    Full Text Available Objectives. The −420C>G polymorphism located in the resistin gene (RETN promoter has recently been suggested to play a potential role in proinflammatory conditions and cardiovascular disease. This study investigated the association of the RETN promoter polymorphism with Kawasaki disease (KD and its clinical parameters in Chinese children. Methods. We compared patients with complete KD to incomplete KD children. Genotyping of the RETN promoter polymorphism was performed using MassARRAY system, and serum resistin levels were estimated using the sandwich enzyme immunoassay method. Results. There was no significant difference in RETN (−420C>G genotypes between KD and control groups. However, the frequency of the G allele was higher in iKD patients than in cKD children due to a significantly increased frequency of the GG genotypes. Serum levels of resistin were significantly higher in KD patients than in controls regardless of the presence of coronary artery lesions (CALs. Conclusion. The present findings suggest that while resistin may play a role in the pathogenesis of KD, there is no apparent association between CAL and the RETN (−420C>G gene polymorphism in KD children. However, the diagnosis of iKD is challenging but can be supported by the presence of the G allele and the GG genotypes.

  12. Transgenic Studies with a Keratin Promoter-Driven Growth Hormone Transgene: Prospects for Gene Therapy

    Science.gov (United States)

    Wang, Xiaoming; Zinkel, Sandra; Polonsky, Kenneth; Fuchs, Elaine

    1997-01-01

    Keratinocytes are potentially appealing vehicles for the delivery of secreted gene products because they can be transferred to human skin by the relatively simple procedure of grafting. Adult human keratinocytes can be efficiently propagated in culture with sufficient proliferative capacity to produce enough epidermis to cover the body surface of an average adult. However, the feasibility of delivering secreted proteins through skin grafting rests upon (i) the strength of the promoter in keratinocytes and (ii) the efficiency of protein transport through the basement membrane of the stratified epithelium and into the bloodstream. In this paper, we use transgenic technology to demonstrate that the activity of the human keratin 14 promoter remains high in adult skin and that keratinocyte-derived human growth hormone (hGH) can be produced, secreted, and transported to the bloodstream of mice with efficiency that is sufficient to exceed by an order of magnitude the circulating hGH concentration in growing children. Transgenic skin grafts from these adults continue to produce and secrete hGH stably, at ≈ 1/10 physiological levels in the bloodstream of nontransgenic recipient mice. These studies underscore the utility of the keratin 14 promoter for expressing foreign transgenes in keratinocytes and demonstrate that keratinocytes can be used as effective vehicles for transporting factors to the bloodstream and for eliciting metabolic changes. These findings have important implications for considering the keratinocyte as a possible vehicle for gene therapy.

  13. Impact of MIF gene promoter polymorphism on F508del cystic fibrosis patients.

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    Paola Melotti

    Full Text Available Macrophage migration Inhibitory Factor (MIF is a pro-inflammatory cytokine sustaining the acute response to gram-negative bacteria and a regulatory role for MIF in Cystic Fibrosis has been suggested by the presence of a functional, polymorphic, four-nucleotide repeat in this gene's promoter at position -794, with the 5-repeat allele displaying lower promoter activity. We aimed at assessing the association of this polymorphism with disease severity in a group of Cystic Fibrosis patients homozygous for F508del CFTR gene mutation. Genotype frequencies were determined in 189 Cystic Fibrosis and 134 control subjects; key clinical features of patients were recorded and compared among homozygous 5-allele patients and the other MIF genotypes. Patients homozygous for the 5-repeat allele of MIF promoter displayed a slower rate of lung function decline (p = 0.027 at multivariate survival analysis. Multiple regression analysis on age-normalized respiratory volume showed no association of the homozygous 5-repeat genotype with lung function under stable conditions and no correlation with P.aeruginosa chronic colonization. Therefore, only the Homozygous 5-repeat genotype at MIF -794 is associated with milder disease in F508del Cystic Fibrosis patients.

  14. Structure of the goat psi beta y beta-globin pseudogene. Analysis of goat pseudogene evolutionary patterns.

    Science.gov (United States)

    Shapiro, S G; Moshirfar, M

    1989-09-20

    The 12-member beta-globin gene locus of the goat contains three beta(adult)-type pseudogenes, one in each of three four-gene subsets of the locus. We have determined the complete nucleotide sequence of psi beta y, the pseudogene present in the most downstream four-gene subset, which also contains the functional fetal gene, beta F. psi beta y contains, throughout its length, numerous incapacitating mutations in common with the previously sequenced goat psi beta x and psi beta z pseudogenes consistent with the model that all were descended from a common pseudogene ancestor which became defective prior to the expansion of the beta-globin locus in the goat lineage. Evolutionary analysis of the psi beta y sequence in comparison to psi beta x and psi beta z provides evidence that nucleotide substitutions were fixed in a random manner within these pseudogenes with respect to polarity, coding versus non-coding regions, and replacement sites versus silent sites. However, substitutions appear to have accumulated asymmetrically between different pseudogenes in a manner that provides evidence for partial gene conversion. Moreover, the presence of deletions in goat psi beta y, which are also observed in the cow pseudogene psi 2, but not in the cow psi 1 pseudogene, indicate that goat psi beta y and cow psi 2 are orthologous but cow psi 1 actually arose prior to the goat/cow divergence. The authentic goat orthologue to cow psi 1 temporarily existed in the goat lineage but was deleted, probably prior to the divergence of goats and sheep.

  15. Primary characterization and basal promoter activity of two hexamerin genes of Musca domestica

    Directory of Open Access Journals (Sweden)

    C.K. Moreira

    2004-02-01

    Full Text Available Hexamerins are high molecular-weight proteins found in the hemolymph of insects and have been proposed to function as storage proteins. In previous studies, two Musca domestica hexamerins, designated Hex-L and Hex-F were characterized. Hex-L is synthesized exclusively by the larval fat bodies, is secreted into the hemolymph and likely provides a source of amino acids and energy during metamorphosis. Hex-F synthesis is induced by a proteinaceous meal and occurs only in the adult insect fat bodies. Hex-F also is secreted into the hemolymph and it has been suggested that in females it may be an amino acid reservoir to be used during the final stages of egg formation. Genomic clones containing full-length copies of the genes MdHexL1 and MdHexF1, encoding subunits of the larval and the adult female hexamerin, respectively, were isolated. Complete nucleotide sequences, including the 5'-end untranscribed regions, were determined and analyzed for each of the genes. Comparisons of the conceptual translation products of the cloned genes indicated that MdHexL1 and MdHexF1 are related to the larval serum proteins (LSP 1 and 2 of Calliphora vicina and Drosophila melanogaster. DNA fragments containing the putative promoters of the two hexamerin genes were compared and cloned into a plasmid vector so as to drive the expression of the GFP reporter gene. The constructs were assayed in vitro in transfected S2 Drosophila melanogaster cells demonstrating that the cloned M. domestica DNA fragments exhibit promoter activity.

  16. 1 kb of the lactase-phlorizin hydrolase promoter directs post-weaning decline and small intestinal-specific expression in transgenic mice

    DEFF Research Database (Denmark)

    Troelsen, J T; Mehlum, A; Spodsberg, N

    1994-01-01

    Adult-type hypolactasia is a genetic condition making approximately one half of the human population intolerant to milk because of abdominal symptoms. The cause is a post-weaning down-regulation of the intestinal-specific enzyme lactase-phlorizin hydrolase (LPH) reducing the intestinal capacity...... to hydrolyze lactose. We here demonstrate that the stretch -17 to -994 in the pig LPH-promoter carries cis-elements which direct a small intestinal-specific expression and a post-weaning decline of a linked rabbit beta-globin gene. These data demonstrate that the post-weaning decline of LPH is mainly due...

  17. Identification and functional characterisation of the promoter of the calcium sensor gene CBL1 from the xerophyte Ammopiptanthus mongolicus

    Directory of Open Access Journals (Sweden)

    Yin Weilun

    2010-01-01

    Full Text Available Abstract Background CBL1 is a calcium sensor that regulates drought, cold and salt signals in Arabidopsis. Overexpression of CBL1 gene in Arabidopsis and in Ammopiptanthus mongolicus showed different tolerant activities. We are interested in understanding the molecular mechanism of the upstream region of the CBL1 gene of A. mongolicus (AmCBL1. We investigated and characterized the promoter of the AmCBL1 gene, for promoters play a very important role in regulating gene expression in eukaryotes. Results A 1683-bp 5' flanking region was isolated from A. mongolicus. The sequence was identified as AmCBL1 promoter. Analysis of the promoter sequence indicated a 690-bp intron and some basic cis-acting elements were related to various environmental stresses and plant hormones. To identify the functional region of the AmCBL1 promoter, five plant expression vectors fused with the GUS (β-glucuronidase gene, driven by series deleted fragments of AmCBL1 promoter at different lengths from -1659, -1414, -1048, -296 to -167 bp relative to the transcriptional start site were constructed and transformed into Nicotiana tabacum L. cv. 89. Functional properties of each promoter segment were examined by GUS staining and fluorescence quantitative analyses using at least three single-copy PCR-positive plants of transgenic tobacco, treated with various environmental stresses and plant hormones for different times. We demonstrated that the AmCBL1 promoter was a vascular-specific and multiple-stress-inducible promoter. Our results further imply that the promoter fragment B1S3 possessed sufficient essential cis-acting elements, accounting for vascular-specific and stress-induced expression patterns. It may also indicate that for response to some stresses certain cis-elements are required in tissues outside the region of the B1S3 construct. Conclusions To help resolve uncertainties about the upstream regulatory mechanism of the CBL1 gene in desert plants, we suggest that

  18. Variants in the dopamine-4-receptor gene promoter are not associated with sensation seeking in skiers.

    Science.gov (United States)

    Thomson, Cynthia J; Rajala, Amelia K; Carlson, Scott R; Rupert, Jim L

    2014-01-01

    Sensation seeking is a personality trait that has been associated with disinhibited behaviours including substance use and gambling, but also with high-risk sport practices including skydiving, paragliding, and downhill skiing. Twin studies have shown that sensation seeking is moderately heritable, and candidate genes encoding components involved in dopaminergic transmission have been investigated as contributing to this type of behaviour. To determine whether variants in the regulatory regions of the dopamine-4-receptor gene (DRD4) influenced sport-specific sensation seeking, we analyzed five polymorphisms (-1106T/C, -906T/C, -809G/A, -291C/T, 120-bp duplication) in the promoter region of the gene in a cohort of skiers and snowboarders (n = 599) that represented a broad range of sensation seeking behaviours. We grouped subjects by genotype at each of the five loci and compared impulsive sensation seeking and domain-specific (skiing) sensation seeking between groups. There were no significant associations between genotype(s) and general or domain-specific sensation seeking in the skiers and snowboarders, suggesting that while DRD4 has previously been implicated in sensation seeking, the promoter variants investigated in this study do not contribute to sensation seeking in this athlete population.

  19. Novel screening assay for in vivo selection of Klebsiella pneumoniae genes promoting gastrointestinal colonisation

    Science.gov (United States)

    2012-01-01

    Background Klebsiella pneumoniae is an important opportunistic pathogen causing pneumonia, sepsis and urinary tract infections. Colonisation of the gastrointestinal (GI) tract is a key step in the development of infections; yet the specific factors important for K. pneumoniae to colonize and reside in the GI tract of the host are largely unknown. To identify K. pneumoniae genes promoting GI colonisation, a novel genomic-library-based approach was employed. Results Screening of a K. pneumoniae C3091 genomic library, expressed in E. coli strain EPI100, in a mouse model of GI colonisation led to the positive selection of five clones containing genes promoting persistent colonisation of the mouse GI tract. These included genes encoding the global response regulator ArcA; GalET of the galactose operon; and a cluster of two putative membrane-associated proteins of unknown function. Both ArcA and GalET are known to be involved in metabolic pathways in Klebsiella but may have additional biological actions beneficial to the pathogen. In support of this, GalET was found to confer decreased bile salt sensitivity to EPI100. Conclusions The present work establishes the use of genomic-library-based in vivo screening assays as a valuable tool for identification and characterization of virulence factors in K. pneumoniae and other bacterial pathogens. PMID:22967317

  20. [Promoter methylation status of SFRP genes and induced apoptosis by demethylation in Jurkat cells].

    Science.gov (United States)

    Xu, Chengbo; Shen, Jianzhen; Liao, Bin; Fu, Haiying; Zhou, Huarong; Qi, Yan; Huangfu, Zhenping; Chen, Yining; Chen, Jiawei

    2016-01-01

    To study the promoter methylation status of SFRP genes and the effect of 5- aza- 2'- deoxycytidine (5- Aza- CdR)induced apoptosis via Wnt/β- catenin pathway by demethylation in Jurkat cells. Jurkat cells were treated with different concentrations of 5- Aza- CdR. The cell proliferation level of Jurkat cells was detected by MTT assay. Apoptosis was evaluated by flow cytometry. Methylation- spcific PCR (MSP) was used to determine the methylation status of SFRP genes. The expressions of SFRP genes were detected by real time fluorescence quantitative PCR. The mRNA expression levels of survivin, c- myc and cyclin- D1 were analyzed by RT- PCR. Western blot was used to detect the levels of β-catenin protein. Compared with control group, the different concentrations of 5-Aza-CdR could significantly inhibit the proliferation of Jurkat cells in a time-dose dependent manner (P<0.05). After being treated by 5- Aza- CdR for 48 hours, the cell early apoptosis rate in experiment group was significantly higher than that in control group (P<0.05). The promoters of SFRP1, SFRP2, SFRP4, SFRP5 genes were hypermethylation state in the control group, after being treated by 5-Aza-CdR for 72 hours, the brightness of SFRP1, SFRP2, SFRP4, SFRP5 genes' methylation strips weakened in a dose- dependent manner. SFRP mRNA expression increased (P<0.05) when 5- Aza- CdR concentration increased, and the level of β- catenin protein was dampened in a dose- dependent manner (P<0.05). As compared to the control group, the mRNA expressions of associated apoptosis genes survivin, c-myc and cyclin- D1, respectively were obviously down- regulated in a dose- dependent manner (P<0.05). The effect of demethylation could up- regulate SFRP genes expressions by reversing its hypermethylation and induced apoptosis by down-regulation of β-catenin and associated apoptosis genes.

  1. Relationship of interleukin-1B gene promoter region polymorphism with Helicobacter pylori infection and gastritis.

    Science.gov (United States)

    Ramis, Ivy Bastos; Vianna, Júlia Silveira; Halicki, Priscila Cristina Bartolomeu; Lara, Caroline; Tadiotto, Thássia Fernanda; da Silva Maciel, João Batista; Gonçalves, Carla Vitola; von Groll, Andrea; Dellagostin, Odir Antônio; da Silva, Pedro Eduardo Almeida

    2015-09-29

    Helicobacter pylori infection is associated with gastritis, peptic ulcer disease and gastric carcinoma. The severity of damage is determined by the interplay between environmental/behavioral factors, bacterial pathogenicity genes and host genetic polymorphisms that can influence the secretion levels of inflammatory cytokines. Accordingly, this study aimed to identify polymorphisms in the IL-1B and IL-1RN genes and their associations with H. pylori infection, cagA gene of H. pylori, and gastroduodenal diseases. Gastric biopsy samples from 151 patients infected with H. pylori and 76 uninfected individuals were analyzed. H. pylori infection was diagnosed by histology and PCR. Polymorphisms at positions -511, -31 and +3954 of the IL-1B gene were detected by PCR-RFLP, and an analysis of the VNTR polymorphism of the IL-1RN gene was performed by PCR. It was observed that the presence of the T/T genotype at position -511 and the C/C genotype at position -31 were associated with H. pylori infection and with an increased risk of gastritis in H. pylori-positive patients. Additionally, strains from patients H. pylori-positive carrying the cagA gene was significantly related with the T/T genotype at position -511 of IL-1B.  No association of polymorphisms at position +3954 of IL-1B and in the IL-1RN with H. pylori infection and with risk of severe gastric diseases was found. We demonstrated that polymorphisms in the promoter region of the IL-1B gene (at positions -511 and -31) are associated with an enhanced risk of H. pylori infection as well as gastritis in H. pylori-positive patients.

  2. Genome-wide analysis of CREB target genes reveals a core promoter requirement for cAMP responsiveness.

    Science.gov (United States)

    Conkright, Michael D; Guzmán, Ernesto; Flechner, Lawrence; Su, Andrew I; Hogenesch, John B; Montminy, Marc

    2003-04-01

    We have employed a hidden Markov model (HMM) based on known cAMP responsive elements to search for putative CREB target genes. The best scoring sites were positionally conserved between mouse and human orthologs, suggesting that this parameter can be used to enrich for true CREB targets. Target validation experiments revealed a core promoter requirement for transcriptional induction via CREB; TATA-less promoters were unresponsive to cAMP compared to TATA-containing genes, despite comparable binding of CREB to both sets of genes in vivo. Indeed, insertion of a TATA box motif rescued cAMP responsiveness on a TATA-less promoter. These results illustrate a mechanism by which subsets of target genes for a transcription factor are differentially regulated depending on core promoter configuration.

  3. Minimal enhancer elements of the leghemoglobin lba and lbc3 gene promoters from Glycine max L. have different properties

    DEFF Research Database (Denmark)

    She, Q; Lauridsen, P; Stougaard, J

    1993-01-01

    The characteristics of the soybean leghemoglobin lba gene promoter were analyzed and important promoter elements from the lba and lbc3 promoters were compared using transgenic Lotus corniculatus plants. A 5' deletion analysis of the lba promoter delimited two cis-acting elements controlling...... expression: a distal positive element (-1254, -884) required for expression and a proximal element (-285, -60) essential for full-level activity. In contrast to the corresponding region of the lbc3 promoter, the lba proximal element is unable to control expression from the heterologous CaMV 35S enhancer....... The upstream positive element of the lba gene contains a position- and orientation-independent enhancer between positions (-1091, -788). The sequence of this enhancer region is conserved in the lbc3 gene upstream (-1333, -1132) of the previously assigned strong positive element (SPE; -1090, -947). The present...

  4. Gene Therapy Approaches to Hemoglobinopathies.

    Science.gov (United States)

    Ferrari, Giuliana; Cavazzana, Marina; Mavilio, Fulvio

    2017-10-01

    Gene therapy for hemoglobinopathies is currently based on transplantation of autologous hematopoietic stem cells genetically modified with a lentiviral vector expressing a globin gene under the control of globin transcriptional regulatory elements. Preclinical and early clinical studies showed the safety and potential efficacy of this therapeutic approach as well as the hurdles still limiting its general application. In addition, for both beta-thalassemia and sickle cell disease, an altered bone marrow microenvironment reduces the efficiency of stem cell harvesting as well as engraftment. These hurdles need be addressed for gene therapy for hemoglobinopathies to become a clinical reality. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Defining the cutoff value of MGMT gene promoter methylation and its predictive capacity in glioblastoma.

    Science.gov (United States)

    Brigliadori, Giovanni; Foca, Flavia; Dall'Agata, Monia; Rengucci, Claudia; Melegari, Elisabetta; Cerasoli, Serenella; Amadori, Dino; Calistri, Daniele; Faedi, Marina

    2016-06-01

    Despite advances in the treatment of glioblastoma (GBM), median survival is 12-15 months. O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status is acknowledged as a predictive marker for temozolomide (TMZ) treatment. When MGMT promoter values fall into a "methylated" range, a better response to chemotherapy is expected. However, a cutoff that discriminates between "methylated" and "unmethylated" status has yet to be defined. We aimed to identify the best cutoff value and to find out whether variability in methylation profiles influences the predictive capacity of MGMT promoter methylation. Data from 105 GBM patients treated between 2008 and 2013 were analyzed. MGMT promoter methylation status was determined by analyzing 10 CpG islands by pyrosequencing. Patients were treated with radiotherapy followed by TMZ. MGMT promoter methylation status was classified into unmethylated 0-9 %, methylated 10-29 % and methylated 30-100 %. Statistical analysis showed that an assumed methylation cutoff of 9 % led to an overestimation of responders. All patients in the 10-29 % methylation group relapsed before the 18-month evaluation. Patients with a methylation status ≥30 % showed a median overall survival of 25.2 months compared to 15.2 months in all other patients, confirming this value as the best methylation cutoff. Despite wide variability among individual profiles, single CpG island analysis did not reveal any correlation between single CpG island methylation values and relapse or death. Specific CpG island methylation status did not influence the predictive value of MGMT. The predictive role of MGMT promoter methylation was maintained only with a cutoff value ≥30 %.

  6. Characterization of the bovine pregnancy-associated glycoprotein gene family – analysis of gene sequences, regulatory regions within the promoter and expression of selected genes

    Directory of Open Access Journals (Sweden)

    Walker Angela M

    2009-04-01

    Full Text Available Abstract Background The Pregnancy-associated glycoproteins (PAGs belong to a large family of aspartic peptidases expressed exclusively in the placenta of species in the Artiodactyla order. In cattle, the PAG gene family is comprised of at least 22 transcribed genes, as well as some variants. Phylogenetic analyses have shown that the PAG family segregates into 'ancient' and 'modern' groupings. Along with sequence differences between family members, there are clear distinctions in their spatio-temporal distribution and in their relative level of expression. In this report, 1 we performed an in silico analysis of the bovine genome to further characterize the PAG gene family, 2 we scrutinized proximal promoter sequences of the PAG genes to evaluate the evolution pressures operating on them and to identify putative regulatory regions, 3 we determined relative transcript abundance of selected PAGs during pregnancy and, 4 we performed preliminary characterization of the putative regulatory elements for one of the candidate PAGs, bovine (bo PAG-2. Results From our analysis of the bovine genome, we identified 18 distinct PAG genes and 14 pseudogenes. We observed that the first 500 base pairs upstream of the translational start site contained multiple regions that are conserved among all boPAGs. However, a preponderance of conserved regions, that harbor recognition sites for putative transcriptional factors (TFs, were found to be unique to the modern boPAG grouping, but not the ancient boPAGs. We gathered evidence by means of Q-PCR and screening of EST databases to show that boPAG-2 is the most abundant of all boPAG transcripts. Finally, we provided preliminary evidence for the role of ETS- and DDVL-related TFs in the regulation of the boPAG-2 gene. Conclusion PAGs represent a relatively large gene family in the bovine genome. The proximal promoter regions of these genes display differences in putative TF binding sites, likely contributing to observed

  7. Study of P14/ARF Gene Promoter Methylation and Effect of Interleukin-17 Gene Polymorphism on this Methylation among Breast Cancer Patients

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    Sirous Naeimi

    2016-11-01

    Full Text Available Background: hyper-methylation in CpG Island is one of the major mechanisms in gene silencing. In many cancers, different genes are experiencing CIHM (CpG island hyper methylation. P14 / ARF regulatory factor, involved in negative regulation of the cell cycle through the effect on P53 factor pathway. On the other hand, Interleukin 17 can be increased methylation by inflammatory effects. The purpose of this study was to study the P14/ARF gene promoter methylation and effect of Interleukin-17 on this methylation among breast cancer patients in the south of the country and its comparison with healthy people. Materials and Methods: in this case–control study, peripheral blood of 40 patients with breast cancer who were referred to hospitals in Shiraz and 40 healthy women was used to DNA extraction by using salt out and K proteinase . Control subjects with a family history of cancer or autoimmune diseases were excluded from the study. We used PCR-RFLP method In order to study of Interleukin-17 gene polymorphism, and MSPCR method was used to study of P14/ARF gene promoter methylation. The results of the study were studied by using SPSS software, Arlequin, chi-square and Hardy-weinberg equilibrium test was used respectively. Results: findings confirms that there was a significant association between P14/ARF gene promoter methylation and disease and mentioned gene promoter was less methylated in healthy subjects compared to patients (p<0.05. On the other hand, there is a significant association between GG genotype in IL17 F gene polymorphisms and P14 /ARF gene methylation (P<0.05. Conclusion: P14/ARF gene promoter’s methylation play an important role in breast cancer. Due to the decline of P14P14/ARF gene promoters methylation and its association with this disease, seems that it could be used as a biomarker for diagnosis.  

  8. Long non-coding RNA ROR decoys gene-specific histone methylation to promote tumorigenesis.

    Science.gov (United States)

    Fan, Jiayan; Xing, Yue; Wen, Xuyang; Jia, Renbin; Ni, Hongyan; He, Jie; Ding, Xia; Pan, Hui; Qian, Guanxiang; Ge, Shengfang; Hoffman, Andrew R; Zhang, He; Fan, Xianqun

    2015-07-14

    Long non-coding RNAs (lncRNAs) are not translated into proteins and were initially considered to be part of the 'dark matter' of the genome. Recently, it has been shown that lncRNAs play a role in the recruitment of chromatin modifying complexes and can influence gene expression. However, it is unknown if lncRNAs function in a similar way in cancer. Here, we show that the lncRNA ROR occupies and activates the TESC promoter by repelling the histone G9A methyltransferase and promoting the release of histone H3K9 methylation. Suppression of ROR in tumors results in silencing of TESC expression, and G9A-mediated histone H3K9 methylation in the TESC promoter is restored, which significantly reduces tumor growth and metastasis. Without ROR silencing, TESC knockdown presents consistent and significant reductions in tumor progression. Our results reveal a novel mechanism by which ROR may serve as a decoy oncoRNA that blocks binding surfaces, preventing the recruitment of histone modifying enzymes, thereby specifying a new pattern of histone modifications that promote tumorigenesis.

  9. Characterization and functional analyses of the human G protein-coupled receptor kinase 4 gene promoter.

    Science.gov (United States)

    Hasenkamp, Sandra; Telgmann, Ralph; Staessen, Jan A; Hagedorn, Claudia; Dördelmann, Corinna; Bek, Martin; Brand-Herrmann, Stefan-Martin; Brand, Eva

    2008-10-01

    The G protein-coupled receptor kinase 4 is involved in renal sodium handling and blood pressure regulation. Missense variants have already been tested functionally and are associated with hypertension, but no data on promoter analyses are yet available. We scanned 94 hypertensive white subjects for genetic variation and performed promoter reporter gene analyses in HEK293T, COS7, and SaOs-2 cells. Transient transfections with various full lengths and wild-type deletion constructs revealed that 1851 bp of the flanking region and 275 bp of the 5'-untranslated region were sufficient for transcriptional activities and composed a powerful cis-active element in the distal 293 bp. The -1702T and +2T alleles resulted in drastic general reductions of promoter function, whereas an activity increasing effect of +268C was cell type specific. Electrophoretic mobility-shift assay, supershift, and cotransfection analyses of transcription factor binding sites predicted in silico (Alibaba2.1/Transfac7) resulted in allele-specific binding patterns of nuclear proteins and identified the participation of CCAAT/enhancer-binding protein transcription factor family members. The G protein-coupled receptor kinase 4 core promoter resides in the first 1851 bp upstream of its transcription start site. The 4 identified genetic variants within this region exert allele-specific impact on both cell type- and stimulation-dependent transcription and may affect the expression balance of renal G protein-coupled receptor kinase 4.

  10. CypA, a gene downstream of HIF-1α, promotes the development of PDAC.

    Directory of Open Access Journals (Sweden)

    Huan Zhang

    Full Text Available Hypoxia-inducible factor-1α (HIF-1α is a highly important transcription factor involved in cell metabolism. HIF-1α promotes glycolysis and inhibits of mitochondrial respiration in pancreatic ductal adenocarcinoma (PDAC. In response to tumor hypoxia, cyclophilin A (CypA is over-expressed in various cancer types, and is associated with cell apoptosis, tumor invasion, metastasis, and chemoresistance in PDAC. In this study, we showed that both HIF-1α and CypA expression were significantly associated with lymph node metastasis and tumor stage. The expression of CypA was correlated with HIF-1α. Moreover, the mRNA and protein expression of CypA markedly decreased or increased following the suppression or over-expression of HIF-1α in vitro. Chromatin immunoprecipitation analysis showed that HIF-1α could directly bind to the hypoxia response element (HRE in the CypA promoter regions and regulated CypA expression. Consistent with other studies, HIF-1α and CypA promoted PDAC cell proliferation and invasion, and suppressed apoptosis in vitro. Furthermore, we proved the combination effect of 2-methoxyestradiol and cyclosporin A both in vitro and in vivo. These results suggested that,CypA, a gene downstream of HIF-1α, could promote the development of PDAC. Thus, CypA might serve as a potential therapeutic target for PDAC.

  11. Distinct promoter activation mechanisms modulate noise-driven HIV gene expression

    Science.gov (United States)

    Chavali, Arvind K.; Wong, Victor C.; Miller-Jensen, Kathryn

    2015-12-01

    Latent human immunodeficiency virus (HIV) infections occur when the virus occupies a transcriptionally silent but reversible state, presenting a major obstacle to cure. There is experimental evidence that random fluctuations in gene expression, when coupled to the strong positive feedback encoded by the HIV genetic circuit, act as a ‘molecular switch’ controlling cell fate, i.e., viral replication versus latency. Here, we implemented a stochastic computational modeling approach to explore how different promoter activation mechanisms in the presence of positive feedback would affect noise-driven activation from latency. We modeled the HIV promoter as existing in one, two, or three states that are representative of increasingly complex mechanisms of promoter repression underlying latency. We demonstrate that two-state and three-state models are associated with greater variability in noisy activation behaviors, and we find that Fano factor (defined as variance over mean) proves to be a useful noise metric to compare variability across model structures and parameter values. Finally, we show how three-state promoter models can be used to qualitatively describe complex reactivation phenotypes in response to therapeutic perturbations that we observe experimentally. Ultimately, our analysis suggests that multi-state models more accurately reflect observed heterogeneous reactivation and may be better suited to evaluate how noise affects viral clearance.

  12. Evaluation of methylation of MGMT (O⁶-methylguanine-DNA methyltransferase) gene promoter in sporadic colorectal cancer.

    Science.gov (United States)

    Farzanehfar, Mohammadreza; Vossoughinia, Hasan; Jabini, Raheleh; Tavassoli, Alireza; Saadatnia, Hasan; Khorashad, Ahmad Khosravi; Ahadi, Mitra; Afzalaghaee, Monavvar; Ghayoor Karimiani, Ehsan; Mirzaei, Farzaneh; Ayatollahi, Hossein

    2013-07-01

    The DNA repair gene O⁶-methylguanine-DNA methyltransferase (MGMT) is frequently methylated in colorectal cancer (CRC). The aim of this study was to demonstrate that MGMT methylation may be one of the candidate mediators of field cancerization in the colon mucosa. Therefore, quantitative methylation-specific polymerase chain reaction was performed on tumor itself and additional samples of 5 and 10 cm away from the tumor in 40 CRC patients. Moreover, colon mucosa was examined from 30 cases with no evidence of cancer as a control. MGMT promoter methylation was present in 27.5% of colorectal tumor specimens. Tumors that showed MGMT promoter methylation had substantial MGMT promoter methylation in their normal adjacent mucosa. The methylation was also observed in 36.36% (4/11) of normal samples with MGMT promoter methylation in the adjacent tumors, in 20.79% (6/29) of samples without MGMT methylation in the adjacent tumors, and in 6.66% (2/30) of control samples (pMGMT methylation levels was significantly higher in the cancerous group than in the control group (6.25±1.702 vs. 0.086±0.036, pMGMT. Detection of such abnormality may ultimately be useful in risk assessment for CRCs.

  13. Effects of dietary intake and genetic factors on hypermethylation of the hMLH1 gene promoter in gastric cancer

    Science.gov (United States)

    Nan, Hong-Mei; Song, Young-Jin; Yun, Hyo-Yung; Park, Joo-Seung; Kim, Heon

    2005-01-01

    AIM: Hypermethylation of the promoter of the hMLH1 gene, which plays an important role in mismatch repair during DNA replication, occurs in more than 30% of human gastric cancer tissues. The purpose of this study was to investigate the effects of environmental factors, genetic polymorphisms of major metabolic enzymes, and microsatellite instability on hypermethylation of the promoter of the hMLH1 gene in gastric cancer. METHODS: Data were obtained from a hospital-based, case-control study of gastric cancer. One hundred and ten gastric cancer patients and 220 age- and sex-matched control patients completed a structured questionnaire regarding their exposure to environmental risk factors. Hypermethylation of the hMLH1 gene promoter, polymorphisms of the GSTM1, GSTT1, CYP1A1, CYP2E1, ALDH2 and L-myc genes, microsatellite instability and mutations of p53 and Ki-ras genes were investigated. RESULTS: Both smoking and alcohol consumption were associated with a higher risk of gastric cancer with hypermethylation of the hMLH1 gene promoter. High intake of vegetables and low intake of potato were associated with increased likelihood of gastric cancer with hypermethylation of the hMLH1 gene promoter. Genetic polymorphisms of the GSTM1, GSTT1, CYP1A1, CYP2E1, ALDH2, and L-myc genes were not significantly associated with the risk of gastric cancer either with or without hypermethylation in the promoter of the hMLH1 gene. Hypermethylation of the hMLH1 promoter was significantly associated with microsatellite instability (MSI): 10 of the 14 (71.4%) MSI-positive tumors showed hypermethylation, whereas 28 of 94 (29.8%) the MSI-negative tumors were hypermethylated at the hMLH1 promoter region. Hypermethylation of the hMLH1 gene promoter was significantly inversely correlated with mutation of the p53 gene. CONCLUSION: These results suggest that cigarette smoking and alcohol consumption may influence the development of hMLH1-positive gastric cancer. Most dietary factors and

  14. Gene promoter methylation and DNA repair capacity in monozygotic twins with discordant smoking habits.

    Science.gov (United States)

    Ottini, Laura; Rizzolo, Piera; Siniscalchi, Ester; Zijno, Andrea; Silvestri, Valentina; Crebelli, Riccardo; Marcon, Francesca

    2015-02-01

    The influence of DNA repair capacity, plasma nutrients and tobacco smoke exposure on DNA methylation was investigated in blood cells of twenty-one couples of monozygotic twins with discordant smoking habits. All study subjects had previously been characterized for mutagen sensitivity with challenge assays with ionizing radiation in peripheral blood lymphocytes. Plasma levels of folic acid, vitamin B12 and homocysteine were also available from a previous investigation. In this work DNA methylation in the promoter region of a panel of ten genes involved in cell cycle control, differentiation, apoptosis and DNA repair (p16, FHIT, RAR, CDH1, DAPK1, hTERT, RASSF1A, MGMT, BRCA1 and PALB2) was assessed in the same batches of cells isolated for previous studies, using the methylation-sensitive high-resolution melting technique. Fairly similar profiles of gene promoter methylation were observed within co-twins compared to unrelated subjects (p= 1.23 × 10(-7)), with no significant difference related to smoking habits (p = 0.23). In a regression analysis the methylation index of study subjects, used as synthetic descriptor of overall promoter methylation, displayed a significant inverse correlation with radiation-induced micronuclei (p = 0.021) and plasma folic acid level (p = 0.007) both in smokers and in non-smokers. The observed association between repair of radiation-induced DNA damage and promoter methylation suggests the involvement of the DNA repair machinery in DNA modification. Data also highlight the possible modulating effect of folate deficiency on DNA methylation and the strong influence of familiarity on the individual epigenetic profile. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. The bidirectional promoter of the divergently transcribed mouse Surf-1 and Surf-2 genes.

    OpenAIRE

    Lennard, A C; Fried, M

    1991-01-01

    The ubiquitously expressed mouse Surf-1 and Surf-2 genes are divergently transcribed, and their heterogeneous start sites are separated by up to a maximum of only 73 bp. By using in vitro DNase I, dimethyl sulfate methylation, and gel retardation assays, we have identified five putative promoter control elements between and around the Surf-1 and Surf-2 start sites. The effects of each site on the regulation of Surf-1 and Surf-2 transcription have been studied in vivo, and four sites were foun...

  16. Promoter decoding of transcription factor dynamics involves a trade-off between noise and control of gene expression.

    Science.gov (United States)

    Hansen, Anders S; O'Shea, Erin K

    2013-11-05

    Numerous transcription factors (TFs) encode information about upstream signals in the dynamics of their activation, but how downstream genes decode these dynamics remains poorly understood. Using microfluidics to control the nucleocytoplasmic translocation dynamics of the budding yeast TF Msn2, we elucidate the principles that govern how different promoters convert dynamical Msn2 input into gene expression output in single cells. Combining modeling and experiments, we classify promoters according to their signal-processing behavior and reveal that multiple, distinct gene expression programs can be encoded in the dynamics of Msn2. We show that both oscillatory TF dynamics and slow promoter kinetics lead to higher noise in gene expression. Furthermore, we show that the promoter activation timescale is related to nucleosome remodeling. Our findings imply a fundamental trade-off: although the cell can exploit different promoter classes to differentially control gene expression using TF dynamics, gene expression noise fundamentally limits how much information can be encoded in the dynamics of a single TF and reliably decoded by promoters.

  17. Mediterranean river buffalo oxytocin-neurophysin I (OXT gene: structure, promoter analysis and allele detection

    Directory of Open Access Journals (Sweden)

    L. Ramunno

    2010-02-01

    Full Text Available Oxytocin (OXT is a very abundant nonapeptide neurohypophysial hormone implicated in several aspects of reproduction, including social, sexual and maternal behaviour, induction of labour and milk ejection. The nucleotide sequence of the whole OXTneurophysin I encoding gene (OXT in Mediterranean river buffalo was determined, plus 993 nucleotides at the 5’ flanking region. Buffalo oxytocin gene sequence analysis showed two transitions in the promoter region (C→T in position – 966 and G→A in position – 790 and one transversion G→T at the 170th nucleotide of the second exon, responsible for the Arg97→Leu aa substitution which identifies an allele named OXT B. A PCR-RFLP based method for a rapid identification of carriers of these alleles has been developed.

  18. A practical platform for blood biomarker study by using global gene expression profiling of peripheral whole blood.

    Directory of Open Access Journals (Sweden)

    Ze Tian

    Full Text Available Although microarray technology has become the most common method for studying global gene expression, a plethora of technical factors across the experiment contribute to the variable of genome gene expression profiling using peripheral whole blood. A practical platform needs to be established in order to obtain reliable and reproducible data to meet clinical requirements for biomarker study.We applied peripheral whole blood samples with globin reduction and performed genome-wide transcriptome analysis using Illumina BeadChips. Real-time PCR was subsequently used to evaluate the quality of array data and elucidate the mode in which hemoglobin interferes in gene expression profiling. We demonstrated that, when applied in the context of standard microarray processing procedures, globin reduction results in a consistent and significant increase in the quality of beadarray data. When compared to their pre-globin reduction counterparts, post-globin reduction samples show improved detection statistics, lowered variance and increased sensitivity. More importantly, gender gene separation is remarkably clearer in post-globin reduction samples than in pre-globin reduction samples. Our study suggests that the poor data obtained from pre-globin reduction samples is the result of the high concentration of hemoglobin derived from red blood cells either interfering with target mRNA binding or giving the pseudo binding background signal.We therefore recommend the combination of performing globin mRNA reduction in peripheral whole blood samples and hybridizing on Illumina BeadChips as the practical approach for biomarker study.

  19. Inference of quantitative models of bacterial promoters from time-series reporter gene data.

    Science.gov (United States)

    Stefan, Diana; Pinel, Corinne; Pinhal, Stéphane; Cinquemani, Eugenio; Geiselmann, Johannes; de Jong, Hidde

    2015-01-01

    The inference of regulatory interactions and quantitative models of gene regulation from time-series transcriptomics data has been extensively studied and applied to a range of problems in drug discovery, cancer research, and biotechnology. The application of existing methods is commonly based on implicit assumptions on the biological processes under study. First, the measurements of mRNA abundance obtained in transcriptomics experiments are taken to be representative of protein concentrations. Second, the observed changes in gene expression are assumed to be solely due to transcription factors and other specific regulators, while changes in the activity of the gene expression machinery and other global physiological effects are neglected. While convenient in practice, these assumptions are often not valid and bias the reverse engineering process. Here we systematically investigate, using a combination of models and experiments, the importance of this bias and possible corrections. We measure in real time and in vivo the activity of genes involved in the FliA-FlgM module of the E. coli motility network. From these data, we estimate protein concentrations and global physiological effects by means of kinetic models of gene expression. Our results indicate that correcting for the bias of commonly-made assumptions improves the quality of the models inferred from the data. Moreover, we show by simulation that these improvements are expected to be even stronger for systems in which protein concentrations have longer half-lives and the activity of the gene expression machinery varies more strongly across conditions than in the FliA-FlgM module. The approach proposed in this study is broadly applicable when using time-series transcriptome data to learn about the structure and dynamics of regulatory networks. In the case of the FliA-FlgM module, our results demonstrate the importance of global physiological effects and the active regulation of FliA and FlgM half-lives for

  20. CASSIS and SMIPS: promoter-based prediction of secondary metabolite gene clusters in eukaryotic genomes.

    Science.gov (United States)

    Wolf, Thomas; Shelest, Vladimir; Nath, Neetika; Shelest, Ekaterina

    2016-04-15

    Secondary metabolites (SM) are structurally diverse natural products of high pharmaceutical importance. Genes involved in their biosynthesis are often organized in clusters, i.e., are co-localized and co-expressed. In silico cluster prediction in eukaryotic genomes remains problematic mainly due to the high variability of the clusters' content and lack of other distinguishing sequence features. We present Cluster Assignment by Islands of Sites (CASSIS), a method for SM cluster prediction in eukaryotic genomes, and Secondary Metabolites by InterProScan (SMIPS), a tool for genome-wide detection of SM key enzymes ('anchor' genes): polyketide synthases, non-ribosomal peptide synthetases and dimethylallyl tryptophan synthases. Unlike other tools based on protein similarity, CASSIS exploits the idea of co-regulation of the cluster genes, which assumes the existence of common regulatory patterns in the cluster promoters. The method searches for 'islands' of enriched cluster-specific motifs in the vicinity of anchor genes. It was validated in a series of cross-validation experiments and showed high sensitivity and specificity. CASSIS and SMIPS are freely available at https://sbi.hki-jena.de/cassis thomas.wolf@leibniz-hki.de or ekaterina.shelest@leibniz-hki.de Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

  1. Hydrogel Design for Supporting Neurite Outgrowth and Promoting Gene Delivery to Maximize Neurite Extension

    Science.gov (United States)

    Shepard, Jaclyn A.; Stevans, Alyson C.; Holland, Samantha; Wang, Christine E.; Shikanov, Ariella; Shea, Lonnie D.

    2012-01-01

    Hydrogels capable of gene delivery provide a combinatorial approach for nerve regeneration, with the hydrogel supporting neurite outgrowth and gene delivery inducing the expression of inductive factors. This report investigates the design of hydrogels that balance the requirements for supporting neurite growth with those requirements for promoting gene delivery. Enzymatically-degradable PEG hydrogels encapsulating dorsal root ganglia explants, fibroblasts, and lipoplexes encoding nerve growth factor were gelled within channels that can physically guide neurite outgrowth. Transfection of fibroblasts increased with increasing concentration of Arg-Gly-Asp (RGD) cell adhesion sites and decreasing PEG content. The neurite length increased with increasing RGD concentration within 10% PEG hydrogels, yet was maximal within 7.5% PEG hydrogels at intermediate RGD levels. Delivering lipoplexes within the gel produced longer neurites than culture in NGF-supplemented media or co-culture with cells exposed to DNA prior to encapsulation. Hydrogels designed to support neurite outgrowth and deliver gene therapy vectors locally may ultimately be employed to address multiple barriers that limit regeneration. PMID:22038654

  2. Promoters Architecture-Based Mechanism for Noise-Induced Oscillations in a Single-Gene Circuit.

    Science.gov (United States)

    Guisoni, N; Monteoliva, D; Diambra, L

    2016-01-01

    It is well known that single-gene circuits with negative feedback loop can lead to oscillatory gene expression when they operate with time delay. In order to generate these oscillations many processes can contribute to properly timing such delay. Here we show that the time delay coming from the transitions between internal states of the cis-regulatory system (CRS) can drive sustained oscillations in an auto-repressive single-gene circuit operating in a small volume like a cell. We found that the cooperative binding of repressor molecules is not mandatory for a oscillatory behavior if there are enough binding sites in the CRS. These oscillations depend on an adequate balance between the CRS kinetic, and the synthesis/degradation rates of repressor molecules. This finding suggest that the multi-site CRS architecture can play a key role for oscillatory behavior of gene expression. Finally, our results can also help to synthetic biologists on the design of the promoters architecture for new genetic oscillatory circuits.

  3. TFPI-2 is a putative tumor suppressor gene frequently inactivated by promoter hypermethylation in nasopharyngeal carcinoma

    International Nuclear Information System (INIS)

    Wang, Shumin; Ma, Ning; Murata, Mariko; Huang, Guangwu; Zhang, Zhe; Xiao, Xue; Zhou, Xiaoying; Huang, Tingting; Du, Chunping; Yu, Nana; Mo, Yingxi; Lin, Longde; Zhang, Jinyan

    2010-01-01

    Epigenetic silencing of tumor suppressor genes play important roles in NPC tumorgenesis. Tissue factor pathway inhibitor-2 (TFPI-2), is a protease inhibitor. Recently, TFPI-2 was suggested to be a tumor suppressor gene involved in tumorigenesis and metastasis in some cancers. In this study, we investigated whether TFPI-2 was inactivated epigenetically in nasopharyngeal carcinoma (NPC). Transcriptional expression levels of TFPI-2 was evaluated by RT-PCR. Methylation status were investigated by methylation specific PCR and bisulfate genomic sequencing. The role of TFPI-2 as a tumor suppressor gene in NPC was addressed by re-introducing TFPI-2 expression into the NPC cell line CNE2. TFPI-2 mRNA transcription was inactivated in NPC cell lines. TFPI-2 was aberrantly methylated in 66.7% (4/6) NPC cell lines and 88.6% (62/70) of NPC primary tumors, but not in normal nasopharyngeal epithelia. TFPI-2 expression could be restored in NPC cells after demethylation treatment. Ectopic expression of TFPI-2 in NPC cells induced apoptosis and inhibited cell proliferation, colony formation and cell migration. Epigenetic inactivation of TFPI-2 by promoter hypermethylation is a frequent and tumor specific event in NPC. TFPI-2 might be considering as a putative tumor suppressor gene in NPC

  4. Targeting gene expression to specific cells of kidney tubules in vivo, using adenoviral promoter fragments.

    Science.gov (United States)

    Watanabe, Sumiyo; Ogasawara, Toru; Tamura, Yoshifuru; Saito, Taku; Ikeda, Toshiyuki; Suzuki, Nobuchika; Shimosawa, Tatsuo; Shibata, Shigeru; Chung, Ung-Il; Nangaku, Masaomi; Uchida, Shunya

    2017-01-01

    Although techniques for cell-specific gene expression via viral transfer have advanced, many challenges (e.g., viral vector design, transduction of genes into specific target cells) still remain. We investigated a novel, simple methodology for using adenovirus transfer to target specific cells of the kidney tubules for the expression of exogenous proteins. We selected genes encoding sodium-dependent phosphate transporter type 2a (NPT2a) in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) in the collecting duct. The promoters of the three genes were linked to a GFP-coding fragment, the final constructs were then incorporated into an adenovirus vector, and this was then used to generate gene-manipulated viruses. After flushing circulating blood, viruses were directly injected into the renal arteries of rats and were allowed to site-specifically expression in tubule cells, and rats were then euthanized to obtain kidney tissues for immunohistochemistry. Double staining with adenovirus-derived EGFP and endogenous proteins were examined to verify orthotopic expression, i.e. "adenovirus driven NPT2a-EGFP and endogenous NHE3 protein", "adenovirus driven NKCC2-EGFP and endogenous NKCC2 protein" and "adenovirus driven AQP2-EGFP and endogenous AQP2 protein". Owing to a lack of finding good working anti-NPT2a antibody, an antibody against a different protein (sodium-hydrogen exchanger 3 or NHE3) that is also specifically expressed in the proximal tubule was used. Kidney structures were well-preserved, and other organ tissues did not show EGFP staining. Our gene transfer method is easier than using genetically engineered animals, and it confers the advantage of allowing the manipulation of gene transfer after birth. This is the first method to successfully target gene expression to specific cells in the kidney tubules. This study may serve as the first step for safe and effective gene

  5. Mutational analysis of the mycobacteriophage BPs promoter PR reveals context-dependent sequences for mycobacterial gene expression.

    Science.gov (United States)

    Oldfield, Lauren M; Hatfull, Graham F

    2014-10-01

    The PR promoter of mycobacteriophage BPs directs early lytic gene expression and is under the control of the BPs repressor, gp33. Reporter gene fusions showed that PR has modest activity in an extrachromosomal context but has activity that is barely detectable in an integrated context, even in the absence of its repressor. Mutational dissection of PR showed that it uses a canonical -10 hexamer recognized by SigA, and mutants with mutations to the sequence 5'-TATAMT had the greatest activities. It does not contain a 5'-TGN-extended -10 sequence, although mutants with mutations creating an extended -10 sequence had substantially increased promoter activity. Mutations in the -35 hexamer also influenced promoter activity but were strongly context dependent, and similar substitutions in the -35 hexamer differentially affected promoter activity, depending on the -10 and extended -10 motifs. This warrants caution in the construction of synthetic promoters or the bioinformatic prediction of promoter activity. Combinations of mutations throughout PR generated a calibrated series of promoters for expression of stably integrated recombinant genes in both Mycobacterium smegmatis and M. tuberculosis, with maximal promoter activity being more than 2-fold that of the strong hsp60 promoter. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  6. Association of CXCL12 gene promoter methylation with periodontitis in patients with diabetes mellitus type 2.

    Science.gov (United States)

    Grdović, Nevena; Rajić, Jovana; Petrović, Sanja Matić; Dinić, Svetlana; Uskoković, Aleksandra; Mihailović, Mirjana; Jovanović, Jelena Arambašić; Tolić, Anja; Pucar, Ana; Milašin, Jelena; Vidaković, Melita

    2016-12-01

    CXCL12 is widely expressed, constitutive chemokine involved in tissue repair and regeneration, while the extent of its expression is important in various chronic inflammatory conditions. Involvement of DNA methylation in CXCL12 gene suppression (CXCL12) has been shown in malignancy and some autoimmune diseases. The aim of this study was to investigate whether the alterations in DNA methylation of CXCL12 are also involved in progression of periodontitis in combination with diabetes, as these chronic inflammatory conditions are strongly interrelated. Study included 72 subjects divided in three groups: healthy control (C, n=21), periodontitis (P, n=29) and diabetes/periodontitis group (D/P, n=22). DNA extracted from epithelial cells obtained by sterile cotton swabs from buccal mucosa was subjected to methylation specific polymerase chain reaction (MSP) to obtain DNA methylation pattern of CXCL12 promoter. CXCL12 promoter was predominantly unmethylated in all groups. However, increase in the frequency of the methylated form and increase in percent of methylation of CXCL12 promoter in periodontitis and diabetes/periodontitis group compared to control group were found, although without statistical significance. However, statistically significant increase in Tm of MSP products in diabetes/periodontitis group was observed. Correlation analysis revealed statistically significant relationship between the extent of DNA methylation of the CXCL12 promoter and periodontal parameters, as well as between DNA methylation of CXCL12 and glycosylated hemoglobin. Presented results suggest that chronic inflammation contributes to the change of CXCL12 DNA methylation in buccal cells and that DNA methylation profile of CXCL12 promoter plays important role in development and progression of periodontal disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Regulatory elements in the promoter region of the rat gene encoding the acyl-CoA-binding protein

    DEFF Research Database (Denmark)

    Elholm, M; Bjerking, G; Knudsen, J

    1996-01-01

    Acyl-CoA-binding protein (ACBP) is an ubiquitously expressed 10-kDa protein which is present in high amounts in cells involved in solute transport or secretion. Rat ACBP is encoded by a gene containing the typical hallmarks of a housekeeping gene. Analysis of the promoter region of the rat ACBP g...

  8. A fungal transcription factor gene is expressed in plants from its own promoter and improves drought tolerance.

    Science.gov (United States)

    Martínez, Félix; Arif, Anjuman; Nebauer, Sergio G; Bueso, Eduardo; Ali, Rashid; Montesinos, Consuelo; Brunaud, Veronique; Muñoz-Bertomeu, Jesús; Serrano, Ramón

    2015-07-01

    A fungal gene encoding a transcription factor is expressed from its own promoter in Arabidopsis phloem and improves drought tolerance by reducing transpiration and increasing osmotic potential. Horizontal gene transfer from unrelated organisms has occurred in the course of plant evolution, suggesting that some foreign genes may be useful to plants. The CtHSR1 gene, previously isolated from the halophytic yeast Candida tropicalis, encodes a heat-shock transcription factor-related protein. CtHSR1, with expression driven by its own promoter or by the Arabidopsis UBQ10 promoter, was introduced into the model plant Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation and the resulting transgenic plants were more tolerant to drought than controls. Fusions of the CtHSR1 promoter with β-glucuronidase reporter gene indicated that this fungal promoter drives expression to phloem tissues. A chimera of CtHSR1 and green fluorescence protein is localized at the cell nucleus. The physiological mechanism of drought tolerance in transgenic plants is based on reduced transpiration (which correlates with decreased opening of stomata and increased levels of jasmonic acid) and increased osmotic potential (which correlates with increased proline accumulation). Transcriptomic analysis indicates that the CtHSR1 transgenic plants overexpressed a hundred of genes, including many relevant to stress defense such as LOX4 (involved in jasmonic acid synthesis) and P5CS1 (involved in proline biosynthesis). The promoters of the induced genes were enriched in upstream activating sequences for water stress induction. These results demonstrate that genes from unrelated organisms can have functional expression in plants from its own promoter and expand the possibilities of useful transgenes for plant biotechnology.

  9. Characterization of promoter of EgPAL1, a novel PAL gene from the oil palm Elaeis guineensis Jacq.

    Science.gov (United States)

    Yusuf, Chong Yu Lok; Abdullah, Janna Ong; Shaharuddin, Noor Azmi; Abu Seman, Idris; Abdullah, Mohd Puad

    2018-02-01

    The oil palm EgPAL1 gene promoter and its regulatory region were functional as a promoter in the heterologous system of Arabidopsis according to the cis-acting elements present in that region. The promoter was developmentally regulated, vascular tissue specific and responsive to water stress agents. Phenylalanine ammonia lyase (PAL, EC 4.3.1.24) is the key enzyme of the phenylpropanoid pathway which plays important roles in plant development and adaptation. To date, there is no report on the study of PAL from oil palm (Elaeis guineensis), an economically important oil crop. In this study, the 5' regulatory sequence of a highly divergent oil palm PAL gene (EgPAL1) was isolated and fused with GUS in Arabidopsis to create two transgenic plants carrying the minimal promoter with (2302 bp) and without its regulatory elements (139 bp). The regulatory sequence contained cis-acting elements known to be important for plant development and stress response including the AC-II element for lignin biosynthesis and several stress responsive elements. The promoter and its regulatory region were fully functional in Arabidopsis. Its activities were characterised by two common fundamental features of PAL which are responsive to plant internal developmental programme and external factors. The promoter was developmentally regulated in certain organs; highly active in young organs but less active or inactive in mature organs. The presence of the AC elements and global activity of the EgPAL1 promoter in all organs resembled the property of lignin-related genes. The existence of the MBS element and enhancement of the promoter activity by PEG reflected the behaviour of drought-responsive genes. Our findings provide a platform for evaluating oil palm gene promoters in the heterologous system of Arabidopsis and give insights into the activities of EgPAL1 promoter in oil palm.

  10. Transcriptional coactivator NT-PGC-1α promotes gluconeogenic gene expression and enhances hepatic gluconeogenesis.

    Science.gov (United States)

    Chang, Ji Suk; Jun, Hee-Jin; Park, Minsung

    2016-10-01

    The transcriptional coactivator PGC-1α plays a central role in hepatic gluconeogenesis. We previously reported that alternative splicing of the PGC-1α gene produces an additional transcript encoding the truncated protein NT-PGC-1α NT-PGC-1α is co-expressed with PGC-1α and highly induced by fasting in the liver. NT-PGC-1α regulates tissue-specific metabolism, but its role in the liver has not been investigated. Thus, the objective of this study was to determine the role of hepatic NT-PGC-1α in the regulation of gluconeogenesis. Adenovirus-mediated expression of NT-PGC-1α in primary hepatocytes strongly stimulated the expression of key gluconeogenic enzyme genes (PEPCK and G6Pase), leading to increased glucose production. To further understand NT-PGC-1α function in hepatic gluconeogenesis in vivo, we took advantage of a previously reported FL-PGC-1α -/- mouse line that lacks full-length PGC-1α (FL-PGC-1α) but retains a slightly shorter and functionally equivalent form of NT-PGC-1α (NT-PGC-1α 254 ). In FL-PGC-1α -/- mice, NT-PGC-1α 254 was induced by fasting in the liver and recruited to the promoters of PEPCK and G6Pase genes. The enrichment of NT-PGC-1α 254 at the promoters was closely associated with fasting-induced increase in PEPCK and G6Pase gene expression and efficient production of glucose from pyruvate during a pyruvate tolerance test in FL-PGC-1α -/- mice. Moreover, FL-PGC-1α -/- primary hepatocytes showed a significant increase in gluconeogenic gene expression and glucose production after treatment with dexamethasone and forskolin, suggesting that NT-PGC-1α 254 is sufficient to stimulate the gluconeogenic program in the absence of FL-PGC-1α Collectively, our findings highlight the role of hepatic NT-PGC-1α in stimulating gluconeogenic gene expression and glucose production. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  11. The importance of conserved amino acids in heme-based globin-coupled diguanylate cyclases.

    Directory of Open Access Journals (Sweden)

    Xuehua Wan

    Full Text Available Globin-coupled diguanylate cyclases contain globin, middle, and diguanylate cyclase domains that sense O2 to synthesize c-di-GMP and regulate bacterial motility, biofilm formation, and virulence. However, relatively few studies have extensively examined the roles of individual residues and domains of globin-coupled diguanylate cyclases, which can shed light on their signaling mechanisms and provide drug targets. Here, we report the critical residues of two globin-coupled diguanylate cyclases, EcGReg from Escherichia coli and BpeGReg from Bordetella pertussis, and show that their diguanylate cyclase activity requires an intact globin domain. In the distal heme pocket of the globin domain, residues Phe42, Tyr43, Ala68 (EcGReg/Ser68 (BpeGReg, and Met69 are required to maintain full diguanylate cyclase activity. The highly conserved amino acids His223/His225 and Lys224/Lys226 in the middle domain of EcGReg/BpeGReg are essential to diguanylate cyclase activity. We also identified sixteen important residues (Leu300, Arg306, Asp333, Phe337, Lys338, Asn341, Asp342, Asp350, Leu353, Asp368, Arg372, Gly374, Gly375, Asp376, Glu377, and Phe378 in the active site and inhibitory site of the diguanylate cyclase domain of EcGReg. Moreover, BpeGReg266 (residues 1-266 and BpeGReg296 (residues 1-296, which only contain the globin and middle domains, can inhibit bacterial motility. Our findings suggest that the distal residues of the globin domain affect diguanylate cyclase activity and that BpeGReg may interact with other c-di-GMP-metabolizing proteins to form mixed signaling teams.

  12. ΔNp73 enhances promoter activity of TGF-β induced genes.

    Directory of Open Access Journals (Sweden)

    Maarten Niemantsverdriet

    Full Text Available The p53 homolog p73 is frequently overexpressed in cancers. Especially the transactivation domain truncated isoform ΔNp73 has oncogenic properties and its upregulation is associated with poor patient survival. It has been shown that ΔNp73 has an inhibitory effect on the transactivation capacity of p53 and other p73 isoforms. Here, we confirm this finding but surprisingly find that ΔNp73 may also stimulate the expression of TGF-β signaling targets. Promoter-reporter analysis indicated that the presence of Smad Binding Elements (SBE in the promoter is sufficient for stimulation of gene expression by ΔNp73. TGF-β signaling was less efficient in ΔNp73 downregulated cells, whereas tetracycline induced ΔNp73 increased expression of endogenous TGF-β regulated genes PAI-1 and Col1a1. Pull-down assays with SBE DNA suggest that ΔNp73 enhances smad3/4 binding to SBEs, thereby stimulating TGF-β signaling. Chromatin immunoprecipitation assays confirmed a direct interaction between ΔNp73 and SBE. Given the role of TGF-β signaling in carcinogenesis, tumor invasion and metastasis via targets like PAI-1 and Col1a1, our data suggest a model on how this effect of ΔNp73 could be a contributing factor in cancer progression.

  13. RN-1, a potent and selective lysine-specific demethylase 1 inhibitor, increases γ-globin expression, F reticulocytes, and F cells in a sickle cell disease mouse model.

    Science.gov (United States)

    Rivers, Angela; Vaitkus, Kestis; Ruiz, Maria Armila; Ibanez, Vinzon; Jagadeeswaran, Ramasamy; Kouznetsova, Tatiana; DeSimone, Joseph; Lavelle, Donald

    2015-07-01

    Increased levels of fetal hemoglobin are associated with decreased symptoms and increased lifespan in patients with sickle cell disease (SCD). Hydroxyurea, the only drug currently approved for SCD, is not effective in a large fraction of patients, and therefore, new agents are urgently needed. Recently it was found that lysine demethylase 1, an enzyme that removes monomethyl and dimethyl residues from the lysine 4 residue of histone H3, is a repressor of γ-globin gene expression. In this article, we have compared the ability of tranylcypromine (TCP) and a more potent TCP derivative, RN-1, to increase γ-globin expression in cultured baboon erythroid progenitor cells and in the SCD mouse model. The results indicate that the ability of RN-1 to induce F cells and γ-globin mRNA in SCD mice is similar to that of decitabine, the most powerful fetal hemoglobin-inducing drug known, and greater than that of either TCP or hydroxyurea. We conclude that RN-1 and other lysine demethylase 1 inhibitors may be promising new γ-globin-inducing agents for the treatment of SCD that warrant further studies in other preclinical models, such as nonhuman primates. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  14. In silico analysis and DHPLC screening strategy identifies novel apoptotic gene targets of aberrant promoter hypermethylation in prostate cancer.

    LENUS (Irish Health Repository)

    Murphy, Therese M

    2011-01-01

    Aberrant DNA methylation has been implicated as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many cellular processes including apoptosis. Limited data is available on the methylation profile of apoptotic genes in prostate cancer (CaP). The aim of this study was to profile methylation of apoptotic-related genes in CaP using denaturing high performance liquid chromatography (DHPLC).

  15. The CHR promoter element controls cell cycle-dependent gene transcription and binds the DREAM and MMB complexes.

    Science.gov (United States)

    Müller, Gerd A; Quaas, Marianne; Schümann, Michael; Krause, Eberhard; Padi, Megha; Fischer, Martin; Litovchick, Larisa; DeCaprio, James A; Engeland, Kurt

    2012-02-01

    Cell cycle-dependent gene expression is often controlled on the transcriptional level. Genes like cyclin B, CDC2 and CDC25C are regulated by cell cycle-dependent element (CDE) and cell cycle genes homology region (CHR) promoter elements mainly through repression in G(0)/G(1). It had been suggested that E2F4 binding to CDE sites is central to transcriptional regulation. However, some promoters are only controlled by a CHR. We identify the DREAM complex binding to the CHR of mouse and human cyclin B2 promoters in G(0). Association of DREAM and cell cycle-dependent regulation is abrogated when the CHR is mutated. Although E2f4 is part of the complex, a CDE is not essential but can enhance binding of DREAM. We show that the CHR element is not only necessary for repression of gene transcription in G(0)/G(1), but also for activation in S, G(2) and M phases. In proliferating cells, the B-myb-containing MMB complex binds the CHR of both promoters independently of the CDE. Bioinformatic analyses identify many genes which contain conserved CHR elements in promoters binding the DREAM complex. With Ube2c as an example from that screen, we show that inverse CHR sites are functional promoter elements that can bind DREAM and MMB. Our findings indicate that the CHR is central to DREAM/MMB-dependent transcriptional control during the cell cycle.

  16. The CHR Promoter Element Controls Cell Cycle-Dependent Gene Transcription and Binds the DREAM and MMB Complexes

    OpenAIRE

    Müller, Gerd A.; Quaas, Marianne; Schümann, Michael; Krause, Eberhard; Fischer, Martin; Engeland, Kurt; Padi, Megha; Litovchick, Larisa; DeCaprio, James A.

    2011-01-01

    Cell cycle-dependent gene expression is often controlled on the transcriptional level. Genes like \\(cyclin B, CDC2\\) and \\(CDC25C\\) are regulated by cell cycle-dependent element (CDE) and cell cycle genes homology region (CHR) promoter elements mainly through repression in \\(G_0/G_1\\). It had been suggested that E2F4 binding to CDE sites is central to transcriptional regulation. However, some promoters are only controlled by a CHR. We identify the DREAM complex binding to the CHR of mouse and...

  17. Characterization of an unusual thyroid response unit in the promoter of the human placental lactogen gene.

    Science.gov (United States)

    Voz, M L; Peers, B; Belayew, A; Martial, J A

    1991-07-15

    The human placental lactogen B (hCS-B) promoter activity is strongly stimulated by thyroid hormones in the rat pituitary GC cell line. The minimal DNA sequence required for stimulation, as determined by transfection with 5' and 3' deletion mutants, spans 67 base pairs, from coordinate -97 to -31. DNase I footprinting experiments show that this thyroid response unit includes two adjacent binding sites: one for the thyroid receptor (-67/-41), the other for the pituitary-specific factor GHF1 (-95/-68). Neither region alone is sufficient to confer thyroid responsiveness. The thyroid receptor binding element (TBE) does not contain any repeats or palindromes but is composed of two different domains, one of which is very similar to the half-palindromic motif described by Glass et al. (Glass, C.K., Holloway, J.M., Devary, O.L., and Rosenfeld, M.G. (1988) Cell 54, 313-323). The other is very rich in purine. The normal human growth hormone (hGH-N) promoter, which is 94% similar to the hCS-B promoter, differs from its hCS-B counterpart precisely in this TBE. This difference may explain the opposite 3,5,3'-triiodothyronine (T3) regulation of these two genes.

  18. Methylation state of the EDA gene promoter in Chinese X-linked hypohidrotic ectodermal dysplasia carriers.

    Directory of Open Access Journals (Sweden)

    Wei Yin

    Full Text Available Hypodontia, hypohidrosis, sparse hair and characteristic faces are the main characters of X-linked hypohidrotic ectodermal dysplasia (XLHED which is caused by genetic ectodysplasin A (EDA deficiency. Heterozygous female carriers tend to have mild to moderate XLHED phenotype, even though 30% of them present no obvious symptom.A large Chinese XLHED family was reported and the entire coding region and exon-intron boundaries of EDA gene were sequenced. To elucidate the mechanism for carriers' tempered phenotype, we analyzed the methylation level on four sites of the promoter of EDA by the pyrosequencing system.A known frameshift mutation (c.573-574 insT was found in this pedigree. Combined with the pedigrees we reported before, 120 samples comprised of 23 carrier females from 11 families and 97 healthy females were analyzed for the methylation state of EDA promoter. Within 95% confidence interval (CI, 18 (78.26% carriers were hypermethylated at these 4 sites.Chinese XLHED carriers often have a hypermethylated EDA promoter.

  19. Human papillomavirus type 16 E7 perturbs DREAM to promote cellular proliferation and mitotic gene expression.

    Science.gov (United States)

    DeCaprio, J A

    2014-07-31

    The study of the small DNA tumor viruses continues to provide valuable new insights into oncogenesis and fundamental biological processes. Although much has already been revealed about how the human papillomaviruses (HPVs) can transform cells and contribute to cervical and oropharyngeal cancer, there clearly is much more to learn. In this issue of Oncogene, Pang et al., doi:10.1038/onc.2013.426, demonstrate that the high-risk HPV16 E7 oncogene can promote cellular proliferation by interacting with the DREAM (DP, RB-like, E2F and MuvB) complex at two distinct phases of the cell cycle. Consistent with earlier work, HPV16 E7 can bind to the retinoblastoma tumor suppressor (RB) family member p130 (RBL2) protein and promote its proteasome-mediated destruction thereby disrupting the DREAM complex and can prevent exit from the cell cycle into quiescence. In addition, they demonstrate that HPV16 E7 can bind to MuvB core complex in association with BMYB and FOXM1 and activate gene expression during the G2 and M phase of the cell cycle. Thus, HPV16 E7 acts to prevent exit from the cell cycle entry and promotes mitotic proliferation and may account for the high levels of FOXM1 often observed in poor-risk cervical cancers.

  20. Regulation of gene expression in the protozoan parasite Entamoeba invadens identification of core promoter elements and promoters with stage-specific expression patterns

    Science.gov (United States)

    Manna, Dipak; Ehrenkaufer, Gretchen M.; Singh, Upinder

    2014-01-01

    Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. Entamoeba histolytica is an important human pathogen and is a leading parasitic cause of death globally. During its life cycle, Entamoeba converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Despite being central to its biology, the triggers that are involved in the developmental pathways of this parasite are not well understood. In order to define the transcriptional network associated with stage conversion we used Entamoeba invadens which serves as a model system for Entamoeba developmental biology, and performed RNA sequencing at different developmental time points . In this study RNA-Seq data was utilized to define basal transcriptional control elements as well as to identify promoters which regulate stage-specific gene expression patterns. We discovered that the 5’ and 3’ untranslated regions of E. invadens genes are short, a median of 20 nucleotides (nt) and 26 nt respectively. Bioinformatics analysis of DNA sequences proximate to the start and stop codons identified two conserved motifs: (i) E. invadens Core Promoter Motif - GAAC-Like (EiCPM-GL) (GAACTACAAA), and (ii) E. invadens 3’- U-Rich Motif (Ei3’-URM) (TTTGTT) in the 5’ and 3’ flanking regions, respectively. Electrophoretic mobility shift assays demonstrated that both motifs specifically bind nuclear protein(s) from E. invadens trophozoites. Additionally, we identified select genes with stage-specific expression patterns and analyzed the ability of each gene promoter to drive a luciferase reporter gene during the developmental cycle. This approach confirmed three trophozoite-specific, four encystation-specific and two excystation-specific promoters. This work lays the framework for use of stage-specific promoters to express proteins of interest in a particular life-cycle stage, adding to the molecular toolbox for genetic

  1. Regulation of gene expression in the protozoan parasite Entamoeba invadens: identification of core promoter elements and promoters with stage-specific expression patterns.

    Science.gov (United States)

    Manna, Dipak; Ehrenkaufer, Gretchen M; Singh, Upinder

    2014-10-01

    Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. Entamoeba histolytica is an important human pathogen and is a leading parasitic cause of death globally. During its life cycle, Entamoeba converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Despite being central to its biology, the triggers that are involved in the developmental pathways of this parasite are not well understood. In order to define the transcriptional network associated with stage conversion we used Entamoeba invadens which serves as a model system for Entamoeba developmental biology, and performed RNA sequencing at different developmental time points. In this study RNA-Seq data was utilised to define basal transcriptional control elements as well as to identify promoters which regulate stage-specific gene expression patterns. We discovered that the 5' and 3' untranslated regions of E. invadens genes are short, a median of 20 nucleotides (nt) and 26 nt respectively. Bioinformatics analysis of DNA sequences proximate to the start and stop codons identified two conserved motifs: (i) E. invadens Core Promoter Motif - GAAC-Like (EiCPM-GL) (GAACTACAAA), and (ii) E. invadens 3'-U-Rich Motif (Ei3'-URM) (TTTGTT) in the 5' and 3' flanking regions, respectively. Electrophoretic mobility shift assays demonstrated that both motifs specifically bind nuclear protein(s) from E. invadens trophozoites. Additionally, we identified select genes with stage-specific expression patterns and analysed the ability of each gene promoter to drive a luciferase reporter gene during the developmental cycle. This approach confirmed three trophozoite-specific, four encystation-specific and two excystation-specific promoters. This work lays the framework for use of stage-specific promoters to express proteins of interest in a particular life-cycle stage, adding to the molecular toolbox for genetic manipulation of E

  2. Molecular analysis of the human SLC13A4 sulfate transporter gene promoter

    Energy Technology Data Exchange (ETDEWEB)

    Jefferis, J. [Mater Medical Research Institute, South Brisbane, Queensland (Australia); Rakoczy, J. [Mater Medical Research Institute, South Brisbane, Queensland (Australia); School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland (Australia); Translational Research Institute, Woolloongabba, Queensland (Australia); Simmons, D.G. [School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland (Australia); Dawson, P.A., E-mail: paul.dawson@mmri.mater.org.au [Mater Medical Research Institute, South Brisbane, Queensland (Australia); Translational Research Institute, Woolloongabba, Queensland (Australia)

    2013-03-29

    Highlights: ► Basal promoter activity of SLC13A4 −57 to −192 nt upstream of transcription initiation site. ► Human SLC13A4 5′-flanking region has conserved motifs with other placental species. ► Putative NFY, SP1 and KLF7 motifs in SLC13A4 5′-flanking region enhance transcription. -- Abstract: The human solute linked carrier (SLC) 13A4 gene is primarily expressed in the placenta where it is proposed to mediate the transport of nutrient sulfate from mother to fetus. The molecular mechanisms involved in the regulation of SLC13A4 expression remain unknown. To investigate the regulation of SLC13A4 gene expression, we analysed the transcriptional activity of the human SLC13A4 5′-flanking region in the JEG-3 placental cell line using luciferase reporter assays. Basal transcriptional activity was identified in the region −57 to −192 nucleotides upstream of the SLC13A4 transcription initiation site. Mutational analysis of the minimal promoter region identified Nuclear factor Y (NFY), Specificity protein 1 (SP1) and Krüppel like factor 7 (KLF7) motifs which conferred positive transcriptional activity, as well as Zinc finger protein of the cerebellum 2 (ZIC2) and helix–loop–helix protein 1 (HEN1) motifs that repressed transcription. The conserved NFY, SP1, KLF7, ZIC2 and HEN1 motifs in the SLC13A4 promoter of placental species but not in non-placental species, suggests a potential role for these putative transcriptional factor binding motifs in the physiological control of SLC13A4 mRNA expression.

  3. Linkage mapping of candidate genes for induce resistance and growth promotion by trichoderma koningiopsis (th003) in tomato solanum lycopersicum

    International Nuclear Information System (INIS)

    Simbaqueba, Jaime; Cotes, Alba Marina; Barrero, Luz Stella

    2011-01-01

    Induced systemic resistance (ISR) is a mechanism by which plants enhance defenses against any stress condition. ISR and growth promotion are enhanced when tomato (Solanum lycopersicum) is inoculated with several strains of Trichoderma ssp. this study aims to genetically map tomato candidate genes involved in ISR and growth promotion induced by the Colombian native isolate Trichoderma koningiopsis th003. Forty-nine candidate genes previously identified on tomato plants treated with th003 and T. hamatum T382 strains were evaluated for polymorphisms and 16 of them were integrated on the highly saturated genetic linkage map named TOMATO EXPEN 2000. The location of six unigenes was similar to the location of resistance gene analogs (RGAS), defense related ests and resistance QTLs previously reported, suggesting new possible candidates for these quantitative trait loci (QTL) regions. The candidate gene-markers may be used for future ISR or growth promotion assisted selection in tomato.

  4. Light response and potential interacting proteins of a grape flavonoid 3'-hydroxylase gene promoter.

    Science.gov (United States)

    Sun, Run-Ze; Pan, Qiu-Hong; Duan, Chang-Qing; Wang, Jun

    2015-12-01

    Flavonoid 3'-hydroxylase (F3'H), a member of cytochrome P450 protein family, introduces B-ring hydroxyl group in the 3' position of the flavonoid. In this study, the cDNA sequence of a F3'H gene (VviF3'H), which contains an open reading frame of 1530 bp encoding a polypeptide of 509 amino acids, was cloned and characterized from Vitis vinifera L. cv. Cabernet Sauvignon. VviF3'H showed high homology to known F3'H genes, especially F3'Hs from the V. vinifera reference genome (Pinot Noir) and lotus. Expression profiling analysis using real-time PCR revealed that VviF3'H was ubiquitously expressed in all tested tissues including berries, leaves, flowers, roots, stems and tendrils, suggesting its important physiological role in plant growth and development. Moreover, the transcript level of VviF3'H gene in grape berries was relatively higher at early developmental stages and gradually decreased during véraison, and then increased in the mature phase. In addition, the promoter of VviF3'H was isolated by using TAIL-PCR. Yeast one-hybrid screening of the Cabernet Sauvignon cDNA library and subsequent in vivo/vitro validations revealed the interaction between VviF3'H promoter and several transcription factors, including members of HD-Zip, NAC, MYB and EIN families. A transcriptional regulation mechanism of VviF3'H expression is proposed for the first time. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  5. Estrogen regulates transcription of the ovine oxytocin receptor gene through GC-rich SP1 promoter elements.

    Science.gov (United States)

    Fleming, Joann G W; Spencer, Thomas E; Safe, Stephen H; Bazer, Fuller W

    2006-02-01

    Establishment of pregnancy in ruminants results from paracrine signaling by interferon tau (IFNT) from the conceptus to uterine endometrial luminal epithelia (LE) that prevents release of luteolytic prostaglandin F(2alpha) pulses. In cyclic and pregnant ewes, progesterone down-regulates progesterone receptor (PGR) gene expression in LE. In cyclic ewes, loss of PGR allows for increases in estrogen receptor alpha (ESR1) and then oxytocin receptor (OXTR) gene expression followed by oxytocin-induced prostaglandin F(2alpha) pulses. In pregnant ewes, IFNT inhibits transcription of the ESR1 gene, which presumably inhibits OXTR gene transcription. Alternatively, IFNT may directly inhibit OXTR gene transcription. The 5' promoter/enhancer region of the ovine OXTR gene was cloned and found to contain predicted binding sites for activator protein 1, SP1, and PGR, but not for ESR1. Deletion analysis showed that the basal promoter activity was dependent on the region from -144 to -4 bp that contained only SP1 sites. IFNT did not affect activity of the OXTR promoter. In cells transfected with ESR1, E2, and ICI 182,780 increased promoter activity due to GC-rich SP1 binding sites at positions -104 and -64. Mutation analyses showed that the proximal SP1 sites mediated ESR1 action as well as basal activity of the promoter. In response to progesterone, progesterone receptor B also increased OXTR promoter activity. SP1 protein was constitutively expressed and abundant in the LE of the ovine uterus. These results support the hypothesis that the antiluteolytic effects of IFNT are mediated by direct inhibition or silencing of ESR1 gene transcription, thereby precluding ESR1/SP1 from stimulating OXTR gene transcription.

  6. Deletion of the Imprinted Gene Grb10 Promotes Hematopoietic Stem Cell Self-Renewal and Regeneration.

    Science.gov (United States)

    Yan, Xiao; Himburg, Heather A; Pohl, Katherine; Quarmyne, Mamle; Tran, Evelyn; Zhang, Yurun; Fang, Tiancheng; Kan, Jenny; Chao, Nelson J; Zhao, Liman; Doan, Phuong L; Chute, John P

    2016-11-01

    Imprinted genes are differentially expressed by adult stem cells, but their functions in regulating adult stem cell fate are incompletely understood. Here we show that growth factor receptor-bound protein 10 (Grb10), an imprinted gene, regulates hematopoietic stem cell (HSC) self-renewal and regeneration. Deletion of the maternal allele of Grb10 in mice (Grb10 m/+ mice) substantially increased HSC long-term repopulating capacity, as compared to that of Grb10 +/+ mice. After total body irradiation (TBI), Grb10 m/+ mice demonstrated accelerated HSC regeneration and hematopoietic reconstitution, as compared to Grb10 +/+ mice. Grb10-deficient HSCs displayed increased proliferation after competitive transplantation or TBI, commensurate with upregulation of CDK4 and Cyclin E. Furthermore, the enhanced HSC regeneration observed in Grb10-deficient mice was dependent on activation of the Akt/mTORC1 pathway. This study reveals a function for the imprinted gene Grb10 in regulating HSC self-renewal and regeneration and suggests that the inhibition of Grb10 can promote hematopoietic regeneration in vivo. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  7. USP7 Attenuates Hepatic Gluconeogenesis Through Modulation of FoxO1 Gene Promoter Occupancy

    Science.gov (United States)

    Hall, Jessica A.; Tabata, Mitsuhisa; Rodgers, Joseph T.

    2014-01-01

    Hepatic forkhead protein FoxO1 is a key component of systemic glucose homeostasis via its ability to regulate the transcription of rate-limiting enzymes in gluconeogenesis. Important in the regulation of FoxO1 transcriptional activity are the modifying/demodifying enzymes that lead to posttranslational modification. Here, we demonstrate the functional interaction and regulation of FoxO1 by herpesvirus-associated ubiquitin-specific protease 7 (USP7; also known as herpesvirus-associated ubiquitin-specific protease, HAUSP), a deubiquitinating enzyme. We show that USP7-mediated mono-deubiquitination of FoxO1 results in suppression of FoxO1 transcriptional activity through decreased FoxO1 occupancy on the promoters of gluconeogenic genes. Knockdown of USP7 in primary hepatocytes leads to increased expression of FoxO1-target gluconeogenic genes and elevated glucose production. Consistent with this, USP7 gain-of-function suppresses the fasting/cAMP-induced activation of gluconeogenic genes in hepatocyte cells and in mouse liver, resulting in decreased hepatic glucose production. Notably, we show that the effects of USP7 on hepatic glucose metabolism depend on FoxO1. Together, these results place FoxO1 under the intimate regulation of deubiquitination and glucose metabolic control with important implication in diseases such as diabetes. PMID:24694308

  8. Klotho gene silencing promotes pathology in the mdx mouse model of Duchenne muscular dystrophy

    Science.gov (United States)

    Wehling-Henricks, Michelle; Li, Zhenzhi; Lindsey, Catherine; Wang, Ying; Welc, Steven S.; Ramos, Julian N.; Khanlou, Négar; Kuro-o, Makoto; Tidball, James G.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a lethal muscle disease involving progressive loss of muscle regenerative capacity and increased fibrosis. We tested whether epigenetic silencing of the klotho gene occurs in the mdx mouse model of DMD and whether klotho silencing is an important feature of the disease. Our findings show that klotho undergoes muscle-specific silencing at the acute onset of mdx pathology. Klotho experiences increased methylation of CpG sites in its promoter region, which is associated with gene silencing, and increases in a repressive histone mark, H3K9me2. Expression of a klotho transgene in mdx mice restored their longevity, reduced muscle wasting, improved function and greatly increased the pool of muscle-resident stem cells required for regeneration. Reductions of fibrosis in late, progressive stages of the mdx pathology achieved by transgene expression were paralleled by reduced expression of Wnt target genes (axin-2), transforming growth factor-beta (TGF-β1) and collagens types 1 and 3, indicating that Klotho inhibition of the profibrotic Wnt/TGFβ axis underlies its anti-fibrotic effect in aging, dystrophic muscle. Thus, epigenetic silencing of klotho during muscular dystrophy contributes substantially to lost regenerative capacity and increased fibrosis of dystrophic muscle during late progressive stages of the disease. PMID:27154199

  9. Allele-specific enzymatic amplification of. beta. -globin genomic DNA for diagnosis of sickle cell anemia

    Energy Technology Data Exchange (ETDEWEB)

    Wu, D.Y.; Ugozzoli, L.; Pal, B.K.; Wallace, B. (Beckman Research Institute of the City of Hope, Duarte, CA (USA))

    1989-04-01

    A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell {beta}-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3{prime} nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing.

  10. Association between promoter polymorphisms of OPN gene and cancer risk: a meta-analysis

    Directory of Open Access Journals (Sweden)

    Liu JW

    2015-12-01

    Full Text Available Jingwei Liu,1–2 Caiyun He,1–2 Quan Yuan,1–2 Zhenning Wang,1–2 Chengzhong Xing,1–2 Yuan Yuan1–2 1Tumor Etiology and Screening Department of Cancer Institute and General Surgery, The First Affiliated Hospital of China Medical University, 2Key Laboratory of Cancer Etiology and Prevention, China Medical University, Liaoning Provincial Education Department, Shenyang, People’s Republic of China Background: Results of the association between polymorphisms of osteopontin (OPN gene promoter region and risk of cancer were inconclusive. The aim of this meta-analysis was to elucidate whether OPN promoter polymorphisms were associated with cancer risk.Methods: Electronic databases including PubMed, Web of Science, and Chinese National Knowledge Infrastructure were systematically searched. Odd ratios (ORs and their 95% confidential interval (CI were used to assess the strength of association between OPN promoter polymorphisms and cancer risks.Results: Nine studies were finally included in this meta-analysis. For OPN rs17524488 polymorphism, carriers of GG or -/G genotype were significantly associated with increased cancer risk compared with wild-type -/- carriers, respectively (GG vs -/-: OR =1.40, 95% CI =1.03–1.91, P=0.033; -/G vs -/-: OR =1.22, 95% CI =1.07–1.40, P=0.002. Additionally, G allele was significantly associated with increased cancer risk compared with (- allele (OR =1.21, 95% CI =1.04–1.40, P=0.016. However, no significant association was observed of OPN rs11730582 polymorphism and cancer risk (CC vs TT: OR =0.98, 95% CI =0.49–1.97, P=0.964; CT vs TT: OR =0.88, 95% CI =0.54–1.43, P=0.610.Conclusion: Carriers of GG or -/G genotype of OPN promoter rs17524488 (-156-/G polymorphism might be associated with increased risk of cancer compared with wild-type -/- carriers, respectively. However, no significant association was observed between OPN promoter rs11730582 (-443C/T polymorphism and risk of cancer. Keywords: OPN

  11. Promoter Analysis Reveals Globally Differential Regulation of Human Long Non-Coding RNA and Protein-Coding Genes

    KAUST Repository

    Alam, Tanvir

    2014-10-02

    Transcriptional regulation of protein-coding genes is increasingly well-understood on a global scale, yet no comparable information exists for long non-coding RNA (lncRNA) genes, which were recently recognized to be as numerous as protein-coding genes in mammalian genomes. We performed a genome-wide comparative analysis of the promoters of human lncRNA and protein-coding genes, finding global differences in specific genetic and epigenetic features relevant to transcriptional regulation. These two groups of genes are hence subject to separate transcriptional regulatory programs, including distinct transcription factor (TF) proteins that significantly favor lncRNA, rather than coding-gene, promoters. We report a specific signature of promoter-proximal transcriptional regulation of lncRNA genes, including several distinct transcription factor binding sites (TFBS). Experimental DNase I hypersensitive site profiles are consistent with active configurations of these lncRNA TFBS sets in diverse human cell types. TFBS ChIP-seq datasets confirm the binding events that we predicted using computational approaches for a subset of factors. For several TFs known to be directly regulated by lncRNAs, we find that their putative TFBSs are enriched at lncRNA promoters, suggesting that the TFs and the lncRNAs may participate in a bidirectional feedback loop regulatory network. Accordingly, cells may be able to modulate lncRNA expression levels independently of mRNA levels via distinct regulatory pathways. Our results also raise the possibility that, given the historical reliance on protein-coding gene catalogs to define the chromatin states of active promoters, a revision of these chromatin signature profiles to incorporate expressed lncRNA genes is warranted in the future.

  12. Characterization of the osmotic response element of the human aldose reductase gene promoter.

    Science.gov (United States)

    Ruepp, B; Bohren, K M; Gabbay, K H

    1996-08-06

    Aldose reductase (EC 1.1.1.21) catalyzes the NADPH-mediated conversion of glucose to sorbitol. The hyperglycemia of diabetes increases sorbitol production primarily through substrate availability and is thought to contribute to the pathogenesis of many diabetic complications. Increased sorbitol production can also occur at normoglycemic levels via rapid increases in aldose reductase transcription and expression, which have been shown to occur upon exposure of many cell types to hyperosmotic conditions. The induction of aldose reductase transcription and the accumulation of sorbitol, an organic osmolyte, have been shown to be part of the physiological osmoregulatory mechanism whereby renal tubular cells adjust to the intraluminal hyperosmolality during urinary concentration. Previously, to explore the mechanism regulating aldose reductase levels, we partially characterized the human aldose reductase gene promoter present in a 4.2-kb fragment upstream of the transcription initiation start site. A fragment (-192 to +31 bp) was shown to contain several elements that control the basal expression of the enzyme. In this study, we examined the entire 4.2-kb human AR gene promoter fragment by deletion mutagenesis and transfection studies for the presence of osmotic response enhancer elements. An 11-bp nucleotide sequence (TGGAAAATTAC) was located 3.7 kb upstream of the transcription initiation site that mediates hypertonicity-responsive enhancer activity. This osmotic response element (ORE) increased the expression of the chloramphenicol acetyltransferase reporter gene product 2-fold in transfected HepG2 cells exposed to hypertonic NaCl media as compared with isoosmotic media. A more distal homologous sequence is also described; however, this sequence has no osmotic enhancer activity in transfected cells. Specific ORE mutant constructs, gel shift, and DNA fragment competition studies confirm the nature of the element and identify specific nucleotides essential for enhancer

  13. Compound A, a Selective Glucocorticoid Receptor Modulator, Enhances Heat Shock Protein Hsp70 Gene Promoter Activation

    Science.gov (United States)

    Beck, Ilse M.; Drebert, Zuzanna J.; Hoya-Arias, Ruben; Bahar, Ali A.; Devos, Michael; Clarisse, Dorien; Desmet, Sofie; Bougarne, Nadia; Ruttens, Bart; Gossye, Valerie; Denecker, Geertrui; Lievens, Sam; Bracke, Marc; Tavernier, Jan; Declercq, Wim; Gevaert, Kris; Berghe, Wim Vanden; Haegeman, Guy; De Bosscher, Karolien

    2013-01-01

    Compound A possesses glucocorticoid receptor (GR)-dependent anti-inflammatory properties. Just like classical GR ligands, Compound A can repress NF-κB-mediated gene expression. However, the monomeric Compound A-activated GR is unable to trigger glucocorticoid response element-regulated gene expression. The heat shock response potently activates heat shock factor 1 (HSF1), upregulates Hsp70, a known GR chaperone, and also modulates various aspects of inflammation. We found that the selective GR modulator Compound A and heat shock trigger similar cellular effects in A549 lung epithelial cells. With regard to their anti-inflammatory mechanism, heat shock and Compound A are both able to reduce TNF-stimulated IκBα degradation and NF-κB p65 nuclear translocation. We established an interaction between Compound A-activated GR and Hsp70, but remarkably, although the presence of the Hsp70 chaperone as such appears pivotal for the Compound A-mediated inflammatory gene repression, subsequent novel Hsp70 protein synthesis is uncoupled from an observed CpdA-induced Hsp70 mRNA upregulation and hence obsolete in mediating CpdA’s anti-inflammatory effect. The lack of a Compound A-induced increase in Hsp70 protein levels in A549 cells is not mediated by a rapid proteasomal degradation of Hsp70 or by a Compound A-induced general block on translation. Similar to heat shock, Compound A can upregulate transcription of Hsp70 genes in various cell lines and BALB/c mice. Interestingly, whereas Compound A-dependent Hsp70 promoter activation is GR-dependent but HSF1-independent, heat shock-induced Hsp70 expression alternatively occurs in a GR-independent and HSF1-dependent manner in A549 lung epithelial cells. PMID:23935933

  14. Mediator subunit MED1 is a T3-dependent and T3-independent coactivator on the thyrotropin β gene promoter

    Energy Technology Data Exchange (ETDEWEB)

    Matsui, Keiji; Oda, Kasumi; Mizuta, Shumpei; Ishino, Ruri; Urahama, Norinaga; Hasegawa, Natsumi [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Roeder, Robert G. [Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Ito, Mitsuhiro, E-mail: itomi@med.kobe-u.ac.jp [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Department of Family and Community Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 654-0142 (Japan); Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan)

    2013-10-11

    Highlights: •MED1 is a bona fide T3-dependent coactivator on TSHB promoter. •Mice with LxxLL-mutant MED1 have attenuated TSHβ mRNA and thyroid hormone levels. •MED1 activates TSHB promoter T3-dependently in cultured cells. •T3-dependent MED1 action is enhanced when SRC1/SRC2 or HDAC2 is downregulated. •MED1 is also a T3-independent GATA2/Pit1 coactivator on TSHB promoter. -- Abstract: The MED1 subunit of the Mediator transcriptional coregulator complex is a nuclear receptor-specific coactivator. A negative feedback mechanism of thyroid-stimulating hormone (TSH, or thyrotropin) expression in the thyrotroph in the presence of triiodothyronine (T3) is employed by liganded thyroid hormone receptor β (TRβ) on the TSHβ gene promoter, where conventional histone-modifying coactivators act as corepressors. We now provide evidence that MED1 is a ligand-dependent positive cofactor on this promoter. TSHβ gene transcription was attenuated in MED1 mutant mice in which the nuclear receptor-binding ability of MED1 was specifically disrupted. MED1 stimulated GATA2- and Pit1-mediated TSHβ gene promoter activity in a ligand-independent manner in cultured cells. MED1 also stimulated transcription from the TSHβ gene promoter in a T3-dependent manner. The transcription was further enhanced when the T3-dependent corepressors SRC1, SRC2, and HDAC2 were downregulated. Hence, MED1 is a T3-dependent and -independent coactivator on the TSHβ gene promoter.

  15. [A meta-analysis of Association between MGMT gene promoter methylation and non-small cell lung cancer].

    Science.gov (United States)

    Fang, Nianzhen; Gu, Jundong; Wei, Huijun; You, Jiacong; Zhou, Qinghua

    2014-08-20

    DNA promoter methylation of the tumor suppressor genes was one of the key mechanism for gene silence. The aim of this study is to investigate the difference of MGMT gene promoter methylation rate in tumor tissue and autologous controls (serum, normal lung tissue and bronchial lavage fluid) in patients with non-small cell lung cancer (NSCLC). The databases of Medline, EMBSE, CNKI and Wanfang were searched for selection of published articles of MGMT gene promoter methylation and non-small cell lung carcinoma risk. The pooled odds ratio (OR) and percentage of MGMT for lung cancer tissue of NSCLC patients compared with normal lung tissue, plasma and the bronchial lavage fluid were pooled. 15 articles of association between MGMT gene promoter methylation and non small cell lung carcinoma risk were included in this meta-analysis. The combined results demonstrated the methylation rate of MGMT in NSCLC cancer tissue was 38% (95%CI: 23%-53%). For normal lung tissue, plasma and the bronchial lavage fluid were 16% (95%CI: 5%-27%), 23% (95%CI: 10%-34%) and 39% (95%CI: 23%-55%) respectively. The OR in cancer tissue was much higher than that in normal lung tissue and plasma odds ratio (OR) 3.98 (95%CI: 2.71-5.84, P0.05). Mehtylation rate in MGMT