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Sample records for adult globin genes

  1. Roles of fetal G gamma-globin promoter elements and the adult beta-globin 3' enhancer in the stage-specific expression of globin genes.

    PubMed

    Perez-Stable, C; Costantini, F

    1990-03-01

    The human fetal G gamma-globin and adult beta-globin genes are expressed in a tissue- and developmental stage-specific pattern in transgenic mice: the G gamma gene in embryonic cells and the beta gene in fetal and adult erythroid cells. Several of the cis-acting DNA sequences thought to be responsible for these patterns of expression are located 5' to the G gamma-globin gene and 3' to the beta-globin gene. To further define the locations and functional roles of these elements, we examined the effects of 5' truncations on the expression of the G gamma-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene. We found that sequences between -201 and -136 are essential for expression of the G gamma-globin gene, whereas those upstream of -201 have little effect on the level or tissue or stage specificity of G gamma-globin expression. The G gamma-globin upstream sequences from -201 to -136 were, furthermore, capable of activating a linked beta-globin gene in embryonic blood cells; however, a G gamma-globin fragment from -383 to -206 was similarly active in this assay, and the complete fragment from -383 to -136 was considerably more active than either of the smaller fragments, suggesting the presence of multiple cis-acting elements for embryonic blood cells. Our data also suggested the possibility of a negative regulatory element between -201 and -136. These results are discussed in relation to several DNA elements in the G gamma-globin upstream region, which have been shown to bind nuclear factors in erythroid cells. Finally, we observed that removal of the beta-globin 3'-flanking sequences, including the 3' enhancer, from the G gamma-globin upstream-beta-globin hybrid gene resulted in a 25-fold reduction in expression in embryonic blood cells. This suggests that the beta-globin

  2. Pharmacological Induction of Human Fetal Globin Gene in Hydroxyurea-Resistant Primary Adult Erythroid Cells.

    PubMed

    Chou, Yu-Chi; Chen, Ruei-Lin; Lai, Zheng-Sheng; Song, Jen-Shin; Chao, Yu-Sheng; Shen, Che-Kun James

    2015-07-01

    Pharmacological induction of the fetal γ globin gene and the consequent formation of HbF (α2/γ2) in adult erythroid cells are one feasible therapeutic strategy for sickle cell disease (SCD) and severe β-thalassemias. Hydroxyurea (HU) is the current drug of choice for SCD, but serious side effects limit its clinical use. Moreover, 30 to 50% of patients are irresponsive to HU treatment. We have used high-throughput screening to identify benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one and its derivatives (compounds I to VI) as potent γ globin inducers. Of the compounds, I to V exert superior γ globin induction and have better therapeutic potential than HU, likely because of their activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway and modulation of expression levels and/or chromosome binding of γ globin gene regulators, including BCL11A, and chromatin structure over the γ globin promoter. Unlike sodium butyrate (NaB), the global levels of acetylated histones H3 and H4 are not changed by compound II treatment. Remarkably, compound II induces the γ globin gene in HU-resistant primary human adult erythroid cells, the p38 signaling pathway of which appears to be irresponsive to HU and NaB as well as compound II. This study provides a new framework for the development of new and superior compounds for treating SCD and severe β-thalassemias. PMID:25986606

  3. Isolation and characterization of the complete chicken beta-globin gene region: frequent deletion of the adult beta-globin genes in lambda.

    PubMed Central

    Villeponteau, B; Martinson, H

    1981-01-01

    A library of bacteriophage lambda clones containing chicken chromosomal DNA was screened, using the adult beta-globin cDNA plasmid pHb 1001 as a probe. Sixteen overlapping clones were isolated containing 35 kilobase pairs (kbp) of chicken DNA. Characterization of these clones revealed four beta-like globin genes, some genomically repeated sequences, but no pseudo-genes. The four beta-like genes have an average intergenic distance of less than half of that found for the mammalian beta-like globin gene clusters so far characterized. The overall features of the map were confirmed by genomic Southern analysis. Frequent deletions were shown to occur between the various beta-like globin genes during phage propagation. The presumptive hatching gene in particular was always associated with abnormal lambda clones although we were able to find one such clone that did contain a normal copy of the hatching gene itself. Probably such deletions explain the failure to recover this gene in previous attempts. Images PMID:6269092

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

    SciTech Connect

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

    1987-06-01

    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.

  5. Silencing of Aγ-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the −566 GATA Site▿ †

    PubMed Central

    Harju-Baker, Susanna; Costa, Flávia C.; Fedosyuk, Halyna; Neades, Renee; Peterson, Kenneth R.

    2008-01-01

    Autonomous silencing of γ-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the Aγ-globin gene was identified between −730 and −378 relative to the mRNA start site. A marked copy of the Aγ-globin gene inserted between locus control region 5′ DNase I-hypersensitive site 1 and the ɛ-globin gene was transcriptionally silenced in adult β-globin locus yeast artificial chromosome (β-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the −566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low γ-globin levels, whereas only a weak signal was detected when γ-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from β-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the Aγ-globin −566 or Gγ-globin −567 GATA site when γ-globin expression was low (day 18) but not when γ-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, γ-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the −566/−567 GATA sites of the proximal γ-globin promoters. PMID:18347053

  6. Perturbation of chromatin structure in the region of the adult beta-globin gene in chicken erythrocyte chromatin.

    PubMed

    Caplan, A; Kimura, T; Gould, H; Allan, J

    1987-01-01

    An EcoRI chromatin fragment containing the adult beta-globin gene and flanking sequences, isolated from chicken erythrocyte nuclei, sediments at a reduced rate relative to bulk chromatin fragments of the same size. We show that the specific retardation cannot be reversed by adding extra linker histones to native chromatin. When the chromatin fragments are unfolded either by removing linker histones or lowering the ionic strength, the difference between globin and bulk chromatin fragments is no longer seen. The refolded chromatin obtained by restoring the linker histones to the depleted chromatin, however, exhibits the original sedimentation difference. This difference is therefore due to a special property of the histone octamers on the active gene that determines the extent of its folding into higher-order structure. That it is not due to the differential binding of linker histones in vitro is shown by measurements of the protein to DNA ratios using CsCl density-gradients. Both before and after selective removal of the linker histones, the globin gene fragment and bulk chromatin fragments exhibit only a marginal difference in buoyant density. In addition, we show that cleavage of the EcoRI fragment by digestion at the 5' and 3' nuclease hypersensitive sites flanking the globin gene liberates a fragment from between these sites that sediments normally. We conclude that the hypersensitive sites per se are responsible for the reduction in sedimentation rate. The non-nucleosomal DNA segments appear to be too long to be incorporated into the chromatin solenoid and thus create spacers between separate solenoidal elements in the chromatin, which can account for its hydrodynamic behaviour. PMID:3586025

  7. Globin gene switching in primates.

    PubMed

    Johnson, Robert M; Gumucio, Deborah; Goodman, Morris

    2002-11-01

    Evolutionary approaches to the identification of DNA sequences required for transcription of the genes of the beta-globin cluster are reviewed. Sequence alignments of non-coding regions from widely divergent species revealed many conserved motifs (phylogenetic footprints) that were putative transcription factor binding sites and candidate binding proteins were identified. The differential timing of the prosimian and simian gamma-globin genes was analyzed by identifying base changes in the vicinity of the phylogenetic footprints. These differential phylogenetic footprints were shown to bind different nuclear factors, and the behavior of constructs with human or galago gamma-promoters in transgenic mice indicated that DNA motifs near the gamma-globin genes are sufficient to determine the developmental stage of expression. Locus control region alignments have identified many conserved sequence differences outside of the hypersensitive sites. Globin protein and mRNA expression profiles during embryological development in a series of catarrhine (Old World monkeys and apes) and platyrrhine (New World monkeys) primates have been determined. While all catarrhines examined to date have globin expression patterns that are highly similar to the well-established human switching behavior, platyrrhines have inactivated their gamma 1 genes by a variety of mechanisms, and have an earlier gamma-beta switch. PMID:12443943

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

    PubMed Central

    Patel, Vidushi S; Cooper, Steven JB; Deakin, Janine E; Fulton, Bob; Graves, Tina; Warren, Wesley C; Wilson, Richard K; Graves, Jennifer AM

    2008-01-01

    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. PMID:18657265

  9. Genomic organization and gene expression of the multiple globins in Atlantic cod: conservation of globin-flanking genes in chordates infers the origin of the vertebrate globin clusters

    PubMed Central

    2010-01-01

    Background The vertebrate globin genes encoding the α- and β-subunits of the tetrameric hemoglobins are clustered at two unlinked loci. The highly conserved linear order of the genes flanking the hemoglobins provides a strong anchor for inferring common ancestry of the globin clusters. In fish, the number of α-β-linked globin genes varies considerably between different sublineages and seems to be related to prevailing physico-chemical conditions. Draft sequences of the Atlantic cod genome enabled us to determine the genomic organization of the globin repertoire in this marine species that copes with fluctuating environments of the temperate and Arctic regions. Results The Atlantic cod genome was shown to contain 14 globin genes, including nine hemoglobin genes organized in two unlinked clusters designated β5-α1-β1-α4 and β3-β4-α2-α3-β2. The diverged cod hemoglobin genes displayed different expression levels in adult fish, and tetrameric hemoglobins with or without a Root effect were predicted. The novel finding of maternally inherited hemoglobin mRNAs is consistent with a potential role played by fish hemoglobins in the non-specific immune response. In silico analysis of the six teleost genomes available showed that the two α-β globin clusters are flanked by paralogs of five duplicated genes, in agreement with the proposed teleost-specific duplication of the ancestral vertebrate globin cluster. Screening the genome of extant urochordate and cephalochordate species for conserved globin-flanking genes revealed linkage of RHBDF1, MPG and ARHGAP17 to globin genes in the tunicate Ciona intestinalis, while these genes together with LCMT are closely positioned in amphioxus (Branchiostoma floridae), but seem to be unlinked to the multiple globin genes identified in this species. Conclusion The plasticity of Atlantic cod to variable environmental conditions probably involves the expression of multiple globins with potentially different properties. The

  10. Sickle cell disorder, beta-globin gene cluster haplotypes and alpha-thalassemia in neonates and adults from Guadeloupe.

    PubMed

    Kéclard, L; Romana, M; Lavocat, E; Saint-Martin, C; Berchel, C; Mérault, G

    1997-05-01

    We have studied haplotype of beta(S) chromosome and alpha-globin gene status in 534 patients (255 adults and 279 children of whom 159 neonates) from Guadeloupe with various sickle cell-related conditions, namely SS (n = 298), SC (n = 170), S-beta-thal (n = 56), and other rare forms (n = 10). Haplotype data on beta(S) chromosomes confirm our previous observation that Benin type is the most prevalent (75%) beta(S) chromosome in Guadeloupe, in disagreement with the historical records. Comparison of the frequency of distribution of various beta(S) haplotypes between neonates and adults on the one hand and between SS and SC cases on the other shows that the current beta(S) haplotype distribution in this island is not distorted by haplotype-related differential survival. We also show that the frequency of alpha-thalassemia (-3.7 kb) in Guadeloupe is one of the highest recorded in this region involved in Atlantic slave trade and also failed to reveal any age-dependent increase in frequency. We conclude that the African component of Guadeloupe is distinct from that of Brazil and Cuba but is close to that of Jamaica. PMID:9136913

  11. Human fetal globin gene expression is regulated by LYAR

    PubMed Central

    Ju, Junyi; Wang, Ying; Liu, Ronghua; Zhang, Yichong; Xu, Zhen; Wang, Yadong; Wu, Yupeng; Liu, Ming; Cerruti, Loretta; Zou, Fengwei; Ma, Chi; Fang, Ming; Tan, Renxiang; Jane, Stephen M.; Zhao, Quan

    2014-01-01

    Human globin gene expression during development is modulated by transcription factors in a stage-dependent manner. However, the mechanisms controlling the process are still largely unknown. In this study, we found that a nuclear protein, LYAR (human homologue of mouse Ly-1 antibody reactive clone) directly interacted with the methyltransferase PRMT5 which triggers the histone H4 Arg3 symmetric dimethylation (H4R3me2s) mark. We found that PRMT5 binding on the proximal γ-promoter was LYAR-dependent. The LYAR DNA-binding motif (GGTTAT) was identified by performing CASTing (cyclic amplification and selection of targets) experiments. Results of EMSA and ChIP assays confirmed that LYAR bound to a DNA region corresponding to the 5′-untranslated region of the γ-globin gene. We also found that LYAR repressed human fetal globin gene expression in both K562 cells and primary human adult erythroid progenitor cells. Thus, these data indicate that LYAR acts as a novel transcription factor that binds the γ-globin gene, and is essential for silencing the γ-globin gene. PMID:25092918

  12. Human fetal globin gene expression is regulated by LYAR.

    PubMed

    Ju, Junyi; Wang, Ying; Liu, Ronghua; Zhang, Yichong; Xu, Zhen; Wang, Yadong; Wu, Yupeng; Liu, Ming; Cerruti, Loretta; Zou, Fengwei; Ma, Chi; Fang, Ming; Tan, Renxiang; Jane, Stephen M; Zhao, Quan

    2014-09-01

    Human globin gene expression during development is modulated by transcription factors in a stage-dependent manner. However, the mechanisms controlling the process are still largely unknown. In this study, we found that a nuclear protein, LYAR (human homologue of mouse Ly-1 antibody reactive clone) directly interacted with the methyltransferase PRMT5 which triggers the histone H4 Arg3 symmetric dimethylation (H4R3me2s) mark. We found that PRMT5 binding on the proximal γ-promoter was LYAR-dependent. The LYAR DNA-binding motif (GGTTAT) was identified by performing CASTing (cyclic amplification and selection of targets) experiments. Results of EMSA and ChIP assays confirmed that LYAR bound to a DNA region corresponding to the 5'-untranslated region of the γ-globin gene. We also found that LYAR repressed human fetal globin gene expression in both K562 cells and primary human adult erythroid progenitor cells. Thus, these data indicate that LYAR acts as a novel transcription factor that binds the γ-globin gene, and is essential for silencing the γ-globin gene. PMID:25092918

  13. Erythroid-Specific Expression of LIN28A Is Sufficient for Robust Gamma-Globin Gene and Protein Expression in Adult Erythroblasts

    PubMed Central

    Byrnes, Colleen; Kaushal, Megha; Rabel, Antoinette; Tumburu, Laxminath; Allwardt, Joshua M.; Miller, Jeffery L.

    2015-01-01

    Increasing fetal hemoglobin (HbF) levels in adult humans remains an active area in hematologic research. Here we explored erythroid-specific LIN28A expression for its effect in regulating gamma-globin gene expression and HbF levels in cultured adult erythroblasts. For this purpose, lentiviral transduction vectors were produced with LIN28A expression driven by erythroid-specific gene promoter regions of the human KLF1 or SPTA1 genes. Transgene expression of LIN28A with a linked puromycin resistance marker was restricted to the erythroid lineage as demonstrated by selective survival of erythroid colonies (greater than 95% of all colonies). Erythroblast LIN28A over-expression (LIN28A-OE) did not significantly affect proliferation or inhibit differentiation. Greater than 70% suppression of total let-7 microRNA levels was confirmed in LIN28A-OE cells. Increases in gamma-globin mRNA and protein expression with HbF levels reaching 30–40% were achieved. These data suggest that erythroblast targeting of LIN28A expression is sufficient for increasing fetal hemoglobin expression in adult human erythroblasts. PMID:26675483

  14. Butyrate Infusions in the Ovine Fetus Delay the Biologic Clock for Globin Gene Switching

    NASA Astrophysics Data System (ADS)

    Perrine, Susan P.; Rudolph, Abraham; Faller, Douglas V.; Roman, Christine; Cohen, Ruth A.; Chen, Shao-Jing; Kan, Yuet Wai

    1988-11-01

    The switch from fetal to adult hemoglobin expression is regulated in many mammalian species by a developmental clock-like mechanism and determined by the gestational age of the fetus. Prolonging fetal globin gene expression is of considerable interest for therapeutic potential in diseases caused by abnormal β -globin genes. Butyric acid, which is found in increased plasma concentrations in infants of diabetic mothers who have delayed globin gene switching, was infused into catheterized fetal lambs in utero during the time of the normal globin gene switch period. The globin gene switch was significantly delayed in three of four butyrate-treated fetuses compared with controls and was entirely prevented in one fetus in whom the infusion was begun before the globin switch was under way. These data provide a model for investigating and arresting the biologic clock of hemoglobin switching.

  15. Genomic evidence for independent origins of β-like globin genes in monotremes and therian mammals

    PubMed Central

    Opazo, Juan C.; Hoffmann, Federico G.; Storz, Jay F.

    2008-01-01

    Phylogenetic reconstructions of the β-globin gene family in vertebrates have revealed that developmentally regulated systems of hemoglobin synthesis have been reinvented multiple times in independent lineages. For example, the functional differentiation of embryonic and adult β-like globin genes occurred independently in birds and mammals. In both taxa, the embryonic β-globin gene is exclusively expressed in primitive erythroid cells derived from the yolk sac. However, the “ε-globin” gene in birds is not orthologous to the ε-globin gene in mammals, because they are independently derived from lineage-specific duplications of a proto β-globin gene. Here, we report evidence that the early and late expressed β-like globin genes in monotremes and therian mammals (marsupials and placental mammals) are the products of independent duplications of a proto β-globin gene in each of these two lineages. Results of our analysis of genomic sequence data from a large number of vertebrate taxa, including sequence from the recently completed platypus genome, reveal that the ε- and β-globin genes of therian mammals arose via duplication of a proto β-globin gene after the therian/monotreme split. Our analysis of genomic sequence from the platypus also revealed the presence of a duplicate pair of β-like globin genes that originated via duplication of a proto β-globin gene in the monotreme lineage. This discovery provides evidence that, in different lineages of mammals, descendent copies of the same proto β-globin gene may have been independently neofunctionalized to perform physiological tasks associated with oxygen uptake and storage during embryonic development. PMID:18216242

  16. Genomic remnants of alpha-globin genes in the hemoglobinless antarctic icefishes.

    PubMed Central

    Cocca, E; Ratnayake-Lecamwasam, M; Parker, S K; Camardella, L; Ciaramella, M; di Prisco, G; Detrich, H W

    1995-01-01

    Alone among piscine taxa, the antarctic icefishes (family Channichthyidae, suborder Notothenioidei) have evolved compensatory adaptations that maintain normal metabolic functions in the absence of erythrocytes and the respiratory oxygen transporter hemoglobin. Although the uniquely "colorless" or "white" condition of the blood of icefishes has been recognized since the early 20th century, the status of globin genes in the icefish genomes has, surprisingly, remained unexplored. Using alpha- and beta-globin cDNAs from the antarctic rockcod Notothenia coriiceps (family Nototheniidae, suborder Notothenioidei), we have probed the genomes of three white-blooded icefishes and four red-blooded notothenioid relatives (three antarctic, one temperate) for globin-related DNA sequences. We detect specific, high-stringency hybridization of the alpha-globin probe to genomic DNAs of both white- and red-blooded species, whereas the beta-globin cDNA hybridizes only to the genomes of the red-blooded fishes. Our results suggest that icefishes retain inactive genomic remnants of alpha-globin genes but have lost, either through deletion or through rapid mutation, the gene that encodes beta-globin. We propose that the hemoglobinless phenotype of extant icefishes is the result of deletion of the single adult beta-globin locus prior to the diversification of the clade. Images Fig. 2 Fig. 3 Fig. 4 PMID:7892183

  17. Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation.

    PubMed

    Ruiz, Maria Armila; Rivers, Angela; Ibanez, Vinzon; Vaitkus, Kestis; Mahmud, Nadim; DeSimone, Joseph; Lavelle, Donald

    2015-01-01

    The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5 hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5 mC) and 5 hmC at a CCGG site within the 5' γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5 mC and 5 hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5 hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5 hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5 hmC and negatively correlated with 5 mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5 hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter. PMID:25932923

  18. Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation

    PubMed Central

    Ruiz, Maria Armila; Rivers, Angela; Ibanez, Vinzon; Vaitkus, Kestis; Mahmud, Nadim; DeSimone, Joseph; Lavelle, Donald

    2015-01-01

    The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5mC) and 5hmC at a CCGG site within the 5′ γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5mC and 5hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5hmC and negatively correlated with 5mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter. PMID:25932923

  19. Globin gene expression in correlation with G protein-related genes during erythroid differentiation

    PubMed Central

    2013-01-01

    Background The guanine nucleotide binding protein (G protein)-coupled receptors (GPCRs) regulate cell growth, proliferation and differentiation. G proteins are also implicated in erythroid differentiation, and some of them are expressed principally in hematopoietic cells. GPCRs-linked NO/cGMP and p38 MAPK signaling pathways already demonstrated potency for globin gene stimulation. By analyzing erythroid progenitors, derived from hematopoietic cells through in vitro ontogeny, our study intends to determine early markers and signaling pathways of globin gene regulation and their relation to GPCR expression. Results Human hematopoietic CD34+ progenitors are isolated from fetal liver (FL), cord blood (CB), adult bone marrow (BM), peripheral blood (PB) and G-CSF stimulated mobilized PB (mPB), and then differentiated in vitro into erythroid progenitors. We find that growth capacity is most abundant in FL- and CB-derived erythroid cells. The erythroid progenitor cells are sorted as 100% CD71+, but we did not find statistical significance in the variations of CD34, CD36 and GlyA antigens and that confirms similarity in maturation of studied ontogenic periods. During ontogeny, beta-globin gene expression reaches maximum levels in cells of adult blood origin (176 fmol/μg), while gamma-globin gene expression is consistently up-regulated in CB-derived cells (60 fmol/μg). During gamma-globin induction by hydroxycarbamide, we identify stimulated GPCRs (PTGDR, PTGER1) and GPCRs-coupled genes known to be activated via the cAMP/PKA (ADIPOQ), MAPK pathway (JUN) and NO/cGMP (PRPF18) signaling pathways. During ontogeny, GPR45 and ARRDC1 genes have the most prominent expression in FL-derived erythroid progenitor cells, GNL3 and GRP65 genes in CB-derived cells (high gamma-globin gene expression), GPR110 and GNG10 in BM-derived cells, GPR89C and GPR172A in PB-derived cells, and GPR44 and GNAQ genes in mPB-derived cells (high beta-globin gene expression). Conclusions These results

  20. Globin gene structure in a reptile supports the transpositional model for amniote α- and β-globin gene evolution.

    PubMed

    Patel, Vidushi S; Ezaz, Tariq; Deakin, Janine E; Graves, Jennifer A Marshall

    2010-12-01

    The haemoglobin protein, required for oxygen transportation in the body, is encoded by α- and β-globin genes that are arranged in clusters. The transpositional model for the evolution of distinct α-globin and β-globin clusters in amniotes is much simpler than the previously proposed whole genome duplication model. According to this model, all jawed vertebrates share one ancient region containing α- and β-globin genes and several flanking genes in the order MPG-C16orf35-(α-β)-GBY-LUC7L that has been conserved for more than 410 million years, whereas amniotes evolved a distinct β-globin cluster by insertion of a transposed β-globin gene from this ancient region into a cluster of olfactory receptors flanked by CCKBR and RRM1. It could not be determined whether this organisation is conserved in all amniotes because of the paucity of information from non-avian reptiles. To fill in this gap, we examined globin gene organisation in a squamate reptile, the Australian bearded dragon lizard, Pogona vitticeps (Agamidae). We report here that the α-globin cluster (HBK, HBA) is flanked by C16orf35 and GBY and is located on a pair of microchromosomes, whereas the β-globin cluster is flanked by RRM1 on the 3' end and is located on the long arm of chromosome 3. However, the CCKBR gene that flanks the β-globin cluster on the 5' end in other amniotes is located on the short arm of chromosome 5 in P. vitticeps, indicating that a chromosomal break between the β-globin cluster and CCKBR occurred at least in the agamid lineage. Our data from a reptile species provide further evidence to support the transpositional model for the evolution of β-globin gene cluster in amniotes. PMID:21116705

  1. Phylogenetic Diversification of the Globin Gene Superfamily in Chordates

    PubMed Central

    Storz, Jay F.; Opazo, Juan C.; Hoffmann, Federico G.

    2015-01-01

    Summary Phylogenetic reconstructions provide a means of inferring the branching relationships among members of multigene families that have diversified via successive rounds of gene duplication and divergence. Such reconstructions can illuminate the pathways by which particular expression patterns and protein functions evolved. For example, phylogenetic analyses can reveal cases in which similar expression patterns or functional properties evolved independently in different lineages, either through convergence, parallelism, or evolutionary reversals. The purpose of this paper is to provide a robust phylogenetic framework for interpreting experimental data and for generating hypotheses about the functional evolution of globin proteins in chordate animals. To do this we present a consensus phylogeny of the chordate globin gene superfamily. We document the relative roles of gene duplication and whole-genome duplication in fueling the functional diversification of vertebrate globins, and we unravel patterns of shared ancestry among globin genes from representatives of the three chordate subphyla (Craniata, Urochordata, and Cephalochordata). Our results demonstrate the value of integrating phylogenetic analyses with genomic analyses of conserved synteny to infer the duplicative origins and evolutionary histories of globin genes. We also discuss a number of case studies that illustrate the importance of phylogenetic information when making inferences about the evolution of globin gene expression and protein function. Finally, we discuss why the globin gene superfamily presents special challenges for phylogenetic analysis, and we describe methodological approaches that can be used to meet those challenges. PMID:21557448

  2. Gene Turnover in the Avian Globin Gene Families and Evolutionary Changes in Hemoglobin Isoform Expression

    PubMed Central

    Opazo, Juan C.; Hoffmann, Federico G.; Natarajan, Chandrasekhar; Witt, Christopher C.; Berenbrink, Michael; Storz, Jay F.

    2015-01-01

    The apparent stasis in the evolution of avian chromosomes suggests that birds may have experienced relatively low rates of gene gain and loss in multigene families. To investigate this possibility and to explore the phenotypic consequences of variation in gene copy number, we examined evolutionary changes in the families of genes that encode the α- and β-type subunits of hemoglobin (Hb), the tetrameric α2β2 protein responsible for blood-O2 transport. A comparative genomic analysis of 52 bird species revealed that the size and membership composition of the α- and β-globin gene families have remained remarkably constant during approximately 100 My of avian evolution. Most interspecific variation in gene content is attributable to multiple independent inactivations of the αD-globin gene, which encodes the α-chain subunit of a functionally distinct Hb isoform (HbD) that is expressed in both embryonic and definitive erythrocytes. Due to consistent differences in O2-binding properties between HbD and the major adult-expressed Hb isoform, HbA (which incorporates products of the αA-globin gene), recurrent losses of αD-globin contribute to among-species variation in blood-O2 affinity. Analysis of HbA/HbD expression levels in the red blood cells of 122 bird species revealed high variability among lineages and strong phylogenetic signal. In comparison with the homologous gene clusters in mammals, the low retention rate for lineage-specific gene duplicates in the avian globin gene clusters suggests that the developmental regulation of Hb synthesis in birds may be more highly conserved, with orthologous genes having similar stage-specific expression profiles and similar functional properties in disparate taxa. PMID:25502940

  3. Mutation of a transcriptional motif of a distant regulatory element reduces the expression of embryonic and fetal globin genes

    PubMed Central

    Navas, Patrick A.; Swank, Richard A.; Yu, Man; Peterson, Kenneth R.; Stamatoyannopoulos, George

    2010-01-01

    High-level β-globin gene expression is dependent on the presence of the locus control region (LCR), a powerful regulatory element physically characterized by five DNase I-hypersensitive sites (HS), designated HS1–HS5. Of these, HS3 contains seven GT motifs that are essential for its activity. One of the motifs, GT6, has been shown by in vivo footprinting to display the largest difference in signal between fetal and adult globin expressing cells. We assessed the contribution of GT6 on the downstream globin gene expression by mutating this motif in a 248 kb β-globin locus yeast artificial chromosome and measuring the activity of β-globin genes in GT6m β-YAC transgenic mice. Seven transgenic lines were established, three of which contained at least one intact copy of the β-globin locus and were further investigated. The mutation of the GT6 motif reduced the expression of ε- and γ-globin genes during embryonic erythropoiesis. During definitive erythropoiesis, γ-globin gene expression was significantly reduced while β-globin gene expression was virtually indistinguishable from wild-type controls. We conclude that the GT6 motif of hypersensitive site 3 of the LCR is required for normal ε- and γ-globin gene expression during embryonic erythropoiesis and for γ-globin gene expression during definitive erythropoiesis in the fetal liver. Our results provide evidence that mutations of single transcriptional motifs of distant regulatory elements can have profound effects on gene expression. PMID:14506128

  4. Characterization of the transcriptome profiles related to globin gene switching during in vitro erythroid maturation

    PubMed Central

    2012-01-01

    Background The fetal and adult globin genes in the human β-globin cluster on chromosome 11 are sequentially expressed to achieve normal hemoglobin switching during human development. The pharmacological induction of fetal γ-globin (HBG) to replace abnormal adult sickle βS-globin is a successful strategy to treat sickle cell disease; however the molecular mechanism of γ-gene silencing after birth is not fully understood. Therefore, we performed global gene expression profiling using primary erythroid progenitors grown from human peripheral blood mononuclear cells to characterize gene expression patterns during the γ-globin to β-globin (γ/β) switch observed throughout in vitro erythroid differentiation. Results We confirmed erythroid maturation in our culture system using cell morphologic features defined by Giemsa staining and the γ/β-globin switch by reverse transcription-quantitative PCR (RT-qPCR) analysis. We observed maximal γ-globin expression at day 7 with a switch to a predominance of β-globin expression by day 28 and the γ/β-globin switch occurred around day 21. Expression patterns for transcription factors including GATA1, GATA2, KLF1 and NFE2 confirmed our system produced the expected pattern of expression based on the known function of these factors in globin gene regulation. Subsequent gene expression profiling was performed with RNA isolated from progenitors harvested at day 7, 14, 21, and 28 in culture. Three major gene profiles were generated by Principal Component Analysis (PCA). For profile-1 genes, where expression decreased from day 7 to day 28, we identified 2,102 genes down-regulated > 1.5-fold. Ingenuity pathway analysis (IPA) for profile-1 genes demonstrated involvement of the Cdc42, phospholipase C, NF-Kβ, Interleukin-4, and p38 mitogen activated protein kinase (MAPK) signaling pathways. Transcription factors known to be involved in γ-and β-globin regulation were identified. The same approach was used to generate profile-2

  5. Asynchronous DNA replication within the human. beta. -globin gene locus

    SciTech Connect

    Epner, E.; Forrester, W.C.; Groudine, M. )

    1988-11-01

    The timing of DNA replication of the human {beta}-globin gene locus has been studied by blot hybridization of newly synthesized BrdUrd-substituted DNA from cells in different stages of the S phase. Using probes that span >120 kilobases across the human {beta}-globin gene locus, the authors show that the majority of this domain replicates in early S phase in the human erythroleukemia cell line K562 and in middle-to-late S phase in the lymphoid cell line Manca. However, in K562 cells three small regions display a strikingly different replication pattern than adjacent sequences. These islands, located in the inter-{gamma}-globin gene region and approximately 20 kilobases 5' to the {epsilon}-globin gene and 20 kilobases 3' to the {beta}-globin gene, replicate later and throughout S phase. A similar area is also present in the {alpha}-globin gene region in K562 cells. They suggest that these regions may represent sites of termination of replication forks.

  6. Distinctive Patterns of Evolution of the δ-Globin Gene (HBD) in Primates

    PubMed Central

    Moleirinho, Ana; Lopes, Alexandra M.; Seixas, Susana; Morales-Hojas, Ramiro; Prata, Maria J.; Amorim, António

    2015-01-01

    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 corroborated a

  7. Erythroid activator NF-E2, TAL1 and KLF1 play roles in forming the LCR HSs in the human adult β-globin locus.

    PubMed

    Kim, Yea Woon; Yun, Won Ju; Kim, AeRi

    2016-06-01

    The β-like globin genes are developmental stage specifically transcribed in erythroid cells. The transcription of the β-like globin genes requires erythroid specific activators such as GATA-1, NF-E2, TAL1 and KLF1. However, the roles of these activators have not fully elucidated in transcription of the human adult β-globin gene. Here we employed hybrid MEL cells (MEL/ch11) where a human chromosome containing the β-globin locus is present and the adult β-globin gene is highly transcribed by induction. The roles of erythroid specific activators were analyzed by inhibiting the expression of NF-E2, TAL1 or KLF1 in MEL/ch11 cells. The loss of each activator decreased the transcription of human β-globin gene, locus wide histone hyperacetylation and the binding of other erythroid specific activators including GATA-1, even though not affecting the expression of other activators. Notably, sensitivity to DNase I was reduced in the locus control region (LCR) hypersensitive sites (HSs) with the depletion of activators. These results indicate that NF-E2, TAL1 and KLF1, all activators play a primary role in HSs formation in the LCR. It might contribute to the transcription of human adult β-globin gene by allowing the access of activators and cofactors. The roles of activators in the adult β-globin locus appear to be different from the roles in the early fetal locus. PMID:27026582

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

    SciTech Connect

    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.

  9. Fetal Globin Gene Inducers: Novel Agents & New Potential

    PubMed Central

    Perrine, Susan P.; Castaneda, Serguei A.; Chui, David H.; Faller, Douglas V.; Berenson, Ronald J.; Fucharoen, Suthat

    2013-01-01

    Inducing expression of endogenous fetal globin (γ-globin) gene expression to 60-70% of alpha globin synthesis produces β-thalassemia trait globin synthetic ratios and can reduce anemia to a mild level. Several classes of therapeutics have induced γ-globin expression in beta thalassemia patients and subsequently raised total hemoglobin levels, demonstrating proof-of-concept of the approach. Butyrate treatment eliminated transfusion requirements in formerly transfusion-dependent patients with treatment for as long as 7 years. However, prior generations were not readily applicable for widespread use. Currently, a novel oral dual-action therapeutic sodium 2,2-dimethylbutyrate is in clinical trials, an oral decitabine formulation is under development, and agents with complementary mechanisms of action can be applied in combined regimens. Identification of 3 major genetic trait loci which modulate clinical severity provides avenues for developing tailored regimens. These refinements offer renewed potential to apply fetal globin induction as a treatment approach in patient-friendly regimens that can be used world-wide. PMID:20712788

  10. Transcriptional interference among the murine β-like globin genes

    PubMed Central

    Hu, Xiao; Eszterhas, Susan; Pallazzi, Nicolas; Bouhassira, Eric E.; Fields, Jennifer; Tanabe, Osamu; Gerber, Scott A.; Bulger, Michael; Engel, James Douglas; Groudine, Mark

    2007-01-01

    Mammalian β-globin loci contain multiple genes that are activated at different developmental stages. Studies have suggested that the transcription of one gene in a locus can influence the expression of the other locus genes. The prevalent model to explain this transcriptional interference is that all potentially active genes compete for locus control region (LCR) activity. To investigate the influence of transcription by the murine embryonic genes on transcription of the other β-like genes, we generated mice with deletions of the promoter regions of Ey and βh1 and measured transcription of the remaining genes. Deletion of the Ey and βh1 promoters increased transcription of βmajor and βminor 2-fold to 3-fold during primitive erythropoiesis. Deletion of Ey did not affect βh1 nor did deletion of βh1 affect Ey, but Ey deletion uniquely activated transcription from βh0, a β-like globin gene immediately downstream of Ey. Protein analysis showed that βh0 encodes a translatable β-like globin protein that can pair with alpha globin. The lack of transcriptional interference between Ey and βh1 and the gene-specific repression of βh0 did not support LCR competition among the embryonic genes and suggested that direct transcriptional interference from Ey suppressed βh0. PMID:17077320

  11. Evolutionary pathway of pseudogenization of globin genes, α5 and β5, in genus Oryzias.

    PubMed

    Maruyama, Kouichi; Wang, Bing; Ishikawa, Yuji; Yasumasu, Shigeki; Iuchi, Ichiro

    2015-09-01

    Hemoglobin transports oxygen in many organisms and consists of α- and β-globin chains. Previously, using molecular phylogenetic analysis, we proposed that both α- and β-globins of teleost could be classified into four groups. We also showed that the Hd-rR strain of medaka (Oryzias latipes) inhabiting southern Japan had all four groups of globin genes but that the α- and β-globin genes of group III were pseudogenized (α5(ψα), β5(ψβ)). Based on the small degree of nucleotide variations, the pseudogenization of β5 was assumed to have occurred at a relatively late stage of evolution. Here, we compared the α5(ψα)-β5(ψβ) of two other strains of O. latipes and found that both α5(ψα) and β5(ψβ) of the northern Japanese and Korean strains were pseudogenized similar to those of Hd-rR. In a Philippine population (Oryzias luzonensis), α5(ψα) was also pseudogenized, but the structure was different from that of O. latipes, and β5(ψβ) was almost deleted. Interestingly, an Indonesian population (Oryzias celebensis) had α5 and β5 genes that were deduced to be functional. Indeed, they were expressed from the young to adult development stages, and this expression pattern was consistent with the expression of α2 and ad.α1 in Hd-rR. Because α2 and ad.α1 in Hd-rR were assigned to groups I and II, respectively, we speculate that their expression patterns might be altered by pseudogenization of group III genes. These results provide a basis for further investigations of recruiting and changing expression patterns of one globin gene after pseudogenization of other globin genes during evolution. PMID:26199047

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

    PubMed

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

    2014-05-01

    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. PMID:24814285

  13. Repeated Evolution of Chimeric Fusion Genes in the β-Globin Gene Family of Laurasiatherian Mammals

    PubMed Central

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

    2014-01-01

    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. PMID:24814285

  14. Tissue specific transcription of the human epsilon-globin gene following transfection into the embryonic erythroid cell line K562.

    PubMed Central

    Allan, M; Montague, P; Grindlay, G J; Sibbet, G; Donovan-Peluso, M; Bank, A; Paul, J

    1985-01-01

    We have introduced a plasmid containing the human epsilon-globin gene either stably or transiently into a number of erythroid or non-erythroid cell lines, and analysed the accuracy and efficiency of transcription. In non-erythroid cells (or in mouse erythroleukaemia (MEL) cells in which adult but not embryonic globin genes are expressed) transcription of the epsilon-globin gene occurs mainly from a site 200 bp upstream of the major cap site (the -200 cap site). In the human K562 cell line, in which the endogenous epsilon-globin gene is transcribed at high levels, transcription initiation from the introduced gene occurs mainly from the major cap site. Transcriptional activity of the epsilon-globin gene introduced into K562 cell is quantitatively similar to that of the endogenous gene. This suggests the presence (or absence) in K562 cells of factor(s) which activate (or repress) the epsilon-globin gene in a tissue specific manner. Images PMID:2995916

  15. Evidence that the recently discovered theta 1-globin gene is functional in higher primates.

    PubMed

    Shaw, J P; Marks, J; Shen, C K

    A new subfamily of the alpha-globin-like family has recently been identified in higher primates, rabbit, galago and possibly the horse. One member of this subfamily, theta 1, is downstream from the adult alpha 1-globin gene. In orang-utan, but not in rabbit or galago, the theta 1-gene appears to be structurally intact, suggesting that it may be functional in this species. The orang-utan theta 1-gene possesses initiation and termination codons, and the predicted polypeptide differs from the orang-utan alpha 1-globin by 55 amino acids. The upstream promoter boxes CCAAT and ATA are present, although approximately 150 base pairs (bp) farther upstream than in the alpha 1-gene. This structural difference in the promoter between the orang-utan theta 1- and alpha 1-genes has led Proudfoot to speculate that the theta 1-gene may be inactive. We have now cloned the theta 1- and alpha 1-globin genes from the olive baboon, and have compared their sequences with those of orang-utan. The unique promoter structure of the orang-utan theta 1-gene is highly conserved in baboon, although the orang-utan and baboon diverged nearly 30 million years ago. The coding sequences of the two theta 1-genes differ by only 6.3% with 22 out of 27 nucleotide substitutions being codon third position silent changes. These data support the view that the theta 1-gene has been functional in the baboon, orang-utan, and by implication, in man. We also estimate that the duplication event generating the theta 1- and alpha-globin-like subfamilies may have occurred as much as 260 million years ago. PMID:3561513

  16. Effect of AGM and fetal liver-derived stromal cell lines on globin expression in adult baboon (P. anubis) bone marrow-derived erythroid progenitors.

    PubMed

    Lavelle, Donald; Vaitkus, Kestutis; Ruiz, Maria Armila; Ibanez, Vinzon; Kouznetsova, Tatiana; Saunthararajah, Yogen; Mahmud, Nadim; DeSimone, Joseph

    2012-01-01

    This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the micro-environment on globin gene expression. Adult progenitors express high levels of γ-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, γ-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in γ-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and γ-globin promoter DNA methylation. We conclude that γ-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction. PMID:22693559

  17. Effect of AGM and Fetal Liver-Derived Stromal Cell Lines on Globin Expression in Adult Baboon (P. anubis) Bone Marrow-Derived Erythroid Progenitors

    PubMed Central

    Lavelle, Donald; Vaitkus, Kestutis; Ruiz, Maria Armila; Ibanez, Vinzon; Kouznetsova, Tatiana; Saunthararajah, Yogen; Mahmud, Nadim; DeSimone, Joseph

    2012-01-01

    This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the micro-environment on globin gene expression. Adult progenitors express high levels of γ-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, γ-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in γ-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and γ-globin promoter DNA methylation. We conclude that γ-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction. PMID:22693559

  18. Versatile Cosmid Vectors for the Isolation, Expression, and Rescue of Gene Sequences: Studies with the Human α -globin Gene Cluster

    NASA Astrophysics Data System (ADS)

    Lau, Yun-Fai; Kan, Yuet Wai

    1983-09-01

    We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells. These cosmids were constructed by inserting one of the SV2-derived selectable gene markers-SV2-gpt, SV2-DHFR, and SV2-neo-in cosmid pJB8. High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases. We isolated recombinant cosmids containing the human α -globin gene cluster from these genomic libraries. The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of α -globin genes in these cells. These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems. Both of the adult α -globin genes were more actively expressed than the embryonic zeta -globin genes in these transformed cell lines. Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA. Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.

  19. Transcriptional activity of the human pseudogene psi alpha globin compared with alpha globin, its functional gene counterpart.

    PubMed Central

    Whitelaw, E; Proudfoot, N J

    1983-01-01

    Transcriptional analysis of the human pseudogene psi alpha globin has revealed the following features: (1) The promoter with a 23 bp deletion between the CCAAT and ATA boxes is functional both in vitro and in vivo, 3 fold and 10 fold less efficient, respectively, than alpha. (2) Both the psi alpha and alpha globin gene promoters are active in the absence of transcriptional enhancers, either a gene-encoded or viral enhancer. (3) The mutated poly(A) addition signal in psi alpha (AATGAA) appears to be completely nonfunctional. This result provides an explanation for the absence of psi alpha transcripts in human erythroid cells. Images PMID:6316269

  20. The Caenorhabditis globin gene family reveals extensive nematode-specific radiation and diversification

    PubMed Central

    2008-01-01

    Background Globin isoforms with variant properties and functions have been found in the pseudocoel, body wall and cuticle of various nematode species and even in the eyespots of the insect-parasite Mermis nigrescens. In fact, much higher levels of complexity exist, as shown by recent whole genome analysis studies. In silico analysis of the genome of Caenorhabditis elegans revealed an unexpectedly high number of globin genes featuring a remarkable diversity in gene structure, amino acid sequence and expression profiles. Results In the present study we have analyzed whole genomic data from C. briggsae, C. remanei, Pristionchus pacificus and Brugia malayi and EST data from several other nematode species to study the evolutionary history of the nematode globin gene family. We find a high level of conservation of the C. elegans globin complement, with even distantly related nematodes harboring orthologs to many Caenorhabditis globins. Bayesian phylogenetic analysis resolves all nematode globins into two distinct globin classes. Analysis of the globin intron-exon structures suggests extensive loss of ancestral introns and gain of new positions in deep nematode ancestors, and mainly loss in the Caenorhabditis lineage. We also show that the Caenorhabditis globin genes are expressed in distinct, mostly non-overlapping, sets of cells and that they are all under strong purifying selection. Conclusion Our results enable reconstruction of the evolutionary history of the globin gene family in the nematode phylum. A duplication of an ancestral globin gene occurred before the divergence of the Platyhelminthes and the Nematoda and one of the duplicated genes radiated further in the nematode phylum before the split of the Spirurina and Rhabditina and was followed by further radiation in the lineage leading to Caenorhabditis. The resulting globin genes were subject to processes of subfunctionalization and diversification leading to cell-specific expression patterns. Strong purifying

  1. Amoebozoa possess lineage-specific globin gene repertoires gained by individual horizontal gene transfers.

    PubMed

    Dröge, Jasmin; Buczek, Dorota; Suzuki, Yutaka; Makałowski, Wojciech

    2014-01-01

    The Amoebozoa represent a clade of unicellular amoeboid organisms that display a wide variety of lifestyles, including free-living and parasitic species. For example, the social amoeba Dictyostelium discoideum has the ability to aggregate into a multicellular fruiting body upon starvation, while the pathogenic amoeba Entamoeba histolytica is a parasite of humans. Globins are small heme proteins that are present in almost all extant organisms. Although several genomes of amoebozoan species have been sequenced, little is known about the phyletic distribution of globin genes within this phylum. Only two flavohemoglobins (FHbs) of D. discoideum have been reported and characterized previously while the genomes of Entamoeba species are apparently devoid of globin genes. We investigated eleven amoebozoan species for the presence of globin genes by genomic and phylogenetic in silico analyses. Additional FHb genes were identified in the genomes of four social amoebas and the true slime mold Physarum polycephalum. Moreover, a single-domain globin (SDFgb) of Hartmannella vermiformis, as well as two truncated hemoglobins (trHbs) of Acanthamoeba castellanii were identified. Phylogenetic evidence suggests that these globin genes were independently acquired via horizontal gene transfer from some ancestral bacteria. Furthermore, the phylogenetic tree of amoebozoan FHbs indicates that they do not share a common ancestry and that a transfer of FHbs from bacteria to amoeba occurred multiple times. PMID:25013378

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

    PubMed Central

    Borgio, J. Francis

    2015-01-01

    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. PMID:26593158

  3. [Joint locus of a/b-globin genes in Danio rerio is segregated into structural subdomains active at different stages of development].

    PubMed

    Dolgushin, K V; Petrova, N V; Iudinkova, E S; Razin, S V; Iarovaia, O V

    2015-01-01

    In the domain model of eukaryotic genome organization, the functional unit of the genome, along with the relevant regulatory elements, is considered to be a gene or a gene family. In hot-blooded vertebrate animals, the domains of a- and b-globin genes are positioned at different chromosomes and are organized and regulated in different fashion. In cold-blooded animals, in particular in tropical fish Danio rerio, a- and b globin genes are located in a common gene cluster. However, the joint a/b-globin gene cluster is subdivided into two development stage-specific subdomains, the adult one and the embryonic-larval one. In an attempt to find out whether this functional segregation correlates with structural segregation of the domain we compared the DNase I sensitivity and profiles of histone modifications of adult and embryonic-larval segments of the domain in cultured D. rerio fibroblasts. We have demonstrated that, in these nonerythroid cells, adult and embryonic- larval subdomains possess different DNase I sensitivities and different profiles of H3K27me3, a histone modification introduced by PRC2 complex. These observations suggest that joint a/b globin gene domain of Danio rerio is segregated into two structural subdomain harboring adult and embryonic-larval globin genes. PMID:26107904

  4. Decitabine Increases Fetal Hemoglobin in P. Anubis by Increasing γ-globin Gene Transcription

    PubMed Central

    Akpan, Imo; Banzon, Virryan; Ibanez, Vinzon; Vaitkus, Kestis; DeSimone, Joseph; Lavelle, Donald

    2014-01-01

    1) Objective The mechanism responsible for increased fetal hemoglobin (HbF) levels following decitabine treatment remains controversial. These experiments were performed to evaluate the role of transcriptional versus translational mechanisms in the ability of decitabine to increase HbF levels in vivo. 2) Methods Three normal, nonanemic baboons were treated with decitabine subcutaneously (0.5mg/kg/d) for 10 days. The effect of decitabine on globin chain synthesis and globin mRNA levels was measured in pre- and post-treatment bone marrow (BM) aspirates by biosynthetic radiolabelling with [3H] leucine followed by separation of globin chains by HPLC, and real time PCR, respectively. The effect on DNA methylation of the ε- and γ-globin gene promoters was determined by bisulfite sequence analysis. 3) Results Decitabine treatment of normal, nonanemic baboons induced similar increases in the γ/γ+β chain synthetic ratio and the γ/total β-like globin RNA ratio and also increased expression of ε-globin transcripts. Increased expression of ε- and γ-globin was associated with decreased DNA methylation of the ε- and γ-globin gene promoters. 4) Conclusion Decitabine increases HbF in vivo by transcriptional activation of the γ-globin gene. PMID:20713129

  5. The 5' flanking region of human epsilon-globin gene.

    PubMed Central

    Baralle, F E; Shoulders, C C; Goodbourn, S; Jeffreys, A; Proudfoot, N J

    1980-01-01

    The structural analysis of the 2.0 kb region upstream from the epsilon-globin gene has been carried out. A genomic DNA map around the gene was worked out in some detail to ensure that the cloned DNA was representative of the actual chromosomal arrangement. Furthermore, a new technique was developed to precisely map a reiterated DNA sequence present 1.5 kb to the 5' side of the gene. The complete nucleotide sequence of the 2.0 kb 5' flanking region was then determined and overlapped with the gene. The sequence included the reiterated DNA sequence which is homologous to the so-called AluI family of repeats. Unusual stretches of sequence 50 nucleotides long, where A + T represent about 90% of the bases, are present at both the 5' and 3' sides of the repeat. Images PMID:6253916

  6. Alpha globin gene analysis in a Sardinian family with interacting alpha and beta thalassaemia genes.

    PubMed

    Melis, M A; Galanello, R; Cao, A

    1983-04-01

    This paper reports the results of alpha globin gene analysis in a Sardinian family with interacting alpha and beta thalassaemia genes. The propositus, who was identified in a newborn survey as he had 26.0% Hb Bart's and 74.0% Hb F, successively developed the clinical and haematological picture of a transfusion-dependent thalassaemia major. According to the haemoglobin pattern, restriction endonuclease analysis of the DNA from this patient showed the deletion of three of the four alpha-globin structural genes. Thus beta 0-thalassaemia homozygotes with the delection of three alpha-structural genes seem to have a severe clinical phenotype similar to that of patients with a full complement of four alpha-globin structural genes. PMID:6299325

  7. Developmental changes in DNA methylation and covalent histone modifications of chromatin associated with the epsilon-, gamma-, and beta-globin gene promoters in Papio anubis.

    PubMed

    Lavelle, Donald; Vaitkus, Kestis; Hankewych, Maria; Singh, Mahipal; DeSimone, Joseph

    2006-01-01

    The baboon is a suitable and relevant animal model to study the mechanism of human globin gene switching. This investigation addresses the role of DNA methylation and histone coding in globin gene switching in the baboon, Papio anubis. Bisulfite sequencing and chromatin immunoprecipitation studies were performed in erythroid cells purified from fetuses of varying gestational ages and from adult bone marrow to analyze the manner that changes in DNA methylation of the epsilon-, gamma-, and beta-globin promoters and association of ac-H3, ac-H4, H3-dimeK4, H3-dimeK36, and H3-dimeK79 with the epsilon-, gamma-, and beta-globin promoters occur during development. Changes in DNA methylation of the epsilon- and gamma-globin gene promoters during transitional stages of globin gene switching were consistent with the stochastic model of methylation and a role of DNA methylation in gene silencing. Enrichment of ac-H3, ac-H4, and pol II at the promoters of developmentally active genes was observed, while the pattern of distribution of H3-dimeK4 and H3-dimeK79 suggests that these modifications are found near both currently and formerly active promoters. Enrichment of H3-dimeK36 at the silenced epsilon-globin gene promoter was observed. These studies demonstrate that coordinated epigenetic modifications in the chromatin structure of the beta-like globin gene promoters accompany the highly regulated changes in expression patterns of these genes during development. PMID:16527500

  8. Origin and Ascendancy of a Chimeric Fusion Gene: The β/δ-Globin Gene of Paenungulate Mammals

    PubMed Central

    Opazo, Juan C.; Sloan, Angela M.; Campbell, Kevin L.

    2009-01-01

    The δ-globin gene (HBD) of eutherian mammals exhibits a propensity for recombinational exchange with the closely linked β-globin gene (HBB) and has been independently converted by the HBB gene in multiple lineages. Here we report the presence of a chimeric β/δ fusion gene in the African elephant (Loxodonta africana) that was created by unequal crossing-over between misaligned HBD and HBB paralogs. The recombinant chromosome that harbors the β/δ fusion gene in elephants is structurally similar to the “anti-Lepore” duplication mutant of humans (the reciprocal exchange product of the hemoglobin Lepore deletion mutant). However, the situation in the African elephant is unique in that the chimeric β/δ fusion gene supplanted the parental HBB gene and is therefore solely responsible for synthesizing the β-chain subunits of adult hemoglobin. A phylogenetic survey of β-like globin genes in afrotherian and xenarthran mammals revealed that the origin of the chimeric β/δ fusion gene and the concomitant inactivation of the HBB gene predated the radiation of “Paenungulata,” a clade of afrotherian mammals that includes three orders: Proboscidea (elephants), Sirenia (dugongs and manatees), and Hyracoidea (hyraxes). The reduced fitness of the human Hb Lepore deletion mutant helps to explain why independently derived β/δ fusion genes (which occur on an anti-Lepore chromosome) have been fixed in a number of mammalian lineages, whereas the reciprocal δ/β fusion gene (which occurs on a Lepore chromosome) has yet to be documented in any nonhuman mammal. This illustrates how the evolutionary fates of chimeric fusion genes can be strongly influenced by their recombinational mode of origin. PMID:19332641

  9. Evolution of the globin gene family in deuterostomes: lineage-specific patterns of diversification and attrition.

    PubMed

    Hoffmann, Federico G; Opazo, Juan C; Hoogewijs, David; Hankeln, Thomas; Ebner, Bettina; Vinogradov, Serge N; Bailly, Xavier; Storz, Jay F

    2012-07-01

    In the Metazoa, globin proteins display an underlying unity in tertiary structure that belies an extraordinary diversity in primary structures, biochemical properties, and physiological functions. Phylogenetic reconstructions can reveal which of these functions represent novel, lineage-specific innovations, and which represent ancestral functions that are shared with homologous globin proteins in other eukaryotes and even prokaryotes. To date, our understanding of globin diversity in deuterostomes has been hindered by a dearth of genomic sequence data from the Ambulacraria (echinoderms + hemichordates), the sister group of chordates, and the phylum Xenacoelomorpha, which includes xenoturbellids, acoelomorphs, and nemertodermatids. Here, we report the results of a phylogenetic and comparative genomic analysis of the globin gene repertoire of deuterostomes. We first characterized the globin genes of the acorn worm, Saccoglossus kowalevskii, a representative of the phylum Hemichordata. We then integrated genomic sequence data from the acorn worm into a comprehensive analysis of conserved synteny and phylogenetic relationships among globin genes from representatives of the eight lineages that comprise the superphylum Deuterostomia. The primary aims were 1) to unravel the evolutionary history of the globin gene superfamily in deuterostomes and 2) to use the estimated phylogeny to gain insights into the functional evolution of deuterostome globins. Results of our analyses indicate that the deuterostome common ancestor possessed a repertoire of at least four distinct globin paralogs and that different subsets of these ancestral genes have been retained in each of the descendant organismal lineages. In each major deuterostome group, a different subset of ancestral precursor genes underwent lineage-specific expansions of functional diversity through repeated rounds of gene duplication and divergence. By integrating results of the phylogenetic analysis with available

  10. Methylation of alpha-type embryonic globin gene alpha pi represses transcription in primary erythroid cells.

    PubMed

    Singal, Rakesh; vanWert, Jane M; Ferdinand, Larry

    2002-12-01

    The inverse relationship between expression and methylation of beta-type globin genes is well established. However, little is known about the relationship between expression and methylation of avian alpha-type globin genes. The embryonic alpha(pi)-globin promoter was unmethylated, and alpha(pi)-globin RNA was easily detected in 5-day chicken erythroid cells. A progressive methylation of the CpG dinucleotides in the alpha(pi) promoter associated with loss of expression of alpha(pi)-globin gene was seen during development in primary erythroid cells. A 315-bp alpha(pi)-globin promoter region was cloned in an expression construct (alpha(pi)pGL3E) containing a luciferase reporter gene and SV40 enhancer. The alpha(pi)pGL3E construct was transfected into primary erythroid cells derived from 5-day-old chicken embryos. Methylation of alpha(pi)pGL3E plasmid and alpha(pi)-globin promoter alone resulted in a 20-fold and 7-fold inhibition of expression, respectively. The fully methylated but not the unmethylated 315-bp alpha(pi)-globin gene promoter fragment formed a methyl cytosine-binding protein complex (MeCPC). Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with the alpha(pi)-globin gene promoter. Slight hyperacetylation of histone H3 but a marked hyperacetylation of histone H4 was seen in 5-day when compared with 14-day erythroid cells. These results demonstrate that methylation can silence transcription of an avian alpha-type embryonic globin gene in homologous primary erythroid cells, possibly by interacting with an MeCPC and histone deacetylase complex. PMID:12393573

  11. Alternative sites of transcription initiation upstream of the canonical cap site in human gamma-globin and beta-globin genes.

    PubMed Central

    Grindlay, G J; Lanyon, W G; Allan, M; Paul, J

    1984-01-01

    Using S1 mapping and primer extension analysis, we have identified a number of human kappa-globin and beta-globin 5' RNA termini originating in the 200 bp upstream of the canonical mRNA cap sites. Upstream initiation sites have previously been reported for the human epsilon-globin gene (4,5) and the present work indicates that this is a general feature of the human beta-type globin genes. We have attempted to identify features common to such sites between the three genes. One site 170 bp upstream of the major beta-globin cap site and a site 1400 bp upstream of the major epsilon-globin cap site are located near putative PolIII promoter sequences and may therefore be transcribed by this enzyme. Alternative initiation sites located 200 bp and 50-100 bp upstream of the epsilon-globin and kappa-globin cap sites respectively are located within S1 hypersensitive regions of chromatin. Images PMID:6701091

  12. Genomic organization and differential signature of positive selection in the alpha and beta globin gene clusters in two cetacean species.

    PubMed

    Nery, Mariana F; Arroyo, José Ignacio; Opazo, Juan C

    2013-01-01

    The hemoglobin of jawed vertebrates is a heterotetramer protein that contains two α- and two β-chains, which are encoded by members of α- and β-globin gene families. Given the hemoglobin role in mediating an adaptive response to chronic hypoxia, it is likely that this molecule may have experienced a selective pressure during the evolution of cetaceans, which have to deal with hypoxia tolerance during prolonged diving. This selective pressure could have generated a complex history of gene turnover in these clusters and/or changes in protein structure themselves. Accordingly, we aimed to characterize the genomic organization of α- and β-globin gene clusters in two cetacean species and to detect a possible role of positive selection on them using a phylogenetic framework. Maximum likelihood and Bayesian phylogeny reconstructions revealed that both cetacean species had retained a similar complement of putatively functional genes. For the α-globin gene cluster, the killer whale presents a complement of genes composed of HBZ, HBK, and two functional copies of HBA and HBQ genes, whereas the dolphin possesses HBZ, HBK, HBA and HBQ genes, and one HBA pseudogene. For the β-globin gene cluster, both species retained a complement of four genes, two early expressed genes-HBE and HBH-and two adult expressed genes-HBD and HBB. Our natural selection analysis detected two positively selected sites in the HBB gene (56 and 62) and four in HBA (15, 21, 49, 120). Interestingly, only the genes that are expressed during the adulthood showed the signature of positive selection. PMID:24259315

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

    PubMed Central

    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

  14. Transcription in vivo of an Alu family member upstream from the human epsilon-globin gene.

    PubMed Central

    Allan, M; Paul, J

    1984-01-01

    The more distal Alu repeat flanking the epsilon-globin gene is transcribed in K562 cells to generate transcripts 350-400 nucleotides in length. Initiation occurs at the start of the repeat, upstream of a putative PolIII control signal. These transcripts originate from the strand which does not code epsilon-globin and are oriented in the opposite direction from the gene. They are non-polyadenylated, nucleus-confined and are only detectable in association with expression of the epsilon-globin gene. Images PMID:6320117

  15. High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range β-Globin Gene Regulation.

    PubMed

    Stees, Jared R; Hossain, Mir A; Sunose, Tomoki; Kudo, Yasushi; Pardo, Carolina E; Nabilsi, Nancy H; Darst, Russell P; Poudyal, Rosha; Igarashi, Kazuhiko; Huang, Suming; Kladde, Michael P; Bungert, Jörg

    2015-01-01

    Enhancers and promoters assemble protein complexes that ultimately regulate the recruitment and activity of RNA polymerases. Previous work has shown that at least some enhancers form stable protein complexes, leading to the formation of enhanceosomes. We analyzed protein-DNA interactions in the murine β-globin gene locus using the methyltransferase accessibility protocol for individual templates (MAPit). The data show that a tandem Maf recognition element (MARE) in locus control region (LCR) hypersensitive site 2 (HS2) reveals a remarkably high degree of occupancy during differentiation of mouse erythroleukemia cells. Most of the other transcription factor binding sites in LCR HS2 or in the adult β-globin gene promoter regions exhibit low fractional occupancy, suggesting highly dynamic protein-DNA interactions. Targeting of an artificial zinc finger DNA-binding domain (ZF-DBD) to the HS2 tandem MARE caused a reduction in the association of MARE-binding proteins and transcription complexes at LCR HS2 and the adult βmajor-globin gene promoter but did not affect expression of the βminor-globin gene. The data demonstrate that a stable MARE-associated footprint in LCR HS2 is important for the recruitment of transcription complexes to the adult βmajor-globin gene promoter during erythroid cell differentiation. PMID:26503787

  16. High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range β-Globin Gene Regulation

    PubMed Central

    Stees, Jared R.; Hossain, Mir A.; Sunose, Tomoki; Kudo, Yasushi; Pardo, Carolina E.; Nabilsi, Nancy H.; Darst, Russell P.; Poudyal, Rosha; Igarashi, Kazuhiko; Kladde, Michael P.

    2015-01-01

    Enhancers and promoters assemble protein complexes that ultimately regulate the recruitment and activity of RNA polymerases. Previous work has shown that at least some enhancers form stable protein complexes, leading to the formation of enhanceosomes. We analyzed protein-DNA interactions in the murine β-globin gene locus using the methyltransferase accessibility protocol for individual templates (MAPit). The data show that a tandem Maf recognition element (MARE) in locus control region (LCR) hypersensitive site 2 (HS2) reveals a remarkably high degree of occupancy during differentiation of mouse erythroleukemia cells. Most of the other transcription factor binding sites in LCR HS2 or in the adult β-globin gene promoter regions exhibit low fractional occupancy, suggesting highly dynamic protein-DNA interactions. Targeting of an artificial zinc finger DNA-binding domain (ZF-DBD) to the HS2 tandem MARE caused a reduction in the association of MARE-binding proteins and transcription complexes at LCR HS2 and the adult βmajor-globin gene promoter but did not affect expression of the βminor-globin gene. The data demonstrate that a stable MARE-associated footprint in LCR HS2 is important for the recruitment of transcription complexes to the adult βmajor-globin gene promoter during erythroid cell differentiation. PMID:26503787

  17. Genomic Organization and Differential Signature of Positive Selection in the Alpha and Beta Globin Gene Clusters in Two Cetacean Species

    PubMed Central

    Nery, Mariana F.; Arroyo, José Ignacio; Opazo, Juan C.

    2013-01-01

    The hemoglobin of jawed vertebrates is a heterotetramer protein that contains two α- and two β-chains, which are encoded by members of α- and β-globin gene families. Given the hemoglobin role in mediating an adaptive response to chronic hypoxia, it is likely that this molecule may have experienced a selective pressure during the evolution of cetaceans, which have to deal with hypoxia tolerance during prolonged diving. This selective pressure could have generated a complex history of gene turnover in these clusters and/or changes in protein structure themselves. Accordingly, we aimed to characterize the genomic organization of α- and β-globin gene clusters in two cetacean species and to detect a possible role of positive selection on them using a phylogenetic framework. Maximum likelihood and Bayesian phylogeny reconstructions revealed that both cetacean species had retained a similar complement of putatively functional genes. For the α-globin gene cluster, the killer whale presents a complement of genes composed of HBZ, HBK, and two functional copies of HBA and HBQ genes, whereas the dolphin possesses HBZ, HBK, HBA and HBQ genes, and one HBA pseudogene. For the β-globin gene cluster, both species retained a complement of four genes, two early expressed genes—HBE and HBH—and two adult expressed genes—HBD and HBB. Our natural selection analysis detected two positively selected sites in the HBB gene (56 and 62) and four in HBA (15, 21, 49, 120). Interestingly, only the genes that are expressed during the adulthood showed the signature of positive selection. PMID:24259315

  18. Identification and characterization of globin genes from two lepidopteran insects, Bombyx mori and Samia cynthia ricini.

    PubMed

    Kawaoka, Shinpei; Katsuma, Susumu; Meng, Yan; Hayashi, Nobumitsu; Mita, Kazuei; Shimada, Toru

    2009-02-15

    We describe the characterization of hemoglobin-like genes from two lepidopteran insects, Bombyx mori (Bmglobin) and Samia cynthia ricini (Scglobin). Bmglobin and Scglobin are predicted to be intracellular proteins and contain amino acids required for heme and oxygen binding. Expression profiles of two lepidopteran globins, especially Bmglobin, were different from that of other insect globins. Although other insect globins are mainly associated with the tracheal system, Bmglobin was expressed almost exclusively in the Malpighian tubules, and the strongest signal for Scglobin was detected in the fat body. Furthermore, biochemical fractionation analysis revealed that both Bmglobin and Scglobin were localized in the cytoplasm. These results suggest that each lepidopteran globin has a distinct role in the tissues in which it is expressed and that the functions of insect globins are more divergent than previously thought. PMID:19059317

  19. Understanding α-globin gene regulation and implications for the treatment of β-thalassemia.

    PubMed

    Mettananda, Sachith; Gibbons, Richard J; Higgs, Douglas R

    2016-03-01

    Over the past three decades, a vast amount of new information has been uncovered describing how the globin genes are regulated. This knowledge has provided significant insights into the general understanding of the regulation of human genes. It is now known that molecular defects within and around the α- and β-globin genes, as well as in the distant regulatory elements, can cause thalassemia. Unbalanced production of globin chains owing to defective synthesis of one, and the continued unopposed synthesis of another, is the central causative factor in the cellular pathology and pathophysiology of thalassemia. A large body of clinical, genetic, and experimental evidence suggests that altering globin chain imbalance by reducing the production of α-globin synthesis ameliorates the disease severity in patients with β-thalassemia. With the development of new genetic-based therapeutic tools that have a potential to decrease the expression of a selected gene in a tissue-specific manner, the possibility of decreasing expression of the α-globin gene to improve the clinical severity of β-thalassemia could become a reality. PMID:26695885

  20. Ancient Duplications and Expression Divergence in the Globin Gene Superfamily of Vertebrates: Insights from the Elephant Shark Genome and Transcriptome

    PubMed Central

    Opazo, Juan C.; Toloza-Villalobos, Jessica; Burmester, Thorsten; Venkatesh, Byrappa; Storz, Jay F.

    2015-01-01

    Comparative analyses of vertebrate genomes continue to uncover a surprising diversity of genes in the globin gene superfamily, some of which have very restricted phyletic distributions despite their antiquity. Genomic analysis of the globin gene repertoire of cartilaginous fish (Chondrichthyes) should be especially informative about the duplicative origins and ancestral functions of vertebrate globins, as divergence between Chondrichthyes and bony vertebrates represents the most basal split within the jawed vertebrates. Here, we report a comparative genomic analysis of the vertebrate globin gene family that includes the complete globin gene repertoire of the elephant shark (Callorhinchus milii). Using genomic sequence data from representatives of all major vertebrate classes, integrated analyses of conserved synteny and phylogenetic relationships revealed that the last common ancestor of vertebrates possessed a repertoire of at least seven globin genes: single copies of androglobin and neuroglobin, four paralogous copies of globin X, and the single-copy progenitor of the entire set of vertebrate-specific globins. Combined with expression data, the genomic inventory of elephant shark globins yielded four especially surprising findings: 1) there is no trace of the neuroglobin gene (a highly conserved gene that is present in all other jawed vertebrates that have been examined to date), 2) myoglobin is highly expressed in heart, but not in skeletal muscle (reflecting a possible ancestral condition in vertebrates with single-circuit circulatory systems), 3) elephant shark possesses two highly divergent globin X paralogs, one of which is preferentially expressed in gonads, and 4) elephant shark possesses two structurally distinct α-globin paralogs, one of which is preferentially expressed in the brain. Expression profiles of elephant shark globin genes reveal distinct specializations of function relative to orthologs in bony vertebrates and suggest hypotheses about

  1. Correction of human. beta. sup S -globin gene by gene targeting

    SciTech Connect

    Shesely, E.G.; Hyungsuk Kim; Shehee, W.R.; Smithies, O. ); Papayannopoulou, T. ); Popovich, B.W. )

    1991-05-15

    As a step toward using gene targeting for gene therapy, the authors have corrected a human {beta}{sup S}-globin gene to the normal {beta}{sup A} allele by homologous recombination in the mouse-human hybrid cell line BSM. BSM is derived from a mouse erythroleukemia cell line and carries a single human chromosome 11 with the {beta}{sup S}-globin allele. A {beta}{sup A}-globin targeting construct containing a unique oligomer and a neomycin-resistance gene was electroporated into the BSM cells, which were then placed under G418 selection. Then 126 resulting pools containing a total {approx}29,000 G418-resistant clones were screened by PCR for the presence of a targeted recombinant: 3 positive pools were identified. A targeted clone was isolated by replating one of the positive pools into smaller pools and rescreening by PCR, followed by dilution cloning. Southern blot analysis demonstrated that the isolated clone had been targeted as planned. The correction of the {beta}{sup S} allele to {beta}{sup A} was confirmed both by allele-specific PCR and by allele-specific antibodies. Expression studies comparing the uninduced and induced RNA levels in unmodified BSM cells and in the targeted clone showed no significant alteration in the ability of the targeted clone to undergo induction, despite the potentially disrupting presence of a transcriptionally active neomycin gene 5{prime} to the human {beta}{sup A}-globin gene. Thus gene targeting can correct a {beta}{sup S} allele to {beta}{sup A}, and the use of a selectable helper gene need not significantly interfere with the induction of the corrected gene.

  2. Gene duplication, genome duplication, and the functional diversification of vertebrate globins

    PubMed Central

    Storz, Jay F.; Opazo, Juan C.; Hoffmann, Federico G.

    2015-01-01

    The functional diversification of the vertebrate globin gene superfamily provides an especially vivid illustration of the role of gene duplication and whole-genome duplication in promoting evolutionary innovation. For example, key globin proteins that evolved specialized functions in various aspects of oxidative metabolism and oxygen signaling pathways (hemoglobin [Hb], myoglobin [Mb], and cytoglobin [Cygb]) trace their origins to two whole-genome duplication events in the stem lineage of vertebrates. The retention of the proto-Hb and Mb genes in the ancestor of jawed vertebrates permitted a physiological division of labor between the oxygen-carrier function of Hb and the oxygen-storage function of Mb. In the Hb gene lineage, a subsequent tandem gene duplication gave rise to the proto α- and β-globin genes, which permitted the formation of multimeric Hbs composed of unlike subunits (α2β2). The evolution of this heteromeric quaternary structure was central to the emergence of Hb as a specialized oxygen-transport protein because it provided a mechanism for cooperative oxygen-binding and allosteric regulatory control. Subsequent rounds of duplication and divergence have produced diverse repertoires of α- and β-like globin genes that are ontogenetically regulated such that functionally distinct Hb isoforms are expressed during different stages of prenatal development and postnatal life. In the ancestor of jawless fishes, the proto Mb and Hb genes appear to have been secondarily lost, and the Cygb homolog evolved a specialized respiratory function in blood-oxygen transport. Phylogenetic and comparative genomic analyses of the vertebrate globin gene superfamily have revealed numerous instances in which paralogous globins have convergently evolved similar expression patterns and/or similar functional specializations in different organismal lineages. PMID:22846683

  3. Differential loss of embryonic globin genes during the radiation of placental mammals

    PubMed Central

    Opazo, Juan C.; Hoffmann, Federico G.; Storz, Jay F.

    2008-01-01

    The differential gain and loss of genes from homologous gene families represents an important source of functional variation among the genomes of different species. Differences in gene content between species are primarily attributable to lineage-specific gene gains via duplication and lineage-specific losses via deletion or inactivation. Here, we use a comparative genomic approach to investigate this process of gene turnover in the β-globin gene family of placental mammals. By analyzing genomic sequence data from representatives of each of the main superordinal clades of placental mammals, we were able to reconstruct pathways of gene family evolution during the basal radiation of this physiologically and morphologically diverse vertebrate group. Our analysis revealed that an initial expansion of the nonadult portion of the β-globin gene cluster in the ancestor of placental mammals was followed by the differential loss and retention of ancestral gene lineages, thereby generating variation in the complement of embryonic globin genes among contemporary species. The sorting of ε-, γ-, and η-globin gene lineages among the basal clades of placental mammals has produced species differences in the functional types of hemoglobin isoforms that can be synthesized during the course of embryonic development. PMID:18755893

  4. A Luciferase Reporter Gene System for High-Throughput Screening of γ-Globin Gene Activators.

    PubMed

    Xie, Wensheng; Silvers, Robert; Ouellette, Michael; Wu, Zining; Lu, Quinn; Li, Hu; Gallagher, Kathleen; Johnson, Kathy; Montoute, Monica

    2016-01-01

    Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic β-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase. PMID:27316998

  5. Molecular cloning and sequencing of mRNAs coding for minor adult globin polypeptides of Xenopus laevis.

    PubMed Central

    Knöchel, W; Meyerhof, W; Hummel, S; Grundmann, U

    1983-01-01

    Globin mRNA was isolated from immature red blood cells of an adult Xenopus laevis female. mRNA/cDNA hybrids were integrated in the Pst I cleavage site of pBR 322 by G/C tailing, and cloned in Escherichia coli strain HB 101. By restriction site analysis as well as hybridization behaviour we identified two clones coding for minor adult alpha and beta globin chains. Nucleotide sequence analysis and derived amino acid sequences are presented. PMID:6298748

  6. Evidence on primate phylogeny from epsilon-globin gene sequences and flanking regions.

    PubMed

    Porter, C A; Sampaio, I; Schneider, H; Schneider, M P; Czelusniak, J; Goodman, M

    1995-01-01

    Phylogenetic relationships among various primate groups were examined based on sequences of epsilon-globin genes. epsilon-globin genes were sequenced from five species of strepsirhine primates. These sequences were aligned and compared with other known primate epsilon-globin sequences, including data from two additional strepsirhine species, one species of tarsier, 19 species of New World monkeys (representing all extant genera), and five species of catarrhines. In addition, a 2-kb segment upstream of the epsilon-globin gene was sequenced in two of the five strepsirhines examined. This upstream sequence was aligned with five other species of primates for which data are available in this segment. Domestic rabbit and goat were used as outgroups. This analysis supports the monophyly of order Primates but does not support the traditional prosimian grouping of tarsiers, lorisoids, and lemuroids; rather it supports the sister grouping of tarsiers and anthropoids into Haplorhini and the sister grouping of lorisoids and lemuroids into Strepsirhini. The mouse lemur (Microcebus murinus) and dwarf lemur (Cheirogaleus medius) appear to be most closely related to each other, forming a clade with the lemuroids, and are probably not closely related to the lorisoids, as suggested by some morphological studies. Analysis of the epsilon-globin data supports the hypothesis that the aye-aye (Daubentonia madagascariensis) shares a sister-group relationship with other Malagasy strepsirhines (all being classified as lemuroids). Relationships among ceboids agree with findings from a previous epsilon-globin study in which fewer outgroup taxa were employed. Rates of molecular evolution were higher in lorisoids than in lemuroids. PMID:7714911

  7. β-Thalassemia Due to Intronic LINE-1 Insertion in the β-Globin Gene (HBB): Molecular Mechanisms Underlying Reduced Transcript Levels of the β-GlobinL1 Allele

    PubMed Central

    Lanikova, Lucie; Kucerova, Jana; Indrak, Karel; Divoka, Martina; Issa, Jean-Pierre; Papayannopoulou, Thalia; Prchal, Josef T.; Divoky, Vladimir

    2016-01-01

    We describe the molecular etiology of β+-thalassemia that is caused by the insertion of the full-length transposable element LINE-1 (L1) into the intron-2 of the β-globin gene (HBB). The transcript level of the affected β-globin gene was severely reduced. The remaining transcripts consisted of full-length, correctly processed β-globin mRNA and a minute amount of three aberrantly spliced transcripts with a decreased half-life due to activation of the nonsense-mediated decay pathway. The lower steady-state amount of mRNA produced by the β-globinL1 allele also resulted from a reduced rate of transcription and decreased production of full-length β-globin primary transcripts. The promoter and enhancer sequences of the β-globinL1 allele were hypermethylated; however, treatment with a demethylating agent did not restore the impaired transcription. A histone deacetylase inhibitor partially reactivated the β-globinL1 transcription despite permanent β-globinL1 promoter CpG methylation. This result indicates that the decreased rate of transcription from the β-globinL1 allele is associated with an altered chromatin structure. Therefore, the molecular defect caused by intronic L1 insertion in the β-globin gene represents a novel etiology of β-thalassemia. PMID:23878091

  8. A Novel Mutation in the Promoter Region of the β-Globin Gene: HBB: c.-127G > C.

    PubMed

    Bilgen, Turker; Canatan, Duran; Delibas, Serpil; Keser, Ibrahim

    2016-08-01

    Novel β-globin gene mutations are still occasionally being reported, especially when evaluating milder phenotypes. We report here a novel putative mutation in the promoter region of the β-globin gene and assess its clinical implications. A family, parents and four siblings, with hematological and clinical features suspected of being β-globin gene mutation(s), were involved in this study. In addition to hematological and clinical evaluations of the whole family, molecular analyses of the β-globin gene were performed by direct sequencing. Sequencing of the β-globin gene revealed a novel genomic alteration in the regulatory region of the gene. This novel genomic alteration was defined as HBB: c.-127G > C according to the Human Genome Variation Society (HGVS) nomenclature. Two siblings were found to be carriers of the HBB: c.-127G > C mutation, while the other two siblings were carriers of the codon 8 (-AA) (HBB: c.25_26delAA) deletion of the β-globin gene. The mother was a compound heterozygote for the codon 8 and HBB: c.-127G > C mutations. Based on hematological and clinical evaluations, we conclude that this novel β-globin gene promoter region change would be associated with a mild phenotype of β-thalassemia (β-thal). PMID:27349616

  9. A phylogenomic profile of globins

    PubMed Central

    Vinogradov, Serge N; Hoogewijs, David; Bailly, Xavier; Arredondo-Peter, Raúl; Gough, Julian; Dewilde, Sylvia; Moens, Luc; Vanfleteren, Jacques R

    2006-01-01

    Background Globins occur in all three kingdoms of life: they can be classified into single-domain globins and chimeric globins. The latter comprise the flavohemoglobins with a C-terminal FAD-binding domain and the gene-regulating globin coupled sensors, with variable C-terminal domains. The single-domain globins encompass sequences related to chimeric globins and «truncated» hemoglobins with a 2-over-2 instead of the canonical 3-over-3 α-helical fold. Results A census of globins in 26 archaeal, 245 bacterial and 49 eukaryote genomes was carried out. Only ~25% of archaea have globins, including globin coupled sensors, related single domain globins and 2-over-2 globins. From one to seven globins per genome were found in ~65% of the bacterial genomes: the presence and number of globins are positively correlated with genome size. Globins appear to be mostly absent in Bacteroidetes/Chlorobi, Chlamydia, Lactobacillales, Mollicutes, Rickettsiales, Pastorellales and Spirochaetes. Single domain globins occur in metazoans and flavohemoglobins are found in fungi, diplomonads and mycetozoans. Although red algae have single domain globins, including 2-over-2 globins, the green algae and ciliates have only 2-over-2 globins. Plants have symbiotic and nonsymbiotic single domain hemoglobins and 2-over-2 hemoglobins. Over 90% of eukaryotes have globins: the nematode Caenorhabditis has the most putative globins, ~33. No globins occur in the parasitic, unicellular eukaryotes such as Encephalitozoon, Entamoeba, Plasmodium and Trypanosoma. Conclusion Although Bacteria have all three types of globins, Archaeado not have flavohemoglobins and Eukaryotes lack globin coupled sensors. Since the hemoglobins in organisms other than animals are enzymes or sensors, it is likely that the evolution of an oxygen transport function accompanied the emergence of multicellular animals. PMID:16600051

  10. Beta-globin gene cluster haplotype frequencies in Khalkhs and Buryats of Mongolia.

    PubMed

    Shimizu, Koji; Tokimasa, Kozue; Takeuchi, Yukiko; Gereksaikhan, Tudevdagva; Tanabe, Yuichi; Omoto, Keiichi; Imanishi, Tadashi; Harihara, Shinji; Hao, Luping; Jing, Feng

    2006-12-01

    Beta-globin gene cluster haplotype frequencies of 169 Khalkhs and 145 Buryats were estimated, and their characteristics were compared with those of Evenkis, Oroqens, Koreans, Japanese, and three Colombian Amerindian groups. The present study suggests that Colombian Amerindians diverged first from Asian populations and then Buryats diverged from other Asian populations. PMID:17564253

  11. Molecular cloning and expression of α-globin and β-globin genes from crocodile (Crocodylus siamensis).

    PubMed

    Anwised, Preeyanan; Kabbua, Thai; Temsiripong, Theeranan; Dhiravisit, Apisak; Jitrapakdee, Sarawut; Araki, Tomohiro; Yoneda, Kazunari; Thammasirirak, Sompong

    2013-03-01

    The first report of complete nucleotide sequences for α- and β-globin chains from the Siamese hemoglobin (Crocodylus siamensis) is given in this study. The cDNAs encoding α- and β-globins were cloned by RT-PCR using the degenerate primers and by the rapid amplification of cDNA ends method. The full-length α-globin cDNA contains an open reading frame of 423 nucleotides encoding 141 amino acid residues, whereas the β-globin cDNA contains an open reading frame of 438 nucleotides encoding 146 amino acid residues. The authenticity of both α- and β-globin cDNA clones were also confirmed by the heterologous expression in Escherichia coli (E. coli). This is the first time that the recombinant C. siamensis globins were produced in prokaryotic system. Additionally, the heme group was inserted into the recombinant proteins and purified heme-bound proteins were performed by affinity chromatography using Co(2+)-charged Talon resins. The heme-bound proteins appeared to have a maximum absorbance at 415 nm, indicated that the recombinant proteins bound to oxygen and formed active oxyhemoglobin (HbO2). The results indicated that recombinant C. siamensis globins were successfully expressed in prokaryotic system and possessed an activity as ligand binding protein. PMID:23463382

  12. Recent advances in globin research using genome-wide association studies and gene editing.

    PubMed

    Orkin, Stuart H

    2016-03-01

    A long-sought goal in the hemoglobin field has been an improved understanding of the mechanisms that regulate the switch from fetal (HbF) to adult (HbA) hemoglobin during development. With such knowledge, the hope is that strategies for directed reactivation of HbF in adults could be devised as an approach to therapy for the β-hemoglobinopathies thalassemia and sickle cell disease. Recent genome-wide association studies (GWAS) led to identification of three loci (BCL11A, HBS1L-MYB, and the β-globin cluster itself) in which natural genetic variation is correlated with different HbF levels in populations. Here, the central role of BCL11A in control of HbF is reviewed from the perspective of how findings may be translated to gene therapy in the not-too-distant future. This summary traces the evolution of recent studies from the initial recognition of BCL11A through GWAS to identification of critical sequences in an enhancer required for its erythroid-specific expression, thereby highlighting an Achilles heel for genome editing. PMID:26866328

  13. Developmental regulation of the human embryonic beta-like globin gene is mediated by synergistic interactions among multiple tissue- and stage-specific elements.

    PubMed Central

    Trepicchio, W L; Dyer, M A; Baron, M H

    1993-01-01

    The stage-specific regulation of mammalian embryonic globin genes has been an experimentally elusive problem, in part because of the developmentally early timing of their expression. We have carried out a systematic analysis of truncation and internal deletion mutations within the 5'-flanking region of the human embryonic beta-like globin gene (epsilon) in erythroid and nonerythroid cell lines. Within a 670-bp region upstream from the constitutive promoter are multiple positive and negative control elements. Of these, a positive regulatory element (epsilon-PRE II) which is active only in embryonic erythroid cells is of particular interest. Remarkably, although it is inactive on its own, in the presence of other sequences located further upstream, it confers tissue- and developmental stage-specific expression on a constitutive epsilon-globin or heterologous promoter. The activity of epsilon-PRE II is also modulated by another positive regulatory domain located further downstream to direct erythroid cell-specific, but little or no embryonic stage-specific, transcription. A nuclear factor highly enriched in embryonic erythroid cells binds specifically within a 19-bp region of epsilon-PRE II. Nuclei from adult erythroid cells also contain a factor that binds to this region but forms a complex of faster electrophoretic mobility. We speculate that interactions between epsilon-PRE II and other upstream control elements play an important role in the developmental regulation of the human embryonic beta-like globin gene. Images PMID:8246963

  14. Pomalidomide reverses γ-globin silencing through the transcriptional reprogramming of adult hematopoietic progenitors.

    PubMed

    Dulmovits, Brian M; Appiah-Kubi, Abena O; Papoin, Julien; Hale, John; He, Mingzhu; Al-Abed, Yousef; Didier, Sebastien; Gould, Michael; Husain-Krautter, Sehba; Singh, Sharon A; Chan, Kyle W H; Vlachos, Adrianna; Allen, Steven L; Taylor, Naomi; Marambaud, Philippe; An, Xiuli; Gallagher, Patrick G; Mohandas, Narla; Lipton, Jeffrey M; Liu, Johnson M; Blanc, Lionel

    2016-03-17

    Current therapeutic strategies for sickle cell anemia are aimed at reactivating fetal hemoglobin. Pomalidomide, a third-generation immunomodulatory drug, was proposed to induce fetal hemoglobin production by an unknown mechanism. Here, we report that pomalidomide induced a fetal-like erythroid differentiation program, leading to a reversion of γ-globin silencing in adult human erythroblasts. Pomalidomide acted early by transiently delaying erythropoiesis at the burst-forming unit-erythroid/colony-forming unit-erythroid transition, but without affecting terminal differentiation. Further, the transcription networks involved in γ-globin repression were selectively and differentially affected by pomalidomide including BCL11A, SOX6, IKZF1, KLF1, and LSD1. IKAROS (IKZF1), a known target of pomalidomide, was degraded by the proteasome, but was not the key effector of this program, because genetic ablation of IKZF1 did not phenocopy pomalidomide treatment. Notably, the pomalidomide-induced reprogramming was conserved in hematopoietic progenitors from individuals with sickle cell anemia. Moreover, multiple myeloma patients treated with pomalidomide demonstrated increased in vivo γ-globin levels in their erythrocytes. Together, these data reveal the molecular mechanisms by which pomalidomide reactivates fetal hemoglobin, reinforcing its potential as a treatment for patients with β-hemoglobinopathies. PMID:26679864

  15. Amelioration of murine sickle cell disease by nonablative conditioning and γ-globin gene-corrected bone marrow cells

    PubMed Central

    Pestina, Tamara I; Hargrove, Phillip W; Zhao, Huifen; Mead, Paul E; Smeltzer, Matthew P; Weiss, Mitchell J; Wilber, Andrew; Persons, Derek A

    2015-01-01

    Patients with severe sickle cell disease (SCD) are candidates for gene therapy using autologous hematopoietic stem cells (HSCs), but concomitant multi-organ disease may contraindicate pretransplant conditioning with full myeloablation. We tested whether nonmyeloablative conditioning, a regimen used successfully for allogeneic bone marrow transplantation of adult SCD patients, allows engraftment of γ-globin gene-corrected cells to a therapeutic level in the Berkeley mouse model of SCD. Animals transplanted according to this regimen averaged 35% engraftment of transduced hematopoietic stem cells with an average vector copy < 2.0. Fetal hemoglobin (HbF) levels ranged from 20 to 44% of total hemoglobin and approximately two-thirds of circulating red blood cells expressed HbF detected by immunofluorescence (F-cells). Gene therapy treatment of SCD mice ameliorated anemia, reduced hyperleukocytosis, improved renal function, and reduced iron accumulation in liver, spleen, and kidneys. Thus, modest levels of chimerism with donor cells expressing high levels of HbF from an insulated γ-globin lentiviral vector can improve the pathology of SCD in mice, thereby illustrating a potentially safe and effective strategy for gene therapy in humans. PMID:26665131

  16. Nonsense mutations in the human. beta. -globin gene affect mRNA metabolism

    SciTech Connect

    Baserga, S.J.; Benz, E.J. Jr. )

    1988-04-01

    A number of premature translation termination mutations (nonsense mutations) have been described in the human {alpha}- and {beta}-globin genes. Studies on mRNA isolated from patients with {beta}{sup 0}-thalassemia have shown that for both the {beta}-17 and the {beta}-39 mutations less than normal levels of {beta}-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human {beta}-globin mRNA). In vitro studies using the cloned {beta}-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. The authors have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, they have studied the effect of five nonsense mutations and two missense mutations on the expression of human {beta}-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation.

  17. Recombination within and between the human insulin and beta-globin gene loci.

    PubMed Central

    Lebo, R V; Chakravarti, A; Buetow, K H; Cheung, M C; Cann, H; Cordell, B; Goodman, H

    1983-01-01

    We detected a large number of polymorphic insulin restriction fragments in black Americans. These different size fragments were probably generated by unequal recombination on both sides of the human insulin gene. Population genetic analysis indicates that recombination occurred 33 times more frequently than expected to generate this large number of polymorphic fragments. Specific properties of the unique repeated 14- to 16-base-pair sequences 5' to the insulin gene suggest that this sequence would promote increased unequal recombination. Additional pedigree analysis showed that the recombination rate between the structural insulin and beta-globin gene loci was 14% with strong evidence for linkage. Since both insulin and beta-globin have been mapped to the short arm of human chromosome 11, this study establishes that the genetic map distance between these genes is 14.2 centimorgans. PMID:6348773

  18. Mi2β Is Required for γ-Globin Gene Silencing: Temporal Assembly of a GATA-1-FOG-1-Mi2 Repressor Complex in β-YAC Transgenic Mice

    PubMed Central

    Costa, Flávia C.; Fedosyuk, Halyna; Chazelle, Allen M.; Neades, Renee Y.; Peterson, Kenneth R.

    2012-01-01

    Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the −566 GATA motif of the Aγ-globin gene. We show that Mi2β is essential for γ-globin gene silencing using Mi2β conditional knockout β-YAC transgenic mice. In addition, increased expression of Aγ-globin was detected in adult blood from β-YAC transgenic mice containing a T>G HPFH point mutation at the −566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type β-YAC transgenic mice. Recruitment of the GATA-1–mediated repressor complex was disrupted by the −566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2β knockout deletion mutation or the cis-acting −566 Aγ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis. PMID:23284307

  19. Oral decitabine reactivates expression of the methylated gamma-globin gene in Papio anubis.

    PubMed

    Lavelle, Donald; Chin, Janet; Vaitkus, Kestis; Redkar, Sanjeev; Phiasivongsa, Pasit; Tang, Chunlin; Will, Roselle; Hankewych, Maria; Roxas, Bryan; Singh, Mahipal; Saunthararajah, Yogen; Desimone, Joseph

    2007-11-01

    The silencing of tumor suppressor genes associated with increased DNA methylation of the promoter regions is a frequent observation in many forms of cancer. Reactivation of these genes using pharmacological inhibitors of DNA methyltransferase such as 5-aza-2'-deoxycytidine (decitabine) is a worthwhile therapeutic goal. The effectiveness and tolerability of low-dose intravenous and subcutaneous decitabine regimens to demethylate and reactivate expression of the methylated gamma-globin gene in baboons and in patients with sickle cell disease led to successful trials of low-dose regimens of this drug in patients with myelodysplastic syndrome. Since these low-dose regimens are well-tolerated with minimal toxicity, they are suitable for chronic dosing to maintain promoter hypomethylation and expression of target genes. The development of an orally administered therapy using DNA methyltransferase inhibitors would facilitate such chronic approaches to therapy. We tested the ability of decitabine and a new salt derivative, decitabine mesylate, to reactivate the methylated gamma-globin gene in baboons when administered orally. Our results demonstrate that oral administration of these drugs at doses 17-34 times optimal subcutaneous doses of decitabine reactivates fetal hemoglobin, demethylates the epsilon- and gamma-globin gene promoters, and increases histone acetylation of these promoters in baboons (Papio anubis). PMID:17696208

  20. Characterization of two globin genes from the malaria mosquito Anopheles gambiae: divergent origin of nematoceran haemoglobins.

    PubMed

    Burmester, Thorsten; Klawitter, Sabine; Hankeln, Thomas

    2007-04-01

    The chironomid midges are the only insects that harbour true haemoglobin in their haemolymph. Here we report the identification of haemoglobin genes in two other nematoceran species. Two paralogous haemoglobin genes (glob1 and glob2) from the malaria mosquito Anopheles gambiae were cloned and sequenced. Furthermore, we identified two orthologous haemoglobin genes in the yellow fever mosquito Aedes aegypti. All four haemoglobins were predicted to be intracellular proteins, with the amino acids required for heme- and oxygen-binding being conserved. In situ-hybridization studies showed that glob1 and glob2 expression in An. gambiae is mainly associated with the tracheal system. This pattern resembles that of other insect intracellular globins. We also observed expression of glob2 in visceral muscles. Phylogenetic analyses showed that the globins of the mosquitoes and the Chironomidae are not orthologous. The chironomid haemoglobins share a recent common origin with the brachyceran glob1 proteins. The mosquito glob1 and glob2 proteins, which separated by gene duplication around 170 million years ago, form a distinct clade of more ancient evolutionary origin within the insects. The glob1 genes have introns in the ancestral globin positions B12.2 and G7.0. An additional intron was observed in Ae. aegypti glob1 helix position E18.0, providing evidence for a recent intron gain event. Both mosquito glob2 genes have lost the B12.2 intron. This pattern must be interpreted in terms of dynamic intron gain and loss events in the globin gene lineage. PMID:17298561

  1. Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin.

    PubMed

    Wienert, Beeke; Funnell, Alister P W; Norton, Laura J; Pearson, Richard C M; Wilkinson-White, Lorna E; Lester, Krystal; Vadolas, Jim; Porteus, Matthew H; Matthews, Jacqueline M; Quinlan, Kate G R; Crossley, Merlin

    2015-01-01

    Genetic disorders resulting from defects in the adult globin genes are among the most common inherited diseases. Symptoms worsen from birth as fetal γ-globin expression is silenced. Genome editing could permit the introduction of beneficial single-nucleotide variants to ameliorate symptoms. Here, as proof of concept, we introduce the naturally occurring Hereditary Persistance of Fetal Haemoglobin (HPFH) -175T>C point mutation associated with elevated fetal γ-globin into erythroid cell lines. We show that this mutation increases fetal globin expression through de novo recruitment of the activator TAL1 to promote chromatin looping of distal enhancers to the modified γ-globin promoter. PMID:25971621

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

    PubMed Central

    Ekwattanakit, Supachai; Monteerarat, Yuwarat; Riolueang, Suchada; Tachavanich, Kalaya; Viprakasit, Vip

    2012-01-01

    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. PMID:23049556

  3. Precise nucleosome positioning in the promoter of the chicken beta A globin gene.

    PubMed

    Kefalas, P; Gray, F C; Allan, J

    1988-01-25

    Histone octamers were reconstituted onto 5' end-labelled DNA fragments derived from the promoter region of the chicken beta A globin gene. The location of the reconstituted histone octamer with respect to the DNA sequence of each fragment was assessed by Exonuclease III digestion of purified nucleosome monomers. By this approach we have found a strong preference for histone octamers to be positioned over nucleotides -206 to -62 relative to the gene cap site. This stretch of DNA contains all those 5' beta globin sequences which, by DNase footprinting, bind specific protein factors and incorporates three promoter consensus sequence motifs. The upstream terminal 32 base pairs of this DNA segment contains the binding sites for the erythrocyte specific G-string binding protein and transcription factor Spl and appears to be relatively weakly bound to the histone octamer. PMID:3340546

  4. Precise nucleosome positioning in the promoter of the chicken beta A globin gene.

    PubMed Central

    Kefalas, P; Gray, F C; Allan, J

    1988-01-01

    Histone octamers were reconstituted onto 5' end-labelled DNA fragments derived from the promoter region of the chicken beta A globin gene. The location of the reconstituted histone octamer with respect to the DNA sequence of each fragment was assessed by Exonuclease III digestion of purified nucleosome monomers. By this approach we have found a strong preference for histone octamers to be positioned over nucleotides -206 to -62 relative to the gene cap site. This stretch of DNA contains all those 5' beta globin sequences which, by DNase footprinting, bind specific protein factors and incorporates three promoter consensus sequence motifs. The upstream terminal 32 base pairs of this DNA segment contains the binding sites for the erythrocyte specific G-string binding protein and transcription factor Spl and appears to be relatively weakly bound to the histone octamer. Images PMID:3340546

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

    PubMed

    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. PMID:15757246

  6. A physiological delay in human fetal hemoglobin switching is associated with specific globin DNA hypomethylation.

    PubMed

    Perrine, S P; Greene, M F; Cohen, R A; Faller, D V

    1988-02-01

    The human fetal-to-adult globin switch normally occurs on a fixed schedule, beginning at 32-34 weeks gestation, and recent studies have suggested an association between this developmental inactivation of the fetal (gamma) globin genes and the appearance of methylation within and around these genes. We have studied a population of infants in whom this switch does not occur before birth (infants of diabetic mothers, IDM) and examined the patterns of methylation surrounding their active gamma-globin genes, in comparison to the gamma-globin genes of age-matched controls who have switched their pattern of globin gene expression on schedule. All genomic DNA samples from infants with delays in the globin switch demonstrated extensive hypomethylation in the region of the gamma-globin genes, comparable to that found in the genomes of fetuses of less than 21 weeks gestation. DNA from the erythroid cells of infants of 32-40 weeks gestation had no detectable hypomethylation in the gamma-globin region. These findings support the concept that hypomethylation is an accurate developmental marker of globin gene switching, and suggest that globin gene expression in IDM may be arrested at an early preswitch stage. PMID:2449361

  7. Analysis of α1 and α2 globin genes among patients with hemoglobin Adana in Malaysia.

    PubMed

    Lee, T Y; Lai, M I; Ismail, P; Ramachandran, V; Tan, J A M A; Teh, L K; Othman, R; Hussein, N H; George, E

    2016-01-01

    Hemoglobin (Hb) Adana [HBA2: c179G>A (or HBA1); p.Gly60Asp] is a non-deletional α-thalassemia variant found in Malaysia. An improvement in the molecular techniques in recent years has made identification of Hb Adana much easier. For this study, a total of 26 Hb Adana α-thalassemia intermedia and 10 Hb Adana trait blood samples were collected from patients. Common deletional and non-deletional α-thalassemia genotypes were determined using multiplex gap polymerase chain reaction (PCR) and multiplex ARMS PCR techniques. Identification of the Hb Adana location on the α-globin gene was carried out using genomic sequencing and the location of the mutation was confirmed via restriction fragment length polymorphism-PCR. Among the 36 samples, 24 (66.7%) had the -α(3.7)/α(Cd59)α mutation, while the -α(3.7)/α(Cd59)α mutation accounted for 2 samples (5.6%) and the remaining 10 (27.8%) samples were α/α(Cd59)α. All 36 samples were found to have the Hb Adana mutation on the α2-globin gene. The position of the α-globin gene mutation found in our cases was similar to that reported in Indonesia (16%) but not to that in Turkey (0.6%). Our results showed that the Hb Adana mutation was preferentially present in the α2-globin genes in Malays compared to the other ethnicities in Malaysia. Thus, the Malays might have similar ancestry based on the similarities in the Hb Adana position. PMID:27173219

  8. Thalassaemia mutations within the 5'UTR of the human beta-globin gene disrupt transcription.

    PubMed

    Sgourou, Argyro; Routledge, Samantha; Antoniou, Michael; Papachatzopoulou, Adamantia; Psiouri, Lambrini; Athanassiadou, Aglaia

    2004-03-01

    The mechanisms by which mutations within the 5' untranslated region (UTR) of the human beta-globin gene (HBB) cause thalassaemia are currently not well understood. We present here the first comprehensive comparative functional analysis of four 'silent' mutations in the human beta-globin 5'UTR, namely: +10(-T), +22(G --> A), +33(C --> G) and +(40-43)(-AAAC), which are present in patients with beta-thalassaemia intermedia. Expression of these genes under the control of the beta-globin locus control region in stable transfected murine erythroleukaemia cells showed that all four mutations decreased steady state levels of mRNA to 61.6%, 68%, 85.2% and 70.6%, respectively, compared with the wildtype gene. These mutations did not interfere with either mRNA transport from the nucleus to the cytoplasm, 3' end processing or mRNA stability. Nuclear run-on experiments demonstrated that mutations +10(-T) and +33(C --> G) reduced the rate of transcription to a degree that fully accounted for the observed lower level of mRNA accumulation, suggesting a disruption of downstream promoter sequences. Interestingly, mutation +22(G --> A) decreased the rate of transcription to a low degree, indicating the existence of a mechanism that acts post-transcriptionally. Generally, our data demonstrated the significance of functionally analysing mutants of this type in the presence of a full complement of transcriptional regulatory elements within a stably integrated chromatin context in an erythroid cell environment. PMID:15009072

  9. Altitudinal Variation at Duplicated β-Globin Genes in Deer Mice: Effects of Selection, Recombination, and Gene Conversion

    PubMed Central

    Storz, Jay F.; Natarajan, Chandrasekhar; Cheviron, Zachary A.; Hoffmann, Federico G.; Kelly, John K.

    2012-01-01

    Spatially varying selection on a given polymorphism is expected to produce a localized peak in the between-population component of nucleotide diversity, and theory suggests that the chromosomal extent of elevated differentiation may be enhanced in cases where tandemly linked genes contribute to fitness variation. An intriguing example is provided by the tandemly duplicated β-globin genes of deer mice (Peromyscus maniculatus), which contribute to adaptive differentiation in blood–oxygen affinity between high- and low-altitude populations. Remarkably, the two β-globin genes segregate the same pair of functionally distinct alleles due to a history of interparalog gene conversion and alleles of the same functional type are in perfect coupling-phase linkage disequilibrium (LD). Here we report a multilocus analysis of nucleotide polymorphism and LD in highland and lowland mice with different genetic backgrounds at the β-globin genes. The analysis of haplotype structure revealed a paradoxical pattern whereby perfect LD between the two β-globin paralogs (which are separated by 16.2 kb) is maintained in spite of the fact that LD within both paralogs decays to background levels over physical distances of less than 1 kb. The survey of nucleotide polymorphism revealed that elevated levels of altitudinal differentiation at each of the β-globin genes drop away quite rapidly in the external flanking regions (upstream of the 5′ paralog and downstream of the 3′ paralog), but the level of differentiation remains unexpectedly high across the intergenic region. Observed patterns of diversity and haplotype structure are difficult to reconcile with expectations of a two-locus selection model with multiplicative fitness. PMID:22042573

  10. Coinheritance of a Rare Nucleotide Substitution on the β-Globin Gene and Other Known Mutations in the Globin Clusters: Management in Genetic Counseling.

    PubMed

    Vinciguerra, Margherita; Passarello, Cristina; Leto, Filippo; Crivello, Anna; Fustaneo, Maria; Cassarà, Filippo; Cannata, Monica; Maggio, Aurelio; Giambona, Antonino

    2016-08-01

    A large number of methods for DNA analysis are available to identify defects in globin genes associated with hemoglobin (Hb) disorders. In this study, we report a rare nucleotide (nt) substitution on the β-globin gene, nt 781 in the second intron [IVS-II-781 (C > G); HBB: c.316-70C > G], identified in four patients. This nt substitution was previously described only as a personal communication to the HbVar database and indicated as a β(0) or β(+) mutation. The purpose of this study was to evaluate the clinical implication of this nt change, particularly when coinherited with severe β-thalassemia (β-thal), in order to be able to conduct appropriate genetic counseling. Genetic studies were performed on two subjects, one carried Hb S [β6(A3)Glu→Val; HBB: c.20A > T], and the other carried IVS-I-110 (G > A) (HBB: c.93-21G > A). All these subjects showed this new β nt substitution in association with Hb A2' (or Hb B2) [δ16(A13)Gly→Arg; HBD: c.49G > C]. Another 16 samples, carrying the same δ variant as the probands, were processed by β-globin gene sequencing in order to better understand the correlation between this Hb variant and the rare nt substitution reported in this study. The present investigation emphasizes the importance of sharing the observed nt changes in the globin gene cluster, especially in the case of new or rare undefined mutations, in order to facilitate the determination of their phenotypic expression, the possible interactions with known molecular defects and to formulate appropriate genetic counseling for at-risk couples. PMID:27258795

  11. Gene Therapy of the β-Hemoglobinopathies by Lentiviral Transfer of the β(A(T87Q))-Globin Gene.

    PubMed

    Negre, Olivier; Eggimann, Anne-Virginie; Beuzard, Yves; Ribeil, Jean-Antoine; Bourget, Philippe; Borwornpinyo, Suparerk; Hongeng, Suradej; Hacein-Bey, Salima; Cavazzana, Marina; Leboulch, Philippe; Payen, Emmanuel

    2016-02-01

    β-globin gene disorders are the most prevalent inherited diseases worldwide and result from abnormal β-globin synthesis or structure. Novel therapeutic approaches are being developed in an effort to move beyond palliative management. Gene therapy, by ex vivo lentiviral transfer of a therapeutic β-globin gene derivative (β(AT87Q)-globin) to hematopoietic stem cells, driven by cis-regulatory elements that confer high, erythroid-specific expression, has been evaluated in human clinical trials over the past 8 years. β(AT87Q)-globin is used both as a strong inhibitor of HbS polymerization and as a biomarker. While long-term studies are underway in multiple centers in Europe and in the United States, proof-of-principle of efficacy and safety has already been obtained in multiple patients with β-thalassemia and sickle cell disease. PMID:26886832

  12. Gene Therapy of the β-Hemoglobinopathies by Lentiviral Transfer of the βA(T87Q)-Globin Gene

    PubMed Central

    Negre, Olivier; Eggimann, Anne-Virginie; Beuzard, Yves; Ribeil, Jean-Antoine; Bourget, Philippe; Borwornpinyo, Suparerk; Hongeng, Suradej; Hacein-Bey, Salima; Cavazzana, Marina; Leboulch, Philippe; Payen, Emmanuel

    2016-01-01

    β-globin gene disorders are the most prevalent inherited diseases worldwide and result from abnormal β-globin synthesis or structure. Novel therapeutic approaches are being developed in an effort to move beyond palliative management. Gene therapy, by ex vivo lentiviral transfer of a therapeutic β-globin gene derivative (βAT87Q-globin) to hematopoietic stem cells, driven by cis-regulatory elements that confer high, erythroid-specific expression, has been evaluated in human clinical trials over the past 8 years. βAT87Q-globin is used both as a strong inhibitor of HbS polymerization and as a biomarker. While long-term studies are underway in multiple centers in Europe and in the United States, proof-of-principle of efficacy and safety has already been obtained in multiple patients with β-thalassemia and sickle cell disease. PMID:26886832

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

    PubMed Central

    Sadelain, M; Wang, C H; Antoniou, M; Grosveld, F; Mulligan, R C

    1995-01-01

    Retrovirus-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 sickle cell anemia), however, has been hampered by the inability to generate recombinant viruses able to efficiently and faithfully transmit the necessary sequences for appropriate gene expression. We have addressed this problem by carefully examining the interactions between retroviral and beta-globin gene sequences which affect vector transmission, stability, and expression. First, we examined the transmission properties of a large number of different recombinant proviral genomes which vary both in the precise nature of vector, beta-globin structural gene, and locus control region (LCR) core sequences incorporated and in the placement and orientation of those sequences. Through this analysis, we identified one specific vector, termed M beta 6L, which carries both the human beta-globin gene and core elements HS2, HS3, and HS4 from the LCR and faithfully transmits recombinant proviral sequences to cells with titers greater than 10(6) per ml. Populations of murine erythroleukemia (MEL) cells transduced by this virus expressed levels of human beta-globin transcript which, on a per gene copy basis, were 78% of the levels detected in an MEL-derived cell line, Hu11, which carries human chromosome 11, the site of the beta-globin locus. Analysis of individual transduced MEL cell clones, however, indicated that, while expression was detected in every clone tested (n = 17), the levels of human beta-globin treatment varied between 4% and 146% of the levels in Hu11. This clonal variation in expression levels suggests that small beta-globin LCR sequences may not provide for as strict chromosomal position-independent expression of beta-globin as previously suspected, at least in the context of

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

    PubMed

    Helder, J; Deisseroth, A

    1987-04-01

    The loss of alpha-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 alpha-globin gene expression (hemoglobin H disease) in these patients is linked with the alpha-globin genes on chromosome 16, we transferred chromosome 16 from preleukemic patients with acquired hemoglobin H disease to mouse erythroleukemia cells and measured the transcriptional activity of the human alpha-globin genes. After transfer to mouse erythroleukemia cells, the expression of human alpha-globin genes from the peripheral blood or marrow cells of preleukemic patients with acquired hemoglobin H disease was similar to that of human alpha-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 alpha-globin gene defect in these preleukemia patients with acquired hemoglobin H disease and suggest that altered expression of a gene in trans to the alpha-globin gene may be responsible for the acquisition of hemoglobin H disease in these patients. PMID:3031681

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

    SciTech Connect

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

    1987-05-01

    The predominant ..beta..-thalassemia in Sardinia is the ..beta../sup 0/ type in which no ..beta..-globin chains are synthesized in the homozygous state. The authors determined the ..beta..-thalassemia mutations in this population by the oligonucleotide-probe method and defined the chromosome haplotypes on which the mutation resides. The same ..beta../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 ..beta..-globin gene region.

  16. Human beta-globin gene polymorphisms characterized in DNA extracted from ancient bones 12,000 years old.

    PubMed Central

    Béraud-Colomb, E; Roubin, R; Martin, J; Maroc, N; Gardeisen, A; Trabuchet, G; Goosséns, M

    1995-01-01

    Analyzing the nuclear DNA from ancient human bones is an essential step to the understanding of genetic diversity in current populations, provided that such systematic studies are experimentally feasible. This article reports the successful extraction and amplification of nuclear DNA from the beta-globin region from 5 of 10 bone specimens up to 12,000 years old. These have been typed for beta-globin frameworks by sequencing through two variable positions and for a polymorphic (AT) chi (T) gamma microsatellite 500 bp upstream of the beta-globin gene. These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably older than those used to study high-copy-number human mtDNA. These results show that the systematic study of nuclear DNA polymorphisms of ancient populations is feasible. Images Figure 2 Figure 3 PMID:8533755

  17. Human {beta}-globin gene polymorphisms characterized in DNA extracted from ancient bones 12,000 years old

    SciTech Connect

    Beraud-Colomb, E. |; Maroc, N.; Roubin, R.

    1995-12-01

    Analyzing the nuclear DNA from ancient human bones is an essential step to the understanding of genetic diversity in current populations, provided that such systematic studies are experimentally feasible. This article reports the successful extraction and amplification of nuclear DNA from the P-globin region from 5 of 10 bone specimens up to 12,000 years old. These have been typed for P-globin frameworks by sequencing through two variable positions and for a polymorphic (AT){sub x}(T){sub y} microsatellite 500 bp upstream of the P-globin gene. These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably older than those used to study high-copy-number human mtDNA. These results show that the systematic study of nuclear DNA polymorphisms of ancient populations is feasible. 34 refs., 3 figs., 2 tabs.

  18. Tissue-specific and developmentally regulated expression of a chimeric actin-globin gene in transgenic mice.

    PubMed Central

    Shani, M

    1986-01-01

    A chimeric plasmid containing about 2/3 of the rat skeletal muscle actin gene plus 730 base pairs of its 5' flanking sequences fused to the 3' end of a human embryonic globin gene (D. Melloul, B. Aloni, J. Calvo, D. Yaffe, and U. Nudel, EMBO J. 3:983-990, 1984) was inserted into mice by microinjection into fertilized eggs. Eleven transgenic mice carrying the chimeric gene with or without plasmid pBR322 DNA sequences were identified. The majority of these mice transmitted the injected DNA to about 50% of their progeny. However, in transgenic mouse CV1, transmission to progeny was associated with amplification or deletion of the injected DNA sequences, while in transgenic mouse CV4 transmission was distorted, probably as a result of insertional mutagenesis. Tissue-specific expression was dependent on the removal of the vector DNA sequences from the chimeric gene sequences prior to microinjection. None of the transgenic mice carrying the chimeric gene together with plasmid pBR322 sequences expressed the introduced gene in striated muscles. In contrast, the six transgenic mice carrying the chimeric gene sequences alone expressed the inserted gene specifically in skeletal and cardiac muscles. Moreover, expression of the chimeric gene was not only tissue specific, but also developmentally regulated. Similar to the endogenous skeletal muscle actin gene, the chimeric gene was expressed at a relatively high level in cardiac muscle of neonatal mice and at a significantly lower level in adult cardiac muscle. These results indicate that the injected DNA included sufficient cis-acting control elements for its tissue-specific and developmentally regulated expression in transgenic mice. Images PMID:3023942

  19. Protein-binding sites within the 5' DNase I-hypersensitive region of the chicken alpha D-globin gene.

    PubMed Central

    Kemper, B; Jackson, P D; Felsenfeld, G

    1987-01-01

    We mapped at high resolution and as a function of development the hypersensitive domain in the 5'-flanking region of the chicken alpha D-globin gene and determined the specific protein-binding sites within the domain. The domain extends from -130 to +80 nucleotides (nt) relative to the cap site. DNase I footprinting within intact embryonic erythrocyte nuclei revealed a strongly protected area from -71 to -52 nt. The same area was weakly protected in adult nuclei. A factor was present in extracts of erythrocyte nuclei from both embryos and adults that protected the sequence AAGATAAGG (-63 to -55 nt) in DNase I footprinting experiments; at higher concentrations of extract, sequences immediately adjacent (-73 to -64 and -53 to -38) were also protected. The same pattern of binding was revealed by gel mobility shift assays. The identical AAGATAAGG sequence is found in the 5'-flanking region of the beta rho gene; it competed for binding of the alpha D-specific factor, suggesting that regulatory elements are shared. Images PMID:3600658

  20. Duplication of the gamma-globin gene mediated by L1 long interspersed repetitive elements in an early ancestor of simian primates.

    PubMed Central

    Fitch, D H; Bailey, W J; Tagle, D A; Goodman, M; Sieu, L; Slightom, J L

    1991-01-01

    Regions surrounding the single gamma-globin gene of galago and the duplicated gamma 1- and gamma 2-globin genes of gibbon, rhesus monkey, and spider monkey were sequenced and aligned with those from humans. Contrary to previous studies, spider monkey was found to have not one but two gamma-globin genes, only one of which (gamma 2) is functional. The reconstructed evolutionary history of the gamma-globin genes and their flanking sequences traces their origin to a tandem duplication of a DNA segment approximately 5.5 kilobases long that occurred before catarrhine primates (humans, apes, and Old World monkeys) diverged from platyrrhines (New World monkeys), much earlier than previously thought. This reconstructed molecular history also reveals that the duplication resulted from an unequal homologous crossover between two related L1 long interspersed repetitive elements, one upstream and one downstream of the single ancestral gamma-globin gene. Perhaps facilitated by the redundancy resulting from the duplication, the gamma-globin genes escaped the selective constraints of embryonically functioning genes and evolved into fetally functioning genes. This view is supported by the finding that a burst of nonsynonymous substitutions occurred in the gamma-globin genes while they became restructured for fetal expression in the common ancestor of platyrrhines and catarrhines. PMID:1908094

  1. Peptide nucleic acids targeting β-globin mRNAs selectively inhibit hemoglobin production in murine erythroleukemia cells.

    PubMed

    Montagner, Giulia; Gemmo, Chiara; Fabbri, Enrica; Manicardi, Alex; Accardo, Igea; Bianchi, Nicoletta; Finotti, Alessia; Breveglieri, Giulia; Salvatori, Francesca; Borgatti, Monica; Lampronti, Ilaria; Bresciani, Alberto; Altamura, Sergio; Corradini, Roberto; Gambari, Roberto

    2015-01-01

    In the treatment of hemoglobinopathies, amending altered hemoglobins and/or globins produced in excess is an important part of therapeutic strategies and the selective inhibition of globin production may be clinically beneficial. Therefore the development of drug-based methods for the selective inhibition of globin accumulation is required. In this study, we employed peptide nucleic acids (PNAs) to alter globin gene expression. The main conclusion of the present study was that PNAs designed to target adult murine β-globin mRNA inhibit hemoglobin accumulation and erythroid differentiation of murine erythroleukemia (MEL) cells with high efficiency and fair selectivity. No major effects were observed on cell proliferation. Our study supports the concept that PNAs may be used to target mRNAs that, similar to globin mRNAs, are expressed at very high levels in differentiating erythroid cells. Our data suggest that PNAs inhibit the excess production of globins involved in the pathophysiology of hemoglobinopathies. PMID:25405921

  2. Bergamot (Citrus bergamia Risso) fruit extracts as γ-globin gene expression inducers: phytochemical and functional perspectives.

    PubMed

    Guerrini, Alessandra; Lampronti, Ilaria; Bianchi, Nicoletta; Zuccato, Cristina; Breveglieri, Giulia; Salvatori, Francesca; Mancini, Irene; Rossi, Damiano; Potenza, Rocco; Chiavilli, Francesco; Sacchetti, Gianni; Gambari, Roberto; Borgatti, Monica

    2009-05-27

    Epicarps of Citrus bergamia fruits from organic farming were extracted with the objective of obtaining derived products differently rich in coumarins and psoralens. The extracts were chemically characterized by (1)H nuclear magnetic resonance (NMR), gas chromatography-flame ionization detection (GC-FID), gas chromatography-mass spectrometry (GC-MS), and high-pressure liquid chromatography (HPLC) for detecting and quantifying the main constituents. Both bergamot extracts and chemical standards corresponding to the main constituents detected were then assayed for their capacity to increase erythroid differentiation of K562 cells and expression of γ-globin genes in human erythroid precursor cells. Three experimental cell systems were employed: (a) the human leukemic K562 cell line, (b) K562 cell clones stably transfected with a pCCL construct carrying green-enhanced green fluorescence protein (EGFP) under the γ-globin gene promoter, and (c) the two-phase liquid culture of human erythroid progenitors isolated from healthy donors. The results suggest that citropten and bergapten are powerful inducers of differentiation and γ-globin gene expression in human erythroid cells. These data could have practical relevance, because pharmacologically mediated regulation of human γ-globin gene expression, with the consequent induction of fetal hemoglobin, is considered to be a potential therapeutic approach in hematological disorders, including β-thalassemia and sickle cell anemia. PMID:19371028

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

    SciTech Connect

    Patrick, N.; Miyakawa, F.; Hunt, J.A.

    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].

  4. Evaluation of α-Globin Gene Mutations Among Different Ethnic Groups in Khuzestan Province, Southwest Iran.

    PubMed

    Khosravi, Abbas; Jalali-Far, Mohammadali; Saki, Najmaldin; Hosseini, Hossein; Galehdari, Hamid; Kiani-Ghalesardi, Omid; Paridar, Mostafa; Azarkeivan, Azita; Magaji-Hamid, Kabir

    2016-01-01

    α-Thalassemia (α-thal) is one of the most common inherited hemoglobin (Hb) disorders in the world. In addition to large deletions, over 50 different α-thal point mutations were detected around the world, thus, patients showed different phenotypes with regard to genotype. This study evaluated the genetic frequency of α-thal in Khuzestan Province, Southwest Iran, to help implement premarital and prenatal screening programs. The study was conducted on couples proposing to get married and parents who were referred to the genetic center of Shafa Hospital, Ahvaz, Iran, for prenatal diagnosis (PND) in 2012. Genomic DNA was purified by the salting-out method and tested using multiplex gap-polymerase chain reaction (gap-PCR), amplification refractory mutation system-PCR (ARMS-PCR), reverse hybridization test strips and DNA sequencing. Overall, 11 mutations were found on the α-globin genes. Based on gene frequency, the most common mutant allele was -α(3.7) (rightward) (71.3%) followed by the two gene deletion - -(MED) (9.7%). Other common mutations were α(codon 19)α (GCG>GC-, α2) (8.4%), the polyadenylation (polyA1) site α(polyA1)α (AATAAA>AATAAG) (2.8%), and α(-5 nt)α (-TGAGG) (2.0%). In addition, an extremely rare mutation at α(codon 21)α [Hb Fontainebleau, HBA2: c.64G > C (or HBA1)] was also found. The results of this study are critical for correct diagnosis of α-thal carriers, premarriage counseling and PND. This study suggests that the distribution of mutations on the α-globin genes differs among the ethnic groups in Khuzestan Province as well as in other provinces. PMID:26878087

  5. Essential role of NF-E2 in remodeling of chromatin structure and transcriptional activation of the epsilon-globin gene in vivo by 5' hypersensitive site 2 of the beta-globin locus control region.

    PubMed Central

    Gong, Q H; McDowell, J C; Dean, A

    1996-01-01

    Much of our understanding of the process by which enhancers activate transcription has been gained from transient-transfection studies in which the DNA is not assembled with histones and other chromatin proteins as it is in the cell nucleus. To study the activation of a mammalian gene in a natural chromatin context in vivo, we constructed a minichromosome containing the human epsilon-globin gene and portions of the beta-globin locus control region (LCR). The minichromosomes replicate and are maintained at stable copy number in human erythroid cells. Expression of the minichromosomal epsilon-globin gene requires the presence of beta-globin LCR elements in cis, as is the case for the chromosomal gene. We determined the chromatin structure of the epsilon-globin gene in both the active and inactive states. The transcriptionally inactive locus is covered by an array of positioned nucleosomes extending over 1,400 bp. In minichromosomes with a (mu)LCR or DNase I-hypersensitive site 2 (HS2) which actively transcribe the epsilon-globin gene, the nucleosome at the promoter is altered or disrupted while positioning of nucleosomes in the rest of the locus is retained. All or virtually all minichromosomes are simultaneously hypersensitive to DNase I both at the promoter and at HS2. Transcriptional activation and promoter remodeling, as well as formation of the HS2 structure itself, depended on the presence of the NF-E2 binding motif in HS2. The nucleosome at the promoter which is altered upon activation is positioned over the transcriptional elements of the epsilon-globin gene, i.e., the TATA, CCAAT, and CACCC elements, and the GATA-1 site at -165. The simple availability of erythroid transcription factors that recognize these motifs is insufficient to allow expression. As in the chromosomal globin locus, regulation also occurs at the level of chromatin structure. These observations are consistent with the idea that one role of the beta-globin LCR is to maintain promoters free

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

    SciTech Connect

    Louie, E.; Dietz, L.; Shafer, F.

    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.

  7. The relationship between the presence of extra α-globin genes and blood cell traits in Altamurana sheep

    PubMed Central

    2003-01-01

    Additional α-globin genes in sheep might produce extra α-globin chains and, consequently, the subject carrying triplicated (ααα) or quadruplicated (αααα) haplotypes may exhibit different hematological phenotypes when compared to the normal duplicated (αα) homozygotes (NN). Both ααα and αααα heterozygous (ND) and ααα and αααα homozygous (DD) individuals were obtained by selection and inbreeding. Chromatographic RP-HPLC analyses of the globin chains of 65 subjects (15 DD, 20 ND and 30 NN) were performed. A highly significant linear regression (r2 = 0.967) of the α/β ratio on the number of α-globin genes was found, and the α/β ratio ranged on average from 1.0 in NN individuals to 1.2 in the ND and 1.6 in the DD subjects. Values for blood fell within the range of normality but were rather peculiar as a whole. When the erythrocytes of individuals carrying normal arrangements were compared with those of subjects with extra α-genes, the latter had fewer erythrocytes that were bigger in size and had a higher Hb content and a greater osmotic fragility. This hematological picture is consistent with the existence of an unbalanced α/β ratio. PMID:12927085

  8. HbVar: A relational database of human hemoglobin variants and thalassemia mutations at the globin gene server.

    PubMed

    Hardison, Ross C; Chui, David H K; Giardine, Belinda; Riemer, Cathy; Patrinos, George P; Anagnou, Nicholas; Miller, Webb; Wajcman, Henri

    2002-03-01

    We have constructed a relational database of hemoglobin variants and thalassemia mutations, called HbVar, which can be accessed on the web at http://globin.cse.psu.edu. Extensive information is recorded for each variant and mutation, including a description of the variant and associated pathology, hematology, electrophoretic mobility, methods of isolation, stability information, ethnic occurrence, structure studies, functional studies, and references. The initial information was derived from books by Dr. Titus Huisman and colleagues [Huisman et al., 1996, 1997, 1998]. The current database is updated regularly with the addition of new data and corrections to previous data. Queries can be formulated based on fields in the database. Tables of common categories of variants, such as all those involving the alpha1-globin gene (HBA1) or all those that result in high oxygen affinity, are maintained by automated queries on the database. Users can formulate more precise queries, such as identifying "all beta-globin variants associated with instability and found in Scottish populations." This new database should be useful for clinical diagnosis as well as in fundamental studies of hemoglobin biochemistry, globin gene regulation, and human sequence variation at these loci. PMID:11857738

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

    PubMed Central

    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 muα-globin2/huβ-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. Recombinant adeno-associated virus (rAAV)-mediated expression of a human gamma-globin gene in human progenitor-derived erythroid cells.

    PubMed Central

    Miller, J L; Donahue, R E; Sellers, S E; Samulski, R J; Young, N S; Nienhuis, A W

    1994-01-01

    Effective gene therapy for the severe hemoglobin (Hb) disorders, sickle-cell anemia and thalassemia, will require an efficient method to transfer, integrate, and express a globin gene in primary erythroid cells. To evaluate recombinant adeno-associated virus (rAAV) for this purpose, we constructed a rAAV vector encoding a human gamma-globin gene (pJM24/vHS432A gamma). Its 4725-nucleotide genome consists of two 180-bp AAV inverted terminal repeats flanking the core elements of hypersensitive sites 2, 3, and 4 from the locus control region of the beta-globin gene cluster, linked to a mutationally marked A gamma-globin gene (A gamma) containing native promoter and RNA processing signals. CD34+ human hematopoietic cells were exposed to rAAV particles at a multiplicity of infection of 500-1000 and cultured in semisolid medium containing several cytokines. A reverse transcriptase polymerase chain reaction assay distinguished mRNA signals derived from transduced and endogenous human gamma-globin genes. Twenty to 40% of human erythroid burst-forming unit-derived colonies expressed the rAAV-transduced A gamma-globin gene at levels 4-71% that of the endogenous gamma-globin genes. The HbF content of pooled control colonies was 26%, whereas HbF was 40% of the total in pooled colonies derived from rAAV transduced progenitors. These data establish that rAAV containing elements from the locus control region linked to a gamma-globin gene are capable of transferring and expressing that gene in primary human hematopoietic cells resulting in a substantial increase in HbF content. Images PMID:7524085

  11. An erythrocyte-specific protein that binds to the poly(dG) region of the chicken beta-globin gene promoter.

    PubMed

    Lewis, C D; Clark, S P; Felsenfeld, G; Gould, H

    1988-07-01

    The promoter region of the chicken adult beta-globin gene contains a sequence of 16 deoxyguanosine residues located at a nucleosome boundary in tissues where the gene is inactive. In definitive erythrocytes that express the beta-globin gene, the nucleosome is displaced, the G-string and adjacent sequences are occupied by sequence-specific DNA-binding proteins, and a nuclease hypersensitive domain is generated in this region. To gain insight into the role of the G-string in this series of events, we have examined the proteins that bind to it. Using the gel mobility shift assay and a monoclonal antibody that blocks specific binding to the G-string, we have identified a specific protein, BGP1, that is found only in chicken erythroid cells and appears at the same time, or shortly before, the changes in chromatin structure. The antibody interacts strongly with BGP1 and cross-reacts weakly with Sp1. Although both BGP1 and Sp1 require Zn2+ for their DNA-binding activity, these proteins differ in their binding-site specificities, chromatographic properties, and molecular weights. In contrast to Sp1, which is found in a wide variety of cell types, BGP1 is restricted to erythrocytes and is most abundant in definitive erythrocytes. Thus, its presence corresponds to the tissue- and stage-specific occupancy of the G-string in vivo. PMID:3209071

  12. Accuracy of Reverse Dot-Blot PCR in Detection of Different β-Globin Gene Mutations.

    PubMed

    El-Fadaly, N; Abd-Elhameed, A; Abd-Elbar, E; El-Shanshory, M

    2016-06-01

    Prevention programs for β-thalassemia based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation screening methods. The aim of this study was to compare between direct DNA sequencing, and reverse dot-blot PCR in detection of different β-globin gene mutations in Egyptian children with β-thalassemia. Forty children with β-thalassemia were subjected to mutation analysis, performed by both direct DNA sequencing and β-globin Strip Assay MED™ (based on reverse dot-blot PCR). The most frequent mutant alleles detected by reverse dot-blot PCR were; IVSI-110 G>A (31.25 %), IVS I-6 T > C (21.25 %), and IVS I-1 G>A (20 %). Relatively less frequent mutant alleles detected by reverse dot-blot PCR were "IVSII-1 G>A (5 %), IVSII-745 C>G (5 %), IVSII-848 C>A (2.5 %), IVSI-5 G>C (2.5 %), -87 C>G(2.5 %), and cd39 C>T (2.5 %)", While the genotypes of three patients (6 alleles 7.5 %) were not detected by reverse dot-blot PCR. Mutant alleles detected by direct DNA sequencing were the same as reverse dot-blot PCR method except it revealed the genotypes of 3 undetected patients (one patient was homozygous IVSI-110 G>A, and two patients were homozygous IVS I-1 G>A. Sensitivity of the reverse dot-blot PCR was 92.5 % when compared to direct DNA sequencing for detecting β-thalassemia mutations. Our results therefore suggest that, direct DNA sequencing may be preferred over reverse dot-blot PCR in critical diagnostic situations like genetic counseling for prenatal diagnosis. PMID:27065589

  13. The three-dimensional folding of the α-globin gene domain reveals formation of chromatin globules.

    PubMed

    Baù, Davide; Sanyal, Amartya; Lajoie, Bryan R; Capriotti, Emidio; Byron, Meg; Lawrence, Jeanne B; Dekker, Job; Marti-Renom, Marc A

    2011-01-01

    We developed a general approach that combines chromosome conformation capture carbon copy (5C) with the Integrated Modeling Platform (IMP) to generate high-resolution three-dimensional models of chromatin at the megabase scale. We applied this approach to the ENm008 domain on human chromosome 16, containing the α-globin locus, which is expressed in K562 cells and silenced in lymphoblastoid cells (GM12878). The models accurately reproduce the known looping interactions between the α-globin genes and their distal regulatory elements. Further, we find using our approach that the domain folds into a single globular conformation in GM12878 cells, whereas two globules are formed in K562 cells. The central cores of these globules are enriched for transcribed genes, whereas nontranscribed chromatin is more peripheral. We propose that globule formation represents a higher-order folding state related to clustering of transcribed genes around shared transcription machineries, as previously observed by microscopy. PMID:21131981

  14. Copy number polymorphism in the α-globin gene cluster of European rabbit (Oryctolagus cuniculus)

    PubMed Central

    Campos, R; Storz, J F; Ferrand, N

    2012-01-01

    Comparative genomic studies have revealed that mammals typically possess two or more tandemly duplicated copies of the α-globin (HBA) gene. The domestic rabbit represents an exception to this general rule, as this species was found to possess a single HBA gene. Previous electrophoretic surveys of HBA polymorphism in natural populations of the European rabbit (Oryctolagus cuniculus) revealed extensive geographic variation in the frequencies of three main electromorphs. The variation in frequency of two electromorphs is mainly partitioned between two distinct subspecies of European rabbit, and a third is restricted to the hybrid zone between the two rabbit subspecies in Iberia. Here we report the results of a survey of nucleotide polymorphism, which revealed HBA copy number polymorphism in Iberian populations of the European rabbit. By characterizing patterns of HBA polymorphism in populations from the native range of the European rabbit, we were able to identify the specific amino-acid substitutions that distinguish the previously characterized electromorphs. Within the hybrid zone, we observed the existence of a second HBA gene duplicate, named HBA2, that mostly represents a novel sequence haplotype, which occurs in higher frequency within the hybrid zone, and thus appears to have arisen in hybrids of the two distinct subspecies. Although this novel gene is also present in other wild Iberian populations, it is almost absent from French populations, which suggest a recent ancestry, associated with the establishment of the post-Pleistocene contact zone between the two European rabbit subspecies. PMID:22146981

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

    PubMed

    Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S; Krause, Diane S; Seidman, Michael M; Peterson, Kenneth R; Nielsen, Peter E; Kole, Ryszard; Glazer, Peter M

    2008-09-01

    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 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 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 protein-beta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose, sequence, cell-cycle stage, and the presence of a homologous donor DNA molecule. Enhanced recombination, with frequencies up to 0.4%, was observed with use of the lysomotropic agent chloroquine. Finally, we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells, including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells. PMID:18757759

  16. Fetal Hemoglobin in Tunisian Sickle Cell Disease Patient: Relationship with Polymorphic Sequences Cis to the β-Globin Gene.

    PubMed

    Moumni, Imen; Ben Mustapha, Maha; Ben Mansour, Ikbel; Zoraï, Amine; Douzi, Kaïs; Sassi, Sarah; Chaouachi, Dorra; Mellouli, Fethi; Bejaoui, Mohamed; Abbes, Salem

    2016-03-01

    Fetal hemoglobin (HbF) plays a dominant role in ameliorating morbidity and mortality of hemoglobinopathies. We evaluated the effects of polymorphic markers within the β-globin gene cluster to identify the genetic mechanics that influence HbF on Tunisian sickling patients (n = 242). Haplotype analysis was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the framework polymorphism was established by PCR-sequencing, four independent regions of interest were identified: the 5' region of β-LCR-HS2 site, the intervening sequence II (IVSII) region of two fetal (Gγ and Aγ) genes and the 5' region of β-globin gene. The correlation of these various Haplotypes and SNPs with HbF expression and clinical data was studied. Our data showed that among the various polymorphic markers analyzed, only the sequence (AT)xN12(AT)y in LCR HS2 region was significantly associated (p < 0.05) with increased HbF levels, suggesting that the β-globin gene cluster exerts a significant effect on HbF in sickle cell patients. This study can improve understanding of the physiopathology of the disease and aid to increase our ability to predict clinical severity. PMID:26855518

  17. Generation of Mice Deficient in both KLF3/BKLF and KLF8 Reveals a Genetic Interaction and a Role for These Factors in Embryonic Globin Gene Silencing

    PubMed Central

    Funnell, Alister P. W.; Mak, Ka Sin; Twine, Natalie A.; Pelka, Gregory J.; Norton, Laura J.; Radziewic, Tania; Power, Melinda; Wilkins, Marc R.; Bell-Anderson, Kim S.; Fraser, Stuart T.; Perkins, Andrew C.; Tam, Patrick P.; Pearson, Richard C. M.

    2013-01-01

    Krüppel-like factors 3 and 8 (KLF3 and KLF8) are highly related transcriptional regulators that bind to similar sequences of DNA. We have previously shown that in erythroid cells there is a regulatory hierarchy within the KLF family, whereby KLF1 drives the expression of both the Klf3 and Klf8 genes and KLF3 in turn represses Klf8 expression. While the erythroid roles of KLF1 and KLF3 have been explored, the contribution of KLF8 to this regulatory network has been unknown. To investigate this, we have generated a mouse model with disrupted KLF8 expression. While these mice are viable, albeit with a reduced life span, mice lacking both KLF3 and KLF8 die at around embryonic day 14.5 (E14.5), indicative of a genetic interaction between these two factors. In the fetal liver, Klf3 Klf8 double mutant embryos exhibit greater dysregulation of gene expression than either of the two single mutants. In particular, we observe derepression of embryonic, but not adult, globin expression. Taken together, these results suggest that KLF3 and KLF8 have overlapping roles in vivo and participate in the silencing of embryonic globin expression during development. PMID:23716600

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

    PubMed

    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. PMID:23457306

  19. The Full Globin Repertoire of Turtles Provides Insights into Vertebrate Globin Evolution and Functions

    PubMed Central

    Schwarze, Kim; Singh, Abhilasha; Burmester, Thorsten

    2015-01-01

    Globins are small heme proteins that play an important role in oxygen supply, but may also have other functions. Globins offer a unique opportunity to study the functional evolution of genes and proteins. We have characterized the globin repertoire of two different turtle species: the Chinese softshell turtle (Pelodiscus sinensis) and the western painted turtle (Chrysemys picta bellii). In the genomes of both species, we have identified eight distinct globin types: hemoglobin (Hb), myoglobin, neuroglobin, cytoglobin, globin E, globin X, globin Y, and androglobin. Therefore, along with the coelacanth, turtles are so far the only known vertebrates with a full globin repertoire. This fact allows for the first time a comparative analysis of the expression of all eight globins in a single species. Phylogenetic analysis showed an early divergence of neuroglobin and globin X before the radiation of vertebrates. Among the other globins, cytoglobin diverged first, and there is a close relationship between myoglobin and globin E; the position of globin Y is not resolved. The globin E gene was selectively lost in the green anole, and the genes coding for globin X and globin Y were deleted in chicken. Quantitative real-time reverse transcription polymerase chain reaction experiments revealed that myoglobin, neuroglobin, and globin E are highly expressed with tissue-specific patterns, which are in line with their roles in the oxidative metabolism of the striated muscles, the brain, and the retina, respectively. Histochemical analyses showed high levels of globin E in the pigment epithelium of the eye. Globin E probably has a myoglobin-like role in transporting O2 across the pigment epithelium to supply in the metabolically highly active retina. PMID:26078264

  20. A bioinformatics insight to rhizobial globins: gene identification and mapping, polypeptide sequence and phenetic analysis, and protein modeling.

    PubMed Central

    Gesto-Borroto, Reinier; Sánchez-Sánchez, Miriam; Arredondo-Peter, Raúl

    2015-01-01

    Globins (Glbs) are proteins widely distributed in organisms. Three evolutionary families have been identified in Glbs: the M, S and T Glb families. The M Glbs include flavohemoglobins (fHbs) and single-domain Glbs (SDgbs); the S Glbs include globin-coupled sensors (GCSs), protoglobins and sensor single domain globins, and the T Glbs include truncated Glbs (tHbs). Structurally, the M and S Glbs exhibit 3/3-folding whereas the T Glbs exhibit 2/2-folding. Glbs are widespread in bacteria, including several rhizobial genomes. However, only few rhizobial Glbs have been characterized. Hence, we characterized Glbs from 62 rhizobial genomes using bioinformatics methods such as data mining in databases, sequence alignment, phenogram construction and protein modeling. Also, we analyzed soluble extracts from Bradyrhizobium japonicum USDA38 and USDA58 by (reduced + carbon monoxide (CO) minus reduced) differential spectroscopy. Database searching showed that only fhb, sdgb, gcs and thb genes exist in the rhizobia analyzed in this work. Promoter analysis revealed that apparently several rhizobial glb genes are not regulated by a -10 promoter but might be regulated by -35 and Fnr (fumarate-nitrate reduction regulator)-like promoters. Mapping analysis revealed that rhizobial fhbs and thbs are flanked by a variety of genes whereas several rhizobial sdgbs and gcss are flanked by genes coding for proteins involved in the metabolism of nitrates and nitrites and chemotaxis, respectively. Phenetic analysis showed that rhizobial Glbs segregate into the M, S and T Glb families, while structural analysis showed that predicted rhizobial SDgbs and fHbs and GCSs globin domain and tHbs fold into the 3/3- and 2/2-folding, respectively. Spectra from B. japonicum USDA38 and USDA58 soluble extracts exhibited peaks and troughs characteristic of bacterial and vertebrate Glbs thus indicating that putative Glbs are synthesized in B. japonicum USDA38 and USDA58. PMID:26594329

  1. The human ankyrin 1 promoter insulator sustains gene expression in a β-globin lentiviral vector in hematopoietic stem cells

    PubMed Central

    Romero, Zulema; Campo-Fernandez, Beatriz; Wherley, Jennifer; Kaufman, Michael L; Urbinati, Fabrizia; Cooper, Aaron R; Hoban, Megan D; Baldwin, Kismet M; Lumaquin, Dianne; Wang, Xiaoyan; Senadheera, Shantha; Hollis, Roger P; Kohn, Donald B

    2015-01-01

    Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term β-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies. PMID:26029723

  2. Expression of a cellular gene cloned in herpes simplex virus: rabbit beta-globin is regulated as an early viral gene in infected fibroblasts.

    PubMed Central

    Smiley, J R; Smibert, C; Everett, R D

    1987-01-01

    We constructed nondefective herpes simplex virus type 1 recombinants bearing the intact rabbit beta-globin gene inserted into the viral gene for thymidine kinase to study the expression of a cellular gene when it is present in the viral genome during lytic viral infections. The globin promoter was activated to high levels during productive infection of Vero cells, giving rise to properly spliced and processed cytoplasmic globin transcripts. Expression of globin RNA occurred with early kinetics, was not affected by blocking viral DNA replication, and was strongly inhibited by preventing viral immediate-early protein synthesis with cycloheximide. These results support the hypothesis that temporal control of herpes simplex virus early gene expression is accomplished by mechanisms that are not restricted to viral promoters. In addition, these data show that a cellular transcript can be correctly processed and can accumulate to high levels during viral infection; this indicates that the mechanisms of virally induced shutoff of host RNA accumulation and degradation of host mRNAs do not depend on sequence-specific differentiation between host and viral RNAs. These findings also suggest that herpesviruses have considerable potential as high-capacity gene transfer vectors for a variety of applications. Images PMID:3037101

  3. alpha-Amanitin-insensitive transcription of mouse beta major-globin 5'-flanking and structural gene sequences correlates with mRNA expression.

    PubMed Central

    Carlson, D P; Ross, J

    1984-01-01

    A small proportion of the RNAs of mouse reticulocytes consists of beta major-globin mRNA sequences linked to sequences transcribed from the 5'-flanking region of the beta major-globin gene. These upstream RNAs are polyadenylylated and contain 700-800 nucleotides, and their 5' regions are heterogeneous. RNAs with similar or identical 5' regions are transcribed in cell-free extracts from a circular mouse beta major-globin gene template. Synthesis of most of the upstream RNAs in vitro is not inhibited by low levels (1 microgram/ml) of alpha-amanitin, indicating that they are transcribed by an enzyme(s) different from RNA polymerase II. During culture of mouse erythroleukemia cells with dimethyl sulfoxide, globin mRNA and upstream RNAs accumulate with similar kinetics. In contrast, upstream RNAs are not detected in hemin-treated cells. Images PMID:6595660

  4. Role of the GATA-1/FOG-1/NuRD Pathway in the Expression of Human β-Like Globin Genes▿

    PubMed Central

    Miccio, Annarita; Blobel, Gerd A.

    2010-01-01

    The human β-globin genes are expressed in a developmentally controlled fashion. Studies on the molecular mechanisms underlying the stage-specific regulation of globin genes have been fueled by the clinical benefit of elevated fetal γ-globin expression in patients with sickle cell anemia and thalassemia. Recent reports suggested a role of the hematopoietic transcription factor GATA-1, its cofactor FOG-1, and the associated chromatin remodeling complex NuRD in the developmental silencing of HBG1 and HBG2 gene expression. To examine whether FOG-1 via NuRD controls HBG1 and HBG2 silencing in vivo, we created mice in which the FOG-1/NuRD complex is disrupted (A. Miccio et al., EMBO J. 29:442-456, 2010) and crossed these with animals carrying the entire human β-globin gene locus as a transgene. We found that the FOG-1/NuRD interaction is dispensable for the silencing of human HBG1 and HBG2 expression. In addition, mutant animals displayed normal silencing of the endogenous embryonic globin genes. In contrast, a significant reduction of adult-type human and murine globin gene expression was found in adult bone marrows of mutant animals. These results suggest that, unexpectedly, NuRD is required for FOG-1-dependent activation of adult-type globin gene expression but is dispensable for human γ-globin silencing in vivo. PMID:20439494

  5. Incidence of Alpha-Globin Gene Defect in the Lebanese Population: A Pilot Study

    PubMed Central

    Daher, Rose; Badra, Rebecca; el Rafei, Rym; Charafeddine, Lama

    2015-01-01

    Background. It is well established that the Mediterranean and Arab populations are at high risk for thalassemias in general and for alpha-thalassemia in particular. Yet, reports on alpha-thalassemia in Lebanon are still lacking. In this study, we aim at assessing the incidence of alpha-thalassemia in the Lebanese population. Methods. 230 newborns' dried blood cards remaining from routine neonatal screening at the American University of Beirut Medical Center were collected for DNA extraction. Samples were screened for the 21 most common α-globin deletions and point mutations reported worldwide, through multiplex Polymerase Chain Reaction (PCR) and Reverse-Hybridization technique. Results. Upon analyses, the carrier rate of α-thalassemia was found to be 8%. Two mutations detected the −α3,7 single gene deletion found in 75% of cases and the nongene deletion α2 IVS1 [−5nt] in the remaining samples. Conclusion. This study is the first dedicated to investigate α-thalassemia trait incidence in Lebanon. Data obtained demonstrates a high carrier rate in a relatively, highly consanguineous population; it also highlighted the presence of two common mutations. These results may be of an important impact on premarital and newborn screening policies in our country. PMID:25834820

  6. Globin gene-associated restriction-fragment-length polymorphisms in southern African peoples.

    PubMed Central

    Ramsay, M; Jenkins, T

    1987-01-01

    The combination of polymorphic restriction-enzyme sites in the 3' region of the beta-globin gene cluster shows very little variation in southern-African Bantu-speaking black and Kalahari !Kung San populations. The sites of the 5' region, on the other hand, show marked variation, and two common haplotypes are present--the "Negro" type (- - - - +) and the "San" type (- + - - +)--in frequencies of .404 and .106, respectively, in the Bantu-speakers and .262 and .405, respectively, in the San. Twenty of 23 beta s-associated haplotypes in southern-African Bantu-speaking black subjects were the same as that found commonly in the Central African Republic (CAR)--i.e., the "Bantu" type--a finding providing the first convincing biological evidence for the common ancestry of geographically widely separated speakers of languages belonging to the Bantu family. The (-alpha) haplotype has a frequency of .21 in the Venda, .07 in both the Sotho-Tswana and the Nguni, and .06 among the !Kung San. These data are interpreted in the light of Plasmodium falciparum malaria selection and population movements in the African subcontinent. PMID:2891298

  7. β-globin gene transfer to human bone marrow for sickle cell disease

    PubMed Central

    Romero, Zulema; Urbinati, Fabrizia; Geiger, Sabine; Cooper, Aaron R.; Wherley, Jennifer; Kaufman, Michael L.; Hollis, Roger P.; Ruiz de Assin, Rafael; Senadheera, Shantha; Sahagian, Arineh; Jin, Xiangyang; Gellis, Alyse; Wang, Xiaoyan; Gjertson, David; DeOliveira, Satiro; Kempert, Pamela; Shupien, Sally; Abdel-Azim, Hisham; Walters, Mark C.; Meiselman, Herbert J.; Wenby, Rosalinda B.; Gruber, Theresa; Marder, Victor; Coates, Thomas D.; Kohn, Donald B.

    2013-01-01

    Autologous hematopoietic stem cell gene therapy is an approach to treating sickle cell disease (SCD) patients that may result in lower morbidity than allogeneic transplantation. We examined the potential of a lentiviral vector (LV) (CCL-βAS3-FB) encoding a human hemoglobin (HBB) gene engineered to impede sickle hemoglobin polymerization (HBBAS3) to transduce human BM CD34+ cells from SCD donors and prevent sickling of red blood cells produced by in vitro differentiation. The CCL-βAS3-FB LV transduced BM CD34+ cells from either healthy or SCD donors at similar levels, based on quantitative PCR and colony-forming unit progenitor analysis. Consistent expression of HBBAS3 mRNA and HbAS3 protein compromised a fourth of the total β-globin–like transcripts and hemoglobin (Hb) tetramers. Upon deoxygenation, a lower percentage of HBBAS3-transduced red blood cells exhibited sickling compared with mock-transduced cells from sickle donors. Transduced BM CD34+ cells were transplanted into immunodeficient mice, and the human cells recovered after 2–3 months were cultured for erythroid differentiation, which showed levels of HBBAS3 mRNA similar to those seen in the CD34+ cells that were directly differentiated in vitro. These results demonstrate that the CCL-βAS3-FB LV is capable of efficient transfer and consistent expression of an effective anti-sickling β-globin gene in human SCD BM CD34+ progenitor cells, improving physiologic parameters of the resulting red blood cells. PMID:23863630

  8. Developmental- and differentiation-specific patterns of human gamma- and beta-globin promoter DNA methylation.

    PubMed

    Mabaera, Rodwell; Richardson, Christine A; Johnson, Kristin; Hsu, Mei; Fiering, Steven; Lowrey, Christopher H

    2007-08-15

    The mechanisms underlying the human fetal-to-adult beta-globin gene switch remain to be determined. While there is substantial experimental evidence to suggest that promoter DNA methylation is involved in this process, most data come from studies in nonhuman systems. We have evaluated human gamma- and beta-globin promoter methylation in primary human fetal liver (FL) and adult bone marrow (ABM) erythroid cells. Our results show that, in general, promoter methylation and gene expression are inversely related. However, CpGs at -162 of the gamma promoter and -126 of the beta promoter are hypomethylated in ABM and FL, respectively. We also studied gamma-globin promoter methylation during in vitro differentiation of erythroid cells. The gamma promoters are initially hypermethylated in CD34(+) cells. The upstream gamma promoter CpGs become hypomethylated during the preerythroid phase of differentiation and are then remethylated later, during erythropoiesis. The period of promoter hypomethylation correlates with transient gamma-globin gene expression and may explain the previously observed fetal hemoglobin production that occurs during early adult erythropoiesis. These results provide the first comprehensive survey of developmental changes in human gamma- and beta-globin promoter methylation and support the hypothesis that promoter methylation plays a role in human beta-globin locus gene switching. PMID:17456718

  9. Phylogenetic comparisons suggest that distance from the locus control region guides developmental expression of primate beta-type globin genes.

    PubMed

    Johnson, Robert M; Prychitko, Tom; Gumucio, Deborah; Wildman, Derek E; Uddin, Monica; Goodman, Morris

    2006-02-28

    Phylogenetic inferences drawn from comparative data on mammalian beta-globin gene clusters indicate that the ancestral primate cluster contained a locus control region (LCR) and five paralogously related beta-type globin loci (5'-LCR-epsilon-gamma-psieta-delta-beta-3'), with epsilon and gamma expressed solely during embryonic life. A gamma locus tandem duplication (5'-gamma(1)-gamma(2)-3') triggered gamma's evolution toward fetal expression but by a different trajectory in platyrrhines (New World monkeys) than in catarrhines (Old World monkeys and apes, including humans). In platyrrhine (e.g., Cebus) fetuses, gamma(1) at the ancestral distance from epsilon is down-regulated, whereas gamma(2) at increased distance is up-regulated. Catarrhine gamma(1) and gamma(2) acquired longer distances from epsilon (14 and 19 kb, respectively), and both are up-regulated throughout fetal life with gamma(1)'s expression predominating over gamma(2)'s. On enlarging the platyrrhine expression data, we find Aotus gamma is embryonic, Alouatta gamma is inactive at term, and in Callithrix, gamma(1) is down-regulated fetally, whereas gamma(2) is up-regulated. Of eight mammalian taxa now represented per taxon by embryonic, fetal, and postnatal beta-type globin gene expression data, four taxa are primates, and data for three of these primates are from this laboratory. Our results support a model in which a short distance (<10 kb) between epsilon and the adjacent gamma is a plesiomorphic character that allows the LCR to drive embryonic expression of both genes, whereas a longer distance (>10 kb) impedes embryonic activation of the downstream gene. PMID:16488971

  10. RNA sequencing for increasing gene discovery and and coverage using globin RNA reduced porcine blood samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Transcriptome analysis in porcine whole blood will provide major insights to decipher genetic mechanisms for host responses to viral infection. The abundance of porcine globin transcripts, however, impedes the ability to detect less abundant transcripts. The objective of our study was to...

  11. Inhibition of G9a methyltransferase stimulates fetal hemoglobin production by facilitating LCR/γ-globin looping.

    PubMed

    Krivega, Ivan; Byrnes, Colleen; de Vasconcellos, Jaira F; Lee, Y Terry; Kaushal, Megha; Dean, Ann; Miller, Jeffery L

    2015-07-30

    Induction of fetal hemoglobin (HbF) production in adult erythrocytes can reduce the severity of sickle cell disease and β-thalassemia. Transcription of β-globin genes is regulated by the distant locus control region (LCR), which is brought into direct gene contact by the LDB1/GATA-1/TAL1/LMO2-containing complex. Inhibition of G9a H3K9 methyltransferase by the chemical compound UNC0638 activates fetal and represses adult β-globin gene expression in adult human hematopoietic precursor cells, but the underlying mechanisms are unclear. Here we studied UNC0638 effects on β-globin gene expression using ex vivo differentiation of CD34(+) erythroid progenitor cells from peripheral blood of healthy adult donors. UNC0638 inhibition of G9a caused dosed accumulation of HbF up to 30% of total hemoglobin in differentiated cells. Elevation of HbF was associated with significant activation of fetal γ-globin and repression of adult β-globin transcription. Changes in gene expression were associated with widespread loss of H3K9me2 in the locus and gain of LDB1 complex occupancy at the γ-globin promoters as well as de novo formation of LCR/γ-globin contacts. Our findings demonstrate that G9a establishes epigenetic conditions preventing activation of γ-globin genes during differentiation of adult erythroid progenitor cells. In this view, manipulation of G9a represents a promising epigenetic approach for treatment of β-hemoglobinopathies. PMID:25979948

  12. siDNMT1 Increases γ-globin Expression in Chemical-Inducer-of-Dimerization (CID)-Dependent Mouse βYAC Bone Marrow Cells and in Baboon Erythroid Progenitor Cell Cultures

    PubMed Central

    Banzon, Virryan; Ibanez, Vinzon; Vaitkus, Kestis; Ruiz, Maria Armila; Peterson, Kenneth; DeSimone, Joseph; Lavelle, Donald

    2014-01-01

    1) Objective These studies were performed to test the hypothesis that DNMT1 is required for maintenance of DNA methylation and repression of the γ-globin gene in adult stage erythroid cells. 2) Methods DNMT1 levels were reduced by nucleofection of siRNA targeting DNMT1 in chemical-inducer-of-dimerization (CID)-dependent multipotential mouse bone marrow (BM) cells containing the human β-globin gene locus in the context of a yeast artificial chromosome (βYAC) and in primary cultures of erythroid progenitor cells derived from CD34+ baboon BM cells. The effect of reduced DNMT1 levels on globin gene expression was measured by real time PCR and the effect on globin chain synthesis in primary erythroid progenitor cell cultures was determined by biosynthetic radiolabelling of globin chains followed by HPLC analysis. The effect on DNA methylation was determined by bisulfite sequence analysis. 3) Results Reduced DNMT1 levels in cells treated with siDNMT1 were associated with increased expression of γ-globin mRNA, an increased γ/γ+β chain ratio in cultured erythroid progenitors, and decreased DNA methylation of the γ-globin promoter. Similar effects were observed in cells treated with decitabine, a pharmacological inhibitor of DNA methyltransferase inhibitor. 4) Conclusion DNMT1 is required to maintain DNA methylation of the γ-globin gene promoters and repress γ-globin gene expression in adult-stage erythroid cells. PMID:20974210

  13. KLF1 stabilizes GATA-1 and TAL1 occupancy in the human β-globin locus.

    PubMed

    Kang, Yujin; Kim, Yea Woon; Yun, Jangmi; Shin, Jongo; Kim, AeRi

    2015-03-01

    KLF1 is an erythroid specific transcription factor that binds to regulatory regions of erythroid genes. Binding sites of KLF1 are often found near binding sites of GATA-1 and TAL1. In the β-globin locus, KLF1 is required for forming active chromatin structure, although its role is unclear. To explore the role of KLF1 in transcribing the human γ-globin genes, we stably reduced the expression of KLF1 in erythroid K562 cells, compromising its association in the β-globin locus. The γ-globin transcription was reduced with disappearance of active chromatin structure of the locus in the KLF1 knockdown cells. Interestingly, GATA-1 and TAL1 binding was reduced in the β-globin locus, even though their expressions were not affected by KLF1 knockdown. The KLF1-dependent GATA-1 and TAL1 binding was observed in the adult locus transcribing the β-globin gene and in several erythroid genes, where GATA-1 occupancy is independent from TAL1. These results indicate that KLF1 plays a role in facilitating and/or stabilizing GATA-1 and TAL1 occupancy in the erythroid genes, contributing to the generation of active chromatin structure such as histone acetylation and chromatin looping. PMID:25528728

  14. Dynamics of α-globin locus chromatin structure and gene expression during erythroid differentiation of human CD34+ cells in culture

    PubMed Central

    Mahajan, Milind C; Karmakar, Subhradip; Krause, Diane; Weissman, Sherman M

    2009-01-01

    Objective The aim of the present study has been to establish serum free culture conditions for the ex vivo expansion and differentiation of human CD34+ cells into erythroid lineage and to study the chromatin structure, gene expression and transcription factor recruitment at the α–globin locus in the developing erythron. Methods A basal IMDM cell culture medium with 1% bovine serum albumin as a serum replacement and a combination of cytokines and growth factors was used for the expansion and differentiation of the CD34+ cells. Expression patterns of the alpha and beta like genes at various stages of erythropoiesis was studied by Reverse transcriptase (RT)-qPCR analysis, profile of key erythroid transcription factors was investigated by western blotting, and the chromatin structure and transcription factor recruitment at the alpha globin locus was investigated by ChIP-qPCR analysis. Results Human CD34+ cells in the serum free medium undergo near synchronous erythroid differentiation to yield large amount of cells at different differentiation stages. We observe distinct patterns of the histone modifications and transcription factor binding at the α-globin locus during erythroid differentiation of CD34+ cells. NF-E2 was present at upstream activator sites even before addition of erythropoietin (Epo), while bound GATA-1 was only detectable after Epo treatment. After seven days of erythropoietin treatment, H3K4Me2 modification uniformly increases throughout the α–globin locus. Acetylation at H3K9 and binding of Pol II, NF-E2 and GATA-1 were restricted to certain HS sites of the enhancer and theta gene, and were conspicuously low at the α-like globin promoters. Rearrangement of the insulator binding factor CTCF took place at and around the α-globin locus as CD34+ cells differentiated into erythroid pathway. Conclusion Our results indicate that remodeling of the upstream elements may be the primary event in activation of α–globin gene expression. Activation of

  15. A novel δ-globin gene mutation (HBD: c.323G>A) masking the diagnosis of β-thalassemia: a first report from India.

    PubMed

    Jain, Sachin; Edison, Eunice S; Mathews, Vikram; Shaji, R V

    2012-05-01

    An elevated HbA(2) (α2δ2) level (>3.5%) is a well-established diagnostic test for heterozygous β-thalassemia. Mutations in the δ-globin gene can cause decreased expression of HbA(2), resulting in heterozygous β-thalassemia with normal levels of HbA(2). In this report, we describe a novel missense mutation in δ-globin (HBD: c.323G>A, Gly > Asp) in an Indian family with heterozygous β-thalassemia with normal HbA(2) levels. PMID:22477537

  16. A New δ-Globin Gene Variant: Hb A2-Fengshun [δ121(GH4)Glu→Lys (HBD: c.364G > A)].

    PubMed

    Yan, Jin-Mei; Zhou, Jian-Ying; Xie, Xing-Mei; Li, Jian; Li, Dong-Zhi

    2016-06-01

    An elevated Hb A2 (α2δ2 level) is a diagnostic marker for heterozygous β-thalassemia (β-thal). Mutations in the δ-globin gene can cause decreased expression of Hb A2, compromising screening for heterozygous β-thal. In this report, we describe a novel missense mutation of the δ-globin [Hb A2-Fengshun or δ121(GH4)Glu→Lys, HBD: c.364G > A] in a Chinese individual who had coinherited a heterozygous β-thal with a normal Hb A2 level. PMID:27117573

  17. A novel deletion/insertion caused by a replication error in the β-globin gene locus control region.

    PubMed

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Meley, Roland; Pondarré, Corinne; Francina, Alain

    2011-01-01

    Deletions in the β-globin locus control region (β-LCR) lead to (εγδβ)(0)-thalassemia [(εγδβ)(0)-thal]. In patients suffering from these rare deletions, a normal hemoglobin (Hb), phenotype is found, contrasting with a hematological thalassemic phenotype. Multiplex-ligation probe amplification (MLPA) is an efficient tool to detect β-LCR deletions combined with long-range polymerase chain reaction (PCR) and DNA sequencing to pinpoint deletion breakpoints. We present here a novel 11,155 bp β-LCR deletion found in a French Caucasian patient which removes DNase I hypersensitive site 2 (HS2) to HS4 of the β-LCR. Interestingly, a 197 bp insertion of two inverted sequences issued from the HS2-HS3 inter-region is present and suggests a complex rearrangement during replication. Carriers of this type of thalassemia can be misdiagnosed as an α-thal trait. Consequently, a complete α- and β-globin gene cluster analysis is required to prevent a potentially damaging misdiagnosis in genetic counselling. PMID:21797698

  18. A new Frameshift mutation on the α2-globin gene causing α⁺-thalassemia: codon 43 (TTC>-TC or TTC>T-C).

    PubMed

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Barro, Claire; Francina, Alain

    2012-01-01

    We report a new mutation on the α2-globin gene causing α(+)-thalassemia (α(+)-thal) with a deletion of a single nucleotide (T) at amino acid residue 43 [HBA2:c.130delT or HBA2:c.131delT]. This frameshift deletion gives rise to a premature termination codon at codon 47. PMID:22738776

  19. Heterozygosis deficit of polymorphic markers linked to the β-globin gene cluster region in the Iranian population

    PubMed Central

    Moradi, Tahereh; Vallian, Reihaneh; Fazeli, Zahra; Haghighatnia, Asieh; Vallian, Sadeq

    2015-01-01

    Objective(s): Iran is considered as one of the high-prevalence areas for β-thalassemia with a rate of about 10% carrier frequency. Molecular diagnosis of the disease is performed both by direct sequencing and indirectly by the use of polymorphic markers present in the beta globin gene cluster. However, to date there is no reliable information on the application of the markers in the Iranian population. Here we report the results of an extended molecular analysis of five RFLP markers, XmnI, HindIIIA, HindIIIG, RsaI and HinfI, located within the β-globin gene cluster region in four subpopulations of Iran. Materials and Methods: A total of 552 blood samples taken from the Iranian subpopulations including Isfahan, Chaharmahal-O-Bakhtiari, Khuzestan and Hormozgan were genotyped using PCR-RFLP and sequencing. The allele frequency, the expected and observed heterozygosity, and Shannon’s information index (I) of these markers were calculated. Results: Distribution of the allele frequencies for XmnI, HindIIIA, HindIIIG, RsaI and HinfI polymorphic markers did not differ significantly among the subpopulations examined. Overall observed heterozygosity ranged from 0.1706 for HindIIIA to 0.4484 for RsaI. The Shannon index was <1 for all the polymorphic markers in the populations studied. The data indicated that heterozygosity of these markers was low in the Iranian population. Conclusion: The results suggested that genotyping of these markers is not informative enough once used as single markers for prenatal diagnosis and carrier detection of β-thalassemia in the Iranian population. However, haplotyping of these markers may provide more useful data in linkage analysis and prenatal diagnosis as well as carrier detections for β-thalassemia in Iranians. PMID:26229579

  20. Krüppel-Like Transcription Factor KLF1 Is Required for Optimal γ- and β-Globin Expression in Human Fetal Erythroblasts

    PubMed Central

    Vinjamur, Divya S.; Alhashem, Yousef N.; Mohamad, Safa F.; Amin, Parth; Williams, David C.; Lloyd, Joyce A.

    2016-01-01

    In human adult erythroid cells, lower than normal levels of Krüppel-like transcription factor 1 (KLF1) are generally associated with decreased adult β- and increased fetal γ-globin gene expression. KLF1 also regulates BCL11A, a known repressor of adult γ-globin expression. In seeming contrast to the findings in adult cells, lower amounts of KLF1 correlate with both reduced embryonic and reduced fetal β-like globin mRNA in mouse embryonic erythroid cells. The role of KLF1 in primary human fetal erythroid cells, which express both γ- and β-globin mRNA, is less well understood. Therefore, we studied the role of KLF1 in ex vivo differentiated CD34+ umbilical cord blood cells (UCB erythroblasts), representing the fetal milieu. In UCB erythroblasts, KLF1 binds to the β-globin locus control region (LCR), and the β-globin promoter. There is very little KLF1 binding detectable at the γ-globin promoter. Correspondingly, when cultured fetal UCB erythroblasts are subjected to lentiviral KLF1 knockdown, the active histone mark H3K4me3 and RNA pol II recruitment are diminished at the β- but not the γ-globin gene. The amount of KLF1 expression strongly positively correlates with β-globin mRNA and weakly positively correlates with BCL11A mRNA. With modest KLF1 knockdown, mimicking haploinsufficiency, γ-globin mRNA is increased in UCB erythroblasts, as is common in adult cells. However, a threshold level of KLF1 is evidently required, or there is no absolute increase in γ-globin mRNA in UCB erythroblasts. Therefore, the role of KLF1 in γ-globin regulation in fetal erythroblasts is complex, with both positive and negative facets. Furthermore, in UCB erythroblasts, diminished BCL11A is not sufficient to induce γ-globin in the absence of KLF1. These findings have implications for the manipulation of BCL11A and/or KLF1 to induce γ-globin for therapy of the β-hemoglobinopathies. PMID:26840243

  1. Krüppel-Like Transcription Factor KLF1 Is Required for Optimal γ- and β-Globin Expression in Human Fetal Erythroblasts.

    PubMed

    Vinjamur, Divya S; Alhashem, Yousef N; Mohamad, Safa F; Amin, Parth; Williams, David C; Lloyd, Joyce A

    2016-01-01

    In human adult erythroid cells, lower than normal levels of Krüppel-like transcription factor 1 (KLF1) are generally associated with decreased adult β- and increased fetal γ-globin gene expression. KLF1 also regulates BCL11A, a known repressor of adult γ-globin expression. In seeming contrast to the findings in adult cells, lower amounts of KLF1 correlate with both reduced embryonic and reduced fetal β-like globin mRNA in mouse embryonic erythroid cells. The role of KLF1 in primary human fetal erythroid cells, which express both γ- and β-globin mRNA, is less well understood. Therefore, we studied the role of KLF1 in ex vivo differentiated CD34+ umbilical cord blood cells (UCB erythroblasts), representing the fetal milieu. In UCB erythroblasts, KLF1 binds to the β-globin locus control region (LCR), and the β-globin promoter. There is very little KLF1 binding detectable at the γ-globin promoter. Correspondingly, when cultured fetal UCB erythroblasts are subjected to lentiviral KLF1 knockdown, the active histone mark H3K4me3 and RNA pol II recruitment are diminished at the β- but not the γ-globin gene. The amount of KLF1 expression strongly positively correlates with β-globin mRNA and weakly positively correlates with BCL11A mRNA. With modest KLF1 knockdown, mimicking haploinsufficiency, γ-globin mRNA is increased in UCB erythroblasts, as is common in adult cells. However, a threshold level of KLF1 is evidently required, or there is no absolute increase in γ-globin mRNA in UCB erythroblasts. Therefore, the role of KLF1 in γ-globin regulation in fetal erythroblasts is complex, with both positive and negative facets. Furthermore, in UCB erythroblasts, diminished BCL11A is not sufficient to induce γ-globin in the absence of KLF1. These findings have implications for the manipulation of BCL11A and/or KLF1 to induce γ-globin for therapy of the β-hemoglobinopathies. PMID:26840243

  2. Characterization of a globin-coupled oxygen sensor with a gene-regulating function.

    PubMed

    Thijs, Liesbet; Vinck, Evi; Bolli, Alessandro; Trandafir, Florin; Wan, Xuehua; Hoogewijs, David; Coletta, Massimiliano; Fago, Angela; Weber, Roy E; Van Doorslaer, Sabine; Ascenzi, Paolo; Alam, Maqsudul; Moens, Luc; Dewilde, Sylvia

    2007-12-28

    Globin-coupled sensors (GCSs) are multiple-domain transducers, consisting of a regulatory globin-like heme-binding domain and a linked transducer domain(s). GCSs have been described in both Archaea and bacteria. They are generally assumed to bind O(2) (and perhaps other gaseous ligands) and to transmit a conformational change signal through the transducer domain in response to fluctuating O(2) levels. In this study, the heme-binding domain, AvGReg178, and the full protein, AvGReg of the Azotobacter vinelandii GCS, were cloned, expressed, and purified. After purification, the heme iron of AvGReg178 was found to bind O(2). This form was stable over many hours. In contrast, the predominant presence of a bis-histidine coordinate heme in ferric AvGReg was revealed. Differences in the heme pocket structure were also observed for the deoxygenated ferrous state of these proteins. The spectra showed that the deoxygenated ferrous derivatives of AvGReg178 and AvGReg are characterized by a penta-coordinate and hexa-coordinate heme iron, respectively. O(2) binding isotherms indicate that AvGReg178 and AvGReg show a high affinity for O(2) with P(50) values at 20 degrees C of 0.04 and 0.15 torr, respectively. Kinetics of CO binding indicate that AvGReg178 carbonylation conforms to a monophasic process, comparable with that of myoglobin, whereas AvGReg carbonylation conforms to a three-phasic reaction, as observed for several proteins with bis-histidine heme iron coordination. Besides sensing ligands, in vitro data suggest that AvGReg(178) may have a role in O(2)-mediated NO-detoxification, yielding metAvGReg(178) and nitrate. PMID:17925395

  3. Regulation of Gγ-Globin Gene by ATF2 and Its Associated Proteins through the cAMP-Response Element

    PubMed Central

    Dhar, Ruby; Mahajan, Milind; Choudhury, Alina; Weissman, Sherman; Pace, Betty S.

    2013-01-01

    The upstream Gγ-globin cAMP-response element (G-CRE) plays an important role in regulating Gγ-globin expression through binding of ATF2 and its DNA-binding partners defined in this study. ATF2 knockdown resulted in a significant reduction of γ-globin expression accompanied by decreased ATF2 binding to the G-CRE. By contrast, stable ATF2 expression in K562 cells increased γ-globin transcription which was reduced by ATF2 knockdown. Moreover, a similar effect of ATF2 on γ-globin expression was observed in primary erythroid progenitors. To understand the role of ATF2 in γ-globin expression, chromatographically purified G-CRE/ATF2-interacting proteins were subjected to mass spectrometry analysis; major binding partners included CREB1, cJun, Brg1, and histone deacetylases among others. Immunoprecipitation assays demonstrated interaction of these proteins with ATF2 and in vivo GCRE binding in CD34+ cells undergoing erythroid differentiation which was correlated with γ-globin expression during development. These results suggest synergism between developmental stage-specific recruitments of the ATF2 protein complex and expression of γ-globin during erythropoiesis. Microarray studies in K562 cells support ATF2 plays diverse roles in hematopoiesis and chromatin remodeling. PMID:24223142

  4. Two novel copy number variations involving the α-globin gene cluster on chromosome 16 cause thalassemia in two Chinese families.

    PubMed

    Hu, Lingling; Shang, Xuan; Yi, Sheng; Cai, Ren; Li, Zhetao; Liu, Cuixian; Liang, Yidan; Cai, Decheng; Zhang, Feng; Xu, Xiangmin

    2016-06-01

    Copy number variations (CNVs) can cause many genetic disorders and the structure analysis of unknown CNVs is important for clinical diagnosis. The human α-globin gene cluster lies close to the telomere of the short arm on chromosome 16. Copy number variations of this region produce excessive or insufficient α-globin chains which imbalances the β-globin chains, resulting in thalassemia. However, these CNVs usually cannot be precisely defined by traditional methods. Here, we designed a technique strategy and applied it to identify two CNVs involving the α-globin gene cluster causing thalassemia in two Chinese families. A novel 282 kb duplication (αααα(282)) was identified in family A and a novel 235 kb deletion (--(235)) in family B. Proband A is a coinheritance of β(CD41-42) and αααα(282) and showed severe β-thalassemia intermedia phenotype. Proband B is a compound heterozygote of --(235)/α(CS)α genotype and was diagnosed with hemoglobin H disease. The clinical phenotypic features of the CNVs carriers were described, together with a complete picture of molecular structure of these rearrangements. Two CNVs are novel rearrangements in α-globin clusters and the αααα(282) is the first to identify the exact insert position of a duplication region from the telomere on chromosome 16. In a conclusion, successful identification and characterization of these two novel CNVs not only demonstrates the precision and effectiveness of our strategy in analyzing the structure of unknown CNVs, but also extended the spectrum of thalassemia and provide new examples for studying genomic recombination. PMID:27000657

  5. Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations.

    PubMed

    Chassanidis, Christos; Boutou, Effrossyni; Voskaridou, Ersi; Balassopoulou, Angeliki

    2016-01-01

    Beta-thalassaemia is one of the most common autosomal recessive disorders worldwide. The disease's high incidence, which is observed in the broader Mediterranean area has led to the establishment of molecular diagnostics' assays to prevent affected births. Therefore, the development of a reliable, cost-effective and rapid scanning method for β globin gene point mutations, easily adapted to a routine laboratory, is absolutely essential. Here, we describe, for the first time, the development of a High-Resolution Melting Analysis (HRMA) approach, suitable for scanning the particularly heterogeneous beta globin gene mutations present in the Greek population, and thus adaptable to the Mediterranean and other areas where these mutations have been identified. Within this context, β globin gene regions containing mutations frequently identified in the Greek population were divided in ten overlapping amplicons. Our reactions' setup allowed for the simultaneous amplification of multiple primer sets and partial multiplexing, thereby resulting in significant reduction of the experimental time. DNA samples from β-thalassaemia patients/carriers with defined genotypes were tested. Distinct genotypes displayed distinguishable melting curves, enabling accurate detection of mutations. The described HRMA can be adapted to a high-throughput level. It represents a rapid, simple, cost-effective, reliable, highly feasible and sensitive method for β-thalassaemia gene scanning. PMID:27351925

  6. Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations

    PubMed Central

    Chassanidis, Christos; Boutou, Effrossyni; Voskaridou, Ersi; Balassopoulou, Angeliki

    2016-01-01

    Beta-thalassaemia is one of the most common autosomal recessive disorders worldwide. The disease’s high incidence, which is observed in the broader Mediterranean area has led to the establishment of molecular diagnostics’ assays to prevent affected births. Therefore, the development of a reliable, cost-effective and rapid scanning method for β globin gene point mutations, easily adapted to a routine laboratory, is absolutely essential. Here, we describe, for the first time, the development of a High-Resolution Melting Analysis (HRMA) approach, suitable for scanning the particularly heterogeneous beta globin gene mutations present in the Greek population, and thus adaptable to the Mediterranean and other areas where these mutations have been identified. Within this context, β globin gene regions containing mutations frequently identified in the Greek population were divided in ten overlapping amplicons. Our reactions’ setup allowed for the simultaneous amplification of multiple primer sets and partial multiplexing, thereby resulting in significant reduction of the experimental time. DNA samples from β-thalassaemia patients/carriers with defined genotypes were tested. Distinct genotypes displayed distinguishable melting curves, enabling accurate detection of mutations. The described HRMA can be adapted to a high-throughput level. It represents a rapid, simple, cost-effective, reliable, highly feasible and sensitive method for β-thalassaemia gene scanning. PMID:27351925

  7. Induction of gamma-globin gene transcription by hydroxycarbamide in primary erythroid cell cultures from Lepore patients.

    PubMed

    Calzolari, Roberta; Pecoraro, Alice; Borruso, Vito; Troia, Antonio; Acuto, Santina; Maggio, Aurelio; Di Marzo, Rosalba

    2008-05-01

    Increased expression of fetal haemoglobin (HbF) may ameliorate the clinical course of beta-thalassemia and sickle cell disease. Some pharmacological agents, such as hydroxycarbamide (HC), can increase fetal haemoglobin synthesis during adult life. Cellular selection and/or molecular mechanisms have been proposed to account for this increase. To explore the mechanism of action of HC we focused on homozygous Hb-Lepore patients that presented with high fetal haemoglobin levels and were good responders to HC treatment "in vivo". We performed primary erythroid cultures from peripheral blood of four homozygous Lepore patients. The increase in HBG (gamma-globin) transcription levels and HbF content in these cultures, after HC treatment, were detected by quantitative real time polymerase chain reaction analysis and flow cytometric analysis. Primary transcript "in-situ" hybridization analysis showed a 2-fold increase in the number of cells expressing both HBG alleles in HC-treated erythroid cultures. These studies, demonstrating the larger number of biallelic HBG expressing cells, suggest that HC is able to stimulate the activation of HBG transcription. These observations provide evidences that the molecular mechanism of action is involved in the increase of fetal haemoglobin production by HC. PMID:18422777

  8. The phylogenetic history of New World monkey beta globin reveals a platyrrhine beta to delta gene conversion in the atelid ancestry.

    PubMed

    Prychitko, Tom; Johnson, Robert M; Wildman, Derek E; Gumucio, Deborah; Goodman, Morris

    2005-04-01

    Orthologues of the beta globin gene locus from 10 New World monkey species were sequenced and aligned against available beta and delta globin sequences from rabbit and other primates. Where needed, additional primate sequencing was performed. Phylogenetic analysis identified a beta to delta conversion in the stem of the Anthropoidea, stretching from the 3' part of the proximal promotor to the 5' start of intron 2, consistent with earlier findings. No further conversion appeared to have occurred in the descent of the catarrhines. Within the New World monkey lineage that led to spider monkey and other atelids, another shorter gene conversion was found, spanning adjacent parts of exon 1 and intron 1. The analysis also confirmed that galago beta had replaced galago delta, that an earlier loriform-specific gene conversion extended over intron 2, and that gene conversion throughout the main gene conversion region occurred in the tarsiiform lineage. Platyrrhine phylogenetic relationships were investigated with beta sequences restricted to those that were not involved in gene conversions. This phylogeny generally agreed with results from other nuclear genes. The one exception was that the beta sequences did not place the callitrichine clade within the Cebidae but weakly joined the callitrichine and atelid clades. PMID:15737593

  9. Chromatin looping as a target for altering erythroid gene expression.

    PubMed

    Krivega, Ivan; Dean, Ann

    2016-03-01

    The β-hemoglobinopathies are the most common monogenic disorders in humans, with symptoms arising after birth when the fetal γ-globin genes are silenced and the adult β-globin gene is activated. There is a growing appreciation that genome organization and the folding of chromosomes are key determinants of gene transcription. Underlying this function is the activity of transcriptional enhancers that increase the transcription of target genes over long linear distances. To accomplish this, enhancers engage in close physical contact with target promoters through chromosome folding or looping that is orchestrated by protein complexes that bind to both sites and stabilize their interaction. We find that enhancer activity can be redirected with concomitant changes in gene transcription. Both targeting the β-globin locus control region (LCR) to the γ-globin gene in adult erythroid cells by tethering and epigenetic unmasking of a silenced γ-globin gene lead to increased frequency of LCR/γ-globin contacts and reduced LCR/β-globin contacts. The outcome of these manipulations is robust, pancellular γ-globin transcription activation with a concomitant reduction in β-globin transcription. These examples show that chromosome looping may be considered a therapeutic target for gene activation in β-thalassemia and sickle cell disease. PMID:26918894

  10. Thalidomide is more efficient than sodium butyrate in enhancing GATA-1 and EKLF gene expression in erythroid progenitors derived from HSCs with β-globin gene mutation

    PubMed Central

    Jalali Far, Mohammad Ali; Dehghani Fard, Ali; Hajizamani, Saiedeh; Mossahebi-Mohammadi, Majid; Yaghooti, Hamid; Saki, Najmaldin

    2016-01-01

    Background: Efficient induction of fetal hemoglobin (HbF) is considered as an effective therapeutic approach in beta thalassemia. HbF inducer agents can induce the expression of γ-globin gene and produce high levels of HbF via different epigenetic and molecular mechanisms. Thalidomide and sodium butyrate are known as HbF inducer drugs. Material and methods: CD133+ stem cells were isolated from umbilical cord blood of a newborn with minor β-thalassemia in order to evaluate the effects of these two drugs on the in vitro expression of GATA-1 and EKLF genes as erythroid transcription factors. CD133+ stem cells were expanded and differentiated into erythroid lineage and then treated with thalidomide and sodium butyrate and finally analyzed by quantitative real-time PCR. Statistical analysis was performed using student’s t-test by SPSS software. Results: Thalidomide and sodium butyrate increased GATA-1 and EKLF gene expression, compared to the non-treated control (P<0.05). Conclusion: Thalidomide was more efficient than sodium butyrate in augmenting expression of GATA-1 and EKLF genes. It seems that GATA-1 and EKLF have crucial roles in the efficient induction of HbF by thalidomide. PMID:27047649

  11. Globin synthesis in hybrid cells constructed by transplantation of dormant avian erythrocyte nuclei into enucleated fibroblasts.

    PubMed Central

    Bruno, J; Reich, N; Lucas, J J

    1981-01-01

    The polypeptides synthesized by mature embryonic erythrocytes prepared from the peripheral blood of 14- to 15-day-old chicken embryos were analyzed by two-dimensional gel electrophoresis. Fewer than 200 species of polypeptides were detected; the major polypeptides made at this time were identified as the alpha A-, alpha D-, and beta-globin chains. The dormant erythrocyte nuclei were next reactivated to transcriptional competence by transplantation into enucleated mouse or chicken embryo fibroblasts, with frequencies of cytoplast renucleation of about 50 and 90%, respectively. Since large numbers of hybrid cells could be constructed, a biochemical analysis was possible. Electrophoretic analysis of the [35S]methionine-labeled polypeptides made in the hybrid cell types showed that polypeptides having the mobilities of only two (alpha A and alpha D) of the three major adult globin chains were made as major constituents of the hybrid cells. However, analysis of 14C-amino acid-labeled polypeptides revealed that a beta-like polypeptide that lacked methionine was also synthesized in large amounts. This polypeptide was tentatively identified as the early embryonic globin species rho. Globin synthesis was detected as early as 3 h after nuclear transplantation and as late as 18 h, the last time measured in these experiments. It appeared that globin polypeptides made at very early times were translated at least partially from chicken messenger ribonucleic acid introduced into the hybrid cells during fusion, whereas those made at later times were translated primarily from newly synthesized globin messenger ribonucleic acid. The potential usefulness of this hybrid cell system in analyzing mechanisms regulating globin gene expression is discussed. Images PMID:7346715

  12. Specific repression of β-globin promoter activity by nuclear ferritin

    PubMed Central

    Broyles, Robert H.; Belegu, Visar; DeWitt, Christina R.; Shah, Sandeep N.; Stewart, Charles A.; Pye, Quentin N.; Floyd, Robert A.

    2001-01-01

    Developmental hemoglobin switching involves sequential globin gene activations and repressions that are incompletely understood. Earlier observations, described herein, led us to hypothesize that nuclear ferritin is a repressor of the adult β-globin gene in embryonic erythroid cells. Our data show that a ferritin-family protein in K562 cell nuclear extracts binds specifically to a highly conserved CAGTGC motif in the β-globin promoter at −153 to −148 bp from the cap site, and mutation of the CAGTGC motif reduces binding 20-fold in competition gel-shift assays. Purified human ferritin that is enriched in ferritin-H chains also binds the CAGTGC promoter segment. Expression clones of ferritin-H markedly repress β-globin promoter-driven reporter gene expression in cotransfected CV-1 cells in which the β-promoter has been stimulated with the transcription activator erythroid Krüppel-like factor (EKLF). We have constructed chloramphenicol acetyltransferase reporter plasmids containing either a wild-type or mutant β-globin promoter for the −150 CAGTGC motif and have compared the constructs for susceptibility to repression by ferritin-H in cotransfection assays. We find that stimulation by cotransfected EKLF is retained with the mutant promoter, whereas repression by ferritin-H is lost. Thus, mutation of the −150 CAGTGC motif not only markedly reduces in vitro binding of nuclear ferritin but also abrogates the ability of expressed ferritin-H to repress this promoter in our cell transfection assay, providing a strong link between DNA binding and function, and strong support for our proposal that nuclear ferritin-H is a repressor of the human β-globin gene. Such a repressor could be helpful in treating sickle cell and other genetic diseases. PMID:11481480

  13. A novel β-globin gene mutation HBB.c.22 G>C produces a hemoglobin variant (Hb Vellore) mimicking HbS in HPLC.

    PubMed

    Edison, E S; Sathya, M; Rajkumar, S V; Nair, S C; Srivastava, A; Shaji, R V

    2012-10-01

    Hemoglobinopathies are highly prevalent in Indian population. DNA analysis to detect causative mutations is required for identifying rare hemoglobin variants or when hematological results are discordant with the clinical phenotype. In this report, we describe a novel hemoglobin variant caused by a mutation in beta-globin gene, Codon 7 GAG→CAG (Glu→Gln) that elutes in the position of sickle haemoglobin (HbS) in cation exchange high performance liquid chromatography. This report highlights possible diagnostic pitfalls in interpreting data solely based on haemoglobin analysis and usefulness of mutation screening in definitive diagnosis of hemoglobinopathies. PMID:22471768

  14. Differential regulation of the α-globin locus by Krüppel-like factor 3 in erythroid and non-erythroid cells

    PubMed Central

    2014-01-01

    Background Krüppel-like Factor 3 (KLF3) is a broadly expressed zinc-finger transcriptional repressor with diverse biological roles. During erythropoiesis, KLF3 acts as a feedback repressor of a set of genes that are activated by Krüppel-like Factor 1 (KLF1). Noting that KLF1 binds α-globin gene regulatory sequences during erythroid maturation, we sought to determine whether KLF3 also interacts with the α-globin locus to regulate transcription. Results We found that expression of a human transgenic α-globin reporter gene is markedly up-regulated in fetal and adult erythroid cells of Klf3−/− mice. Inspection of the mouse and human α-globin promoters revealed a number of canonical KLF-binding sites, and indeed, KLF3 was shown to bind to these regions both in vitro and in vivo. Despite these observations, we did not detect an increase in endogenous murine α-globin expression in Klf3 −/− erythroid tissue. However, examination of murine embryonic fibroblasts lacking KLF3 revealed significant de-repression of α-globin gene expression. This suggests that KLF3 may contribute to the silencing of the α-globin locus in non-erythroid tissue. Moreover, ChIP-Seq analysis of murine fibroblasts demonstrated that across the locus, KLF3 does not occupy the promoter regions of the α-globin genes in these cells, but rather, binds to upstream, DNase hypersensitive regulatory regions. Conclusions These findings reveal that the occupancy profile of KLF3 at the α-globin locus differs in erythroid and non-erythroid cells. In erythroid cells, KLF3 primarily binds to the promoters of the adult α-globin genes, but appears dispensable for normal transcriptional regulation. In non-erythroid cells, KLF3 distinctly binds to the HS-12 and HS-26 elements and plays a non-redundant, albeit modest, role in the silencing of α-globin expression. PMID:24885809

  15. Identification of a stage selector element in the human gamma-globin gene promoter that fosters preferential interaction with the 5' HS2 enhancer when in competition with the beta-promoter.

    PubMed

    Jane, S M; Ney, P A; Vanin, E F; Gumucio, D L; Nienhuis, A W

    1992-08-01

    The erythroid-specific enhancer within hypersensitivity site 2 (HS2) of the human beta-globin locus control region is required for high level globin gene expression. We investigated interaction between HS2 and the gamma- and beta-promoters using reporter constructs in transient assays in human erythroleukemia (K562) cells. The beta-promoter, usually silent in K562 cells, was activated by HS2. This activity was abolished when a gamma-promoter was linked in cis. Analysis of truncation mutants suggested that sequences conveying the competitive advantage of the gamma-promoter for HS2 included those between positions -53 and -35 relative to the transcriptional start site. This sequence, when used to replace the corresponding region of the beta-promoter, increased beta-promoter activity 10-fold when linked to HS2. The modified beta-promoter was also capable of competing with a gamma-promoter modified internally in the -53 to -35 region, when the two promoters were linked to HS2 in a single plasmid. The corresponding sequences from the Galago gamma-promoter, a species which lacks fetal gamma-gene expression, were inactive in analogous assays. We have identified and partially purified a nuclear protein found in human (fetal stage) erythroleukemia cells, but present in much lower concentration in murine (adult stage) erythroleukemia cells, that binds the -53 to -35 sequence of the gamma-promoter. We speculate that this region of the gamma-promoter functions as a stage selector element in the regulation of hemoglobin switching in humans. PMID:1639067

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

    PubMed Central

    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

  17. A Phylogenetic Analysis of the Globins in Fungi

    PubMed Central

    Hoogewijs, David; Dewilde, Sylvia; Vierstraete, Andy; Moens, Luc; Vinogradov, Serge N.

    2012-01-01

    Background All globins belong to one of three families: the F (flavohemoglobin) and S (sensor) families that exhibit the canonical 3/3 α-helical fold, and the T (truncated 3/3 fold) globins characterized by a shortened 2/2 α-helical fold. All eukaryote 3/3 hemoglobins are related to the bacterial single domain F globins. It is known that Fungi contain flavohemoglobins and single domain S globins. Our aims are to provide a census of fungal globins and to examine their relationships to bacterial globins. Results Examination of 165 genomes revealed that globins are present in >90% of Ascomycota and ∼60% of Basidiomycota genomes. The S globins occur in Blastocladiomycota and Chytridiomycota in addition to the phyla that have FHbs. Unexpectedly, group 1 T globins were found in one Blastocladiomycota and one Chytridiomycota genome. Phylogenetic analyses were carried out on the fungal globins, alone and aligned with representative bacterial globins. The Saccharomycetes and Sordariomycetes with two FHbs form two widely divergent clusters separated by the remaining fungal sequences. One of the Saccharomycete groups represents a new subfamily of FHbs, comprising a previously unknown N-terminal and a FHb missing the C-terminal moiety of its reductase domain. The two Saccharomycete groups also form two clusters in the presence of bacterial FHbs; the surrounding bacterial sequences are dominated by Proteobacteria and Bacilli (Firmicutes). The remaining fungal FHbs cluster with Proteobacteria and Actinobacteria. The Sgbs cluster separately from their bacterial counterparts, except for the intercalation of two Planctomycetes and a Proteobacterium between the Fungi incertae sedis and the Blastocladiomycota and Chytridiomycota. Conclusion Our results are compatible with a model of globin evolution put forward earlier, which proposed that eukaryote F, S and T globins originated via horizontal gene transfer of their bacterial counterparts to the eukaryote ancestor, resulting from

  18. The Prevalence and Molecular Spectrum of α- and β-Globin Gene Mutations in 14,332 Families of Guangdong Province, China

    PubMed Central

    Xu, Longchang; Wu, Li; Zhang, Liang; Ma, Yuanzhu; Chen, Tingting; Gao, Shuang; Liang, Juqing; Guo, Hao; Qin, Danqing; Wang, Jicheng; Yuan, Tenglong; Wang, Yixia; Huang, Wei-wei; He, Wen-Fei; Zhang, Yanxia; Liu, Chang; Xia, Sujian; Chen, Qingshan; Zhao, Qingguo; Zhang, Xiaozhuang

    2014-01-01

    Objective To reveal the familial prevalence and molecular variation of α- and β-globin gene mutations in Guangdong Province. Methods A total of 40,808 blood samples from 14,332 families were obtained and analyzed for both hematological and molecular parameters. Results A high prevalence of α- and β-globin gene mutations was found. Overall, 17.70% of pregnant women, 15.94% of their husbands, 16.03% of neonates, and 16.83% of couples (pregnant women and their husbands) were heterozygous carriers of α- or β-thalassemia. The regions with the highest prevalence were the mountainous and western regions, followed by the Pearl River Delta; the region with the lowest prevalence was Chaoshan. The total familial carrier rate (both spouses were α- or β-thalassemia carriers) was 1.87%, and the individual carrier rates of α- and β-thalassemia were 1.68% and 0.20%, respectively. The total rate of moderate-to-severe fetal thalassemia was 12.78% among couples in which both parents were carriers. Conclusions There was a high prevalence of α- and β-thalassemia in Guangdong Province. This study will contribute to the development of thalassemia prevention and control strategies in Guangdong Province. PMID:24587075

  19. The Role of Crowding Forces in Juxtaposing β-Globin Gene Domain Remote Regulatory Elements in Mouse Erythroid Cells.

    PubMed

    Golov, Arkadiy K; Gavrilov, Alexey A; Razin, Sergey V

    2015-01-01

    The extremely high concentration of macromolecules in a eukaryotic cell nucleus indicates that the nucleoplasm is a crowded macromolecular solution in which large objects tend to gather together due to crowding forces. It has been shown experimentally that crowding forces support the integrity of various nuclear compartments. However, little is known about their role in control of chromatin dynamics in vivo. Here, we experimentally addressed the possible role of crowding forces in spatial organization of the eukaryotic genome. Using the mouse β-globin domain as a model, we demonstrated that spatial juxtaposition of the remote regulatory elements of this domain in globin-expressing cells may be lost and restored by manipulation of the level of macromolecular crowding. In addition to proving the role of crowding forces in shaping interphase chromatin, our results suggest that the folding of the chromatin fiber is a major determinant in juxtaposing remote genomic elements. PMID:26436546

  20. The T----C substitution at -198 of the A gamma-globin gene associated with the British form of HPFH generates overlapping recognition sites for two DNA-binding proteins.

    PubMed Central

    Fischer, K D; Nowock, J

    1990-01-01

    Defects in the developmental changes of human hemoglobin production characterized by the continued expression of fetal globin during adult life are classified as hereditary persistence of fetal hemoglobin (HPFH). Among the various molecular lesions associated with this phenotype, the non-deletion forms with point mutations in the promoter region are thought to provide mechanistic clues for gamma-globin gene regulation. The natural occurrence of four different base substitutions mapping within six nucleotides of a homopurine.homopyrimidine motif in the upstream promoter region demarcate a potential control element. To assess its importance for transcriptional activity, we compared the -202 (C----G), -198 (T----C) and -196 (C----T) HPFH mutations with the normal sequence in binding studies with nuclear proteins from erythroid and non-erythroid cells. Wildtype DNA and HPFH mutations at -202 or -196 showed only a weak protein interaction of unclear functional significance. In contrast, -198 (T----C) generated overlapping, high-affinity binding sites for two ubiquitous nuclear proteins. One cognate protein was identified as the transcription factor Sp1. The second one was termed NF-G.C as it interacted strongly with the homopolymer poly(dG).poly(dC). The generation of additional recognition sites for trans-acting factors by the -198 HPFH mutation correlated with a modest increase in promoter activity in vitro specifically with nuclear extracts from erythroid cells. The activation appears to be mediated by binding of Sp1, but it requires interaction with an erythroid-specific factor, most likely GF-1. Templates containing the -196 HPFH mutation showed a transcriptional activity identical to wildtype. This suggests that despite the topological proximity of the mutations, the HPFH phenotype may be established by different mechanisms. Images PMID:1699206

  1. Evolution of the primate beta-globin gene region: nucleotide sequence of the delta-beta-globin intergenic region of gorilla and phylogenetic relationships between African apes and man.

    PubMed

    Perrin-Pecontal, P; Gouy, M; Nigon, V M; Trabuchet, G

    1992-01-01

    A 6.0-kb DNA fragment from Gorilla gorilla including the 5' part of the beta-globin gene and about 4.5 kb of its upstream flanking region was cloned and sequenced. The sequence was compared to the human, chimpanzee, and macaque delta-beta intergenic region. This analysis reveals four tandemly repeated sequences (RS), at the same location in the four species, showing a variable number of repeats generating both intraspecific (polymorphism) and interspecific variability. These tandem arrays delimit five regions of unique sequence called IG for intergenic. The divergence for these IG sequences is 1.85 +/- 0.22% between human and gorilla, which is not significantly different from the value estimated in the same region between chimpanzee and human (1.62 +/- 0.21%). The CpG and TpA dinucleotides are avoided. CpGs evolve faster than other sequence sites but do not confuse phylogenetic inferences by producing parallel mutations in different lineages. About 75% of CpG doublets have become TpG or CpA since the common ancestor, in agreement with the methylation/deamination pattern. Comparison of this intergenic region gives information on branching order within Hominoidea. Parsimony and distance-based methods when applied to the delta-beta intergenic region provide evidence (although not statistically significant) that human and chimpanzee are more closely related to each other than to gorilla. CpG sites are indeed rich in information by carrying substitutions along the short internal branch. Combining these results with those on the psi eta-delta intergenic region, shows in a statistically significant way that chimpanzee is the closest relative of human. PMID:1556740

  2. Duplication and Divergence: The Evolution of Nematode Globins

    PubMed Central

    McNally, J.; Barris, W.; Blaxter, M. L.

    2009-01-01

    In common with many other groups, nematodes express globins with unknown functions. Nematode globin-like genes can be divided into class 1 globins, similar to vertebrate myoglobins, and a wide range of additional classes. Here we show that class 1 nematode globins possess a huge amount of diversity in gene sequence and structure. There is evidence for multiple events of gene duplication, intron insertion and loss between species, and for allelic variation effecting both synonymous and non-synonymous sites within species. We have also examined gene expression patterns in class I globins from a variety of species. The results show variation in the degree of gene expression, but the tissue specificity and temporal specificity of expression may be more conserved in the phylum. Because the structure-function relationships for the binding and transport of oxygen by globins are well understood, the consequences of genetic variation causing amino acid changes are explored. The gene family shows great promise for discovering unique insights into both structure-function relationships of globins and their physiologial roles. PMID:22661776

  3. Triplex-forming Peptide Nucleic Acids Induce Heritable Elevations in Gamma-globin Expression in Hematopoietic Progenitor Cells

    PubMed Central

    Chin, Joanna Y; Reza, Faisal; Glazer, Peter M

    2013-01-01

    Potentiating homologous recombination using triplex-forming peptide nucleic acids (PNAs) can be used to mediate targeted sequence editing by donor DNAs and thereby induce functional gene expression to supplant non-functional counterparts. Mutations that disrupt the normal function of the β-globin subunit cause hemoglobinopathies such as sickle cell disease and β-thalassemias. However, expression of the functional γ-globin subunit in adults, a benign condition called hereditary persistence of fetal hemoglobin (HPFH), can ameliorate the severity of these disorders, but this expression is normally silenced. Here, we harness triplex-forming PNA-induced donor DNA recombination to create HPFH mutations that increase the expression of γ-globin in adult mammalian cells, including β-yeast artificial chromosome (YAC) bone marrow and hematopoietic progenitor cells (HPCs). Transfection of human cells led to site-specific modification frequencies of 1.63% using triplex-forming PNA γ-194-3K in conjunction with donor DNAs, compared with 0.29% using donor DNAs alone. We also concurrently modified the γ-globin promoter to insert both HPFH-associated point mutations and a hypoxia-responsive element (HRE), conferring increased expression that was also regulated by oxygen tension. This work demonstrates application of oligonucleotide-based gene therapy to induce a quiescent gene promoter in mammalian cells and regulate its expression via an introduced HRE transcription factor binding site for potential therapeutic purposes. PMID:23337982

  4. Globin chain synthesis in single erythroid bursts from cord blood: studies on gamma leads to beta and G gamma leads to A gamma switches.

    PubMed

    Comi, P; Giglioni, B; Ottolenghi, S; Gianni, A M; Polli, E; Barba, P; Covelli, A; Migliaccio, G; Condorelli, M; Peschle, C

    1980-01-01

    Erythroid bursts from cord or adult blood were grown in methylcellulose cultures (3 international units of erythropoietin per plate). On day 13, single bursts were picked up and reincubated for 16-24 hr with [3H]leucine. Radioactive globin chains [alpha,beta,G gamma, and A gamma (Ala-136)] were analyzed by either isoelectric focusing on polyacrylamide gels and fluorography or carboxymethylcellulose chromatography. In all cases, alpha to non-alpha globin radioactivity ratios were close to 1. In single cord blood bursts, the values of both gamma-to-beta and G gamma-to-A gamma ratios were spread over a large spectrum and further characterized by a continuous rather than a bimodal distribution. Morever, the G gamma-to-A gamma ratios demonstrated in single bursts appeared to be directly correlated with the respective gamma-to-beta ratios. These data suggest that both the gamma leads to beta and the G gamma leads to A gamma switches are mediated via mechanisms modulating the relative activities of the different genes in the non-alpha globin gene cluster rather than via selection of clones committed to the preferential synthesis of beta and A gamma globins. In contrast with the results obtained with cord blood, individual adult blood bursts synthesize a lower and hence relatively more uniform amount of gamma globin chains. PMID:6153796

  5. Prevalence of β(S)-globin gene haplotypes, α-thalassemia (3.7 kb deletion) and redox status in patients with sickle cell anemia in the state of Paraná, Brazil.

    PubMed

    Shimauti, Eliana LitsukoTomimatsu; Silva, Danilo Grunig Humberto; de Souza, Eniuce Menezes; de Almeida, Eduardo Alves; Leal, Francismar Prestes; Bonini-Domingos, Claudia Regina

    2015-01-01

    The aim of this study was to determine the frequency of beta S-globin gene (β(S) globin) haplotypes and alpha thalassemia with 3.7 kb deletion (-α(3.7kb) thalassemia) in the northwest region of Paraná state, and to investigate the oxidative and clinical-hematological profile of β(S) globin carriers in this population. Of the 77 samples analyzed, 17 were Hb SS, 30 were Hb AS and 30 were Hb AA. The β(S)globin haplotypes and -α(3.7kb) thalassemia were identified using polymerase chain reaction.Trolox equivalent antioxidant capacity (TEAC) and lipid peroxidation (LPO) were assessed spectophotometrically. Serum melatonin levels were determined using high-performance liquid chromatography coupled to coulometric electrochemical detection. The haplotype frequencies in the SS individuals were as follows: Bantu- 21 (62%), Benin - 11 (32%) and Atypical- 2 (6%). Bantu/Benin was the most frequent genotype. Of the 47 SS and AS individuals assessed, 17% (n = 8) had the -α(3.7kb) mutation. Clinical manifestations, as well as serum melatonin, TEAC and LPO levels did not differ between Bantu/Bantu and Bantu/Benin individuals (p > 0.05). Both genotypes were associated with high LPO and TEAC levels and decreased melatonin concentration. These data suggest that the level of oxidative stress in patients with Bantu/Bantu and Bantu/Benin genotypes may overload the antioxidant capacity. PMID:26500435

  6. 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.

    PubMed

    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. PMID:25222042

  7. Zebrafish globin Switching Occurs in Two Developmental Stages and is Controlled by the LCR

    PubMed Central

    Ganis, Jared J.; Hsia, Nelson; Trompouki, Eirini; de Jong, Jill L.O.; DiBiase, Anthony; Lambert, Janelle S.; Jia, Zhiying; Sabo, Peter J.; Weaver, Molly; Sandstrom, Richard; Stamatoyannopoulos, John A.; Zhou, Yi; Zon, Leonard I.

    2012-01-01

    Globin gene switching is a complex, highly regulated process allowing expression of distinct globin genes at specific developmental stages. Here, for the first time, we have characterized all of the zebrafish globins based on the completed genomic sequence. Two distinct chromosomal loci, termed major (chromosome 3) and minor (chromosome 12), harbor the globin genes containing α/β pairs in a 5′-3′ to 3′-5′ orientation. Both these loci share synteny with the mammalian α-globin locus. Zebrafish globin expression was assayed during development and demonstrated two globin switches, similar to human development. A conserved regulatory element, the locus control region (LCR), was revealed by analyzing DNase I hypersensitive sites, H3K4 trimethylation marks and GATA1 binding sites. Surprisingly, the position of these sites with relation to the globin genes is evolutionarily conserved, despite a lack of overall sequence conservation. Motifs within the zebrafish LCR include CACCC, GATA, and NFE2 sites, suggesting functional interactions with known transcription factors but not the same LCR architecture. Functional homology to the mammalian α-LCR MCS-R2 region was confirmed by robust and specific reporter expression in erythrocytes of transgenic zebrafish. Our studies provide a comprehensive characterization of the zebrafish globin loci and clarify the regulation of globin switching. PMID:22537494

  8. Detection of β-globin Gene Mutations Among β-thalassaemia Carriers and Patients in Malaysia: Application of Multiplex Amplification Refractory Mutation System–Polymerase Chain Reaction

    PubMed Central

    Hassan, Syahzuwan; Ahmad, Rahimah; Zakaria, Zubaidah; Zulkafli, Zefarina; Abdullah, Wan Zaidah

    2013-01-01

    Background: β-thalassaemia is one of the most common single-gene disorders worldwide. Each ethnic population has its own common mutations, accounting for the majority of cases, with a small number of mutations for the rarer alleles. Due to the heterogeneity of β-thalassaemia and the multi-ethnicity of Malaysians, molecular diagnostics may be expensive and time consuming. Methods: A simple polymerase chain reaction (PCR) approach involving a multiplex amplification refractory mutation system (MARMS) and one amplification refractory mutation system (ARMS), consisting of 20 β-globin gene mutations, were designed and employed to investigate β-thalassaemia patients and carriers. Results: Out of 169 carriers tested with the MARMS, Cd 41/42 (–TTCT), Cd 26 (A–G) HbE, IVS 1–1 (G–T), and IVS 1–5 (G–C) were the most common mutations, accounting for 78.1%. Among the Malays, Cd 26 (A–G) HbE, Cd 41/42 (–TTCT), IVS 1–1 (G–T), and IVS 1–5 (G–C) were the most common mutations, accounting for 81.4%, whereas Cd 41/42 (–TTCT) and IVS 2–654 (C–T) were most common among the Chinese (79.1%). Conclusion: We propose the use of this cheap, easy to interpret, and simple system for the molecular diagnostics of β-thalassaemia among Malaysians at the Institute for Medical Research (IMR). PMID:23613656

  9. Characteristics of the beta-globin gene cluster haplotypes of three Han Chinese populations at Beijing, Xi'an, and Kunming as compared with those of other Asian populations.

    PubMed

    Shimizu, Koji; Nagaoka, Erika; Okada, Yusuke; Takeuchi, Yukiko; Harihara, Shinji; Omoto, Keiichi; Imanishi, Tadashi; Kim, Wook; Shin, Dong-Jik; Hao, Luping; Jin, Feng

    2008-10-01

    Haplotype frequencies of the beta-globin gene cluster of Han Chinese at Beijing, Xi'an, and Kunming were estimated, and their mutual genetic relationships were examined and compared to those of Buryats, Khalkhs, Evenkis, Oroqens, Koreans, and Colombian Amerindians. A major 5' subhaplotype (5' to the delta-globin gene), a major 3' subhaplotype (in and 3' to the beta-globin gene), and a major haplotype (combination of 5' and 3' subhaplotypes) are represented as + - - - -, - +, and + - - - - - +, respectively, and found in all three Han Chinese. A rare 5' subhaplotype, - - - - -, which is one of the possible ancestral types, was found only in Han Chinese at Kunming at low frequency (0.013), and a rare 3' subhaplotype, - -, was also observed in all three Han Chinese at low frequencies (0.009-0.014). The present haplotype frequency study suggested that the highest genetic affinity was found between Han Chinese at Beijing and those at Xi'an; the next highest was between Han Chinese at Beijing and Koreans, followed by that between Han Chinese at Beijing and Khalkhs, then that between Han Chinese at Xi'an and those at Kunming or Khalkhs, and finally that between Han Chinese at Beijing and those at Kunming. A genetic boundary between northern and southern Han Chinese was not evident in the present study. PMID:18553219

  10. Fannin-Lubbock-I [α₂β₂¹¹⁹(GLY>ASP)], a rare mutation in the beta-globin gene, has been detected for the first time in a Hindu Brahmin family in West Bengal, India.

    PubMed

    Basak, Jayasri; Bhattacharyya, Deboshree M; Mukhopadhyay, Ashis

    2014-06-01

    This study aims to describe the hemoglobin Fannin-Lubbock-I, which has a rare mutation substituting the amino acid glycine with aspartic acid at codon 119 of the β-globin chain. A Bengalee Hindu Brahmin family from Kolkata in West Bengal was the focus of this study. Molecular analysis using ARMS-PCR and direct DNA sequencing revealed the presence of a GGC > GAC mutation in codon 119 of the β-globin gene in a heterozygote state in three women of the same family. This is the first report of the hemoglobin Fannin-Lubbock-I from India. Our results will help to identify this mutation, which is relatively infrequent in our population. PMID:24802353

  11. Confirmation of the potential usefulness of two human beta globin pseudogene markers to estimate gene flows to and from sub-Saharan Africans.

    PubMed

    Ciminelli, Bianca Maria; Pompei, Fiorenza; Relucenti, Michela; Lum, J Koji; Simporé, Jacques; Spedini, Gabriella; Martínez-Labarga, Cristina; Pardo, Miguel G

    2002-04-01

    Two polymorphic sites, -107 and -100 with respect to the "cap" site of the human beta globin pseudogene, recently discovered in our laboratory, turned out to have an ethnically complementary distribution. The first site is polymorphic in Europeans, North Africans, Indians (Hindu), and Oriental Asians, and monomorphic in sub-Saharan Africans. Conversely, the second site is polymorphic in sub-Saharan African populations and monomorphic in the aforementioned populations. Here we report the gene frequencies of these two polymorphic sites in nine additional populations (Egyptians, Spaniards, Japanese, Chinese, Filipinos, Vietnamese, Africans from Togo and from Benin, and Pygmies), confirming their ethnospecificity and, through the analysis of these two markers in Oromo and Amhara of Ethiopia (two mixed populations), their usefulness in genetic admixture studies. Moreover, we studied another marker polymorphic in sub-Saharan African populations only, a TaqI restriction fragment length polymorphism located in the same region as the present markers, demonstrating the absence of linkage disequilibrium between it and the -100 site, so that we can exclude that the information they provide is redundant. PMID:12030652

  12. Targeted cross-linking of the human beta-globin gene in living cells mediated by a triple helix forming oligonucleotide.

    PubMed

    Shahid, Kazi Abdus; Majumdar, Alokes; Alam, Rowshon; Liu, Su-Ting; Kuan, Jean Y; Sui, Xuifen; Cuenoud, Bernard; Glazer, Peter M; Miller, Paul S; Seidman, Michael M

    2006-02-14

    Triple helix forming oligonucleotides (TFOs) may have utility as gene targeting reagents for "in situ" gene therapy of genetic disorders. Triplex formation is challenged by negative charge repulsion between third strand and duplex phosphates, and destabilizing positive charge repulsion between adjacent protonated cytosines within pyrimidine motif third strands. Here we describe the synthesis of TFOs designed to target a site in the human beta-globin gene, which is the locus for mutations that underlie the beta-globinopathies, including sickle cell anemia. The target is an uninterrupted polypurine:polypyrimidine sequence, containing four adjacent cytosines, next to a psoralen cross-link site. Pyrimidine motif TFOs that contained four adjacent cytosines or 5-methylcytosines did not form stable triplexes at physiological pH, despite the introduction of otherwise stabilizing base and sugar analogues. We synthesized a series of pso-TFOs containing 2'-O-methyl (OMe) and 2'-O-aminoethoxy substitutions (AE), as well as 8-oxo-adenine (A8) and 2'-O-methylpseudoisocytidine (P) as neutral cytosine replacements. Thermal stability measurements indicated that TFOs with A8 did not meet criteria established in previous work. However, TFOs with P did form triplexes with appropriate T(m) and k(ON) values. A pso-TFO with AE and P residues was sufficiently active to permit the determination of targeting in living cells by direct measurement of cross-link formation at the target site. Our results validate the modification format described in our previous studies and indicate that P substitutions are an effective solution to the problem of targeting genomic sequences containing adjacent cytosines. PMID:16460044

  13. Prevalence of βS-globin gene haplotypes, α-thalassemia (3.7 kb deletion) and redox status in patients with sickle cell anemia in the state of Paraná, Brazil

    PubMed Central

    Shimauti, Eliana LitsukoTomimatsu; Silva, Danilo Grunig Humberto; de Souza, Eniuce Menezes; de Almeida, Eduardo Alves; Leal, Francismar Prestes; Bonini-Domingos, Claudia Regina

    2015-01-01

    The aim of this study was to determine the frequency of beta S-globin gene (βS globin) haplotypes and alpha thalassemia with 3.7 kb deletion (−α3.7kb thalassemia) in the northwest region of Paraná state, and to investigate the oxidative and clinical-hematological profile of βS globin carriers in this population. Of the 77 samples analyzed, 17 were Hb SS, 30 were Hb AS and 30 were Hb AA. The βSglobin haplotypes and −α3.7kb thalassemia were identified using polymerase chain reaction.Trolox equivalent antioxidant capacity (TEAC) and lipid peroxidation (LPO) were assessed spectophotometrically. Serum melatonin levels were determined using high-performance liquid chromatography coupled to coulometric electrochemical detection. The haplotype frequencies in the SS individuals were as follows: Bantu- 21 (62%), Benin - 11 (32%) and Atypical- 2 (6%). Bantu/Benin was the most frequent genotype. Of the 47 SS and AS individuals assessed, 17% (n = 8) had the −α3.7kb mutation. Clinical manifestations, as well as serum melatonin, TEAC and LPO levels did not differ between Bantu/Bantu and Bantu/Benin individuals (p > 0.05). Both genotypes were associated with high LPO and TEAC levels and decreased melatonin concentration. These data suggest that the level of oxidative stress in patients with Bantu/Bantu and Bantu/Benin genotypes may overload the antioxidant capacity. PMID:26500435

  14. Differential expression of murine adult hemoglobins in early ontogeny

    SciTech Connect

    Wawrzyniak, C.J.; Lewis, S.E.; Popp, R.A.

    1985-01-01

    A hemoglobin mutation is described that permits study of the expression of the two adult ..beta..-globin genes throughout fetal and postnatal development. Mice with a mutation at the Hbb/sup s/, ..beta..-globin locus, were used to study the relative levels of ..beta..-s2major and ..beta..-sminor globins specified by the mutant Hbb/sup s2/ haplotype during development. At 11.5 days of gestation ..beta..-sminor comprised over 80% and ..beta..-s2major under 20% of the adult beta-globin. The relative level of ..beta..-sminor decreased through fetal development; at birth ..beta..-sminor represented 33.7% of the ..beta..-globin. The adult values of 71.0% ..beta..-s2major and 29.0% ..beta..-sminor globin are expressed in mice six days after birth. Because the two ..beta..-globin genes are expressed in mice of the Hbb/sup 2s/ haplotype, both the ..beta..-smajor and ..beta..-sminor genes must be expressed in mice of the Hbb/sup s/ haplotype. Expression of the ..beta..-sminor gene is elevated to 35.6% in Hbb/sup s2/ mice that have been bled repeatedly. Thus, the 5' ..beta..-s2major and 3' ..beta..-sminor genes of the Hbb/sup s2/ haplotype and, presumably the 5' ..beta..-smajor and 3' ..beta..-sminor genes of the Hbb/sup s/ haplotype, are regulated independently and are homologous to the 5' ..beta..-dmajor and 3' ..beta..-dminor genes of the Hbb/sup d/ haplotype. Mice of the Hbb/sup s2/ haplotype are better than mice of the Hbb/sup d/ haplotytpe for studying the mechanisms of hemoglobin switching because the Hbb/sup s2/ each of the three embryonic and two adult hemoglobins can be separated by electrophoresis. 17 refs., 3 figs.

  15. Globin-coupled sensors, protoglobins, and the last universal common ancestor.

    PubMed

    Freitas, Tracey Allen K; Saito, Jennifer A; Hou, Shaobin; Alam, Maqsudul

    2005-01-01

    The strategy for detecting oxygen, carbon monoxide, nitric oxide, and sulfides is predominantly through heme-based sensors utilizing either a globin domain or a PAS domain. Whereas PAS domains bind various cofactors, globins bind only heme. Globin-coupled sensors (GCSs) were first described as regulators of the aerotactic responses in Bacillus subtilis and Halobacterium salinarum. GCSs were also identified in diverse microorganisms that appear to have roles in regulating gene expression. Functional and evolutionary analyses of the GCSs, their protoglobin ancestor, and their relationship to the last universal common ancestor (LUCA) are discussed in the context of globin-based signal transduction. PMID:15598488

  16. Alpha-globin loci in homozygous beta-thalassemia intermedia.

    PubMed

    Triadou, P; Lapoumeroulie, C; Girot, R; Labie, D

    1983-01-01

    Homozygous beta-thalassemia intermediate (TI) differs from thalassemia major (TM) in being less severe clinically. Associated alpha-thalassemia could account for the TI phenotype by reducing the alpha/non-alpha chain imbalance. We have analyzed the alpha loci of 9 TI and 11 TM patients by restriction endonuclease mapping. All the TM and 7 of the TI patients have the normal complement of four alpha-globin genes (alpha alpha/alpha alpha). One TI patient has three alpha-globin genes (alpha alpha/-alpha), and another TI patient has five alpha genes (alpha alpha/alpha alpha alpha). PMID:6305827

  17. Hairpin-duplex equilibrium reflected in the A-->B transition in an undecamer quasi-palindrome present in the locus control region of the human beta-globin gene cluster.

    PubMed

    Kaushik, Mahima; Kukreti, Ritushree; Grover, Deepak; Brahmachari, Samir K; Kukreti, Shrikant

    2003-12-01

    Our recent work on an A-->G single nucleotide polymorphism (SNP) at the quasi-palindromic sequence d(TGGGG[A/G]CCCCA) of HS4 of the human beta-globin locus control region in an Indian population showed a significant association between the G allele and the occurrence of beta-thalassemia. Using UV-thermal denaturation, gel assay, circular dichroism (CD) and nuclease digestion experiments we have demonstrated that the undecamer quasi- palindromic sequence d(TGGGGACCCCA) (HPA11) and its reported polymorphic (SNP) version d(TGG GGGCCCCA) (HPG11) exist in hairpin-duplex equilibria. The biphasic nature of the melting profiles for both the oligonucleotides persisted at low as well as high salt concentrations. The HPG11 hairpin showed a higher T(m) than HPA11. The presence of unimolecular and bimolecular species was also shown by non-denaturating gel electrophoresis experiments. The CD spectra of both oligonucleotides showed features of the A- as well as B-type conformations and, moreover, exhibited a concentration dependence. The disappearance of the 265 nm positive CD signal in an oligomer concentration-dependent manner is indicative of an A-->B transition. The results give unprecedented insight into the in vitro structure of the quasi-palindromic sequence and provide the first report in which a hairpin-duplex equilibrium has been correlated with an A-->B interconversion of DNA. The nuclease-dependent degradation suggests that HPG11 is more resistant to nuclease than HPA11. Multiple sequence alignment of the HS4 region of the beta-globin gene cluster from different organisms revealed that this quasi-palindromic stretch is unique to Homo sapiens. We propose that quasi-palindromic sequences may form stable mini- hairpins or cruciforms in the HS4 region and might play a role in regulating beta-globin gene expression by affecting the binding of transcription factors. PMID:14627823

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

    SciTech Connect

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

    1988-06-01

    Haplotype analysis of the {beta}-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){sub n} and (AT){sub x}T{sub 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){sub n} and (AT){sub x}T{sub y} repeats. The authors found three additional structures for (AT){sub x}T{sub y} correlating with the geographic origin of the patients. Ten other nucleotide positions, 5{prime} and 3{prime} to the (AT){sub x}T{sub 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{prime} flanking region of the {beta}-globin gene. The results strongly support the hypothesis of three origins for the sickle mutation in Africa.

  19. The gene for familial Mediterranean fever in both Armenians and non-Ashkenazi Jews is linked to the alpha-globin complex on 16p: evidence for locus homogeneity.

    PubMed Central

    Shohat, M; Bu, X; Shohat, T; Fischel-Ghodsian, N; Magal, N; Nakamura, Y; Schwabe, A D; Schlezinger, M; Danon, Y; Rotter, J I

    1992-01-01

    Familial Mediterranean fever (FMF) is a recurrent inflammatory disorder characterized by short episodes of fever, peritonitis, pleuritis, and arthritis. While FMF has been shown to be inherited in an autosomal recessive fashion in both non-Ashkenazi Jews and Armenian families, clinical differences have raised the possibility of genetic heterogeneity. As its pathogenesis is unknown, mapping of the gene for FMF may provide the first objective method for early and accurate diagnosis of this disease. After excluding 45% of the entire human genome, we studied 14 Armenian and 9 non-Ashkenazi Jewish families with FMF and tested linkage with the alpha-globin locus on chromosome 16. Analysis of the PvuII length polymorphism of the 3' HVR (hypervariable region) probe showed significant linkage with the FMF gene (maximum lod score [lodmax] = 9.76 at maximum recombination fraction [theta] = .076). In the Armenians, the lodmax = 3.61 at theta = .10; and for the non-Ashkenazi Jews, lodmax = 6.28 at theta = .06. There was no evidence for genetic heterogeneity between the Armenians and the non-Ashkenazi Jews (chi 2 = 1.28; P = .26) or within either ethnic group (chi 2 = .00; P = .50). Thus, the gene for FMF is linked to the alpha-globin complex on chromosome 16p in both non-Ashkenazi Jews and Armenians. PMID:1463015

  20. Original Research: Generation of non-deletional hereditary persistence of fetal hemoglobin β-globin locus yeast artificial chromosome transgenic mouse models: -175 Black HPFH and -195 Brazilian HPFH.

    PubMed

    Braghini, Carolina A; Costa, Flavia C; Fedosyuk, Halyna; Neades, Renee Y; Novikova, Lesya V; Parker, Matthew P; Winefield, Robert D; Peterson, Kenneth R

    2016-04-01

    Fetal hemoglobin is a major genetic modifier of the phenotypic heterogeneity in patients with sickle cell disease and certain β-thalassemias. Normal levels of fetal hemoglobin postnatally are approximately 1% of total hemoglobin. Patients who have hereditary persistence of fetal hemoglobin, characterized by elevated synthesis of γ-globin in adulthood, show reduced disease pathophysiology. Hereditary persistence of fetal hemoglobin is caused by β-globin locus deletions (deletional hereditary persistence of fetal hemoglobin) or γ-globin gene promoter point mutations (non-deletional hereditary persistence of fetal hemoglobin). Current research has focused on elucidating the pathways involved in the maintenance/reactivation of γ-globin in adult life. To better understand these pathways, we generated new β-globin locus yeast artificial chromosome transgenic mice bearing the (A)γ-globin -175 T > C or -195 C > G hereditary persistence of fetal hemoglobin mutations to model naturally occurring hereditary persistence of fetal hemoglobin. Adult -175 and -195 mutant β-YAC mice displayed a hereditary persistence of fetal hemoglobin phenotype, as measured at the mRNA and protein levels. The molecular basis for these phenotypes was examined by chromatin immunoprecipitation of transcription factor/co-factor binding, including YY1, PAX1, TAL1, LMO2, and LDB1. In -175 HPFH versus wild-type samples, the occupancy of LMO2, TAL1 and LDB1 proteins was enriched in HPFH mice (5.8-fold, 5.2-fold and 2.7-fold, respectively), a result that concurs with a recent study in cell lines showing that these proteins form a complex with GATA-1 to mediate long-range interactions between the locus control region and the (A)γ-globin gene. Both hereditary persistence of fetal hemoglobin mutations result in a gain of (A)γ-globin activation, in contrast to other hereditary persistence of fetal hemoglobin mutations that result in a loss of repression. The mice provide additional tools to

  1. Generation of non-deletional hereditary persistence of fetal hemoglobin β-globin locus yeast artificial chromosome transgenic mouse models: −175 Black HPFH and −195 Brazilian HPFH

    PubMed Central

    Braghini, Carolina A; Costa, Flavia C; Fedosyuk, Halyna; Neades, Renee Y; Novikova, Lesya V; Parker, Matthew P; Winefield, Robert D; Peterson, Kenneth R

    2016-01-01

    Fetal hemoglobin is a major genetic modifier of the phenotypic heterogeneity in patients with sickle cell disease and certain β-thalassemias. Normal levels of fetal hemoglobin postnatally are approximately 1% of total hemoglobin. Patients who have hereditary persistence of fetal hemoglobin, characterized by elevated synthesis of γ-globin in adulthood, show reduced disease pathophysiology. Hereditary persistence of fetal hemoglobin is caused by β-globin locus deletions (deletional hereditary persistence of fetal hemoglobin) or γ-globin gene promoter point mutations (non-deletional hereditary persistence of fetal hemoglobin). Current research has focused on elucidating the pathways involved in the maintenance/reactivation of γ-globin in adult life. To better understand these pathways, we generated new β-globin locus yeast artificial chromosome transgenic mice bearing the Aγ-globin −175 T >C or −195 C >G hereditary persistence of fetal hemoglobin mutations to model naturally occurring hereditary persistence of fetal hemoglobin. Adult −175 and −195 mutant β-YAC mice displayed a hereditary persistence of fetal hemoglobin phenotype, as measured at the mRNA and protein levels. The molecular basis for these phenotypes was examined by chromatin immunoprecipitation of transcription factor/co-factor binding, including YY1, PAX1, TAL1, LMO2, and LDB1. In −175 HPFH versus wild-type samples, the occupancy of LMO2, TAL1 and LDB1 proteins was enriched in HPFH mice (5.8-fold, 5.2-fold and 2.7-fold, respectively), a result that concurs with a recent study in cell lines showing that these proteins form a complex with GATA-1 to mediate long-range interactions between the locus control region and the Aγ-globin gene. Both hereditary persistence of fetal hemoglobin mutations result in a gain of Aγ-globin activation, in contrast to other hereditary persistence of fetal hemoglobin mutations that result in a loss of repression. The mice provide additional tools to study

  2. Repair of Thalassemic Human β -globin mRNA in Mammalian Cells by Antisense Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Sierakowska, Halina; Sambade, Maria J.; Agrawal, Sudhir; Kole, Ryszard

    1996-11-01

    In one form of β -thalassemia, a genetic blood disorder, a mutation in intron 2 of the β -globin gene (IVS2-654) causes aberrant splicing of β -globin pre-mRNA and, consequently, β -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

  3. Treatment of sickle cell anemia with 5-azacytidine results in increased fetal hemoglobin production and is associated with nonrandom hypomethylation of DNA around the gamma-delta-beta-globin gene complex.

    PubMed Central

    Charache, S; Dover, G; Smith, K; Talbot, C C; Moyer, M; Boyer, S

    1983-01-01

    Increased production of fetal hemoglobin (HbF) was observed in a patient with sickle cell anemia treated with 5-azacytidine. Each of four courses of therapy resulted in a rapid and prolonged increase in the percentage of HbF containing reticulocytes (F reticulocytes) and HbF containing erythrocytes (F cells). The percentage of HbF in peripheral blood rose from 1.8 to 8.9%. The rise in HbF production was accompanied by an increase in peripheral blood hemoglobin concentration from 8 to 12 g/dl and an increase in mean erythrocyte volume. Treatment with 5-azacytidine resulted in hypomethylation of total genomic and a Y-chromosome-specific DNA fragment isolated from both peripheral blood and bone marrow. Of 15 restriction enzyme sites around the gamma-delta-beta-globin gene complex, only 2 became hypomethylated: one 107 bases 5' to the gamma G and the other 107 bases 5' to the gamma A globin genes. Images PMID:6192443

  4. 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.

    PubMed

    Fong, Cristian; Lizarralde-Iragorri, María Alejandra; Rojas-Gallardo, Diana; Barreto, Guillermo

    2013-12-01

    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. PMID:24385850

  5. Locus assignment of alpha-globin structural mutations by hybrid-selected translation.

    PubMed Central

    Liebhaber, S A; Cash, F E

    1985-01-01

    The two human alpha-globin genes, alpha 1 and alpha 2 located 3.4 kilobases apart on chromosome 16, encode identical alpha-globin proteins. A mutation in either gene could result in a structural hemoglobinopathy. It has only recently become possible to assign an alpha-chain mutant to one of these two loci by using recombinant DNA technology. While definitive, this approach has necessitated the cloning and sequencing of the specific gene in question. We present an alternative approach which results in rapid and definitive assignment of an alpha-globin mutation to its encoding genetic locus. This approach uses the technique of hybrid-selected translation. Reticulocyte RNA from individuals with alpha-globin mutations can be fractionated into beta-, alpha 9 (total)-, alpha 1-, and alpha 2-globin mRNA by selective hybridization of each mRNA species to its respective complementary DNA (cDNA) immobilized on nitrocellulose paper. Each mRNA purified in this way can be translated in vitro, and the mRNA species (and hence gene locus) encoding the globin mutant can then be directly identified by gel analysis of the radiolabeled translation products. This procedure can be used to identify globin mutants as alpha or beta and to localize alpha-globin mutants to the alpha 1 or alpha 2 gene. We have used this technique to localize the two alpha-globin mutants, alpha 125Pro (Hb Quong Sze) and alpha 47HIS (Hb Hasharon), to the alpha 2 locus. This approach could potentially be expanded to serve as an alternative to peptide analysis for the initial characterization of all globin structural mutants. Images PMID:2981252

  6. A novel 26 bp deletion [HBB: c.20_45del26bp] in exon 1 of the β-globin gene causing β-thalassemia major.

    PubMed

    Edison, Eunice S; Venkatesan, Rajkumar S; Govindanattar, Sankari Devi; George, Biju; Shaji, Ramachandran V

    2012-01-01

    Molecular characterization of β-thalassemia (β-thal) is essential in prevention and in understanding the biology of the disease. Deletion mutations are relatively uncommon in β-thal. In this report, we describe a novel 26 bp deletion from codon 6 to codon 14 in the β-globin in a consanguineous family from Tamil Nadu, India. This novel mutation causes a shift in the normal reading frame of the β-globin coding sequence, and consequently, a premature chain termination of translation due to the creation of a stop codon at the position of codon 21. The identification of this novel deletional mutation adds to the repertoire of β-thal mutations in India. PMID:22233277

  7. Forced chromatin looping raises fetal hemoglobin in adult sickle cells to higher levels than pharmacologic inducers.

    PubMed

    Breda, Laura; Motta, Irene; Lourenco, Silvia; Gemmo, Chiara; Deng, Wulan; Rupon, Jeremy W; Abdulmalik, Osheiza Y; Manwani, Deepa; Blobel, Gerd A; Rivella, Stefano

    2016-08-25

    Overcoming the silencing of the fetal γ-globin gene has been a long-standing goal in the treatment of sickle cell disease (SCD). The major transcriptional enhancer of the β-globin locus, called the locus control region (LCR), dynamically interacts with the developmental stage-appropriate β-type globin genes via chromatin looping, a process requiring the protein Ldb1. In adult erythroid cells, the LCR can be redirected from the adult β- to the fetal γ-globin promoter by tethering Ldb1 to the human γ-globin promoter with custom-designed zinc finger (ZF) proteins (ZF-Ldb1), leading to reactivation of γ-globin gene expression. To compare this approach to pharmacologic reactivation of fetal hemoglobin (HbF), hematopoietic cells from patients with SCD were treated with a lentivirus expressing the ZF-Ldb1 or with chemical HbF inducers. The HbF increase in cells treated with ZF-Ldb1 was more than double that observed with decitabine and pomalidomide; butyrate had an intermediate effect whereas tranylcypromine and hydroxyurea showed relatively low HbF reactivation. ZF-Ldb1 showed comparatively little toxicity, and reduced sickle hemoglobin (HbS) synthesis as well as sickling of SCD erythroid cells under hypoxic conditions. The efficacy and low cytotoxicity of lentiviral-mediated ZF-Ldb1 gene transfer compared with the drug regimens support its therapeutic potential for the treatment of SCD. PMID:27405777

  8. Two genetic markers closely linked to adult polycystic kidney disease on chromosome 16.

    PubMed Central

    Reeders, S T; Breuning, M H; Corney, G; Jeremiah, S J; Meera Khan, P; Davies, K E; Hopkinson, D A; Pearson, P L; Weatherall, D J

    1986-01-01

    The genetic locus for autosomal dominant adult polycystic kidney disease was recently assigned to chromosome 16 by the finding of genetic linkage to the alpha globin gene cluster. Further study showed that the phosphoglycolate phosphatase locus is also closely linked to both the locus for adult polycystic kidney disease and the alpha globin gene cluster. These findings have important implications for the prenatal and presymptomatic diagnosis of adult polycystic kidney disease and for a better understanding of its pathogenesis. Images FIG 1 PMID:3008903

  9. Two new δ-globin gene variants: Hb A(2)-Saint-Etienne [δ14(A11)Leu→Pro (HBD: c.44T>C)] and Hb A(2)-Marseille [δ22(B4) Ala→Lys (HBD: c.67G>A;68C>A)].

    PubMed

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Desbrée, Aurélie; Couprie, Nicole; Francina, Alain

    2013-01-01

    We report two new variants of the δ-globin gene: Hb A(2)-Saint-Etienne [δ14(A11)Leu→Pro] and Hb A(2)-Marseille [δ22(B4)Ala→Lys]. The first variant has a low rate of expression, the second results from a double nucleotide mutation on the same codon. PMID:23227922

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

    SciTech Connect

    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.

  11. Hb St. Jozef, A Val-->Leu N-terminal mutation leading to retention of the methionine, and partial acetylation found in the globin gene in Cis with a -alpha3.7 thalassemia deletion.

    PubMed

    Harteveld, Cornelis L; Versteegh, Florens G A; van Leer, Eduard H G; Starreveld, Jaap S; Kok, Peter J M J; van Rooijen-Nijdam, Irene; van Delft, Peter; Zanella-Cleon, Isabelle; Becchi, Michel; Wajcman, Henri; Giordano, Piero C

    2007-01-01

    We report a new hemoglobin (Hb) variant found in a 6-year-old girl of Moroccan origin, living in the Dutch city of Gouda. The child was referred because of microcytic and hypochromic parameters. A normal zinc protoporphyirin (ZPP) value excluded iron deficiency and gap-polymerase chain reaction (gap-PCR) revealed a heterozygosity for the common -alpha(3.7) thalassemia deletion, partially justifying the hematological picture. The Hb pattern on alkaline electrophoresis and capillary electrophoresis was normal, while a fraction of 9% preceding the Hb A peak, remained visible on different high performance liquid chromatography (HPLC) devices. This fraction, located in front of the Hb A peak, is usually considered as a Hb A derivate that becomes more expressed in older samples. However, the sample was freshly collected and the peak unusually evident. Therefore, direct sequencing of the alpha-globin genes was performed revealing a GTG-->CTG transversion at codon 1 of the alpha1-globin gene or of the hybrid gene. This point mutation induces a single amino acid substitution from valine to leucine. Electrospray-mass spectrometry (ES-MS) analysis revealed, in addition to this substitution, that the N-terminal methionine was retained and that about 20% of the variant was acetylated. As expected for an association with a -alpha(3.7)-thalassemia (thal) deletion, the non acetylated and acetylated abnormal alpha chain amounted to 32% of the total alpha chains. Family studies revealed that the mutated codon was located in cis of the deletion. PMID:17654068

  12. Polymorphism and divergence in the beta-globin replication origin initiation region.

    PubMed

    Fullerton, S M; Bond, J; Schneider, J A; Hamilton, B; Harding, R M; Boyce, A J; Clegg, J B

    2000-01-01

    DNA sequence polymorphism and divergence was examined in the vicinity of the human beta-globin gene cluster origin of replication initiation region (IR), a 1.3-kb genomic region located immediately 5' of the adult-expressed beta-globin gene. DNA sequence variation in the replication origin IR and 5 kb of flanking DNA was surveyed in samples drawn from two populations, one African (from the Gambia, West Africa) and the other European (from Oxford, England). In these samples, levels of nucleotide and length polymorphism in the IR were found to be more than two times as high as adjacent non-IR-associated regions (estimates of per-nucleotide heterozygosity were 0.30% and 0.12%, respectively). Most polymorphic positions identified in the origin IR fall within or just adjacent to a 52-bp alternating purine-pyrimidine ((RY)n) sequence repeat. Within- and between-populations divergence is highest in this portion of the IR, and interspecific divergence in the same region, determined by comparison with an orthologous sequence from the chimpanzee, is also pronounced. Higher levels of diversity in this subregion are not, however, primarily attributable to slippage-mediated repeat unit changes, as nucleotide substitution contributes disproportionately to allelic heterogeneity. An estimate of helical stability in the sequenced region suggests that the hypervariable (RY)n constitutes the major DNA unwinding element (DUE) of the replication origin IR, the location at which the DNA duplex first unwinds and new strand synthesis begins. These findings suggest that the beta-globin IR experiences a higher underlying rate of neutral mutation than do adjacent genomic regions and that enzyme fidelity associated with the initiation of DNA replication at this origin may be compromised. The significance of these findings for our understanding of eukaryotic replication origin biology is discussed. PMID:10666717

  13. Characterization of histone H3K27 modifications in the {beta}-globin locus

    SciTech Connect

    Kim, Yea Woon; Kim, AeRi

    2011-02-11

    Research highlights: {yields} The {beta}-globin locus control region is hyperacetylated and monomethylated at histone H3K27. {yields} Highly transcribed globin genes are marked by H3K27ac, but H3K27me2 is remarkable at silent globin genes in erythroid K562 cells. {yields} Association of PRC2 subunits is comparable with H3K27me3 pattern. {yields} 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 {beta}-globin locus using the ChIP assay. The LCR of the human {beta}-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 {beta}-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.

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

    PubMed

    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. PMID:25976463

  15. alpha-Thalassemia caused by an unstable alpha-globin mutant.

    PubMed Central

    Liebhaber, S A; Kan, Y W

    1983-01-01

    In a previous study, molecular cloning of the alpha-globin genes from a patient with nondeletion Hb-H disease (genotype--/alpha alpha) showed that a single nucleotide mutation (CTG to CCG) in one of the genes resulted in a leucine to proline substitution. This paper describes the approach we used to detect the abnormal alpha-globin chain. The chain was identified using a cell-free translation system. It turned over rapidly both in vitro and in vivo in the patient's reticulocytes. The unusual feature of this unstable alpha-globin is that the alpha-globin deficiency causes alpha-thalassemia. Simple heterozygotes for this lesion (alpha Pro alpha/alpha alpha) resemble alpha-thalassemia carriers and do not exhibit the hemolytic anemia usually associated with unstable hemoglobins. Images PMID:6826718

  16. Therapeutic fetal-globin inducers reduce transcriptional repression in hemoglobinopathy erythroid progenitors through distinct mechanisms.

    PubMed

    Dai, Yan; Sangerman, Jose; Luo, Hong Yuan; Fucharoen, Suthat; Chui, David H K; Faller, Douglas V; Perrine, Susan P

    2016-01-01

    Pharmacologic augmentation of γ-globin expression sufficient to reduce anemia and clinical severity in patients with diverse hemoglobinopathies has been challenging. In studies here, representative molecules from four chemical classes, representing several distinct primary mechanisms of action, were investigated for effects on γ-globin transcriptional repressors, including components of the NuRD complex (LSD1 and HDACs 2-3), and the downstream repressor BCL11A, in erythroid progenitors from hemoglobinopathy patients. Two HDAC inhibitors (MS-275 and SB939), a short-chain fatty acid derivative (sodium dimethylbutyrate [SDMB]), and an agent identified in high-throughput screening, Benserazide, were studied. These therapeutics induced γ-globin mRNA in progenitors above same subject controls up to 20-fold, and increased F-reticulocytes up to 20%. Cellular protein levels of BCL11A, LSD-1, and KLF1 were suppressed by the compounds. Chromatin immunoprecipitation assays demonstrated a 3.6-fold reduction in LSD1 and HDAC3 occupancy in the γ-globin gene promoter with Benserazide exposure, 3-fold reduction in LSD-1 and HDAC2 occupancy in the γ-globin gene promoter with SDMB exposure, while markers of gene activation (histone H3K9 acetylation and H3K4 demethylation), were enriched 5.7-fold. These findings identify clinical-stage oral therapeutics which inhibit or displace major co-repressors of γ-globin gene transcription and may suggest a rationale for combination therapy to produce enhanced efficacy. PMID:26603726

  17. An embryonic stage–specific enhancer within the murine β-globin locus mediates domain-wide histone hyperacetylation

    PubMed Central

    Fromm, George; Cadiz-Rivera, Brenda; de Vries, Christina; Getman, Michael; McGrath, Kathleen E.; Kingsley, Paul D.; Fields, Jennifer; Fiering, Steven

    2011-01-01

    In mammalian nuclei, a select number of tissue-specific gene loci exhibit broadly distributed patterns of histone modifications, such as histone hyperacetylation, that are normally associated with active gene promoters. Previously, we characterized such hyperacetylated domains within mammalian β-globin gene loci, and determined that within the murine locus, neither the β-globin locus control region nor the gene promoters were required for domain formation. Here, we identify a developmentally specific erythroid enhancer, hypersensitive site-embryonic 1 (HS-E1), located within the embryonic β-globin domain in mouse, which is homologous to a region located downstream of the human embryonic ϵ-globin gene. This sequence exhibits nuclease hypersensitivity in primitive erythroid cells and acts as an enhancer in gain-of-function assays. Deletion of HS-E1 from the endogenous murine β-globin locus results in significant decrease in the expression of the embryonic β-globin genes and loss of the domain-wide pattern of histone hyperacetylation. The data suggest that HS-E1 is an enhancer that is uniquely required for β-like globin expression in primitive erythroid cells, and that it defines a novel class of enhancer that works in part by domain-wide modulation of chromatin structure. PMID:21321362

  18. Correlation of BACH1 and Hemoglobin E/Beta-Thalassemia Globin Expression

    PubMed Central

    Lee, Tze Yan; Muniandy, Logeswaran; Teh, Lai Kuan; Abdullah, Maha; George, Elizabeth; Sathar, Jameela; Lai, Mei I

    2016-01-01

    Objective: The diverse clinical phenotype of hemoglobin E (HbE)/β-thalassemia has not only confounded clinicians in matters of patient management but has also led scientists to investigate the complex mechanisms involved in maintaining the delicate red cell environment where, even with apparent similarities of α- and β-globin genotypes, the phenotype tells a different story. The BTB and CNC homology 1 (BACH1) protein is known to regulate α- and β-globin gene transcriptions during the terminal differentiation of erythroid cells. With the mutations involved in HbE/β-thalassemia disorder, we studied the role of BACH1 in compensating for the globin chain imbalance, albeit for fine-tuning purposes. Materials and Methods: A total of 47 HbE/β-thalassemia samples were analyzed using real-time quantitative polymerase chain reaction and correlated with age, sex, red blood cell parameters, globin gene expressions, and some clinical data. Results: The BACH1 expression among the β-thalassemia intermedia patients varied by up to 2-log differences and was positively correlated to age; α-, β-, and γ-globin gene expression level; and heme oxygenase 1 protein. BACH1 was also negatively correlated to reticulocyte level and had a significant correlation with splenectomy. Conclusion: This study indicates that the expression of BACH1 could be elevated as a compensatory mechanism to decrease the globin chain imbalance as well as to reduce the oxidative stress found in HbE/β-thalassemia. PMID:26377036

  19. [γ-Globin Inductive Therapy of β-thalassemia and Its Relationship with MicroRNA].

    PubMed

    Li, Yao-Yao; Gu, Jian; Yu, Duo-Nan

    2016-04-01

    β-thalassemia is a chronic hemolytic anemia characterized by the reduction or absence of synthesis of β-globin chains because of the β-globin gene mutations. β-thalassemia belongs to the inherited hemoglobin disease, and occurs in some provinces of China, such as in Guangdong, Guangxi, Fujian, its prevalence is about 2%. The treatment of this disease include transfusion, iron chelating agent, hematopoietic stem cell transplantation, splenectomy, induced expression of Fetal Hemoglobin (HbF) and gene therapies. However, the mortality rate of this disease is still higher, thus some new treatments are urgently needed. In recent years, the study was mainly concentrated in 2 aspects: the normal β-globin gene transfer and endogenous γ-globin re-activation. Some studies showed that the expression of miRNAs was dysregulated in β-thalassemia. Some miRNAs could regulate γ-globin at posttranscriptional level, thus, the clarification of relationship between miRNAs and β-thalassemia is expected to provide experimental bases to β-thalassemia therapy. In this review, the induced therapy of γ-globin for β-thalassemia and its relationship with the miRNA are summarized. PMID:27151042

  20. Structure and Properties of a Bis-Histidyl Ligated Globin from Caenorhabditis elegans†

    PubMed Central

    Yoon, Jungjoo; Herzik, Mark A.; Winter, Michael B.; Tran, Rosalie; Olea, Charles; Marletta, Michael A.

    2010-01-01

    Globins are heme-containing proteins that are best known for their roles in oxygen (O2) transport and storage. However, more diverse roles of globins in biology are being revealed, including gas and redox sensing. In the nematode Caenorhabditis elegans, 33 globin or globin-like genes have been recently identified, some of which are known to be expressed in the sensory neurons of the worm and linked to O2 sensing behavior. Here, we describe GLB-6, a novel globin-like protein expressed in the neurons of C. elegans. Recombinantly expressed full-length GLB-6 contains a heme site with spectral features that are similar to those of other bis-histidyl ligated globins, such as neuroglobin and cytoglobin. In contrast to these globins, however, ligands such as CO, NO, and CN− do not bind to the heme in GLB-6, demonstrating that the endogenous histidine ligands are likely very tightly coordinated. Additionally, GLB-6 exhibits rapid two-state autoxidation kinetics in the presence of physiological O2 levels as well as a low redox potential (−193 ± 2 mV). A high resolution (1.40 Å) crystal structure of the ferric form of the heme-domain of GLB-6 confirms both the putative globin-fold and bis-histidyl ligation and also demonstrates key structural features that can be correlated with the unusual ligand binding and redox properties exhibited by the full-length protein. Taken together, the biochemical properties of GLB-6 suggest that this neural protein would most likely serve as a physiological sensor for O2 in C. elegans via redox signaling and/or electron transfer. PMID:20518498

  1. Gene Duplication and the Evolution of Hemoglobin Isoform Differentiation in Birds*

    PubMed Central

    Grispo, Michael T.; Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E.; Storz, Jay F.

    2012-01-01

    The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes as follows: HbA (the major adult Hb isoform, with α-chain subunits encoded by the αA-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the αD-globin gene). The αD-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. Functional experiments involving purified HbA and HbD isoforms from 11 different bird species revealed that HbD is characterized by a consistently higher O2 affinity in the presence of allosteric effectors such as organic phosphates and Cl− ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH dependence of Hb-O2 affinity stems primarily from changes in the O2 association constant of deoxy (T-state)-Hb. Ancestral sequence reconstructions revealed that the amino acid substitutions that distinguish the adult-expressed Hb isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the αD-globin gene that is shared with the embryonic α-like globin gene. PMID:22962007

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

    PubMed Central

    Moon, A M; Ley, T J

    1990-01-01

    DNA sequences located in a region 6-18 kilobases (kb) upstream from the human epsilon-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 beta-like globin gene cluster during erythroid development. The beta-globin LAR activates linked globin genes in transiently or stably transfected erythroleukemia cells and in erythroid cells of transgenic mice. Since the human beta-globin LAR is functional in mice, we reasoned that critical LAR sequence elements might be conserved between mice and humans. We therefore cloned murine genomic sequences homologous to one portion of the human LAR (site II, positions -11,054 to -10,322 with respect to the human epsilon gene). We 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 beta-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 approximately 800 base pairs. Multiple core sequences with greater than 80% identity were present within this region. Transient and stable transfection assays of K562 erythroleukemia cells demonstrated that both human and mouse LAR elements contain enhancer activity and confer hemin inducibility on a linked human gamma-globin promoter. These results suggest that primary structural elements--and the spatial organization of these elements--are important for function of the beta-globin LAR. Images PMID:2217202

  3. Analysis of β-globin chromatin micro-environment using a novel 3C variant, 4Cv.

    PubMed

    Pink, Ryan C; Eskiw, Christopher H; Caley, Daniel P; Carter, David R F

    2010-01-01

    Higher order chromatin folding is critical to a number of developmental processes, including the regulation of gene expression. Recently developed biochemical techniques such as RNA TRAP and chromosome conformation capture (3C) have provided us with the tools to probe chromosomal structures. These techniques have been applied to the β-globin locus, revealing a complex pattern of interactions with regions along the chromosome that the gene resides on. However, biochemical and microscopy data on the nature of β-globin interactions with other chromosomes is contradictory. Therefore we developed a novel 4C variant, Complete-genome 3C by vectorette amplification (4Cv), which allows an unbiased and quantitative method to examine chromosomal structure. We have used 4Cv to study the microenvironment of the β-globin locus in mice and show that a significant proportion of the interactions of β-globin are inter-chromosomal. Furthermore, our data show that in the liver, where the gene is active, β-globin is more likely to interact with other chromosomes, compared to the brain where the gene is silent and is more likely to interact with other regions along the same chromosome. Our data suggest that transcriptional activation of the β-globin locus leads to a change in nuclear position relative to the chromosome territory. PMID:20927371

  4. Compounds of the anthracycline family of antibiotics elevate human gamma-globin expression both in erythroid cultures and in a transgenic mouse model.

    PubMed

    Spyrou, Pandelis; Phylactides, Marios; Lederer, Carsten W; Kithreotis, Lucas; Kirri, Andriani; Christou, Soteroulla; Kkolou, Elena; Kanavakis, Emanuel; Anagnou, Nicholas P; Stamatoyannopoulos, George; Kleanthous, Marina

    2010-01-01

    We examined the effect of the anthracyclines aclarubicin, bleomycin, daunorubicin, doxorubicin and idarubicin on human gamma- and beta-globin promoter activity in an in vitro luciferase assay, ex vivo in erythroid cultures and in vivo in transgenic mice carrying the human gamma-globin gene. Effects in erythroid liquid cultures derived from healthy donors were assayed by evaluating HbF production with high performance liquid chromatography and by measuring mRNA levels of the globin genes and the proportion of erythroblasts containing HbF. Compounds testing positive in the in vitro and ex vivo assays were applied to erythroid cultures derived from thalassaemic patients. Doxorubicin, idarubicin and daunorubicin increased HbF production in cultures of both, healthy and thalassaemic donors. Daunorubicin induced HbF in thalassaemic cells ex vivo with the highest statistical significance and, importantly and in contrast to the clinical HbF inducer hydroxyurea, showed specific induction of gamma-globin without associated induction of alpha-globin. Daunorubicin was screened in transgenic mice carrying the human (A)gamma-globin gene, and it resulted in increased (A)gamma-globin mRNA levels. Our results indicate that anthracyclines are a promising group of compounds with the potential to provide lead substances for the synthesis of new agents with clinical applications as gamma-globin gene inducers. In parallel, future studies of the epigenetic effects of the five anthracyclines on the beta-globin locus will generate possible mechanistic leads on the regulation of the globin genes. PMID:19914848

  5. Recent trends in the gene therapy of β-thalassemia

    PubMed Central

    Finotti, Alessia; Breda, Laura; Lederer, Carsten W; Bianchi, Nicoletta; Zuccato, Cristina; Kleanthous, Marina; Rivella, Stefano; Gambari, Roberto

    2015-01-01

    The β-thalassemias are a group of hereditary hematological diseases caused by over 300 mutations of the adult β-globin gene. Together with sickle cell anemia, thalassemia syndromes are among the most impactful diseases in developing countries, in which the lack of genetic counseling and prenatal diagnosis have contributed to the maintenance of a very high frequency of these genetic diseases in the population. Gene therapy for β-thalassemia has recently seen steadily accelerating progress and has reached a crossroads in its development. Presently, data from past and ongoing clinical trials guide the design of further clinical and preclinical studies based on gene augmentation, while fundamental insights into globin switching and new technology developments have inspired the investigation of novel gene-therapy approaches. Moreover, human erythropoietic stem cells from β-thalassemia patients have been the cellular targets of choice to date whereas future gene-therapy studies might increasingly draw on induced pluripotent stem cells. Herein, we summarize the most significant developments in β-thalassemia gene therapy over the last decade, with a strong emphasis on the most recent findings, for β-thalassemia model systems; for β-, γ-, and anti-sickling β-globin gene addition and combinatorial approaches including the latest results of clinical trials; and for novel approaches, such as transgene-mediated activation of γ-globin and genome editing using designer nucleases. PMID:25737641

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

    PubMed

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

    2016-04-01

    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 24h 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 Δ(9) desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ(9)-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

  7. Beta-Globin Gene Haplotypes Among Cameroonians and Review of the Global Distribution: Is There a Case for a Single Sickle Mutation Origin in Africa?

    PubMed Central

    Bitoungui, Valentina J. Ngo; Pule, Gift D.; Hanchard, Neil; Ngogang, Jeanne

    2015-01-01

    Abstract Studies of hemoglobin S haplotypes in African subpopulations have potential implications for patient care and our understanding of genetic factors that have shaped the prevalence of sickle cell disease (SCD). We evaluated HBB gene cluster haplotypes in SCD patients from Cameroon, and reviewed the literature for a global distribution. We reviewed medical records to obtain pertinent socio-demographic and clinical features for 610 Cameroonian SCD patients, including hemoglobin electrophoresis and full blood counts. RFLP-PCR was used to determine the HBB gene haplotype on 1082 chromosomes. A systematic review of the current literature was undertaken to catalogue HBB haplotype frequencies in SCD populations around the world. Benin (74%; n=799) and Cameroon (19%; n=207) were the most prevalent haplotypes observed among Cameroonian patients. There was no significant association between HBB haplotypes and clinical life events, anthropometric measures, hematological parameters, or fetal hemoglobin (HbF) levels. The literature review of the global haplotype distributions was consistent with known historical migrations of the people of Africa. Previously reported data from Sudan showed a distinctly unusual pattern; all four classical haplotypes were reported, with an exceptionally high proportion of the Senegal, Cameroon, and atypical haplotypes. We did not observe any significant associations between HBB haplotype and SCD disease course in this cohort. Taken together, the data from Cameroon and from the wider literature suggest that a careful reassessment of African HBB haplotypes may shed further light on the evolutionary dynamics of the sickle allele, which could suggest a single origin of the sickle mutation. PMID:25748438

  8. Reversal of hemochromatosis by apotransferrin in non-transfused and transfused Hbbth3/+ (heterozygous B1/B2 globin gene deletion) mice.

    PubMed

    Gelderman, Monique P; Baek, Jin Hyen; Yalamanoglu, Ayla; Puglia, Michele; Vallelian, Florence; Burla, Bo; Vostal, Jaroslav; Schaer, Dominik J; Buehler, Paul W

    2015-05-01

    Intermediate beta-thalassemia has a broad spectrum of sequelae and affected subjects may require occasional blood transfusions over their lifetime to correct anemia. Iron overload in intermediate beta-thalassemia results from a paradoxical intestinal absorption, iron release from macrophages and hepatocytes, and sporadic transfusions. Pathological iron accumulation in parenchyma is caused by chronic exposure to non-transferrin bound iron in plasma. The iron scavenger and transport protein transferrin is a potential treatment being studied for correction of anemia. However, transferrin may also function to prevent or reduce iron loading of tissues when exposure to non-transferrin bound iron increases. Here we evaluate the effects of apotransferrin administration on tissue iron loading and early tissue pathology in non-transfused and transfused Hbb(th3/+) mice. Mice with the Hbb(th3/+) phenotype have mild to moderate anemia and consistent tissue iron accumulation in the spleen, liver, kidneys and myocardium. Chronic apotransferrin administration resulted in normalization of the anemia. Furthermore, it normalized tissue iron content in the liver, kidney and heart and attenuated early tissue changes in non-transfused Hbb(th3/+) mice. Apotransferrin treatment was also found to attenuate transfusion-mediated increases in plasma non-transferrin bound iron and associated excess tissue iron loading. These therapeutic effects were associated with normalization of transferrin saturation and suppressed plasma non-transferrin bound iron. Apotransferrin treatment modulated a fundamental iron regulatory pathway, as evidenced by decreased erythroid Fam132b gene (erythroferrone) expression, increased liver hepcidin gene expression and plasma hepcidin-25 levels and consequently reduced intestinal ferroportin-1 in apotransferrin-treated thalassemic mice. PMID:25616571

  9. Beta-globin gene haplotypes among cameroonians and review of the global distribution: is there a case for a single sickle mutation origin in Africa?

    PubMed

    Bitoungui, Valentina J Ngo; Pule, Gift D; Hanchard, Neil; Ngogang, Jeanne; Wonkam, Ambroise

    2015-03-01

    Studies of hemoglobin S haplotypes in African subpopulations have potential implications for patient care and our understanding of genetic factors that have shaped the prevalence of sickle cell disease (SCD). We evaluated HBB gene cluster haplotypes in SCD patients from Cameroon, and reviewed the literature for a global distribution. We reviewed medical records to obtain pertinent socio-demographic and clinical features for 610 Cameroonian SCD patients, including hemoglobin electrophoresis and full blood counts. RFLP-PCR was used to determine the HBB gene haplotype on 1082 chromosomes. A systematic review of the current literature was undertaken to catalogue HBB haplotype frequencies in SCD populations around the world. Benin (74%; n = 799) and Cameroon (19%; n = 207) were the most prevalent haplotypes observed among Cameroonian patients. There was no significant association between HBB haplotypes and clinical life events, anthropometric measures, hematological parameters, or fetal hemoglobin (HbF) levels. The literature review of the global haplotype distributions was consistent with known historical migrations of the people of Africa. Previously reported data from Sudan showed a distinctly unusual pattern; all four classical haplotypes were reported, with an exceptionally high proportion of the Senegal, Cameroon, and atypical haplotypes. We did not observe any significant associations between HBB haplotype and SCD disease course in this cohort. Taken together, the data from Cameroon and from the wider literature suggest that a careful reassessment of African HBB haplotypes may shed further light on the evolutionary dynamics of the sickle allele, which could suggest a single origin of the sickle mutation. PMID:25748438

  10. Adult mouse brain gene expression patterns bear an embryologic imprint

    PubMed Central

    Zapala, Matthew A.; Hovatta, Iiris; Ellison, Julie A.; Wodicka, Lisa; Del Rio, Jo A.; Tennant, Richard; Tynan, Wendy; Broide, Ron S.; Helton, Rob; Stoveken, Barbara S.; Winrow, Christopher; Lockhart, Daniel J.; Reilly, John F.; Young, Warren G.; Bloom, Floyd E.; Lockhart, David J.; Barlow, Carrolee

    2005-01-01

    The current model to explain the organization of the mammalian nervous system is based on studies of anatomy, embryology, and evolution. To further investigate the molecular organization of the adult mammalian brain, we have built a gene expression-based brain map. We measured gene expression patterns for 24 neural tissues covering the mouse central nervous system and found, surprisingly, that the adult brain bears a transcriptional “imprint” consistent with both embryological origins and classic evolutionary relationships. Embryonic cellular position along the anterior–posterior axis of the neural tube was shown to be closely associated with, and possibly a determinant of, the gene expression patterns in adult structures. We also observed a significant number of embryonic patterning and homeobox genes with region-specific expression in the adult nervous system. The relationships between global expression patterns for different anatomical regions and the nature of the observed region-specific genes suggest that the adult brain retains a degree of overall gene expression established during embryogenesis that is important for regional specificity and the functional relationships between regions in the adult. The complete collection of extensively annotated gene expression data along with data mining and visualization tools have been made available on a publicly accessible web site (www.barlow-lockhart-brainmapnimhgrant.org). PMID:16002470

  11. Characterization of Clinical and Laboratory Profiles of the Deletional α2-Globin Gene Polyadenylation Signal Sequence (AATAAA > AATA- -) in an Indian Population.

    PubMed

    Deshpande, Prashant; Kamalanathan, Neelagandan; Sampath, Eswari; George, Biju; Shaji, Ramachandran V; Edison, Eunice S

    2015-01-01

    α-Thalassemia (α-thal) is characterized by large deletions involving the variable regions of α2 and/or α1 genes. Nondeletional mutations and polyadenylation (polyA) signal sequence motif mutations are less common. In this retrospective study, we describe a fragment length analysis-based polymerase chain reaction (PCR) assay for screening the T(Indian) (AATAAA > AATA- -; HBA2: c.*93_*94delAA) polyA signal deletion along with its clinical and laboratory presentation in 21 patients. Most of the patients were diagnosed in early adulthood with a clinical presentation ranging from asymptomatic in the heterozygous state to severe Hb H disease with a prominent hemolytic component in the homozygous state. On genetic analysis, 14 patients were found to be homozygotes, five were compound heterozygotes and two were heterozygotes. Thus, the T(Indian) polyA signal deletion is common in the Indian population and should be screened for in patients with nondeletional α-thal mutations. PMID:26365411

  12. A New Intergenic α-Globin Deletion (α-α(Δ125)) Found in a Kabyle Population.

    PubMed

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

    2016-03-01

    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. PMID:26911300

  13. The relative cellular levels of CP2a and CP2b potentiates erythroid cell-specific expression of the {alpha}-globin gene by regulating the nuclear localization of CP2c

    SciTech Connect

    Chae, Ji Hyung; Kang, Ho Chul; Kim, Chul Geun

    2009-03-20

    CP2b activates {alpha}-globin expression in an erythroid cell-specific manner, through interaction with CP2c and PIAS1. Although CP2a is identical to CP2b except for lacking an exon encoding additional 36 amino acids and has the intrinsic DNA binding and transactivation properties, it does not exert any role in {alpha}-globin expression. Investigation of subcellular localization of exogenous CP2 proteins revealed that CP2a and CP2b were exclusively localized in the cytosol and nucleus, respectively. The CP2b-specific exon was in charge of the nuclear localization of CP2b. Interestingly, subcellular localization of CP2c was either in the nucleus or cytosol depending on the relative level of CP2a and CP2b although CP2c intrinsically localized in the cytosol in the absence of CP2a/CP2b. Finally, dramatic increment of hemoglobin expression was correlated with nuclear translocation of CP2c during MEL cell differentiation. Our data suggest that CP2b potentiate erythroid cell-specific {alpha}-globin expression by recruiting CP2c into the nucleus.

  14. Hemoglobin Agenogi--A rare abnormal beta globin chain variant.

    PubMed

    Sharma, Sunita; Sharma, Geetika; Chandra, Jagdish; Colah, Roshan

    2016-01-01

    Haemoglobin (Hb) Agenogi is clinically asymptomatic, rare β-globin chain variant characterized by a substitution of glutamic acid by lysine at position 90 of β-chain. It elutes in the C-window on high-performance liquid chromatography (HPLC). We report a 10-year-old male with easy fatigability, lethargy, pallor, and mild splenomegaly. Hematological parameters revealed microcytic hypochromic anemia and mildly raised red blood cells count, suggestive of thalassemia trait. On HPLC, a predominant peak was observed in the C-window (82.6%) along with raised HbA 2 level (9.3%). Based on these findings, a possibility of HbC disease/β-thalassemia trait doubly heterozygous was considered. Family studies were advised. HPLC findings in father were suggestive of β-thalassemia trait, while both his mother and brother had an abnormal peak in the C-window of 42.7% and 40.8%, respectively, with elevated HbA 2 values of 5% and 4.9%, respectively. Direct DNA sequencing revealed intervening sequences 1-5 (G ; C) in father, confirming β-thalassemia trait. His mother and brother had heterozygous gene mutation at codon 90 of β-globin chain (G ; A) suggestive of Hb Agenogi. The child carried mutations for both β-thalassemia trait as well as Hb Agenogi. PMID:26960650

  15. Age-Related Gene Expression Differences in Monocytes from Human Neonates, Young Adults, and Older Adults.

    PubMed

    Lissner, Michelle M; Thomas, Brandon J; Wee, Kathleen; Tong, Ann-Jay; Kollmann, Tobias R; Smale, Stephen T

    2015-01-01

    A variety of age-related differences in the innate and adaptive immune systems have been proposed to contribute to the increased susceptibility to infection of human neonates and older adults. The emergence of RNA sequencing (RNA-seq) provides an opportunity to obtain an unbiased, comprehensive, and quantitative view of gene expression differences in defined cell types from different age groups. An examination of ex vivo human monocyte responses to lipopolysaccharide stimulation or Listeria monocytogenes infection by RNA-seq revealed extensive similarities between neonates, young adults, and older adults, with an unexpectedly small number of genes exhibiting statistically significant age-dependent differences. By examining the differentially induced genes in the context of transcription factor binding motifs and RNA-seq data sets from mutant mouse strains, a previously described deficiency in interferon response factor-3 activity could be implicated in most of the differences between newborns and young adults. Contrary to these observations, older adults exhibited elevated expression of inflammatory genes at baseline, yet the responses following stimulation correlated more closely with those observed in younger adults. Notably, major differences in the expression of constitutively expressed genes were not observed, suggesting that the age-related differences are driven by environmental influences rather than cell-autonomous differences in monocyte development. PMID:26147648

  16. O-Linked N-Acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Interact with Mi2β Protein at the Aγ-Globin Promoter.

    PubMed

    Zhang, Zhen; Costa, Flávia C; Tan, Ee Phie; Bushue, Nathan; DiTacchio, Luciano; Costello, Catherine E; McComb, Mark E; Whelan, Stephen A; Peterson, Kenneth R; Slawson, Chad

    2016-07-22

    One mode of γ-globin gene silencing involves a GATA-1·FOG-1·Mi2β repressor complex that binds to the -566 GATA site relative to the (A)γ-globin gene cap site. However, the mechanism of how this repressor complex is assembled at the -566 GATA site is unknown. In this study, we demonstrate that the O-linked N-acetylglucosamine (O-GlcNAc) processing enzymes, O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA), interact with the (A)γ-globin promoter at the -566 GATA repressor site; however, mutation of the GATA site to GAGA significantly reduces OGT and OGA promoter interactions in β-globin locus yeast artificial chromosome (β-YAC) bone marrow cells. When WT β-YAC bone marrow cells are treated with the OGA inhibitor Thiamet-G, the occupancy of OGT, OGA, and Mi2β at the (A)γ-globin promoter is increased. In addition, OGT and Mi2β recruitment is increased at the (A)γ-globin promoter when γ-globin becomes repressed in postconception day E18 human β-YAC transgenic mouse fetal liver. Furthermore, we show that Mi2β is modified with O-GlcNAc, and both OGT and OGA interact with Mi2β, GATA-1, and FOG-1. Taken together, our data suggest that O-GlcNAcylation is a novel mechanism of γ-globin gene regulation mediated by modulating the assembly of the GATA-1·FOG-1·Mi2β repressor complex at the -566 GATA motif within the promoter. PMID:27231347

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

    SciTech Connect

    Filon, D.; Oron, V.; Krichevski, S.; Shaag, A.; Goldfarb, A.; Aker, M.; Rachmilewitz, E.A.; Rund, D.; Oppenheim, A. )

    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. Spectrum of Common α-Globin Deletion Mutations in the Southern Region of Vietnam.

    PubMed

    Bui Thi Kim, Ly; Phu Chi, Dung; Hoang Thanh, Chi

    2016-06-01

    The common deletion mutations of α-globin genes in the Vietnamese population is not well known. Here we report the presence of five deletional mutations of Southeast Asia in the southern region of Vietnam. The - -(SEA) (NG_000006.1: g.26264_45564del19301) mutation is the most common type of deletion (87.35%), followed by the -α(3.7) (rightward) (NG_000006.1: g.34164_37967del3804) deletion (9.64%), -α(4.2) (leftward) (AF221717) deletion (2.41%) and - -(THAI) (NG_000006.1: g.10664_44164del33501) (0.6%) mutation in this region. The - -(FIL) (NG_000006.1: g.11684_43534del31581) mutation was not detected in this study. This result provided a view of the distribution of common α-globin gene mutations in Vietnam and could serve as a baseline for further investigations into these genetic defects. PMID:27117571

  19. Why the DNA self-depurination mechanism operates in HB-β but not in β-globin paralogs HB-δ, HB-ɛ1, HB-γ1 and HB-γ2.

    PubMed

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

    2015-08-01

    The human β-globin, δ-globin and ɛ-globin genes contain almost identical coding strand sequences centered about codon 6 having potential to form a stem-loop with a 5'GAGG loop. Provided with a sufficiently stable stem, such a structure can self-catalyze depurination of the loop 5'G residue, leading to a potential mutation hotspot. Previously, we showed that such a hotspot exists about codon 6 of β-globin, with by far the highest incidence of mutations across the gene, including those responsible for 6 anemias (notably Sickle Cell Anemia) and β-thalassemias. In contrast, we show here that despite identical loop sequences, there is no mutational hotspot in the δ- or ɛ1-globin potential self-depurination sites, which differ by only one or two base pairs in the stem region from that of the β-globin gene. These differences result in either one or two additional mismatches in the potential 7-base pair-forming stem region, thereby weakening its stability, so that either DNA cruciform extrusion from the duplex is rendered ineffective or the lifetime of the stem-loop becomes too short to permit self-catalysis to occur. Having that same loop sequence, paralogs HB-γ1 and HB-γ2 totally lack stem-forming potential. Hence the absence in δ- and ɛ1-globin genes of a mutational hotspot in what must now be viewed as non-functional homologs of the self-depurination site in β-globin. Such stem-destabilizing variants appeared early among vertebrates and remained conserved among mammals and primates. Thus, this study has revealed conserved sequence determinants of self-catalytic DNA depurination associated with variability of mutation incidence among human β-globin paralogs. PMID:26042536

  20. Regulation and Function of Adult Neurogenesis. From Genes to Cognition

    DOE PAGESBeta

    Aimone, J. B.; Li, Y.; Lee, S. W.; Clemenson, G. D.; Deng, W.; Gage, F. H.

    2014-10-01

    Adult neurogenesis in the hippocampus is a notable process due not only to its uniqueness and potential impact on cognition but also to its localized vertical integration of different scales of neuroscience, ranging from molecular and cellular biology to behavior. Our review summarizes the recent research regarding the process of adult neurogenesis from these different perspectives, with particular emphasis on the differentiation and development of new neurons, the regulation of the process by extrinsic and intrinsic factors, and their ultimate function in the hippocampus circuit. Arising from a local neural stem cell population, new neurons progress through several stages ofmore » maturation, ultimately integrating into the adult dentate gyrus network. Furthermore, the increased appreciation of the full neurogenesis process, from genes and cells to behavior and cognition, makes neurogenesis both a unique case study for how scales in neuroscience can link together and suggests neurogenesis as a potential target for therapeutic intervention for a number of disorders.« less

  1. Regulation and Function of Adult Neurogenesis. From Genes to Cognition

    SciTech Connect

    Aimone, J. B.; Li, Y.; Lee, S. W.; Clemenson, G. D.; Deng, W.; Gage, F. H.

    2014-10-01

    Adult neurogenesis in the hippocampus is a notable process due not only to its uniqueness and potential impact on cognition but also to its localized vertical integration of different scales of neuroscience, ranging from molecular and cellular biology to behavior. Our review summarizes the recent research regarding the process of adult neurogenesis from these different perspectives, with particular emphasis on the differentiation and development of new neurons, the regulation of the process by extrinsic and intrinsic factors, and their ultimate function in the hippocampus circuit. Arising from a local neural stem cell population, new neurons progress through several stages of maturation, ultimately integrating into the adult dentate gyrus network. Furthermore, the increased appreciation of the full neurogenesis process, from genes and cells to behavior and cognition, makes neurogenesis both a unique case study for how scales in neuroscience can link together and suggests neurogenesis as a potential target for therapeutic intervention for a number of disorders.

  2. Regulation and Function of Adult Neurogenesis: From Genes to Cognition

    PubMed Central

    Aimone, James B.; Li, Yan; Lee, Star W.; Clemenson, Gregory D.; Deng, Wei; Gage, Fred H.

    2014-01-01

    Adult neurogenesis in the hippocampus is a notable process due not only to its uniqueness and potential impact on cognition but also to its localized vertical integration of different scales of neuroscience, ranging from molecular and cellular biology to behavior. This review summarizes the recent research regarding the process of adult neurogenesis from these different perspectives, with particular emphasis on the differentiation and development of new neurons, the regulation of the process by extrinsic and intrinsic factors, and their ultimate function in the hippocampus circuit. Arising from a local neural stem cell population, new neurons progress through several stages of maturation, ultimately integrating into the adult dentate gyrus network. The increased appreciation of the full neurogenesis process, from genes and cells to behavior and cognition, makes neurogenesis both a unique case study for how scales in neuroscience can link together and suggests neurogenesis as a potential target for therapeutic intervention for a number of disorders. PMID:25287858

  3. Widespread occurrence of N-terminal acylation in animal globins and possible origin of respiratory globins from a membrane-bound ancestor.

    PubMed

    Blank, Miriam; Burmester, Thorsten

    2012-11-01

    Proteins of the (hemo-)globin superfamily have been identified in many different animals but also occur in plants, fungi, and bacteria. Globins are renowned for their ability to store and to transport oxygen, but additional globin functions such as sensing, signaling, and detoxification have been proposed. Recently, we found that the zebrafish globin X protein is myristoylated and palmitoylated at its N-terminus. The addition of fatty acids results in an association with the cellular membranes, suggesting a previously unrecognized globin function. In this study, we show that N-terminal acylation likely occurs in globin proteins from a broad range of phyla. An N-terminal myristoylation site was identified in 90 nonredundant globins from Chlorophyta, Heterokontophyta, Cnidaria, Mollusca, Arthropoda, Nematoda, Echinodermata, Hemichordata, and Chordata (including Cephalochordata), of which 66 proteins carry an additional palmitoylation site. Bayesian phylogenetic analyses identified five major globin families, which may mirror the ancient globin diversity of the Metazoa. Globin X-like proteins form two related clades, which diverged before the radiation of the Eumetazoa. Vertebrate hemoglobin (Hb), myoglobin, cytoglobin, globin E, and globin Y form a strongly supported common clade, which is the sister group of a clade consisting of invertebrate Hbs and relatives. The N-terminally acylated globins do not form a single monophyletic group but are distributed to four distinct clades. This pattern may be either explained by multiple introduction of an N-terminal acylation site into distinct globin lineages or by the origin of animal respiratory globins from a membrane-bound ancestor. Similarly, respiratory globins were not monophyletic. This suggests that respiratory globins might have emerged independently several times and that the early metazoan globins might have been associated with a membrane and carried out a function that was related to lipid protection or

  4. Homologous globin cell-free transcription system with comparison of heterologous factors.

    PubMed Central

    Tolunay, H E; Yang, L; Kemper, W M; Safer, B; Anderson, W F

    1984-01-01

    Mouse erythroleukemia (MEL) cells provide a useful model system to examine the regulation of globin gene expression. MEL cells ordinarily do not express globin genes, but in the presence of inducers, such as dimethyl sulfoxide or hexamethylene bisacetamide, they mimic erythroid differentiation. We have developed a cell-free transcription system from uninduced MEL cells to determine the requirements for mRNA synthesis. The MEL system directs accurate transcription of adenovirus type 2 major late DNA and mouse betamaj-globin with an efficiency comparable to those of HeLa and KB cell extracts. Using the procedure of Matsui et al. (T. Matsui, J. Segall, P.A. Weil, and R.G. Roeder, J. Biol. Chem. 255:11992-11996, 1980), we have isolated three active fractions from both MEL and HeLa cell extracts which are required for accurate transcription and have shown that equivalent fractions from MEL and HeLa cell extracts are interchangeable. Our findings suggest that the components required for initiation of transcription are similar in different cell types, at least to the extent that they can be assayed in these in vitro systems. Images PMID:6583493

  5. GATA-1 modulates the chromatin structure and activity of the chicken alpha-globin 3' enhancer.

    PubMed

    Escamilla-Del-Arenal, Martín; Recillas-Targa, Félix

    2008-01-01

    Long-distance regulatory elements and local chromatin structure are critical for proper regulation of gene expression. Here we characterize the chromatin conformation of the chicken alpha-globin silencer-enhancer elements located 3' of the domain. We found a characteristic and erythrocyte-specific structure between the previously defined silencer and the enhancer, defined by two nuclease hypersensitive sites, which appear when the enhancer is active during erythroid differentiation. Fine mapping of these sites demonstrates the absence of a positioned nucleosome and the association of GATA-1. Functional analyses of episomal vectors, as well as stably integrated constructs, revealed that GATA-1 plays a major role in defining both the chromatin structure and the enhancer activity. We detected a progressive enrichment of histone acetylation on critical enhancer nuclear factor binding sites, in correlation with the formation of an apparent nucleosome-free region. On the basis of these results, we propose that the local chromatin structure of the chicken alpha-globin enhancer plays a central role in its capacity to differentially regulate alpha-globin gene expression during erythroid differentiation and development. PMID:17984219

  6. α-Globin as a molecular target in the treatment of β-thalassemia

    PubMed Central

    Mettananda, Sachith; Gibbons, Richard J.

    2015-01-01

    The thalassemias, together with sickle cell anemia and its variants, are the world’s most common form of inherited anemia, and in economically undeveloped countries, they still account for tens of thousands of premature deaths every year. In developed countries, treatment of thalassemia is also still far from ideal, requiring lifelong transfusion or allogeneic bone marrow transplantation. Clinical and molecular genetic studies over the course of the last 50 years have demonstrated how coinheritance of modifier genes, which alter the balance of α-like and β-like globin gene expression, may transform severe, transfusion-dependent thalassemia into relatively mild forms of anemia. Most attention has been paid to pathways that increase γ-globin expression, and hence the production of fetal hemoglobin. Here we review the evidence that reduction of α-globin expression may provide an equally plausible approach to ameliorating clinically severe forms of β-thalassemia, and in particular, the very common subgroup of patients with hemoglobin E β-thalassemia that makes up approximately half of all patients born each year with severe β-thalassemia. PMID:25869286

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

    PubMed Central

    Kimura, Elza Miyuki; Oliveira, Denise Madureira; Jorge, Susan Elisabeth; Ribeiro, Daniela Maria; Zaccariotto, Tânia Regina; Santos, Magnun Nueldo Nunes; Almeida, Vanessa; Albuquerque, Dulcinéia Martins; Costa, Fernando Ferreira; Sonati, Maria de Fátima

    2015-01-01

    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. PMID:25818820

  8. A five prime splice-region G yields C mutation in exon 1 of the human. beta. -globin gene inhibits pre-mRNA splicing: A mechanism for. beta. sup + -thalassemia

    SciTech Connect

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

    1989-02-01

    The authors have characterized a Mediterranean {beta}-thalassemia allele containing a sequence change at codon 30 that alters both {beta}-globin pre-mRNA splicing and the structure of the homoglobin product. Presumably, this G {yields} C transversion at position {minus}1 of intron 1 reduces severely the utilization of the normal 5{prime} splice site since the level of the Arg {yields} 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 {minus}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{prime} 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.

  9. Determination of Ligand Pathways in Globins

    PubMed Central

    Salter, Mallory D.; Blouin, George C.; Soman, Jayashree; Singleton, Eileen W.; Dewilde, Sylvia; Moens, Luc; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; Olson, John S.

    2012-01-01

    Although molecular dynamics simulations suggest multiple interior pathways for O2 entry into and exit from globins, most experiments indicate well defined single pathways. In 2001, we highlighted the effects of large-to-small amino acid replacements on rates for ligand entry and exit onto the three-dimensional structure of sperm whale myoglobin. The resultant map argued strongly for ligand movement through a short channel from the heme iron to solvent that is gated by the distal histidine (His-64(E7)) near the solvent edge of the porphyrin ring. In this work, we have applied the same mutagenesis mapping strategy to the neuronal mini-hemoglobin from Cerebratulus lacteus (CerHb), which has a large internal tunnel from the heme iron to the C-terminal ends of the E and H helices, a direction that is 180° opposite to the E7 channel. Detailed comparisons of the new CerHb map with expanded results for Mb show unambiguously that the dominant (>90%) ligand pathway in CerHb is through the internal tunnel, and the major (>75%) ligand pathway in Mb is through the E7 gate. These results demonstrate that: 1) mutagenesis mapping can identify internal pathways when they exist; 2) molecular dynamics simulations need to be refined to address discrepancies with experimental observations; and 3) alternative pathways have evolved in globins to meet specific physiological demands. PMID:22859299

  10. Analysis of beta-globin mutations shows stable mixed chimerism in patients with thalassemia after bone marrow transplantation.

    PubMed

    Kapelushnik, J; Or, R; Filon, D; Nagler, A; Cividalli, G; Aker, M; Naparstek, E; Slavin, S; Oppenheim, A

    1995-10-15

    Beta-thalassemia major (TM) is caused by any of approximately 150 mutations within the beta-globin gene. To establish the degree of chimerism after bone marrow transplantation (BMT), we have performed molecular analysis of beta-globin mutations in 14 patients with TM over a period of 10 years. All patients underwent T cell-depleted allogeneic BMT from HLA-identical related donors, using either in vitro T-cell depletion with CAMPATH 1M and complement or in vivo depletion using CAMPATH 1G in the bone marrow collection bag. To date, at different time periods after BMT, seven patients have some degree of chimerism; six of these patients, all blood transfusion-independent, have donor cells in the range of 70% to 95%, with stable mixed chimerism (MC). The seventh patient has less than 10% donor cells with, surprisingly, only minimal transfusion requirements. The detection of beta-globin gene point mutation, as used here, is a highly specific and sensitive marker for engraftment and MC in patients with thalassemia. In light of its specificity, the method is applicable in all cases of TM, as it is independent of sex and other non-globin-related DNA markers. The high incidence of MC found in our patients may be a consequence of the pre-BMT T-cell depletion. Because MC was associated with transfusion independence, complete eradication of residual host cells for effective treatment of TM and possibly other genetic diseases may prove not to be essential. PMID:7579421

  11. ATM localization and gene expression in the adult mouse eye

    PubMed Central

    Leemput, Julia; Masson, Christel; Bigot, Karine; Errachid, Abdelmounaim; Dansault, Anouk; Provost, Alexandra; Gadin, Stéphanie; Aoufouchi, Said; Menasche, Maurice

    2009-01-01

    Purpose High levels of metabolism and oxygen consumption in most adult murine ocular compartments, combined with exposure to light and ultraviolet (UV) radiation, are major sources of oxidative stress, causing DNA damage in ocular cells. Of all mammalian body cells, photoreceptor cells consume the largest amount of oxygen and generate the highest levels of oxidative damage. The accumulation of such damage throughout life is a major factor of aging tissues. Several multiprotein complexes have recently been identified as the major sensors and mediators involved in the maintenance of DNA integrity. The activity of these complexes initially seemed to be restricted to dividing cells, given their ultimate role in major cell cycle checkpoints. However, it was later established that they are also active in post-mitotic cells. Recent findings demonstrate that the DNA damage response (DDR) is essential for the development, maintenance, and normal functioning of the adult central nervous system. One major molecular factor in the DDR is the protein, ataxia telangiectasia mutated (ATM). It is required for the rapid induction of cellular responses to DNA double-strand breaks. These cytotoxic DNA lesions may be caused by oxidative damage. To understand how ATM prevents oxidative stress and participates in the maintenance of genomic integrity and cell viability of the adult retina, we determined the ATM expression patterns and studied its localization in the adult mouse eye. Methods Atm gene expression was analyzed by RT–PCR experiments and its localization by in situ hybridization on adult mouse ocular and cerebellar tissue sections. ATM protein expression was determined by western blot analysis of proteins homogenates extracted from several mouse tissues and its localization by immunohistochemistry experiments performed on adult mouse ocular and cerebellar tissue sections. In addition, subcellular localization was realized by confocal microscopy imaging of ocular tissue

  12. Globin X is a six-coordinate globin that reduces nitrite to nitric oxide in fish red blood cells.

    PubMed

    Corti, Paola; Xue, Jianmin; Tejero, Jesús; Wajih, Nadeem; Sun, Ming; Stolz, Donna B; Tsang, Michael; Kim-Shapiro, Daniel B; Gladwin, Mark T

    2016-07-26

    The discovery of novel globins in diverse organisms has stimulated intense interest in their evolved function, beyond oxygen binding. Globin X (GbX) is a protein found in fish, amphibians, and reptiles that diverged from a common ancestor of mammalian hemoglobins and myoglobins. Like mammalian neuroglobin, GbX was first designated as a neuronal globin in fish and exhibits six-coordinate heme geometry, suggesting a role in intracellular electron transfer reactions rather than oxygen binding. Here, we report that GbX to our knowledge is the first six-coordinate globin and the first globin protein apart from hemoglobin, found in vertebrate RBCs. GbX is present in fish erythrocytes and exhibits a nitrite reduction rate up to 200-fold faster than human hemoglobin and up to 50-fold higher than neuroglobin or cytoglobin. Deoxygenated GbX reduces nitrite to form nitric oxide (NO) and potently inhibits platelet activation in vitro, to a greater extent than hemoglobin. Fish RBCs also reduce nitrite to NO and inhibit platelet activation to a greater extent than human RBCs, whereas GbX knockdown inhibits this nitrite-dependent NO signaling. The description of a novel, six-coordinate globin in RBCs with dominant electron transfer and nitrite reduction functionality provides new insights into the evolved signaling properties of ancestral heme-globins. PMID:27407144

  13. Experience-dependent gene expression in adult visual cortex.

    PubMed

    Chen, Jiabin; Yamahachi, Homare; Gilbert, Charles D

    2010-03-01

    Experience-dependent plasticity of the adult visual cortex underlies perceptual learning and recovery of function following central nervous system lesions. To reveal the signal transduction cascades involved in adult cortical plasticity, we utilized a model of remapping of cortical topography following binocular retinal lesions. In this model, the lesion projection zone (LPZ) of primary visual cortex (V1) recovers visually driven activity by the sprouting of horizontal axonal connections originating from the cells in the surrounding region. To explore the molecular mechanism underlying this process, we used gene microarrays from an expression library prepared from Macaque V1. By microarray analysis of gene expression levels in the LPZ and the surrounding region, and subsequent confirmation with Quantitative Real-Time polymerase chain reaction and in situ hybridization, the participation of a number of genes was observed, including the Rho GTPase family. Its role in regulation of cytoskeleton assembly provides a possible link between the alteration of neural activity and cortical functional reorganization. PMID:19571270

  14. An anatomic gene expression atlas of the adult mouse brain.

    PubMed

    Ng, Lydia; Bernard, Amy; Lau, Chris; Overly, Caroline C; Dong, Hong-Wei; Kuan, Chihchau; Pathak, Sayan; Sunkin, Susan M; Dang, Chinh; Bohland, Jason W; Bokil, Hemant; Mitra, Partha P; Puelles, Luis; Hohmann, John; Anderson, David J; Lein, Ed S; Jones, Allan R; Hawrylycz, Michael

    2009-03-01

    Studying gene expression provides a powerful means of understanding structure-function relationships in the nervous system. The availability of genome-scale in situ hybridization datasets enables new possibilities for understanding brain organization based on gene expression patterns. The Anatomic Gene Expression Atlas (AGEA) is a new relational atlas revealing the genetic architecture of the adult C57Bl/6J mouse brain based on spatial correlations across expression data for thousands of genes in the Allen Brain Atlas (ABA). The AGEA includes three discovery tools for examining neuroanatomical relationships and boundaries: (1) three-dimensional expression-based correlation maps, (2) a hierarchical transcriptome-based parcellation of the brain and (3) a facility to retrieve from the ABA specific genes showing enriched expression in local correlated domains. The utility of this atlas is illustrated by analysis of genetic organization in the thalamus, striatum and cerebral cortex. The AGEA is a publicly accessible online computational tool integrated with the ABA (http://mouse.brain-map.org/agea). PMID:19219037

  15. Accurate initiation of human epsilon-globin RNA synthesis by Escherichia coli RNA polymerase in isolated nuclei of K562 erythroleukemia cells.

    PubMed Central

    Gilmour, R S; Allan, M; Paul, J

    1984-01-01

    The human epsilon-globin gene was transcribed in vitro in isolated K562 cell nuclei by using exogenous Escherichia coli RNA polymerase (EC 2.7.7.6). Newly formed RNA transcripts were distinguished from nuclear RNA molecules by (i) incorporating mercurated UTP into RNA under conditions in which the endogenous polymerase II is inactive and (ii) subsequently isolating the mercurated RNA by affinity chromatography on thiolated Sepharose. A specific 5'-end-labeled probe spanning the epsilon-globin gene cap site was used in nuclease S1 mapping studies to examine the in vitro initiation site of the isolated transcripts. It was found that transcription occurred from the coding strand only and originated almost entirely from a point that was identical to that of the major cap site for epsilon-globin mRNA in vivo. Images PMID:6330734

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

    MedlinePlus

    ... blood cells. Specifically, it alters a molecule called hemoglobin within these cells. Hemoglobin within red blood cells attaches (binds) to oxygen ... in tissues throughout the body. Instead of normal hemoglobin, people with methemoglobinemia, beta-globin type have an ...

  17. Gene Test Might One Day Gauge Alzheimer's Risk in Younger Adults

    MedlinePlus

    ... 159737.html Gene Test Might One Day Gauge Alzheimer's Risk in Younger Adults But doctors say the ... day be able to predict the risk for Alzheimer's disease in young adults, a new study suggests. ...

  18. Gene Test Might One Day Gauge Alzheimer's Risk in Younger Adults

    MedlinePlus

    ... news/fullstory_159737.html Gene Test Might One Day Gauge Alzheimer's Risk in Younger Adults But doctors ... 2016 (HealthDay News) -- A gene test may one day be able to predict the risk for Alzheimer's ...

  19. Bone status of adult female butyrylcholinesterase gene-deficient mice.

    PubMed

    Haupt, Malte; Kauschke, Vivien; Sender, Jonas; Kampschulte, Marian; Kovtun, Anna; Dürselen, Lutz; Heiss, Christian; Lips, Katrin Susanne

    2015-11-01

    Butyrylcholinesterase (BChE) degrades acetylcholine in addition to acetylcholinesterase (AChE) which is involved in embryonic development of limbs. Since BChE is expressed by osteoblast-like cells we asked whether it is functional in adult bone remodeling. We addressed this issue by analyzing BChE gene-deficient mice (BChE-KO). Bones were extracted from 16-week old female BChE-KO and corresponding wild type mice (WT). Femoral bones were used for biomechanical testing and μCT evaluation of cancellous and cortical bone. Also vertebrae Th12 and L1 were investigated with μCT while L3 was used for tartrate-resistant acidic phosphatase (TRAP) histomorphometry and Th10 for gene expression analysis by means of real-time RT-PCR. BChE-KO did not reveal significant differences in biomechanical bone strength and bone mineral density determined by μCT. Microarchitecture of cancellous and cortical bone showed an increase in μCT parameters like trabecular thickness, trabecular separation, and relative cortical bone area of femoral BChE-KO bone compared to WT. In vertebrae no changes of microstructure and mRNA expression were detected. However, osteoclast histomorphometry with TRAP stained sections demonstrated a significant increase in relative osteoclast number. In conclusion, in adult murine bone the role of BChE is limited to bone specific changes in microarchitecture and to an increase in relative number of bone resorbing osteoclasts whereas the main collagen resorbing enzyme Cathepsin-K (CtsK) was stably expressed. Besides, AChE might be able to compensate the lack of BChE. Thus, further analyses using bone tissue specific AChE BChE cre-lox double knockout mice would be helpful. PMID:26138460

  20. A Digital Gene Expression-Based Bovine Gene Atlas Evaluating 92 Adult, Juvenile and Fetal Cattle Tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A comprehensive transcriptome survey, or “Gene Atlas,” provides information essential for a complete understanding of the genomic biology of an organism. Using a digital gene expression approach, we developed a Gene Atlas of RNA abundance in 92 adult, juvenile and fetal cattle tissues. The samples...

  1. Insulation of the Chicken β-Globin Chromosomal Domain from a Chromatin-Condensing Protein, MENT

    PubMed Central

    Istomina, Natalia E.; Shushanov, Sain S.; Springhetti, Evelyn M.; Karpov, Vadim L.; A. Krasheninnikov, Igor; Stevens, Kimberly; Zaret, Kenneth S.; Singh, Prim B.; Grigoryev, Sergei A.

    2003-01-01

    Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken β-globin domain. We observed two sharp transitions of MENT concentration coinciding with the β-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation. PMID:12944473

  2. Complexity of the alpha-globin genotypes identified with thalassemia screening in Sardinia.

    PubMed

    Origa, Raffaella; Paglietti, Maria E; Sollaino, Maria C; Desogus, Maria F; Barella, Susanna; Loi, Daniela; Galanello, Renzo

    2014-01-01

    α-Thalassemia commonly results from deletions or point mutations in one or both α-globin genes located on chromosome 16p13.3 giving rise to complex and variable genotypes and phenotypes. Rarely, unusual non-deletion defects or atypical deletions down-regulate the expression of the α-globin gene. In the last decade of the program for β-thalassemia carrier screening and genetic counseling in Sardinia, the association of new techniques of molecular biology such as gene sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) to conventional methods has allowed to better define several thalassemic genotypes and the complex variability of the α-cluster with its flanking regions, with a high frequency of different genotypes and compound heterozygosity for two α mutations even in the same family. The exact molecular definition of the genotypes resulting from the interactions among the large number of α-thalassemia determinants and with β-thalassemia, is important for a correct correlation of genotype-phenotype and to prevent underdiagnosis of carrier status which could hamper the effectiveness of a screening program particularly in those regions where a high frequency of hemoglobinopathies is present. PMID:23896219

  3. Gene-environment contributions to young adult sexual partnering.

    PubMed

    Halpern, Carolyn T; Kaestle, Christine E; Guo, Guang; Hallfors, Denise D

    2007-08-01

    To date, there has been relatively little work on gene-environment contributions to human sexuality, especially molecular analyses examining the potential contributions of specific polymorphisms in conjunction with life experiences. Using Wave III data from 717 heterozygous young adult sibling pairs included in the National Longitudinal Study of Adolescent Health, this article examined the combined contributions of attendance at religious services and three genetic polymorphisms (in the dopamine D4 receptor [DRD4]), dopamine D2 receptor [DRD2]), and the serotonin transporter promoter [5HTT]) to sensation seeking, a personality construct related to sexual behavior, and the number of vaginal sex partners participants had in the year before interview. Data analyses used an Allison mixed model approach to account for population stratification and correlated observations. DRD4 was unrelated to sensation seeking and to the number of sex partners in tests of both main effects and in interaction with religious attendance. Contrary to hypothesis, presence of the A1 DRD2 allele was associated with having had fewer sex partners in the past year. Associations between the 5HTT allele and sex partners varied by religious attendance, but again the patterns of associations were contrary to hypothesized relationships and were small in magnitude. These findings underscore the necessity of using more comprehensive multiple gene-multiple life experience approaches to investigations of complex behaviors such as sexual patterns. PMID:17186131

  4. Cooperativeness of the Higher Chromatin Structure of the β-Globin Locus Revealed by the Deletion Mutations of DNase I Hypersensitive site 3 of the LCR

    PubMed Central

    Fang, Xiangdong; Xiang, Ping; Yin, Wenxuan; Stamatoyannopoulos, George; Li, Qiliang

    2010-01-01

    High-level transcription of the globin genes requires the enhancement by a distant element, the locus control region (LCR). Such long-range regulation in vivo involves spatial interaction between transcriptional elements, with intervening chromatin looping out. It has been proposed that the clustering of the HS sites of the LCR, the active globin genes, as well as the remote 5′ hypersensitive sites (HSs) (HS-60/-62 in mouse, HS-110 in human) and 3′HS1 forms a specific spatial chromatin structure, termed active chromatin hub (ACH). Here we report the effects of the HS3 deletions of the LCR on the spatial chromatin structure of the β-globin locus as revealed by the chromatin conformation capture (3C) technology. The small HS3 core deletion (0.23 kb), but not the large HS3 deletion (2.3 kb), disrupted the spatial interactions among all the HS sites of the LCR, the β-globin gene and 3′HS1. We have previously demonstrated that the large HS3 deletion barely impairs the structure of the LCR holocomplex, while the structure is significantly disrupted by the HS3 core deletion. Taken together, these results suggest that the formation of the ACH is dependent on a largely intact LCR structure. We propose that the ACH indeed is an extension of the LCR holocomplex. PMID:17056066

  5. Identification of a Novel β-Globin Mutation (HBB: C.189_195delTCATGGC) in a Chinese Family.

    PubMed

    He, Sheng; Lin, Li; Wei, Yuan; Chen, Biyan; Yi, Shang; Chen, Qiuli; Qiu, XiaoXia; Wei, Hongwei; Li, Guojian; Zheng, Chenguang

    2016-08-01

    β-Thalassemia (β-thal) is one of the most common genetic disorders worldwide. Molecular characterization of β-thal is essential for prevention and understanding the biology of the disease. More and more rare and novel mutations are being reported. Here, we report a novel 7 bp deletion at codons 63-65 (HBB: c.189_195delTCATGGC) in exon 2 of the β-globin gene in a family from Guangxi Province, China. This novel mutation causes a shift in the normal reading frame of the β-globin coding sequence and created a stop codon at codon 87 in exon 2, which leads to a β(0)-thal phenotype. PMID:27492766

  6. Association study of the C3 gene with adult and childhood asthma.

    PubMed

    Inoue, Hiroki; Mashimo, Yoichi; Funamizu, Makiko; Shimojo, Naoki; Hasegawa, Koichi; Hirota, Tomomitsu; Doi, Satoru; Kameda, Makoto; Miyatake, Akihiko; Kohno, Yoichi; Okamoto, Yoshitaka; Tamari, Mayumi; Hata, Akira; Suzuki, Yoichi

    2008-01-01

    Bronchial asthma (BA) is a multifactorial disorder, the development of which is affected by both environmental and genetic factors. The complement system plays an important role in immunological response against invading microorganisms. It has been shown that complement-C3-deficient mice have reduced inflammation of asthmatic airways. Previously, we reported the association of four single nuclear proteins (SNPs) in the exons of the C3 gene with childhood and adult BA. The C3 gene, however, is a large gene, and functional SNPs associated with susceptibility to BA have not yet been identified. We analyzed 26 SNPs in the C3 gene and its promoter region to narrow down the regions showing association with childhood and adult BA. Childhood and adult atopic BA patients and healthy child and adult controls were recruited from urban cities in Japan and genotyped. In SNP analysis, an SNP (SNP24, rs11569562) located in intron 31 of the C3 gene was associated with adult BA [corrected P (Pcor) = 0.030]. In linkage disequilibrium (LD) block 4 spanning exons 24-41, the frequency of the CCC haplotype in adult BA was significantly higher than that in adult controls (Pcor = 0.038). Neither the SNP nor the haplotype showing association with adult BA demonstrated a significant association with serum total immunoglobulin E (IgE) level in BA patients and controls. Our results suggest that LD block 4 confers susceptibility to adult BA with mechanisms relevant to the effector phase of allergic inflammation. PMID:18566738

  7. The Role of Hox Genes in Female Reproductive Tract Development, Adult Function, and Fertility.

    PubMed

    Du, Hongling; Taylor, Hugh S

    2016-01-01

    HOX genes convey positional identity that leads to the proper partitioning and adult identity of the female reproductive track. Abnormalities in reproductive tract development can be caused by HOX gene mutations or altered HOX gene expression. Diethylstilbestrol (DES) and other endocrine disruptors cause Müllerian defects by changing HOX gene expression. HOX genes are also essential regulators of adult endometrial development. Regulated HOXA10 and HOXA11 expression is necessary for endometrial receptivity; decreased HOXA10 or HOXA11 expression leads to decreased implantation rates. Alternation of HOXA10 and HOXA11 expression has been identified as a mechanism of the decreased implantation associated with endometriosis, polycystic ovarian syndrome, leiomyoma, polyps, adenomyosis, and hydrosalpinx. Alteration of HOX gene expression causes both uterine developmental abnormalities and impaired adult endometrial development that prevent implantation and lead to female infertility. PMID:26552702

  8. The Drosophila Couch Potato Gene: An Essential Gene Required for Normal Adult Behavior

    PubMed Central

    Bellen, H. J.; Vaessin, H.; Bier, E.; Kolodkin, A.; D'Evelyn, D.; Kooyer, S.; Jan, Y. N.

    1992-01-01

    Through enhancer detection screens we have isolated 14 insertions in an essential gene that is expressed in embryonic sensory mother cells (SMC), in most cells of the mature embryonic peripheral nervous system (PNS), and in glial cells of the PNS and the central nervous system (CNS). Embryos homozygote for amorphic alleles die, but show no obvious defects in their cuticle, PNS or CNS. The gene has been named couch potato (cpo) because several insertional alleles alter adult behavior. Homozygous hypomorphic cpo flies recover slowly from ether anaesthesia, show aberrant flight behavior, fail to move toward light and do not exhibit normal negative geotactic behavior. However, the flies are able to groom and walk, and some are able to fly when prodded, indicating that not all processes required for behavior are severely affected. A molecular analysis shows that the 14 insertions are confined to a few hundred nucleotides which probably contain key regulatory sequences of the gene. The orientation of these insertions and their position within this DNA fragment play an important role in the couch potato phenotype. In situ hybridization to whole mount embryos suggest that some insertions affect the levels of transcription of cpo in most cells in which it is expressed. PMID:1644278

  9. In vivo binding of trimethylpsoralen detects DNA structural alterations associated with transcribing regions in the human beta-globin cluster.

    PubMed

    Jiménez-Ruiz, A; Zhang, Q; Shen, C K

    1995-12-01

    In order to increase our knowledge about the mechanisms that regulate expression of human beta-like globin genes, we have used a novel technique to analyze the chromatin structure in living cells. This approach allowed us to detect specific DNA regions in vivo where nucleosome folding or unconstrained DNA supercoiling in erythroid cells differs from that in non-erythroid cells. In this method, we use 4,5',8-trimethylpsoralen (TMP) as a probe capable of detecting altered chromatin conformations. Our results show that TMP binds to DNA with a higher affinity over the regions in the locus that are actively expressed, including both the promoter and the transcribed region. This higher affinity detected when comparing erythroid cells with non-erythroid cells does not extend to other regions inside the beta-globin cluster. Our data suggest that the observed effect is likely due to nucleosome displacement. Alternatively, it could result from localized DNA supercoiling, but not from widespread torsional stress across the entire beta-like globin locus as hypothesized previously. PMID:7499429

  10. Distribution of beta-globin haplotypes among the tribes of southern Gujarat, India.

    PubMed

    Aggarwal, Aastha; Khurana, Priyanka; Mitra, Siuli; Raicha, Bhavesh; Saraswathy, K N; Italia, Yazdi M; Kshatriya, Gautam K

    2013-06-01

    The present study was carried out in Indo-European speaking tribal population groups of southern Gujarat (India) to elucidate the allelic and haplotypic content of β-globin system in individuals with HbAA genotypes. 6 neutral restriction sites of the β-globin system were analysed and various statistical parameters were estimated to draw meaningful interpretations. All the 6 sites were found to be polymorphic and most were in Hardy-Weinberg Equilibrium in the studied group. Haplotypes were constructed using two different combinations of the 6 restriction sites analysed. Analysis of the 5 sites revealed a set of three predominant haplotypes, '+----', '-++-+' and '-+-++'; and haplotypes '+--', '++-' and '+++' were found to be the most frequent when the 3 sites were used to construct the haplotypes. Haplotypic heterozygosity levels (>83%) observed in the present study group were comparable to those observed in African and Afro-American populations and greater than other world populations. All the ancestral haplotypes, +-----, -++-+, -+-++ and ----+ were found in the study group. The distribution pattern of various haplotypes was consistent with the global pattern. The paucity of comparable data from other Indian populations restricted one from making interpretations about the study group's relationships with other Indian populations but the results were indicative of older population histories or experience of gene flow by the study group and their affinities with populations of southern India. PMID:23500448

  11. Genetic dissection of the α-globin super-enhancer in vivo.

    PubMed

    Hay, Deborah; Hughes, Jim R; Babbs, Christian; Davies, James O J; Graham, Bryony J; Hanssen, Lars L P; Kassouf, Mira T; Oudelaar, A Marieke; Sharpe, Jacqueline A; Suciu, Maria C; Telenius, Jelena; Williams, Ruth; Rode, Christina; Li, Pik-Shan; Pennacchio, Len A; Sloane-Stanley, Jacqueline A; Ayyub, Helena; Butler, Sue; Sauka-Spengler, Tatjana; Gibbons, Richard J; Smith, Andrew J H; Wood, William G; Higgs, Douglas R

    2016-08-01

    Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. These super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation. PMID:27376235

  12. Analysis of gene expression in fetal and adult cells infected with rubella virus

    SciTech Connect

    Adamo, Maria Pilar; Zapata, Marta; Frey, Teryl K.

    2008-01-05

    Congenital infection with rubella virus (RUB) leads to persistent infection and congenital defects and we showed previously that primary human fetal fibroblasts did not undergo apoptosis when infected with RUB, which could promote fetal virus persistence [Adamo, P., Asis, L., Silveyra, P., Cuffini, C., Pedranti, M., Zapata, M., 2004. Rubella virus does not induce apoptosis in primary human embryo fibroblasts cultures: a possible way of viral persistence in congenital infection. Viral Immunol. 17, 87-100]. To extend this observation, gene chip analysis was performed on a line of primary human fetal fibroblasts (10 weeks gestation) and a line of human adult lung fibroblasts (which underwent apoptosis in response to RUB infection) to compare gene expression in infected and uninfected cells. A total of 632 and 516 genes were upregulated or downregulated in the infected fetal and adult cells respectively in comparison to uninfected cells, however only 52 genes were regulated in both cell types. Although the regulated genes were different, across functional gene categories the patterns of gene regulation were similar. In general, regulation of pro- and anti-apoptotic genes following infection appeared to favor apoptosis in the adult cells and lack of apoptosis in the fetal cells, however there was a greater relative expression of anti-apoptotic genes and reduced expression of pro-apoptotic genes in uninfected fetal cells versus uninfected adult cells and thus the lack of apoptosis in fetal cells following RUB infection was also due to the prevailing background of gene expression that is antagonistic to apoptosis. In support of this hypothesis, it was found that of a battery of five chemicals known to induce apoptosis, two induced apoptosis in the adult cells, but not in fetal cells, and two induced apoptosis more rapidly in the adult cells than in fetal cells (the fifth did not induce apoptosis in either). A robust interferon-stimulated gene response was induced

  13. Rapid and efficient gene delivery into the adult mouse brain via focal electroporation

    PubMed Central

    Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

    2016-01-01

    In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

  14. Rapid and efficient gene delivery into the adult mouse brain via focal electroporation.

    PubMed

    Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

    2016-01-01

    In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

  15. Acute chest syndrome is associated with single nucleotide polymorphism-defined beta globin cluster haplotype in children with sickle cell anaemia.

    PubMed

    Bean, Christopher J; Boulet, Sheree L; Yang, Genyan; Payne, Amanda B; Ghaji, Nafisa; Pyle, Meredith E; Hooper, W Craig; Bhatnagar, Pallav; Keefer, Jeffrey; Barron-Casella, Emily A; Casella, James F; Debaun, Michael R

    2013-10-01

    Genetic diversity at the human β-globin locus has been implicated as a modifier of sickle cell anaemia (SCA) severity. However, haplotypes defined by restriction fragment length polymorphism sites across the β-globin locus have not been consistently associated with clinical phenotypes. To define the genetic structure at the β-globin locus more thoroughly, we performed high-density single nucleotide polymorphism (SNP) mapping in 820 children who were homozygous for the sickle cell mutation (HbSS). Genotyping results revealed very high linkage disequilibrium across a large region spanning the locus control region and the HBB (β-globin gene) cluster. We identified three predominant haplotypes accounting for 96% of the β(S) -carrying chromosomes in this population that could be distinguished using a minimal set of common SNPs. Consistent with previous studies, fetal haemoglobin level was significantly associated with β(S) -haplotypes. After controlling for covariates, an association was detected between haplotype and rate of hospitalization for acute chest syndrome (ACS) (incidence rate ratio 0·51, 95% confidence interval 0·29-0·89) but not incidence rate of vaso-occlusive pain or presence of silent cerebral infarct (SCI). Our results suggest that these SNP-defined β(S) -haplotypes may be associated with ACS, but not pain or SCI in a study population of children with SCA. PMID:23952145

  16. Gene action for adult-plant resistance to powdery mildew in wheat.

    PubMed

    Das, M K; Griffey, C A

    1995-04-01

    Gene action for adult-plant resistance to powdery mildew was studied using generation mean analyses of parents and of F1, F2, and backcross populations derived from a diallel cross of one susceptible and three adult-plant resistant wheat cultivars. Joint scaling tests showed that an additive-dominance model was sufficient to explain the variability in the expression of adult-plant resistance in one cross, while digenic epistasis was involved in the other five crosses. Additive gene effects were predominant; however, dominance was significant in four crosses, additive x additive interaction was significant in three crosses, additive x dominance interaction was significant in three crosses, and dominance x dominance interaction was significant in one cross. Therefore, selection for adult-plant resistance would likely be most effective in advanced generations derived from crosses among the adult-plant resistant cultivars Redcoat, Houser, and Massey. PMID:18470166

  17. Growth factor enhanced retroviral gene transfer to the adult central nervous system.

    PubMed

    King, L A; Mitrophanous, K A; Clark, L A; Kim, V N; Rohll, J B; Kingsman, A J; Colello, R J

    2000-07-01

    The use of viral vectors for gene delivery into mammalian cells provides a new approach in the treatment of many human diseases. The first viral vector approved for human clinical trials was murine leukemia virus (MLV), which remains the most commonly used vector in clinical trials to date. However, the application of MLV vectors is limited since MLV requires cells to be actively dividing in order for transduction and therefore gene delivery to occur. This limitation precludes the use of MLV for delivering genes to the adult CNS, where very little cell division is occurring. However, we speculated that this inherent limitation of ML V may be overcome by utilizing the known mitogenic effect of growth factors on cells of the CNS. Specifically, an in vivo application of growth factor to the adult brain, if able to induce cell division, could enhance MLV-based gene transfer to the adult brain. We now show that an exogenous application of basic fibroblast growth factor induces cell division in vivo. Under these conditions, where cells of the adult brain are stimulated to divide, MLV-based gene transfer is significantly enhanced. This novel approach precludes any vector modifications and provides a simple and effective way of delivering genes to cells of the adult brain utilizing MLV-based retroviral vectors. PMID:10918476

  18. The Evolution of the Globins: We Thought We Understood It

    NASA Astrophysics Data System (ADS)

    Lesk, Arthur M.

    Protein crystallography achieved its first results in the late 1950s with the structure determinations of sperm whale myoglobin and human haemoglobin. These gave us our first glimpse of the structural changes that take place during protein evolution. Many other structures of proteins in the globin family have continued to reveal interesting and important details of the coordinated divergence during evolution of amino acid sequences and protein structures and functions.

  19. Different Gene Expression Signatures in Children and Adults with Celiac Disease.

    PubMed

    Pascual, V; Medrano, L M; López-Palacios, N; Bodas, A; Dema, B; Fernández-Arquero, M; González-Pérez, B; Salazar, I; Núñez, C

    2016-01-01

    Celiac disease (CD) is developed after gluten ingestion in genetically susceptible individuals. It can appear at any time in life, but some differences are commonly observed between individuals with onset early in life or in adulthood. We aimed to investigate the molecular basis underlying those differences. We collected 19 duodenal biopsies of children and adults with CD and compared the expression of 38 selected genes between each other and with the observed in 13 non-CD controls matched by age. A Bayesian methodology was used to analyze the differences of gene expression between groups. We found seven genes with a similarly altered expression in children and adults with CD when compared to controls (C2orf74, CCR6, FASLG, JAK2, IL23A, TAGAP and UBE2L3). Differences were observed in 13 genes: six genes being altered only in adults (IL1RL1, CD28, STAT3, TMEM187, VAMP3 and ZFP36L1) and two only in children (TNFSF18 and ICOSLG); and four genes showing a significantly higher alteration in adults (CCR4, IL6, IL18RAP and PLEK) and one in children (C1orf106). This is the first extensive study comparing gene expression in children and adults with CD. Differences in the expression level of several genes were found between groups, being notorious the higher alteration observed in adults. Further research is needed to evaluate the possible genetic influence underlying these changes and the specific functional consequences of the reported differences. PMID:26859134

  20. Different Gene Expression Signatures in Children and Adults with Celiac Disease

    PubMed Central

    López-Palacios, N.; Bodas, A.; Dema, B.; Fernández-Arquero, M.; González-Pérez, B.; Salazar, I.; Núñez, C.

    2016-01-01

    Celiac disease (CD) is developed after gluten ingestion in genetically susceptible individuals. It can appear at any time in life, but some differences are commonly observed between individuals with onset early in life or in adulthood. We aimed to investigate the molecular basis underlying those differences. We collected 19 duodenal biopsies of children and adults with CD and compared the expression of 38 selected genes between each other and with the observed in 13 non-CD controls matched by age. A Bayesian methodology was used to analyze the differences of gene expression between groups. We found seven genes with a similarly altered expression in children and adults with CD when compared to controls (C2orf74, CCR6, FASLG, JAK2, IL23A, TAGAP and UBE2L3). Differences were observed in 13 genes: six genes being altered only in adults (IL1RL1, CD28, STAT3, TMEM187, VAMP3 and ZFP36L1) and two only in children (TNFSF18 and ICOSLG); and four genes showing a significantly higher alteration in adults (CCR4, IL6, IL18RAP and PLEK) and one in children (C1orf106). This is the first extensive study comparing gene expression in children and adults with CD. Differences in the expression level of several genes were found between groups, being notorious the higher alteration observed in adults. Further research is needed to evaluate the possible genetic influence underlying these changes and the specific functional consequences of the reported differences. PMID:26859134

  1. PROGRESSION OF REGULATORY GENE EXPRESSION STATES IN FETAL AND ADULT PRO-T CELL DEVELOPMENT

    PubMed Central

    David-Fung, Elizabeth-Sharon; Yui, Mary A.; Morales, Marissa; Wang, Hua; Taghon, Tom; Diamond, Rochelle A.; Rothenberg, Ellen V.

    2014-01-01

    Precursors entering the T-cell developmental pathway traverse a progression of states characterized by distinctive patterns of gene expression. Of particular interest are regulatory genes, which ultimately control the dwell time of cells in each state and establish the mechanisms that propel them forward to subsequent states. Under particular genetic and developmental circumstances, the transitions between these states occur with different timing, and environmental feedbacks may shift the steady-state accumulations of cells in each state. The fetal transit through pro-T cell stages is faster than in the adult, and subject to somewhat different genetic requirements. To explore causes of such variation, this review presents previously unpublished data on differentiation gene activation in pro-T cells of pre-TCR deficient mutant mice, and a quantitative comparison of the profiles of transcription factor gene expression in pro-T cell subsets of fetal and adult wildtype mice. Against a background of consistent gene expression, several regulatory genes show marked differences between fetal and adult expression profiles, including those encoding two bHLH antagonist Id factors, the Ets family factor SpiB, and the Notch target gene Deltex1. The results also reveal global differences in regulatory alterations triggered by the first TCR-dependent selection events in fetal and adult thymopoiesis. PMID:16448545

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

    PubMed

    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. PMID:23346994

  3. Analysis of Gene Expression and Ultrastructure of Stifle Menisci from Juvenile and Adult Pigs.

    PubMed

    Kreinest, Michael; Reisig, Gregor; Ströbel, Philipp; Fickert, Stefan; Brade, Joachim; Wennemuth, Gunther; Lipp, Peter; Schwarz, Markus L

    2016-01-01

    The origin of the age-associated degenerative processes in meniscal tissue is poorly understood and may be related to an imbalance of anabolic and catabolic metabolism. The aim of the current study was to compare medial menisci isolated from juvenile pigs and degenerated medial menisci from adult pigs in terms of gene expression profile and ultrastructure. Medial menisci were isolated from the knee joints of juvenile and adult pigs (n = 8 for each group). Degeneration was determined histologically according to a scoring system. In addition, the gene expression profiles of 14 genes encoding extracellular matrix proteins, catabolic matrix metalloproteinases and mediators of inflammation were analyzed. Changes in the ultrastructure of the collagen network of the meniscal tissue were analyzed by using transmission electron microscopy. The histologic analysis of menisci showed significantly higher grade of degeneration in tissue isolated from adult porcine knee joints compared with menisci isolated from juvenile knee joints. In particular, destruction of the collagen network was greater in adult menisci than in juvenile menisci. Degenerated menisci showed significantly decreased gene expression of COL1A1 and increased expression of MMP2, MMP13, and IL8. The menisci from adult porcine knee joints can serve as a model for meniscal degeneration. Degenerative changes were manifested as differences in histopathology, gene expression and ultrastructure of collagen network. PMID:26884408

  4. β-globin matrix attachment region improves stable genomic expression of the Sleeping Beauty transposon.

    PubMed

    Sjeklocha, Lucas; Chen, Yixin; Daly, Meghan C; Steer, Clifford J; Kren, Betsy T

    2011-09-01

    The liver is an attractive target for gene therapy due to its extensive capability for protein production and the numerous diseases resulting from a loss of gene function it normally provides. The Sleeping Beauty Transposon (SB-Tn)(1) system is a non-viral vector capable of delivering and mediating therapeutic transgene(s) insertion into the host genome for long-term expression. A current challenge for this system is the low efficiency of integration of the transgene. In this study we use a human hepatoma cell line (HuH-7) and primary human blood outgrowth endothelial cells (BOECs) to test vectors containing DNA elements to enhance transposition without integrating themselves. We employed the human β-globin matrix attachment region (MAR) and the Simian virus 40 (SV40) nuclear translocation signal to increase the percent of HuH-7 cells persistently expressing a GFP::Zeo reporter construct by ∼50% for each element; while combining both did not show an additive effect. Interestingly, both elements together displayed an additive effect on the number of insertion sites, and in BOECs the SV40 alone appeared to have an inhibitory effect on transposition. In long-term cultures the loss of plasmid DNA, transposase expression and mapping of insertion sites demonstrated bona fide transposition without episomal expression. These results show that addition of the β-globin MAR and potentially other elements to the backbone of SB-Tn system can enhance transposition and expression of therapeutic transgenes. These findings may have a significant influence on the use of SB transgene delivery to liver for the treatment of a wide variety of disorders. PMID:21520245

  5. Understanding the contrasting spatial haplotype patterns of malaria-protective β-globin polymorphisms.

    PubMed

    Hockham, Carinna; Piel, Frédéric B; Gupta, Sunetra; Penman, Bridget S

    2015-12-01

    The malaria-protective β-globin polymorphisms, sickle-cell (β(S)) and β(0)-thalassaemia, are canonical examples of human adaptation to infectious disease. Occurring on distinct genetic backgrounds, they vary markedly in their patterns of linked genetic variation at the population level, suggesting different evolutionary histories. β(S) is associated with five classical restriction fragment length polymorphism haplotypes that exhibit remarkable specificity in their geographical distributions; by contrast, β(0)-thalassaemia mutations are found on haplotypes whose distributions overlap considerably. Here, we explore why these two polymorphisms display contrasting spatial haplotypic distributions, despite having malaria as a common selective pressure. We present a meta-population genetic model, incorporating individual-based processes, which tracks the evolution of β-globin polymorphisms on different haplotypic backgrounds. Our simulations reveal that, depending on the rate of mutation, a large population size and/or high population growth rate are required for both the β(S)- and the β(0)-thalassaemia-like patterns. However, whilst the β(S)-like pattern is more likely when population subdivision is high, migration low and long-distance migration absent, the opposite is true for β(0)-thalassaemia. Including gene conversion has little effect on the overall probability of each pattern; however, when inter-haplotype fitness variation exists, gene conversion is more likely to have contributed to the diversity of haplotypes actually present in the population. Our findings highlight how the contrasting spatial haplotype patterns exhibited by β(S) and β(0)-thalassaemia may provide important indications as to the evolution of these adaptive alleles and the demographic history of the populations in which they have evolved. PMID:26394108

  6. Understanding the contrasting spatial haplotype patterns of malaria-protective β-globin polymorphisms

    PubMed Central

    Hockham, Carinna; Piel, Frédéric B.; Gupta, Sunetra; Penman, Bridget S.

    2015-01-01

    The malaria-protective β-globin polymorphisms, sickle-cell (βS) and β0-thalassaemia, are canonical examples of human adaptation to infectious disease. Occurring on distinct genetic backgrounds, they vary markedly in their patterns of linked genetic variation at the population level, suggesting different evolutionary histories. βS is associated with five classical restriction fragment length polymorphism haplotypes that exhibit remarkable specificity in their geographical distributions; by contrast, β0-thalassaemia mutations are found on haplotypes whose distributions overlap considerably. Here, we explore why these two polymorphisms display contrasting spatial haplotypic distributions, despite having malaria as a common selective pressure. We present a meta-population genetic model, incorporating individual-based processes, which tracks the evolution of β-globin polymorphisms on different haplotypic backgrounds. Our simulations reveal that, depending on the rate of mutation, a large population size and/or high population growth rate are required for both the βS- and the β0-thalassaemia-like patterns. However, whilst the βS-like pattern is more likely when population subdivision is high, migration low and long-distance migration absent, the opposite is true for β0-thalassaemia. Including gene conversion has little effect on the overall probability of each pattern; however, when inter-haplotype fitness variation exists, gene conversion is more likely to have contributed to the diversity of haplotypes actually present in the population. Our findings highlight how the contrasting spatial haplotype patterns exhibited by βS and β0-thalassaemia may provide important indications as to the evolution of these adaptive alleles and the demographic history of the populations in which they have evolved. PMID:26394108

  7. Mapping a gene for adult-onset primary open-angle glaucoma to chromosome 3q

    SciTech Connect

    Wirtz, M.K.; Samples, J.R.; Kramer, P.L.

    1997-02-01

    Glaucoma is the third-leading cause of blindness in the world, affecting >13.5 million people. Adult-on-set primary open-angle glaucoma (POAG) is the most common form of glaucoma in the United States. We present a family in which adult-onset POAG is inherited as an autosomal dominant trait. Twelve affected family members were identified from 44 at-risk individuals. The disease-causing gene was mapped to chromosome 3q21-24, with analysis of recombinant haplotypes suggesting a total inclusion region of 11.1 cM between markers D3S3637 and D3S1744. This is the first report of mapping of an adult-onset POAG gene to chromosome 3q, gene symbol GLC1C. 57 refs., 3 figs., 3 tabs.

  8. The 3' noncoding region of beta-globin mRNA is not essential for in vitro translation.

    PubMed Central

    Kronenberg, M N; Roberts, B E; Efstratiadis, A

    1979-01-01

    Rabbit beta globin DNA sequence, excised from plasmid pbetaG1, directs in vitro synthesis of beta-globin in a transcription-translation cell-free system, even after specific elimination of the entire 3'-noncoding region. A DNA restriction fragment carrying this 3' noncoding region and hybridized to globin mRNA cannot arrest the cell-free translation of beta-globin mRNA. Images PMID:424286

  9. Adipose tissue gene expression and metabolic health of obese adults.

    PubMed

    Das, S K; Ma, L; Sharma, N K

    2015-05-01

    Obese subjects with a similar body mass index (BMI) exhibit substantial heterogeneity in gluco- and cardiometabolic heath phenotypes. However, defining genes that underlie the heterogeneity of metabolic features among obese individuals and determining metabolically healthy and unhealthy phenotypes remain challenging. We conducted unsupervised hierarchical clustering analysis of subcutaneous adipose tissue transcripts from 30 obese men and women ⩾40 years old. Despite similar BMIs in all subjects, we found two distinct subgroups, one metabolically healthy (group 1) and one metabolically unhealthy (group 2). Subjects in group 2 showed significantly higher total cholesterol (P=0.005), low-density lipoprotein cholesterol (P=0.006), 2-h insulin during oral glucose tolerance test (P=0.015) and lower insulin sensitivity (SI, P=0.029) compared with group 1. We identified significant upregulation of 141 genes (for example, MMP9 and SPP1) and downregulation of 17 genes (for example, NDRG4 and GINS3) in group 2 subjects. Intriguingly, these differentially expressed transcripts were enriched for genes involved in cardiovascular disease-related processes (P=2.81 × 10(-11)-3.74 × 10(-02)) and pathways involved in immune and inflammatory response (P=8.32 × 10(-5)-0.04). Two downregulated genes, NDRG4 and GINS3, have been located in a genomic interval associated with cardiac repolarization in published GWASs and zebra fish knockout models. Our study provides evidence that perturbations in the adipose tissue gene expression network are important in defining metabolic health in obese subjects. PMID:25520251

  10. Brain-expressed imprinted genes and adult behaviour: the example of Nesp and Grb10.

    PubMed

    Dent, Claire L; Isles, Anthony R

    2014-02-01

    Imprinted genes are defined by their parent-of-origin-specific monoallelic expression. Although the epigenetic mechanisms regulating imprinted gene expression have been widely studied, their functional importance is still unclear. Imprinted genes are associated with a number of physiologies, including placental function and foetal growth, energy homeostasis, and brain and behaviour. This review focuses on genomic imprinting in the brain and on two imprinted genes in particular, Nesp and paternal Grb10, which, when manipulated in animals, have been shown to influence adult behaviour. These two genes are of particular interest as they are expressed in discrete and overlapping neural regions, recognised as key "imprinting hot spots" in the brain. Furthermore, these two genes do not appear to influence placental function and/or maternal provisioning of offspring. Consequently, by understanding their behavioural function we may begin to shed light on the evolutionary significance of imprinted genes in the adult brain, independent of the recognised role in maternal care. In addition, we discuss the potential future directions of research investigating the function of these two genes and the behavioural role of imprinted genes more generally. PMID:23974804

  11. Expression pattern of Piwi-like genes in adult Myzostoma cirriferum (Annelida).

    PubMed

    Weigert, Anne; Helm, Conrad; Hausen, Harald; Zakrzewski, Anne-C; Bleidorn, Christoph

    2013-09-01

    Piwi-like genes are a subgroup of Argonaute genes which participate as gene regulators by gene silencing. In most bilaterians, such as mouse, human, insects, and zebrafish, their expression is mostly limited to gonadal stem cells. But there are some striking exceptions to this pattern; flatworms and acoels also express Piwi-like genes in somatic stem cells, due to their unique replacement system. Annelid species like Capitella teleta and Platynereis dumerilii express these genes in cells of the posterior growth zone as well as in gonadal stem cells. To investigate the expression pattern of Piwi-like genes in another annelid, we established in situ hybridization for adult Myzostoma cirriferum. Piwi-like gene transcripts recovered in an mRNA-seq library of pooled adult stages of M. cirriferum were expanded using RACE PCR, cloned and sequenced. ML analysis confirmed the identity of both transcripts as part of the Piwi1-like or Piwi2-like subfamily of Argonaute proteins. The results of in situ hybridization studies show that the expression of both Piwi-like genes, Mc-Piwi1 and Mc-Piwi2, is clearly located only in gonadal stem cells, and as such we did not find any evidence for the existence of a posterior growth zone nor expression in somatic stem cells. PMID:23609434

  12. Gene by Neuroticism Interaction and Cognitive Function among Older Adults

    PubMed Central

    Dar-Nimrod, Ilan; Chapman, Benjamin P.; Robbins, John A.; Porsteinsson, Anton; Mapstone, Mark; Duberstein, Paul R.

    2012-01-01

    Objectives Both ApoE (apolipoprotein E) ε-4 allele(s) and elevated trait neuroticism, the tendency to experience distress, are associated with cognitive function among older adults. We predicted that neuroticism moderates the association between ApoE and cognitive function and also explored whether other personality dimensions (openness to experience, agreeableness, extraversion, and conscientiousness) affect the association between ApoE status and cognitive function. Method Five-hundred and ninety-seven older adults (mean age of 78) enrolled in the Ginkgo Evaluation of Memory (GEM) study completed the NEO-Five Factor Inventory of personality. Cognitive function was assessed via the cognitive portion of the Alzheimer’s Disease Assessment Scale (ADAS-cog), and a blood sample for ApoE genotyping was drawn. Results As hypothesized, regression analysis indicated that neuroticism moderated the relationship between the presence of ApoE ε-4 and cognitive function. Individuals with high neuroticism scores had significantly lower ADAS-cog scores compared with individual with low neuroticism scores, but this was true only among carriers of ApoE ε-4 (interaction effect β = .124, p = .028). There was scant evidence that other personality dimensions moderate the association between ApoE ε-4 and cognitive function. Conclusions Cognitive function may be affected by ApoE and neuroticism acting in tandem. Research on the underlying physiological mechanisms by which neuroticism amplifies the effect of ApoE ε-4 is warranted. The study of genotype by phenotype interactions provides an important and useful direction for the study of cognitive function among older adults and for the development of novel prevention programs. PMID:23042108

  13. Health and population effects of rare gene knockouts in adult humans with related parents.

    PubMed

    Narasimhan, Vagheesh M; Hunt, Karen A; Mason, Dan; Baker, Christopher L; Karczewski, Konrad J; Barnes, Michael R; Barnett, Anthony H; Bates, Chris; Bellary, Srikanth; Bockett, Nicholas A; Giorda, Kristina; Griffiths, Christopher J; Hemingway, Harry; Jia, Zhilong; Kelly, M Ann; Khawaja, Hajrah A; Lek, Monkol; McCarthy, Shane; McEachan, Rosie; O'Donnell-Luria, Anne; Paigen, Kenneth; Parisinos, Constantinos A; Sheridan, Eamonn; Southgate, Laura; Tee, Louise; Thomas, Mark; Xue, Yali; Schnall-Levin, Michael; Petkov, Petko M; Tyler-Smith, Chris; Maher, Eamonn R; Trembath, Richard C; MacArthur, Daniel G; Wright, John; Durbin, Richard; van Heel, David A

    2016-04-22

    Examining complete gene knockouts within a viable organism can inform on gene function. We sequenced the exomes of 3222 British adults of Pakistani heritage with high parental relatedness, discovering 1111 rare-variant homozygous genotypes with predicted loss of function (knockouts) in 781 genes. We observed 13.7% fewer homozygous knockout genotypes than we expected, implying an average load of 1.6 recessive-lethal-equivalent loss-of-function (LOF) variants per adult. When genetic data were linked to the individuals' lifelong health records, we observed no significant relationship between gene knockouts and clinical consultation or prescription rate. In this data set, we identified a healthy PRDM9-knockout mother and performed phased genome sequencing on her, her child, and control individuals. Our results show that meiotic recombination sites are localized away from PRDM9-dependent hotspots. Thus, natural LOF variants inform on essential genetic loci and demonstrate PRDM9 redundancy in humans. PMID:26940866

  14. Health and population effects of rare gene knockouts in adult humans with related parents

    PubMed Central

    Narasimhan, Vagheesh M.; Hunt, Karen A.; Mason, Dan; Baker, Christopher L.; Karczewski, Konrad J.; Barnes, Michael R.; Barnett, Anthony H.; Bates, Chris; Bellary, Srikanth; Bockett, Nicholas A.; Giorda, Kristina; Griffiths, Christopher J.; Hemingway, Harry; Jia, Zhilong; Kelly, M. Ann; Khawaja, Hajrah A.; Lek, Monkol; McCarthy, Shane; McEachan, Rosie; O’Donnell-Luria, Anne; Paigen, Kenneth; Parisinos, Constantinos A.; Sheridan, Eamonn; Southgate, Laura; Tee, Louise; Thomas, Mark; Xue, Yali; Schnall-Levin, Michael; Petkov, Petko M.; Tyler-Smith, Chris; Maher, Eamonn R.; Trembath, Richard C.; MacArthur, Daniel G.; Wright, John; Durbin, Richard; van Heel, David A.

    2016-01-01

    Examining complete gene knockouts within a viable organism can inform on gene function. We sequenced the exomes of 3,222 British Pakistani-heritage adults with high parental relatedness, discovering 1,111 rare-variant homozygous genotypes with predicted loss of gene function (knockouts) in 781 genes. We observed 13.7% fewer than expected homozygous knockout genotypes, implying an average load of 1.6 recessive-lethal-equivalent LOF variants per adult. Linking genetic data to lifelong health records, knockouts were not associated with clinical consultation or prescription rate. In this dataset we identified a healthy PRDM9 knockout mother, and performed phased genome sequencing on her, her child and controls, which showed meiotic recombination sites localised away from PRDM9-dependent hotspots. Thus, natural LOF variants inform upon essential genetic loci, and demonstrate PRDM9 redundancy in humans. PMID:26940866

  15. Comparisons of the polypeptide chains of globins.

    NASA Technical Reports Server (NTRS)

    Jukes, T. H.

    1971-01-01

    Discussion of the amino acid differences and minimum base differences per codon due to mutations which took place during divergent evolution of vertebrates from a common ancestral gene. The ?random mutation model' of evolution is examined by comparing a carp alpha Hb chain and six mammalian alpha Hb chains in terms of the genetic code. The occurrence of recognizable three-base changes is analyzed and a summary of the distribution of changes in the hemoglobin and myoglobin chains is given for 148 sites.

  16. Hierarchy in gene expression is predictive of risk, progression, and outcome in adult acute myeloid leukemia

    NASA Astrophysics Data System (ADS)

    Tripathi, Shubham; Deem, Michael W.

    2015-02-01

    Cancer progresses with a change in the structure of the gene network in normal cells. We define a measure of organizational hierarchy in gene networks of affected cells in adult acute myeloid leukemia (AML) patients. With a retrospective cohort analysis based on the gene expression profiles of 116 AML patients, we find that the likelihood of future cancer relapse and the level of clinical risk are directly correlated with the level of organization in the cancer related gene network. We also explore the variation of the level of organization in the gene network with cancer progression. We find that this variation is non-monotonic, which implies the fitness landscape in the evolution of AML cancer cells is non-trivial. We further find that the hierarchy in gene expression at the time of diagnosis may be a useful biomarker in AML prognosis.

  17. Adult Plant Phenotype of the Rp1-DJ Compound Rust Resistance Gene in Maize.

    PubMed

    Hu, G; Webb, C A; Hulbert, S H

    1997-03-01

    ABSTRACT The complex structure of the rp1 rust resistance locus of maize allows two or more resistance genes to be recombined together in coupling phase. The phenotypic effects of the Rp1-DJ compound gene, which carries both Rp1-D and Rp1-J, were analyzed. The Rp1-DJ compound gene was associated with a chlorotic spotting phenotype in some genetic backgrounds. At the seedling stage, lines carrying Rp1-DJ are fully susceptible to Puccinia sorghi biotype HI1, which is virulent on lines with the two genes singly. At later stages of growth, however, Rp1-DJ lines show partial resistance when compared with sibling lines not carrying the compound gene. The Rp1-DJ gene also confers partial resistance to P. polysora in adult plants. PMID:18945165

  18. Hierarchy in Gene Expression is Predictive of Risk, Progression, and Outcome in Adult Acute Myeloid Leukemia

    PubMed Central

    Tripathi, Shubham; Deem, Michael W.

    2015-01-01

    Cancer progresses with a change in the structure of the gene network in normal cells. We define a measure of organizational hierarchy in gene networks of affected cells in adult acute myeloid leukemia (AML) patients. With a retrospective cohort analysis based on the gene expression profiles of 116 acute myeloid leukemia patients, we find that the likelihood of future cancer relapse and the level of clinical risk are directly correlated with the level of organization in the cancer related gene network. We also explore the variation of the level of organization in the gene network with cancer progression. We find that this variation is non-monotonic, which implies the fitness landscape in the evolution of AML cancer cells is nontrivial. We further find that the hierarchy in gene expression at the time of diagnosis may be a useful biomarker in AML prognosis. PMID:25685944

  19. Differential expression of immune genes of adult honey bee (Apis mellifera) after inoculated by Nosema ceranae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nosema ceranae is a microsporidium parasite infecting adult honey bees (Apis mellifera) and is known to have affects at both the individual and colony level. In this study, the expression levels were measured for four antimicrobial peptide encoding genes that are associated with bee humoral immunity...

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

    ERIC Educational Resources Information Center

    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…

  1. THE EFFECTS OF HYPERTHERMIA ON SPERMATOGENESIS, APOPTOSIS, GENE EXPRESSION AND FERTILITY IN ADULT MALE MICE

    EPA Science Inventory

    The effects of hyperthermia on spermatogenesis, apoptosis, gene expression and fertility in adult male mice
    John C. Rockett1, Faye L. Mapp1, J. Brian Garges1, J. Christopher Luft1, Chisato Mori2 and David J. Dix1.
    1Reproductive Toxicology Division, National Health and Envir...

  2. Cortisol-treated zebrafish embryos develop into pro-inflammatory adults with aberrant immune gene regulation.

    PubMed

    Hartig, Ellen I; Zhu, Shusen; King, Benjamin L; Coffman, James A

    2016-01-01

    Chronic early-life stress increases adult susceptibility to numerous health problems linked to chronic inflammation. One way that this may occur is via glucocorticoid-induced developmental programming. To gain insight into such programming we treated zebrafish embryos with cortisol and examined the effects on both larvae and adults. Treated larvae had elevated whole-body cortisol and glucocorticoid signaling, and upregulated genes associated with defense response and immune system processes. In adulthood the treated fish maintained elevated basal cortisol levels in the absence of exogenous cortisol, and constitutively mis-expressed genes involved in defense response and its regulation. Adults derived from cortisol-treated embryos displayed defective tailfin regeneration, heightened basal expression of pro-inflammatory genes, and failure to appropriately regulate those genes following injury or immunological challenge. These results support the hypothesis that chronically elevated glucocorticoid signaling early in life directs development of a pro-inflammatory adult phenotype, at the expense of immunoregulation and somatic regenerative capacity. PMID:27444789

  3. Cortisol-treated zebrafish embryos develop into pro-inflammatory adults with aberrant immune gene regulation

    PubMed Central

    Hartig, Ellen I.; Zhu, Shusen; King, Benjamin L.

    2016-01-01

    ABSTRACT Chronic early-life stress increases adult susceptibility to numerous health problems linked to chronic inflammation. One way that this may occur is via glucocorticoid-induced developmental programming. To gain insight into such programming we treated zebrafish embryos with cortisol and examined the effects on both larvae and adults. Treated larvae had elevated whole-body cortisol and glucocorticoid signaling, and upregulated genes associated with defense response and immune system processes. In adulthood the treated fish maintained elevated basal cortisol levels in the absence of exogenous cortisol, and constitutively mis-expressed genes involved in defense response and its regulation. Adults derived from cortisol-treated embryos displayed defective tailfin regeneration, heightened basal expression of pro-inflammatory genes, and failure to appropriately regulate those genes following injury or immunological challenge. These results support the hypothesis that chronically elevated glucocorticoid signaling early in life directs development of a pro-inflammatory adult phenotype, at the expense of immunoregulation and somatic regenerative capacity. PMID:27444789

  4. Gene-environment interaction in programming hippocampal plasticity: focus on adult neurogenesis

    PubMed Central

    Koehl, Muriel

    2015-01-01

    Interactions between genes and environment are a critical feature of development and both contribute to shape individuality. They are at the core of vulnerability resiliency for mental illnesses. During the early postnatal period, several brain structures involved in cognitive and emotional processing, such as the hippocampus, still develop and it is likely that interferences with this neuronal development, which is genetically determined, might lead to long-lasting structural and functional consequences and increase the risk of developing psychopathology. One particular target is adult neurogenesis, which is involved in the regulation of cognitive and emotional processes. Insights into the dynamic interplay between genes and environmental factors in setting up individual rates of neurogenesis have come from laboratory studies exploring experience-dependent changes in adult neurogenesis as a function of individual’s genetic makeup. These studies have implications for our understanding of the mechanisms regulating adult neurogenesis, which could constitute a link between environmental challenges and psychopathology. PMID:26300723

  5. Unpredictable neonatal stress enhances adult anxiety and alters amygdala gene expression related to serotonin and GABA.

    PubMed

    Sarro, E C; Sullivan, R M; Barr, G

    2014-01-31

    Anxiety-related disorders are among the most common psychiatric illnesses, thought to have both genetic and environmental causes. Early-life trauma, such as abuse from a caregiver, can be predictable or unpredictable, each resulting in increased prevalence and severity of a unique set of disorders. In this study, we examined the influence of early unpredictable trauma on both the behavioral expression of adult anxiety and gene expression within the amygdala. Neonatal rats were exposed to unpaired odor-shock conditioning for 5 days, which produces deficits in adult behavior and amygdala dysfunction. In adulthood, we used the Light/Dark box test to measure anxiety-related behaviors, measuring the latency to enter the lit area and quantified urination and defecation. The amygdala was then dissected and a microarray analysis was performed to examine changes in gene expression. Animals that had received early unpredictable trauma displayed significantly longer latencies to enter the lit area and more defecation and urination. The microarray analysis revealed over-represented genes related to learning and memory, synaptic transmission and trans-membrane transport. Gene ontology and pathway analysis identified highly represented disease states related to anxiety phenotypes, including social anxiety, obsessive-compulsive disorders, post-traumatic stress disorder and bipolar disorder. Addiction-related genes were also overrepresented in this analysis. Unpredictable shock during early development increased anxiety-like behaviors in adulthood with concomitant changes in genes related to neurotransmission, resulting in gene expression patterns similar to anxiety-related psychiatric disorders. PMID:24240029

  6. Sequencing and mapping hemoglobin gene clusters in the australian model dasyurid marsupial sminthopsis macroura

    SciTech Connect

    De Leo, A.A.; Wheeler, D.; Lefevre, C.; Cheng, Jan-Fang; Hope, R.; Kuliwaba, J.; Nicholas, K.R.; Westermanc, M.; Graves, J.A.M.

    2004-07-26

    Comparing globin genes and their flanking sequences across many species has allowed globin gene evolution to be reconstructed in great detail. Marsupial globin sequences have proved to be of exceptional significance. A previous finding of a beta-like omega gene in the alpha cluster in the tammar wallaby suggested that the alpha and beta cluster evolved via genome duplication and loss rather than tandem duplication. To confirm and extend this important finding we isolated and sequenced BACs containing the alpha and beta loci from the distantly related Australian marsupial Sminthopsis macroura. We report that the alpha gene lies in the same BAC as the beta-like omega gene, implying that the alpha-omega juxtaposition is likely to be conserved in all marsupials. The LUC7L gene was found 3' of the S. macroura alpha locus, a gene order shared with humans but not mouse, chicken or fugu. Sequencing a BAC contig that contained the S. macroura beta globin and epsilon globin loci showed that the globin cluster is flanked by olfactory genes, demonstrating a gene arrangement conserved for over 180 MY. Analysis of the region 5' to the S. macroura epsilon globin gene revealed a region similar to the eutherian LCR, containing sequences and potential transcription factor binding sites with homology to eutherian hypersensitive sites 1 to 5. FISH mapping of BACs containing S. macroura alpha and beta globin genes located the beta globin cluster on chromosome 3q and the alpha locus close to the centromere on 1q, resolving contradictory map locations obtained by previous radioactive in situ hybridization.

  7. Comparison of tetrodotoxin uptake and gene expression in the liver between juvenile and adult tiger pufferfish, Takifugu rubripes.

    PubMed

    Kiriake, Aya; Ohta, Akira; Suga, Emi; Matsumoto, Takuya; Ishizaki, Shoichiro; Nagashima, Yuji

    2016-03-01

    Marine pufferfish of the family Tetraodontidae accumulate high levels of tetrodotoxin (TTX). The profile of TTX accumulation is reported to differ between tiger pufferfish Takifugu rubripes juveniles and adults administered TTX. Adults mainly accumulate TTX in liver, while juveniles transfer TTX from the liver to the skin. In the present study, we investigated TTX uptake into liver tissue slices of T. rubripes juveniles (4-month-old) and adults (18-month-old) in an in vitro incubation experiment, and compared their differential gene expression profiles in the liver by suppression subtracted hybridization (SSH). The tissue culture experiment revealed that TTX uptake in the liver itself was indistinguishable between the juveniles and the adults. In SSH analysis, a total of 176 clones were upregulated in the juvenile liver, the majority of which comprised hemoglobin subunit alpha-2-like gene (53 clones), hemoglobin subunit beta-like gene (40 clones), and type-4 ice-structuring protein LS-12-like gene (20 clones). A total of 211 clones were upregulated in the adult liver, including serotransferrin-like gene (84 clones), fibrinogen beta chain-like gene (15 clones), and 14 kDa apolipoprotein gene (10 clones). Based on these and previous findings on genes related to TTX intoxication in pufferfish, serotransferrin-like gene, complement C3-like gene, water-temperature-acclimation-related-65 kDa-protein-like gene, and chymotrypsin elastase family member 2A-like gene appear to be involved in TTX toxification of the T. rubripes liver. PMID:26708657

  8. Schistosoma mansoni Egg, Adult Male and Female Comparative Gene Expression Analysis and Identification of Novel Genes by RNA-Seq

    PubMed Central

    Anderson, Letícia; Amaral, Murilo S.; Beckedorff, Felipe; Silva, Lucas F.; Dazzani, Bianca; Oliveira, Katia C.; Almeida, Giulliana T.; Gomes, Monete R.; Pires, David S.; Setubal, João C.; DeMarco, Ricardo; Verjovski-Almeida, Sergio

    2015-01-01

    Background Schistosomiasis is one of the most prevalent parasitic diseases worldwide and is a public health problem. Schistosoma mansoni is the most widespread species responsible for schistosomiasis in the Americas, Middle East and Africa. Adult female worms (mated to males) release eggs in the hepatic portal vasculature and are the principal cause of morbidity. Comparative separate transcriptomes of female and male adult worms were previously assessed with using microarrays and Serial Analysis of Gene Expression (SAGE), thus limiting the possibility of finding novel genes. Moreover, the egg transcriptome was analyzed only once with limited bacterially cloned cDNA libraries. Methodology/Principal findings To compare the gene expression of S. mansoni eggs, females, and males, we performed RNA-Seq on these three parasite forms using 454/Roche technology and reconstructed the transcriptome using Trinity de novo assembly. The resulting contigs were mapped to the genome and were cross-referenced with predicted Smp genes and H3K4me3 ChIP-Seq public data. For the first time, we obtained separate, unbiased gene expression profiles for S. mansoni eggs and female and male adult worms, identifying enriched biological processes and specific enriched functions for each of the three parasite forms. Transcripts with no match to predicted genes were analyzed for their protein-coding potential and the presence of an encoded conserved protein domain. A set of 232 novel protein-coding genes with putative functions related to reproduction, metabolism, and cell biogenesis was detected, which contributes to the understanding of parasite biology. Conclusions/Significance Large-scale RNA-Seq analysis using de novo assembly associated with genome-wide information for histone marks in the vicinity of gene models constitutes a new approach to transcriptome analysis that has not yet been explored in schistosomes. Importantly, all data have been consolidated into a UCSC Genome Browser search

  9. Intronless β-Globin Reporter: A Tool for Studying Nuclear RNA Stability Elements.

    PubMed

    Brown, Jessica A; Steitz, Joan A

    2016-01-01

    The intronless β-globin reporter, whose mRNA is intrinsically unstable due to the lack of introns, is a useful tool to study RNA stability elements in a heterologous transcript. Insertion of a stability element leads to the accumulation of intronless β-globin mRNA that can be visualized by conventional Northern blot analyses. In this chapter, we explain how to perform the β-globin reporter assay using the ENE (expression and nuclear retention element), a triple-helix-forming RNA stability element that protects reporter mRNA from 3'- 5' decay. A list of considerations is included for the use of ENEs as a tool to stabilize other RNAs. In this chapter, we provide a brief description of how to insert an ENE sequence into the 3'-untranslated region of an intronless β-globin reporter plasmid using basic cloning technology. Then, we provide a detailed protocol for quantitative measurements of steady-state levels of β-globin mRNA. This entails the transient transfection of mammalian cells with β-globin reporter plasmids, isolation of total cellular RNA, and detection of reporter mRNA via Northern blot. This methodology can be applied for the study of any nuclear RNA stability element using the intronless β-globin reporter. PMID:27236793

  10. Somatic stem cells express Piwi and Vasa genes in an adult ctenophore: ancient association of "germline genes" with stemness.

    PubMed

    Alié, Alexandre; Leclère, Lucas; Jager, Muriel; Dayraud, Cyrielle; Chang, Patrick; Le Guyader, Hervé; Quéinnec, Eric; Manuel, Michaël

    2011-02-01

    Stem cells are essential for animal development and adult tissue homeostasis, and the quest for an ancestral gene fingerprint of stemness is a major challenge for evolutionary developmental biology. Recent studies have indicated that a series of genes, including the transposon silencer Piwi and the translational activator Vasa, specifically involved in germline determination and maintenance in classical bilaterian models (e.g., vertebrates, fly, nematode), are more generally expressed in adult multipotent stem cells in other animals like flatworms and hydras. Since the progeny of these multipotent stem cells includes both somatic and germinal derivatives, it remains unclear whether Vasa, Piwi, and associated genes like Bruno and PL10 were ancestrally linked to stemness, or to germinal potential. We have investigated the expression of Vasa, two Piwi paralogues, Bruno and PL10 in Pleurobrachia pileus, a member of the early-diverging phylum Ctenophora, the probable sister group of cnidarians. These genes were all expressed in the male and female germlines, and with the exception of one of the Piwi paralogues, they showed similar expression patterns within somatic territories (tentacle root, comb rows, aboral sensory complex). Cytological observations and EdU DNA-labelling and long-term retention experiments revealed concentrations of stem cells closely matching these gene expression areas. These stem cell pools are spatially restricted, and each specialised in the production of particular types of somatic cells. These data unveil important aspects of cell renewal within the ctenophore body and suggest that Piwi, Vasa, Bruno, and PL10 belong to a gene network ancestrally acting in two distinct contexts: (i) the germline and (ii) stem cells, whatever the nature of their progeny. PMID:21036163

  11. Different sensitivity of PPARalpha gene expression to nutritional changes in liver of suckling and adult rats.

    PubMed

    Panadero, Maribel; Herrera, Emilio; Bocos, Carlos

    2005-01-14

    The amount of peroxisome proliferator-activated receptor-alpha (PPARalpha) protein was markedly augmented in the liver of suckling rats compared to adult rats. This different PPARalpha abundance was used to study the sensitivity to nutritional changes in the expression and activity of this receptor. Thus, 10-day-old and adult rats were orally given either glucose, Intralipid or a combination of both diets, and liver mRNA levels of PPARalpha and the PPAR related genes, acyl-CoA oxidase (ACO) and phosphoenolpyruvate carboxykinase (PEPCK), and plasma metabolites were measured. In neonates, the expression of PPARalpha and ACO was seen to increase when the level of FFA in plasma was also high, unless an elevated level of insulin was also present. However, this fatty acid-induced effect was not detected in adult rats. On the contrary, the hepatic expression of PEPCK was modulated by the nutritional changes similarly in both neonates and adult rats. Thus, it may be concluded that the expression of the PPARalpha gene in adult rats seems to be less sensitive to nutritional changes than in neonates. PMID:15607334

  12. [Expression of PRAME gene in adult acute leukemia and its significance in prognosis].

    PubMed

    Zhou, Pei-Yi; Li, Wei-Jia; Wei, Cai-Xia; Zhou, Zhi

    2007-12-01

    The study was aimed to investigate the expression of preferentially expressed antigen of melanoma (PRAME) gene in adult acute leukemia and its clinical significance. The expression of the PRAME gene of bone marrow was measured by reverse transcriptase polymerase chain reaction (RT-PCR) in 73 adult newly diagnosed acute leukemia patients, 3 relapsed patients, 7 patients with idiopathic thrombocytopenic purpura (ITP) and 8 healthy donors, as well as two AL cell-lines (K562 and U937). The results indicated that PRAME mRNA was expressed in 42.9% AML patients (n=24) and 20% ALL patients (n=4), also in two leukemia cell-lines K562 and U937, but not in eight health donors and seven ITP patients. PRAME expression not correlated to the white blood count, hemoglobin level, platelet count and the percentage of blasts at diagnosis, yet independent of age, sex, and FAB type. PRAME mRNA expression in complete remission group seems much higher than those in partial complete remission group and death group. The increased levels of expression could be found prior to the relapse in one patient being regularly monitored. PRAME gene was overexpressed in adult acute leukemia patients and leukemia cell-lines. It is concluded that the expression of PRAME is an indicator of favorable prognosis and can be a useful tool for monitoring minimal residual disease (MRD) in adult acute leukemia. Differential expression between adult acute leukemia patients and healthy volunteers suggests that the immunogenic antigens PRAME are potential candidates for immunotherapy in adult acute leukemia. PMID:18088461

  13. Dynamic Gene Expression in the Human Cerebral Cortex Distinguishes Children from Adults

    PubMed Central

    Sterner, Kirstin N.; Weckle, Amy; Chugani, Harry T.; Tarca, Adi L.; Sherwood, Chet C.; Hof, Patrick R.; Kuzawa, Christopher W.; Boddy, Amy M.; Abbas, Asad; Raaum, Ryan L.; Grégoire, Lucie; Lipovich, Leonard; Grossman, Lawrence I.; Uddin, Monica; Wildman, Derek E.

    2012-01-01

    In comparison with other primate species, humans have an extended juvenile period during which the brain is more plastic. In the current study we sought to examine gene expression in the cerebral cortex during development in the context of this adaptive plasticity. We introduce an approach designed to discriminate genes with variable as opposed to uniform patterns of gene expression and found that greater inter-individual variance is observed among children than among adults. For the 337 transcripts that show this pattern, we found a significant overrepresentation of genes annotated to the immune system process (pFDR≅0). Moreover, genes known to be important in neuronal function, such as brain-derived neurotrophic factor (BDNF), are included among the genes more variably expressed in childhood. We propose that the developmental period of heightened childhood neuronal plasticity is characterized by more dynamic patterns of gene expression in the cerebral cortex compared to adulthood when the brain is less plastic. That an overabundance of these genes are annotated to the immune system suggests that the functions of these genes can be thought of not only in the context of antigen processing and presentation, but also in the context of nervous system development. PMID:22666384

  14. Gene × Smoking Interactions on Human Brain Gene Expression: Finding Common Mechanisms in Adolescents and Adults

    ERIC Educational Resources Information Center

    Wolock, Samuel L.; Yates, Andrew; Petrill, Stephen A.; Bohland, Jason W.; Blair, Clancy; Li, Ning; Machiraju, Raghu; Huang, Kun; Bartlett, Christopher W.

    2013-01-01

    Background: Numerous studies have examined gene × environment interactions (G × E) in cognitive and behavioral domains. However, these studies have been limited in that they have not been able to directly assess differential patterns of gene expression in the human brain. Here, we assessed G × E interactions using two publically available datasets…

  15. PW1 gene/paternally expressed gene 3 (PW1/Peg3) identifies multiple adult stem and progenitor cell populations

    PubMed Central

    Besson, Vanessa; Smeriglio, Piera; Wegener, Amélie; Relaix, Frédéric; Nait Oumesmar, Brahim; Sassoon, David A.; Marazzi, Giovanna

    2011-01-01

    A variety of markers are invaluable for identifying and purifying stem/progenitor cells. Here we report the generation of a murine reporter line driven by Pw1 that reveals cycling and quiescent progenitor/stem cells in all adult tissues thus far examined, including the intestine, blood, testis, central nervous system, bone, skeletal muscle, and skin. Neurospheres generated from the adult PW1-reporter mouse show near 100% reporter-gene expression following a single passage. Furthermore, epidermal stem cells can be purified solely on the basis of reporter-gene expression. These cells are clonogenic, repopulate the epidermal stem-cell niches, and give rise to new hair follicles. Finally, we demonstrate that only PW1 reporter-expressing epidermal cells give rise to follicles that are capable of self-renewal following injury. Our data demonstrate that PW1 serves as an invaluable marker for competent self-renewing stem cells in a wide array of adult tissues, and the PW1-reporter mouse serves as a tool for rapid stem cell isolation and characterization. PMID:21709251

  16. The polyoma virus enhancer cannot substitute for DNase I core hypersensitive sites 2-4 in the human beta-globin LCR.

    PubMed Central

    Tanimoto, K; Liu, Q; Bungert, J; Engel, J D

    1999-01-01

    The polyoma virus enhancer (PyE) is capable of conferring integration position-independent expression to linked genes in stably transfected erythroid cells after joining to DNase I hypersensitive site (HS) 5 of the human beta-globin locus control region (LCR). In attempting to separate the chromatin opening activity of the LCR from its enhancer activity and to investigate contributions of the individual HS core elements to LCR function, the human beta-globin LCR HS2, HS3 and HS4 core elements were replaced with the PyE within the context of a yeast artificial chromosome (YAC) bearing the whole locus. We show here that, in contrast to its function in cultured cells, the PyE is unable to replace HS core element function in vivo. We found that the PyE substitution mutant LCR is unable to provide either chromatin opening or transcriptional potentiating activity at any erythroid developmental stage in transgenic mice. These data provide direct evidence that the human beta-globin LCR core elements specify unique functions that cannot be replaced by a ubiquitous enhancer activity. PMID:10454609

  17. Hb Wilde and Hb Patagonia: two novel elongated beta-globin variants causing dominant beta-thalassemia.

    PubMed

    Scheps, Karen G; Hasenahuer, Marcia A; Parisi, Gustavo; Fornasari, María S; Pennesi, Sandra P; Erramouspe, Beatriz; Basack, Felisa N; Veber, Ernesto S; Aversa, Luis; Elena, Graciela; Varela, Viviana

    2015-06-01

    We describe here the molecular and hematological characteristics of novel frameshift mutations in exon 2 of the HBB gene (in heterozygous state) found in two Argentinean pediatric patients with dominant β-thalassemia-like features. In Hb Wilde, HBB:c.270_273delTGAG(p.Glu90Cysfs*67), we detected the deletion of the third base of the codon 89 (T) and the codon 90 (GAG), whereas in Hb Patagonia, HBB:c.296_297dupGT(p.Asp99Trpfs*59), the frameshift mutation was due to a duplication of a 'GT' dinucleotide after the second base of codon 98 (GTG). The Hb Patagonia and Hb Wilde mutations would result in elongated β-globin chains with modified C-terminal sequences and a total of 155 and 157 amino acids residues, respectively. Based on bioinformatics and structural analysis, as well as protein modeling, we predict that the elongated β-globins would affect the formation of the αβ dimers and their stability, which would further support the mechanism for the observed clinical features in both patients. PMID:25284604

  18. Characterization of a DNA binding activity in DNAse I hypersensitive site 4 of the human globin locus control region.

    PubMed Central

    Walters, M; Kim, C; Gelinas, R

    1991-01-01

    A portion of the beta-globin Locus Control Region (LCR), which included DNAse I hypersensitive site 4 (HS4), was analyzed for its interactions with nuclear extracts and its contribution to LCR activity in a functional assay. In gel retardation assays, a short fragment from HS4 formed complexes with nuclear extracts from both erythroid and nonerythroid cells, and a core protected sequence 5'GACTGGC3' was revealed by DNAse I protection and methylation interference studies. This sequence resembles the binding sites of CCAAT-family members. Purified CP-2 but not CP-1 was shown to bind this HS4 sequence in a gel shift reaction, suggesting that the HS4 binding activity shares some sequence specificity with the CCAAT-factor family. Utilizing a transient expression assay in murine erythroleukemia cells, steady-state RNA levels were measured from pairs of LCR constructs linked to distinguishable beta-globin reporter genes. A short DNA fragment from HS4 which included the binding site for this novel binding activity accounted for most of the contribution to high level expression made by the entire HS4 region. Images PMID:1923823

  19. Polygenic risk for externalizing disorders: Gene-by-development and gene-by-environment effects in adolescents and young adults

    PubMed Central

    Salvatore, Jessica E.; Aliev, Fazil; Bucholz, Kathleen; Agrawal, Arpana; Hesselbrock, Victor; Hesselbrock, Michie; Bauer, Lance; Kuperman, Samuel; Schuckit, Marc A.; Kramer, John; Edenberg, Howard J.; Foroud, Tatiana M.; Dick, Danielle M.

    2014-01-01

    In this project, we aimed to bring large-scale gene identification findings into a developmental psychopathology framework. Using a family-based sample, we tested whether polygenic scores for externalizing disorders—based on single nucleotide polymorphism weights derived from genome-wide association study results in adults (n = 1,249)—predicted externalizing disorders, subclinical externalizing behavior, and impulsivity-related traits adolescents (n = 248) and young adults (n = 207), and whether parenting and peer factors in adolescence moderated polygenic risk to predict externalizing disorders. Polygenic scores predicted externalizing disorders in adolescents and young adults, even after controlling for parental externalizing disorder history. Polygenic scores also predicted subclinical externalizing behavior and impulsivity traits in the adolescents and young adults. Adolescent parental monitoring and peer substance use moderated polygenic scores to predict externalizing disorders. This illustrates how state of the science genetics can be integrated with psychological science to identify how genetic risk contributes to the development of psychopathology. PMID:25821660

  20. The nuclear receptor gene nhr-25 plays multiple roles in the C. elegans heterochronic gene network to control the larva-to-adult transition

    PubMed Central

    Hada, Kazumasa; Asahina, Masako; Hasegawa, Hiroshi; Kanaho, Yasunori; Slack, Frank J.; Niwa, Ryusuke

    2010-01-01

    Developmental timing in the nematode Caenorhabditis elegans is controlled by heterochronic genes, mutations in which cause changes in the relative timing of developmental events. One of the heterochronic genes, let-7, encodes a microRNA that is highly evolutionarily conserved, suggesting that similar genetic pathways control developmental timing across phyla. Here we report that the nuclear receptor nhr-25, which belongs to the evolutionarily conserved fushi tarazu-factor 1/nuclear receptor NR5A subfamily, interacts with heterochronic genes that regulate the larva-to-adult transition in C. elegans. We identified nhr-25 as a regulator of apl-1, a homolog of the Alzheimer’s amyloid precursor protein-like gene that is downstream of let-7 family microRNAs. NHR-25 controls not only apl-1 expression but also regulates developmental progression in the larva-to-adult transition. NHR-25 negatively regulates the expression of the adult-specific collagen gene col-19 in lateral epidermal seam cells. In contrast, NHR-25 positively regulates the larva-to-adult transition for other timed events in seam cells, such as cell fusion, cell division and alae formation. The genetic relationships between nhr-25 and other heterochronic genes are strikingly varied among several adult developmental events. We propose that nhr-25 has multiple roles in both promoting and inhibiting the C. elegans heterochronic gene pathway controlling adult differentiation programs. PMID:20678979

  1. High-resolution gene expression atlases for adult and developing mouse brain and spinal cord.

    PubMed

    Henry, Alex M; Hohmann, John G

    2012-10-01

    Knowledge of the structure, genetics, circuits, and physiological properties of the mammalian brain in both normal and pathological states is ever increasing as research labs worldwide probe the various aspects of brain function. Until recently, however, comprehensive cataloging of gene expression across the central nervous system has been lacking. The Allen Institute for Brain Science, as part of its mission to propel neuroscience research, has completed several large gene-mapping projects in mouse, nonhuman primate, and human brain, producing informative online public resources and tools. Here we present the Allen Mouse Brain Atlas, covering ~20,000 genes throughout the adult mouse brain; the Allen Developing Mouse Brain Atlas, detailing expression of approximately 2,000 important developmental genes across seven embryonic and postnatal stages of brain growth; and the Allen Spinal Cord Atlas, revealing expression for ~20,000 genes in the adult and neonatal mouse spinal cords. Integrated data-mining tools, including reference atlases, informatics analyses, and 3-D viewers, are described. For these massive-scale projects, high-throughput industrial techniques were developed to standardize and reliably repeat experimental goals. To verify consistency and accuracy, a detailed analysis of the 1,000 most viewed genes for the adult mouse brain (according to website page views) was performed by comparing our data with peer-reviewed literature and other databases. We show that our data are highly consistent with independent sources and provide a comprehensive compendium of information and tools used by thousands of researchers each month. All data and tools are freely available via the Allen Brain Atlas portal (www.brain-map.org). PMID:22832508

  2. High diversity of alpha-globin haplotypes in a Senegalese population, including many previously unreported variants.

    PubMed Central

    Martinson, J J; Excoffier, L; Swinburn, C; Boyce, A J; Harding, R M; Langaney, A; Clegg, J B

    1995-01-01

    RFLP haplotypes at the alpha-globin gene complex have been examined in 190 individuals from the Niokolo Mandenka population of Senegal: haplotypes were assigned unambiguously for 210 chromosomes. The Mandenka share with other African populations a sample size-independent haplotype diversity that is much greater than that in any non-African population: the number of haplotypes observed in the Mandenka is typically twice that seen in the non-African populations sampled to date. Of these haplotypes, 17.3% had not been observed in any previous surveys, and a further 19.1% have previously been reported only in African populations. The haplotype distribution shows clear differences between African and non-African peoples, but this is on the basis of population-specific haplotypes combined with haplotypes common to all. The relationship of the newly reported haplotypes to those previously recorded suggests that several mutation processes, particularly recombination as homologous exchange or gene conversion, have been involved in their production. A computer program based on the expectation-maximization (EM) algorithm was used to obtain maximum-likelihood estimates of haplotype frequencies for the entire data set: good concordance between the unambiguous and EM-derived sets was seen for the overall haplotype frequencies. Some of the low-frequency haplotypes reported by the estimation algorithm differ greatly, in structure, from those haplotypes known to be present in human populations, and they may not represent haplotypes actually present in the sample. PMID:7485171

  3. Adult rat bone marrow stromal cells express genes associated with dopamine neurons

    SciTech Connect

    Kramer, Brian C.; Woodbury, Dale . E-mail: WOODBURYDL@AOL.COM; Black, Ira B.

    2006-05-19

    An intensive search is underway to identify candidates to replace the cells that degenerate in Parkinson's disease (PD). To date, no suitable substitute has been found. We have recently found that adult rat bone marrow stromal cells (MSCs) can be induced to assume a neuronal phenotype in vitro. These findings may have particular relevance to the treatment of PD. We now report that adult MSCs express multiple dopaminergic genes, suggesting that they are potential candidates for cell therapy. Using RT-PCR, we have examined families of genes that are associated with the development and/or survival of dopaminergic neurons. MSCs transcribe a variety of dopaminergic genes including patched and smoothened (components of the Shh receptor), Gli-1 (downstream mediator of Shh), and Otx-1, a gene associated with formation of the mesencephalon during development. Furthermore, Shh treatment elicits a 1.5-fold increase in DNA synthesis in cultured MSCs, suggesting the presence of a functional Shh receptor complex. We have also found that MSCs transcribe and translate Nurr-1, a nuclear receptor essential for the development of dopamine neurons. In addition, MSCs express a variety of growth factor receptors including the glycosyl-phosphatidylinositol-anchored ligand-binding subunit of the GDNF receptor, GFR{alpha}1, as well as fibroblast growth factor receptors one and four. The expression of genes that are associated with the development and survival of dopamine neurons suggests a potential role for these cells in the treatment of Parkinson's disease.

  4. Gene expression patterns underlying the reinstatement of plasticity in the adult visual system.

    PubMed

    Tiraboschi, Ettore; Guirado, Ramon; Greco, Dario; Auvinen, Petri; Maya-Vetencourt, Jose Fernando; Maffei, Lamberto; Castrén, Eero

    2013-01-01

    The nervous system is highly sensitive to experience during early postnatal life, but this phase of heightened plasticity decreases with age. Recent studies have demonstrated that developmental-like plasticity can be reactivated in the visual cortex of adult animals through environmental or pharmacological manipulations. These findings provide a unique opportunity to study the cellular and molecular mechanisms of adult plasticity. Here we used the monocular deprivation paradigm to investigate large-scale gene expression patterns underlying the reinstatement of plasticity produced by fluoxetine in the adult rat visual cortex. We found changes, confirmed with RT-PCRs, in gene expression in different biological themes, such as chromatin structure remodelling, transcription factors, molecules involved in synaptic plasticity, extracellular matrix, and excitatory and inhibitory neurotransmission. Our findings reveal a key role for several molecules such as the metalloproteases Mmp2 and Mmp9 or the glycoprotein Reelin and open up new insights into the mechanisms underlying the reopening of the critical periods in the adult brain. PMID:23936678

  5. Detection of a major gene for heterocellular hereditary persistence of fetal hemoglobin after accounting for genetic modifiers.

    PubMed

    Thein, S L; Sampietro, M; Rohde, K; Rochette, J; Weatherall, D J; Lathrop, G M; Demenais, F

    1994-02-01

    "Heterocellular hereditary persistence of fetal hemoglobin" (HPFH) is the term used to describe the genetically determined persistence of fetal hemoglobin (Hb F) production into adult life, in the absence of any related hematological disorder. Whereas some forms are caused by mutations in the beta-globin gene cluster on chromosome 11, others segregate independently. While the latter are of particular interest with respect to the regulation of globin gene switching, it has not been possible to determine their chromosomal location, mainly because their mode of inheritance is not clear, but also because several other factors are known to modify Hb F production. We have examined a large Asian Indian pedigree which includes individuals with heterocellular HPFH associated with beta-thalassemia and/or alpha-thalassemia. Segregation analysis was conducted on the HPFH trait FC, defined to be the percentage of Hb F-containing cells (F-cells), using the class D regressive model. Our results provide evidence for the presence of a major gene, dominant or codominant, which controls the FC values with residual familial correlations. The major gene was detected when the effects of genetic modifiers, notably beta-thalassemia and the XmnI-G gamma polymorphism, are accounted for in the analysis. Linkage with the beta-globin gene cluster is excluded. The transmission of the FC values in this pedigree is informative enough to allow detection of linkage with an appropriate marker(s). The analytical approach outlined in this study, using simple regression to allow for genetic modifiers and thus allowing the mode of inheritance of a trait to be dissected out, may be useful as a model for segregation and linkage analyses of other complex phenotypes. PMID:7508182

  6. Gene polymorphisms in association with emerging cardiovascular risk markers in adult women

    PubMed Central

    2010-01-01

    Background Evidence on the associations of emerging cardiovascular disease risk factors/markers with genes may help identify intermediate pathways of disease susceptibility in the general population. This population-based study is aimed to determine the presence of associations between a wide array of genetic variants and emerging cardiovascular risk markers among adult US women. Methods The current analysis was performed among the National Health and Nutrition Examination Survey (NHANES) III phase 2 samples of adult women aged 17 years and older (sample size n = 3409). Fourteen candidate genes within ADRB2, ADRB3, CAT, CRP, F2, F5, FGB, ITGB3, MTHFR, NOS3, PON1, PPARG, TLR4, and TNF were examined for associations with emerging cardiovascular risk markers such as serum C-reactive protein, homocysteine, uric acid, and plasma fibrinogen. Linear regression models were performed using SAS-callable SUDAAN 9.0. The covariates included age, race/ethnicity, education, menopausal status, female hormone use, aspirin use, and lifestyle factors. Results In covariate-adjusted models, serum C-reactive protein concentrations were significantly (P value controlling for false-discovery rate ≤ 0.05) associated with polymorphisms in CRP (rs3093058, rs1205), MTHFR (rs1801131), and ADRB3 (rs4994). Serum homocysteine levels were significantly associated with MTHFR (rs1801133). Conclusion The significant associations between certain gene variants with concentration variations in serum C-reactive protein and homocysteine among adult women need to be confirmed in further genetic association studies. PMID:20078877

  7. 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.

    PubMed

    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. PMID:25931013

  8. Effects of Doxycycline on gene expression in Wolbachia and Brugia malayi adult female worms in vivo

    PubMed Central

    2012-01-01

    Background Most filarial nematodes contain Wolbachia symbionts. The purpose of this study was to examine the effects of doxycycline on gene expression in Wolbachia and adult female Brugia malayi. Methods Brugia malayi infected gerbils were treated with doxycycline for 6-weeks. This treatment largely cleared Wolbachia and arrested worm reproduction. RNA recovered from treated and control female worms was labeled by random priming and hybridized to the Version 2- filarial microarray to obtain expression profiles. Results and discussion Results showed significant changes in expression for 200 Wolbachia (29% of Wolbachia genes with expression signals in untreated worms) and 546 B. malayi array elements after treatment. These elements correspond to known genes and also to novel genes with unknown biological functions. Most differentially expressed Wolbachia genes were down-regulated after treatment (98.5%). In contrast, doxycycline had a mixed effect on B. malayi gene expression with many more genes being significantly up-regulated after treatment (85% of differentially expressed genes). Genes and processes involved in reproduction (gender-regulated genes, collagen, amino acid metabolism, ribosomal processes, and cytoskeleton) were down-regulated after doxycycline while up-regulated genes and pathways suggest adaptations for survival in response to stress (energy metabolism, electron transport, anti-oxidants, nutrient transport, bacterial signaling pathways, and immune evasion). Conclusions Doxycycline reduced Wolbachia and significantly decreased bacterial gene expression. Wolbachia ribosomes are believed to be the primary biological target for doxycycline in filarial worms. B. malayi genes essential for reproduction, growth and development were also down-regulated; these changes are consistent with doxycycline effects on embryo development and reproduction. On the other hand, many B. malayi genes involved in energy production, electron-transport, metabolism, anti

  9. Nucleotide sequence of 3' untranslated portion of human alpha globin mRNA.

    PubMed Central

    Wilson, J T; deRiel, J K; Forget, B G; Marotta, C A; Weissman, S M

    1977-01-01

    We have determined the nucleotide sequence of 75 nucleotides of the 3'-untranslated portion of normal human alpha globin mRNA which corresponds to the elongated amino acid sequence of the chain termination mutant Hb Constant Spring. This was accomplished by sequence analysis of cDNA fragments obtained by restriction endonuclease or T4 endonuclease IV cleavage of human globin cDNA synthesized from globin mRNA by use of viral reverse transcriptase. Analysis of cRNA synthesized from cDNA by use of RNA polymerase provided additional confirmatory sequence information. Possible polymorphism has been identified at one site of the sequence. Our sequence overlaps with, and extends the sequence of 43 nucleotides determined by Proudfood and coworkers for the very 3'-terminal portion of human alpha globin mRNA. The complete 3'-untranslated sequence of human alpha globin mRNA (112 nucleotides including termination codon) shows little homology to that of the human or rabbit beta globin mRNAs except for the presence of the hexanucleotide sequence AAUAAA which is found in most eukaryotic mRNAs near the 3'-terminal poly (A). Images PMID:909779

  10. Dose-Responsive Gene Expression Changes in Juvenile and Adult Mummichogs (Fundulus heteroclitus) After Arsenic Exposure

    PubMed Central

    Gonzalez, Horacio O.; Hu, Jianjun; Gaworecki, Kristen M.; Roling, Jonathan A.; Baldwin, William S.; Gardea-Torresdey, Jorge L.; Bain, Lisa J.

    2010-01-01

    The present study investigated arsenic's effects on mummichogs (Fundulus heteroclitus), while also examining what role that gender or exposure age might play. Adult male and female mummichogs were exposed to 172ppb, 575ppb, or 1,720ppb arsenic as sodium arsenite for 10 days immediately prior to spawning. No differences were noted in the number or viability of eggs between the groups, but there was a significant increase in deformities in 1,720ppb arsenic exposure group. Total RNA from adult livers or 6-week old juveniles was used to probe custom macroarrays for changes in gene expression. In females, 3% of the genes were commonly differentially expressed in the 172 and 575ppb exposure groups compared to controls. In the males, between 1.1-3% of the differentially expressed genes were in common between the exposure groups. Several genes, including apolipoprotein and serum amyloid precursor were commonly expressed in either a dose-responsive manner or were dose-specific, but consistent across genders. These patterns of regulation were confirmed by QPCR. These findings will provide us with a better understanding of the effects of dose, gender, and exposure age on the response to arsenic. PMID:20451245

  11. A mouse model for adult cardiac-specific gene deletion with CRISPR/Cas9.

    PubMed

    Carroll, Kelli J; Makarewich, Catherine A; McAnally, John; Anderson, Douglas M; Zentilin, Lorena; Liu, Ning; Giacca, Mauro; Bassel-Duby, Rhonda; Olson, Eric N

    2016-01-12

    Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)9 genomic editing has revolutionized the generation of mutant animals by simplifying the creation of null alleles in virtually any organism. However, most current approaches with this method require zygote injection, making it difficult to assess the adult, tissue-specific functions of genes that are widely expressed or which cause embryonic lethality when mutated. Here, we describe the generation of cardiac-specific Cas9 transgenic mice, which express high levels of Cas9 in the heart, but display no overt defects. In proof-of-concept experiments, we used Adeno-Associated Virus 9 (AAV9) to deliver single-guide RNA (sgRNA) that targets the Myh6 locus exclusively in cardiomyocytes. Intraperitoneal injection of postnatal cardiac-Cas9 transgenic mice with AAV9 encoding sgRNA against Myh6 resulted in robust editing of the Myh6 locus. These mice displayed severe cardiomyopathy and loss of cardiac function, with elevation of several markers of heart failure, confirming the effectiveness of this method of adult cardiac gene deletion. Mice with cardiac-specific expression of Cas9 provide a tool that will allow rapid and accurate deletion of genes following a single injection of AAV9-sgRNAs, thereby circumventing embryonic lethality. This method will be useful for disease modeling and provides a means of rapidly editing genes of interest in the heart. PMID:26719419

  12. Mammalian Fetal Cardiac Regeneration Following Myocardial Infarction is Associated with Differential Gene Expression Compared to the Adult

    PubMed Central

    Zgheib, Carlos; Allukian, Myron W.; Xu, Junwang; Morris, Michael W.; Caskey, Robert C.; Herdrich, Benjamin J.; Hu, Junyi; Gorman, Joseph H.; Gorman, Robert C.; Liechty, Kenneth W.

    2014-01-01

    Background In adults, MI results in a brisk inflammatory response, myocardium loss and scar formation. We have recently reported the first mammalian large animal model of cardiac regeneration following MI in fetal sheep. We hypothesize that the fetus ability to regenerate functional myocardium following MI is due to differential gene expression regulating the response to MI in the fetus compared to the adult. Methods MI was created in adult (n=4) or early gestation fetal (n=4) sheep. Tissue harvested after 3 or 30 days, RNA extracted for microarray, followed by PCA and global gene expression analysis for the gene ontology (GO) terms: “response to wounding”, “inflammatory response”, “extracellular matrix”, “cell cycle”, “cell migration”, “cell proliferation” and “apoptosis”. Results PCA demonstrated that the global gene expression pattern in adult infarcts was distinctly different from uninfarcted region at 3 days and remained different 30 days post-MI. In contrast, gene expression in the fetal infarct was different from the uninfarcted region at 3 days, but by 30 days it returned to a baseline expression pattern similar to the uninfarcted region. 3 days post-MI there was an increase in the expression of genes related to all GO terms in fetal and adult infarcts, but this increase was much more pronounced in adults. By 30 days, the fetal gene expression returned to baseline, whereas in the adult remained significantly elevated. Conclusions These data demonstrate that the global gene expression pattern is dramatically different in the fetal regenerative response to MI compared to the adult response and may partly be responsible for the regeneration. PMID:24792251

  13. A Stem Cell-Like Chromatin Pattern May Predispose Tumor Suppressor Genes to DNA Hypermethylation and Silencing in Adult Cancers

    PubMed Central

    Ohm, Joyce E.; McGarvey, Kelly M.; Yu, Xiaobing; Cheng, Linzhao; Schuebel, Kornel E.; Cope, Leslie; Mohammad, Helai P.; Chen, Wei; Daniel, Vincent C.; Yu, Wayne; Berman, David M.; Jenuwein, Thomas; Pruitt, Kevin; Sharkis, Saul J.; Watkins, D. Neil; Herman, James G.; Baylin, Stephen B.

    2009-01-01

    Adult cancers may derive from stem or early progenitor cells1,2. Epigenetic modulation of gene expression is essential for normal function of these early cells, but is highly abnormal in cancers, which often exhibit aberrant promoter CpG island hypermethylation and transcriptional silencing of tumor suppressor genes and pro-differentiation factors3-5. We find that, for such genes, both normal and malignant embryonic cells generally lack the gene DNA hypermethylation found in adult cancers. In embryonic stem (ES) cells, these genes are held in a “transcription ready” state mediated by a “bivalent” promoter chromatin pattern consisting of the repressive polycomb group (PcG) H3K27me mark plus the active mark, H3K4me. However, embryonic carcinoma (EC) cells add two key repressive marks, H3K9me2 and H3K9me3, both associated with DNA hypermethylated genes in adult cancers6-8. We hypothesize that cell chromatin patterns and transient silencing of these important growth regulatory genes in stem or progenitor cells of origin for cancer may leave these genes vulnerable to aberrant DNA hypermethylation and heritable gene silencing in adult tumors. PMID:17211412

  14. Allele-specific enzymatic amplification of. beta. -globin genomic DNA for diagnosis of sickle cell anemia

    SciTech Connect

    Wu, D.Y.; Ugozzoli, L.; Pal, B.K.; Wallace, B. )

    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.

  15. Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs

    PubMed Central

    Miyanohara, Atsushi; Kamizato, Kota; Juhas, Stefan; Juhasova, Jana; Navarro, Michael; Marsala, Silvia; Lukacova, Nada; Hruska-Plochan, Marian; Curtis, Erik; Gabel, Brandon; Ciacci, Joseph; Ahrens, Eric T; Kaspar, Brian K; Cleveland, Don; Marsala, Martin

    2016-01-01

    Effective in vivo use of adeno-associated virus (AAV)-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal). Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i) potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii) delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii) potent retrograde transgene expression in brain motor centers (motor cortex and brain stem); and (iv) the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients. PMID:27462649

  16. Gene therapy for red-green colour blindness in adult primates.

    PubMed

    Mancuso, Katherine; Hauswirth, William W; Li, Qiuhong; Connor, Thomas B; Kuchenbecker, James A; Mauck, Matthew C; Neitz, Jay; Neitz, Maureen

    2009-10-01

    Red-green colour blindness, which results from the absence of either the long- (L) or the middle- (M) wavelength-sensitive visual photopigments, is the most common single locus genetic disorder. Here we explore the possibility of curing colour blindness using gene therapy in experiments on adult monkeys that had been colour blind since birth. A third type of cone pigment was added to dichromatic retinas, providing the receptoral basis for trichromatic colour vision. This opened a new avenue to explore the requirements for establishing the neural circuits for a new dimension of colour sensation. Classic visual deprivation experiments have led to the expectation that neural connections established during development would not appropriately process an input that was not present from birth. Therefore, it was believed that the treatment of congenital vision disorders would be ineffective unless administered to the very young. However, here we show that the addition of a third opsin in adult red-green colour-deficient primates was sufficient to produce trichromatic colour vision behaviour. Thus, trichromacy can arise from a single addition of a third cone class and it does not require an early developmental process. This provides a positive outlook for the potential of gene therapy to cure adult vision disorders. PMID:19759534

  17. Adult-Onset Presentations of Genetic Immunodeficiencies: Genes Can Throw Slow Curves

    PubMed Central

    Nelson, Katharine S.; Lewis, David B.

    2016-01-01

    Purpose of Review The molecular and genetic mechanisms behind adult presentations of primary immunodeficiency diseases are examined, with particular emphasis on cases where this was heralded by severe, recurrent or opportunistic infection. Recent Findings A detailed analysis over the last two decades of the relationship between genotype and clinical phenotype for a number of genetic immunodeficiencies has revealed multiple mechanisms that can account for the delayed presentation of genetic disorders that typically present in childhood, including hypomorphic gene mutations and X-linked gene mutations with age-related skewing in random X-chromosome inactivation. Adult-onset presentations of chronic granulomatous disease, X-linked agammaglobulinemia, interleukin-12/T helper 1/interferon-gamma and interleukin-23/T helper 17/interleukin-17 pathway defects, and X-linked lymphoproliferative disorder are used to illustrate these mechanisms. Finally, certain genetic types of common variable immunodeficiency are used to illustrate that inherited null mutations can take decades to manifest immunologically. Summary Both genetic mechanisms and environmental factors can account for adult-onset infectious and non-infectious complications as manifestations of disorders that typically present in childhood. This emphasizes the potential complexity in the relationship between genotype and phenotype with natural human mutations. PMID:20581672

  18. Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs.

    PubMed

    Miyanohara, Atsushi; Kamizato, Kota; Juhas, Stefan; Juhasova, Jana; Navarro, Michael; Marsala, Silvia; Lukacova, Nada; Hruska-Plochan, Marian; Curtis, Erik; Gabel, Brandon; Ciacci, Joseph; Ahrens, Eric T; Kaspar, Brian K; Cleveland, Don; Marsala, Martin

    2016-01-01

    Effective in vivo use of adeno-associated virus (AAV)-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal). Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i) potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii) delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii) potent retrograde transgene expression in brain motor centers (motor cortex and brain stem); and (iv) the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients. PMID:27462649

  19. Correlation of a set of gene variants, life events and personality features on adult ADHD severity.

    PubMed

    Müller, Daniel J; Chiesa, Alberto; Mandelli, Laura; De Luca, Vincenzo; De Ronchi, Diana; Jain, Umesh; Serretti, Alessandro; Kennedy, James L

    2010-07-01

    Increasing evidence suggests that symptoms of attention deficit hyperactivity disorder (ADHD) could persist into adult life in a substantial proportion of cases. The aim of the present study was to investigate the impact of (1) adverse events, (2) personality traits and (3) genetic variants chosen on the basis of previous findings and (4) their possible interactions on adult ADHD severity. One hundred and ten individuals diagnosed with adult ADHD were evaluated for occurrence of adverse events in childhood and adulthood, and personality traits by the Temperament and Character Inventory (TCI). Common polymorphisms within a set of nine important candidate genes (SLC6A3, DBH, DRD4, DRD5, HTR2A, CHRNA7, BDNF, PRKG1 and TAAR9) were genotyped for each subject. Life events, personality traits and genetic variations were analyzed in relationship to severity of current symptoms, according to the Brown Attention Deficit Disorder Scale (BADDS). Genetic variations were not significantly associated with severity of ADHD symptoms. Life stressors displayed only a minor effect as compared to personality traits. Indeed, symptoms' severity was significantly correlated with the temperamental trait of Harm avoidance and the character trait of Self directedness. The results of the present work are in line with previous evidence of a significant correlation between some personality traits and adult ADHD. However, several limitations such as the small sample size and the exclusion of patients with other severe comorbid psychiatric disorders could have influenced the significance of present findings. PMID:20006992

  20. Schizophrenia susceptibility alleles are enriched for alleles that affect gene expression in adult human brain

    PubMed Central

    Richards, Alexander L; Jones, Lesley; Moskvina, Valentina; Kirov, George; Gejman, Pablo V; Levinson, Douglas F; Sanders, Alan R; Purcell, Shaun; Visscher, Peter M; Craddock, Nick; Owen, Michael J; Holmans, Peter; O’Donovan, Michael C

    2016-01-01

    It is widely thought that alleles that influence susceptibility to common diseases, including schizophrenia, will frequently do so through effects on gene expression. Since only a small proportion of the genetic variance for schizophrenia has been attributed to specific loci, this remains an unproven hypothesis. The International Schizophrenia Consortium (ISC) recently reported a substantial polygenic contribution to that disorder, and that schizophrenia risk alleles are enriched among SNPs selected for marginal evidence for association (p<0.5) from genome wide association studies (GWAS). It follows that if schizophrenia susceptibility alleles are enriched for those that affect gene expression, those marginally associated SNPs which are also eQTLs should carry more true association signals compared with SNPs which are not. To test this, we identified marginally associated (p<0.5) SNPs from two of the largest available schizophrenia GWAS datasets. We assigned eQTL status to those SNPs based upon an eQTL dataset derived from adult human brain. Using the polygenic score method of analysis reported by the ISC, we observed and replicated the observation that higher probability cis-eQTLs predicted schizophrenia better than those with a lower probability for being a cis-eQTL. Our data support the hypothesis that alleles conferring risk of schizophrenia are enriched among those that affect gene expression. Moreover, our data show that notwithstanding the likely developmental origin of schizophrenia, studies of adult brain tissue can in principle allow relevant susceptibility eQTLs to be identified. PMID:21339752

  1. Catalog of gene expression in adult neural stem cells and their in vivo microenvironment

    SciTech Connect

    Williams, Cecilia; Wirta, Valtteri; Meletis, Konstantinos; Wikstroem, Lilian; Carlsson, Leif; Frisen, Jonas; Lundeberg, Joakim . E-mail: joakim.lundeberg@biotech.kth.se

    2006-06-10

    Stem cells generally reside in a stem cell microenvironment, where cues for self-renewal and differentiation are present. However, the genetic program underlying stem cell proliferation and multipotency is poorly understood. Transcriptome analysis of stem cells and their in vivo microenvironment is one way of uncovering the unique stemness properties and provides a framework for the elucidation of stem cell function. Here, we characterize the gene expression profile of the in vivo neural stem cell microenvironment in the lateral ventricle wall of adult mouse brain and of in vitro proliferating neural stem cells. We have also analyzed an Lhx2-expressing hematopoietic-stem-cell-like cell line in order to define the transcriptome of a well-characterized and pure cell population with stem cell characteristics. We report the generation, assembly and annotation of 50,792 high-quality 5'-end expressed sequence tag sequences. We further describe a shared expression of 1065 transcripts by all three stem cell libraries and a large overlap with previously published gene expression signatures for neural stem/progenitor cells and other multipotent stem cells. The sequences and cDNA clones obtained within this framework provide a comprehensive resource for the analysis of genes in adult stem cells that can accelerate future stem cell research.

  2. Identification of Susceptibility Genes of Adult Asthma in French Canadian Women

    PubMed Central

    Bérubé, Jean-Christophe; Gaudreault, Nathalie; Lavoie-Charland, Emilie; Sbarra, Laura; Henry, Cyndi; Madore, Anne-Marie; Paré, Peter D.; van den Berge, Maarten; Nickle, David; Laviolette, Michel; Laprise, Catherine; Boulet, Louis-Philippe; Bossé, Yohan

    2016-01-01

    Susceptibility genes of asthma may be more successfully identified by studying subgroups of phenotypically similar asthma patients. This study aims to identify single nucleotide polymorphisms (SNPs) associated with asthma in French Canadian adult women. A pooling-based genome-wide association study was performed in 240 allergic asthmatic and 120 allergic nonasthmatic women. The top associated SNPs were selected for individual genotyping in an extended cohort of 349 asthmatic and 261 nonasthmatic women. The functional impact of asthma-associated SNPs was investigated in a lung expression quantitative trait loci (eQTL) mapping study (n = 1035). Twenty-one of the 38 SNPs tested by individual genotyping showed P values lower than 0.05 for association with asthma. Cis-eQTL analyses supported the functional contribution of rs17801353 associated with C3AR1 (P = 7.90E − 10). The asthma risk allele for rs17801353 is associated with higher mRNA expression levels of C3AR1 in lung tissue. In silico functional characterization of the asthma-associated SNPs also supported the contribution of C3AR1 and additional genes including SYNE1, LINGO2, and IFNG-AS1. This pooling-based GWAS in French Canadian adult women followed by lung eQTL mapping suggested C3AR1 as a functional locus associated with asthma. Additional susceptibility genes were suggested in this homogenous subgroup of asthma patients.

  3. Rare Copy Number Variations in Adults with Tetralogy of Fallot Implicate Novel Risk Gene Pathways

    PubMed Central

    Costain, Gregory; Merico, Daniele; Migita, Ohsuke; Liu, Ben; Yuen, Tracy; Rickaby, Jessica; Thiruvahindrapuram, Bhooma; Marshall, Christian R.; Scherer, Stephen W.; Bassett, Anne S.

    2012-01-01

    Structural genetic changes, especially copy number variants (CNVs), represent a major source of genetic variation contributing to human disease. Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart disease, but to date little is known about the role of CNVs in the etiology of TOF. Using high-resolution genome-wide microarrays and stringent calling methods, we investigated rare CNVs in a prospectively recruited cohort of 433 unrelated adults with TOF and/or pulmonary atresia at a single centre. We excluded those with recognized syndromes, including 22q11.2 deletion syndrome. We identified candidate genes for TOF based on converging evidence between rare CNVs that overlapped the same gene in unrelated individuals and from pathway analyses comparing rare CNVs in TOF cases to those in epidemiologic controls. Even after excluding the 53 (10.7%) subjects with 22q11.2 deletions, we found that adults with TOF had a greater burden of large rare genic CNVs compared to controls (8.82% vs. 4.33%, p = 0.0117). Six loci showed evidence for recurrence in TOF or related congenital heart disease, including typical 1q21.1 duplications in four (1.18%) of 340 Caucasian probands. The rare CNVs implicated novel candidate genes of interest for TOF, including PLXNA2, a gene involved in semaphorin signaling. Independent pathway analyses highlighted developmental processes as potential contributors to the pathogenesis of TOF. These results indicate that individually rare CNVs are collectively significant contributors to the genetic burden of TOF. Further, the data provide new evidence for dosage sensitive genes in PLXNA2-semaphorin signaling and related developmental processes in human cardiovascular development, consistent with previous animal models. PMID:22912587

  4. Apoptosis-Related Gene Expression in an Adult Cohort with Crimean-Congo Hemorrhagic Fever

    PubMed Central

    Guler, Nil; Eroglu, Cafer; Yilmaz, Hava; Karadag, Adil; Alacam, Hasan; Sunbul, Mustafa; Fletcher, Tom E.; Leblebicioglu, Hakan

    2016-01-01

    Crimean-Congo Hemorrhagic Fever (CCHF) is a life threatening acute viral infection characterized by fever, bleeding, leukopenia and thrombocytopenia. It is a major emerging infectious diseases threat, but its pathogenesis remains poorly understood and few data exist for the role of apoptosis in acute infection. We aimed to assess apoptotic gene expression in leukocytes in a cross-sectional cohort study of adults with CCHF. Twenty participants with CCHF and 10 healthy controls were recruited at a tertiary CCHF unit in Turkey; at admission baseline blood tests were collected and total RNA was isolated. The RealTime ready Human Apoptosis Panel was used for real-time PCR, detecting differences in gene expression. Participants had CCHF severity grading scores (SGS) with low risk score (10 out of 20) and intermediate or high risk scores (10 out of 20) for mortality. Five of 20 participants had a fatal outcome. Gene expression analysis showed modulation of pro-apoptotic and anti-apoptotic genes that facilitate apoptosis in the CCHF patient group. Dominant extrinsic pathway activation, mostly related with TNF family members was observed. Severe and fatal cases suggest additional intrinsic pathway activation. The clinical significance of relative gene expression is not clear, and larger longitudinal studies with simultaneous measurement of host and viral factors are recommended. PMID:27304063

  5. Apoptosis-Related Gene Expression in an Adult Cohort with Crimean-Congo Hemorrhagic Fever.

    PubMed

    Guler, Nil; Eroglu, Cafer; Yilmaz, Hava; Karadag, Adil; Alacam, Hasan; Sunbul, Mustafa; Fletcher, Tom E; Leblebicioglu, Hakan

    2016-01-01

    Crimean-Congo Hemorrhagic Fever (CCHF) is a life threatening acute viral infection characterized by fever, bleeding, leukopenia and thrombocytopenia. It is a major emerging infectious diseases threat, but its pathogenesis remains poorly understood and few data exist for the role of apoptosis in acute infection. We aimed to assess apoptotic gene expression in leukocytes in a cross-sectional cohort study of adults with CCHF. Twenty participants with CCHF and 10 healthy controls were recruited at a tertiary CCHF unit in Turkey; at admission baseline blood tests were collected and total RNA was isolated. The RealTime ready Human Apoptosis Panel was used for real-time PCR, detecting differences in gene expression. Participants had CCHF severity grading scores (SGS) with low risk score (10 out of 20) and intermediate or high risk scores (10 out of 20) for mortality. Five of 20 participants had a fatal outcome. Gene expression analysis showed modulation of pro-apoptotic and anti-apoptotic genes that facilitate apoptosis in the CCHF patient group. Dominant extrinsic pathway activation, mostly related with TNF family members was observed. Severe and fatal cases suggest additional intrinsic pathway activation. The clinical significance of relative gene expression is not clear, and larger longitudinal studies with simultaneous measurement of host and viral factors are recommended. PMID:27304063

  6. Association of VAMP-2 and Syntaxin 1A Genes with Adult Attention Deficit Hyperactivity Disorder

    PubMed Central

    Kenar, Aẙe Nur Inci; Ay, Özlem İzci; Erdal, Mehmet Emin

    2014-01-01

    Objective The etiology of attention deficit hyperactivity disorder (ADHD) has not been entirely clarified yet. Structural and metabolic differences at the prefrontal striatal cerebellary system and the interaction of gene and environment are the main factors that thought to play roles in the etiology. Genetic investigations are performed especially about the dopamine pathways and receptors. In this study; it was aimed to investigate the association of the synaptobrevin-2 (VAMP-2) gene Ins/Del polymorphism and syntaxin 1A gene intron 7 polymorphism, which take place in encoding presynaptic protein, with adult ADHD. Methods One hundred thirty-nine patients, having ADHD aging between 18 and 60 years and 106 healthy people as controls were included into the study. DNA samples were extracted from whole blood and genetic analysis were performed. Results A significant difference was determined between ADHD and VAMP-2 Ins/Del polymorphism and syntaxin 1A intron 7 polymorphism according to the control group. These polymorphisms were found not to be associated with subtypes of ADHD. Conclusion It is supposed that synaptic protein genes together with dopaminergic genes might have roles in the etiology of ADHD. PMID:24605127

  7. Molecular Mapping of Adult-Plant Race-Specific Leaf Rust Resistance Gene Lr12 in Bread Wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wheat (Triticum aestivum) gene Lr12 provides adult-plant race-specific resistance to leaf rust caused by Puccinia triticina. It is completely linked or identical to Lr31, which confers seedling resistance only when the complementary gene Lr27 is also present. F2 and F2-derived F3 families were devel...

  8. Seasonal effects of UCP1 gene polymorphism on visceral fat accumulation in Japanese adults.

    PubMed

    Nakayama, Kazuhiro; Miyashita, Hiroshi; Yanagisawa, Yoshiko; Iwamoto, Sadahiko

    2013-01-01

    Uncoupling protein 1 (UCP1) and β3 adrenergic receptor (ADRB3) genes play central roles in the thermogenesis of brown adipose tissue (BAT) in adult humans. However, the importance of single-nucleotide polymorphisms (SNPs) in both genes during the development of obesity is controversial. Although active BAT in adult humans is frequently observed in the winter season, the effects of sampling season have not been taken into consideration in previous association studies. Here, we tested the associations of UCP1 -3826A/G and ADRB3 Trp64Arg with body mass index (BMI) and visceral fat area (VFA) in 3013 Japanese adults sampled during different seasons. Association between SNPs and the obesity-related traits were assessed using multiple linear regression models, including sex, age, physical activity, and genotypes. Both SNPs did not show significant associations in the models based on the entire cohort. However, in subsets comprising individuals mainly sampled from winter to spring, UCP1 showed significant associations with VFA (P = 0.0098) and VFA adjusted for BMI (P = 0.0128). Moreover, the effects of UCP1 on VFA were strongly negatively correlated with outdoor temperature (P = 0.00011), but not with night length (P = 0.039). ADRB3 did not show these associations, but an additive effect with UCP1 was observed for VFA adjusted for BMI (P = 0.0067). Subsets sampled in the hot season did not show significant associations for both SNPs. The season-specific effects of UCP1 on VFA were consistent with a previous finding that active BAT was more frequently found in winter than in summer, and supported the importance of cold stress in BAT activation and the significance of BAT in the development of obesity in adult humans. PMID:24086366

  9. Globin mRNAs are primers for the transcription of influenza viral RNA in vitro

    PubMed Central

    Bouloy, Michele; Plotch, Stephen J.; Krug, Robert M.

    1978-01-01

    Because influenza viral RNA transcription in vitro is greatly enhanced by the addition of a primer dinucleotide, ApG or GpG, we have proposed that viral RNA transcription in vivo requires initiation by primer RNAs synthesized by the host cell, specifically by RNA polymerase II, thereby explaining the α-amanitin sensitivity of viral RNA transcription in vivo. Here, we identify such primer RNAs, initially in reticulocyte extracts, where they are shown to be globin mRNAs. Purified globin mRNAs very effectively stimulated viral RNA transcription in vitro, and the resulting transcripts directed the synthesis of all the nonglycosylated virus-specific proteins in micrococcal nuclease-treated L cell extracts. The viral RNA transcripts synthesized in vitro primed by ApG also directed the synthesis of the nonglycosylated virus-specific proteins, but the globin mRNA-primed transcripts were translated about 3 times more efficiently. The translation of the globin mRNA-primed, but not the ApG-primed, viral RNA transcripts was inhibited by 7-methylguanosine 5′-phosphate in the presence of S-adenosylhomocysteine, suggesting that the globin mRNA-primed transcripts contained a 5′-terminal methylated cap structure. We propose that this cap was transferred from the globin mRNA primer to the newly synthesized viral RNA transcripts, because no detectable de novo synthesis of a methylated cap occurred during globin mRNA-primed viral RNA transcription. Preliminary experiments indicate that other purified eukaryotic mRNAs also stimulate influenza viral RNA transcription in vitro. Images PMID:283399

  10. Evolution of insect metamorphosis: a microarray-based study of larval and adult gene expression in the ant Camponotus festinatus.

    PubMed

    Goodisman, Michael A D; Isoe, Jun; Wheeler, Diana E; Wells, Michael A

    2005-04-01

    Holometabolous insects inhabit almost every terrestrial ecosystem. The evolutionary success of holometabolous insects stems partly from their developmental program, which includes discrete larval and adult stages. To gain an understanding of how development differs among holometabolous insect taxa, we used cDNA microarray technology to examine differences in gene expression between larval and adult Camponotus festinatus ants. We then compared expression patterns obtained from our study to those observed in the fruitfly Drosophila melanogaster. We found that many genes showed distinct patterns of expression between the larval and adult ant life stages, a result that was confirmed through quantitative reverse-transcriptase polymerase chain reaction. Genes involved in protein metabolism and possessing structural activity tended to be more highly expressed in larval than adult ants. In contrast, genes relatively upregulated in adults possessed a greater diversity of functions and activities. We also discovered that patterns of expression observed for homologous genes in D. melanogaster differed substantially from those observed in C. festinatus. Our results suggest that the specific molecular mechanisms involved in metamorphosis will differ substantially between insect taxa. Systematic investigation of gene expression during development of other taxa will provide additional information on how developmental pathways evolve. PMID:15926695

  11. Requirements for Hedgehog, a Segmental Polarity Gene, in Patterning Larval and Adult Cuticle of Drosophila

    PubMed Central

    Mohler, J.

    1988-01-01

    Mutations of the hedgehog gene are generally embryonic lethal, resulting in a lawn of denticles on the ventral surface. In strong alleles, no segmentation is obvious and the anteroposterior polarity of ventral denticles is lost. Temperature shift analysis of a temperature-sensitive allele indicates an embryonic activity period for hedgehog between 2.5 and 6 hr of embryonic development (at 25°) and a larval/pupal period from 4 to 7 days of development (at 25°). Mosaic analysis of hedgehog mutations in the adult cuticle indicates a series of defined defects associated with the failure of appropriate hedgehog expression. In particular, defects in the distal portions of the legs and antenna occur in association with homozygous hedgehog clones in the posterior compartment of those structures. Because the defects are associated with homozygous clones, but are not co-extensive, a type of ``domineering'' nonautonomy is proposed for the activity of the hedgehog gene. PMID:3147217

  12. PRENATAL ALCOHOL EXPOSURE ALTERS STEADY-STATE AND ACTIVATED GENE EXPRESSION IN THE ADULT RAT BRAIN

    PubMed Central

    Stepien, Katarzyna A.; Lussier, Alexandre A.; Neumann, Sarah M.; Pavlidis, Paul; Kobor, Michael S.; Weinberg, Joanne

    2016-01-01

    Background Prenatal alcohol exposure (PAE) is associated with alterations in numerous physiological systems, including the stress and immune systems . We have previously shown that PAE increases the course and severity of arthritis in an adjuvant-induced arthritis (AA) model. While the molecular mechanisms underlying these effects are not fully known, changes in neural gene expression are emerging as important factors in the etiology of PAE effects. As the prefrontal cortex (PFC) and hippocampus (HPC) play key roles in neuroimmune function, PAE-induced alterations to their transcriptome may underlie abnormal steady-state functions and responses to immune challenge. The current study examined brains from adult PAE and control females from our recent AA study to determine whether PAE causes long-term alterations in gene expression and whether these mediate the altered severity and course of arthritis in PAE females Methods Adult females from PAE, pair-fed [PF], and ad libitum-fed control [C]) groups were injected with either saline or complete Freund’s adjuvant. Animals were terminated at the peak of inflammation or during resolution (days 16 and 39 post-injection, respectively); cohorts of saline-injected PAE, PF and C females were terminated in parallel. Gene expression was analyzed in the PFC and HPC using whole genome mRNA expression microarrays. Results Significant changes in gene expression in both the PFC and HPC were found in PAE compared to controls in response to ethanol exposure alone (saline-injected females), including genes involved in neurodevelopment, apoptosis, and energy metabolism. Moreover, in response to inflammation (adjuvant-injected females), PAE animals showed unique expression patterns, while failing to exhibit the activation of genes and regulators involved in the immune response observed in control and pair-fed animals. Conclusions These results support the hypothesis that PAE affects neuroimmune function at the level of gene expression

  13. The alpha-globin genotype does not influence sickle cell disease severity in a retrospective cross-validation study of the pediatric severity score.

    PubMed

    Joly, Philippe; Pondarré, Corinne; Bardel, Claire; Francina, Alain; Martin, Cyril

    2012-01-01

    To validate the recently proposed pediatric severity score (PSS) for sickle cell disease (SCD), we retrospectively assembled clinical data from a cohort of 122 patients with SCD (105 S/S or S/β(0) -thal. and 17 S/C) followed up for at least 2 years. Besides age and α- and β-globin genotypes, four new parameters were also tested against the PSS: duration of data assembly, neonatal screening, use of transcranial Doppler ultrasound to prevent vasculopathies and β-globin gene cluster haplotype. Once again, the PSS clearly differentiated patients by their β-globin genotype (P=0.004) but not by their age during data assembly (P=0.159). But, surprisingly, alpha-gene deletions were not associated with a lower PSS (P=0.604), possibly reflecting the opposite effects of α-thalassemia on global SCD severity. As for the newly tested parameters, the PSS appeared not to be influenced by the duration of data assembly (P=0.071) and neonatal screening (P=0.678) but rather by the introduction of transcranial Doppler ultrasound (P=0.006). Moreover, the Senegal haplotype at the homozygous state may be associated with a lower PSS. Methodologically, our data globally confirm the usefulness of the PSS to identify major etiological factors of SCD gravity. Nevertheless, the score is surely underestimated for patients who have been switched to a chronic therapy before the main SCD complications. Biologically, our study questions about the exact influence of α-thalassemia on global SCD severity. PMID:21910753

  14. Translation of globin messenger RNA by the mouse ovum

    PubMed Central

    Brinster, R. L.; Chen, H. Y.; Trumbauer, M. E.; Avarbock, M. R.

    2016-01-01

    It has been demonstrated that the Xenopus oocyte can translate rabbit haemoglobin messenger RNA (mRNA) following microinjection of the message into the cell1. The Xenopus oocyte has since been shown to be capable of translating a variety of messenger RNAs from different species2–4. This system has proved useful in understanding the mechanism of message translation and has also provided information about the translation capability of the Xenopus oocyte5,6. Several other cell types, including HeLa cells and fibroblasts, can also translate exogenous message injected into the cell7,8. However, there have been no reports of injection of mRNA into oocytes or fertilised one-cell ova of mammalian species. Nevertheless, the latter system could be of considerable use in studying the processing of exogenous messages in a mammalian system undergoing development, as well as providing insight into the way the early embryo processes injected messages and the protein products of such messages. We report here the results of injecting message into the fertilised one-cell mouse ovum and show that both mouse and rabbit globin mRNA are translated in this system. PMID:7352032

  15. Reversible suppression of an essential gene in adult mice using transgenic RNA interference

    PubMed Central

    McJunkin, Katherine; Mazurek, Anthony; Premsrirut, Prem K.; Zuber, Johannes; Dow, Lukas E.; Simon, Janelle; Stillman, Bruce; Lowe, Scott W.

    2011-01-01

    RNAi has revolutionized loss-of-function genetics by enabling sequence-specific suppression of virtually any gene. Furthermore, tetracycline response elements (TRE) can drive expression of short hairpin RNAs (shRNAs) for inducible and reversible target gene suppression. Here, we demonstrate the feasibility of transgenic inducible RNAi for suppression of essential genes. We set out to directly target cell proliferation by screening an RNAi library against DNA replication factors and identified multiple shRNAs against Replication Protein A, subunit 3 (RPA3). We generated transgenic mice with TRE-driven Rpa3 shRNAs whose expression enforced a reversible cell cycle arrest. In adult mice, the block in cell proliferation caused rapid atrophy of the intestinal epithelium which led to weight loss and lethality within 8–11 d of shRNA induction. Upon shRNA withdrawal, villus atrophy and weight loss were fully reversible. Thus, shRpa3 transgenic mice provide an interesting tool to study tissue maintenance and regeneration. Overall, we have established a robust system that serves the purpose of temperature-sensitive alleles in other model organisms, enabling inducible and reversible suppression of essential genes in a mammalian system. PMID:21482754

  16. The Protein Kinase KIS Impacts Gene Expression during Development and Fear Conditioning in Adult Mice

    PubMed Central

    Manceau, Valérie; Kremmer, Elisabeth; Nabel, Elizabeth G.; Maucuer, Alexandre

    2012-01-01

    The brain-enriched protein kinase KIS (product of the gene UHMK1) has been shown to phosphorylate the human splicing factor SF1 in vitro. This phosphorylation in turn favors the formation of a U2AF65-SF1-RNA complex which occurs at the 3′ end of introns at an early stage of spliceosome assembly. Here, we analyzed the effects of KIS knockout on mouse SF1 phosphorylation, physiology, adult behavior, and gene expression in the neonate brain. We found SF1 isoforms are differently expressed in KIS-ko mouse brains and fibroblasts. Re-expression of KIS in fibroblasts restores a wild type distribution of SF1 isoforms, confirming the link between KIS and SF1. Microarray analysis of transcripts in the neonate brain revealed a subtle down-regulation of brain specific genes including cys-loop ligand-gated ion channels and metabolic enzymes. Q-PCR analyses confirmed these defects and point to an increase of pre-mRNA over mRNA ratios, likely due to changes in splicing efficiency. While performing similarly in prepulse inhibition and most other behavioral tests, KIS-ko mice differ in spontaneous activity and contextual fear conditioning. This difference suggests that disregulation of gene expression due to KIS inactivation affects specific brain functions. PMID:22937132

  17. Effects of postnatal alcohol exposure on hippocampal gene expression and learning in adult mice.

    PubMed

    Lee, Dong Hoon; Moon, Jihye; Ryu, Jinhyun; Jeong, Joo Yeon; Roh, Gu Seob; Kim, Hyun Joon; Cho, Gyeong Jae; Choi, Wan Sung; Kang, Sang Soo

    2016-04-28

    Fetal alcohol syndrome (FAS) is a condition resulting from excessive drinking by pregnant women. Symptoms of FAS include abnormal facial features, stunted growth, intellectual deficits and attentional dysfunction. Many studies have investigated FAS, but its underlying mechanisms remain unknown. This study evaluated the relationship between alcohol exposure during the synaptogenesis period in postnatal mice and subsequent cognitive function in adult mice. We delivered two injections, separated by 2 h, of ethanol (3 g/kg, ethanol/saline, 20% v/v) to ICR mice on postnatal day 7. After 10 weeks, we conducted a behavioral test, sacrificed the animals, harvested brain tissue and analyzed hippocampal gene expression using a microarray. In ethanol-treated mice, there was a reduction in brain size and decreased neuronal cell number in the cortex, and also cognitive impairment. cDNA microarray results indicated that 1,548 genes showed a > 2-fold decrease in expression relative to control, whereas 974 genes showed a > 2-fold increase in expression relative to control. Many of these genes were related to signal transduction, synaptogenesis and cell membrane formation, which are highlighted in our findings. PMID:26960969

  18. Membrane-bound globin X protects the cell from reactive oxygen species.

    PubMed

    Koch, Jonas; Burmester, Thorsten

    2016-01-01

    Globin X (GbX) is a member of the globin family that emerged early in the evolution of Metazoa. In vertebrates, GbX is restricted to lampreys, fish, amphibians and some reptiles, and is expressed in neurons. Unlike any other metazoan globin, GbX is N-terminally acylated and anchored in the cell membrane via myristoyl and palmitoyl groups, suggesting a unique function. Here, we compared the capacity of GbX to protect a mouse neuronal cell line from hypoxia and reactive oxygen species (ROS) with that of myoglobin. To evaluate the contribution of membrane-binding, we generated a mutated version of GbX without acyl groups. All three globins enhanced cell viability under hypoxia, with myoglobin having the most pronounced effect. GbX but not myoglobin protected the cells from hydrogen peroxide (H2O2)-induced stress. Membrane-bound GbX was significantly more efficient than its mutated, soluble form. Furthermore, myoglobin and mutated GbX increased production of ROS upon H2O2-treatment, while membrane-bound GbX did not. The results indicate that myoglobin enhances O2 supply while GbX protects the cell membrane from ROS-stress. The ancient origin of GbX suggests that ROS-protection reflects the function of the early globins before they acquired a respiratory role. PMID:26631962

  19. Genes and Pathways Involved in Adult Onset Disorders Featuring Muscle Mitochondrial DNA Instability

    PubMed Central

    Ahmed, Naghia; Ronchi, Dario; Comi, Giacomo Pietro

    2015-01-01

    Replication and maintenance of mtDNA entirely relies on a set of proteins encoded by the nuclear genome, which include members of the core replicative machinery, proteins involved in the homeostasis of mitochondrial dNTPs pools or deputed to the control of mitochondrial dynamics and morphology. Mutations in their coding genes have been observed in familial and sporadic forms of pediatric and adult-onset clinical phenotypes featuring mtDNA instability. The list of defects involved in these disorders has recently expanded, including mutations in the exo-/endo-nuclease flap-processing proteins MGME1 and DNA2, supporting the notion that an enzymatic DNA repair system actively takes place in mitochondria. The results obtained in the last few years acknowledge the contribution of next-generation sequencing methods in the identification of new disease loci in small groups of patients and even single probands. Although heterogeneous, these genes can be conveniently classified according to the pathway to which they belong. The definition of the molecular and biochemical features of these pathways might be helpful for fundamental knowledge of these disorders, to accelerate genetic diagnosis of patients and the development of rational therapies. In this review, we discuss the molecular findings disclosed in adult patients with muscle pathology hallmarked by mtDNA instability. PMID:26251896

  20. Telomerase gene therapy in adult and old mice delays aging and increases longevity without increasing cancer

    PubMed Central

    Bernardes de Jesus, Bruno; Vera, Elsa; Schneeberger, Kerstin; Tejera, Agueda M; Ayuso, Eduard; Bosch, Fatima; Blasco, Maria A

    2012-01-01

    A major goal in aging research is to improve health during aging. In the case of mice, genetic manipulations that shorten or lengthen telomeres result, respectively, in decreased or increased longevity. Based on this, we have tested the effects of a telomerase gene therapy in adult (1 year of age) and old (2 years of age) mice. Treatment of 1- and 2-year old mice with an adeno associated virus (AAV) of wide tropism expressing mouse TERT had remarkable beneficial effects on health and fitness, including insulin sensitivity, osteoporosis, neuromuscular coordination and several molecular biomarkers of aging. Importantly, telomerase-treated mice did not develop more cancer than their control littermates, suggesting that the known tumorigenic activity of telomerase is severely decreased when expressed in adult or old organisms using AAV vectors. Finally, telomerase-treated mice, both at 1-year and at 2-year of age, had an increase in median lifespan of 24 and 13%, respectively. These beneficial effects were not observed with a catalytically inactive TERT, demonstrating that they require telomerase activity. Together, these results constitute a proof-of-principle of a role of TERT in delaying physiological aging and extending longevity in normal mice through a telomerase-based treatment, and demonstrate the feasibility of anti-aging gene therapy. PMID:22585399

  1. Expression of five acetylcholine receptor subunit genes in Brugia malayi adult worms

    PubMed Central

    Li, Ben-Wen; Rush, Amy C.; Weil, Gary J.

    2015-01-01

    Acetylcholine receptors (AChRs) are required for body movement in parasitic nematodes and are targets of “classical” anthelmintic drugs such as levamisole and pyrantel and of newer drugs such as tribendimidine and derquantel. While neurotransmission explains the effects of these drugs on nematode movement, their effects on parasite reproduction are unexplained. The levamisole AChR type (L-AChRs) in Caenorhabditis elegans is comprised of five subunits: Cel-UNC-29, Cel-UNC-38, Cel-UNC-63, Cel-LEV-1 and Cel-LEV-8. The genome of the filarial parasite Brugia malayi contains nine AChRs subunits including orthologues of Cel-unc-29, Cel-unc-38, and Cel-unc-63. We performed in situ hybridization with RNA probes to localize the expression of five AChR genes (Bm1_35890-Bma-unc-29, Bm1_20330-Bma-unc-38, Bm1_38195-Bma-unc-63, Bm1_48815-Bma-acr-26 and Bm1_40515-Bma-acr-12) in B. malayi adult worms. Four of these genes had similar expression patterns with signals in body muscle, developing embryos, spermatogonia, uterine wall adjacent to stretched microfilariae, wall of Vas deferens, and lateral cord. Three L-AChR subunit genes (Bma-unc-29, Bma-unc-38 and Bma-unc-63) were expressed in body muscle, which is a known target of levamisole. Bma-acr-12 was co-expressed with these levamisole subunit genes in muscle, and this suggests that its protein product may form receptors with other alpha subunits. Bma-acr-26 was expressed in male muscle but not in female muscle. Strong expression signals of these genes in early embryos and gametes in uterus and testis suggest that AChRs may have a role in nervous system development of embryogenesis and spermatogenesis. This would be consistent with embryotoxic effects of drugs that target these receptors in filarial worms. Our data show that the expression of these receptor genes is tightly regulated with regard to localization in adult worms and developmental stage in embryos and gametes. These results may help to explain the broad effects

  2. Relationship of a variant in the NTRK1 gene to white matter microstructure in young adults

    PubMed Central

    Braskie, Meredith N; Jahanshad, Neda; Stein, Jason L; Barysheva, Marina; Johnson, Kori; McMahon, Katie L; de Zubicaray, Greig I; Martin, Nicholas G; Wright, Margaret J; Ringman, John M; Toga, Arthur W; Thompson, Paul M

    2012-01-01

    The NTRK1 gene (also known as TRKA) encodes a high affinity receptor for NGF, a neurotrophin involved in nervous system development and myelination. NTRK1 has been implicated in neurological function via links between the T allele at rs6336 (NTRK1-T) and schizophrenia risk. A variant in the neurotrophin gene, BDNF, was previously associated with white matter integrity in young adults, highlighting the importance of neurotrophins to white matter development. We hypothesized that NTRK1-T would relate to lower FA in healthy adults. We scanned 391 healthy adult human twins and their siblings (mean age: 23.6 ± 2.2 years; 31 NTRK1-T carriers, 360 non-carriers) using 105-gradient diffusion tensor imaging at 4 Tesla. We evaluated in brain white matter how NTRK1-T and NTRK1 rs4661063 allele A (rs4661063-A, which is in moderate linkage disequilibrium with rs6336) related to voxelwise fractional anisotropy – a common diffusion tensor imaging measure of white matter microstructure. We used mixed-model regression to control for family relatedness, age, and sex. The sample was split in half to test results reproducibility. The false discovery rate method corrected for voxelwise multiple comparisons. NTRK1-T and rs4661063-A correlated with lower white matter fractional anisotropy, independent of age and sex (multiple comparisons corrected: false discovery rate critical p = 0.038 for NTRK1-T and 0.013 for rs4661063-A). In each half-sample, the NTRK1-T effect was replicated in the cingulum, corpus callosum, superior and inferior longitudinal fasciculi, inferior fronto-occipital fasciculus, superior corona radiata, and uncinate fasciculus. Our results suggest that NTRK1-T is important for developing white matter microstructure. PMID:22539856

  3. α:Non-α and Gγ:Aγ globin chain ratios in thalassemia intermedia patients treated with hydroxyurea

    PubMed Central

    Najjari, Abbas; Asouri, Mohsen; Gouhari, Ladan Hosseini; Niaki, Haleh Akhavan; Nejad, Amir Sasan Mozaffari; Eslami, Seyyedeh Masoumeh; Abolghasemi, Hassan; Ataee, Ramin; Ebrahimi, Abdol Ali; Moshaei, Masoumeh Rezaei; Ahmadi, Ali Asghar

    2014-01-01

    Objectives To elucidate the possible ways by which hydroxyurea molecules affect globin chain (α or β-like) synthesis. Methods A total of 23 thalassemia intermedia patients (13 male and 10 female) aged between 5 and 26 years were treated for five months with 15 mg/(kg·day) of hydroxyurea. Hemoglobins electrophoresis and globin chain electrophoresis was performed on each sample at different time points before and during the treatment. Results Fetal hemoglobin increased significantly in most patients and average episodes of transfusion decreased. Both Gγ and Aγ-globin chains increased significantly and α-globin:Nonα-globin chain as well as Gγ-globin:Aγ globin chains ratios decreased. Conclusions Improvement in α:non-α ratio and consequent decrease of free α-globin chain might be the cause of beneficial effects of hydroxyurea therapy. Two patients who felt better didn't show significant increase in their fetal hemoglobin level, and this is in contradiction with the hypothesis claiming that the HbF level increase is the cause of such therapeutic effect. In spite of the unclear mechanism of action of this drug, hydroxyurea therapy had noticeable impacts on thalassemia intermedia and also sickle cell disease and even patients suffering from thalassemia major. PMID:25183077

  4. Autoinduction, purification, and characterization of soluble α-globin chains of crocodile (Crocodylus siamensis) hemoglobin in Escherichia coli.

    PubMed

    Kabbua, Thai; Anwised, Preeyanan; Boonmee, Atcha; Subedi, Bishnu P; Pierce, Brad S; Thammasirirak, Sompong

    2014-11-01

    We have established a method to express soluble heme-bound recombinant crocodile (Crocodylus siamensis) α-globin chain holo-protein in bacteria (Escherichia coli) using an autoinduction system without addition of exogenous heme. This is the first time that heme-bound crocodile α-globin chains have been expressed in bacteria without in vitro heme reconstitution. The observed molecular mass of purified recombinant α-globin is consistent with that calculated from the primary amino acid sequence of native crocodile (C. siamensis) α-globin. Both the monomeric and the dimeric protein configuration formed by intermolecular disulfide bond could be purified as soluble protein. Spectroscopic characterization [UV-visible, circular dichroism (CD), and electron paramagnetic resonance (EPR)] of purified recombinant α-globin demonstrates nearly identical properties as reported for hemoglobin and myoglobin isolated from other organisms. For comparison, cyanide and nitric oxide binding of purified α-globin was also investigated. These results suggested that C. siamensis α-globin expressed in E. coli was folded correctly with proper incorporation of the heme cofactor. The expression method we now describe can facilitate production and isolation of individual globin chains in order to further study the mechanism and assembly of crocodile hemoglobin. PMID:25175288

  5. Differential expression of id genes and their potential regulator znf238 in zebrafish adult neural progenitor cells and neurons suggests distinct functions in adult neurogenesis.

    PubMed

    Diotel, Nicolas; Beil, Tanja; Strähle, Uwe; Rastegar, Sepand

    2015-01-01

    Teleost fish display a remarkable ability to generate new neurons and to repair brain lesions during adulthood. They are, therefore, a very popular model to investigate the molecular mechanisms of constitutive and induced neurogenesis in adult vertebrates. In this study, we investigated the expression patterns of inhibitor of DNA binding (id) genes and of their potential transcriptional repressor, znf238, in the whole brain of adult zebrafish. We show that while id1 is exclusively expressed in ventricular cells in the whole brain, id2a, id3 and id4 genes are expressed in broader areas. Interestingly, znf238 was also detected in these regions, its expression overlapping with id2a, id3 and id4 expression. Further detailed characterization of the id-expressing cells demonstrated that (a) id1 is expressed in type 1 and type 2 neural progenitors as previously published, (b) id2a in type 1, 2 and 3 neural progenitors, (c) id3 in type 3 neural progenitors and (d) id4 in postmitotic neurons. Our data provide a detailed map of id and znf238 expression in the brain of adult zebrafish, supplying a framework for studies of id genes function during adult neurogenesis and brain regeneration in the zebrafish. PMID:26107416

  6. A redox signalling globin is essential for reproduction in Caenorhabditis elegans

    NASA Astrophysics Data System (ADS)

    de Henau, Sasha; Tilleman, Lesley; Vangheel, Matthew; Luyckx, Evi; Trashin, Stanislav; Pauwels, Martje; Germani, Francesca; Vlaeminck, Caroline; Vanfleteren, Jacques R.; Bert, Wim; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; de Wael, Karolien; Moens, Luc; Dewilde, Sylvia; Braeckman, Bart P.

    2015-12-01

    Moderate levels of reactive oxygen species (ROS) are now recognized as redox signalling molecules. However, thus far, only mitochondria and NADPH oxidases have been identified as cellular sources of ROS in signalling. Here we identify a globin (GLB-12) that produces superoxide, a type of ROS, which serves as an essential signal for reproduction in C. elegans. We find that GLB-12 has an important role in the regulation of multiple aspects in germline development, including germ cell apoptosis. We further describe how GLB-12 displays specific molecular, biochemical and structural properties that allow this globin to act as a superoxide generator. In addition, both an intra- and extracellular superoxide dismutase act as key partners of GLB-12 to create a transmembrane redox signal. Our results show that a globin can function as a driving factor in redox signalling, and how this signal is regulated at the subcellular level by multiple control layers.

  7. Complete amino acid sequence of globin chains and biological activity of fragmented crocodile hemoglobin (Crocodylus siamensis).

    PubMed

    Srihongthong, Saowaluck; Pakdeesuwan, Anawat; Daduang, Sakda; Araki, Tomohiro; Dhiravisit, Apisak; Thammasirirak, Sompong

    2012-08-01

    Hemoglobin, α-chain, β-chain and fragmented hemoglobin of Crocodylus siamensis demonstrated both antibacterial and antioxidant activities. Antibacterial and antioxidant properties of the hemoglobin did not depend on the heme structure but could result from the compositions of amino acid residues and structures present in their primary structure. Furthermore, thirteen purified active peptides were obtained by RP-HPLC analyses, corresponding to fragments in the α-globin chain and the β-globin chain which are mostly located at the N-terminal and C-terminal parts. These active peptides operate on the bacterial cell membrane. The globin chains of Crocodylus siamensis showed similar amino acids to the sequences of Crocodylus niloticus. The novel amino acid substitutions of α-chain and β-chain are not associated with the heme binding site or the bicarbonate ion binding site, but could be important through their interactions with membranes of bacteria. PMID:22648692

  8. A redox signalling globin is essential for reproduction in Caenorhabditis elegans

    PubMed Central

    De Henau, Sasha; Tilleman, Lesley; Vangheel, Matthew; Luyckx, Evi; Trashin, Stanislav; Pauwels, Martje; Germani, Francesca; Vlaeminck, Caroline; Vanfleteren, Jacques R.; Bert, Wim; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; De Wael, Karolien; Moens, Luc; Dewilde, Sylvia; Braeckman, Bart P.

    2015-01-01

    Moderate levels of reactive oxygen species (ROS) are now recognized as redox signalling molecules. However, thus far, only mitochondria and NADPH oxidases have been identified as cellular sources of ROS in signalling. Here we identify a globin (GLB-12) that produces superoxide, a type of ROS, which serves as an essential signal for reproduction in C. elegans. We find that GLB-12 has an important role in the regulation of multiple aspects in germline development, including germ cell apoptosis. We further describe how GLB-12 displays specific molecular, biochemical and structural properties that allow this globin to act as a superoxide generator. In addition, both an intra- and extracellular superoxide dismutase act as key partners of GLB-12 to create a transmembrane redox signal. Our results show that a globin can function as a driving factor in redox signalling, and how this signal is regulated at the subcellular level by multiple control layers. PMID:26621324

  9. The Drosophila melanogaster Muc68E Mucin Gene Influences Adult Size, Starvation Tolerance, and Cold Recovery.

    PubMed

    Reis, Micael; Silva, Ana C; Vieira, Cristina P; Vieira, Jorge

    2016-01-01

    Mucins have been implicated in many different biological processes, such as protection from mechanical damage, microorganisms, and toxic molecules, as well as providing a luminal scaffold during development. Nevertheless, it is conceivable that mucins have the potential to modulate food absorption as well, and thus contribute to the definition of several important phenotypic traits. Here we show that the Drosophila melanogaster Muc68E gene is 40- to 60-million-yr old, and is present in Drosophila species of the subgenus Sophophora only. The central repeat region of this gene is fast evolving, and shows evidence for repeated expansions/contractions. This and/or frequent gene conversion events lead to the homogenization of its repeats. The amino acid pattern P[ED][ED][ST][ST][ST] is found in the repeat region of Muc68E proteins from all Drosophila species studied, and can occur multiple times within a single conserved repeat block, and thus may have functional significance. Muc68E is a nonessential gene under laboratory conditions, but Muc68E mutant flies are smaller and lighter than controls at birth. However, at 4 d of age, Muc68E mutants are heavier, recover faster from chill-coma, and are more resistant to starvation than control flies, although they have the same percentage of lipids as controls. Mutant flies have enlarged abdominal size 1 d after chill-coma recovery, which is associated with higher lipid content. These results suggest that Muc68E has a role in metabolism modulation, food absorption, and/or feeding patterns in larvae and adults, and under normal and stress conditions. Such biological function is novel for mucin genes. PMID:27172221

  10. The Drosophila melanogaster Muc68E Mucin Gene Influences Adult Size, Starvation Tolerance, and Cold Recovery

    PubMed Central

    Reis, Micael; Silva, Ana C.; Vieira, Cristina P.; Vieira, Jorge

    2016-01-01

    Mucins have been implicated in many different biological processes, such as protection from mechanical damage, microorganisms, and toxic molecules, as well as providing a luminal scaffold during development. Nevertheless, it is conceivable that mucins have the potential to modulate food absorption as well, and thus contribute to the definition of several important phenotypic traits. Here we show that the Drosophila melanogaster Muc68E gene is 40- to 60-million-yr old, and is present in Drosophila species of the subgenus Sophophora only. The central repeat region of this gene is fast evolving, and shows evidence for repeated expansions/contractions. This and/or frequent gene conversion events lead to the homogenization of its repeats. The amino acid pattern P[ED][ED][ST][ST][ST] is found in the repeat region of Muc68E proteins from all Drosophila species studied, and can occur multiple times within a single conserved repeat block, and thus may have functional significance. Muc68E is a nonessential gene under laboratory conditions, but Muc68E mutant flies are smaller and lighter than controls at birth. However, at 4 d of age, Muc68E mutants are heavier, recover faster from chill-coma, and are more resistant to starvation than control flies, although they have the same percentage of lipids as controls. Mutant flies have enlarged abdominal size 1 d after chill-coma recovery, which is associated with higher lipid content. These results suggest that Muc68E has a role in metabolism modulation, food absorption, and/or feeding patterns in larvae and adults, and under normal and stress conditions. Such biological function is novel for mucin genes. PMID:27172221

  11. PRMT5-mediated methylation of histone H4R3 recruits DNMT3A, coupling histone and DNA methylation in gene silencing.

    PubMed

    Zhao, Quan; Rank, Gerhard; Tan, Yuen T; Li, Haitao; Moritz, Robert L; Simpson, Richard J; Cerruti, Loretta; Curtis, David J; Patel, Dinshaw J; Allis, C David; Cunningham, John M; Jane, Stephen M

    2009-03-01

    Mammalian gene silencing is established through methylation of histones and DNA, although the order in which these modifications occur remains contentious. Using the human beta-globin locus as a model, we demonstrate that symmetric methylation of histone H4 arginine 3 (H4R3me2s) by the protein arginine methyltransferase PRMT5 is required for subsequent DNA methylation. H4R3me2s serves as a direct binding target for the DNA methyltransferase DNMT3A, which interacts through the ADD domain containing the PHD motif. Loss of the H4R3me2s mark through short hairpin RNA-mediated knockdown of PRMT5 leads to reduced DNMT3A binding, loss of DNA methylation and gene activation. In primary erythroid progenitors from adult bone marrow, H4R3me2s marks the inactive methylated globin genes coincident with localization of PRMT5. Our findings define DNMT3A as both a reader and a writer of repressive epigenetic marks, thereby directly linking histone and DNA methylation in gene silencing. PMID:19234465

  12. Genes involved in thoracic exoskeleton formation during the pupal-to-adult molt in a social insect model, Apis mellifera

    PubMed Central

    2013-01-01

    Background The insect exoskeleton provides shape, waterproofing, and locomotion via attached somatic muscles. The exoskeleton is renewed during molting, a process regulated by ecdysteroid hormones. The holometabolous pupa transforms into an adult during the imaginal molt, when the epidermis synthe3sizes the definitive exoskeleton that then differentiates progressively. An important issue in insect development concerns how the exoskeletal regions are constructed to provide their morphological, physiological and mechanical functions. We used whole-genome oligonucleotide microarrays to screen for genes involved in exoskeletal formation in the honeybee thoracic dorsum. Our analysis included three sampling times during the pupal-to-adult molt, i.e., before, during and after the ecdysteroid-induced apolysis that triggers synthesis of the adult exoskeleton. Results Gene ontology annotation based on orthologous relationships with Drosophila melanogaster genes placed the honeybee differentially expressed genes (DEGs) into distinct categories of Biological Process and Molecular Function, depending on developmental time, revealing the functional elements required for adult exoskeleton formation. Of the 1,253 unique DEGs, 547 were upregulated in the thoracic dorsum after apolysis, suggesting induction by the ecdysteroid pulse. The upregulated gene set included 20 of the 47 cuticular protein (CP) genes that were previously identified in the honeybee genome, and three novel putative CP genes that do not belong to a known CP family. In situ hybridization showed that two of the novel genes were abundantly expressed in the epidermis during adult exoskeleton formation, strongly implicating them as genuine CP genes. Conserved sequence motifs identified the CP genes as members of the CPR, Tweedle, Apidermin, CPF, CPLCP1 and Analogous-to-Peritrophins families. Furthermore, 28 of the 36 muscle-related DEGs were upregulated during the de novo formation of striated fibers attached to the

  13. A Globin Domain in a Neuronal Transmembrane Receptor of Caenorhabditis elegans and Ascaris suum

    PubMed Central

    Tilleman, Lesley; Germani, Francesca; De Henau, Sasha; Helbo, Signe; Desmet, Filip; Berghmans, Herald; Van Doorslaer, Sabine; Hoogewijs, David; Schoofs, Liliane; Braeckman, Bart P.; Moens, Luc; Fago, Angela; Dewilde, Sylvia

    2015-01-01

    We report the structural and biochemical characterization of GLB-33, a putative neuropeptide receptor that is exclusively expressed in the nervous system of the nematode Caenorhabditis elegans. This unique chimeric protein is composed of a 7-transmembrane domain (7TM), GLB-33 7TM, typical of a G-protein-coupled receptor, and of a globin domain (GD), GLB-33 GD. Comprehensive sequence similarity searches in the genome of the parasitic nematode, Ascaris suum, revealed a chimeric protein that is similar to a Phe-Met-Arg-Phe-amide neuropeptide receptor. The three-dimensional structures of the separate domains of both species and of the full-length proteins were modeled. The 7TM domains of both proteins appeared very similar, but the globin domain of the A. suum receptor surprisingly seemed to lack several helices, suggesting a novel truncated globin fold. The globin domain of C. elegans GLB-33, however, was very similar to a genuine myoglobin-type molecule. Spectroscopic analysis of the recombinant GLB-33 GD showed that the heme is pentacoordinate when ferrous and in the hydroxide-ligated form when ferric, even at neutral pH. Flash-photolysis experiments showed overall fast biphasic CO rebinding kinetics. In its ferrous deoxy form, GLB-33 GD is capable of reversibly binding O2 with a very high affinity and of reducing nitrite to nitric oxide faster than other globins. Collectively, these properties suggest that the globin domain of GLB-33 may serve as a highly sensitive oxygen sensor and/or as a nitrite reductase. Both properties are potentially able to modulate the neuropeptide sensitivity of the neuronal transmembrane receptor. PMID:25666609

  14. Decreased Globin Messenger RNA in Thalassemia Detected by Molecular Hybridization

    PubMed Central

    Kacian, D. L.; Gambino, R.; Dow, L. W.; Grossbard, E.; Natta, C.; Ramirez, F.; Spiegelman, S.; Marks, P. A.; Bank, A.

    1973-01-01

    In previous studies of patients with β thalassemia, mRNA extracted from reticulocytes in peripheral blood when added to cell-free systems reproduces the deficient β-chain synthesis characteristic of intact cells. The present studies with specific probes for α and β mRNA were designed to decide whether the decreased β mRNA activity is due to the presence of abnormal or reduced β globin mRNA in these cells. Purified α and β complementary DNAs (cDNAs) have been synthesized with RNA-instructed DNA polymerase; α and β mRNAs isolated from heavy (β-producing) and light (α-producing) polyribosomes of rabbit reticulocytes were used as templates. Each of the cDNAs is more than 80% pure by the criterion of biological activity. The α cDNA labeled with [32P]dCTP and the β cDNA labeled with [3H]dCTP have been added simultaneously to reaction mixtures containing various concentrations of mRNA from thalassemic and nonthalassemic subjects. The extent and rate of hybridization were determined, permitting a comparison of relative α and β mRNA content in the same annealing mixture. In six nonthalassemic patients, relatively equal amounts of hybridizable α and β mRNA appear to be present. In five of seven patients with β-thalassemia, significantly decreased amounts of β mRNA compared to α mRNA can be demonstrated. In two patients with Hemoglobin H disease, there is a decreased amount of α mRNA compared to β mRNA. PMID:4124307

  15. Confirmation of a founder effect in a Northern European population of a new β-globin variant: HBB:c.23_26dup (codons 8/9 (+AGAA)).

    PubMed

    Marchi, Nina; Pissard, Serge; Cliquennois, Manuel; Vasseur, Christian; Le Metayer, Nathalie; Mereau, Claude; Jouet, Jean Pierre; Georgel, Anne-France; Genin, Emmanuelle; Rose, Christian

    2015-09-01

    β-Thalassemia is a genetic disease caused by a defect in the production of the β-like globin chain. More than 200 known different variants can lead to the disease and are mainly found in populations that have been exposed to malaria parasites. We recently described a duplication of four nucleotides in the first exon of β-globin gene in several families of patients living in Nord-Pas-de-Calais (France). Using the genotypes at 12 microsatellite markers surrounding the β-globin gene of four unrelated variant carriers plus an additional one recently discovered, we found that they shared a common haplotype indicating a founder effect that was estimated to have taken place 225 years ago (nine generations). In order to determine whether this variant arose in this region of Northern Europe or was introduced by migrants from regions of the world where thalassemia is endemic, we genotyped the first 4 unrelated variant carriers and 32 controls from Nord-Pas-de-Calais for 97 European ancestry informative markers (EAIMs). Using these EAIMs and comparing with population reference panels, we demonstrated that the variant carriers were very similar to the controls and were closer to North European populations than to South European or Middle-East populations. Rare β-thalassemia variants have already been described in patients sampled in non-endemic regions, but it is the first proof of a founder effect in Northern Europe. PMID:25469539

  16. Controlled delivery of β-globin-targeting TALENs and CRISPR/Cas9 into mammalian cells for genome editing using microinjection

    PubMed Central

    Cottle, Renee N.; Lee, Ciaran M.; Archer, David; Bao, Gang

    2015-01-01

    Tal-effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) proteins are genome editing tools with unprecedented potential. However, the ability to deliver optimal amounts of these nucleases into mammalian cells with minimal toxicity poses a major challenge. Common delivery approaches are transfection- and viral-based methods; each associated with significant drawbacks. An alternative method for directly delivering genome-editing reagents into single living cells with high efficiency and controlled volume is microinjection. Here, we characterize a glass microcapillary-based injection system and demonstrate controlled co-injection of TALENs or CRISPR/Cas9 together with donor template into single K562 cells for targeting the human β-globin gene. We quantified nuclease induced insertions and deletions (indels) and found that, with β-globin-targeting TALENs, similar levels of on- and off-target activity in cells could be achieved by microinjection compared with nucleofection. Furthermore, we observed 11% and 2% homology directed repair in single K562 cells co-injected with a donor template along with CRISPR/Cas9 and TALENs respectively. These results demonstrate that a high level of targeted gene modification can be achieved in human cells using glass-needle microinjection of genome editing reagents. PMID:26558999

  17. Controlled delivery of β-globin-targeting TALENs and CRISPR/Cas9 into mammalian cells for genome editing using microinjection.

    PubMed

    Cottle, Renee N; Lee, Ciaran M; Archer, David; Bao, Gang

    2015-01-01

    Tal-effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) proteins are genome editing tools with unprecedented potential. However, the ability to deliver optimal amounts of these nucleases into mammalian cells with minimal toxicity poses a major challenge. Common delivery approaches are transfection- and viral-based methods; each associated with significant drawbacks. An alternative method for directly delivering genome-editing reagents into single living cells with high efficiency and controlled volume is microinjection. Here, we characterize a glass microcapillary-based injection system and demonstrate controlled co-injection of TALENs or CRISPR/Cas9 together with donor template into single K562 cells for targeting the human β-globin gene. We quantified nuclease induced insertions and deletions (indels) and found that, with β-globin-targeting TALENs, similar levels of on- and off-target activity in cells could be achieved by microinjection compared with nucleofection. Furthermore, we observed 11% and 2% homology directed repair in single K562 cells co-injected with a donor template along with CRISPR/Cas9 and TALENs respectively. These results demonstrate that a high level of targeted gene modification can be achieved in human cells using glass-needle microinjection of genome editing reagents. PMID:26558999

  18. Molecular cloning of the alpha-globin transcription factor CP2.

    PubMed Central

    Lim, L C; Swendeman, S L; Sheffery, M

    1992-01-01

    CP2, a transcription factor that binds the murine alpha-globin promoter, was purified and subjected to amino acid sequence analysis. Oligonucleotide primers derived from the sequence were used to obtain murine and human cDNA clones for the factor. The murine cDNA spans approximately 4 kb and contains two coextensive open reading frames (ORFs) which encode deduced polypeptides of 529 (ORF-1; molecular weight, 59,802) and 502 (ORF-2; molecular weight, 56,957) amino acids, slightly smaller than the purified factor as estimated from its mobility in sodium dodecyl sulfate-polyacrylamide gels (64,000 to 66,000). The human cDNA contains a single ORF of 501 amino acids that is nearly contiguous with murine ORF-2. Indeed, comparison of deduced human and murine amino acid sequences shows that the two polypeptides are 96.4% identical. A strictly conserved region is rich in serine and threonine (17.5%) and in proline (11%) residues (S-T-P domain). This S-T-P domain is immediately amino terminal to a string of 10 glutamines (in the human sequence) or a tract of alternating glutamine and proline residues (in the mouse sequence). Bacterial expression of the full-length (502-amino-acid) murine factor or of a core region comprising amino acids 133 to 395 generated polypeptides with the DNA binding specificity of CP2. These results confirmed the cloning of CP2 and delimited the region sufficient for specific DNA sequence recognition. Antisera produced against the core region recognized polypeptide species with Mrs of 64,000 and 66,000 in immune blots of nuclear extracts prepared from both murine and human cell lines, consistent with the size of the purified factor. Lastly, a data base search revealed that amino acids 63 to 270 of the murine factor are distantly related to a domain in the Drosophila gene regulatory factor Elf-1. Images PMID:1732747

  19. β-globin haplotypes in normal and hemoglobinopathic individuals from Reconcavo Baiano, State of Bahia, Brazil

    PubMed Central

    2010-01-01

    Five restriction site polymorphisms in the β-globin gene cluster (HincII-5‘ ε, HindIII-G γ, HindIII-A γ, HincII- ψβ1 and HincII-3‘ ψβ1) were analyzed in three populations (n = 114) from Reconcavo Baiano, State of Bahia, Brazil. The groups included two urban populations from the towns of Cachoeira and Maragojipe and one rural Afro-descendant population, known as the “quilombo community”, from Cachoeira municipality. The number of haplotypes found in the populations ranged from 10 to 13, which indicated higher diversity than in the parental populations. The haplotypes 2 (+ - - - -), 3 (- - - - +), 4 (- + - - +) and 6 (- + + - +) on the βA chromosomes were the most common, and two haplotypes, 9 (- + + + +) and 14 (+ + - - +), were found exclusively in the Maragojipe population. The other haplotypes (1, 5, 9, 11, 12, 13, 14 and 16) had lower frequencies. Restriction site analysis and the derived haplotypes indicated homogeneity among the populations. Thirty-two individuals with hemoglobinopathies (17 sickle cell disease, 12 HbSC disease and 3 HbCC disease) were also analyzed. The haplotype frequencies of these patients differed significantly from those of the general population. In the sickle cell disease subgroup, the predominant haplotypes were BEN (Benin) and CAR (Central African Republic), with frequencies of 52.9% and 32.4%, respectively. The high frequency of the BEN haplotype agreed with the historical origin of the afro-descendant population in the state of Bahia. However, this frequency differed from that of Salvador, the state capital, where the CAR and BEN haplotypes have similar frequencies, probably as a consequence of domestic slave trade and subsequent internal migrations to other regions of Brazil. PMID:21637405

  20. DAT1 and DRD4 genes involved in key dimensions of adult ADHD.

    PubMed

    Hasler, R; Salzmann, A; Bolzan, T; Zimmermann, J; Baud, P; Giannakopoulos, P; Perroud, N

    2015-06-01

    Attention-deficit hyperactivity disorder (ADHD) is a highly heritable neurodevelopmental disorder often persisting in adulthood. Genetic studies of ADHD mainly focused on the Dopamine Transporter (DAT1) and the Dopamine Receptor 4 (DRD4) genes. Nevertheless, polymorphisms of these genes explain only a small fraction of the assigned risk, suggesting that intermediate dimensions and environmental factors should also be considered. We investigated in 77 adult ADHD subjects compared to 474 controls, how polymorphisms within the genes coding for DAT1 (40-bp VNTR in 3'UTR), the Dopamine Receptor 2 (DRD2) (rs1799732) and DRD4 (48-bp VNTR in exon 3), may modulate the expression of the disorder. By genotyping DAT1, we detected a new 9.5R allele showing a deletion of 40 bp and also an insertion of 19 bp compared to the 10R allele. This novel allele was found to be significantly protective for ADHD (p < 0.0001). Another significant difference was found in the distribution of DRD4 48-bp VNTR 6R allele when comparing patients and controls (p = 0.0007). In addition significant results were also found for DAT1 9.5R allele, which was associated with impulsiveness (p = 1.98 × 10(-4)) and trait anger scores (p = 7.66 × 10(-4)). Moreover, impulsiveness scores were partly modulated by an interaction between the DRD4 48-bp VNTR 6R allele and childhood maltreatment (p = 0.01), however, this result did not resist correction for multiple comparisons. Altogether, our results show the putative involvement of DAT1 and DRD4 genes in the aetiology of ADHD with a main role in modulation of key dimensions of the disorder. PMID:25555995

  1. Fetal gene therapy of α-thalassemia in a mouse model

    PubMed Central

    Han, Xiao-Dong; Lin, Chin; Chang, Judy; Sadelain, Michel; Kan, Y. W.

    2007-01-01

    Fetuses with homozygous α-thalassemia usually die at the third trimester of pregnancy or soon after birth. Hence, the disease could potentially be a target for fetal gene therapy. We have previously established a mouse model of α-thalassemia. These mice mimic the human α-thalassemic conditions and can be used as preclinical models for fetal gene therapy. We tested a lentiviral vector containing the HS 2, 3, and 4 of the β-LCR, a central polypurine tract element, and the β-globin gene promoter directing either the EGFP or the human α-globin gene. We showed that the GFP expression was erythroid-specific and detected in BFU-E colonies and the erythroid progenies of CFU-GEMM. For in utero gene delivery, we did yolk sac vessel injection at midgestation of mouse embryos. The recipient mice were analyzed after birth for human α-globin gene expression. In the newborn, human α-globin gene expression was detected in the liver, spleen, and peripheral blood. The human α-globin gene expression was at the peak at 3–4 months, when it reached 20% in some recipients. However, the expression declined at 7 months. Colony-forming assays in these mice showed low abundance of the transduced human α-globin gene in their BFU-E and CFU-GEMM and the lack of its transcript. Thus, lentiviral vectors can be an effective vehicle for delivering the human α-globin gene into erythroid cells in utero, but, in the mouse model, delivery at late midgestation could not transduce hematopoietic stem cells adequately to sustain gene expression. PMID:17496141

  2. Circadian Clock Genes Are Essential for Normal Adult Neurogenesis, Differentiation, and Fate Determination.

    PubMed

    Malik, Astha; Kondratov, Roman V; Jamasbi, Roudabeh J; Geusz, Michael E

    2015-01-01

    Adult neurogenesis creates new neurons and glia from stem cells in the human brain throughout life. It is best understood in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Circadian rhythms have been identified in the hippocampus, but the role of any endogenous circadian oscillator cells in hippocampal neurogenesis and their importance in learning or memory remains unclear. Any study of stem cell regulation by intrinsic circadian timing within the DG is complicated by modulation from circadian clocks elsewhere in the brain. To examine circadian oscillators in greater isolation, neurosphere cultures were prepared from the DG of two knockout mouse lines that lack a functional circadian clock and from mPer1::luc mice to identify circadian oscillations in gene expression. Circadian mPer1 gene activity rhythms were recorded in neurospheres maintained in a culture medium that induces neurogenesis but not in one that maintains the stem cell state. Although the differentiating neural stem progenitor cells of spheres were rhythmic, evidence of any mature neurons was extremely sparse. The circadian timing signal originated in undifferentiated cells within the neurosphere. This conclusion was supported by immunocytochemistry for mPER1 protein that was localized to the inner, more stem cell-like neurosphere core. To test for effects of the circadian clock on neurogenesis, media conditions were altered to induce neurospheres from BMAL1 knockout mice to differentiate. These cultures displayed unusually high differentiation into glia rather than neurons according to GFAP and NeuN expression, respectively, and very few BetaIII tubulin-positive, immature neurons were observed. The knockout neurospheres also displayed areas visibly devoid of cells and had overall higher cell death. Neurospheres from arrhythmic mice lacking two other core clock genes, Cry1 and Cry2, showed significantly reduced growth and increased astrocyte proliferation during

  3. Circadian Clock Genes Are Essential for Normal Adult Neurogenesis, Differentiation, and Fate Determination

    PubMed Central

    Kondratov, Roman V.; Jamasbi, Roudabeh J.

    2015-01-01

    Adult neurogenesis creates new neurons and glia from stem cells in the human brain throughout life. It is best understood in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Circadian rhythms have been identified in the hippocampus, but the role of any endogenous circadian oscillator cells in hippocampal neurogenesis and their importance in learning or memory remains unclear. Any study of stem cell regulation by intrinsic circadian timing within the DG is complicated by modulation from circadian clocks elsewhere in the brain. To examine circadian oscillators in greater isolation, neurosphere cultures were prepared from the DG of two knockout mouse lines that lack a functional circadian clock and from mPer1::luc mice to identify circadian oscillations in gene expression. Circadian mPer1 gene activity rhythms were recorded in neurospheres maintained in a culture medium that induces neurogenesis but not in one that maintains the stem cell state. Although the differentiating neural stem progenitor cells of spheres were rhythmic, evidence of any mature neurons was extremely sparse. The circadian timing signal originated in undifferentiated cells within the neurosphere. This conclusion was supported by immunocytochemistry for mPER1 protein that was localized to the inner, more stem cell-like neurosphere core. To test for effects of the circadian clock on neurogenesis, media conditions were altered to induce neurospheres from BMAL1 knockout mice to differentiate. These cultures displayed unusually high differentiation into glia rather than neurons according to GFAP and NeuN expression, respectively, and very few BetaIII tubulin-positive, immature neurons were observed. The knockout neurospheres also displayed areas visibly devoid of cells and had overall higher cell death. Neurospheres from arrhythmic mice lacking two other core clock genes, Cry1 and Cry2, showed significantly reduced growth and increased astrocyte proliferation during

  4. A Comparison of the Olfactory Gene Repertoires of Adults and Larvae in the Noctuid Moth Spodoptera littoralis

    PubMed Central

    Poivet, Erwan; Gallot, Aurore; Montagné, Nicolas; Glaser, Nicolas; Legeai, Fabrice; Jacquin-Joly, Emmanuelle

    2013-01-01

    To better understand the olfactory mechanisms in a lepidopteran pest model species, the cotton leafworm Spodoptera littoralis, we have recently established a partial transcriptome from adult antennae. Here, we completed this transcriptome using next generation sequencing technologies, namely 454 and Illumina, on both adult antennae and larval tissues, including caterpillar antennae and maxillary palps. All sequences were assembled in 77,643 contigs. Their analysis greatly enriched the repertoire of chemosensory genes in this species, with a total of 57 candidate odorant-binding and chemosensory proteins, 47 olfactory receptors, 6 gustatory receptors and 17 ionotropic receptors. Using RT-PCR, we conducted the first exhaustive comparison of olfactory gene expression between larvae and adults in a lepidopteran species. All the 127 candidate olfactory genes were profiled for expression in male and female adult antennae and in caterpillar antennae and maxillary palps. We found that caterpillars expressed a smaller set of olfactory genes than adults, with a large overlap between these two developmental stages. Two binding proteins appeared to be larvae-specific and two others were adult-specific. Interestingly, comparison between caterpillar antennae and maxillary palps revealed numerous organ-specific transcripts, suggesting the complementary involvement of these two organs in larval chemosensory detection. Adult males and females shared the same set of olfactory transcripts, except two male-specific candidate pheromone receptors, two male-specific and two female-specific odorant-binding proteins. This study identified transcripts that may be important for sex-specific or developmental stage-specific chemosensory behaviors. PMID:23565215

  5. Embryonic Atrazine Exposure Elicits Alterations in Genes Associated with Neuroendocrine Function in Adult Male Zebrafish.

    PubMed

    Wirbisky, Sara E; Sepúlveda, Maria S; Weber, Gregory J; Jannasch, Amber S; Horzmann, Katharine A; Freeman, Jennifer L

    2016-09-01

    The developmental origins of health and disease (DOHaD) hypothesis states that exposure to environmental stressors early in life can elicit genome and epigenome changes resulting in an increased susceptibility of a disease state during adulthood. Atrazine, a common agricultural herbicide used throughout the Midwestern United States, frequently contaminates potable water supplies and is a suspected endocrine disrupting chemical. In our previous studies, zebrafish was exposed to 0, 0.3, 3, or 30 parts per billion (μg/l) atrazine through embryogenesis, rinsed, and allowed to mature to adulthood. A decrease in spawning was observed with morphological alterations in offspring. In addition, adult females displayed an increase in ovarian progesterone and follicular atresia, alterations in levels of a serotonin metabolite and serotonin turnover in brain tissue, and transcriptome changes in brain and ovarian tissue supporting neuroendocrine alterations. As reproductive dysfunction is also influenced by males, this study assessed testes histology, hormone levels, and transcriptomic profiles of testes and brain tissue in the adult males. The embryonic atrazine exposure resulted in no alterations in body or testes weight, gonadosomatic index, testes histology, or levels of 11-ketotestosterone or testosterone. To further investigate potential alterations, transcriptomic profiles of adult male testes and brain tissue was completed. This analysis demonstrated alterations in genes associated with abnormal cell and neuronal growth and morphology; molecular transport, quantity, and production of steroid hormones; and neurotransmission with an emphasis on the hypothalamus-pituitary-adrenal and hypothalamus-pituitary-thyroid axes. Overall, this data indicate future studies should focus on additional neuroendocrine endpoints to determine potential functional impairments. PMID:27413107

  6. Intermittent rhabdomyolysis with adult onset associated with a mutation in the ACADVL gene.

    PubMed

    Antunes, Ana Patrícia; Nogueira, Célia; Rocha, Hugo; Vilarinho, Laura; Evangelista, Teresinha

    2013-12-01

    Deficiency of very-long-chain acyl-CoA dehydrogenase (VLCAD) is an autosomal recessive disease. Most common phenotypes occur in the neonatal period or in childhood with cardiomyopathy, hepatomegaly, and hypoketogenic hypoglycemia. Juvenile/adult-onset is characterized by exercise intolerance and recurrent rhabdomyolysis triggered by prolonged exercise or fasting. This article reports a patient with the homozygous mutation c.1097G>A (p.R366H) in the ACADVL gene. In Portugal, VLCAD deficiency became part of the neonatal screening plan in 2004, and as of 2012, 8 early-onset cases have been diagnosed, giving an incidence rate of 1:97.238 per 737.902 newborns. This patient was diagnosed outside of the neonatal screening plan. Beta-oxidation defects pose a diagnostic challenge because of their transient clinical and laboratorial manifestations and the absence of morphological changes in muscle biopsy further complicate matters, especially in the late-onset forms of the disease. The adult phenotype of VLCAD deficiency is highlighted, emphasizing the need for a high suspicion index and the value of tandem mass spectrometry for the diagnosis. PMID:24263034

  7. Anatomical localization, gene expression profiling and functional characterization of adult human neck brown fat.

    PubMed

    Cypess, Aaron M; White, Andrew P; Vernochet, Cecile; Schulz, Tim J; Xue, Ruidan; Sass, Christina A; Huang, Tian Liang; Roberts-Toler, Carla; Weiner, Lauren S; Sze, Cathy; Chacko, Aron T; Deschamps, Laura N; Herder, Lindsay M; Truchan, Nathan; Glasgow, Allison L; Holman, Ashley R; Gavrila, Alina; Hasselgren, Per-Olof; Mori, Marcelo A; Molla, Michael; Tseng, Yu-Hua

    2013-05-01

    The imbalance between energy intake and expenditure is the underlying cause of the current obesity and diabetes pandemics. Central to these pathologies is the fat depot: white adipose tissue (WAT) stores excess calories, and brown adipose tissue (BAT) consumes fuel for thermogenesis using tissue-specific uncoupling protein 1 (UCP1). BAT was once thought to have a functional role in rodents and human infants only, but it has been recently shown that in response to mild cold exposure, adult human BAT consumes more glucose per gram than any other tissue. In addition to this nonshivering thermogenesis, human BAT may also combat weight gain by becoming more active in the setting of increased whole-body energy intake. This phenomenon of BAT-mediated diet-induced thermogenesis has been observed in rodents and suggests that activation of human BAT could be used as a safe treatment for obesity and metabolic dysregulation. In this study, we isolated anatomically defined neck fat from adult human volunteers and compared its gene expression, differentiation capacity and basal oxygen consumption to different mouse adipose depots. Although the properties of human neck fat vary substantially between individuals, some human samples share many similarities with classical, also called constitutive, rodent BAT. PMID:23603815

  8. Effects of tamoxifen on autosomal genes regulating ovary maintenance in adult mice.

    PubMed

    Yu, Mingxi; Liu, Wei; Wang, Jingyun; Qin, Junwen; Wang, Yongan; Wang, Yu

    2015-12-01

    Environmental endocrine-disrupting chemicals (EDCs), known to bind to estrogen/androgen receptors and mimic native estrogens, have been implicated as a main source for increasing human reproductive and developmental deficiencies and diseases. Tamoxifen (TAM) is one of the most well-known antiestrogens with defined adverse effects on the female reproductive tract, but the mechanisms related to autosomal gene regulation governing ovary maintenance in mammals remain unclear. The expression pattern and levels of key genes and proteins involved in maintaining the ovarian phenotype in mice were analyzed. The results showed that TAM induced significant upregulation of Sox9, which is the testis-determining factor gene. The results showed that TAM induced significant upregulation of Sox9, the testis-determining factor gene, and the expression level of Sox9 mRNA in the ovaries of mice exposed to 75 or 225 mg/kg bw TAM was 2- and 10-fold that in the control group, respectively (p < 0.001). Furthermore, the testicular fibroblast growth factor gene, Fgf9, was also elevated in TAM-treated ovaries. Accordingly, expression of the ovary development marker, forkhead transcription factor (FOXL2), and WNT4/FST signaling, were depressed. The levels of protein expression changed consistently with the target genes. Moreover, the detection of platelet/endothelial cell adhesion molecule 1 (PECAM-1) in TAM-treated ovaries suggested the formation of vascular endothelial cells, which is a further evidence for the differentiation of the ovaries to a testis-like phenotype. During this period, the level of 17β-estradiol, progesterone, and luteinizing hormone decreased, while that of testosterone increased by 3.3-fold (p = 0.013). The activation of a testis-specific molecular signaling cascade was a potentially important mechanism contributing to the gender disorder induced by TAM, which resulted in the differentiation of the ovaries to a testis-like phenotype in adult mice. Limited with

  9. A six gene expression signature defines aggressive subtypes and predicts outcome in childhood and adult acute lymphoblastic leukemia

    PubMed Central

    Wang, Jin; Mi, Jian-Qing; Debernardi, Alexandra; Vitte, Anne-Laure; Emadali, Anouk; Meyer, Julia A.; Charmpi, Konstantina; Ycart, Bernard; Callanan, Mary B.; Carroll, William L.; Khochbin, Saadi; Rousseaux, Sophie

    2015-01-01

    Abnormal gene expression in cancer represents an under-explored source of cancer markers and therapeutic targets. In order to identify gene expression signatures associated with survival in acute lymphoblastic leukemia (ALL), a strategy was designed to search for aberrant gene activity, which consists of applying several filters to transcriptomic datasets from two pediatric ALL studies. Six genes whose expression in leukemic blasts was associated with prognosis were identified:three genes predicting poor prognosis (AK022211, FASTKD1 and STARD4) and three genes associated with a favorable outcome (CAMSAP1, PCGF6 and SH3RF3). Combining the expression of these 6 genes could successfully predict prognosis not only in the two discovery pediatric ALL studies, but also in two independent validation cohorts of adult patients, one from a publicly available study and one consisting of 62 newly recruited Chinese patients. Moreover, our data demonstrate that our six gene based test is particularly efficient in stratifying MLL or BCR.ABL negative patients. Finally, common biological traits characterizing aggressive forms of ALL in both children and adults were found, including features of dormant hematopoietic stem cells, suggesting new therapeutic strategies. PMID:26001296

  10. Open and Lys–His Hexacoordinated Closed Structures of a Globin with Swapped Proximal and Distal Sites

    PubMed Central

    Teh, Aik-Hong; Saito, Jennifer A.; Najimudin, Nazalan; Alam, Maqsudul

    2015-01-01

    Globins are haem-binding proteins with a conserved fold made up of α-helices and can possess diverse properties. A putative globin-coupled sensor from Methylacidiphilum infernorum, HGbRL, contains an N-terminal globin domain whose open and closed structures reveal an untypical dimeric architecture. Helices E and F fuse into an elongated helix, resulting in a novel site-swapped globin fold made up of helices A–E, hence the distal site, from one subunit and helices F–H, the proximal site, from another. The open structure possesses a large cavity binding an imidazole molecule, while the closed structure forms a unique Lys–His hexacoordinated species, with the first turn of helix E unravelling to allow Lys52(E10) to bind to the haem. Ligand binding induces reorganization of loop CE, which is stabilized in the closed form, and helix E, triggering a large conformational movement in the open form. These provide a mechanical insight into how a signal may be relayed between the globin domain and the C-terminal domain of HGbRL, a Roadblock/LC7 domain. Comparison with HGbI, a closely related globin, further underlines the high degree of structural versatility that the globin fold is capable of, enabling it to perform a diversity of functions. PMID:26094577

  11. Transcriptome Analysis and Identification of Differentially Expressed Transcripts of Immune-Related Genes in Spleen of Gosling and Adult Goose

    PubMed Central

    Wang, Anqi; Liu, Fei; Chen, Shun; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Sun, Kunfeng; Wu, Ying; Chen, Xiaoyue; Cheng, Anchun

    2015-01-01

    The goose (Anser cygnoides), having high nutritional value, high-quality feathers and high economic benefit, is an economically important poultry species. However, the molecular mechanisms underlying the higher susceptibility to pathogens in goslings than in adult geese remains poorly understood. In this study, the histological sections of spleen tissue from a two-week-old gosling and an adult goose, respectively, were subjected to comparative analysis. The spleen of gosling was mainly composed of mesenchyma, accompanied by scattered lymphocytes, whereas the spleen parenchyma was well developed in the adult goose. To investigate goose immune-related genes, we performed deep transcriptome and gene expression analyses of the spleen samples using paired-end sequencing technology (Illumina). In total, 50,390 unigenes were assembled using Trinity software and TGICL software. Moreover, these assembled unigenes were annotated with gene descriptions and gene ontology (GO) analysis was performed. Through Kyoto encyclopedia of genes and genomes (KEGG) analysis, we investigated 558 important immune-relevant unigenes and 23 predicted cytokines. In addition, 22 immune-related genes with differential expression between gosling and adult goose were identified, among which the three genes showing largest differences in expression were immunoglobulin alpha heavy chain (IgH), mannan-binding lectin serine protease 1 isoform X1 (MASP1) and C–X–C chemokine receptor type 4 (CXCR4). Finally, of these 22 differentially expressed immune-related genes, seven genes, including tumor necrosis factor receptor superfamily member 13B (TNFRSF13B), C-C motif chemokine 4-like (CCL4), CXCR4, interleukin 2 receptor alpha (IL2RA), MHC class I heavy chain (MHCIα), transporter of antigen processing 2 (TAP2) IgH, were confirmed by quantitative real-time PCR (qRT-PCR). The expression levels of all the candidate unigenes were up-regulated in adult geese other than that of TNFRSF13B. The comparative

  12. Transcriptome Analysis and Identification of Differentially Expressed Transcripts of Immune-Related Genes in Spleen of Gosling and Adult Goose.

    PubMed

    Wang, Anqi; Liu, Fei; Chen, Shun; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Sun, Kunfeng; Wu, Ying; Chen, Xiaoyue; Cheng, Anchun

    2015-01-01

    The goose (Anser cygnoides), having high nutritional value, high-quality feathers and high economic benefit, is an economically important poultry species. However, the molecular mechanisms underlying the higher susceptibility to pathogens in goslings than in adult geese remains poorly understood. In this study, the histological sections of spleen tissue from a two-week-old gosling and an adult goose, respectively, were subjected to comparative analysis. The spleen of gosling was mainly composed of mesenchyma, accompanied by scattered lymphocytes, whereas the spleen parenchyma was well developed in the adult goose. To investigate goose immune-related genes, we performed deep transcriptome and gene expression analyses of the spleen samples using paired-end sequencing technology (Illumina). In total, 50,390 unigenes were assembled using Trinity software and TGICL software. Moreover, these assembled unigenes were annotated with gene descriptions and gene ontology (GO) analysis was performed. Through Kyoto encyclopedia of genes and genomes (KEGG) analysis, we investigated 558 important immune-relevant unigenes and 23 predicted cytokines. In addition, 22 immune-related genes with differential expression between gosling and adult goose were identified, among which the three genes showing largest differences in expression were immunoglobulin alpha heavy chain (IgH), mannan-binding lectin serine protease 1 isoform X1 (MASP1) and C-X-C chemokine receptor type 4 (CXCR4). Finally, of these 22 differentially expressed immune-related genes, seven genes, including tumor necrosis factor receptor superfamily member 13B (TNFRSF13B), C-C motif chemokine 4-like (CCL4), CXCR4, interleukin 2 receptor alpha (IL2RA), MHC class I heavy chain (MHCIα), transporter of antigen processing 2 (TAP2) IgH, were confirmed by quantitative real-time PCR (qRT-PCR). The expression levels of all the candidate unigenes were up-regulated in adult geese other than that of TNFRSF13B. The comparative

  13. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.

    PubMed

    Deverman, Benjamin E; Pravdo, Piers L; Simpson, Bryan P; Kumar, Sripriya Ravindra; Chan, Ken Y; Banerjee, Abhik; Wu, Wei-Li; Yang, Bin; Huber, Nina; Pasca, Sergiu P; Gradinaru, Viviana

    2016-02-01

    Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics. Here we describe a capsid selection method, called Cre recombination-based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV9 (refs. 14,15,16,17), and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications. PMID:26829320

  14. Novel Cryptic Rearrangements in Adult B-Cell Precursor Acute Lymphoblastic Leukemia Involving the MLL Gene.

    PubMed

    Othman, Moneeb A K; Grygalewicz, Beata; Pienkowska-Grela, Barbara; Rincic, Martina; Rittscher, Katharina; Melo, Joana B; Carreira, Isabel M; Meyer, Britta; Marzena, Watek; Liehr, Thomas

    2015-05-01

    MLL (mixed-lineage-leukemia) gene rearrangements are typical for acute leukemia and are associated with an aggressive course of disease, with a worse outcome than comparable case, and thus require intensified treatment. Here we describe a 69-year-old female with adult B cell precursor acute lymphoblastic leukemia (BCP-ALL) with hyperleukocytosis and immunophenotype CD10- and CD19+ with cryptic MLL rearrangements. G-banding at the time of diagnosis showed a normal karyotype: 46,XX. Molecular cytogenetics using multitude multicolor banding (mMCB) revealed a complex rearrangement of the two copies of chromosome 11. However, a locus-specific probe additionally identified that the MLL gene at 11q23.3 was disrupted, and that the 5' region was inserted into the chromosomal sub-band 4q21; thus the aberration involved three chromosomes and five break events. Unfortunately, the patient died six months after the initial diagnosis from serious infections and severe complications. Overall, the present findings confirm that, by far not all MLL aberrations are seen by routine chromosome banding techniques and that fluorescence in situ hybridization (FISH) should be regarded as standard tool to access MLL rearrangements in patients with BCP-ALL. PMID:25699572

  15. Transposable elements and their KRAB-ZFP controllers regulate gene expression in adult tissues

    PubMed Central

    Ecco, Gabriela; Cassano, Marco; Kauzlaric, Annamaria; Duc, Julien; Coluccio, Andrea; Offner, Sandra; Imbeault, Michaël; Rowe, Helen M.; Turelli, Priscilla; Trono, Didier

    2016-01-01

    Summary KRAB-containing zinc finger proteins (KRAB-ZFPs) are early embryonic controllers of transposable elements (TEs), which they repress with their cofactor KAP1 through histone and DNA methylation, a process thought to result in irreversible silencing. Using a target-centered functional screen, we matched murine TEs with their cognate KRAB-ZFP. We found the paralogs ZFP932 and Gm15446 to bind overlapping but distinguishable subsets of ERVK (endogenous retrovirus K), to repress these elements in embryonic stem cells, and to regulate secondarily the expression of neighboring genes. Most importantly, we uncovered that these KRAB-ZFPs and KAP1 control TEs in adult tissues, in cell culture and in vivo, where they partner up to modulate cellular genes. Therefore, TEs and KRAB-ZFPs establish transcriptional networks that regulate not only development but probably many physiological events. Given the high degree of species-specificity of TEs and KRAB-ZFPs, these results have important implications for understanding the biology of higher vertebrates, including humans. PMID:27003935

  16. Running, swimming and diving modifies neuroprotecting globins in the mammalian brain

    PubMed Central

    Williams, Terrie M; Zavanelli, Mary; Miller, Melissa A; Goldbeck, Robert A; Morledge, Michael; Casper, Dave; Pabst, D. Ann; McLellan, William; Cantin, Lucas P; Kliger, David S

    2007-01-01

    The vulnerability of the human brain to injury following just a few minutes of oxygen deprivation with submergence contrasts markedly with diving mammals, such as Weddell seals (Leptonychotes weddellii), which can remain underwater for more than 90 min while exhibiting no neurological or behavioural impairment. This response occurs despite exposure to blood oxygen levels concomitant with human unconsciousness. To determine whether such aquatic lifestyles result in unique adaptations for avoiding ischaemic–hypoxic neural damage, we measured the presence of circulating (haemoglobin) and resident (neuroglobin and cytoglobin) oxygen-carrying globins in the cerebral cortex of 16 mammalian species considered terrestrial, swimming or diving specialists. Here we report a striking difference in globin levels depending on activity lifestyle. A nearly 9.5-fold range in haemoglobin concentration (0.17–1.62 g Hb 100 g brain wet wt−1) occurred between terrestrial and deep-diving mammals; a threefold range in resident globins was evident between terrestrial and swimming specialists. Together, these two globin groups provide complementary mechanisms for facilitating oxygen transfer into neural tissues and the potential for protection against reactive oxygen and nitrogen groups. This enables marine mammals to maintain sensory and locomotor neural functions during prolonged submergence, and suggests new avenues for averting oxygen-mediated neural injury in the mammalian brain. PMID:18089537

  17. Vascular damage by unstable hemoglobins: the role of heme-depleted globin.

    PubMed

    Tsemakhovich, V A; Bamm, V V; Shaklai, M; Shaklai, N

    2005-04-15

    The study compared the damage inflicted to endothelial cells (ECs) by intact hemoglobin (Hb) and isolated chains. To compare optional in vivo contact of acellular Hb with the endothelium, oxy-forms of Hb and its isolated alpha- and beta-chains existing in the thalassemias were added to primary confluent cultures of bovine aorta EC. Cell damage was followed by morphological changes or leakage of lactic dehydrogenase and pre-inserted 51Cr from the cells, followed for 27 h. Under these experimental conditions, Hb did not affect the cells but its chains inflicted damage, beta- more than alpha-chains. Based on the literature and our data, we hypothesized that hemin and/or globin should be responsible for the increased endothelial damage by beta-chains. While hemin hardly affected ECs, globin, unlike the plasma protein hemopexin, was harmful. Since hemin release leaves globin with a large hydrophobic surface, the globin-damage appears to result from adsorptive pinocytosis to endothelial membrane. PMID:15797243

  18. Plerixafor+G-CSF–mobilized CD34+ cells represent an optimal graft source for thalassemia gene therapy

    PubMed Central

    Karponi, Garyfalia; Psatha, Nikoletta; Lederer, Carsten Werner; Adair, Jennifer Eileen; Zervou, Fani; Zogas, Nikolaos; Kleanthous, Marina; Tsatalas, Constantinos; Anagnostopoulos, Achilles; Sadelain, Michel; Rivière, Isabelle; Stamatoyannopoulos, George

    2015-01-01

    Globin gene therapy requires abundant numbers of highly engraftable, autologous hematopoietic stem cells expressing curative levels of β-globin on differentiation. In this study, CD34+ cells from 31 thalassemic patients mobilized with hydroxyurea+granulocyte colony-stimulating factor (G-CSF), G-CSF, Plerixafor, or Plerixafor+G-CSF were transduced with the TNS9.3.55 β-globin lentivector and compared for transducibility and globin expression in vitro, as well as engraftment potential in a xenogeneic model after partial myeloablation. Transduction efficiency and vector copy number (VCN) averaged 48.4 ± 2.8% and 1.91 ± 0.04, respectively, whereas expression approximated the one-copy normal β-globin output. Plerixafor+G-CSF cells produced the highest β-globin expression/VCN. Long-term multilineage engraftment and persistent VCN and vector expression was encountered in all xenografted groups, with Plerixafor+G-CSF–mobilized cells achieving superior short-term engraftment rates, with similar numbers of CD34+ cells transplanted. Overall, Plerixafor+G-CSF not only allows high CD34+ cell yields but also provides increased β-globin expression/VCN and enhanced early human chimerism under nonmyeloablative conditions, thus representing an optimal graft for thalassemia gene therapy. PMID:26089395

  19. Angiotensin-Converting Enzyme Gene Polymophism in Adult Primary Focal Segmental Glomerulosclerosis

    PubMed Central

    Mohd, Rozita; Wahab, Zaimi Abdul; Cader, Rizna; Gafor, Halim A.; Radzi, Azizah Md; Shah, Shamsul Azhar; Tong, Norella Kong Chiew

    2014-01-01

    Background Primary focal segmental glomerulosclerosis (FSGS) accounts for a third of biopsy-proven primary glomerulonephritis in Malaysia. Pediatric studies have found the insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene to be associated with renal disease progression. The aim of this study was to determine the prevalence of the ACE (I/D) genotypes in adult primary FSGS and its association with renal outcome on follow-up. Methods Prospective observational study involving primary FSGS patients was conducted. Biochemical and urine tests at the time of study were compared to the time of the diagnosis and disease progression analyzed. ACE gene polymorphism was identified using polymerase chain reaction amplification technique and categorized into II, ID and DD genotypes. Results Forty-five patients with a median follow-up of 3.8 years (interquartile range: 1.8 - 5.6) were recruited. The commonest genotype was II (n = 23, 51.1%) followed by ID (n = 19, 42.2%) and DD (n = 3, 6.7%). The baseline characteristics were comparable between the II and non-II groups at diagnosis and at study recruitment except that the median urine protein-creatinine index was significantly lower in the II group compared to the non-II group (0.02 vs. 0.04 g/mmol (P = 0.03). Regardless of genotypes, all parameters of renal outcome improved after treatment. Conclusion The II followed by ID genotypes were the predominant ACE gene alleles in our FSGS. Although the D allele has been reported to have a negative impact on renal outcome, treatment appeared to be more important than genotype in preserving renal function in this cohort. PMID:24883149

  20. Gene Expression Profile of Adult Human Olfactory Bulb and Embryonic Neural Stem Cell Suggests Distinct Signaling Pathways and Epigenetic Control

    PubMed Central

    Marei, Hany E. S.; Ahmed, Abd-Elmaksoud; Michetti, Fabrizio; Pescatori, Mario; Pallini, Roberto; Casalbore, Patricia; Cenciarelli, Carlo; Elhadidy, Mohamed

    2012-01-01

    Global gene expression profiling was performed using RNA from human embryonic neural stem cells (hENSC), and adult human olfactory bulb-derived neural stem cells (OBNSCs), to define a gene expression pattern and signaling pathways that are specific for each cell lineage. We have demonstrated large differences in the gene expression profile of human embryonic NSC, and adult human OBNSCs, but less variability between parallel cultures. Transcripts of genes involved in neural tube development and patterning (ALDH1A2, FOXA2), progenitor marker genes (LMX1a, ALDH1A1, SOX10), proliferation of neural progenitors (WNT1 and WNT3a), neuroplastin (NPTN), POU3F1 (OCT6), neuroligin (NLGN4X), MEIS2, and NPAS1 were up-regulated in both cell populations. By Gene Ontology, 325 out of 3875 investigated gene sets were scientifically different. 41 out of the 307 investigated Cellular Component (CC) categories, 45 out of the 620 investigated Molecular Function (MF) categories, and 239 out of the 2948 investigated Biological Process (BP) categories were significant. KEGG Pathway Class Comparison had revealed that 75 out of 171 investigated gene sets passed the 0.005 significance threshold. Levels of gene expression were explored in three signaling pathways, Notch, Wnt, and mTOR that are known to be involved in NS cell fates determination. The transcriptional signature also deciphers the role of genes involved in epigenetic modifications. SWI/SNF DNA chromatin remodeling complex family, including SMARCC1 and SMARCE1, were found specifically up-regulated in our OBNSC but not in hENSC. Differences in gene expression profile of transcripts controlling epigenetic modifications, and signaling pathways might indicate differences in the therapeutic potential of our examined two cell populations in relation to in cell survival, proliferation, migration, and differentiation following engraftments in different CNS insults. PMID:22485144

  1. Pharmacological and gene modification-based models for studying the impact of perinatal metabolic disturbances in adult life.

    PubMed

    Villarroya, Francesc; Bocos, Carlos; Giralt, Marta; Pilar Ramos, Maria; Herrera, Emilio; Sevillano, Julio; Gual, Margalida; Rosell, Meritxell; Iglesias, Roser

    2009-01-01

    Genetic modification approaches or pharmacological interventions may be useful for understanding the molecular mechanisms by which nutrient derivatives and metabolites exert their effects in the perinatal period and how they may influence longterm metabolism in adults. Examples for such experimental settings in rodents are targeted disruption of the gene for peroxisome proliferator-activated receptor (PPAR)-a, a lipid sensor and master regulator of lipid catabolism, or maternal treatment with agonists of PPARgamma, a master regulator of adipogenesis and target of insulin sensitizing drugs in adults. All these interventions show differential effects in the perinatal period compared to adults and indicate that altered activity of master regulators of metabolism results in profound metabolic alterations in the perinatal period that may influence adult metabolism. PMID:19536673

  2. Detection of two tissue-specific DNA-binding proteins with affinity for sites in the mouse beta-globin intervening sequence 2.

    PubMed Central

    Galson, D L; Housman, D E

    1988-01-01

    To identify proteins from uninduced murine erythroleukemia nuclear extracts which specifically bind to sequences from the DNase I-hypersensitive region within the mouse beta-globin intervening sequence 2 (IVS2), a gel electrophoretic mobility shift assay was used. Two distinct sequence-specific binding proteins were detected. The specific binding sites for these factors were delineated by both DNase I protection footprinting and methylation interference. Factor B1 bound specifically to two homologous sites, B1-A and B1-B, approximately 100 base pairs apart within the IVS2 and on opposite strands. These two regions could interact with factor B1 independently. Factor B1 was limited to cells of hematopoietic lineages. Factor B2 bound to a site approximately 5 base pairs away from the B1-A site and was limited to cells of the erythroid lineage. The limited tissue distribution of these factors and the locations of their binding sites suggest that one or both of these factors may be involved in the formation of the tissue-specific DNase I-hypersensitive site in the IVS2 of the mouse beta-globin gene. Images PMID:3422099

  3. Conservation and expression of PIWI-interacting RNA pathway genes in male and female adult gonad of amniotes.

    PubMed

    Lim, Shu Ly; Tsend-Ayush, Enkhjargal; Kortschak, R Daniel; Jacob, Reuben; Ricciardelli, Carmela; Oehler, Martin K; Grützner, Frank

    2013-12-01

    The PIWI-interacting RNA (piRNA) pathway is essential for germline development and transposable element repression. Key elements of this pathway are members of the piRNA-binding PIWI/Argonaute protein family and associated factors (e.g., VASA, MAELSTROM, and TUDOR domain proteins). PIWI-interacting RNAs have been identified in mouse testis and oocytes, but information about the expression of the different piRNA pathway genes, in particular in the mammalian ovary, remains incomplete. We investigated the evolution and expression of piRNA pathway genes in gonads of amniote species (chicken, platypus, and mouse). Database searches confirm a high level of conservation and revealed lineage-specific gain and loss of Piwi genes in vertebrates. Expression analysis in mammals shows that orthologs of Piwi-like (Piwil) genes, Mael (Maelstrom), Mvh (mouse vasa homolog), and Tdrd1 (Tudor domain-containing protein 1) are expressed in platypus adult testis. In contrast to mouse, Piwil4 is expressed in platypus and human adult testis. We found evidence for Mael and Piwil2 expression in mouse Sertoli cells. Importantly, we show mRNA expression of Piwil2, Piwil4, and Mael in oocytes and supporting cells of human, mouse, and platypus ovary. We found no Piwil1 expression in mouse and chicken ovary. The conservation of gene expression in somatic parts of the gonad and germ cells of species that diverged over 800 million yr ago indicates an important role in adult male and female gonad. PMID:24108303

  4. Utility of heme analogues to intentionally modify heme-globin interactions in myoglobin.

    PubMed

    Neya, Saburo; Nagai, Masako; Nagatomo, Shigenori; Hoshino, Tyuji; Yoneda, Tomoki; Kawaguchi, Akira T

    2016-05-01

    Myoglobin reconstitution with various synthetic heme analogues was reviewed to follow the consequences of modified heme-globin interactions. Utility of dimethyl sulfoxide as the solvent for water-insoluble hemes was emphasized. Proton NMR spectroscopy revealed that loose heme-globin contacts in the heme pocket eventually caused the dynamic heme rotation around the iron-histidine bond. The full rotational rate was estimated to be about 1400 s(-1) at room temperature for 1,4,5,8-tetramethylhemin. The X-ray analysis of the myoglobin containing iron porphine, the smallest heme without any side chains, showed that the original globin fold was well conserved despite the serious disruption of native heme-globin contacts. Comparison between the two myoglobins with static and rotatory prosthetic groups indicated that the oxygen and carbon monoxide binding profiles were almost unaffected by the heme motion. On the other hand, altered tetrapyrrole array of porphyrin dramatically changed the dissociation constant of oxygen from 0.0005 mm Hg of porphycene-myoglobin to ∞ in oxypyriporphyrin-myoglobin. Heme-globin interactions in myoglobin were also monitored with circular dichroism spectroscopy. The observation on several reconstituted protein revealed an unrecognized role of the propionate groups in protoheme. Shortening of heme 6,7-propionates to carboxylates resulted in almost complete disappearance of the positive circular dichroism band in the Soret region. The theoretical analysis suggested that the disappeared circular dichroism band reflected the cancellation effects between different conformers of the carboxyl groups directly attached to heme periphery. The above techniques were proposed to be applicable to other hemoproteins to create new biocatalysts. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson

  5. Pluripotent embryonic stem cells and multipotent adult germline stem cells reveal similar transcriptomes including pluripotency-related genes.

    PubMed

    Meyer, S; Nolte, J; Opitz, L; Salinas-Riester, G; Engel, W

    2010-11-01

    DNA microarray analysis was performed with mouse multipotent adult germline stem cells (maGSCs) and embryonic stem cells (ESCs) from different genetic backgrounds cultured under standard ESC-culture conditions and under differentiation-promoting conditions by the withdrawal of the leukemia inhibitory factor (LIF) and treatment with retinoic acid (RA). The analyzed undifferentiated cell lines are very similar based on their global gene expression pattern and show 97-99% identity dependent on the analyzed background. Only 621 genes are differentially expressed in cells derived from mouse 129SV-background and 72 genes show differences in expression in cells generated from transgenic Stra8-EGFP/Rosa26-LacZ-background. Both maGSCs and ESCs express the same genes involved in the regulation of pluripotency and even show no differences in the expression level of these genes. When comparing maGSCs with previously published signature genes of other pluripotent cell lines, we found that maGSCs shared a very similar gene expression pattern with embryonic germ cells (EGCs). Also after differentiation of maGSCs and ESCs the transcriptomes of the cell lines are nearly identical which suggests that both cell types differentiate spontaneously in a very similar way. This is the first study, at transcriptome level, to compare ESCs and a pluripotent cell line derived from an adult organism (maGSCs). PMID:20624824

  6. LIN28B-mediated expression of fetal hemoglobin and production of fetal-like erythrocytes from adult human erythroblasts ex vivo

    PubMed Central

    Lee, Y. Terry; de Vasconcellos, Jaira F.; Yuan, Joan; Byrnes, Colleen; Noh, Seung-Jae; Meier, Emily R.; Kim, Ki Soon; Rabel, Antoinette; Kaushal, Megha; Muljo, Stefan A.

    2013-01-01

    Reactivation of fetal hemoglobin (HbF) holds therapeutic potential for sickle cell disease and β-thalassemias. In human erythroid cells and hematopoietic organs, LIN28B and its targeted let-7 microRNA family, demonstrate regulated expression during the fetal-to-adult developmental transition. To explore the effects of LIN28B in human erythroid cell development, lentiviral transduction was used to knockdown LIN28B expression in erythroblasts cultured from human umbilical cord CD34+ cells. The subsequent reduction in LIN28B expression caused increased expression of let-7 and significantly reduced HbF expression. Conversely, LIN28B overexpression in cultured adult erythroblasts reduced the expression of let-7 and significantly increased HbF expression. Cellular maturation was maintained including enucleation. LIN28B expression in adult erythroblasts increased the expression of γ-globin, and the HbF content of the cells rose to levels >30% of their hemoglobin. Expression of carbonic anhydrase I, glucosaminyl (N-acetyl) transferase 2, and miR-96 (three additional genes marking the transition from fetal-to-adult erythropoiesis) were reduced by LIN28B expression. The transcription factor BCL11A, a well-characterized repressor of γ-globin expression, was significantly down-regulated. Independent of LIN28B, experimental suppression of let-7 also reduced BCL11A expression and significantly increased HbF expression. LIN28B expression regulates HbF levels and causes adult human erythroblasts to differentiate with a more fetal-like phenotype. PMID:23798711

  7. Developmental methoxychlor exposure affects multiple reproductive parameters and ovarian folliculogenesis and gene expression in adult rats

    SciTech Connect

    Armenti, AnnMarie E.; Zama, Aparna Mahakali; Passantino, Lisa; Uzumcu, Mehmet

    2008-12-01

    Methoxychlor (MXC) is an organochlorine pesticide with estrogenic, anti-estrogenic, and anti-androgenic properties. To investigate whether transient developmental exposure to MXC could cause adult ovarian dysfunction, we exposed Fischer rats to 20 {mu}g/kg/day (low dose; environmentally relevant dose) or 100 mg/kg/day (high dose) MXC between 19 days post coitum and postnatal day 7. Multiple reproductive parameters, serum hormone levels, and ovarian morphology and molecular markers were examined from prepubertal through adult stages. High dose MXC accelerated pubertal onset and first estrus, reduced litter size, and increased irregular cyclicity (P < 0.05). MXC reduced superovulatory response to exogenous gonadotropins in prepubertal females (P < 0.05). Rats exposed to high dose MXC had increasing irregular estrous cyclicity beginning at 4 months of age, with all animals showing abnormal cycles by 6 months. High dose MXC reduced serum progesterone, but increased luteinizing hormone (LH). Follicular composition analysis revealed an increase in the percentage of preantral and early antral follicles and a reduction in the percentage of corpora lutea in high dose MXC-treated ovaries (P < 0.05). Immunohistochemical staining and quantification of the staining intensity showed that estrogen receptor {beta} was reduced by high dose MXC while anti-Mullerian hormone was upregulated by both low- and high dose MXC in preantral and early antral follicles (P < 0.05). High dose MXC significantly reduced LH receptor expression in large antral follicles (P < 0.01), and down-regulated cytochrome P450 side-chain cleavage. These results demonstrated that developmental MXC exposure results in reduced ovulation and fertility and premature aging, possibly by altering ovarian gene expression and folliculogenesis.

  8. Early developmental gene enhancers affect subcortical volumes in the adult human brain.

    PubMed

    Becker, Martin; Guadalupe, Tulio; Franke, Barbara; Hibar, Derrek P; Renteria, Miguel E; Stein, Jason L; Thompson, Paul M; Francks, Clyde; Vernes, Sonja C; Fisher, Simon E

    2016-05-01

    Genome-wide association screens aim to identify common genetic variants contributing to the phenotypic variability of complex traits, such as human height or brain morphology. The identified genetic variants are mostly within noncoding genomic regions and the biology of the genotype-phenotype association typically remains unclear. In this article, we propose a complementary targeted strategy to reveal the genetic underpinnings of variability in subcortical brain volumes, by specifically selecting genomic loci that are experimentally validated forebrain enhancers, active in early embryonic development. We hypothesized that genetic variation within these enhancers may affect the development and ultimately the structure of subcortical brain regions in adults. We tested whether variants in forebrain enhancer regions showed an overall enrichment of association with volumetric variation in subcortical structures of >13,000 healthy adults. We observed significant enrichment of genomic loci that affect the volume of the hippocampus within forebrain enhancers (empirical P = 0.0015), a finding which robustly passed the adjusted threshold for testing of multiple brain phenotypes (cutoff of P < 0.0083 at an alpha of 0.05). In analyses of individual single nucleotide polymorphisms (SNPs), we identified an association upstream of the ID2 gene with rs7588305 and variation in hippocampal volume. This SNP-based association survived multiple-testing correction for the number of SNPs analyzed but not for the number of subcortical structures. Targeting known regulatory regions offers a way to understand the underlying biology that connects genotypes to phenotypes, particularly in the context of neuroimaging genetics. This biology-driven approach generates testable hypotheses regarding the functional biology of identified associations. Hum Brain Mapp 37:1788-1800, 2016. © 2016 Wiley Periodicals, Inc. PMID:26890892

  9. Developmental Methoxychlor Exposure Affects Multiple Reproductive Parameters and Ovarian: Folliculogenesis and Gene Expression in Adult Rats

    PubMed Central

    Armenti, AnnMarie E.; Zama, Aparna Mahakali; Passantino, Lisa; Uzumcu, Mehmet

    2008-01-01

    Methoxychlor (MXC) is an organochlorine pesticide with estrogenic, anti-estrogenic, and anti-androgenic properties. To investigate whether transient developmental exposure to MXC could cause adult ovarian dysfunction, we exposed Fischer rats to 20 μg/kg/day (low dose; environmentally relevant dose) or 100 mg/kg/day (high dose) MXC between 19 days post-coitum and postnatal day 7. Multiple reproductive parameters, serum hormone levels, and ovarian morphology and molecular markers were examined from prepubertal through adult stages. High dose MXC accelerated pubertal onset and first estrus, reduced litter size, and increased irregular cyclicity (P < 0.05). MXC reduced superovulatory response to exogenous gonadotropins in prepubertal females (P < 0.05). Rats exposed to high dose MXC had increasing irregular estrous cyclicity beginning at 4 months of age, with all animals showing abnormal cycles by 6 months. High dose MXC reduced serum progesterone, but increased luteinizing hormone (LH). Follicular composition analysis revealed an increase in the percentage of preantral and early antral follicles and a reduction in the percentage of corpora lutea in high dose MXC-treated ovaries (P < 0.05). Immunohistochemical staining and quantification of the staining intensity showed that estrogen receptor β was reduced by high dose MXC while anti-Mullerian hormone was upregulated by both low- and high dose MXC in preantral and early antral follicles (P < 0.05). High dose MXC significantly reduced LH receptor expression in large antral follicles (P < 0.01), and down-regulated cytochrome P450 side-chain cleavage. These results demonstrated that developmental MXC exposure results in reduced ovulation and fertility and premature aging, possibly by altering ovarian gene expression and folliculogenesis. PMID:18848953

  10. Brain white matter structure and COMT gene are linked to second-language learning in adults.

    PubMed

    Mamiya, Ping C; Richards, Todd L; Coe, Bradley P; Eichler, Evan E; Kuhl, Patricia K

    2016-06-28

    Adult human brains retain the capacity to undergo tissue reorganization during second-language learning. Brain-imaging studies show a relationship between neuroanatomical properties and learning for adults exposed to a second language. However, the role of genetic factors in this relationship has not been investigated. The goal of the current study was twofold: (i) to characterize the relationship between brain white matter fiber-tract properties and second-language immersion using diffusion tensor imaging, and (ii) to determine whether polymorphisms in the catechol-O-methyltransferase (COMT) gene affect the relationship. We recruited incoming Chinese students enrolled in the University of Washington and scanned their brains one time. We measured the diffusion properties of the white matter fiber tracts and correlated them with the number of days each student had been in the immersion program at the time of the brain scan. We found that higher numbers of days in the English immersion program correlated with higher fractional anisotropy and lower radial diffusivity in the right superior longitudinal fasciculus. We show that fractional anisotropy declined once the subjects finished the immersion program. The relationship between brain white matter fiber-tract properties and immersion varied in subjects with different COMT genotypes. Subjects with the Methionine (Met)/Valine (Val) and Val/Val genotypes showed higher fractional anisotropy and lower radial diffusivity during immersion, which reversed immediately after immersion ended, whereas those with the Met/Met genotype did not show these relationships. Statistical modeling revealed that subjects' grades in the language immersion program were best predicted by fractional anisotropy and COMT genotype. PMID:27298360

  11. Brain white matter structure and COMT gene are linked to second-language learning in adults

    PubMed Central

    Mamiya, Ping C.; Richards, Todd L.; Coe, Bradley P.; Eichler, Evan E.; Kuhl, Patricia K.

    2016-01-01

    Adult human brains retain the capacity to undergo tissue reorganization during second-language learning. Brain-imaging studies show a relationship between neuroanatomical properties and learning for adults exposed to a second language. However, the role of genetic factors in this relationship has not been investigated. The goal of the current study was twofold: (i) to characterize the relationship between brain white matter fiber-tract properties and second-language immersion using diffusion tensor imaging, and (ii) to determine whether polymorphisms in the catechol-O-methyltransferase (COMT) gene affect the relationship. We recruited incoming Chinese students enrolled in the University of Washington and scanned their brains one time. We measured the diffusion properties of the white matter fiber tracts and correlated them with the number of days each student had been in the immersion program at the time of the brain scan. We found that higher numbers of days in the English immersion program correlated with higher fractional anisotropy and lower radial diffusivity in the right superior longitudinal fasciculus. We show that fractional anisotropy declined once the subjects finished the immersion program. The relationship between brain white matter fiber-tract properties and immersion varied in subjects with different COMT genotypes. Subjects with the Methionine (Met)/Valine (Val) and Val/Val genotypes showed higher fractional anisotropy and lower radial diffusivity during immersion, which reversed immediately after immersion ended, whereas those with the Met/Met genotype did not show these relationships. Statistical modeling revealed that subjects’ grades in the language immersion program were best predicted by fractional anisotropy and COMT genotype. PMID:27298360

  12. Desensitization and Incomplete Recovery of Hepatic Target Genes After Chronic Thyroid Hormone Treatment and Withdrawal in Male Adult Mice.

    PubMed

    Ohba, Kenji; Leow, Melvin Khee-Shing; Singh, Brijesh Kumar; Sinha, Rohit Anthony; Lesmana, Ronny; Liao, Xiao-Hui; Ghosh, Sujoy; Refetoff, Samuel; Sng, Judy Chia Ghee; Yen, Paul Michael

    2016-04-01

    Clinical symptoms may vary and not necessarily reflect serum thyroid hormone (TH) levels during acute and chronic hyperthyroidism as well as recovery from hyperthyroidism. We thus examined changes in hepatic gene expression and serum TH/TSH levels in adult male mice treated either with a single T3 (20 μg per 100 g body weight) injection (acute T3) or daily injections for 14 days (chronic T3) followed by 10 days of withdrawal. Gene expression arrays from livers harvested at these time points showed that among positively-regulated target genes, 320 were stimulated acutely and 429 chronically by T3. Surprisingly, only 69 of 680 genes (10.1%) were induced during both periods, suggesting desensitization of the majority of acutely stimulated target genes. About 90% of positively regulated target genes returned to baseline expression levels after 10 days of withdrawal; however, 67 of 680 (9.9%) did not return to baseline despite normalization of serum TH/TSH levels. Similar findings also were observed for negatively regulated target genes. Chromatin immunoprecipitation analysis of representative positively regulated target genes suggested that acetylation of H3K9/K14 was associated with acute stimulation, whereas trimethylation of H3K4 was associated with chronic stimulation. In an in vivo model of chronic intrahepatic hyperthyroidism since birth, adult male monocarboxylate transporter-8 knockout mice also demonstrated desensitization of most acutely stimulated target genes that were examined. In summary, we have identified transcriptional desensitization and incomplete recovery of gene expression during chronic hyperthyroidism and recovery. Our findings may be a potential reason for discordance between clinical symptoms and serum TH levels observed in these conditions. PMID:26866609

  13. A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level γ-Globin Inducing Compounds

    PubMed Central

    Durlak, Marta; Fugazza, Cristina; Elangovan, Sudharshan; Marini, Maria Giuseppina; Marongiu, Maria Franca; Moi, Paolo; Fraietta, Ivan; Cappella, Paolo; Barbarani, Gloria; Font-Monclus, Isaura; Mauri, Mario; Ottolenghi, Sergio; Gasparri, Fabio; Ronchi, Antonella

    2015-01-01

    The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect variation in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ- and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and dacinostat) and identified Heme-oxygenases as novel candidate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other drugs) to improve pharmacological γ-globin reactivation for the treatment of β-hemoglobinopathies. PMID:26509275

  14. β-Globin Sleeping Beauty Transposon Reduces Red Blood Cell Sickling in a Patient-Derived CD34+-Based In Vitro Model

    PubMed Central

    Sjeklocha, Lucas M.; Wong, Phillip Y.-P.; Belcher, John D.; Vercellotti, Gregory M.; Steer, Clifford J.

    2013-01-01

    The ultimate goal of gene therapy for sickle cell anemia (SCA) is an improved phenotype for the patient. In this study, we utilized bone marrow from a sickle cell patient as a model of disease in an in vitro setting for the hyperactive Sleeping Beauty transposon gene therapy system. We demonstrated that mature sickle red blood cells containing hemoglobin-S and sickling in response to metabisulfite can be generated in vitro from SCA bone marrow. These cells showed the characteristic morphology and kinetics of hemoglobin-S polymerization, which we quantified using video microscopy and imaging cytometry. Using video assessment, we showed that delivery of an IHK-βT87Q antisickling globin gene by Sleeping Beauty via nucleofection improves metrics of sickling, decreasing percent sickled from 53.2 ± 2.2% to 43.9 ± 2.0%, increasing the median time to sickling from 8.5 to 9.6 min and decreasing the maximum rate of sickling from 2.3 x 10-3 sickling cells/total cells/sec in controls to 1.26 x 10-3 sickling cells/total cells/sec in the IHK-βT87Q-globin group (p < 0.001). Using imaging cytometry, the percentage of elongated sickled cells decreased from 34.8 ± 4.5% to 29.5 ± 3.0% in control versus treated (p < 0.05). These results support the potential use of Sleeping Beauty as a clinical gene therapy vector and provide a useful tool for studying sickle red blood cells in vitro. PMID:24260386

  15. cap alpha. -skeletal and. cap alpha. -cardiac actin genes are coexpressed in adult human skeletal muscle and heart

    SciTech Connect

    Gunning, P.; Ponte, P.; Blau, H.; Kedes, L.

    1983-11-01

    The authors determined the actin isotypes encoded by 30 actin cDNA clones previously isolated from an adult human muscle cDNA library. Using 3' untranslated region probes, derived from ..cap alpha.. skeletal, ..beta..- and ..gamma..-actin cDNAs and from an ..cap alpha..-cardiac actin genomic clone, they showed that 28 of the cDNAs correspond to ..cap alpha..-skeletal actin transcripts. Unexpectedly, however, the remaining two cDNA clones proved to derive from ..cap alpha..-cardiac actin mRNA. Sequence analysis confirmed that the two skeletal muscle ..cap alpha..-cardiac actin cDNAs are derived from transcripts of the cloned ..cap alpha..-cardiac actin gene. Comparison of total actin mRNA levels in adult skeletal muscle and adult heart revealed that the steady-state levels in skeletal muscle are about twofold greater, per microgram of total cellular RNA, than those in heart. Thus, in skeletal muscle and in heart, both of the sarcomeric actin mRNA isotypes are quite abundant transcripts. They conclude that ..cap alpha..-skeletal and ..cap alpha..-cardiac actin genes are coexpressed as an actin pair in human adult striated muscles. Since the smooth-muscle actins (aortic and stomach) and the cytoplasmic actins (..beta.. and ..gamma..) are known to be coexpressed in smooth muscle and nonmuscle cells, respectively, they postulate that coexpression of actin pairs may be a common feature of mammalian actin gene expression in all tissues.

  16. A gain-of-function screen for genes that affect the development of the Drosophila adult external sensory organ.

    PubMed Central

    Abdelilah-Seyfried, S; Chan, Y M; Zeng, C; Justice, N J; Younger-Shepherd, S; Sharp, L E; Barbel, S; Meadows, S A; Jan, L Y; Jan, Y N

    2000-01-01

    The Drosophila adult external sensory organ, comprising a neuron and its support cells, is derived from a single precursor cell via several asymmetric cell divisions. To identify molecules involved in sensory organ development, we conducted a tissue-specific gain-of-function screen. We screened 2293 independent P-element lines established by P. Rorth and identified 105 lines, carrying insertions at 78 distinct loci, that produced misexpression phenotypes with changes in number, fate, or morphology of cells of the adult external sensory organ. On the basis of the gain-of-function phenotypes of both internal and external support cells, we subdivided the candidate lines into three classes. The first class (52 lines, 40 loci) exhibits partial or complete loss of adult external sensory organs. The second class (38 lines, 28 loci) is associated with increased numbers of entire adult external sensory organs or subsets of sensory organ cells. The third class (15 lines, 10 loci) results in potential cell fate transformations. Genetic and molecular characterization of these candidate lines reveals that some loci identified in this screen correspond to genes known to function in the formation of the peripheral nervous system, such as big brain, extra macrochaetae, and numb. Also emerging from the screen are a large group of previously uncharacterized genes and several known genes that have not yet been implicated in the development of the peripheral nervous system. PMID:10835395

  17. Faecal Microbiota Composition in Adults Is Associated with the FUT2 Gene Determining the Secretor Status

    PubMed Central

    Wacklin, Pirjo; Tuimala, Jarno; Nikkilä, Janne; Sebastian Tims; Mäkivuokko, Harri; Alakulppi, Noora; Laine, Pia; Rajilic-Stojanovic, Mirjana; Paulin, Lars; de Vos, Willem M.; Mättö, Jaana

    2014-01-01

    The human intestine is colonised with highly diverse and individually defined microbiota, which likely has an impact on the host well-being. Drivers of the individual variation in the microbiota compositions are multifactorial and include environmental, host and dietary factors. We studied the impact of the host secretor status, encoded by fucosyltransferase 2 (FUT2) -gene, on the intestinal microbiota composition. Secretor status determines the expression of the ABH and Lewis histo-blood group antigens in the intestinal mucosa. The study population was comprised of 14 non-secretor (FUT2 rs601338 genotype AA) and 57 secretor (genotypes GG and AG) adult individuals of western European descent. Intestinal microbiota was analyzed by PCR-DGGE and for a subset of 12 non-secretor subjects and 12 secretor subjects additionally by the 16S rRNA gene pyrosequencing and the HITChip phylogenetic microarray analysis. All three methods showed distinct clustering of the intestinal microbiota and significant differences in abundances of several taxa representing dominant microbiota between the non-secretors and the secretors as well as between the FUT2 genotypes. In addition, the non-secretors had lower species richness than the secretors. The soft clustering of microbiota into enterotypes (ET) 1 and 3 showed that the non-secretors had a higher probability of belonging to ET1 and the secretors to ET3. Our study shows that secretor status and FUT2 polymorphism are associated with the composition of human intestinal microbiota, and appears thus to be one of the key drivers affecting the individual variation of human intestinal microbiota. PMID:24733310

  18. Enantio-alteration of gene transcription associated with bioconcentration in adult zebrafish (Danio rerio) exposed to chiral PCB149

    PubMed Central

    Chai, Tingting; Cui, Feng; Mu, Pengqian; Yang, Yang; Xu, Nana; Yin, Zhiqiang; Jia, Qi; Yang, Shuming; Qiu, Jing; Wang, Chengju

    2016-01-01

    Enantioselective enrichment of chiral PCB149 (2,2’,3,4’,5’,6-hexachlorobiphenyl) was analysed in adult zebrafish (Danio rerio) exposed to the racemate, (−)-PCB149, and (+)-PCB149. Greater enrichment of (−)-PCB149 compared to (+) PCB149 was observed following 0.5 ng/L exposure; however, as the exposure time and concentration increased, racemic enrichment was observed in adult fish exposed to the racemate. No biotransformation between the two isomers was observed in fish exposed to single enantiomers. When zebrafish were exposed to different forms of chiral PCB149, enantioselective expression of genes associated with polychlorinated biphenyls (PCBs) was observed in brain and liver tissues and enantioselective correlations between bioconcentration and target gene expression levels were observed in brain and liver tissues. The strong positive correlations between expression levels of target genes (alox5a and alox12) and PCB149 bioconcentration suggest that prolonged exposure to the racemate of chiral PCB149 may result in inflammation-associated diseases. Prolonged exposure to (−)-PCB149 may also affect metabolic pathways such as dehydrogenation and methylation in the brain tissues of adult zebrafish. Hepatic expression levels of genes related to the antioxidant system were significantly negatively correlated with bioconcentration following exposure to (+)-PCB149. PMID:26786282

  19. Enantio-alteration of gene transcription associated with bioconcentration in adult zebrafish (Danio rerio) exposed to chiral PCB149.

    PubMed

    Chai, Tingting; Cui, Feng; Mu, Pengqian; Yang, Yang; Xu, Nana; Yin, Zhiqiang; Jia, Qi; Yang, Shuming; Qiu, Jing; Wang, Chengju

    2016-01-01

    Enantioselective enrichment of chiral PCB149 (2,2',3,4',5',6-hexachlorobiphenyl) was analysed in adult zebrafish (Danio rerio) exposed to the racemate, (-)-PCB149, and (+)-PCB149. Greater enrichment of (-)-PCB149 compared to (+) PCB149 was observed following 0.5 ng/L exposure; however, as the exposure time and concentration increased, racemic enrichment was observed in adult fish exposed to the racemate. No biotransformation between the two isomers was observed in fish exposed to single enantiomers. When zebrafish were exposed to different forms of chiral PCB149, enantioselective expression of genes associated with polychlorinated biphenyls (PCBs) was observed in brain and liver tissues and enantioselective correlations between bioconcentration and target gene expression levels were observed in brain and liver tissues. The strong positive correlations between expression levels of target genes (alox5a and alox12) and PCB149 bioconcentration suggest that prolonged exposure to the racemate of chiral PCB149 may result in inflammation-associated diseases. Prolonged exposure to (-)-PCB149 may also affect metabolic pathways such as dehydrogenation and methylation in the brain tissues of adult zebrafish. Hepatic expression levels of genes related to the antioxidant system were significantly negatively correlated with bioconcentration following exposure to (+)-PCB149. PMID:26786282

  20. Enantio-alteration of gene transcription associated with bioconcentration in adult zebrafish (Danio rerio) exposed to chiral PCB149

    NASA Astrophysics Data System (ADS)

    Chai, Tingting; Cui, Feng; Mu, Pengqian; Yang, Yang; Xu, Nana; Yin, Zhiqiang; Jia, Qi; Yang, Shuming; Qiu, Jing; Wang, Chengju

    2016-01-01

    Enantioselective enrichment of chiral PCB149 (2,2’,3,4’,5’,6-hexachlorobiphenyl) was analysed in adult zebrafish (Danio rerio) exposed to the racemate, (-)-PCB149, and (+)-PCB149. Greater enrichment of (-)-PCB149 compared to (+) PCB149 was observed following 0.5 ng/L exposure; however, as the exposure time and concentration increased, racemic enrichment was observed in adult fish exposed to the racemate. No biotransformation between the two isomers was observed in fish exposed to single enantiomers. When zebrafish were exposed to different forms of chiral PCB149, enantioselective expression of genes associated with polychlorinated biphenyls (PCBs) was observed in brain and liver tissues and enantioselective correlations between bioconcentration and target gene expression levels were observed in brain and liver tissues. The strong positive correlations between expression levels of target genes (alox5a and alox12) and PCB149 bioconcentration suggest that prolonged exposure to the racemate of chiral PCB149 may result in inflammation-associated diseases. Prolonged exposure to (-)-PCB149 may also affect metabolic pathways such as dehydrogenation and methylation in the brain tissues of adult zebrafish. Hepatic expression levels of genes related to the antioxidant system were significantly negatively correlated with bioconcentration following exposure to (+)-PCB149.

  1. Adult midgut expressed sequence tags from the tsetse fly Glossina morsitans morsitans and expression analysis of putative immune response genes

    PubMed Central

    Lehane, M J; Aksoy, S; Gibson, W; Kerhornou, A; Berriman, M; Hamilton, J; Soares, M B; Bonaldo, M F; Lehane, S; Hall, N

    2003-01-01

    Background Tsetse flies transmit African trypanosomiasis leading to half a million cases annually. Trypanosomiasis in animals (nagana) remains a massive brake on African agricultural development. While trypanosome biology is widely studied, knowledge of tsetse flies is very limited, particularly at the molecular level. This is a serious impediment to investigations of tsetse-trypanosome interactions. We have undertaken an expressed sequence tag (EST) project on the adult tsetse midgut, the major organ system for establishment and early development of trypanosomes. Results A total of 21,427 ESTs were produced from the midgut of adult Glossina morsitans morsitans and grouped into 8,876 clusters or singletons potentially representing unique genes. Putative functions were ascribed to 4,035 of these by homology. Of these, a remarkable 3,884 had their most significant matches in the Drosophila protein database. We selected 68 genes with putative immune-related functions, macroarrayed them and determined their expression profiles following bacterial or trypanosome challenge. In both infections many genes are downregulated, suggesting a malaise response in the midgut. Trypanosome and bacterial challenge result in upregulation of different genes, suggesting that different recognition pathways are involved in the two responses. The most notable block of genes upregulated in response to trypanosome challenge are a series of Toll and Imd genes and a series of genes involved in oxidative stress responses. Conclusions The project increases the number of known Glossina genes by two orders of magnitude. Identification of putative immunity genes and their preliminary characterization provides a resource for the experimental dissection of tsetse-trypanosome interactions. PMID:14519198

  2. Differential expression of the FMRF gene in adult and hatchling stellate ganglia of the squid Loligo pealei

    PubMed Central

    Burbach, J. Peter H.; Grant, Philip; Hellemons, Anita J. C. G. M.; Degiorgis, Joseph A.; Li, Ka Wan; Pant, Harish C.

    2014-01-01

    Summary The giant fiber system of the squid Loligo pealei mediates the escape response and is an important neurobiological model. Here, we identified an abundant transcript in the stellate ganglion (SG) that encodes a FMRFamide precursor, and characterized FMRFamide and FI/LRF-amide peptides. To determine whether FMRFamide plays a role in the adult and hatchling giant fiber system, we studied the expression of the Fmrf gene and FMRFamide peptides. In stage 29 embryos and stage 30 hatchlings, Ffmr transcripts and FMRFamide peptide were low to undetectable in the SG, in contrast to groups of neurons intensely expressing the Fmrf gene in several brain lobes, including those that innervate the SG. In the adult SG the Fmrf gene was highly expressed, but the FMRFamide peptide was in low abundance. Intense staining for FMRFamide in the adult SG was confined to microneurons and fibers in the neuropil and to small fibers surrounding giant axons in stellar nerves. This shows that the Fmrf gene in the SG is strongly regulated post-hatching, and suggests that the FMRFamide precursor is incompletely processed in the adult SG. The data suggest that the SG only employs the Fmrf gene post-hatching and restricts the biosynthesis of FMRFamide, demonstrating that this peptide is not a major transmitter of the giant fiber system. This contrasts with brain lobes that engage FMRFamide embryonically as a regulatory peptide in multiple neuronal systems, including the afferent fibers that innervate the SG. The biological significance of these mechanisms may be to generate diversity within Fmrf-expressing systems in cephalopods. PMID:24326188

  3. A comparison of the multiple oocyte maturation gene expression patterns between the newborn and adult mouse ovary

    PubMed Central

    Bahmanpour, Soghra; Talaei Khozani, Tahereh; Zarei fard, Nehleh; Jaberipour, Mansoureh; Hosseini, Ahmah; Esmaeilpour, Tahereh

    2013-01-01

    Background: The interaction between follicular cells and oocyte leads to a change in gene expression involved in oocyte maturation processes. Objective: The purpose of this study was to quantify the expression of more common genes involved in follicular growth and oocyte developmental competence. Materials and Methods: In this experimental study, the expression of genes was evaluated with qRT-PCR assay in female BALB/c mice pups at 3-day of pre-pubertal and 8 week old virgin adult ovaries. The tissue was prepared by H&E staining for normal morphological appearance. The data were calculated with the 2-∆Ct formula and assessed using non-parametric two-tailed Mann-Whitney test. The p<0.05 was considered as significant. Results: The data showed a significant increase in the level of Stra8 and GDF9 in adult compared with newborn mice ovaries (p=0.049). In contrast, a significant decrease in the level of Mvh, REC8, SCP1, SCP3, and ZP2 was observed in adult mice ovaries compared to those in the newborn mice ovaries (all p=0.049 except SCP1: p=0.046). There was no significant difference in the level of OCT4 and Cx37 expression between adult and newborn mice ovaries. Conclusion: The modifications in gene expression patterns coordinate the follicular developmental processes. Furthermore, the findings showed higher expression level of premeiotic gene (Stra8) and lower level of meiotic entry markers (SCP1, SCP3, and REC8) in juvenile than newborn mouse ovaries. This article extracted from Ph.D. thesis. (Nehleh Zarei fard) PMID:24639702

  4. The association between romantic relationship status and 5-HT1A gene in young adults.

    PubMed

    Liu, Jinting; Gong, Pingyuan; Zhou, Xiaolin

    2014-01-01

    What factors determine whether or not a young adult will fall in love? Sociological surveys and psychological studies have shown that non-genetic factors, such as socioeconomic status, external appearance, and personality attributes, are crucial components in romantic relationship formation. Here we demonstrate that genetic variants also contribute to romantic relationship formation. As love-related behaviors are associated with serotonin levels in the brain, this study investigated to what extent a polymorphism (C-1019G, rs6295) of 5-HT1A gene is related to relationship status in 579 Chinese Han people. We found that 50.4% of individuals with the CC genotype and 39.0% with CG/GG genotype were in romantic relationship. Logistic regression analysis indicated that the C-1019G polymorphism was significantly associated with the odds of being single both before and after controlling for socioeconomic status, external appearance, religious beliefs, parenting style, and depressive symptoms. These findings provide, for the first time, direct evidence for the genetic contribution to romantic relationship formation. PMID:25412229

  5. Fat brains, greedy genes, and parent power: a biobehavioural risk model of child and adult obesity.

    PubMed

    Carnell, Susan; Kim, Yale; Pryor, Katherine

    2012-06-01

    We live in a world replete with opportunities to overeat highly calorific, palatable foods - yet not everyone becomes obese. Why? We propose that individuals show differences in appetitive traits (e.g. food cue responsiveness, satiety sensitivity) that manifest early in life and predict their eating behaviours and weight trajectories. What determines these traits? Parental feeding restriction is associated with higher child adiposity, pressure to eat with lower adiposity, and both strategies with less healthy eating behaviours, while authoritative feeding styles coincide with more positive outcomes. But, on the whole, twin and family studies argue that nature has a greater influence than nurture on adiposity and eating behaviour, and behavioural investigations of genetic variants that are robustly associated with obesity (e.g. FTO) confirm that genes influence appetite. Meanwhile, a growing body of neuroimaging studies in adults, children and high risk populations suggests that structural and functional variation in brain networks associated with reward, emotion and control might also predict appetite and obesity, and show genetic influence. Together these different strands of evidence support a biobehavioural risk model of obesity development. Parental feeding recommendations should therefore acknowledge the powerful - but modifiable - contribution of genetic and neurological influences to children's eating behaviour. PMID:22724640

  6. An N-Myristoylated Globin with a Redox-Sensing Function That Regulates the Defecation Cycle in Caenorhabditis elegans

    PubMed Central

    Tilleman, Lesley; De Henau, Sasha; Pauwels, Martje; Nagy, Nora; Pintelon, Isabel; Braeckman, Bart P.; De Wael, Karolien; Van Doorslaer, Sabine; Adriaensen, Dirk; Timmermans, Jean-Pierre; Moens, Luc; Dewilde, Sylvia

    2012-01-01

    Globins occur in all kingdoms of life where they fulfill a wide variety of functions. In the past they used to be primarily characterized as oxygen transport/storage proteins, but since the discovery of new members of the globin family like neuroglobin and cytoglobin, more diverse and complex functions have been assigned to this heterogeneous family. Here we propose a function for a membrane-bound globin of C. elegans, GLB-26. This globin was predicted to be myristoylated at its N-terminus, a post-translational modification only recently described in the globin family. In vivo, this globin is found in the membrane of the head mesodermal cell and in the tail stomato-intestinal and anal depressor muscle cells. Since GLB-26 is almost directly oxidized when exposed to oxygen, we postulate a possible function as electron transfer protein. Phenotypical studies show that GLB-26 takes part in regulating the length of the defecation cycle in C. elegans under oxidative stress conditions. PMID:23251335

  7. An association analysis of the HLA gene region in latent autoimmune diabetes in adults

    PubMed Central

    2011-01-01

    Aims/hypothesis Pathophysiological similarities between latent autoimmune diabetes in adults (LADA) and type 1 diabetes indicate an overlap in genetic susceptibility. HLA-DRB1 and HLA-DQB1 are major susceptibility genes for type 1 diabetes but studies of these genes in LADA have been limited. Our aim was to define patterns of HLA-encoded susceptibility/protection in a large, well characterised LADA cohort, and to establish association with disease and age at diagnosis. Materials and methods Patients with LADA (n=387, including 211 patients from the UK Prospective Diabetes Study) and non-diabetic control subjects (n=327) were of British/Irish European origin. The HLA-DRB1 and -DQB1 genes were genotyped by sequence-specific PCR. Results As in type 1 diabetes mellitus, DRB1*0301_DQB1*0201 (odds ratio [OR]=3.08, 95% CI 2.32–4.12, p=1.2× 10−16) and DRB1*0401_DQB1*0302 (OR=2.57, 95% CI 1.80–3.73, p=4.5×10−8) were the main susceptibility haplotypes in LADA, and DRB1*1501_DQB1*0602 was protective (OR=0.21, 95% CI 0.13–0.34, p=4.2×10−13). Differential susceptibility was conferred by DR4 subtypes: DRB1*0401 was predisposing (OR=1.79, 95% CI 1.35–2.38, p=2.7×10−5) whereas DRB1*0403 was protective (OR=0.37, 95% CI 0.13–0.97, p=0.033). The highest-risk genotypes were DRB1*0301/DRB1*0401 and DQB1*0201/DQB1*0302 (OR=5.14, 95% CI 2.68–10.69, p=1.3×10−8; and OR=6.88, 95% CI 3.54–14.68, p=1.2×10−11, respectively). These genotypes and those containing DRB1*0401 and DQB1*0302 associated with a younger age at diagnosis in LADA, whereas genotypes containing DRB1*1501 and DQB1*0602 associated with an older age at diagnosis. Conclusions/interpretation Patterns of susceptibility at the HLA-DRB1 and HLA-DQB1 loci in LADA are similar to those reported for type 1 diabetes, supporting the hypothesis that autoimmune diabetes occurring in adults is an age-related extension of the pathophysiological process presenting as childhood-onset type 1 diabetes. PMID

  8. [Correlation of adult AML Npm1 mutations with prognosis and its relationship with gene mutation of FLT3 and CEBPA].

    PubMed

    Bao, Li-Yan; Wang, Ji-Shi

    2010-02-01

    This study was aimed to investigate the correlation of 12th exon mutations in the npm1 gene with prognosis of adult AML patients and to explore the relationship of 12th exon mutation with other gene mutations. The specimen of bone marrow and peripheral blood from AML patients, the informations of medical history, symptoms, related image examinations, blood routine examination, NAP, oxygen saturation level in artery blood and EPO level in serum were collected; the bcr/abl fusion gene was detected by routine examination of bone marrow + biopsy + chromosome mapping + FISH. The patients were typed according to WHO classification. The DNA in cells was extracted, the npm1 gene mutation was detected by allele specific PCR combined were the sequencing. The results indicated that the npm1 heterozygote gene mutation was found in 72 out of 150 AML patients with normal cytogenetics (48%, 72/150). 48% patients showed a frameshift mutation in the C-terminal region of the NPM1 protein. The AML patients with npm1 gene mutation had specific clinical, phenotypic and genetic characteristics. The statistical analysis demonstrated the relationship between npm1 and flt3 ITDs. The patients with npm1 mutation showed a better response to induction therapy, furthermore, the overall survival (OS) rate of patients without flt3 ITD mutation was enhanced. The multivariate analysis demonstrated that the npm1 gene mutation and cebpa mutation positively correlated to the OS rate, and the correlation of flt3 mutation to OS rate showed negative. It is concluded that npm1 mutation is a favorable independent prognostic factor for adult AML patients with normal cytogenetics under conditions without FIT3 gene mutation. PMID:20137111

  9. Comparative Transcriptomics Reveals Key Gene Expression Differences between Diapausing and Non-Diapausing Adults of Culex pipiens

    PubMed Central

    Kang, David S.; Denlinger, David L.; Sim, Cheolho

    2016-01-01

    Diapause is a critical eco-physiological adaptation for winter survival in the West Nile Virus vector, Culex pipiens, but little is known about the molecular mechanisms that distinguish diapause from non-diapause in this important mosquito species. We used Illumina RNA-seq to simultaneously identify and quantify relative transcript levels in diapausing and non-diapausing adult females. Among 65,623,095 read pairs, we identified 41 genes with significantly different transcript abundances between these two groups. Transcriptome divergences between these two phenotypes include genes related to juvenile hormone synthesis, anaerobic metabolism, innate immunity and cold tolerance. PMID:27128578

  10. Parent-of-Origin Effects of the APOB Gene on Adiposity in Young Adults.

    PubMed

    Hochner, Hagit; Allard, Catherine; Granot-Hershkovitz, Einat; Chen, Jinbo; Sitlani, Colleen M; Sazdovska, Sandra; Lumley, Thomas; McKnight, Barbara; Rice, Kenneth; Enquobahrie, Daniel A; Meigs, James B; Kwok, Pui; Hivert, Marie-France; Borecki, Ingrid B; Gomez, Felicia; Wang, Ting; van Duijn, Cornelia; Amin, Najaf; Rotter, Jerome I; Stamatoyannopoulos, John; Meiner, Vardiella; Manor, Orly; Dupuis, Josée; Friedlander, Yechiel; Siscovick, David S

    2015-10-01

    Loci identified in genome-wide association studies (GWAS) of cardio-metabolic traits account for a small proportion of the traits' heritability. To date, most association studies have not considered parent-of-origin effects (POEs). Here we report investigation of POEs on adiposity and glycemic traits in young adults. The Jerusalem Perinatal Family Follow-Up Study (JPS), comprising 1250 young adults and their mothers was used for discovery. Focusing on 18 genes identified by previous GWAS as associated with cardio-metabolic traits, we used linear regression to examine the associations of maternally- and paternally-derived offspring minor alleles with body mass index (BMI), waist circumference (WC), fasting glucose and insulin. We replicated and meta-analyzed JPS findings in individuals of European ancestry aged ≤50 belonging to pedigrees from the Framingham Heart Study, Family Heart Study and Erasmus Rucphen Family study (total N≅4800). We considered p<2.7x10-4 statistically significant to account for multiple testing. We identified a common coding variant in the 4th exon of APOB (rs1367117) with a significant maternally-derived effect on BMI (β = 0.8; 95%CI:0.4,1.1; p = 3.1x10-5) and WC (β = 2.7; 95%CI:1.7,3.7; p = 2.1x10-7). The corresponding paternally-derived effects were non-significant (p>0.6). Suggestive maternally-derived associations of rs1367117 were observed with fasting glucose (β = 0.9; 95%CI:0.3,1.5; p = 4.0x10-3) and insulin (ln-transformed, β = 0.06; 95%CI:0.03,0.1; p = 7.4x10-4). Bioinformatic annotation for rs1367117 revealed a variety of regulatory functions in this region in liver and adipose tissues and a 50% methylation pattern in liver only, consistent with allelic-specific methylation, which may indicate tissue-specific POE. Our findings demonstrate a maternal-specific association between a common APOB variant and adiposity, an association that was not previously detected in GWAS. These results provide evidence for the role of

  11. Parent-of-Origin Effects of the APOB Gene on Adiposity in Young Adults

    PubMed Central

    Hochner, Hagit; Allard, Catherine; Granot-Hershkovitz, Einat; Chen, Jinbo; Sitlani, Colleen M.; Sazdovska, Sandra; Lumley, Thomas; McKnight, Barbara; Rice, Kenneth; Enquobahrie, Daniel A.; Meigs, James B.; Kwok, Pui; Hivert, Marie-France; Borecki, Ingrid B.; Gomez, Felicia; Wang, Ting; van Duijn, Cornelia; Amin, Najaf; Rotter, Jerome I.; Stamatoyannopoulos, John; Meiner, Vardiella; Manor, Orly; Dupuis, Josée; Friedlander, Yechiel; Siscovick, David S.

    2015-01-01

    Loci identified in genome-wide association studies (GWAS) of cardio-metabolic traits account for a small proportion of the traits' heritability. To date, most association studies have not considered parent-of-origin effects (POEs). Here we report investigation of POEs on adiposity and glycemic traits in young adults. The Jerusalem Perinatal Family Follow-Up Study (JPS), comprising 1250 young adults and their mothers was used for discovery. Focusing on 18 genes identified by previous GWAS as associated with cardio-metabolic traits, we used linear regression to examine the associations of maternally- and paternally-derived offspring minor alleles with body mass index (BMI), waist circumference (WC), fasting glucose and insulin. We replicated and meta-analyzed JPS findings in individuals of European ancestry aged ≤50 belonging to pedigrees from the Framingham Heart Study, Family Heart Study and Erasmus Rucphen Family study (total N≅4800). We considered p<2.7x10-4 statistically significant to account for multiple testing. We identified a common coding variant in the 4th exon of APOB (rs1367117) with a significant maternally-derived effect on BMI (β = 0.8; 95%CI:0.4,1.1; p = 3.1x10-5) and WC (β = 2.7; 95%CI:1.7,3.7; p = 2.1x10-7). The corresponding paternally-derived effects were non-significant (p>0.6). Suggestive maternally-derived associations of rs1367117 were observed with fasting glucose (β = 0.9; 95%CI:0.3,1.5; p = 4.0x10-3) and insulin (ln-transformed, β = 0.06; 95%CI:0.03,0.1; p = 7.4x10-4). Bioinformatic annotation for rs1367117 revealed a variety of regulatory functions in this region in liver and adipose tissues and a 50% methylation pattern in liver only, consistent with allelic-specific methylation, which may indicate tissue-specific POE. Our findings demonstrate a maternal-specific association between a common APOB variant and adiposity, an association that was not previously detected in GWAS. These results provide evidence for the role of

  12. Sensitive Tumorigenic Potential Evaluation of Adult Human Multipotent Neural Cells Immortalized by hTERT Gene Transduction

    PubMed Central

    Jeong, Da Eun; Kim, Sung Soo; Song, Hye Jin; Pyeon, Hee Jang; Kang, Kyeongjin; Hong, Seung-Cheol; Nam, Do-Hyun; Joo, Kyeung Min

    2016-01-01

    Stem cells and therapeutic genes are emerging as a new therapeutic approach to treat various neurodegenerative diseases with few effective treatment options. However, potential formation of tumors by stem cells has hampered their clinical application. Moreover, adequate preclinical platforms to precisely test tumorigenic potential of stem cells are controversial. In this study, we compared the sensitivity of various animal models for in vivo stem cell tumorigenicity testing to identify the most sensitive platform. Then, tumorigenic potential of adult human multipotent neural cells (ahMNCs) immortalized by the human telomerase reverse transcriptase (hTERT) gene was examined as a stem cell model with therapeutic genes. When human glioblastoma (GBM) cells were injected into adult (4–6-week-old) Balb/c-nu, adult NOD/SCID, adult NOG, or neonate (1–2-week-old) NOG mice, the neonate NOG mice showed significantly faster tumorigenesis than that of the other groups regardless of intracranial or subcutaneous injection route. Two kinds of ahMNCs (682TL and 779TL) were primary cultured from surgical samples of patients with temporal lobe epilepsy. Although the ahMNCs were immortalized by lentiviral hTERT gene delivery (hTERT-682TL and hTERT-779TL), they did not form any detectable masses, even in the most sensitive neonate NOG mouse platform. Moreover, the hTERT-ahMNCs had no gross chromosomal abnormalities on a karyotype analysis. Taken together, our data suggest that neonate NOG mice could be a sensitive animal platform to test tumorigenic potential of stem cell therapeutics and that ahMNCs could be a genetically stable stem cell source with little tumorigenic activity to develop regenerative treatments for neurodegenerative diseases. PMID:27391353

  13. Sensitive Tumorigenic Potential Evaluation of Adult Human Multipotent Neural Cells Immortalized by hTERT Gene Transduction.

    PubMed

    Lee, Kee Hang; Nam, Hyun; Jeong, Da Eun; Kim, Sung Soo; Song, Hye Jin; Pyeon, Hee Jang; Kang, Kyeongjin; Hong, Seung-Cheol; Nam, Do-Hyun; Joo, Kyeung Min

    2016-01-01

    Stem cells and therapeutic genes are emerging as a new therapeutic approach to treat various neurodegenerative diseases with few effective treatment options. However, potential formation of tumors by stem cells has hampered their clinical application. Moreover, adequate preclinical platforms to precisely test tumorigenic potential of stem cells are controversial. In this study, we compared the sensitivity of various animal models for in vivo stem cell tumorigenicity testing to identify the most sensitive platform. Then, tumorigenic potential of adult human multipotent neural cells (ahMNCs) immortalized by the human telomerase reverse transcriptase (hTERT) gene was examined as a stem cell model with therapeutic genes. When human glioblastoma (GBM) cells were injected into adult (4-6-week-old) Balb/c-nu, adult NOD/SCID, adult NOG, or neonate (1-2-week-old) NOG mice, the neonate NOG mice showed significantly faster tumorigenesis than that of the other groups regardless of intracranial or subcutaneous injection route. Two kinds of ahMNCs (682TL and 779TL) were primary cultured from surgical samples of patients with temporal lobe epilepsy. Although the ahMNCs were immortalized by lentiviral hTERT gene delivery (hTERT-682TL and hTERT-779TL), they did not form any detectable masses, even in the most sensitive neonate NOG mouse platform. Moreover, the hTERT-ahMNCs had no gross chromosomal abnormalities on a karyotype analysis. Taken together, our data suggest that neonate NOG mice could be a sensitive animal platform to test tumorigenic potential of stem cell therapeutics and that ahMNCs could be a genetically stable stem cell source with little tumorigenic activity to develop regenerative treatments for neurodegenerative diseases. PMID:27391353

  14. Analysis of histone gene expression in adult tissues of the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus: tissue-specific expression of sperm histone genes.

    PubMed Central

    Lieber, T; Weisser, K; Childs, G

    1986-01-01

    We analyzed the histone mRNA population found in several adult tissues of the sea urchin Strongylocentrotus purpuratus and in testis of Lytechinus pictus. Unique species of H1 and H2b mRNAs encoding the sperm-specific histone subtypes can be found exclusively in testis RNA. S. purpuratus contains two distinct testis-specific H1 transcripts, while L. pictus contains one such transcript. Each of these mRNAs is larger than either early or late embryonic H1 mRNAs. Other somatic adult tissues contain transcripts derived from members of the late embryonic H1 histone gene family. S. purpuratus contains one H2b transcript found exclusively in testis, while L. pictus contains two such H2b mRNAs. Similarly, in tissues other than testis, late H2b transcripts were found. While there is no sperm-specific H2a protein, a limited set of late histone H2a genes encoding primarily the H2a-beta subtype is expressed in testis. The majority of the H2a protein found in diploid adult tissues is also the H2a-beta subtype; however, the size of the H2a transcripts differs between testis and other tissues. We conclude that different members of the late H2a gene family are differentially expressed in embryos and adult tissues. We prepared and characterized cDNA clones encoding the sperm-specific H2b protein as well as the H2a-beta protein found in testis. Images PMID:3785204

  15. Globin mRNA reduction for whole-blood transcriptome sequencing.

    PubMed

    Krjutškov, Kaarel; Koel, Mariann; Roost, Anne Mari; Katayama, Shintaro; Einarsdottir, Elisabet; Jouhilahti, Eeva-Mari; Söderhäll, Cilla; Jaakma, Ülle; Plaas, Mario; Vesterlund, Liselotte; Lohi, Hannes; Salumets, Andres; Kere, Juha

    2016-01-01

    The transcriptome analysis of whole-blood RNA by sequencing holds promise for the identification and tracking of biomarkers; however, the high globin mRNA (gmRNA) content of erythrocytes hampers whole-blood and buffy coat analyses. We introduce a novel gmRNA locking assay (GlobinLock, GL) as a robust and simple gmRNA reduction tool to preserve RNA quality, save time and cost. GL consists of a pair of gmRNA-specific oligonucleotides in RNA initial denaturation buffer that is effective immediately after RNA denaturation and adds only ten minutes of incubation to the whole cDNA synthesis procedure when compared to non-blood RNA analysis. We show that GL is fully effective not only for human samples but also for mouse and rat, and so far incompletely studied cow, dog and zebrafish. PMID:27515369

  16. Binding of Protein Factor CTCF within Chicken Genome Alpha-Globin Locus.

    PubMed

    Kotova, E S; Akopov, S B; Didych, D A; Petrova, N V; Iarovaia, O V; Razin, S V; Nikolaev, L G

    2016-01-01

    A systematic search for DNA fragments containing potential CTCF transcription factor binding sites in the chicken alpha-globin domain and its flanking regions was performed by means of the two-dimension electrophoretic mobility shift assay. For the alpha-globin domain fragments selected, the occupancy by the CTCF in erythroid and lymphoid chicken cells was tested by chromatin immunoprecipitation. Only one of 13 DNA fragments capable of CTCF binding in vitro was efficiently bound to this protein in vivo in erythroid cells, and somewhat less efficiently - in lymphoid cells. So, binding of CTCF to the DNA fragment in vitro in most cases does not mean that this fragment will be occupied by CTCF in the cell nucleus. Yet, CTCF binding in vivo, as a rule, is accompanied by the binding of the protein to this DNA region in vitro. During the erythroid differentiation, no significant changes in CTCF binding to the DNA fragments studied were detected. PMID:27099788

  17. Binding of Protein Factor CTCF within Chicken Genome Alpha-Globin Locus

    PubMed Central

    Kotova, E. S.; Akopov, S. B.; Didych, D. A.; Petrova, N. V.; Iarovaia, O. V.; Razin, S. V.; Nikolaev, L. G.

    2016-01-01

    A systematic search for DNA fragments containing potential CTCF transcription factor binding sites in the chicken alpha-globin domain and its flanking regions was performed by means of the two-dimension electrophoretic mobility shift assay. For the alpha-globin domain fragments selected, the occupancy by the CTCF in erythroid and lymphoid chicken cells was tested by chromatin immunoprecipitation. Only one of 13 DNA fragments capable of CTCF binding in vitro was efficiently bound to this protein in vivo in erythroid cells, and somewhat less efficiently – in lymphoid cells. So, binding of CTCF to the DNA fragment in vitro in most cases does not mean that this fragment will be occupied by CTCF in the cell nucleus. Yet, CTCF binding in vivo, as a rule, is accompanied by the binding of the protein to this DNA region in vitro. During the erythroid differentiation, no significant changes in CTCF binding to the DNA fragments studied were detected. PMID:27099788

  18. Nitrite binding to globins: linkage isomerism, EPR silence and reductive chemistry

    PubMed Central

    Silaghi-Dumitrescu, Radu; Svistunenko, Dimitri A.; Cioloboc, Daniela; Bischin, Cristina; Scurtu, Florina; Cooper, Chris E.

    2014-01-01

    The nitrite adducts of globins can potentially bind via O- or N- linkage to the heme iron. We have used EPR (electron paramagnetic resonance) and DFT (density functional theory) to explore these binding modes to myoglobin and hemoglobin. We demonstrate that the nitrite adducts of both globins have detectable EPR signals; we provide an explanation for the difficulty in detecting these EPR features, based on uniaxial state considerations. The EPR and DFT data show that both nitrite linkage isomers can be present at the same time and that the two isomers are readily interconvertible in solution. The millisecond-scale process of nitrite reduction by Hb is investigated in search of the elusive Fe(II)-nitrite adduct. PMID:25172022

  19. Globin mRNA reduction for whole-blood transcriptome sequencing

    PubMed Central

    Krjutškov, Kaarel; Koel, Mariann; Roost, Anne Mari; Katayama, Shintaro; Einarsdottir, Elisabet; Jouhilahti, Eeva-Mari; Söderhäll, Cilla; Jaakma, Ülle; Plaas, Mario; Vesterlund, Liselotte; Lohi, Hannes; Salumets, Andres; Kere, Juha

    2016-01-01

    The transcriptome analysis of whole-blood RNA by sequencing holds promise for the identification and tracking of biomarkers; however, the high globin mRNA (gmRNA) content of erythrocytes hampers whole-blood and buffy coat analyses. We introduce a novel gmRNA locking assay (GlobinLock, GL) as a robust and simple gmRNA reduction tool to preserve RNA quality, save time and cost. GL consists of a pair of gmRNA-specific oligonucleotides in RNA initial denaturation buffer that is effective immediately after RNA denaturation and adds only ten minutes of incubation to the whole cDNA synthesis procedure when compared to non-blood RNA analysis. We show that GL is fully effective not only for human samples but also for mouse and rat, and so far incompletely studied cow, dog and zebrafish. PMID:27515369

  20. Phylogenetic relations of humans and African apes from DNA sequences in the Psi eta-globin region

    SciTech Connect

    Miyamoto, M.M.; Slightom, J.L.; Goodman, M.

    1987-10-16

    Sequences from the upstream and downstream flanking DNA regions of the Psi eta-globin locus in Pan troglodytes (common chimpanzee), Gorilla gorilla (gorilla), and Pongo pygmaeus (orangutan, the closest living relative to Homo, Pan, and Gorilla) provided further data for evaluating the phylogenetic relations of humans and African apes. These newly sequenced orthologs (an additional 4.9 kilobase pairs (kbp) for each species) were combined with published Psi eta-gene sequences and then compared to the same orthologous stretch (a continuous 7.1-kbp region) available for humans. Phylogenetic analysis of these nucleotide sequences by the parsimony method indicated (i) that human and chimpanzee are more closely related to each other than either is to gorilla and (ii) that the slowdown in the rate of sequence evolution evident in higher primates is especially pronounced in humans. These results indicate that features unique to African apes (but not to humans) are primitive and that even local molecular clocks should be applied with caution.

  1. Patterns of differential gene expression in adult rotation-resistant and wild-type western corn rootworm digestive tracts

    PubMed Central

    Chu, Chia-Ching; Zavala, Jorge A; Spencer, Joseph L; Curzi, Matías J; Fields, Christopher J; Drnevich, Jenny; Siegfried, Blair D; Seufferheld, Manfredo J

    2015-01-01

    The western corn rootworm (WCR,Diabrotica virgifera virgifera LeConte) is an important pest of corn. Annual crop rotation between corn and soybean disrupts the corn-dependent WCR life cycle and is widely adopted to manage this pest. This strategy selected for rotation-resistant (RR) WCR with reduced ovipositional fidelity to corn. Previous studies revealed that RR-WCR adults exhibit greater tolerance of soybean diets, different gut physiology, and host–microbe interactions compared to rotation-susceptible wild types (WT). To identify the genetic mechanisms underlying these phenotypic changes, a de novo assembly of the WCR adult gut transcriptome was constructed and used for RNA-sequencing analyses of RNA libraries from different WCR phenotypes fed with corn or soybean diets. Global gene expression profiles of WT- and RR-WCR were similar when feeding on corn diets, but different when feeding on soybean. Using network-based methods, we identified gene modules transcriptionally correlated with the RR phenotype. Gene ontology enrichment analyses indicated that the functions of these modules were related to metabolic processes, immune responses, biological adhesion, and other functions/processes that appear to correlate to documented traits in RR populations. These results suggest that gut transcriptomic divergence correlated with brief soybean feeding and other physiological traits may exist between RR- and WT-WCR adults. PMID:26240606

  2. oct4-EGFP reporter gene expression marks the stem cells in embryonic development and in adult gonads of transgenic medaka.

    PubMed

    Froschauer, Alexander; Khatun, Mst Muslima; Sprott, David; Franz, Alexander; Rieger, Christiane; Pfennig, Frank; Gutzeit, Herwig O

    2013-01-01

    Maintenance of pluripotency in stem cells is tightly regulated among vertebrates. One of the key genes in this process is oct4, also referred to as pou5f1 in mammals and pou2 in teleosts. Pou5f1 evolved by duplication of pou2 early in the tetrapod lineage, but only monotremes and marsupials retained both genes. Either pou2 or pou5f1 was lost from the genomes of the other tetrapods that have been analyzed to date. Consequently, these two homologous genes are often designated oct4 in functional studies. In most vertebrates oct4 is expressed in pluripotent cells of the early embryo until the blastula stage, and later persist in germline stem cells until adulthood. The isolation and analysis of stem cells from embryo or adult individuals is hampered by the need for reliable markers that can identify and define the cell populations. Here, we report the faithful expression of EGFP under the control of endogenous pou2/oct4 promoters in transgenic medaka (Oryzias latipes). In vivo imaging in oct4-EGFP transgenic medaka reveals the temporal and spatial expression of pou2 in embryos and adults alike. We describe the temporal and spatial patterns of endogenous pou2 and oct4-EGFP expression in medaka with respect to germline and adult stem cells, and discuss applications of oct4-EGFP transgenic medaka in reproductive and stem cell biology. PMID:23139203

  3. Neofunctionalization of embryonic head patterning genes facilitates the positioning of novel traits on the dorsal head of adult beetles.

    PubMed

    Zattara, Eduardo E; Busey, Hannah A; Linz, David M; Tomoyasu, Yoshinori; Moczek, Armin P

    2016-07-13

    The origin and integration of novel traits are fundamental processes during the developmental evolution of complex organisms. Yet how novel traits integrate into pre-existing contexts remains poorly understood. Beetle horns represent a spectacular evolutionary novelty integrated within the context of the adult dorsal head, a highly conserved trait complex present since the origin of insects. We investigated whether otd1/2 and six3, members of a highly conserved gene network that instructs the formation of the anterior end of most bilaterians, also play roles in patterning more recently evolved traits. Using ablation-based fate-mapping, comparative larval RNA interference (RNAi) and transcript sequencing, we found that otd1/2, but not six3, play a fundamental role in the post-embryonic formation of the adult dorsal head and head horns of Onthophagus beetles. By contrast, neither gene appears to pattern the adult head of Tribolium flour beetles even though all are expressed in the dorsal head epidermis of both Onthophagus and Tribolium We propose that, at least in beetles, the roles of otd genes during post-embryonic development are decoupled from their embryonic functions, and that potentially non-functional post-embryonic expression in the dorsal head facilitated their co-option into a novel horn-patterning network during Onthophagus evolution. PMID:27412276

  4. Patterns of differential gene expression in adult rotation-resistant and wild-type western corn rootworm digestive tracts.

    PubMed

    Chu, Chia-Ching; Zavala, Jorge A; Spencer, Joseph L; Curzi, Matías J; Fields, Christopher J; Drnevich, Jenny; Siegfried, Blair D; Seufferheld, Manfredo J

    2015-08-01

    The western corn rootworm (WCR,Diabrotica virgifera virgifera LeConte) is an important pest of corn. Annual crop rotation between corn and soybean disrupts the corn-dependent WCR life cycle and is widely adopted to manage this pest. This strategy selected for rotation-resistant (RR) WCR with reduced ovipositional fidelity to corn. Previous studies revealed that RR-WCR adults exhibit greater tolerance of soybean diets, different gut physiology, and host-microbe interactions compared to rotation-susceptible wild types (WT). To identify the genetic mechanisms underlying these phenotypic changes, a de novo assembly of the WCR adult gut transcriptome was constructed and used for RNA-sequencing analyses of RNA libraries from different WCR phenotypes fed with corn or soybean diets. Global gene expression profiles of WT- and RR-WCR were similar when feeding on corn diets, but different when feeding on soybean. Using network-based methods, we identified gene modules transcriptionally correlated with the RR phenotype. Gene ontology enrichment analyses indicated that the functions of these modules were related to metabolic processes, immune responses, biological adhesion, and other functions/processes that appear to correlate to documented traits in RR populations. These results suggest that gut transcriptomic divergence correlated with brief soybean feeding and other physiological traits may exist between RR- and WT-WCR adults. PMID:26240606

  5. CONVECTION-ENHANCED DELIVERY AND SYSTEMIC MANNITOL INCREASE GENE PRODUCT DISTRIBUTION OF AAV VECTORS 5, 8, AND 9 AND INCREASE GENE PRODUCT IN THE ADULT MOUSE BRAIN

    PubMed Central

    Carty, Nikisha; Lee, Daniel; Dickey, Chad; Ceballos-Diaz, Carolina; Jansen-West, Karen; Golde, Todd E.; Gordon, Marcia N.; Morgan, Dave; Nash, Kevin

    2010-01-01

    The use of recombinant adeno-associated viral (rAAV) vectors as a means of gene delivery to the central nervous system has emerged as a potentially viable method for the treatment of several types of degenerative brain diseases. However, a limitation of typical intracranial injections into the adult brain parenchyma is the relatively restricted distribution of the delivered gene to large brain regions such as the cortex, presumably due to confined dispersion of the injected particles. Optimizing the administration techniques to maximize gene distribution and gene expression is an important step in developing gene therapy studies. Here, we have found additive increases in distribution when 3 methods to increase brain distribution of rAAV were combined. The convection enhanced delivery (CED) method with the step-design cannula was used to deliver rAAV vector serotypes 5, 8 and 9 encoding GFP into the hippocampus of the mouse brain. While the CED method improved distribution of all 3 serotypes, the combination of rAAV9 and CED was particularly effective. Systemic mannitol administration, which reduces intracranial pressure, also further expanded distribution of GFP expression, in particular, increased expression on the contralateral hippocampi. These data suggest that combining advanced injection techniques with newer rAAV serotypes greatly improves viral vector distribution, which could have significant benefits for implementation of gene therapy strategies. PMID:20951738

  6. Enlarged striatal volume in adults with ADHD carrying the 9-6 haplotype of the dopamine transporter gene DAT1.

    PubMed

    Onnink, A Marten H; Franke, Barbara; van Hulzen, Kimm; Zwiers, Marcel P; Mostert, Jeanette C; Schene, Aart H; Heslenfeld, Dirk J; Oosterlaan, Jaap; Hoekstra, Pieter J; Hartman, Catharina A; Vasquez, Alejandro Arias; Kan, Cornelis C; Buitelaar, Jan; Hoogman, Martine

    2016-08-01

    The dopamine transporter gene, DAT1 (SLC6A3), has been studied extensively as a candidate gene for attention-deficit/hyperactivity disorder (ADHD). Different alleles of variable number of tandem repeats (VNTRs) in this gene have been associated with childhood ADHD (10/10 genotype and haplotype 10-6) and adult ADHD (haplotype 9-6). This suggests a differential association depending on age, and a role of DAT1 in modulating the ADHD phenotype over the lifespan. The DAT1 gene may mediate susceptibility to ADHD through effects on striatal volumes, where it is most highly expressed. In an attempt to clarify its mode of action, we examined the effect of three DAT1 alleles (10/10 genotype, and the haplotypes 10-6 and 9-6) on bilateral striatal volumes (nucleus accumbens, caudate nucleus, and putamen) derived from structural magnetic resonance imaging scans using automated tissue segmentation. Analyses were performed separately in three cohorts with cross-sectional MRI data, a childhood/adolescent sample (NeuroIMAGE, 301 patients with ADHD and 186 healthy participants) and two adult samples (IMpACT, 118 patients with ADHD and 111 healthy participants; BIG, 1718 healthy participants). Regression analyses revealed that in the IMpACT cohort, and not in the other cohorts, carriers of the DAT1 adult ADHD risk haplotype 9-6 had 5.9 % larger striatum volume relative to participants not carrying this haplotype. This effect varied by diagnostic status, with the risk haplotype affecting striatal volumes only in patients with ADHD. An explorative analysis in the cohorts combined (N = 2434) showed a significant gene-by-diagnosis-by-age interaction suggesting that carriership of the 9-6 haplotype predisposes to a slower age-related decay of striatal volume specific to the patient group. This study emphasizes the need of a lifespan approach in genetic studies of ADHD. PMID:26935821

  7. DNA repair gene polymorphisms and risk of adult meningioma, glioma, and acoustic neuroma.

    PubMed

    Rajaraman, Preetha; Hutchinson, Amy; Wichner, Sara; Black, Peter M; Fine, Howard A; Loeffler, Jay S; Selker, Robert G; Shapiro, William R; Rothman, Nathaniel; Linet, Martha S; Inskip, Peter D

    2010-01-01

    Although the etiology of primary brain tumors is largely unknown, prior studies suggest that DNA repair polymorphisms may influence risk of glioma. Altered DNA repair is also likely to affect the risk of meningioma and acoustic neuroma, but these tumors have not been well studied. We estimated the risk of glioma (n = 362), meningioma (n = 134), and acoustic neuroma (n = 69) in non-Hispanic whites with respect to 36 single nucleotide polymorphisms from 26 genes involved in DNA repair in a hospital-based, case-control study conducted by the National Cancer Institute. We observed significantly increased risk of meningioma with the T variant of GLTSCR1 rs1035938 (OR(CT/TT) = 3.5; 95% confidence interval: 1.8-6.9; P(trend) .0006), which persisted after controlling for multiple comparisons (P = .019). Significantly increased meningioma risk was also observed for the minor allele variants of ERCC4 rs1800067 (P(trend) .01); MUTYH rs3219466 (P(trend) .02), and PCNA rs25406 (P(trend) .03). The NBN rs1805794 minor allele variant was associated with decreased meningioma risk (P(trend) .006). Risk of acoustic neuroma was increased for the ERCC2 rs1799793 (P(trend) .03) and ERCC5 rs17655 (P(trend) .05) variants and decreased for the PARP1 rs1136410 (P(trend) .03). Decreased glioma risk was observed with the XRCC1 rs1799782 variant (P(trend) .04). Our results suggest that common DNA repair variants may affect the risk of adult brain tumors, especially meningioma. PMID:20150366

  8. Association of autophagy related gene polymorphisms with neutrophilic airway inflammation in adult asthma

    PubMed Central

    Pham, Duy Le; Kim, Seung-Hyun; Losol, Purevsuren; Yang, Eun-Mi; Shin, Yoo Seob; Ye, Young-Min; Park, Hae-Sim

    2016-01-01

    Background/Aims: Role of autophagy in neutrophil function and the association of autophagy and autophagy related (ATG) gene polymorphisms with asthma susceptibility were suggested. In this study, we investigated the genetic association of ATG5 and ATG7 polymorphisms with asthma risk, severity and neutrophilic airway inflammation. Methods: We recruited 408 asthma patients and 201 healthy controls. Sputum neutrophil counts were determined by H&E staining. Serum interleukin 8 (IL-8) levels were measured by enzyme-linked immunosorbent assay (ELISA). Genetic polymorphisms of ATG5 (–769T>C, –335G>A, and 8830C>T) and ATG7 (–100A>G and 25108G>C) were genotyped. The functional activities of ATG5 –769T>C and –335G>A variants were investigated by luciferase reporter assays. Results: No associations of ATG5 and ATG7 polymorphisms with asthma susceptibility and severity were found. ATG5 –769T>C and –335G>A were in complete linkage disequilibrium. In the asthma group, GA/AA genotypes at ATG5 –335G>A were associated with higher neutrophil counts in sputum (p < 0.05); CC/TT genotype at ATG5 8830C>T associated with lower FEV1% predicted value (p < 0.05). DNA fragments containing ATG5 –769T and –335G alleles had higher promoter activities compared to those with –769C and –335A in both human airway epithelial cells (A549, p < 0.01) and human mast cell (HMC-1, p < 0.001). GG and CC genotype at ATG7 –100A>G and 25108G>C were significantly associated with high serum levels of IL-8 (p < 0.05 for both variants). Conclusions: Genetic polymorphisms of ATG5 and ATG7 could contribute to neutrophilic airway inflammation in the pathogenesis of adult asthma. PMID:26701229

  9. Quantification of tertiary structural conservation despite primary sequence drift in the globin fold.

    PubMed

    Aronson, H E; Royer, W E; Hendrickson, W A

    1994-10-01

    The globin family of protein structures was the first for which it was recognized that tertiary structure can be highly conserved even when primary sequences have diverged to a virtually undetectable level of similarity. This principle of structural inertia in molecular evolution is now evident for many other protein families. We have performed a systematic comparison of the sequences and structures of 6 representative hemoglobin subunits as diverse in origin as plants, clams, and humans. Our analysis is based on a 97-residue helical core in common to all 6 structures. Amino acid sequence identities range from 12.4% to 42.3% in pairwise comparisons, and, despite these variations, the maximal RMS deviation in alpha-carbon positions is 3.02 A. Overall, sequence similarity and structural deviation are significantly anticorrelated, with a correlation coefficient of -0.71, but for a set of structures having under 20% pairwise identity, this anticorrelation falls to -0.38, which emphasizes the weak connection between a specific sequence and the tertiary fold. There is substantial variability in structure outside the helical core, and functional characteristics of these globins also differ appreciably. Nevertheless, despite variations in detail that the sequence dissimilarities and functional differences imply, the core structures of these globins remain remarkably preserved. PMID:7849587

  10. Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

    PubMed

    Fantoni, A; Bozzoni, I; Ullu, E; Farace, M G

    1979-08-10

    Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z. PMID:493112

  11. Comparison of ligand migration and binding in heme proteins of the globin family

    NASA Astrophysics Data System (ADS)

    Karin, Nienhaus; Ulrich Nienhaus, G.

    2015-12-01

    The binding of small diatomic ligands such as carbon monoxide or dioxygen to heme proteins is among the simplest biological processes known. Still, it has taken many decades to understand the mechanistic aspects of this process in full detail. Here, we compare ligand binding in three heme proteins of the globin family, myoglobin, a dimeric hemoglobin, and neuroglobin. The combination of structural, spectroscopic, and kinetic experiments over many years by many laboratories has revealed common properties of globins and a clear mechanistic picture of ligand binding at the molecular level. In addition to the ligand binding site at the heme iron, a primary ligand docking site exists that ensures efficient ligand binding to and release from the heme iron. Additional, secondary docking sites can greatly facilitate ligand escape after its dissociation from the heme. Although there is only indirect evidence at present, a preformed histidine gate appears to exist that allows ligand entry to and exit from the active site. The importance of these features can be assessed by studies involving modified proteins (via site-directed mutagenesis) and comparison with heme proteins not belonging to the globin family.

  12. Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

    PubMed Central

    Fantoni, A; Bozzoni, I; Ullu, E; Farace, M G

    1979-01-01

    Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z. Images PMID:493112

  13. Modulation of retinoblastoma gene in normal adult hematopoiesis: peak expression and functional role in advanced erythroid differentiation.

    PubMed Central

    Condorelli, G L; Testa, U; Valtieri, M; Vitelli, L; De Luca, A; Barberi, T; Montesoro, E; Campisi, S; Giordano, A; Peschle, C

    1995-01-01

    The retinoblastoma (RB) gene specifies a nuclear phosphoprotein (pRb 105), which is a prototype tumor suppressor inactivated in a variety of human tumors. Recent studies suggest that RB is also involved in embryonic development of murine central nervous and hematopoietic systems. We have investigated RB expression and function in human adult hematopoiesis--i.e., in liquid suspension culture of purified quiescent hematopoietic progenitor cells (HPCs) induced by growth factor stimulus to proliferation and unilinage differentiation/maturation through the erythroid or granulocytic lineage. In the initial HPC differentiation stages, the RB gene is gradually induced at the mRNA and protein level in both erythroid and granulopoietic cultures. In late HPC differentiation and then precursor maturation, RB gene expression is sustained in the erythroid lineage, whereas it is sharply downmodulated in the granulocytic series. Functional studies were performed by treatment of HPC differentiation culture with phosphorothioate antisense oligomer targeting Rb mRNA; coherent with the expression pattern, oligomer treatment of late HPCs causes a dose-dependent and selective inhibition of erythroid colony formation. These observations suggest that the RB gene plays an erythroid- and stage-specific functional role in normal adult hematopoiesis, particularly at the level of late erythroid HPCs. Images Fig. 2 Fig. 3 Fig. 4 PMID:7761404

  14. The promoter polymorphism of the interleukin-6 gene regulates interleukin-6 production in neonates but not in adults.

    PubMed

    Kilpinen, S; Hulkkonen, J; Wang, X Y; Hurme, M

    2001-03-01

    In the promoter region of the IL-6 gene there is a single base exchange (G --> C) polymorphism at position -174. Recent findings suggest that this polymorphism may affect the transcription rate of the IL-6 gene and IL-6 plasma levels. To analyse its biological significance, we examined IL-6 plasma levels in cord blood and IL-6 production by neonatal cells after LPS-stimulation in relation to the presence of the IL-6G and IL-6C alleles. We hypothesized that since healthy neonates lack a previous exposure to exogenous antigens, their cytokine production could be genetically regulated. We also assumed that the normal labour-related stress could provide a physiological stimulus for IL-6 production. Cord blood was collected from 50 healthy, full-term neonates after normal vaginal delivery (VD) and from 42 healthy, full-term neonates after elective caesarean section (ECS). Adult samples were obtained from 450 healthy adult controls. The -174 polymorphism was analysed using PCR. IL-6 plasma levels and in vitro IL-6 production were measured using an ELISA method. Generally, IL-6 plasma levels in neonates were significantly higher than those in adults (neonates born by VD versus adults p < 0.001 and neonates born by ECS versus adults p < 0.001); the median value for neonates born by VD was 11.4 pg/ml (4.5-45.9), for neonates born by ECS 2.9 pg/ml (1.9-6.4) and for adults, 1.2 pg/ml (0.7-2.0). Surprisingly, cord blood IL-6 levels after VD differed significantly from those after ECS (p < 0.001). An analysis was carried out to ascertain if there was a genetic association between different IL-6 genotypes and IL-6 plasma levels in neonates. In the group of VD neonates with the CC genotype, non-carriers of the G allele, secreted significantly more IL-6 than carriers of the G allele (p < 0.03); 21.4 pg/ml (9.5-81.3) and 9.6 pg/ml (3.5-36.2) respectively. In line with this, ECS newborns with the CC genotype had higher IL-6 plasma levels than carriers of the G allele (p < 0

  15. Deletion and deletion/insertion mutations in the juxtamembrane domain of the FLT3 gene in adult acute myeloid leukemia

    PubMed Central

    Deeb, Kristin K.; Smonskey, Matthew T.; DeFedericis, HanChun; Deeb, George; Sait, Sheila N.J.; Wetzler, Meir; Wang, Eunice S.; Starostik, Petr

    2014-01-01

    In contrast to FLT3 ITD mutations, in-frame deletions in the FLT3 gene have rarely been described in adult acute leukemia. We report two cases of AML with uncommon in-frame mutations in the juxtamembrane domain of the FLT3 gene: a 3-bp (c.1770_1774delCTACGinsGT; p.F590_V592delinsLF) deletion/insertion and a 12-bp (c.1780_1791delTTCAGAGAATAT; p.F594_Y597del) deletion. We verified by sequencing that the reading frame of the FLT3 gene was preserved and by cDNA analysis that the mRNA of the mutant allele was expressed in both cases. Given the recent development of FLT3 inhibitors, our findings may be of therapeutic value for AML patients harboring similar FLT3 mutations. PMID:25379410

  16. Gene polymorphisms and gene scores linked to low serum carotenoid status and their associations with metabolic disturbance and depressive symptoms in African-American adults

    PubMed Central

    Beydoun, May A.; Nalls, Michael A.; Canas, J. Atilio; Evans, Michele K.; Zonderman, Alan B.

    2016-01-01

    Gene polymorphisms provide means to obtain unconfounded associations between carotenoids and various health outcomes. We tested whether gene polymophorisms and gene scores linked to serum carotenoid status are related to metabolic disturbance and depressive symptoms in African-American adults residing in Baltimore city, MD, using cross-sectional data from the Healthy Aging in Neighborhood of Diversity Across the Lifespan (HANDLS) study (Age range:30–64y, N=873–994). We examined 24 single nucleotide polymorphisms of various gene loci that were previously shown to be associated with low serum carotenoid status (SNPlcar). Genetic risk scores (5 low specific-carotenoid risk scores (LSCRS: α-carotene, β-carotene, lutein+zeaxanthin, β-cryptoxanthin, lycopene) and 1 low total-carotenoid risk score (LTCRS: total carotenoids)) were created. SNPlcar, LSCRS and LTCRS were entered as predictors for a number of health outcomes. Those included obesity, National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP) III metabolic syndrome (MetS) and its components, elevated homeostatic model assessment, Insulin Resistance (HOMA-IR), C-reactive protein (CRP), hyperuricemia and elevated depressive symptoms (EDS, Center for Epidemiologic Studies-Depression (CES-D) score≥16). Among key findings, SNPlcar were not associated with the main outcomes after correction for multiple testing. However, an inverse association was found between LTCRS and HDL-C dyslipidemia. Specifically, the α-carotene and β-cryptoxanthin LSCRS were associated with lower odds of HDL-C dyslipidemia. However, the β-cryptoxanthin LSCRS was linked to a higher odds of EDS, with a linear dose-response relationship. In sum, gene risk scores linked to low serum carotenoids had mixed effects on HDL-C dyslipidemia and EDS. Further studies using larger African-American samples are needed. PMID:25201307

  17. Non-covalent and covalent modifications modulate the reactivity of monomeric mammalian globins.

    PubMed

    Ascenzi, Paolo; Marino, Maria; Polticelli, Fabio; Coletta, Massimo; Gioia, Magda; Marini, Stefano; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; Reeder, Brandon J; Wilson, Michael T

    2013-09-01

    Multimeric globins (e.g., hemoglobin) are considered to be the prototypes of allosteric enzymes, whereas monomeric globins (e.g., myoglobin; Mb) usually are assumed to be non-allosteric. However, the modulation of the functional properties of monomeric globins by non-covalent (or allosteric) and covalent modifications casts doubts on this general assumption. Here, we report examples referable to these two extreme mechanisms modulating the reactivity of three mammalian monomeric globins. Sperm whale Mb, which acts as a reserve supply of O2 and facilitates the O2 flux within a myocyte, displays the allosteric modulation of the O2 affinity on lactate, an obligatory product of glycolysis under anaerobic conditions, thus facilitating O2 diffusion to the mitochondria in supporting oxidative phosphorylation. Human neuroglobin (NGB), which appears to protect neurons from hypoxia in vitro and in vivo, undergoes hypoxia-dependent phosphorylation (i.e., covalent modulation) affecting the coordination equilibrium of the heme-Fe atom and, in turn, the heme-protein reactivity. This facilitates heme-Fe-ligand binding and enhances the rate of anaerobic nitrite reduction to form NO, thus contributing to cellular adaptation to hypoxia. The reactivity of human cytoglobin (CYGB), which has been postulated to protect cells against oxidative stress, depends on both non-covalent and covalent mechanisms. In fact, the heme reactivity of CYGB depends on the lipid, such as oleate, binding which stabilizes the penta-coordination geometry of the heme-Fe atom. Lastly, the reactivity of NGB and CYGB is modulated by the redox state of the intramolecular CysCD7/CysD5 and CysB2/CysE9 residue pairs, respectively, affecting the heme-Fe atom coordination state. In conclusion, the modulation of monomeric globins reactivity by non-covalent and covalent modifications appears a very widespread phenomenon, opening new perspectives in cell survival and protection. This article is part of a Special Issue

  18. Lack of Association between a 3'UTR VNTR Polymorphism of Dopamine Transporter Gene (SLC6A3) and ADHD in a Brazilian Sample of Adult Patients

    ERIC Educational Resources Information Center

    Aperecida da Silva, Maria; Cordeiro, Quirino; Louza, Mario; Vallada, Homero

    2011-01-01

    Objective: To investigate a possible association between a 3'UTR VNTR polymorphism of the dopamine transporter gene (SLC6A3) and ADHD in a Brazilian sample of adult patients. Method: Study Case-control with 102 ADHD adult outpatients ("DSM-IV" criteria) and 479 healthy controls. The primers' sequence used were: 3'UTR-Forward: 5' TGT GGT GAT GGG…

  19. The Drosophila Hand gene is required for remodeling of the developing adult heart and midgut during metamorphosis

    PubMed Central

    Lo, Patrick C.H.; Zaffran, Stéphane; Sénatore, Sébastien; Frasch, Manfred

    2007-01-01

    The Hand proteins of the bHLH family of transcriptional factors play critical roles in vertebrate cardiogenesis. In Drosophila, the single orthologous Hand gene is expressed in the developing embryonic dorsal vessel (heart), lymph glands, circular visceral musculature, and a subset of CNS cells. We demonstrate that the absence of Hand activity causes semilethality during the early larval instars. The dorsal vessel and midgut musculature are unaffected in null mutant embryos, but in a large fraction the lymph glands are missing. However, homozygous adult flies lacking Hand possess morphologically abnormal dorsal vessels characterized by a disorganized myofibrillar structure, reduced systolic and diastolic diameter, abnormal heartbeat contractions, and suffer from premature lethality. In addition, their midguts are highly deformed; in the most severe cases, there is midgut blockage and a massive excess of ectopic peritrophic membrane tubules exiting a rupture in an anterior midgut bulge. Nevertheless, the visceral musculature appears to be relatively normal. Based on these phenotypes, we conclude that the expression of the Drosophila Hand gene in the dorsal vessel and circular visceral muscles is mainly required during pupal stages, when Hand participates in the proper hormone-dependent remodeling of the larval aorta into the adult heart and in the normal morphogenesis of the adult midgut endoderm during metamorphosis. PMID:17904115

  20. Interactions between beta-2 adrenoceptor gene variation, cardiovascular control and dietary sodium in healthy young adults

    PubMed Central

    Eisenach, John H; Schroeder, Darrell R; Pavey, Emily S; Penheiter, Alan R; Knutson, Jean N; Turner, Stephen T; Joyner, Michael J

    2014-01-01

    Dietary sodium affects function of the beta-2 adrenoceptor (ADRB2). We tested the hypothesis that haplotype variation in the ADRB2 gene would influence the cardiovascular and regional vasodilator responses to sympathoexcitatory manoeuvres following low, normal and high sodium diets, and ADRB2-mediated forearm vasodilation in the high sodium condition. Seventy-one healthy young adults were grouped by double homozygous haplotypes: Arg16+Gln27 (n = 31), the rare Gly16+Gln27 (n = 10) and Gly16+Glu27 (n = 30). Using a randomized cross-over design, subjects were studied following 5 days of controlled low, normal and high sodium with 1 month or longer between diets (and low hormone phase of the menstrual cycle). All three visits utilized ECG and finger plethysmography for haemodynamic measures, and the high sodium visit included a brachial arterial catheter for forearm vasodilator responses to isoprenaline with plethysmography. Lymphocytes were sampled for ex vivo analysis of ADRB2 density and binding conformation. We found a main effect of haplotype on ADRB2 density (P = 0.03) with the Gly16+Glu27 haplotype having the greatest density (low, normal, high sodium: 12.9 ± 0.9, 13.5 ± 0.9 and 13.6 ± 0.8 fmol mg−1 protein, respectively) and Arg16+Gln27 having the least (9.3 ± 0.6, 10.1 ± 0.5 and 10.3 ± 0.6  fmol mg−1 protein, respectively), but there were no sodium or haplotype effects on receptor binding conformation. In the mental stress trial, there was a main effect of haplotype on cardiac output (P = 0.04), as Arg16+Gln27 had the lowest responses. Handgrip and forearm vasodilation yielded no haplotype differences, and no correlations were present for ADRB2 density and haemodynamics. Our findings support cell-based evidence that ADRB2 haplotype influences ADRB2 protein expression independent of dietary sodium, yet the haemodynamic consequences appear modest in healthy humans. PMID:25260632

  1. Expression and Regulation of the Fkbp5 Gene in the Adult Mouse Brain

    PubMed Central

    Scharf, Sebastian H.; Liebl, Claudia; Binder, Elisabeth B.

    2011-01-01

    Background Chronic stress has been found to be a major risk factor for various human pathologies. Stress activates the hypothalamic-pituitary-adrenal (HPA) axis, which is tightly regulated via, among others, the glucocorticoid receptor (GR). The activity of the GR is modulated by a variety of proteins, including the co-chaperone FK506 binding protein 51 (FKBP5). Although FKBP5 has been associated with risk for affective disorders and has been implicated in GR sensitivity, previous studies focused mainly on peripheral blood, while information about basal distribution and induction in the central nervous system are sparse. Methodology/Principal Findings In the present study, we describe the basal expression pattern of Fkbp5 mRNA in the brain of adult male mice and show the induction of Fkbp5 mRNA via dexamethasone treatment or different stress paradigms. We could show that Fkbp5 is often, but not exclusively, expressed in regions also known for GR expression, for example the hippocampus. Furthermore, we were able to induce Fkbp5 expression via dexamethasone in the CA1 and DG subregions of the hippocampus, the paraventricular nucleus (PVN) and the central amygdala (CeA). Increase of Fkbp5 mRNA was also found after restrained stress and 24 hours of food deprivation in the PVN and the CeA, while in the hippocampus only food deprivation caused an increase in Fkbp5 mRNA. Conclusions/Significance Interestingly, regions with a low basal expression showed higher increase in Fkbp5 mRNA following induction than regions with high basal expression, supporting the hypothesis that GR sensitivity is, at least partly, mediated via Fkbp5. In addition, this also supports the use of Fkbp5 gene expression as a marker for GR sensitivity. In summary, we were able to give an overview of the basal expression of fkbp5 mRNA as well as to extend the findings of induction of Fkbp5 and its regulatory influence on GR sensitivity from peripheral blood to the brain. PMID:21347384

  2. Circadian regulation gene polymorphisms are associated with sleep disruption and duration, and circadian phase and rhythm in adults with HIV.

    PubMed

    Lee, Kathryn A; Gay, Caryl; Byun, Eeeseung; Lerdal, Anners; Pullinger, Clive R; Aouizerat, Bradley E

    2015-01-01

    Genes involved in circadian regulation, such as circadian locomotor output cycles kaput [CLOCK], cryptochrome [CRY1] and period [PER], have been associated with sleep outcomes in prior animal and human research. However, it is unclear whether polymorphisms in these genes are associated with the sleep disturbances commonly experienced by adults living with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). Thus, the purpose of this study was to describe polymorphisms in selected circadian genes that are associated with sleep duration or disruption as well as the sleep-wake rhythm strength and phase timing among adults living with HIV/AIDS. A convenience sample of 289 adults with HIV/AIDS was recruited from HIV clinics and community sites in the San Francisco Bay Area. A wrist actigraph was worn for 72 h on weekdays to estimate sleep duration or total sleep time (TST), sleep disruption or percentage of wake after sleep onset (WASO) and several circadian rhythm parameters: mesor, amplitude, the ratio of mesor to amplitude (circadian quotient), and 24-h autocorrelation. Circadian phase measures included clock time for peak activity (acrophase) from actigraphy movement data, and bed time and final wake time from actigraphy and self-report. Genotyping was conducted for polymorphisms in five candidate genes involved in circadian regulation: CLOCK, CRY1, PER1, PER2 and PER3. Demographic and clinical variables were evaluated as potential covariates. Interactions between genotype and HIV variables (i.e. viral load, years since HIV diagnosis) were also evaluated. Controlling for potentially confounding variables (e.g. race, gender, CD4+ T-cell count, waist circumference, medication use, smoking and depressive symptoms), CLOCK was associated with WASO, 24-h autocorrelation and objectively-measured bed time; CRY1 was associated with circadian quotient; PER1 was associated with mesor and self-reported habitual wake time; PER2 was associated with TST

  3. Glucocorticoids inhibit coordinated translation of. cap alpha. - and. beta. -globin mRNAs in Friend erythroleukemia cells

    SciTech Connect

    Papaconstantinou, J.; Stewart, J.A.; Rabek, J.P.; McClintock, P.R.; Wong, E.Y.

    1983-12-01

    The dimethylsulfoxide (Me/sub 2/SO)-mediated induction of hemoglobin synthesis in Friend erythroleukemia cells is inhibited by the glucocorticoids hydrocortisone, dexamethasone, and fluocinolone acetonide; hydrocortisone, at concentrations of 10/sup -5/ to 10/sup -8/ M inhibits by 90-30% and fluocinolone acetonide at concentrations of 10/sup -8/ to 10/sup -11/ M shows a greater than 90% inhibition. At these concentrations the hormones have no effect on cell growth or