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Sample records for embryonic globin genes

  1. Characterization, expression, and evolution of the mouse embryonic zeta-globin gene.

    PubMed Central

    Leder, A; Weir, L; Leder, P

    1985-01-01

    We have determined the complete sequence of the embryonic alpha-like, zeta (zeta)-globin gene of the BALB/c mouse. The structure of this gene establishes the amino acid sequence of the mouse embryonic zeta-globin polypeptide chain and allows us to identify sequences within the gene that may be important for its expression. One of these is a 300-base segment that is tightly conserved between mice and humans and is located at the 5' end of the zeta-globin gene. By introducing the cloned gene into permanently transfected mouse erythroleukemic cell lines and comparing its transcript with that of zeta-globin mRNA derived from embryonic yolk sac erythrocytes, we are able to show that the cloned gene is transcriptionally active and that its transcript is correctly initiated and processed. Interestingly, the zeta-globin gene is also active when permanently transfected into an immunoglobulin-producing B-cell, a cell that presumably has tissue-specific requirements for gene expression. Further, a comparison of the amino acid coding sequence of the mouse zeta-globin gene to that of zeta-like globin genes of other species supports a revised evolutionary lineage in which goats and humans are closely related, whereas mice are further removed. Images PMID:4000117

  2. FKLF, a Novel Krüppel-Like Factor That Activates Human Embryonic and Fetal β-Like Globin Genes

    PubMed Central

    Asano, Haruhiko; Li, Xi Susan; Stamatoyannopoulos, George

    1999-01-01

    A cDNA encoding a novel Krüppel-type zinc finger protein, FKLF, was cloned from fetal globin-expressing human fetal erythroid cells. The deduced polypeptide sequence composed of 512 amino acids revealed that, like Sp1 and EKLF, FKLF has three contiguous zinc fingers at the near carboxyl-terminal end. A long amino-terminal domain is characterized by the presence of two acidic and two proline-rich regions. Reverse transcription (RT)-PCR assays using various cell lines demonstrated that the FKLF mRNA is expressed predominantly in erythroid cells. FKLF message is detectable by RT-PCR in fetal liver but not in adult bone marrow cells. As predicted from its structural features, FKLF is a transcriptional activator. In luciferase assays FKLF activated the γ- and ɛ-globin gene promoters, and, to a much lower degree, the β-globin promoter. Studies of HS2-γ gene reporter constructs carrying CACCC box deletions revealed that the CACCC box sequence of the γ gene promoter mediates the activation of the γ gene by FKLF. Other erythroid promoters (GATA-1, glycophorin B, ferrochelatase, porphobilinogen deaminase, and 5-aminolevulinate synthase) containing CACCC elements or GC-rich potential Sp1-binding sites were activated minimally, if at all, by FKLF, indicating that FKLF is not a general activator of genes carrying the CACCC motifs. Transfection of K562 cells with FKLF cDNA enhanced the expression of the endogenous ɛ- and γ-globin genes, suggesting an in vivo role of FKLF in fetal and embryonic globin gene expression. Our results indicate that the protein potentially encoded by the FKLF cDNA acts as a transcriptional activator of embryonic and fetal β-like globin genes. PMID:10207080

  3. Proximal promoter elements of the human zeta-globin gene confer embryonic-specific expression on a linked reporter gene in transgenic mice.

    PubMed

    Pondel, M D; Sharpe, J A; Clark, S; Pearson, L; Wood, W G; Proudfoot, N J

    1996-11-01

    We have investigated the transcriptional regulation of the human embryonic zeta-globin gene promoter. First, we examined the effect that deletion of sequences 5' to zeta-globin's CCAAT box have on zeta-promoter activity in erythroid cell lines. Deletions of sequences between -116 and -556 (cap = 0) had little effect while further deletion to -84 reduced zeta-promoter activity by only 2-3-fold in both transiently and stably transfected erythroid cells. Constructs containing 67, 84 and 556 bp of zeta-globin 5' flanking region linked to a beta-galactosidase reporter gene (lacZ) and hypersensitive site -40 (HS-40) of the human alpha-globin gene cluster were then employed for the generation of transgenic mice. LacZ expression from all constructs, including a 67 bp zeta-globin promoter, was erythroid-specific and most active between 8.5 and 10.5 days post-fertilisation. By 16.5 days gestation, lacZ expression dropped 40-100-fold. These results suggest that embryonic-specific activation of the human zeta-globin promoter is conferred by a 67 bp zeta-promoter fragment containing only a CCAAT and TATA box. PMID:8932366

  4. 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

  5. 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

  6. 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

  7. Histone acetylation and globin gene switching.

    PubMed Central

    Hebbes, T R; Thorne, A W; Clayton, A L; Crane-Robinson, C

    1992-01-01

    An affinity-purified antibody that recognises the epitope epsilon-acetyl lysine has been used to fractionate chicken erythrocyte mononucleosomes obtained from 5 and 15 day embryos. The antibody bound chromatin was enriched in multiply acetylated forms of the core histones H3, H4 and H2B, but not in ubiquitinated H2A. The DNA of these modified nucleosomes was probed with genomic sequences from the embryonic beta rho gene (active at 5 days) and from the adult beta A gene (active at 15 days). Both genes were found to be highly enriched in the acetylated nucleosomes fractionated from both 5 day and from 15 day erythrocytes. We conclude that globin switching is not linked to a change in acetylation status of the genes and that a 'poised' gene carries histones acetylated to a similar level as a transcriptionally active gene. Core histone acetylation is not therefore a direct consequence of the transcriptional process and might operate at the level of the globin locus as a general enabling step for transcription. Images PMID:1549462

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

    PubMed

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

    2008-02-01

    Phylogenetic reconstructions of the beta-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 beta-like globin genes occurred independently in birds and mammals. In both taxa, the embryonic beta-globin gene is exclusively expressed in primitive erythroid cells derived from the yolk sac. However, the "epsilon-globin" gene in birds is not orthologous to the epsilon-globin gene in mammals, because they are independently derived from lineage-specific duplications of a proto beta-globin gene. Here, we report evidence that the early and late expressed beta-like globin genes in monotremes and therian mammals (marsupials and placental mammals) are the products of independent duplications of a proto beta-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 epsilon- and beta-globin genes of therian mammals arose via duplication of a proto beta-globin gene after the therian/monotreme split. Our analysis of genomic sequence from the platypus also revealed the presence of a duplicate pair of beta-like globin genes that originated via duplication of a proto beta-globin gene in the monotreme lineage. This discovery provides evidence that, in different lineages of mammals, descendent copies of the same proto beta-globin gene may have been independently neofunctionalized to perform physiological tasks associated with oxygen uptake and storage during embryonic development.

  9. Conservation of globin genes in the "living fossil" Latimeria chalumnae and reconstruction of the evolution of the vertebrate globin family.

    PubMed

    Schwarze, Kim; Burmester, Thorsten

    2013-09-01

    The (hemo-)globins are among the best-investigated proteins in biomedical sciences. These small heme-proteins play an important role in oxygen supply, but may also have other functions. In addition to well known hemoglobin and myoglobin, six other vertebrate globin types have been identified in recent years: neuroglobin, cytoglobin, globin E, globin X, globin Y, and androglobin. Analyses of the genome of the "living fossil" Latimeria chalumnae show that the coelacanth is the only known vertebrate that includes all eight globin types. Thus, Latimeria can also be considered as a "globin fossil". Analyses of gene synteny and phylogenetic reconstructions allow us to trace the evolution and the functional changes of the vertebrate globin family. Neuroglobin and globin X diverged from the other globin types before the separation of Protostomia and Deuterostomia. The cytoglobins, which are unlikely to be involved in O2 supply, form the earliest globin branch within the jawed vertebrates (Gnathostomata), but do not group with the agnathan hemoglobins, as it has been proposed before. There is strong evidence from phylogenetic reconstructions and gene synteny that the eye-specific globin E and muscle-specific myoglobin constitute a common clade, suggesting a similar role in intracellular O2 supply. Latimeria possesses two α- and two β-hemoglobin chains, of which one α-chain emerged prior to the divergence of Actinopterygii and Sarcopterygii, but has been retained only in the coelacanth. Notably, the embryonic hemoglobin α-chains of Gnathostomata derive from a common ancestor, while the embryonic β-chains - with the exception of a more complex pattern in the coelacanth and amphibians - display a clade-specific evolution. Globin Y is associated with the hemoglobin gene cluster, but its phylogenetic position is not resolved. Our data show an early divergence of distinct globin types in the vertebrate evolution before the emergence of tetrapods. The subsequent loss of

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

    PubMed Central

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

    1995-01-01

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

  12. Evolution and molecular characterization of a beta-globin gene from the Australian Echidna Tachyglossus aculeatus (Monotremata).

    PubMed

    Lee, M H; Shroff, R; Cooper, S J; Hope, R

    1999-07-01

    Coinciding with a period in evolution when monotremes, marsupials, and eutherians diverged from a common ancestor, a proto-beta-globin gene duplicated, producing the progenitors of mammalian embryonic and adult beta-like globin genes. To determine whether monotremes contain orthologues of these genes and to further investigate the evolutionary relationships of monotremes, marsupials, and eutherians, we have determined the complete DNA sequence of an echidna (Tachyglossus aculeatus) beta-like globin gene. Conceptual translation of the gene and sequence comparisons with eutherian and marsupial beta-like globin genes and echidna adult beta-globin indicate that the gene is adult expressed. Phylogenetic analyses do not clearly resolve the branching pattern of mammalian beta-like globin gene lineages and it is therefore uncertain whether monotremes have orthologues of the embryonic beta-like globin genes of marsupials and eutherians. Four models are proposed that provide a framework for interpreting further studies on the evolution of beta-like globin genes in the context of the evolution of monotremes, marsupials, and eutherians.

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

    PubMed

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

    1996-11-15

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

  14. Activation of Developmentally Mutated Human Globin Genes by Cell Fusion

    NASA Astrophysics Data System (ADS)

    Papayannopoulou, Thalia; Enver, Tariq; Takegawa, Susumu; Anagnou, Nicholas P.; Stamatoyannopoulos, George

    1988-11-01

    Human fetal globin genes are not expressed in hybrid cells produced by the fusion of normal human lymphocytes with mouse erythroleukemia cells. In contrast, when lymphocytes from persons with globin gene developmental mutations (hereditary persistence of fetal hemoglobin) are used for these fusions, fetal globin is expressed in the hybrid cells. Thus, mutations of developmental origin can be reconstituted in vitro by fusing mutant lymphoid cells with differentiated cell lines of the proper lineage. This system can readily be used for analyses, such as globin gene methylation, that normally require large numbers of pure nucleated erythroid cells, which are difficult to obtain.

  15. 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

  16. Lineage-Specific Patterns of Functional Diversification in the α- and β-Globin Gene Families of Tetrapod Vertebrates

    PubMed Central

    Storz, Jay F.; Gorr, Thomas A.; Opazo, Juan C.

    2010-01-01

    The α- and β-globin gene families of jawed vertebrates have diversified with respect to both gene function and the developmental timing of gene expression. Phylogenetic reconstructions of globin gene family evolution have provided suggestive evidence that the developmental regulation of hemoglobin synthesis has evolved independently in multiple vertebrate lineages. For example, the embryonic β-like globin genes of birds and placental mammals are not 1:1 orthologs. Despite the similarity in developmental expression profiles, the genes are independently derived from lineage-specific duplications of a β-globin pro-ortholog. This suggests the possibility that other vertebrate taxa may also possess distinct repertoires of globin genes that were produced by repeated rounds of lineage-specific gene duplication and divergence. Until recently, investigations into this possibility have been hindered by the dearth of genomic sequence data from nonmammalian vertebrates. Here, we report new insights into globin gene family evolution that were provided by a phylogenetic analysis of vertebrate globins combined with a comparative genomic analysis of three key sauropsid taxa: a squamate reptile (anole lizard, Anolis carolinensis), a passeriform bird (zebra finch, Taeniopygia guttata), and a galliform bird (chicken, Gallus gallus). The main objectives of this study were 1) to characterize evolutionary changes in the size and membership composition of the α- and β-globin gene families of tetrapod vertebrates and 2) to test whether functional diversification of the globin gene clusters occurred independently in different tetrapod lineages. Results of our comparative genomic analysis revealed several intriguing patterns of gene turnover in the globin gene clusters of different taxa. Lineage-specific differences in gene content were especially pronounced in the β-globin gene family, as phylogenetic reconstructions revealed that amphibians, lepidosaurs (as represented by anole

  17. Modification of Globin Gene Expression by RNA Targeting Strategies

    PubMed Central

    Shen, Tong-Jian; Rogers, Heather; Yu, Xiaobing; Lin, Felix; Noguchi, Constance T.; Ho, Chien

    2007-01-01

    Objective Sickle cell anemia is a genetic blood disease resulting from production of mutant β-globin (βS) and has severe clinical consequences. It is known that a higher cellular γ-globin level, e.g., higher ratio of cellular γ-globin to βS-globin (γ/βS ratio), inhibits sickle hemoglobin (HbS) polymerization tendency. Hence, therapeutic treatment of sickle cell anemia has been focused on introducing γ-globin gene into red blood cells to increase the cellular γ/βS ratio. Here, we have introduced ribozymes and small interfering RNAs (siRNAs) against βS-globin mRNA into blood cells as a means to increase the γ/βS ratio. Methods Single and multi-ribozymes against βS-globin mRNA have been tested in vitro and in human erythroleukemia K562βS cells that stably express exogenous βS-globin gene. Primary human hematopoietic progenitor cells were also transfected with multi-ribozyme and the γ/(γ+β) ratio determined and compared with cells transfected with long hairpin β-globin cDNA and synthetic siRNA genes. Results We have found that the multi-ribozyme zb21A containing two ribozyme units effectively reduces βS-globin mRNA both in vitro and in K562βS cells. The γ-globin mRNA to βS-globin mRNA ratio in the multi-ribozyme transfected cells is about a factor of 2 more than that in the control cells. We have also found that the γ/(γ+β) ratio in the transfected hematopoietic progenitor cells is increased by more than 2-fold in cells treated with multi-ribozyme zb21A or siRNA ib5. Conclusion Our results suggest that introducing multi-ribozymes or siRNAs into red blood cells are comparable in their effectiveness to increase the ratio of cellular γ-globin mRNA to β- or βS-globin mRNA, providing possible strategies to increase the effectiveness of γ-globin gene transfer as gene therapy for treatment of patients with sickle cell anemia. PMID:17662889

  18. Recruitment of Transcription Complexes to the β-Globin Gene Locus in Vivo and in Vitro*

    PubMed Central

    Vieira, Karen F.; Levings, Padraic P.; Hill, Meredith A.; Crusselle, Valerie J.; Kang, Sung-Hae Lee; Engel, James Douglas; Bungert, Jörg

    2013-01-01

    Erythroid-specific, high level expression of the β-globin genes is regulated by the locus control region (LCR), composed of multiple DNase I-hypersensitive sites and located far upstream of the genes. Recent studies have shown that LCR core elements recruit RNA polymerase II (pol II). In the present study we demonstrate the following: 1) pol II and other basal transcription factors are recruited to LCR core hypersensitive elements; 2) pol II dissociates from and re-associates with the globin gene locus during replication; 3) pol II interacts with the LCR but not with the β-globin gene prior to erythroid differentiation in embryonic stem cells; and 4) the erythroid transcription factor NF-E2 facilitates the transfer of pol II from immobilized LCR constructs to a β-globin gene in vitro. The data are consistent with the hypothesis that the LCR serves as the primary attachment site for the recruitment of macromolecular complexes involved in chromatin structure alterations and transcription of the globin genes. PMID:15385559

  19. Binding of HMG 17 to mononucleosomes of the avian beta-globin gene cluster in erythroid and non-erythroid cells.

    PubMed Central

    Brotherton, T W; Reneker, J; Ginder, G D

    1990-01-01

    The binding of HMG 17 to stripped core mononucleosomes containing DNA from the avian beta-globin gene cluster was examined to determine whether binding in vitro in this developmentally-regulated gene domain was associated with transcriptional activity or DNaseI-sensitivity in intact nuclei. Mononucleosomes were prepared from primitive and definitive stage embryonic red blood cells of chick embryos, adult reticulocytes, adult reticulocytes in which embryonic rho-globin transcription was induced, and adult thymus cells. Preferential binding by HMG 17 to mononucleosomes containing the beta-globin gene cluster was confined to erythroid-derived mononucleosomes that contain the embryonic rho-globin gene, the adult beta-globin gene, and DNA sequences located between these two genes, but not to those that contain the embryonic epsilon-globin gene. Comparison of these results to the known patterns of transcription and DNaseI-sensitivity within the beta-globin gene cluster shows that HMG 17 binding, although tissue-specific, does not correlate directly with either DNaseI-sensitivity or active gene transcription, but is dependent on other factors present in core mononucleosomes from this active gene domain. Images PMID:1692412

  20. Organization, structure, and evolution of the nonadult rat beta-globin gene cluster.

    PubMed

    Satoh, H; Inokuchi, N; Nagae, Y; Okazaki, T

    1999-07-01

    The beta-globin gene cluster of Wistar rat was extensively cloned and the embryonic genes were mapped and sequenced. Four overlapping lambda Dash recombinant clones cover about 31 kb and contain four nonadult beta-globin genes, 5'-epsilon1-gamma1-gamma2-psigamma3-3'. The epsilon1 and gamma2 are active genes, since their protein products were detected in the fetal stage of the rat (Iwahara et al., J Biochem 119:360-366, 1996). The gamma1 locus might be a pseudogene, since the ATA box in the promoter region is mutated to GTA; however, no other defect is observed. The psigamma3 locus is a truncated pseudogene because a 19-base deletion, which causes a shift of the reading frame, is observed between the second nucleotide of the putative codon 68 and codon 76. A sequence comparison suggests that the psigamma3 might be produced by a gene conversion event of the proto-gamma-globin gene set. Possible histories of the evolution of rat nonadult beta-globin genes are discussed.

  1. Analysis of the human alpha-globin gene cluster in transgenic mice.

    PubMed Central

    Sharpe, J A; Wells, D J; Whitelaw, E; Vyas, P; Higgs, D R; Wood, W G

    1993-01-01

    A 350-bp segment of DNA associated with an erythroid-specific DNase I-hypersensitive site (HS-40), upstream of the alpha-globin gene cluster, has been identified as the major tissue-specific regulator of the alpha-globin genes. However, this element does not direct copy number-dependent or developmentally stable expression of the human genes in transgenic mice. To determine whether additional upstream hypersensitive sites could provide more complete regulation of alpha gene expression we have studied 17 lines of transgenic mice bearing various DNA fragments containing HSs -33, -10, -8, and -4, in addition to HS -40. Position-independent, high-level expression of the human zeta- and alpha-globin genes was consistently observed in embryonic erythroid cells. However, the additional HSs did not confer copy-number dependence, alter the level of expression, or prevent the variable down-regulation of expression in adults. These results suggest that the region upstream of the human alpha-globin genes is not equivalent to that upstream of the beta locus and that although the two clusters are coordinately expressed, there may be differences in their regulation. Images Fig. 2 Fig. 4 Fig. 6 PMID:8248238

  2. 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.

  3. 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

  4. Organization, structure, and expression of the goat globin genes.

    PubMed

    Lingrel, J B; Townes, T M; Shapiro, S G; Spence, S E; Liberator, P A; Wernke, S M

    1983-01-01

    Several hemoglobin switches occur during the development of the goat, making this a useful animal for the study of globin gene expression. In order to help understand the basis for these switches, we have isolated the beta-globin genes of the goat by recombinant DNA technology and characterized these genes with respect to linkage, nucleotide sequence, and expression. The linkage arrangement so far established is epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V. It is proposed that epsilon V is followed by epsilon VI-psi beta-gamma, but so far this linkage has not been established. Several conclusions can be drawn from our findings to date. First, the beta- and gamma-globin genes of the goat have a very different evolutionary history from the beta- and gamma-globin genes of humans. While the beta and gamma genes of the human can be traced to a duplication of the ancestral epsilon/beta-globin gene before the mammalian radiation, the goat beta and gamma genes have arisen much later, and are probably the results of a duplication of a four-gene set, namely the epsilon-epsilon-psi beta-beta primordial linkage group. The beta C gene probably arose from a similar, even later duplication of the non-gamma quadruplet. Because the beta C, beta A, and gamma genes of the goat have diverged much more recently in evolution, they are much more homologous than the equivalent genes in other species. In fact, there are large regions of these genes that share identical sequences. This is meaningful in that regions of sequence identity define areas that cannot be involved in the developmental regulation of these genes.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. 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.

  6. [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

  7. β-Globin-Expressing Definitive Erythroid Progenitor Cells Generated from Embryonic and Induced Pluripotent Stem Cell-Derived Sacs.

    PubMed

    Fujita, Atsushi; Uchida, Naoya; Haro-Mora, Juan J; Winkler, Thomas; Tisdale, John

    2016-06-01

    Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell transfusion. However, when using traditional methods with embryoid bodies, ES cell-derived erythroid cells predominantly express embryonic type ɛ-globin, with lesser fetal type γ-globin and very little adult type β-globin. Furthermore, no β-globin expression is detected in iPS cell-derived erythroid cells. ES cell-derived sacs (ES sacs) have been recently used to generate functional platelets. Due to its unique structure, we hypothesized that ES sacs serve as hemangioblast-like progenitors capable to generate definitive erythroid cells that express β-globin. With our ES sac-derived erythroid differentiation protocol, we obtained ∼120 erythroid cells per single ES cell. Both primitive (ɛ-globin expressing) and definitive (γ- and β-globin expressing) erythroid cells were generated from not only ES cells but also iPS cells. Primitive erythropoiesis is gradually switched to definitive erythropoiesis during prolonged ES sac maturation, concurrent with the emergence of hematopoietic progenitor cells. Primitive and definitive erythroid progenitor cells were selected on the basis of glycophorin A or CD34 expression from cells within the ES sacs before erythroid differentiation. This selection and differentiation strategy represents an important step toward the development of in vitro erythroid cell production systems from pluripotent stem cells. Further optimization to improve expansion should be required for clinical application. Stem Cells 2016;34:1541-1552.

  8. Identification of patients with defects in the globin genes

    PubMed Central

    Dell’Edera, Domenico; Epifania, Annunziata Anna; Milazzo, Giusi Natalia; Leo, Manuela; Santacesaria, Carmela; Allegretti, Arianna; Mazzone, Eleonora; Panetta, Paolo; Iammarino, Giovanna; Lupo, Maria Giovanna; Barbieri, Rocchina; Lioi, Maria Brigida

    2013-01-01

    Summary Introduction hemoglobinopathies constitute a major health problem worldwide. These disorders are characterized by a clinical and hematological phenotypic heterogeneity. The increase of HbA2 is an invaluable hematological marker of the beta-thalassemia heterozygosis and of double heterozygosis for the alleles of delta and alpha globin genes or for the alleles of delta and beta globin genes which can cause the increase of HbA2 up to normal or borderline values. Case Report we report the case of a 30-year-old woman (first pregnant) who was admitted to our Unit at 12 weeks for a screening for thalassemia. The outcomes of the biochemical and haematological exams (MCV, MCH, HbA2, HbF) highlighted that the patient was a carrier of a beta-thalassemic trait. Molecular analysis of the beta globin genes highlighted a β039C>T heterozygous mutation. Biochemical and hematological parameters of the husband (MCV, MCH, HbA2, HbF) were normal except for the level of HbA2 (3,6%). The molecular analysis of the beta globin genes highlighted a IVS2 nt844 C>G heterozygous mutation. Furthermore, the heterozygous mutation δ+cod.27G>T was detected in his δ globin gene. For this reason, he was diagnosed a δ+β Thal. Conclusions the aim of this paper is to highlight that biochemical diagnosis could not exhaustive and a molecular diagnostic widening is required to detect the genetic deficiency causing the thalassemic trait. PMID:24611095

  9. Arrangements of alpha-globin gene cluster in Taiwan.

    PubMed

    Peng, H W; Choo, K B; Ho, C H; Yen, M S; Liung, W Y; Lin, C K; Yang, Z L; Ng, H T; Ching, K N; Han, S H

    1989-01-01

    In a gene mapping study on 217 newborn babies in Taiwan with alpha- and zeta-globin probes, we have observed 4 cases (1.84%) of alpha-thalassemia-2 heterozygotes (zeta zeta-alpha/zeta zeta alpha alpha) without increased levels of hemoglobin (Hb) Bart's in the cord blood. Eleven subjects (5.07%) were found to have the South East Asian alpha-thalassemia-1 haplotype (zeta zeta--SEA/zeta zeta alpha alpha) with increased Hb Bart's levels ranging from 2.2 to 9%. One case, with Hb Bart's level of 14% in the cord blood, was found to have the genotype of zeta zeta--SEA/zeta zeta alpha alpha T (0.46%). Four heterozygotes (1.84%) were found with the triple alpha gene anti-rightward arrangement (zeta zeta alpha alpha alpha 3.7/zeta zeta alpha alpha). Twenty-one heterozygotes (9.68%) were found to have the triple zeta-globin gene arrangement (zeta zeta zeta alpha alpha/zeta zeta alpha alpha). A new triple zeta-globin gene variant with a BamHI polymorphism was also observed in this study.

  10. 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.

  11. Intervening sequence of DNA identified in the structural portion of a mouse beta-globin gene.

    PubMed Central

    Tilghman, S M; Tiemeier, D C; Seidman, J G; Peterlin, B M; Sullivan, M; Maizel, J V; Leder, P

    1978-01-01

    The unusual electron microscopic appearance of a hybrid formed between 9S mouse beta-globin mRNA and its corresponding cloned gene segment is caused by at least one, and possibly two, intervening sequences of DNA that interrupt the mouse beta-globin gene. Such an interpretation is consistent with a paradoxical restriction site pattern previously noted in this gene and with the nucleotide sequence of that portion of the gene that spans both structural and intervening sequences. The large intervening sequence, approximately 550 base pairs in length, occurs in the structural globin sequence and immediately follows the beta-globin codon corresponding to amino acid 104. A smaller, putative intervening sequence is located close to the 5' end of the beta-globin-coding sequence but may reside beyond its initiation codon. The beta-globin gene thus appears to be encoded in two, and possibly three, discontinuous segments. Images PMID:273235

  12. Deletion and replacement of the mouse adult beta-globin genes by a "plug and socket" repeated targeting strategy.

    PubMed

    Detloff, P J; Lewis, J; John, S W; Shehee, W R; Langenbach, R; Maeda, N; Smithies, O

    1994-10-01

    We describe a two-step strategy to alter any mouse locus repeatedly and efficiently by direct positive selection. Using conventional targeting for the first step, a functional neo gene and a nonfunctional HPRT minigene (the "socket") are introduced into the genome of HPRT- embryonic stem (ES) cells close to the chosen locus, in this case the beta-globin locus. For the second step, a targeting construct (the "plug") that recombines homologously with the integrated socket and supplies the remaining portion of the HPRT minigene is used; this homologous recombination generates a functional HPRT gene and makes the ES cells hypoxanthine-aminopterin-thymidine resistant. At the same time, the plug provides DNA sequences that recombine homologously with sequences in the target locus and modifies them in the desired manner; the plug is designed so that correctly targeted cells also lose the neo gene and become G418 sensitive. We have used two different plugs to make alterations in the mouse beta-globin locus starting with the same socket-containing ES cell line. One plug deleted 20 kb of DNA containing the two adult beta-globin genes. The other replaced the same region with the human beta-globin gene containing the mutation responsible for sickle cell anemia.

  13. 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

  14. Transcriptional activation of human adult alpha-globin genes by hypersensitive site-40 enhancer: function of nuclear factor-binding motifs occupied in erythroid cells.

    PubMed Central

    Rombel, I; Hu, K Y; Zhang, Q; Papayannopoulou, T; Stamatoyannopoulos, G; Shen, C K

    1995-01-01

    The developmental stage- and erythroid lineage-specific activation of the human embryonic zeta- and fetal/adult alpha-globin genes is controlled by an upstream regulatory element [hypersensitive site (HS)-40] with locus control region properties, a process mediated by multiple nuclear factor-DNA complexes. In vitro DNase I protection experiments of the two G+C-rich, adult alpha-globin promoters have revealed a number of binding sites for nuclear factors that are common to HeLa and K-562 extracts. However, genomic footprinting analysis has demonstrated that only a subset of these sites, clustered between -130 and +1, is occupied in an erythroid tissue-specific manner. The function of these in vivo-occupied motifs of the alpha-globin promoters, as well as those previously mapped in the HS-40 region, is assayed by site-directed mutagenesis and transient expression in embryonic/fetal erythroid K-562 cells. These studies, together with our expression data on the human embryonic zeta-globin promoter, provide a comprehensive view of the functional roles of individual nuclear factor-DNA complexes in the final stages of transcriptional activation of the human alpha-like globin promoters by the HS-40 element. Images Fig. 2 Fig. 3 Fig. 5 Fig. 6 PMID:7604012

  15. Structure and function of the globin and globin gene from the Antarctic mollusc Yoldia eightsi.

    PubMed Central

    Dewilde, Sylvia; Angelini, Elisa; Kiger, Laurent; Marden, Michael C; Beltramini, Mariano; Salvato, Benedetto; Moens, Luc

    2003-01-01

    The mechanism of adaptation of haemoglobin from the Antarctic mollusc Yoldia eightsi to its low-temperature environment is a decrease in the oxygen affinity via an increased ligand-dissociation rate. At 2 degrees C this haemoglobin has an oxygen affinity similar to other haemoglobins at 25 degrees C. At 25 degrees C, Yoldia haemoglobin shows a low oxygen affinity, resembling that of human deoxyhaemoglobin. The mechanism involves a lower binding energy to oxygen, suggesting a loss or weakening of the usual hydrogen bond, leading to a higher oxygen-dissociation rate. However, Yoldia haemoglobin has the usual distal and proximal histidines, so the primary structure alone does not provide an obvious explanation for the low affinity. The CO-binding kinetics are biphasic, with the fraction of slow phase increasing at higher protein concentrations, indicating the formation of dimers or a higher level of polymerization. The protein-protein interaction appears to be of hydrophobic nature, since it can be partially reversed by addition of ethylene glycol as co-solvent. While the CO-association rates differ by a factor of 10, the oxygen equilibrium data could be simulated with a single affinity. The Yoldia haemoglobin gene contains three introns, interrupting the coding region at position NA1.2, B12.2 and G7.0. The conservation of the B12.2 and G7.0 introns is in contrast with the unprecedented NA1.2 intron. Phylogenetic analyses reveal a gene tree where the Yoldia haemoglobin gene is separated from other mollusc globin genes, confirming the specific adaptation of the Yoldia haemoglobin. PMID:12423205

  16. CACCC and GATA-1 sequences make the constitutively expressed alpha-globin gene erythroid-responsive in mouse erythroleukemia cells.

    PubMed Central

    Ren, S; Li, J; Atweh, G F

    1996-01-01

    Although the human alpha-globin and beta-globin genes are co-regulated in adult life, they achieve the same end by very different mechanisms. For example, a transfected beta-globin gene is expressed in an inducible manner in mouse erythroleukemia (MEL) cells while a transfected alpha-globin gene is constitutively expressed at a high level in induced and uninduced MEL cells. Interestingly, when the alpha-globin gene is transferred into MEL cells as part of human chromosome 16, it is appropriately expressed in an inducible manner. We explored the basis for the lack of erythroid-responsiveness of the proximal regulatory elements of the human alpha-globin gene. Since the alpha-globin gene is the only functional human globin gene that lacks CACCC and GATA-1 motifs, we asked whether their addition to the alpha-globin promoter would make the gene erythroid-responsive in MEL cells. The addition of each of these binding sites to the alpha-globin promoter separately did not result in inducibility in MEL cells. However, when both sites were added together, the alpha-globin gene became inducible in MEL cells. This suggests that erythroid non-responsiveness of the alpha-globin gene results from the lack of erythroid binding sites and is not necessarily a function of the constitutively active, GC rich promoter. PMID:8628660

  17. Peptide nucleic acid (PNA) binding-mediated induction of human gamma-globin gene expression.

    PubMed

    Wang, G; Xu, X; Pace, B; Dean, D A; Glazer, P M; Chan, P; Goodman, S R; Shokolenko, I

    1999-07-01

    Peptide nucleic acids (PNAs) can bind to homopurine/homopyrimidine sequences of double-stranded DNA targets in a sequence-specific manner and form [PNA]2/DNA triplexes with single-stranded DNA D-loop structures at the PNA binding sites. These D-loop structures have been found to have a capacity to initiate transcription in vitro. If this strategy can be used to induce transcription of endogenous genes, it may provide a novel approach for gene therapy of many human diseases. Human [beta] globin disorders such as sickle cell anemia and beta-thalassemia are very common genetic diseases that are caused by mutations in the beta-globin gene. When gamma-globin genes are highly expressed in sickle cell patients, the presence of high levels of fetal hemoglobin (HbF, alpha2gamma2) can compensate for the defective beta-globin gene product and such patients have much improved symptoms or are free of disease. However, the gamma-globin genes are developmentally regulated and normally expressed at very low levels (>1%) in adult blood cells. We have investigated the possibility of inducing gamma-globin gene expression with PNAs. Using PNAs designed to bind to the 5' flanking region of the gamma-globin gene, induction of expression of a reporter gene construct was demonstrated both in vitro and in vivo. More importantly, PNA-mediated induction of endogenous gamma-globin gene expression was also demonstrated in K562 human erythroleukemia cells. This result suggests that induction of gamma-globin gene expression with PNAs might provide a new approach for the treatment of sickle cell disease. PNA-induced gene expression strategy also may have implications in gene therapy of other diseases such as genetic diseases, cancer and infectious diseases.

  18. 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

  19. 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

  20. Evolution of the Globin Gene Family in Deuterostomes: Lineage-Specific Patterns of Diversification and Attrition

    PubMed Central

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

    2012-01-01

    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

  1. 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

  2. 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

  3. Identification of a recent recombination event within the human beta-globin gene cluster.

    PubMed Central

    Gerhard, D S; Kidd, K K; Kidd, J R; Egeland, J A; Housman, D E

    1984-01-01

    In a detailed study of inheritance of DNA sequence polymorphism in a large reference pedigree, an individual was identified with an apparent genetic recombination event within the human beta-globin gene cluster. Analysis of the haplotypes of relevant individuals within this pedigree suggested that the meiotic crossing-over event is likely to have occurred within a 19.8-kilobase-pair region of the beta-globin gene cluster. Analysis of other DNA markers closely linked to the beta-globin gene cluster--segment 12 of chromosome 11 (D11S12) and loci for insulin, the cellular oncogene c-Ha-ras, and preproparathyroid hormone--confirmed that a crossover event must have occurred within the region of chromosome 11 between D11S12 and the beta-globin gene cluster. It is suggested that the event observed has occurred within a DNA region compatible with recombinational "hot spots" suggested by population studies. PMID:6096866

  4. Established epigenetic modifications determine the expression of developmentally regulated globin genes in somatic cell hybrids.

    PubMed Central

    Stanworth, S J; Roberts, N A; Sharpe, J A; Sloane-Stanley, J A; Wood, W G

    1995-01-01

    Somatic cell hybrids generated from transgenic mouse cells have been used to examine the developmental regulation of human gamma-to-beta-globin gene switching. In hybrids between mouse erythroleukemia (MEL) cells and transgenic erythroblasts taken at various stages of development, there was regulated expression of the human fetal gamma and adult beta genes, reproducing the in vivo pattern prior to fusion. Hybrids formed from embryonic blood cells produced predominantly gamma mRNA, whereas beta gene expression was observed in adult hybrids and a complete range of intermediate patterns was found in fetal liver hybrids. The adult environment of the MEL cells, therefore, did not appear to influence selective transcription from this gene complex. Irradiation of the embryonic erythroid cells prior to fusion resulted in hybrids containing only small fragments of donor chromosomes, but the pattern of gene expression did not differ from that of unirradiated hybrids. This finding suggests that continued expression of trans-acting factors from the donor erythroblasts is not necessary for continued expression of the human gamma gene in MEL cells. These results contrast with the lack of developmental regulation of the cluster after transfection of naked DNA into MEL cells and suggest that epigenetic processes established during normal development result in the gene cluster adopting a developmental stage-specific, stable conformation which is maintained through multiple rounds of replication and transcription in the MEL cell hybrids. On prolonged culture, hybrids that initially expressed only the gamma transgene switched to beta gene expression. The time period of switching, from approximately 10 to > 40 weeks, was similar to that seen previously in human fetal erythroblast x MEL cell hybrids but in this case bore no relationship to the time of in vivo switching. It seems unlikely, therefore, that switching in these hybrids is regulated by a developmental clock. PMID:7623793

  5. Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector.

    PubMed Central

    Cone, R D; Weber-Benarous, A; Baorto, D; Mulligan, R C

    1987-01-01

    We introduced a human beta-globin gene into murine erythroleukemia (MEL) cells by infection with recombinant retroviruses containing the complete genomic globin sequence. The beta-globin gene was correctly regulated during differentiation, steady-state mRNA levels being induced 5- to 30-fold after treatment of the cells with the chemical inducer dimethyl sulfoxide. Studies using vectors which yield integrated proviruses lacking transcriptional enhancer sequences indicated that neither retroviral transcription nor the retroviral enhancer sequences themselves had any obvious effect on expression of the globin gene. Viral RNA expression also appeared inducible, being considerably depressed in uninduced MEL cells but approaching normal wild-type levels after dimethyl sulfoxide treatment. We provide data which suggest that the control point for both repression and subsequent activation of virus expression in MEL cells lies in the viral enhancer element. Images PMID:3029570

  6. 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.

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

    PubMed

    Opazo, Juan C; Lee, Alison P; Hoffmann, Federico G; Toloza-Villalobos, Jessica; Burmester, Thorsten; Venkatesh, Byrappa; Storz, Jay F

    2015-07-01

    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

  8. 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

  9. Multiple physical stresses induce γ-globin gene expression and fetal hemoglobin production in erythroid cells.

    PubMed

    Schaeffer, Emily K; West, Rachel J; Conine, Sarah J; Lowrey, Christopher H

    2014-04-01

    Increased fetal hemoglobin (HbF) expression is beneficial for β-hemoglobinopathy patients; however, current inducing agents do not possess the ideal combination of efficacy, safety and ease of use. Better understanding the mechanisms involved in γ-globin gene induction is critical for designing improved therapies, as no complete mechanism for any inducing agent has been identified. Given the cytotoxic nature of most known inducing drugs, we hypothesized that γ-globin is a cell stress response gene, and that induction occurs via activation of cell stress signaling pathways. We tested this hypothesis by investigating the ability of physical stresses including heat-shock (HS), UV- and X-irradiation and osmotic shock to increase γ-globin gene expression in erythroid cells. Experiments in K562 and KU812 cells showed that each of these stresses increased steady-state γ-globin mRNA levels, but only after 3-5days of treatments. HS and UV also increased γ-globin mRNA and HbF levels in differentiating primary human erythroid cells. Mechanistic studies showed that HS affects γ-globin mRNA at multiple levels, including nascent transcription and transcript stability, and that induction is dependent on neither the master regulator of the canonical HS response, HSF1, nor p38 MAPK. Inhibitor panel testing identified PI3K inhibitor LY294002 as a novel inducing agent and revealed potential roles for NFκB and VEGFR/PDGFR/Raf kinases in HS-mediated γ-globin gene induction. These findings suggest that cell stress signaling pathways play an important role in γ-globin gene induction and may provide novel targets for the pharmacologic induction of fetal hemoglobin.

  10. Recombinant plasmids containing Xenopus laevis globin structural genes derived from complementary DNA.

    PubMed Central

    Humphries, P; Old, R; Coggins, L W; McShane, T; Watson, C; Paul, J

    1978-01-01

    Details are presented of the in vitro synthesis of double-stranded DNA complementary to purified Xenopus globin messenger RNA, using a combination of reverse transcriptase, fragment 'A' of E. coli DNA polymerase 1 and S1 endonuclease. After selection of duplex DNA molecules approaching the length of Xenopus globin messenger RNA by sedimentation of the DNA through neutral sucrose gradients, the 3'-OH termini of the synthetic globin gene sequences were extended with short tracts of oligo dGMP using terminal transferase. This material was integrated into oligo dCMP-extended linear pCR1 plasmid DNA and amplified by transfection of E. coli. Plasmids carrying globin sequences were identified by hybridization of 32P-labelled globin mRNA to total cellular DNA in situ, by hybridization of purified plasmids to globin cDNA in solution, by analysis of recombinant DNA on polyacrylamide and agarose gels, and by heteroduplex mapping. The results show that extensive DNA copies of Xenopus globin mRNA have been integrated into recombinant plasmids. Images PMID:347404

  11. 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.

  12. Transgenic mice containing a 248-kb yeast artificial chromosome carrying the human beta-globin locus display proper developmental control of human globin genes.

    PubMed Central

    Peterson, K R; Clegg, C H; Huxley, C; Josephson, B M; Haugen, H S; Furukawa, T; Stamatoyannopoulos, G

    1993-01-01

    Transgenic mice were generated using a purified 248-kb yeast artificial chromosome (YAC) bearing an intact 82-kb human beta-globin locus and 148 kb of flanking sequence. Seventeen of 148 F0 pups were transgenic. RNase protection analysis of RNA isolated from the blood of 13 gamma- and beta-globin-positive founders showed that only the human beta-globin gene was expressed in the adult founders. Studies of F1 and F2 fetuses demonstrated that the genes of the beta-locus YAC displayed the proper developmental switches in beta-like globin gene expression. Expression of epsilon- and gamma-globin, but not beta-globin, was observed in the yolk sac, there was only minor gamma and mostly beta expression in the 14-day liver, and only beta mRNA in the blood of the adult animals. Structural data showed that the locus was intact. These results indicate that it is now possible to dissect regulatory mechanisms within the context of an entire locus in vivo by using the ability to perform mutagenesis efficiently in yeast via homologous recombination, followed by purification of the altered YAC and its introduction into mice. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8356061

  13. Developmental and species-divergent globin switching are driven by BCL11A

    PubMed Central

    Sankaran, Vijay G.; Xu, Jian; Ragoczy, Tobias; Ippolito, Gregory C.; Walkley, Carl R.; Maika, Shanna D.; Fujiwara, Yuko; Ito, Masafumi; Groudine, Mark; Bender, M.A.; Tucker, Philip W.; Orkin, Stuart H.

    2013-01-01

    The contribution of changes in cis-regulatory elements or trans-acting factors to interspecies differences in gene expression is not well understood. The mammalian β-globin loci have served as a paradigm for gene regulation during development. Transgenic mice harboring the human β-globin locus, consisting of the linked embryonic (ε), fetal (γ) and adult (β) genes, have been used as a model system to study the temporal switch from fetal to adult hemoglobin, as occurs in humans. We show that the human γ-globin genes in these mice behave as murine embryonic globin genes, revealing a limitation of the model and demonstrating that critical differences in the trans-acting milieu have arisen during mammalian evolution. We show that the expression of BCL11A, a repressor of human γ-globin expression identified through genome-wide association studies, differs between mouse and human. Developmental silencing of the mouse embryonic globin and human γ-globin genes fails to occur in mice in the absence of BCL11A. Thus, BCL11A is a critical mediator of species-divergent globin switching. By comparing the ontogeny of β-globin gene regulation in mice and humans, we have shown that alterations in expression of a trans-acting factor constitute a critical driver of gene expression changes during evolution. PMID:19657335

  14. Stimulated stromal cells induce gamma-globin gene expression in erythroid cells via nitric oxide production

    PubMed Central

    Čokić, Vladan P.; Beleslin-Čokić, Bojana B.; Smith, Reginald D.; Economou, Antaeus P.; Wahl, Larry M.; Noguchi, Constance T.; Schechter, Alan N.

    2009-01-01

    Objective We have previously shown that nitric oxide (NO) is involved in the hydroxyurea-induced increase of gamma-globin gene expression in cultured human erythroid progenitor cells and that hydroxyurea increases NO production in endothelial cells via endothelial NO synthase (NOS). We have now expanded those studies to demonstrate that the stimulation of gamma-globin gene expression is also mediated by NOS induction in stromal cells within the bone marrow microenvironment. Materials and Methods Using NO analyzer, we measured NO production in endothelial and macrophage cell cultures. In co-culture studies of erythroid and stromal cells we measured globin gene expression during stimulation by NO inducers. Results Hydroxyurea (30–100 μM) induced NOS-dependent production of NO in human macrophages (up to 1.2 μM). Co-culture studies of human macrophages with erythroid progenitor cells also resulted in induction of gamma-globin mRNA expression (up to 3 fold) in the presence of hydroxyurea. NOS-dependent stimulation of NO by lipopolysaccharide (up to 0.6 μM) has been observed in human macrophages. We found that lipopolysaccharide and interferon-gamma together increased gamma-globin gene expression (up to 2 fold) in human macrophage/erythroid cell co-cultures. Co-culture of human bone marrow endothelial cells with erythroid progenitor cells also induced gamma-globin mRNA expression (2.4 fold) in the presence of hydroxyurea (40 μM). Conclusion These results demonstrate an arrangement by which NO and fetal hemoglobin inducers may stimulate globin genes in erythroid cells via the common paracrine effect of bone marrow stromal cells. PMID:19576950

  15. 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

  16. 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

    2014-05-09

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

  17. Retroviral transfer of a human beta-globin/delta-globin hybrid gene linked to beta locus control region hypersensitive site 2 aimed at the gene therapy of sickle cell disease.

    PubMed

    Takekoshi, K J; Oh, Y H; Westerman, K W; London, I M; Leboulch, P

    1995-03-28

    Human gamma-globin and delta-globin chains have been previously identified as strong inhibitors of the polymerization of hemoglobin S, in contrast to the beta-globin chain, which exerts only a moderate antisickling effect. However, gamma-globin and delta-globin are normally expressed at very low levels in adult erythroid cells, in contrast to beta-globin. We report the design of a beta-globin/delta-globin hybrid gene, beta/delta-sickle cell inhibitor 1 (beta/delta-SCI1) and its transduction by retrovirus-mediated gene transfer. The beta/delta-SCI1-encoding gene retains the overall structure of the human beta-globin gene, while incorporating specific amino acid residues from the delta chain previously found responsible for its enhanced antisickling properties. To achieve high expression levels of beta/delta-SCI1 in adult erythrocytes, the hybrid gene was placed under the transcriptional control of the human beta-globin promoter and the DNase I hypersensitive site 2 of the human beta locus control region. High-titer retroviruses were generated, and stable proviral transmission was achieved in infected cells. The mRNA expression levels of the beta/delta-SCI1 gene in infected, dimethyl sulfoxide-induced murine erythroleukemia cells approached 85% of the endogenous murine beta maj-globin mRNA, on a per gene basis, evidence that high gene expression levels were achieved in adult erythroid cells. Further evaluation of this strategy in transgenic animal models of sickle cell disease should assess its efficacy for the gene therapy of human patients.

  18. Polymorphism of DNA sequence adjacent to human beta-globin structural gene: relationship to sickle mutation.

    PubMed

    Kan, Y W; Dozy, A M

    1978-11-01

    Restriction endonuclease mapping of the human globin genes revealed a genetic variation in a Hpa I recognition site about 5000 nucleotides from the 3' end of the beta-globin structural gene. Instead of a normal 7.6-kilobase (kb) fragment which contains the beta-globin structural gene, 7.0-kb and 13.0-kb variants were detected. Both variants were found in people of African origin and were not detected in Asians or Caucasians. The 13.0-kb variant is frequently associated with the sickle hemoglobin mutation and may be useful for the prediction of the sickle cell gene in prenatal diagnosis. Polymorphism in a restriction enzyme site could be considered as a new class of genetic marker and may offer a new approach to linkage analysis and anthropological studies.

  19. Globin genes transcriptional switching, chromatin structure and linked lessons to epigenetics in cancer: a comparative overview.

    PubMed

    Guerrero, Georgina; Delgado-Olguín, Paul; Escamilla-Del-Arenal, Martín; Furlan-Magaril, Mayra; Rebollar, Eria; De La Rosa-Velázquez, Inti A; Soto-Reyes, Ernesto; Rincón-Arano, Héctor; Valdes-Quezada, Christian; Valadez-Graham, Viviana; Recillas-Targa, Félix

    2007-07-01

    At the present time research situates differential regulation of gene expression in an increasingly complex scenario based on interplay between genetic and epigenetic information networks, which need to be highly coordinated. Here we describe in a comparative way relevant concepts and models derived from studies on the chicken alpha- and beta-globin group of genes. We discuss models for globin switching and mechanisms for coordinated transcriptional activation. A comparative overview of globin genes chromatin structure, based on their genomic domain organization and epigenetic components is presented. We argue that the results of those studies and their integrative interpretation may contribute to our understanding of epigenetic abnormalities, from beta-thalassemias to human cancer. Finally we discuss the interdependency of genetic-epigenetic components and the need of their mutual consideration in order to visualize the regulation of gene expression in a more natural context and consequently better understand cell differentiation, development and cancer.

  20. 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

  1. 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

  2. Role of upstream DNase I hypersensitive sites in the regulation of human alpha globin gene expression.

    PubMed

    Sharpe, J A; Summerhill, R J; Vyas, P; Gourdon, G; Higgs, D R; Wood, W G

    1993-09-01

    Erythroid-specific DNase 1 hypersensitive sites have been identified at the promoters of the human alpha-like genes and within the region from 4 to 40 kb upstream of the gene cluster. One of these sites, HS-40, has been shown previously to be the major regulator of tissue-specific alpha-globin gene expression. We have now examined the function of other hypersensitive sites by studying the expression in mouse erythroleukemia (MEL) cells of various fragments containing these sites attached to HS-40 and an alpha-globin gene. High level expression of the alpha gene was observed in all cases. When clones of MEL cells bearing a single copy of the alpha-globin gene fragments were examined, expression levels were similar to those of the endogenous mouse alpha genes and similar to MEL cells bearing beta gene constructs under the control of the beta-globin locus control region. However, there was no evidence that the additional hypersensitive sites increased the level of expression or conferred copy number dependence on the expression of a linked alpha gene in MEL cells. PMID:7689876

  3. The globin gene repertoire of lampreys: convergent evolution of hemoglobin and myoglobin in jawed and jawless vertebrates.

    PubMed

    Schwarze, Kim; Campbell, Kevin L; Hankeln, Thomas; Storz, Jay F; Hoffmann, Federico G; Burmester, Thorsten

    2014-10-01

    Agnathans (jawless vertebrates) occupy a key phylogenetic position for illuminating the evolution of vertebrate anatomy and physiology. Evaluation of the agnathan globin gene repertoire can thus aid efforts to reconstruct the origin and evolution of the globin genes of vertebrates, a superfamily that includes the well-known model proteins hemoglobin and myoglobin. Here, we report a comprehensive analysis of the genome of the sea lamprey (Petromyzon marinus) which revealed 23 intact globin genes and two hemoglobin pseudogenes. Analyses of the genome of the Arctic lamprey (Lethenteron camtschaticum) identified 18 full length and five partial globin gene sequences. The majority of the globin genes in both lamprey species correspond to the known agnathan hemoglobins. Both genomes harbor two copies of globin X, an ancient globin gene that has a broad phylogenetic distribution in the animal kingdom. Surprisingly, we found no evidence for an ortholog of neuroglobin in the lamprey genomes. Expression and phylogenetic analyses identified an ortholog of cytoglobin in the lampreys; in fact, our results indicate that cytoglobin is the only orthologous vertebrate-specific globin that has been retained in both gnathostomes and agnathans. Notably, we also found two globins that are highly expressed in the heart of P. marinus, thus representing functional myoglobins. Both genes have orthologs in L. camtschaticum. Phylogenetic analyses indicate that these heart-expressed globins are not orthologous to the myoglobins of jawed vertebrates (Gnathostomata), but originated independently within the agnathans. The agnathan myoglobin and hemoglobin proteins form a monophyletic group to the exclusion of functionally analogous myoglobins and hemoglobins of gnathostomes, indicating that specialized respiratory proteins for O2 transport in the blood and O2 storage in the striated muscles evolved independently in both lineages. This dual convergence of O2-transport and O2-storage proteins in

  4. The nucleotide sequence of the mouse embryonic beta-like y-globin messenger RNA as determined from cloned cDNA.

    PubMed

    Vanin, E F; Farace, M G; Gambari, R; Fantoni, A

    1981-12-01

    We have determined the nucleotide sequence of two cloned cDNAs corresponding to the mRNA of mouse embryonic y2 globin. The combined overlapping sequences span a total of 480 bp, beginning at the codon corresponding to amino acido residue 21 and extending to the AATAAA sequence in the 3' untranslated region. Therefore, when the amino acid sequence encoded by the cDNA is combined with the available amino acid sequence, a complete y2 protein sequence can be obtained. Comparisons, at the nucleotide level, between the known beta- and beta-like globin sequences and the y2 sequence show that the embryonic, fetal-adult duplication occurred approx. 160 million years (MY) ago and that the embryonic-fetal duplication occurred approx. 100 MY ago.

  5. Human globin gene analysis for a patient with beta-o/delta beta-thalassemia.

    PubMed Central

    Ottolenghi, S; Lanyon, W G; Williamson, R; Weatherall, D J; Clegg, J B; Pitcher, C S

    1975-01-01

    Complementary DNA (cDNA) was prepared with RNA-dependent DNA polymerase from human globin messenger RNA (mRNA). Annealing and translation experimenta with total mRNA from circulating cells from a patient with heterozygous beta/heterozygous beta-delta-o thalassemia (beta-o/delta beta-o-thalassemia) demonstrated no detectable mRNA for beta-globin. cDNA enriched in sequences homologous to beta-globin mRNA was prepared by hydroxylapatite fractionation of hybrids formed between beta-o/delta beta-o-thalassemic mRNA and cDNA made from mRNA from a patient with alpha-thalassemia (hemoglobin H disease). The rate of annealing of this beta-enriched cDNA to normal human nuclear DNA was that of a sequence present as only a single copy per haploid genome. The beta-enriched cDNA annealed to the beta-o-delta beta-o-thalassemia total DNA with approximately the same kinetics as to normal DNA, indicating that no total gene deletion of beta-globin genes from the diploid genome has occurred, although the accuracy of the technique could not exclude with certainty a partial deletion or a deletion of a beta-globin gene from only one of the haploid genomes. This demonstrates that at least one of the beta-o- or the delta beta-o-thalassemia haploid genomes in this case contains a substantially intact beta-globin gene. PMID:49057

  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. Identification and molecular characterization of four new large deletions in the beta-globin gene cluster.

    PubMed

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

    2009-01-01

    Despite the fact that mutations in the human beta-globin gene cluster are essentially point mutations, a significant number of large deletions have also been described. We present here four new large deletions in the beta-globin gene cluster that have been identified on patients displaying an atypical hemoglobin phenotype (high HbF) at routine analysis. The first deletion, which spreads over 2.0 kb, removes the entire beta-globin gene, including its promoter, and is associated with a typical beta-thal minor phenotype. The three other deletions are larger (19.7 to 23.9 kb) and remove both the delta and beta-globin genes. Phenotypically, they look like an HPFH-deletion as they are associated with normal hematological parameters. The precise localization of their 5' and 3' breakpoints gives new insights about the differences between HPFH and (deltabeta)(0)-thalassemia at the molecular level. The importance of detection of these deletions in prenatal diagnosis and newborn screening of hemoglobinopathies is also discussed.

  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. 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.

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

    PubMed

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

    1995-07-18

    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

  11. 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.

  12. Whole-Genome Duplications Spurred the Functional Diversification of the Globin Gene Superfamily in Vertebrates

    PubMed Central

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

    2012-01-01

    It has been hypothesized that two successive rounds of whole-genome duplication (WGD) in the stem lineage of vertebrates provided genetic raw materials for the evolutionary innovation of many vertebrate-specific features. However, it has seldom been possible to trace such innovations to specific functional differences between paralogous gene products that derive from a WGD event. Here, we report genomic evidence for a direct link between WGD and key physiological innovations in the vertebrate oxygen transport system. Specifically, we demonstrate that key globin proteins that evolved specialized functions in different aspects of oxidative metabolism (hemoglobin, myoglobin, and cytoglobin) represent paralogous products of two WGD events in the vertebrate common ancestor. Analysis of conserved macrosynteny between the genomes of vertebrates and amphioxus (subphylum Cephalochordata) revealed that homologous chromosomal segments defined by myoglobin + globin-E, cytoglobin, and the α-globin gene cluster each descend from the same linkage group in the reconstructed proto-karyotype of the chordate common ancestor. The physiological division of labor between the oxygen transport function of hemoglobin and the oxygen storage function of myoglobin played a pivotal role in the evolution of aerobic energy metabolism, supporting the hypothesis that WGDs helped fuel key innovations in vertebrate evolution. PMID:21965344

  13. Globin genes in Micronesia: origins and affinities of Pacific Island peoples.

    PubMed Central

    O'Shaughnessy, D F; Hill, A V; Bowden, D K; Weatherall, D J; Clegg, J B

    1990-01-01

    DNA polymorphisms and copy-number variants of alpha-, zeta-, and gamma-globin genes have been studied in seven Micronesian island populations and have been compared with those in populations from Southeast Asia, Melanesia, and Polynesia. Micronesians are not significantly different from Polynesians at these loci and appear to be intermediate between Southeast Asians and Melanesians. There is evidence of significant Melanesian input into the Micronesian gene pool and of substantial proto-Polynesian contact with Melanesia. PMID:1967206

  14. Activation of delta-globin gene expression by erythroid Krupple-like factor: a potential approach for gene therapy of sickle cell disease.

    PubMed

    Donze, D; Jeancake, P H; Townes, T M

    1996-11-15

    Hemoglobin A2 (HbA2; alpha 2 delta 2) is a powerful inhibitor of HbS (alpha 2 beta 2(3)) polymerization. However, HbA2 levels are normally low in sickle cell patients. We show that a major reason for low delta-globin gene expression is the defective CACCC box at -90 in the delta-globin promoter. When the CACCC box defect in delta is corrected, expression of an HS2 delta /Luciferase reporter is equivalent to HS2 beta /Luciferase. Erythroid Krupple-like factor (EKLF), which binds to the CACCC box of the beta-globin gene and activates high-level expression, does not bind to the normal delta-globin promoter. Our goal is to design a modified EKLF that binds to the defective delta-globin promoter and enhances delta-globin gene expression. To test the feasibility of this strategy, we inserted the beta-globin CACCC box at -90 of the delta-globin gene promoter to produce an HS2 delta CAC-beta construct and quantitated human delta- and beta-globin mRNA in stably transformed murine erythroleukemia (MEL) cells. delta- Globin mRNA in these cells was 22.0% +/- 9.0% of total human globin mRNA (delta/delta + beta) as compared with 3.0% +/- 1.3% in the HS2 delta-beta control. In a second set of experiments a GAL4 DNA-binding site was inserted at -90 of the delta-globin gene to produce an HS2 delta GAL4-beta construct. This construct and a GAL4(1-147)/EKLF expression vector were stably transfected into MEL cells. delta-Globin mRNA in these cells was 27.8% +/- 7.1% of total human globin mRNA as compared with 9.9% +/- 2.5% in the HS2 delta GAL4-beta plus GAL4(1-147) control. These results show that delta-globin gene expression can be significantly increased by a modified EKLF. Based on these results, we suggest that modified EKLFs, which contain zinc fingers designed to bind specifically to the defective delta-globin CACCC box, may be useful in gene therapy approaches to increase HbA2 levels and inhibit HbS polymerization.

  15. A phase I/II clinical trial of beta-globin gene therapy for beta-thalassemia.

    PubMed

    Bank, Arthur; Dorazio, Ronald; Leboulch, Philippe

    2005-01-01

    Recent success in the long-term correction of mouse models of human beta-thalassemia and sickle cell anemia by lentiviral vectors and evidence of high gene transfer and expression in transduced human hematopoietic cells have led to a first clinical trial of gene therapy for the disease. A LentiGlobin vector containing a beta-globin gene (beta(A-T87Q)) that produces a hemoglobin (Hbbeta(A-T87Q)) that can be distinguished from normal hemoglobin will be used. The LentiGlobin vector is self-inactivating and contains large elements of the beta-globin locus control region as well as chromatin insulators and other features that should prevent untoward events. The study will be done in Paris with Eliane Gluckman as the principal investigator and Philippe Leboulch as scientific director. PMID:16339679

  16. 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

  17. 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

    2016-04-07

    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.

  18. 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

  19. 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

  20. 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

  1. 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

  2. Heterogeneity of DNA fragments associated with the sickle-globin gene.

    PubMed

    Feldenzer, J; Mears, J G; Burns, A L; Natta, C; Bank, A

    1979-09-01

    We have examined the genetic polymorphism previously reported to be associated with the sickle-cell (beta s) gene. The polymorphism involves an alteration of the DNA sequence 3' to the beta-globin gene as detected with the restriction endonuclease, Hpa I. In normal individuals, the beta-globin gene is contained within a DNA fragment of 7.6 kilobases (kb), whereas 87% of individuals with sickle-cell anemia have been reported to have the beta s-gene associated with a 13.0-kb Hpa I fragment. We have studied this polymorphism in 31 New York Black individuals homozygous for sickle-cell anemia to ascertain its genetic and biochemical significance and to evaluate its potential use in the prenatal diagnosis of sickle-cell disease. Our results show only a 58% association of the beta s-gene and the 13.0-kb Hpa I fragment, as well as the presence of additional variants involving the Hpa I site. In addition, the 13.0-kb fragment is also found associated with the beta c- and beta A-genes. Thus, the Hpa I polymorphism probably represents a change in DNA not specifically associated with the beta s-gene, and appears to antedate the beta s-and beta c-mutations.

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

    SciTech Connect

    Hunt, J.A.; Haruyama, A.Z.; Chu, B.M.

    1994-09-01

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

  4. A four base pair deletion 5' to the A gamma T gene is associated not only with decreased expression of the A gamma T-globin gene, but also of the G gamma-globin gene in cis.

    PubMed

    Coleman, M B; Adams, J G; Steinberg, M H; Winter, W P

    1994-12-01

    A four base pair deletion 5' to A gamma T-globin gene at positions -222 to -225 has been reported to reduce the expression of this gene. To evaluate the prevalence and effect of this deletion, PCR-based methods were employed. The deletion had a gene frequency of 0.06 in a sample of African-American individuals with sickle cell trait, 0.18 in adult African-Americans with normal Hb AA, and 0.36 in caucasians. Seventy cord blood samples from African-American newborns with Hb AA were evaluated by both HPLC and PCR. The frequency of the A gamma T allele was 0.13. The A gamma T-globin chain was always present in a lower proportion than the A gamma I allele (70% of A gamma I), but the percentage of A gamma-globin was the same whether or not A gamma T was present. The total percentage of Hb F, however, was significantly lower in the group with the A gamma T allele (77.1% vs. 87.4%, P < 0.01). These results indicate that the four base pair deletion is not only associated with reduced expression of the A gamma T allele, but also of the G gamma allele in cis, further suggesting a possible role of this region in the modulation of the expression of the linked gamma-globin genes.

  5. Restoration of the CCAAT box or insertion of the CACCC motif activates [corrected] delta-globin gene expression.

    PubMed

    Tang, D C; Ebb, D; Hardison, R C; Rodgers, G P

    1997-07-01

    Hemoglobin A2 (HbA2), which contains delta-globin as its non-alpha-globin, represents a minor fraction of the Hb found in normal adults. It has been shown recently that HbA2 is as potent as HbF in inhibiting intracellular deoxy-HbS polymerization, and its expression is therefore relevant to sickle cell disease treatment strategies. To elucidate the mechanisms responsible for the low-level expression of the delta-globin gene in adult erythroid cells, we first compared promoter sequences and found that the delta-globin gene differs from the beta-globin gene in the absence of an erythroid Krüppel-like factor (EKLF) binding site, the alteration of the CCAAT box to CCAAC, and the presence of a GATA-1 binding site. Second, serial deletions of the human delta-globin promoter sequence fused to a luciferase (LUC) reporter gene were transfected into K562 cells. We identified both positive and negative regulatory regions in the 5' flanking sequence. Furthermore, a plasmid containing a single base pair (bp) mutation in the CCAAC box of the delta promoter, restoring the CCAAT box, caused a 5.6-fold and 2.4-fold (P < .05) increase of LUC activity in transfected K562 cells and MEL cells, respectively, in comparison to the wild-type delta promoter. A set of substitutions that create an EKLF binding site centered at -85 bp increased the expression by 26.8-fold and 6.5-fold (P < .05) in K562 and MEL cells, respectively. These results clearly demonstrate that the restoration of either an EKLF binding site or the CCAAT box can increase delta-globin gene expression, with potential future clinical benefit.

  6. 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

  7. Chromatin looping and eRNA transcription precede the transcriptional activation of gene in the β-globin locus

    PubMed Central

    Kim, Yea Woon; Lee, Sungkung; Yun, Jangmi; Kim, AeRi

    2015-01-01

    Enhancers are closely positioned with actively transcribed target genes by chromatin looping. Non-coding RNAs are often transcribed on active enhancers, referred to as eRNAs (enhancer RNAs). To explore the kinetics of enhancer–promoter looping and eRNA transcription during transcriptional activation, we induced the β-globin locus by chemical treatment and analysed cross-linking frequency between the β-globin gene and locus control region (LCR) and the amount of eRNAs transcribed on the LCR in a time course manner. The cross-linking frequency was increased after chemical induction but before the transcriptional activation of gene in the β-globin locus. Transcription of eRNAs was increased in concomitant with the increase in cross-linking frequency. These results show that chromatin looping and eRNA transcription precedes the transcriptional activation of gene. Concomitant occurrence of the two events suggests functional relationship between them. PMID:25588787

  8. Chromatin looping and eRNA transcription precede the transcriptional activation of gene in the β-globin locus.

    PubMed

    Kim, Yea Woon; Lee, Sungkung; Yun, Jangmi; Kim, AeRi

    2015-03-18

    Enhancers are closely positioned with actively transcribed target genes by chromatin looping. Non-coding RNAs are often transcribed on active enhancers, referred to as eRNAs (enhancer RNAs). To explore the kinetics of enhancer-promoter looping and eRNA transcription during transcriptional activation, we induced the β-globin locus by chemical treatment and analysed cross-linking frequency between the β-globin gene and locus control region (LCR) and the amount of eRNAs transcribed on the LCR in a time course manner. The cross-linking frequency was increased after chemical induction but before the transcriptional activation of gene in the β-globin locus. Transcription of eRNAs was increased in concomitant with the increase in cross-linking frequency. These results show that chromatin looping and eRNA transcription precedes the transcriptional activation of gene. Concomitant occurrence of the two events suggests functional relationship between them.

  9. 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

  10. Gamma thalassemia resulting from the deletion of a gamma-globin gene.

    PubMed Central

    Sukumaran, P K; Nakatsuji, T; Gardiner, M B; Reese, A L; Gilman, J G; Huisman, T H

    1983-01-01

    The first example of a deletion of one of the two gamma globin genes has been characterized through an analysis of the DNA of the heterozygous parent of a homozygous newborn, using restriction endonuclease mapping techniques. A deletion of approximately 5 kb was observed which was probably caused by an unequal crossing-over between the -G gamma- and -A gamma- genes resulting in the formation of a -G gamma A gamma- hybrid gene. Data on proportions of G gamma and A gamma chains in newborn babies assumed to be heterozygous for the hybrid and normal genes suggest that this hybrid gene may be producing its A gamma chain at levels normally seen only for the G gamma chain. Images PMID:6306596

  11. 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.

  12. 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

  13. 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.

  14. Beta-globin gene evolution in the ruminants: evidence for an ancient origin of sheep haplotype B.

    PubMed

    Jiang, Y; Wang, X; Kijas, J W; Dalrymple, B P

    2015-10-01

    Domestic sheep (Ovis aries) can be divided into two groups with significantly different responses to hypoxic environments, determined by two allelic beta-globin haplotypes. Haplotype A is very similar to the goat beta-globin locus, whereas haplotype B has a deletion spanning four globin genes, including beta-C globin, which encodes a globin with high oxygen affinity. We surveyed the beta-globin locus using resequencing data from 70 domestic sheep from 42 worldwide breeds and three Ovis canadensis and two Ovis dalli individuals. Haplotype B has an allele frequency of 71.4% in O. aries and was homozygous (BB) in all five wild sheep. This shared ancestry indicates haplotype B is at least 2-3 million years old. Approximately 40 kb of the sequence flanking the ~37-kb haplotype B deletion had unexpectedly low identity between haplotypes A and B. Phylogenetic analysis showed that the divergent region of sheep haplotype B is remarkably distinct from the beta-globin loci in goat and cattle but still groups with the Ruminantia. We hypothesize that this divergent ~40-kb region in haplotype B may be from an unknown ancestral ruminant and was maintained in the lineage to O. aries, but not other Bovidae, evolving independently of haplotype A. Alternatively, the ~40-kb sequence in haplotype B was more recently acquired by an ancestor of sheep from an unknown non-Bovidae ruminant, replacing part of haplotype A. Haplotype B has a lower nucleotide diversity than does haplotype A, suggesting a recent bottleneck, whereas the higher frequency of haplotype B suggests a subsequent spread through the global population of O. aries.

  15. Beta-globin gene evolution in the ruminants: evidence for an ancient origin of sheep haplotype B.

    PubMed

    Jiang, Y; Wang, X; Kijas, J W; Dalrymple, B P

    2015-10-01

    Domestic sheep (Ovis aries) can be divided into two groups with significantly different responses to hypoxic environments, determined by two allelic beta-globin haplotypes. Haplotype A is very similar to the goat beta-globin locus, whereas haplotype B has a deletion spanning four globin genes, including beta-C globin, which encodes a globin with high oxygen affinity. We surveyed the beta-globin locus using resequencing data from 70 domestic sheep from 42 worldwide breeds and three Ovis canadensis and two Ovis dalli individuals. Haplotype B has an allele frequency of 71.4% in O. aries and was homozygous (BB) in all five wild sheep. This shared ancestry indicates haplotype B is at least 2-3 million years old. Approximately 40 kb of the sequence flanking the ~37-kb haplotype B deletion had unexpectedly low identity between haplotypes A and B. Phylogenetic analysis showed that the divergent region of sheep haplotype B is remarkably distinct from the beta-globin loci in goat and cattle but still groups with the Ruminantia. We hypothesize that this divergent ~40-kb region in haplotype B may be from an unknown ancestral ruminant and was maintained in the lineage to O. aries, but not other Bovidae, evolving independently of haplotype A. Alternatively, the ~40-kb sequence in haplotype B was more recently acquired by an ancestor of sheep from an unknown non-Bovidae ruminant, replacing part of haplotype A. Haplotype B has a lower nucleotide diversity than does haplotype A, suggesting a recent bottleneck, whereas the higher frequency of haplotype B suggests a subsequent spread through the global population of O. aries. PMID:26096044

  16. Molecular spectrum of α-globin gene mutations in the Aegean region of Turkey: first observation of three α-globin gene mutations in the Turkish population.

    PubMed

    Onay, Hüseyin; Aykut, Ayça; Karaca, Emin; Durmaz, Asude; Solmaz, Aslı Ece; Çoğulu, Özgür; Aydınok, Yeşim; Vergin, Canan; Özkınay, Ferda

    2015-07-01

    Molecular test results of 231 individuals referred to our molecular genetics laboratory for analysis of α-globin gene mutations between the years 2007 and 2013 were evaluated. Analysis of α-thalassemia gene mutations was performed using reverse dot-blot hybridisation, which includes 21 common mutations. Twelve distinct α-thalassemia mutations and 23 different genotypes have been detected in the Aegean region of Turkey. The most frequent mutations were -α3.7 (52.28 %), -(α)20.5 (14.74 %), --MED (10.53 %), and αPA-1α (8.77 %). Three α-thalassemia mutations (αcd142α, --SEA, and αICα), which are more prevalent in Southeast Asia, are identified for the first time in Turkey in this study. We find that a broad spectrum of α-thalassemia mutations is present in the Aegean region of Turkey. The results obtained in this study may help inform decisions in the design and implementation of prevention strategies and diagnostic approaches.

  17. 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].

  18. A mutation of the beta-globin gene initiation codon, ATG-->AAG, found in a French Caucasian man.

    PubMed

    Lacan, Philippe; Aubry, Martine; Couprie, Nicole; Francina, Alain

    2005-01-01

    A new mutation of the beta-globin gene initiation codon, ATG-->AAG (Met-->Tyr), is reported in a man originating from the southeast of France. Typical hematological findings of beta-thalassemia (thal) trait were found. We emphasize the importance of characterizing uncommon beta-thal mutations for genetic counseling.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. Screening for trans-acting factors and other factors involved in the activating or silencing of the gamma-globin gene during human ontogeny.

    PubMed

    Ma, Yan-Ni; Zhang, Xin; Zhang, Jun-Wu; Zhang, Xin-Hua; Wang, Rong-Xin

    2007-06-01

    Researchers hope to increase gamma-globin expression by controlling potential trans-acting factors that specifically activate the gamma-globin gene in fetuses or silence this gene in adults to potentially treat sickle cell disease and beta-thalassemias. To characterize genes encoding such factors, we analyzed the differential expression of mRNAs in erythroid induction cultures of CD34+ cells derived from normal adult bone marrow, umbilical cord blood, and bone marrow from a patient with heterocellular hereditary persistence of fetal hemoglobin. Using differential-display - reverse-transcription PCR analysis, we identified a number of genes with differential expression in the above-mentioned cells. The differential expression of some genes was also confirmed by quantitative real-time PCR. Our data provide important clues for identifying and validating trans-activators that activate the gamma-globin gene in fetuses, and trans-acting factors involved in silencing the gamma-globin gene in adults.

  4. 1990 Mack Forster Prize lecture. The molecular genetics of the alpha globin gene family.

    PubMed

    Higgs, D R

    1990-08-01

    The naturally occurring mutants described here provide an excellent opportunity for elucidating the relationship between structure and function of the alpha globin complex and the larger chromosomal region 16p13.3. From a practical point of view it is important to remember that millions of individuals throughout the world are carriers for alpha thalassaemia and every year many thousands of pregnancies are at risk of producing children with the severe alpha thalassaemia syndromes. The data summarized here provide the basis for accurately predicting the genotype in such cases and thus enabling appropriate prenatal testing. The less common larger rearrangements involving chromosomal band 16p13.3 may provide information on the nature of other genes that surround the alpha complex. Furthermore, the mechanism by which they have occurred provide some new and more general insights into the possible causes of other forms of unexplained mental handicap.

  5. 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.

  6. 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

  7. Sequential changes in chromatin structure during transcriptional activation in the beta globin LCR and its target gene.

    PubMed

    Kim, Kihoon; Kim, AeRi

    2010-09-01

    Chromatin structure is modulated during transcriptional activation. The changes include the association of transcriptional activators, formation of hypersensitive sites and covalent modifications of histones. To understand the order of the various changes accompanying transcriptional activation, we analyzed the mouse beta globin gene, which is transcriptionally inducible in erythroid MEL cells over a time course of HMBA treatment. Transcription of the globin genes requires the locus control region (LCR) consisting of several hypersensitive sites (HSs). Erythroid specific transcriptional activators such as NF-E2, GATA-1, TAL1 and EKLF were associated with the LCR in the uninduced state before transcriptional activation. The HSs of the LCR were formed in this state as revealed by high sensitivity to DNase I and MNase attack. However the binding of transcriptional activators and the depletion of histones were observed in the promoter of the beta globin gene only after transcriptional activation. In addition, various covalent histone modifications were sequentially detected in lysine residues of histone H3 during the activation. Acetylation of K9, K36 and K27 was notable in both LCR HSs and gene after induction but before transcriptional initiation. Inactive histone marks such as K9me2, K36me2 and K27me2 were removed coincident with transcriptional initiation in the gene region. Taken together, these results indicate that LCR has a substantially active structure in the uninduced state while transcriptional activation serially adds active marks, including histone modifications, and removes inactive marks in the target gene of the LCR.

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

    PubMed

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

    1994-10-11

    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.

  9. 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

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

    PubMed

    Das, S K; Talukder, G

    2001-01-01

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

  11. 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.

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

    PubMed

    Schwarze, Kim; Singh, Abhilasha; Burmester, Thorsten

    2015-07-01

    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

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

    PubMed

    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

  14. 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

  15. Genetic Heterogeneity of the β-Globin Gene in Various Geographic Populations of Yunnan in Southwestern China

    PubMed Central

    Zhang, Jie; He, Jing; Zeng, Xiao-Hong; Ge, Shi-Jun; Huang, Yu; Su, Jie; Ding, Xue-Mei; Yang, Ji-Qing; Cao, Yong-Jiu; Chen, Hong; Zhang, Ying-Hong; Zhu, Bao-Sheng

    2015-01-01

    Objectives The aim of this study was to investigate the geographic distribution of β-globin gene mutations in different ethnic groups in Yunnan province. Methods From 2004 to 2014, 1,441 subjects with hemoglobin disorders, identified by PCR-reverse dot blot and DNA sequencing, were studied according to ethnicity and geographic origin. Haplotypes were examined among 41 unrelated thalassemia chromosomes. Results Eighteen β-thalassemia mutations and seven hemoglobin variants were identified for 1,616 alleles in 22 different ethnic groups from all 16 prefecture-level divisions of Yunnan. The prevalence of β-thalassemia was heterogeneous and regionally specific. CD 41-42 (-TCTT) was the most prevalent mutation in the populations of northeastern Yunnan. CD 17 (A>T) was the most common mutation in the populations of southeastern Yunnan, especially for the Zhuang minority, whereas Hb E (CD 26, G>A) was the most prevalent mutation in populations of southwestern Yunnan, especially for the Dai minority. Among the seven types of haplotypes identified, CD 17 (A>T) was mainly linked to haplotype VII (+ - - - - - +) and IVS-II-654 (C>T) was only linked to haplotype I (+ - - - - + +). Conclusion Our data underline the heterogeneity of β-globin gene mutations in Yunnan. This distribution of β-globin mutations in the geographic regions and ethnic populations provided a detailed ethnic basis and evolutionary view of humans in southern China, which will be beneficial for genetic counseling and prevention strategies. PMID:25849334

  16. Strategy for a multicenter phase I clinical trial to evaluate globin gene transfer in beta-thalassemia.

    PubMed

    Sadelain, Michel; Rivière, Isabelle; Wang, Xiuyan; Boulad, Farid; Prockop, Susan; Giardina, Patricia; Maggio, Aurelio; Galanello, Renzo; Locatelli, Franco; Yannaki, Evangelia

    2010-08-01

    Globin gene transfer in autologous hematopoietic stem cells offers a potentially curative treatment option for patients suffering from beta-thalassemia major who lack an HLA-matched hematopoietic stem cell donor. Based on extensive preclinical investigation, we are initiating a phase I clinical trial using G-CSF mobilized, autologous CD34(+) cells transduced with a vector similar to the original TNS9 vector. Our first mobilizations in adult beta-thalassemic subjects have been well tolerated and yielded the required CD34(+) cell dose. To minimize toxicity to enrolled subjects, and in the absence of a demonstrated requirement for myeloablative conditioning, our trial will use a reduced intensity conditioning regimen. Because low vector titers may adversely affect efficacy and safety, we have focused on vector manufacturing processes. We are now in a position to transfer our globin lentiviral vectors in a clinically relevant dosage (averaging 0.8 vector copy per cell in bulk CD34(+) cells) and to supply clinical grade vector to collaborating centers in the U.S.A. and in Europe. We anticipate that the first U.S. trial of globin gene transfer will start in 2010.

  17. 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.

  18. 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

  19. Spectrum of alpha-globin gene mutations among premarital Baluch couples in southeastern Iran

    PubMed Central

    Miri-Moghaddam, Ebrahim; Nikravesh, Abass; Gasemzadeh, Negin; Badaksh, Mahin; Rakhshi, Nahid

    2015-01-01

    Background: Alpha thalassemia (α-thal) is one of the most common hemoglobinopathies worldwide. The aim of this study was to investigate the spectrum of α-thal mutations among premarital Baluch couples in southeastern Iran. Subjects and Methods: We assessed 1215 individuals by multiplex gap polymerase chain reaction (gap-PCR) and amplification refractory mutation system (ARMS-PCR). Results: Of the 1215 participants with mean age of 23±5.7 years, 62.3% lived in urban areas, and the rate of consanguineous marriage was 68.1%. Five mutations were identified, the most frequent one was –α3.7 (rightward) with a frequency of 76.5%, followed by α−5 nt (16.8%), α2/ Codon 19(-G) (4%), –α4.2 (leftward)(2.4%), – –MED (0.3%) among mutated alleles of the α -globin gene. Conclusion : Knowing the alpha-genotype is helpful for genetic counseling, microcytic anemia discrimination and hemoglobinopathy prevention. PMID:26261699

  20. Novel technique for rapid detection of alpha-globin gene mutations and deletions.

    PubMed

    Liu, Jingzhong; Jia, Xingyuan; Tang, Ning; Zhang, Xu; Wu, Xiaoyi; Cai, Ren; Wang, Lirong; Liu, Quanzhang; Xiao, Bai; Zhu, Jim; Wang, Qingtao

    2010-03-01

    Populations in Southeast Asia and South China have high frequencies of alpha-thalassemia caused by alpha-globin gene mutations and/or deletions. This study was designed to find an efficient and simple diagnostic test for the mutations and deletions. A duplex polymerase chain reaction (PCR)/denaturing high-pressure liquid chromatography (DHPLC) was used to detect the mutations and deletions. A blinded study of 110 samples, which included 92 alpha-thalassemia samples with various genotypes and 18 normal DNA samples, was carried out by the methods. The duplex PCR products of the sample with known Constand spring mutation (CS)/alphaalpha, Quonsze mutation (QS)/alphaalpha, and Weastmead mutation (WS)/alphaalpha DNA showed significantly different profiles, which suggests that DHPLC analysis at 63.8 degrees C can detect potential mutations directly. The DHPLC at 50 degrees C analysis can distinguish the --SEA and nondeletional alleles. The new assay is 100% concordant with the original genotype. In conclusion, the technique including the duplex PCR assay followed by DHPLC analysis can be used to diagnose alpha-thalassemia; this methodology is simple, rapid, accurate, semiautomatic, and high output, and thus, it is suitable for large-scale screening.

  1. A globin enhancer acts by increasing the proportion of erythrocytes expressing a linked transgene.

    PubMed Central

    Sutherland, H G; Martin, D I; Whitelaw, E

    1997-01-01

    Enhancer elements have been shown to affect the probability of a gene establishing an active transcriptional state and suppress the silencing of reporter genes in cell lines, but their effect in transgenic mice has been obscured by the use of assays that do not assess expression on a cell-by-cell basis. We have examined the effect of a globin enhancer on the variegation of lacZ expression in erythrocytes of transgenic mice. Mice carrying lacZ driven by the alpha-globin promoter exhibit beta-galactosidase (beta-Gal) expression in only a very small proportion of embryonic erythrocytes. When the transgenic construct also contains the (alphaHS-40 enhancer, which controls expression of the alpha-globin gene, expression is seen in a high proportion of embryonic erythrocytes, although there are variations between transgenic lines which can be attributed to different sites of integration. Analysis of beta-Gal expression levels suggests that expressing cells in lines carrying only the alpha-globin promoter express as much beta-Gal as those in which the transgene also contains alphaHS-40. A marked decline in transgene expression occurs as mice age, which is mainly due to a decrease in the proportion of cells expressing the transgene. Thus, a globin enhancer can act to suppress variegation of a linked transgene; this result is consistent with a model in which enhancers act to establish and maintain an active domain without directly affecting the transcriptional rate. PMID:9032288

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

    PubMed

    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

  3. 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

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

    PubMed

    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.

  5. (AC)n dinucleotide repeat polymorphism in 5' beta-globin gene in native and Mestizo Mexican populations.

    PubMed

    Peñaloza, R; Delgado, P; Arenas, D; Barrientos, C; Buentello, L; Loeza, F; Salamanca, F

    2001-12-01

    Repeated sequences are dispersed along the human genome. These sequences are useful as markers in diagnosis of inherited diseases, in forensic medicine, and in tracking the origin and evolution of human populations. The (AC)n repeated element is the most frequent in the human genome. In this paper, the (AC)n repeated element located in the 5' flanking region of the beta-globin gene was studied by single-strand conformation polymorphism (SSCP). Four ethnic Mexican groups (Mixteca, Nahua, Otomí, Purépecha) and a Mestizo population were analyzed. We observed three alleles, A [(AC)16, B [(AC)14], and C [(AC)18], with a frequency of between 68.2% and 86.9%, 13.1% and 18.2%, and 6.7% and 13.7%, respectively. Allele C was present only in Purépecha and Mestizo groups. The absence of this allele in the other ethnic groups studied suggests that there is low genetic admixture of Purépecha and that this is a relatively isolated population. However, it could be that the C allele occurs in low frequencies in the other groups as a result of small sample sizes. The (AC)n repeat polymorphism in the beta-globin gene has not been previously studied in Amerindian populations.

  6. 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

  7. The beta-globin gene cluster haplotypes in sickle cell anemia patients from Northeast Brazil: a clinical and molecular view.

    PubMed

    Adorno, Elisângela Vitória; Zanette, Angela; Lyra, Isa; Souza, Cyntia Cajado; Santos, Leandro Ferraz; Menezes, Joelma Figueiredo; Dupuit, Marie France; Almeida, Mari Ney Tavares; Reis, Mitermayer Galvão; Gonçalves, Marilda Souza

    2004-08-01

    The beta(S)-globin haplotypes were studied in 78 sickle cell Brazilian patients from Bahia, Northeast Brazil, that has a large population of African origin. Hemoglobin (Hb) profiles were developed by high-performance liquid chromatography (HPLC), and beta(S)-globin gene haplotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. We identified 44 (55.0%) patients with the CAR/Ben (Central African Republic/Benin) genotype, 16 (20.0%) Ben/Ben, 13 (16.2%) CAR/CAR and seven (8.8%) with other genotypes. Analyses of the phenotypes showed clinical differences related only to Hb F levels and blood transfusion therapy; the presence of -alpha(-3.7)-thalassemia (thal) demonstrated statistical significance when associated with hematocrit (p=0.044), MCV (p=0.0007), MCH (p=0.012) and spleen sequestration events. The haplotype diversity found in the present study can be justified by information about the origin of the slave traffic period in Bahia during the 19th century. The specific characteristics described among the Bahian sickle cell patients could be confirmed by increasing the number of patients with specific genotypes and further studies of genetic markers.

  8. 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.

  9. 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

  10. Hb Filottrano [codon 120 (-A)]: a novel frameshift mutation in exon 3 of the β-globin gene causing dominantly inherited β-thalassemia intermedia.

    PubMed

    Amato, Antonio; Cappabianca, Maria Pia; Perri, Maria; Zaghis, Ivo; Mastropietro, Fabrizio; Ponzini, Donatella; Di Biagio, Paola; Piscitelli, Roberta

    2012-01-01

    We report a novel frameshift mutation in exon 3 of the β-globin gene, that, in the heterozygous state, leads to a β-thalassemia intermedia (β-TI) phenotype (marked anemia, splenomegaly, hyperbilirubinemia, jaundice, unbalanced synthesis of α/non-α chains in a 34-year-old Italian woman. This frameshift mutation, due to the deletion of the first nucleotide (-A) at codon 120, results in a β-globin chain that is elongated to 156 amino acid residues. These highly unstable abnormal chains precipitate in the erythroblasts as inclusion bodies, thus causing inefficient erythropoiesis and ultimately resulting in the observed dominant clinical phenotype.

  11. 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

  12. Site-specific base changes in the coding or promoter region of the human beta- and gamma-globin genes by single-stranded oligonucleotides.

    PubMed

    Yin, Wenxuan; Kren, Betsy T; Steer, Clifford J

    2005-08-15

    SSOs (single-stranded oligonucleotides) can mediate site-specific alteration of base-pairs in episomal and chromosomal target genes in mammalian cells. The TNE (targeted nucleotide exchange) can result in either repair or mutation of a gene sequence and is mediated through endogenous DNA repair pathway(s). Thus the approach provides a technique for the treatment of monogenic disorders associated with specific point mutations such as SCD (sickle cell disease). We studied the potential application of SSOs for SCD by introducing either an A to T substitution at the sixth codon of the human beta-globin gene (sickle locus) or a C to G mutation at -202 of the Ggamma-globin gene promoter region. The latter TNE is an alternative strategy to ameliorate the clinical manifestations of sickle cell anaemia by re-activating fetal haemoglobin gene expression in adult erythrocytes. A sensitive and valid PCR assay system was developed, which allows detection of point mutations as low as 0.01% at these sites. Using this system, TNE between 0.01 and 0.1% at the sickle locus or gamma-globin gene promoter region was detected after transfection with SSOs in cultured human cell lines. TNE in the Ggamma-globin promoter region exhibited varying degrees of strand bias that was dependent on SSO design and the cell's DNA mismatch repair activity. The results suggest that the endogenous DNA repair machinery may permit SSO correction of the sickle defect by modification of the beta- and/or gamma-globin genes.

  13. 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.

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

    PubMed

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

    1998-04-15

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

  15. 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

  16. 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.

  17. 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

  18. Potentially therapeutic levels of anti-sickling globin gene expression following lentivirus-mediated gene transfer in sickle cell disease bone marrow CD34+ cells.

    PubMed

    Urbinati, Fabrizia; Hargrove, Phillip W; Geiger, Sabine; Romero, Zulema; Wherley, Jennifer; Kaufman, Michael L; Hollis, Roger P; Chambers, Christopher B; Persons, Derek A; Kohn, Donald B; Wilber, Andrew

    2015-05-01

    Sickle cell disease (SCD) can be cured by allogeneic hematopoietic stem cell transplant. However, this is only possible when a matched donor is available, making the development of gene therapy using autologous hematopoietic stem cells a highly desirable alternative. We used a culture model of human erythropoiesis to directly compare two insulated, self-inactivating, and erythroid-specific lentiviral vectors, encoding for γ-globin (V5m3-400) or a modified β-globin (βAS3-FB) for production of antisickling hemoglobin (Hb) and correction of red cell deformability after deoxygenation. Bone marrow CD34+ cells from three SCD patients were transduced using V5m3-400 or βAS3-FB and compared with mock-transduced SCD or healthy donor CD34+ cells. Lentiviral transduction did not impair cell growth or differentiation, as gauged by proliferation and acquisition of erythroid markers. Vector copy number averaged approximately one copy per cell, and corrective globin mRNA levels were increased more than sevenfold over mock-transduced controls. Erythroblasts derived from healthy donor and mock-transduced SCD cells produced a low level of fetal Hb that was increased to 23.6 ± 4.1% per vector copy for cells transduced with V5m3-400. Equivalent levels of modified normal adult Hb of 17.6 ± 3.8% per vector copy were detected for SCD cells transduced with βAS3-FB. These levels of antisickling Hb production were sufficient to reduce sickling of terminal-stage red blood cells upon deoxygenation. We concluded that the achieved levels of fetal Hb and modified normal adult Hb would likely prove therapeutic to SCD patients who lack matched donors.

  19. α(+)-Thalassemia Due to a Frameshift Mutation of the α2-Globin Gene [codons 55/56 (+T) or HBA2: c.168dup].

    PubMed

    Waye, John S; Eng, Barry; Hanna, Meredith; Hohenadel, Betty-Ann; Nakamura, Lisa M; Walker, Lynda

    2015-01-01

    We report a case of α(+)-thalassemia (α(+)-thal) trait in a Chinese-Canadian family caused by a novel frameshift mutation of the α2-globin gene, specifically the duplication of a single nucleotide at amino acid codon 56 [HBA2: c.168dup]. The mutation results in substitution of a termination codon (TAA) for lysine (AAG) at amino acid position 56.

  20. Concordance of a point mutation 5' to the A gamma-globin gene with A gamma beta + hereditary persistence of fetal hemoglobin in Greeks.

    PubMed

    Waber, P G; Bender, M A; Gelinas, R E; Kattamis, C; Karaklis, A; Sofroniadou, K; Stamatoyannopoulos, G; Collins, F S; Forget, B G; Kazazian, H H

    1986-02-01

    In the Greek A gamma beta + type of hereditary persistence of fetal hemoglobin (HPFH), adult heterozygotes produce about 20% fetal hemoglobin (HbF), which is predominantly of the A gamma chain variety. The affected beta-globin gene cluster produces near normal amounts of beta-like globin, but in a A gamma to beta ratio of 20:80 instead of 0.5:99.5. Gelinas et al and Collins et al have shown a G to A change 117 nucleotides 5' to the A gamma gene in two Greeks with A gamma beta + HPFH. To demonstrate that this change is not a neutral polymorphism, we carried out hybridization with oligonucleotide probes (19mers) specific for the normal and the mutant sequences. While normal probe identified the A gamma fragment in genomic DNA of all subjects studied, mutant probe was positive only in Greeks with A gamma beta + HPFH. In sum, 108 beta-globin gene clusters of individuals without HPFH were negative when tested with mutant probe, but all 11 affected individuals of six families with Greek A gamma beta + HPFH (two previously sequenced and four new families) were positive with mutant probe. These data support the conclusion that the -117 mutation is causative of A gamma beta + HPFH in Greeks.

  1. Microbial eukaryote globins.

    PubMed

    Vinogradov, Serge N; Bailly, Xavier; Smith, David R; Tinajero-Trejo, Mariana; Poole, Robert K; Hoogewijs, David

    2013-01-01

    A bioinformatics survey of about 120 protist and 240 fungal genomes and transcriptomes revealed a broad array of globins, representing five of the eight subfamilies identified in bacteria. Most conspicuous is the absence of protoglobins and globin-coupled sensors, except for a two-domain globin in Leishmanias, that comprises a nucleotidyl cyclase domain, and the virtual absence of truncated group 3 globins. In contrast to bacteria, co-occurrence of more than two globin subfamilies appears to be rare in protists. Although globins were lacking in the Apicomplexa and the Microsporidia intracellular pathogens, they occurred in the pathogenic Trypanosomatidae, Stramenopiles and certain fungi. Flavohaemoglobins (FHbs) and related single-domain globins occur across the protist groups. Fungi are unique in having FHbs co-occurring with sensor single-domain globins (SSDgbs). Obligately biotrophic fungi covered in our analysis lack globins. Furthermore, SSDgbs occur only in a heterolobosean amoeba, Naegleria and the stramenopile Hyphochytrium. Of the three subfamilies of truncated Mb-fold globins, TrHb1s appear to be the most widespread, occurring as multiple copies in chlorophyte and ciliophora genomes, many as multidomain proteins. Although the ciliates appear to have only TrHb1s, the chlorophytes have Mb-like globins and TrHb2s, both closely related to the corresponding plant globins. The presently available number of protist genomes is inadequate to provide a definitive census of their globins. Bayesian molecular analyses of single-domain 3/3 Mb-fold globins suggest a close relationship of chlorophyte and haptophyte globins, including choanoflagellate and Capsaspora globins to land plant symbiotic and non-symbiotic haemoglobins and to vertebrate neuroglobins.

  2. Caffeine exposure alters cardiac gene expression in embryonic cardiomyocytes.

    PubMed

    Fang, Xiefan; Mei, Wenbin; Barbazuk, William B; Rivkees, Scott A; Wendler, Christopher C

    2014-12-15

    Previous studies demonstrated that in utero caffeine treatment at embryonic day (E) 8.5 alters DNA methylation patterns, gene expression, and cardiac function in adult mice. To provide insight into the mechanisms, we examined cardiac gene and microRNA (miRNA) expression in cardiomyocytes shortly after exposure to physiologically relevant doses of caffeine. In HL-1 and primary embryonic cardiomyocytes, caffeine treatment for 48 h significantly altered the expression of cardiac structural genes (Myh6, Myh7, Myh7b, Tnni3), hormonal genes (Anp and BnP), cardiac transcription factors (Gata4, Mef2c, Mef2d, Nfatc1), and microRNAs (miRNAs; miR208a, miR208b, miR499). In addition, expressions of these genes were significantly altered in embryonic hearts exposed to in utero caffeine. For in utero experiments, pregnant CD-1 dams were treated with 20-60 mg/kg of caffeine, which resulted in maternal circulation levels of 37.3-65.3 μM 2 h after treatment. RNA sequencing was performed on embryonic ventricles treated with vehicle or 20 mg/kg of caffeine daily from E6.5-9.5. Differential expression (DE) analysis revealed that 124 genes and 849 transcripts were significantly altered, and differential exon usage (DEU) analysis identified 597 exons that were changed in response to prenatal caffeine exposure. Among the DE genes identified by RNA sequencing were several cardiac structural genes and genes that control DNA methylation and histone modification. Pathway analysis revealed that pathways related to cardiovascular development and diseases were significantly affected by caffeine. In addition, global cardiac DNA methylation was reduced in caffeine-treated cardiomyocytes. Collectively, these data demonstrate that caffeine exposure alters gene expression and DNA methylation in embryonic cardiomyocytes.

  3. Beta-globin gene cluster haplotypes and HbF levels are not the only modulators of sickle cell disease in Lebanon.

    PubMed

    Inati, A; Taher, A; Bou Alawi, W; Koussa, S; Kaspar, H; Shbaklo, H; Zalloua, P A

    2003-02-01

    Sickle cell disease (SCD) is an inherited autosomal recessive disorder of the beta-globin chain. Despite the fact that all subjects with SCD have the same single base pair mutation, the severity of the clinical and hematological manifestations is extremely variable. This study examined for the first time in Lebanon the correlation between the clinical manifestation of SCD and the beta-globin gene haplotypes. The haplotypes of 50 patients diagnosed with SCD were determined using polymerase chain reaction amplification of fragments containing nine polymorphic restriction sites around and within the epsilon-Ggamma-Agamma-psibeta-delta-beta-globin gene complex. Most reported haplotypes were found in our population with the Benin haplotype as the most prevalent one. When the patients were divided according to their HbF levels into three groups (Group A: HbF < 5%, Group B: HbF between 5 and 15%, and Group C: HbF > 15%), surprisingly, the highest levels of HbF were associated with the most severe clinical cases. Our findings suggest that fetal hemoglobin levels are important but not the only parameters that affect the severity of the disease. In addition, the high levels of HbF in patients with CAR haplotypes did not seem to ameliorate the severity of symptoms, suggesting that genetic factors other than haplotypes are the major determinants of increased HbF levels in Lebanon.

  4. Mutation in the US -globin gene detected in the progeny of a female mouse treated with ethylnitrosourea

    SciTech Connect

    Lewis, S.E.; Johnson, F.M.; Skow, L.C.; Popp, D.; Barnett, L.B.; Popp, R.A.

    1985-09-01

    A mouse with a variant hemoglobin was discovered during electrophoretic screening of (C57BL/6J x DBA/2J) F1 progeny of females treated with ethylnitrosourea. The variant trait was transmitted as a simple Mendelian alternate at the Hbb locus in all crosses except those involving the original carrier of the mutation. The proband mouse which received the mutation directly from the mutagen-treated parent was a germinal mosaic for the mutant and normal Hbb/sup s/ alleles. The mutant allele was designated Hbb/sup s2/. The mutant haplotype specifies both an electrophoretically fast hemoglobin band and a hemoglobin band in the normal US /sup single/ hemoglobin position. A comparison of the relative concentrations of the two hemoglobins in Hbb/sup s2/ mice demonstrates preferential expression of the mutant gene. Analysis of the amino acid sequence of the variant US -globin revealed that the valine at position 60 was changed to glutamic acid. The simplest mutation mechanisms for such an alteration is A x T T x A transversion.

  5. Evolution of the mammalian embryonic pluripotency gene regulatory network.

    PubMed

    Fernandez-Tresguerres, Beatriz; Cañon, Susana; Rayon, Teresa; Pernaute, Barbara; Crespo, Miguel; Torroja, Carlos; Manzanares, Miguel

    2010-11-16

    Embryonic pluripotency in the mouse is established and maintained by a gene-regulatory network under the control of a core set of transcription factors that include octamer-binding protein 4 (Oct4; official name POU domain, class 5, transcription factor 1, Pou5f1), sex-determining region Y (SRY)-box containing gene 2 (Sox2), and homeobox protein Nanog. Although this network is largely conserved in eutherian mammals, very little information is available regarding its evolutionary conservation in other vertebrates. We have compared the embryonic pluripotency networks in mouse and chick by means of expression analysis in the pregastrulation chicken embryo, genomic comparisons, and functional assays of pluripotency-related regulatory elements in ES cells and blastocysts. We find that multiple components of the network are either novel to mammals or have acquired novel expression domains in early developmental stages of the mouse. We also find that the downstream action of the mouse core pluripotency factors is mediated largely by genomic sequence elements nonconserved with chick. In the case of Sox2 and Fgf4, we find that elements driving expression in embryonic pluripotent cells have evolved by a small number of nucleotide changes that create novel binding sites for core factors. Our results show that the network in charge of embryonic pluripotency is an evolutionary novelty of mammals that is related to the comparatively extended period during which mammalian embryonic cells need to be maintained in an undetermined state before engaging in early differentiation events.

  6. Gene targeting in embryonic stem cells, II: conditional technologies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genome modification via transgenesis has allowed researchers to link genotype and phenotype as an alternative approach to the characterization of random mutations through evolution. The synergy of technologies from the fields of embryonic stem (ES) cells, gene knockouts, and protein-mediated recombi...

  7. Cancer Genes Hypermethylated in Human Embryonic Stem Cells

    PubMed Central

    Calvanese, Vincenzo; Horrillo, Angelica; Hmadcha, Abdelkrim; Suarez-Álvarez, Beatriz; Fernandez, Agustín F.; Lara, Ester; Casado, Sara; Menendez, Pablo; Bueno, Clara; Garcia-Castro, Javier; Rubio, Ruth; Lapunzina, Pablo; Alaminos, Miguel; Borghese, Lodovica; Terstegge, Stefanie; Harrison, Neil J.; Moore, Harry D.; Brüstle, Oliver; Lopez-Larrea, Carlos; Andrews, Peter W.; Soria, Bernat; Esteller, Manel; Fraga, Mario F.

    2008-01-01

    Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation. PMID:18820729

  8. Developmental regulation of embryonic genes in plants

    SciTech Connect

    Borkird, C.; Choi, Jung, H.; Jin, Zhenghua; Franz, G.; Hatzopoulos, P.; Chorneaus, R.; Bonas, U.; Pelegri, F.; Sung, Z.R.

    1988-09-01

    Somatic embryogenesis from cultured carrot cells progresses through successive morphogenetic stages termed globular, heart, and torpedo. To understand the molecular mechanisms underlying plant embryogenesis, the authors isolated two genes differentially expressed during embryo development. The expression of these two genes is associated with heart-stage embryogenesis. By altering the culture conditions and examining their expressions in a developmental variant cell line, they found that these genes were controlled by the developmental program of embryogenesis and were not directly regulated by 2,4-dichlorophenoxyacetic acid, the growth regulator that promotes unorganized growth of cultured cells and suppresses embryo morphogenesis. These genes are also expressed in carrot zygotic embryos but not in seedlings or mature plants.

  9. 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

  10. 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

  11. Telencephalic embryonic subtractive sequences: a unique collection of neurodevelopmental genes.

    PubMed

    Bulfone, Alessandro; Carotenuto, Pietro; Faedo, Andrea; Aglio, Veruska; Garzia, Livia; Bello, Anna Maria; Basile, Andrea; Andrè, Alessandra; Cocchia, Massimo; Guardiola, Ombretta; Ballabio, Andrea; Rubenstein, John L R; Zollo, Massimo

    2005-08-17

    The vertebrate telencephalon is composed of many architectonically and functionally distinct areas and structures, with billions of neurons that are precisely connected. This complexity is fine-tuned during development by numerous genes. To identify genes involved in the regulation of telencephalic development, a specific subset of differentially expressed genes was characterized. Here, we describe a set of cDNAs encoded by genes preferentially expressed during development of the mouse telencephalon that was identified through a functional genomics approach. Of 832 distinct transcripts found, 223 (27%) are known genes. Of the remaining, 228 (27%) correspond to expressed sequence tags of unknown function, 58 (7%) are homologs or orthologs of known genes, and 323 (39%) correspond to novel rare transcripts, including 48 (14%) new putative noncoding RNAs. As an example of this latter group of novel precursor transcripts of micro-RNAs, telencephalic embryonic subtractive sequence (TESS) 24.E3 was functionally characterized, and one of its targets was identified: the zinc finger transcription factor ZFP9. The TESS transcriptome has been annotated, mapped for chromosome loci, and arrayed for its gene expression profiles during neural development and differentiation (in Neuro2a and neural stem cells). Within this collection, 188 genes were also characterized on embryonic and postnatal tissue by in situ hybridization, demonstrating that most are specifically expressed in the embryonic CNS. The full information has been organized into a searchable database linked to other genomic resources, allowing easy access to those who are interested in the dissection of the molecular basis of telencephalic development.

  12. 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

  13. A chromosome 11-linked determinant controls fetal globin expression and the fetal-to-adult globin switch.

    PubMed Central

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

    1987-01-01

    Hybrids formed by fusing mouse erythroleukemia (MEL) cells with human fetal erythroid cells produce human fetal globin, but they switch to adult globin production as culture time advances. To obtain information on the chromosomal assignment of the elements that control gamma-to-beta switching, we analyzed the chromosomal composition of hybrids producing exclusively or predominantly human fetal globin and hybrids producing only adult human globin. No human chromosome was consistently present in hybrids expressing fetal globin and consistently absent in hybrids expressing adult globin. Subcloning experiments demonstrated identical chromosomal compositions in subclones displaying the fetal globin program and those that had switched to expression of the adult globin program. These data indicate that retention of only one human chromosome-i.e., chromosome 11--sufficient for expression of human fetal globin and the subsequent gamma-to-beta switch. The results suggest that the gamma-to-beta switch is controlled either cis to the beta-globin locus or by a trans-acting mechanism, the genes of which reside on human chromosome 11. Images PMID:3479779

  14. 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

  15. Molecular and Functional Characterization of Broccoli EMBRYONIC FLOWER 2 Genes

    PubMed Central

    Chen, Long-Fang O.; Lin, Chun-Hung; Lai, Ying-Mi; Huang, Jia-Yuan; Sung, Zinmay Renee

    2012-01-01

    Polycomb group (PcG) proteins regulate major developmental processes in Arabidopsis. EMBRYONIC FLOWER 2 (EMF2), the VEFS domain-containing PcG gene, regulates diverse genetic pathways and is required for vegetative development and plant survival. Despite widespread EMF2-like sequences in plants, little is known about their function other than in Arabidopsis and rice. To study the role of EMF2 in broccoli (Brassica oleracea var. italica cv. Elegance) development, we identified two broccoli EMF2 (BoEMF2) genes with sequence homology to and a similar gene expression pattern to that in Arabidopsis (AtEMF2). Reducing their expression in broccoli resulted in aberrant phenotypes and gene expression patterns. BoEMF2 regulates genes involved in diverse developmental and stress programs similar to AtEMF2 in Arabidopsis. However, BoEMF2 differs from AtEMF2 in the regulation of flower organ identity, cell proliferation and elongation, and death-related genes, which may explain the distinct phenotypes. The expression of BoEMF2.1 in the Arabidopsis emf2 mutant (Rescued emf2) partially rescued the mutant phenotype and restored the gene expression pattern to that of the wild type. Many EMF2-mediated molecular and developmental functions are conserved in broccoli and Arabidopsis. Furthermore, the restored gene expression pattern in Rescued emf2 provides insights into the molecular basis of PcG-mediated growth and development. PMID:22537758

  16. Prediction of gene expression in embryonic structures of Drosophila melanogaster.

    PubMed

    Samsonova, Anastasia A; Niranjan, Mahesan; Russell, Steven; Brazma, Alvis

    2007-07-01

    Understanding how sets of genes are coordinately regulated in space and time to generate the diversity of cell types that characterise complex metazoans is a major challenge in modern biology. The use of high-throughput approaches, such as large-scale in situ hybridisation and genome-wide expression profiling via DNA microarrays, is beginning to provide insights into the complexities of development. However, in many organisms the collection and annotation of comprehensive in situ localisation data is a difficult and time-consuming task. Here, we present a widely applicable computational approach, integrating developmental time-course microarray data with annotated in situ hybridisation studies, that facilitates the de novo prediction of tissue-specific expression for genes that have no in vivo gene expression localisation data available. Using a classification approach, trained with data from microarray and in situ hybridisation studies of gene expression during Drosophila embryonic development, we made a set of predictions on the tissue-specific expression of Drosophila genes that have not been systematically characterised by in situ hybridisation experiments. The reliability of our predictions is confirmed by literature-derived annotations in FlyBase, by overrepresentation of Gene Ontology biological process annotations, and, in a selected set, by detailed gene-specific studies from the literature. Our novel organism-independent method will be of considerable utility in enriching the annotation of gene function and expression in complex multicellular organisms.

  17. Reliability of detection by polymerase chain reaction of the sickle cell-containing region of the beta-globin gene in single human blastomeres.

    PubMed

    Pickering, S J; McConnell, J M; Johnson, M H; Braude, P R

    1992-05-01

    Human preimplantation embryos at various stages of development have been analysed using the polymerase chain reaction to amplify a 680 base pair fragment of the beta-globin gene. Successful amplification was achieved more frequently with DNA from intact embryos containing between one and 11 cells, single cumulus cells, oocytes which had failed to fertilize and polar bodies than from single blastomeres disaggregated from intact embryos and treated in an identical manner. The distribution of nuclei demonstrated using the nuclear chromophore diamino-phenyl-indole showed considerable inter-blastomere variation; however, no clear correlation between staining pattern and successful amplification was observed. The reason for the unreliable amplification of DNA from single blastomeres is unclear but this finding has important implications for preimplantation diagnosis of genetic disease.

  18. Identification of a novel mutation in the β-globin gene 3' untranslated region (HBB: c.*+118A > G) in Spain.

    PubMed

    Herrera, Maria Ascensión; De La Fuente-Gonzalo, Félix; González, Fernando Ataúlfo; Nieto, Jorge M; Dominguez, Alejandra Blum; Villegas, Ana; Ropero, Paloma

    2015-01-01

    The 3' untranslated region (3'UTR) region is well known to be associated with mRNA stability because of its associations with 3' end processing, polyadenylation and mRNA capping. Mutations located in this area cause a β-thalassemia (β-thal) phenotype compatible with β(+)-thal. Two brothers, 49- and 41-years-old, were diagnosed with β-thal intermedia (β-TI) at 2 years of age. The β-globin gene from the promoter region to the 3'UTR was sequenced and both brothers were diagnosed to be compound heterozygotes for the 3'UTR +1592 (A > G) (HBB: c.*+118A > G) and codon 39 (C > T) (HBB: c.118C > T) mutations. Their mother was a carrier of the nonsense codon 39 mutation and her hematological data suggested β-thal trait; their father was a carrier of the 3'UTR +1592 mutation, though he did not have hematological parameters associated with β-thal. The adenine at position +1592 or +118 bases downstream of the termination codon is highly conserved among primates and placental mammals, as it is located between the polyadenylation A signal (PAS) and the polyadenylation A cleavage (PAC) sites. Given its location, it is likely that this mutation would interfere with mRNA maturation; however, the clinical data of the heterozygous carriers show virtually no significant alterations. Therefore, we suggest that the impact on cleavage-stimulation factor (CstF) recognition of the mRNA sequence would be minimal and not significantly alter polyadenylation. Although the mechanism is not known, and because the carrier has no β-thal minor, the mRNA is stable enough that the synthesis of the β-globin chain is unaffected. PMID:25572186

  19. A small set of extra-embryonic genes defines a new landmark for bovine embryo staging.

    PubMed

    Degrelle, Séverine A; Lê Cao, Kim-Anh; Heyman, Yvan; Everts, Robin E; Campion, Evelyne; Richard, Christophe; Ducroix-Crépy, Céline; Tian, X Cindy; Lewin, Harris A; Renard, Jean-Paul; Robert-Granié, Christèle; Hue, Isabelle

    2011-01-01

    Axis specification in mouse is determined by a sequence of reciprocal interactions between embryonic and extra-embryonic tissues so that a few extra-embryonic genes appear as 'patterning' the embryo. Considering these interactions as essential, but lacking in most mammals the genetically driven approaches used in mouse and the corresponding patterning mutants, we examined whether a molecular signature originating from extra-embryonic tissues could relate to the developmental stage of the embryo proper and predict it. To this end, we have profiled bovine extra-embryonic tissues at peri-implantation stages, when gastrulation and early neurulation occur, and analysed the subsequent expression profiles through the use of predictive methods as previously reported for tumour classification. A set of six genes (CALM1, CPA3, CITED1, DLD, HNRNPDL, and TGFB3), half of which had not been previously associated with any extra-embryonic feature, appeared significantly discriminative and mainly dependent on embryonic tissues for its faithful expression. The predictive value of this set of genes for gastrulation and early neurulation stages, as assessed on naive samples, was remarkably high (93%). In silico connected to the bovine orthologues of the mouse patterning genes, this gene set is proposed as a new trait for embryo staging. As such, this will allow saving the bovine embryo proper for molecular or cellular studies. To us, it offers as well new perspectives for developmental phenotyping and modelling of embryonic/extra-embryonic co-differentiation.

  20. 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

  1. Toxic responses in rat embryonic cells to silver nanoparticles and released silver ions as analyzed via gene expression profiles and transmission electron microscopy.

    PubMed

    Xu, Liming; Shi, Chang; Shao, Anliang; Li, Xuefei; Cheng, Xiang; Ding, Rigao; Wu, Gang; Chou, Laisheng Lee

    2015-05-01

    After exposing rat embryonic cells to 20 μg/mL of silver nanoparticle (NP) suspension and their released ions for different time periods, silver nanoparticles were found in cellular nuclei, mitochondria, cytoplasm and lysosomes by transmission electron microscopy (TEM). We also observed mitochondrial destruction, distension of endoplasmic reticulum and apoptotic bodies. Global gene expression analysis showed a total of 279 genes that were up-regulated and 389 genes that were down-regulated in the silver-NP suspension exposure group, while 3 genes were up-regulated and 41 genes were down-regulated in the silver ion exposure group. Further, the GO pathway analysis suggested that these differentially expressed genes are involved in several biological processes, such as energy metabolism, oxygen transport, enzyme activities, molecular binding, etc. It is possible that inhibition of oxygen transport is mediated by the significant down-regulation of genes of the globin family, which might play an important role in silver ion-induced toxicity. KEGG pathway analysis showed that there were 23 signal pathways that were affected in the cells after exposure to silver-NP suspension, but not silver ion alone. The most significant change concerned inflammatory signal pathways, which were only found in silver-NP suspension exposed cells, indicating that inflammatory response might play an important role in the mechanism(s) of silver-NP-induced toxicity. The significant up-regulation of matrix metalloproteinases 3 and 9 suggests that silver NPs could induce extracellular matrix degradation via an inflammatory signaling pathway. The significant up-regulation of secretory leukocyte peptidase inhibitor and serine protease inhibitor 2c was considered to be an embryonic cellular defense mechanism in response to silver-NP-induced inflammation.

  2. Origin and spread of beta-globin gene mutations in India, Africa, and Mediterranea: analysis of the 5' flanking and intragenic sequences of beta S and beta C genes.

    PubMed

    Trabuchet, G; Elion, J; Baudot, G; Pagnier, J; Bouhass, R; Nigon, V M; Labie, D; Krishnamoorthy, R

    1991-06-01

    Nucleotide polymorphisms of both the 5' flanking and intragenic regions of the human beta-globin gene were investigated by directly sequencing genomic DNA after amplification by the polymerase chain reaction in 47 subjects homozygous for the beta S or the beta C mutation. The sickle-cell mutation was found in the context of five different haplotypes defined by eight nucleotide substitutions and various structures of a region of the simple repeated sequence (AT) chi Ty. All subjects from the same geographic origin bear an identical chromosomal structure, defining the Senegal-, Bantu-, Benin-, Cameroon-, and Indian-type chromosomes. These results strengthen our previous conclusions about the multiple occurrence of the sickle-cell mutation. The Benin-type chromosome was also found among Algerian and Sicilian sickle-cell patients, whereas the Indian-type chromosome was observed in two geographically distant tribes, illustrating the spread of these sickle-cell genes. We also found that the intragenic sequence polymorphisms (frameworks) are not always in linkage disequilibrium with the BamH I polymorphism downstream from the beta-globin gene, as had been previously observed. Finally, we present a tentative phylogenetic tree of the different alleles at this locus. Some polymorphisms of this sequence might be contemporary with our last common ancestor, the great apes, that is, about 4-6 millions years old.

  3. 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

  4. 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

  5. Inhibitors of histone deacetylases and DNA methyltransferases alter imprinted gene regulation in embryonic stem cells.

    PubMed

    Baqir, Senan; Smith, Lawrence C

    2006-01-01

    Pluripotent embryonic stem cells are able to differentiate into a variety of cell types, thereby making them a valuable source for transplantation medicine. Recent studies have reported the use of pharmacological agents, namely 5-Aza-Cytidine (5AzaC) and Trichostatin A (TSA), to guide embryonic stem (ES) cells to differentiate into specific cellular lineages. However, those drugs are known to be potent inhibitors of DNA methyltransferases and/or histone deacetylases. Since both epigenetic mechanisms are involved in the expression of imprinted genes in fetal and adult somatic tissues, it is essential to investigate further the role of these agents in regulating imprinted gene expression in embryonic cells. Embryonic stem cells were exposed to 5AzaC and TSA and analyzed for transcript abundance of a number of imprinted and non-imprinted marker genes. Most imprinted gene transcripts increased following exposure to 5AzaC or TSA alone and responded in either an additive or synergistic manner when exposed to both drugs together. Interestingly, transcript levels of several imprinted genes remained high and in some cases, increased further after drug removal or even after passaging the cells, indicating a long lasting and retarded effect on gene expression. Together, our results suggest that DNA methylation and histone acetylation play jointly an important epigenetic role in governing imprinted gene expression in embryonic stem cells. Moreover, these results describe the sensitivity and irreversibility of embryonic stem cells to epigenetic modifiers, highlighting potential risks for their use in therapeutic applications.

  6. 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.

  7. An erythroid chaperone that facilitates folding of α-globin subunits for hemoglobin synthesis

    PubMed Central

    Yu, Xiang; Kong, Yi; Dore, Louis C.; Abdulmalik, Osheiza; Katein, Anne M.; Zhou, Suiping; Choi, John K.; Gell, David; Mackay, Joel P.; Gow, Andrew J.; Weiss, Mitchell J.

    2007-01-01

    Erythrocyte precursors produce abundant α- and β-globin proteins, which assemble with each other to form hemoglobin A (HbA), the major blood oxygen carrier. αHb-stabilizing protein (AHSP) binds free α subunits reversibly to maintain their structure and limit their ability to generate reactive oxygen species. Accordingly, loss of AHSP aggravates the toxicity of excessive free α-globin caused by β-globin gene disruption in mice. Surprisingly, we found that AHSP also has important functions when free α-globin is limited. Thus, compound mutants lacking both Ahsp and 1 of 4 α-globin genes (genotype Ahsp–/–α-globin*α/αα) exhibited more severe anemia and Hb instability than mice with either mutation alone. In vitro, recombinant AHSP promoted folding of newly translated α-globin, enhanced its refolding after denaturation, and facilitated its incorporation into HbA. Moreover, in erythroid precursors, newly formed free α-globin was destabilized by loss of AHSP. Therefore, in addition to its previously defined role in detoxification of excess α-globin, AHSP also acts as a molecular chaperone to stabilize nascent α-globin for HbA assembly. Our findings illustrate what we believe to be a novel adaptive mechanism by which a specialized cell coordinates high-level production of a multisubunit protein and protects against various synthetic imbalances. PMID:17607360

  8. Parental vitamin deficiency affects the embryonic gene expression of immune-, lipid transport- and apolipoprotein genes

    NASA Astrophysics Data System (ADS)

    Skjærven, Kaja H.; Jakt, Lars Martin; Dahl, John Arne; Espe, Marit; Aanes, Håvard; Hamre, Kristin; Fernandes, Jorge M. O.

    2016-10-01

    World Health Organization is concerned for parental vitamin deficiency and its effect on offspring health. This study examines the effect of a marginally dietary-induced parental one carbon (1-C) micronutrient deficiency on embryonic gene expression using zebrafish. Metabolic profiling revealed a reduced 1-C cycle efficiency in F0 generation. Parental deficiency reduced the fecundity and a total of 364 genes were differentially expressed in the F1 embryos. The upregulated genes (53%) in the deficient group were enriched in biological processes such as immune response and blood coagulation. Several genes encoding enzymes essential for the 1-C cycle and for lipid transport (especially apolipoproteins) were aberrantly expressed. We show that a parental diet deficient in micronutrients disturbs the expression in descendant embryos of genes associated with overall health, and result in inherited aberrations in the 1-C cycle and lipid metabolism. This emphasises the importance of parental micronutrient status for the health of the offspring.

  9. Parental vitamin deficiency affects the embryonic gene expression of immune-, lipid transport- and apolipoprotein genes

    PubMed Central

    Skjærven, Kaja H.; Jakt, Lars Martin; Dahl, John Arne; Espe, Marit; Aanes, Håvard; Hamre, Kristin; Fernandes, Jorge M. O.

    2016-01-01

    World Health Organization is concerned for parental vitamin deficiency and its effect on offspring health. This study examines the effect of a marginally dietary-induced parental one carbon (1-C) micronutrient deficiency on embryonic gene expression using zebrafish. Metabolic profiling revealed a reduced 1-C cycle efficiency in F0 generation. Parental deficiency reduced the fecundity and a total of 364 genes were differentially expressed in the F1 embryos. The upregulated genes (53%) in the deficient group were enriched in biological processes such as immune response and blood coagulation. Several genes encoding enzymes essential for the 1-C cycle and for lipid transport (especially apolipoproteins) were aberrantly expressed. We show that a parental diet deficient in micronutrients disturbs the expression in descendant embryos of genes associated with overall health, and result in inherited aberrations in the 1-C cycle and lipid metabolism. This emphasises the importance of parental micronutrient status for the health of the offspring. PMID:27731423

  10. Embryonic cerebrospinal fluid collaborates with the isthmic organizer to regulate mesencephalic gene expression.

    PubMed

    Parada, Carolina; Martín, Cristina; Alonso, María I; Moro, José A; Bueno, David; Gato, Angel

    2005-11-01

    Early in development, the behavior of neuroepithelial cells is controlled by several factors acting in a developmentally regulated manner. Recently it has been shown that diffusible factors contained within embryonic cerebrospinal fluid (CSF) promote neuroepithelial cell survival, proliferation, and neurogenesis in mesencephalic explants lacking any known organizing center. In this paper, we show that mesencephalic and mesencephalic+isthmic organizer explants cultured only with basal medium do not express the typically expressed mesencephalic or isthmic organizer genes analyzed (otx2 and fgf8, respectively) and that mesencephalic explants cultured with embryonic CSF-supplemented medium do effect such expression, although they exhibit an altered pattern of gene expression, including ectopic shh expression domains. Other trophic sources that are able to maintain normal neuroepithelial cell behavior, i.e., fibroblast growth factor-2, fail to activate this ectopic shh expression. Conversely, the expression pattern of the analyzed genes in mesencephalic+isthmic organizer explants cultured with embryonic cerebrospinal fluid-supplemented medium mimics the pattern for control embryos developed in ovo. We demonstrate that embryonic CSF collaborates with the isthmic organizer in regulation of the expression pattern of some characteristic neuroectodermal genes during early stages of central nervous system (CNS) development, and we suggest that this collaboration is not restricted to the maintenance of neuroepithelial cell survival. Data reported in this paper corroborate the hypothesis that factors contained within embryonic CSF contribute to the patterning of the CNS during early embryonic development. PMID:16180222

  11. A Concise Protocol for siRNA-Mediated Gene Suppression in Human Embryonic Stem Cells.

    PubMed

    Renz, Peter F; Beyer, Tobias A

    2016-01-01

    Human embryonic stem cells hold great promise for future biomedical applications such as disease modeling and regenerative medicine. However, these cells are notoriously difficult to culture and are refractory to common means of genetic manipulation, thereby limiting their range of applications. In this protocol, we present an easy and robust method of gene repression in human embryonic stem cells using lipofection of small interfering RNA (siRNA).

  12. [Teratogenesis and gene targets of 17alpha-ethynylestradiol on embryonic development in zebrafish].

    PubMed

    Tong, Jun-Wei; Zhang, Jing-Pu; Meng, Jie

    2011-01-01

    The pharmaceutical ethynylestradiol (EE) is a potent endocrine modulator. Application enlargement of ethynylestradiol in clinics and abuse in livestock farming and fishing make it important to explore ethynylestradiol toxicological action on vertebrate embryonic development and to establish an in vivo method for EE toxicity detection efficiently and conveniently. In the present study, using a model animal zebrafish and 17alpha-ethynylestradiol as a representative compound, we have investigated EE2 teratogenicity, target tissues and target genes on zebrafish embryo. The results show that median teratogenesis concentration (TC50) of EE2 is 0.8 microg x mL(-1), and median lethal dose (LD50) is 3.3 microg x mL(-1). Targets of EE2 action were implicated in brain, eyes, heart, muscle, skeleton, pigment and viscera. Embryonic cardiac arrhythmia caused by EE2 is probably resulted from heart abnormal structure. The embryonic stage sensitive to EE2 mainly started at cleavage and last up to the organogenesis with time-accumulating effect. RT-PCR results indicate that EE2 treatment disturbed gene expression pattern at the early period of zebrafish embryonic development by suppressing transcription of gene boz that promotes brain development, upregulating genes for trunk and tail, such as ntl, spt, shh, and perturbing Nodal signal expression of TGFbeta superfamily, for example, cyc, sqt and oep. Using zebrafish, an efficient in vivo method for quick evaluation of EE toxicity on embryonic development has been developed.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. Erythroid-specific expression of beta-globin by the sleeping beauty transposon for Sickle cell disease.

    PubMed

    Zhu, Jianhui; Kren, Betsy T; Park, Chang Won; Bilgim, Rasim; Wong, Phillip Y-P; Steer, Clifford J

    2007-06-12

    Sickle cell disease (SCD) results predominately from a single monogenic mutation that affects thousands of individuals worldwide. Gene therapy approaches have focused on using viral vectors to transfer wild-type beta- or gamma-globin transgenes into hematopoietic stem cells for long-term expression of the recombinant globins. In this study, we investigated the use of a novel nonviral vector system, the Sleeping Beauty (SB) transposon (Tn) to insert a wild-type beta-globin expression cassette into the human genome for sustained expression of beta-globin. We initially constructed a beta-globin expression vector composed of the hybrid cytomegalovirus (CMV) enhancer chicken beta-actin promoter (CAGGS) and full-length beta-globin cDNA, as well as truncated forms lacking either the 3' or 3' and 5' untranslated regions (UTRs), to optimize expression of beta-globin. Beta-globin with its 5' UTR was efficiently expressed from its cDNA in K-562 cells induced with hemin. However, expression was constitutive and not erythroid-specific. We then constructed cis SB-Tn-beta-globin plasmids using a minimal beta-globin gene driven by hybrid promoter IHK (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, ankyrin-1 promoter), IHbetap (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, beta-globin promoter), or HS3betap (HS3 core element from human betaLCR, beta-globin promoter) to establish erythroid-specific expression of beta-globin. Stable genomic insertion of the minimal gene and expression of the beta-globin transgene for >5 months at a level comparable to that of the endogenous gamma-globin gene were achieved using a SB-Tn beta-globin cis construct. Interestingly, erythroid-specific expression of beta-globin driven by IHK was regulated primarily at the translational level, in contrast to post-transcriptional regulation in non-erythroid cells. The SB-Tn system is a promising nonviral vector for efficient

  18. 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

  19. The organization, structure, and in vitro transcription of Alu family RNA polymerase III transcription units in the human alpha-like globin gene cluster: precipitation of in vitro transcripts by lupus anti-La antibodies.

    PubMed

    Shen, C K; Maniatis, T

    1982-01-01

    We have studied the location, structure, and in vitro transcription of repetitive DNA sequences within the human alpha-like globin gene cluster. At least eight different Alu family repeats were identified, each of which is transcribed in vitro to produce discrete RNA transcripts. The nucleotide sequence of one Alu repeat sequence, located on the 3' side of the alpha l globin gene (3'-alpha l), was determined and compared to published Alu repeat sequences. In vitro transcription of this repeat sequence generates RNA fragments of approximately 410, 260, 160, and 86 nucleotides. To determine whether these transcripts associate with specific proteins in vitro, we carried out immunoprecipitation experiments using an antiserum from systemic lupus erythematosus (SLE) patients. We find that the antiserum anti-La, which was shown to precipitate ribonucleoproteins (RNPs) containing the adenovirus VAI RNA from virus infected cells, preferentially precipitates the smallest two in vitro transcripts of the 3'-alpha l Alu repeat. These results suggest that the RNAs interact with specific factors in the in vitro transcription reaction mix to form RNP.

  20. Developmental effect of the XmnI site on Ggamma-globin gene expression among newborn Hb F-Malta-I [Ggamma117(G19)His-->Arg, CAT-->CGT] heterozygotes and adult beta+ -Thalassemia homozygotes.

    PubMed

    Pulis, Svetlana; Scerri, Christian A; Wismayer, Pierre Schembri; Galdies, Ruth; Wettinger, Stephanie Bezzina; Felice, Alex E

    2007-01-01

    Hb F-Malta-I [Ggamma117(19)His-->Arg, CAT-->CGT] is a stable and benign variant of Hb F found in 1.8% of Maltese newborn. We studied 120 Hb F-Malta-I heterozygotes and four Hb F-Malta-I homozygotes. The mean proportion of Ggamma-F-Malta-I in Hb F was 0.26 +/- 0.03 for the Hb F-Malta-I heterozygotes and 0.58 +/- 0.06 for the Hb F-Malta-I homozygotes. The Hb F-Malta-I allele was shown to occur on a background of the common Mediterranean haplotype Va [+ + - - - - - + + -]. Furthermore, the common Mediterranean haplotypes Va, IIIb [- + + + - + + + + -], I [+ + - - - - - + + +] and II [- + - + + - + + + +] accounted for most (66.2%) of the wild-type alleles among the tested Hb F-Malta-I heterozygotes. Different genotypes at the 5' epsilon HincII, Ggamma and Agamma HindIII, and 3'psibeta HincII sites (but not at the 5' Ggamma XmnI site) were found to be linked to significant variations in the proportion of Ggamma-F-Malta-I and Ggamma-globins in the Hb F of newborn Hb F-Malta-I heterozygotes. Moreover, the 5' Ggamma XmnI site was found to be associated with variations in Hb F and Ggamma-globin levels in a population of adult Maltese beta-thalassemia (thal) homozygotes. This implies that a determinant linked to the XmnI site which effects Ggamma-globin gene expression is active in anemic adults but not in normal infants.

  1. Beta-globin locus activation regions: conservation of organization, structure, and function.

    PubMed

    Li, Q L; Zhou, B; Powers, P; Enver, T; Stamatoyannopoulos, G

    1990-11-01

    The human beta-globin locus activation region (LAR) comprises four erythroid-specific DNase I hypersensitive sites (I-IV) thought to be largely responsible for activating the beta-globin domain and facilitating high-level erythroid-specific globin gene expression. We identified the goat beta-globin LAR, determined 10.2 kilobases of its sequence, and demonstrated its function in transgenic mice. The human and goat LARs share 6.5 kilobases of homologous sequences that are as highly conserved as the epsilon-globin gene promoters. Furthermore, the overall spatial organization of the two LARs has been conserved. These results suggest that the functionally relevant regions of the LAR are large and that in addition to their primary structure, the spatial relationship of the conserved elements is important for LAR function.

  2. Characterization and embryonic expression of four amphioxus Frizzled genes with important functions during early embryogenesis.

    PubMed

    Qian, Guanghui; Li, Guang; Chen, Xiaoying; Wang, Yiquan

    2013-12-01

    The Wnt signaling pathway plays crucial roles in the embryonic patterning of all metazoans. Recent studies on Wnt genes in amphioxus have shed important insights into the evolution of the vertebrate Wnt gene family and their functions. Nevertheless, the potential roles of Wnt family receptors encoded by Frizzled (Fz) genes in amphioxus embryonic development remain to be investigated. In the present study, we identified four amphioxus Fz genes-AmphiFz1/2/7, AmphiFz4, AmphiFz5/8, and AmphiFz9/10-and analyzed their expression patterns during amphioxus embryogenesis. We found that these four Fz genes were maternally expressed and might be involved in early animal-vegetal axis establishment. The AmphiFz1/2/7 transcripts were detected in the central dorsal neural plate, mesoderm, the Hatschek's pit, and rim of the mouth, whereas those of AmphiFz4 were detected in the mesoderm, pharyngeal endoderm, and entire gut region. AmphiFz5/8 was exclusively expressed in the anterior-most region, whereas AmphiFz9/10 was expressed in the neural plate, somites, and tail bud. The dynamic and diverse expression patterns of amphioxus Fz genes suggest that these genes are not only associated with early embryonic axis establishment but also are involved in the development of several organs in amphioxus.

  3. Characterization and embryonic expression of four amphioxus Frizzled genes with important functions during early embryogenesis.

    PubMed

    Qian, Guanghui; Li, Guang; Chen, Xiaoying; Wang, Yiquan

    2013-12-01

    The Wnt signaling pathway plays crucial roles in the embryonic patterning of all metazoans. Recent studies on Wnt genes in amphioxus have shed important insights into the evolution of the vertebrate Wnt gene family and their functions. Nevertheless, the potential roles of Wnt family receptors encoded by Frizzled (Fz) genes in amphioxus embryonic development remain to be investigated. In the present study, we identified four amphioxus Fz genes-AmphiFz1/2/7, AmphiFz4, AmphiFz5/8, and AmphiFz9/10-and analyzed their expression patterns during amphioxus embryogenesis. We found that these four Fz genes were maternally expressed and might be involved in early animal-vegetal axis establishment. The AmphiFz1/2/7 transcripts were detected in the central dorsal neural plate, mesoderm, the Hatschek's pit, and rim of the mouth, whereas those of AmphiFz4 were detected in the mesoderm, pharyngeal endoderm, and entire gut region. AmphiFz5/8 was exclusively expressed in the anterior-most region, whereas AmphiFz9/10 was expressed in the neural plate, somites, and tail bud. The dynamic and diverse expression patterns of amphioxus Fz genes suggest that these genes are not only associated with early embryonic axis establishment but also are involved in the development of several organs in amphioxus. PMID:24012522

  4. Detection of sickle cell beta S-globin allele by hybridization with synthetic oligonucleotides.

    PubMed

    Conner, B J; Reyes, A A; Morin, C; Itakura, K; Teplitz, R L; Wallace, R B

    1983-01-01

    Two 19-base-long oligonucleotides were synthesized, one complementary to the normal human beta-globin gene (beta A) and one complementary to the sickle cell beta-globin gene (beta S). The nonadecanucleotides were radioactively labeled and used as probes in DNA hybridization. Under appropriate hybridization conditions, these probes can be used to distinguish the beta A gene from the beta S allele. The DNA from individuals homozygous for the normal beta-globin gene (beta A beta A) only hybridized with the beta A specific probe; the DNA from those homozygous for the sickle cell beta-globin gene (beta S beta S) only hybridized with the beta S specific probe. The DNA from heterozygous individuals (beta A beta S) hybridized with both probes. This allele-specific hybridization behavior of oligonucleotides provides a general method for diagnosis of any genetic disease which involves a point mutation in the DNA sequence of a single-copy gene.

  5. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells

    SciTech Connect

    Gao, Xiugong Sprando, Robert L.; Yourick, Jeffrey J.

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72 h after exposure to 0.25 mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. - Highlights: • Studied genomic changes in mouse embryonic stem cells upon thalidomide exposure • Identified gene expression changes that may represent thalidomide embryotoxicity • The toxicogenomic changes coincide well with known thalidomide clinical outcomes. • The mouse embryonic stem cell model is suitable for developmental toxicity testing. • The model has the potential for high-throughput screening of a multitude of compounds.

  6. Gene duplication and the evolution of hemoglobin isoform differentiation in birds.

    PubMed

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

    2012-11-01

    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 α(A)-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the α(D)-globin gene). The α(D)-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 O(2) 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-O(2) affinity stems primarily from changes in the O(2) 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 α(D)-globin gene that is shared with the embryonic α-like globin gene.

  7. 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

  8. Identification of Estrogen Target Genes during Zebrafish Embryonic Development through Transcriptomic Analysis

    PubMed Central

    Hao, Ruixin; Bondesson, Maria; Singh, Amar V.; Riu, Anne; McCollum, Catherine W.; Knudsen, Thomas B.; Gorelick, Daniel A.; Gustafsson, Jan-Åke

    2013-01-01

    Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio) as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 µM 17β-estradiol (E2) or vehicle from 3 hours to 4 days post fertilization (dpf), harvested at 1, 2, 3 and 4 dpf, and subjected to RNA extraction for transcriptome analysis using microarrays. Differentially expressed genes by E2-treatment were analyzed with hierarchical clustering followed by biological process and tissue enrichment analysis. Markedly distinct sets of genes were up and down-regulated by E2 at the four different time points. Among these genes, only the well-known estrogenic marker vtg1 was co-regulated at all time points. Despite this, the biological functional categories targeted by E2 were relatively similar throughout zebrafish development. According to knowledge-based tissue enrichment, estrogen responsive genes were clustered mainly in the liver, pancreas and brain. This was in line with the developmental dynamics of estrogen-target tissues that were visualized using transgenic zebrafish containing estrogen responsive elements driving the expression of GFP (Tg(5xERE:GFP)). Finally, the identified embryonic estrogen-responsive genes were compared to already published estrogen-responsive genes identified in male adult zebrafish (Gene Expression Omnibus database). The expressions of a few genes were co-regulated by E2 in both embryonic and adult zebrafish. These could potentially be used as estrogenic biomarkers for exposure to estrogens or estrogenic endocrine disruptors in zebrafish. In conclusion, our data suggests that estrogen effects on early embryonic zebrafish development are stage- and tissue- specific. PMID:24223173

  9. Surface functionalities of gold nanoparticles impact embryonic gene expression responses

    PubMed Central

    Truong, Lisa; Tilton, Susan C.; Zaikova, Tatiana; Richman, Erik; Waters, Katrina M.; Hutchison, James E.; Tanguay, Robert L.

    2012-01-01

    Incorporation of gold nanoparticles (AuNPs) into consumer products is increasing; however, there is a gap in available toxicological data to determine the safety of AuNPs. In this study, we utilised the embryonic zebrafish to investigate how surface functionalisation and charge influence molecular responses. Precisely engineered AuNPs with 1.5 nm cores were synthesised and functionalized with three ligands: 2-mercaptoethanesulfonic acid (MES), N,N,N-trimethylammoniumethanethiol (TMAT), or 2-(2-(2-mercaptoethoxy)ethoxy)ethanol. Developmental assessments revealed differential biological responses when embryos were exposed to the functionalised AuNPs at the same concentration. Using inductively coupled plasma–mass spectrometry, AuNP uptake was confirmed in exposed embryos. Following exposure to MES- and TMAT-AuNPs from 6 to 24 or 6 to 48 h post fertilisation, pathways involved in inflammation and immune response were perturbed. Additionally, transport mechanisms were misregulated after exposure to TMAT and MES-AuNPs, demonstrating that surface functionalisation influences many molecular pathways. PMID:22263968

  10. 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.

  11. An embryonic story: analysis of the gene regulative network controlling Xist expression in mouse embryonic stem cells.

    PubMed

    Navarro, Pablo; Avner, Philip

    2010-07-01

    In mice, dosage compensation of X-linked gene expression is achieved through the inactivation of one of the two X-chromosomes in XX female cells. The complex epigenetic process leading to X-inactivation is largely controlled by Xist and Tsix, two non-coding genes of opposing function. Xist RNA triggers X-inactivation by coating the inactive X, while Tsix is critical for the designation of the active X-chromosome through cis-repression of Xist RNA accumulation. Recently, a plethora of trans-acting factors and cis-regulating elements have been suggested to act as key regulators of either Xist, Tsix or both; these include ubiquitous factors such as Yy1 and Ctcf, developmental proteins such as Nanog, Oct4 and Sox2, and X-linked regulators such as Rnf12. In this paper we summarise recent advances in our knowledge of the regulation of Xist and Tsix in embryonic stem (ES) and differentiating ES cells.

  12. Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells

    PubMed Central

    Soucie, Erinn L.; Weng, Ziming; Geirsdóttir, Laufey; Molawi, Kaaweh; Maurizio, Julien; Fenouil, Romain; Mossadegh-Keller, Noushine; Gimenez, Gregory; VanHille, Laurent; Beniazza, Meryam; Favret, Jeremy; Berruyer, Carole; Perrin, Pierre; Hacohen, Nir; Andrau, J.-C.; Ferrier, Pierre; Dubreuil, Patrice; Sidow, Arend; Sieweke, Michael H.

    2016-01-01

    Differentiated macrophages can self-renew in tissues and expand long-term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network controlling self-renewal. Single cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells. PMID:26797145

  13. Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells.

    PubMed

    Soucie, Erinn L; Weng, Ziming; Geirsdóttir, Laufey; Molawi, Kaaweh; Maurizio, Julien; Fenouil, Romain; Mossadegh-Keller, Noushine; Gimenez, Gregory; VanHille, Laurent; Beniazza, Meryam; Favret, Jeremy; Berruyer, Carole; Perrin, Pierre; Hacohen, Nir; Andrau, J-C; Ferrier, Pierre; Dubreuil, Patrice; Sidow, Arend; Sieweke, Michael H

    2016-02-12

    Differentiated macrophages can self-renew in tissues and expand long term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network that controls self-renewal. Single-cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells.

  14. Methodology matters: IVF versus ICSI and embryonic gene expression.

    PubMed

    Bridges, Phillip J; Jeoung, Myoungkun; Kim, Heyoung; Kim, Jung Ho; Lee, Dong Ryul; Ko, CheMyong; Baker, Doris J

    2011-08-01

    The use of assisted reproduction treatment, especially intracytoplasmic sperm injection (ICSI), is now linked to a range of adverse consequences, the aetiology of which remains largely undefined. Our objective of this study was to determine differences in gene expression of blastocysts generated by ICSI as well as ICSI with artificial oocyte activation (ICSI-A) versus the less manipulative IVF, providing fundamental genetic information that can be used to aid in the diagnosis or treatment of those adversely affected by assisted reproduction treatment, as well as stimulate research to further refine these techniques. Murine blastocysts were generated by ICSI, ICSI-A and IVF, and processed for a microarray-based analysis of gene expression. Ten blastocysts were pooled for each procedure and three independent replicates generated. The data were then processed to determine differential gene expression and to identify biological pathways affected by the procedures. In blastocysts derived by ICSI versus IVF, the expression of 197 genes differed (P < 0.01). In blastocysts derived by ICSI-A versus IVF and ICSI-A versus ICSI, the expression of 132 and 65 genes differed respectively (P < 0.01). Procedural-induced changes in genes regulating specific biological pathways revealed some consistency to known adverse consequences. Detailed investigation of procedure-specific dysfunction is therefore warranted.

  15. Discriminating classes of developmental toxicants using gene expression profiling in the embryonic stem cell test.

    PubMed

    van Dartel, Dorien A M; Pennings, Jeroen L A; Robinson, Joshua F; Kleinjans, Jos C S; Piersma, Aldert H

    2011-03-01

    The embryonic stem cell test (EST) has been shown to be a promising in vitro method for the prediction of developmental toxicity. In our previous studies, we demonstrated that the implementation of gene expression analysis in the EST may further improve the identification of developmental toxicants. In the present study, we investigated if gene expression profiling could be used to discriminate compound classes with distinct modes of action (MoA) using the EST protocol. Gene expression data of our previous study were used and were analyzed of embryonic stem cell (ESC) differentiation cultures exposed to six compounds belonging to two classes with distinct MoA, namely phthalates and triazoles. We used three approaches to study class-characteristic gene regulation that may be useful for discrimination of compound classes. First, at the individual gene level, gene signatures characteristic for each class were identified that successfully discriminated both classes using principal component analysis. Second, at the functional level, enriched gene ontology (GO) biological processes showed their usefulness for class discrimination via hierarchical clustering. Third, two previously identified gene sets, which we designed to predict developmental toxicity, appeared successful in separating phthalate from triazole compounds. In summary, we established the possibility to discriminate between compound classes in the EST system using three different specific transcriptomics-based approaches. Differential gene expression information specific for the class of compound under study may be employed to optimize prioritization of compounds within that class for further testing.

  16. 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.

  17. Structural analysis of the 5' 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.

    PubMed

    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)n and (AT)xTy, 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. Nevertheless, the haplotype determination does not give any information about the variable segment and does not totally exclude the possibility of recombination leading to different haplotypes linked to the mutation. The structure of the variable segment in the three African populations was studied by S1 nuclease mapping of genomic DNA, which allows a comparison of several samples. A 1080-base-pair DNA segment was sequenced for one sample from each population. S1 nuclease mapping confirmed the homogeneity of each population with regard to both (ATTTT)n and (AT)xTy repeats. We found three additional structures for (AT)xTy correlating with the geographic origin of the patients. Ten other nucleotide positions, 5' and 3' to the (AT)xTy copies, were found to be variable when compared to homologous sequences from human and monkey DNAs. These results allow us to propose an evolutionary scheme for the polymorphisms in the 5' flanking region of the beta-globin gene. The results strongly support the hypothesis of three origins for the sickle mutation in Africa.

  18. Sequence of the 3'-noncoding and adjacent coding regions of human gamma-globin mRNA.

    PubMed Central

    Poon, R; Kan, Y W; Boyer, H W

    1978-01-01

    In cloning human fetal globin cDNA in bacterial plasmids, we obtained a recombinant which contained a fragment of gammg-globin cDNA corresponding to the region from amino acid 99 to the poly A. We determined a sequence of 169 nucleotides which included the complete 3' non-coding region of the gamma-globin mRNA. The codon for amino acid 136 was GCA, indicating that this cloned fragment was derived from the Agamma-globin gene. In conjunction with the surrounding sequences, the GCA codon provides the Agamma-species with a unique CTGCAG hexanucleotide that is recognized by the restriction enzyme Pst I. The 3'-untranslated region of the gamma-globin mRNA consists of 90 nucleotides, and shares little homology with that of the human beta-globin mRNA. As in other mammalian mRNAs, a symmetrical sequence and the hexanucleotide AAUAAA are present. Images PMID:318163

  19. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells.

    PubMed

    Gao, Xiugong; Sprando, Robert L; Yourick, Jeffrey J

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72h after exposure to 0.25mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment.

  20. Prominent expression of transforming growth factor beta2 gene in the chicken embryonic gonad as revealed by suppressive subtraction cloning.

    PubMed

    Hattori Ma, Masa-aki; Furuta, Hiroki; Hiyama, Yoshio; Kato, Yukio; Fujihara, Noboru

    2002-02-01

    cDNA cloning from chicken embryonic gonad subtracted from tissues of the brain, heart, liver, gizzard, mesonephros, and muscle was performed to identify growth factor genes with expression unique to embryonic ovary and testis. We obtained several cDNA clones encoding known and many unknown genes. We found for the first time that the transforming growth factor beta2 (TGF-beta2) is preferentially expressed in the chicken embryonic ovary and testis. cDNA subtraction cloning with respect to the selective expression of TGF-beta2 in the ovary and testis was further analyzed by reverse transcription-polymerase chain reaction analyses of other embryonic tissues. The ontogeny of TGF-beta2 was evaluated in chicken embryonic ovary and testis. In both testis and ovary, the levels of TGF-beta2 transcripts were high during the early period of embryonic development (E7), gradually decreased until the late embryonic days (E14--E17), and then slightly increased at the last embryonic day (E21). There was no difference in the TGF-beta2 transcripts per RNA between the left and the right ovaries. TGF-beta2 may have a critical role in the regulation of the development of chicken ovarian and testicular germ cells during the embryonic period.

  1. Preliminary identification of hemoglobin q-iran in an Iranian family from central province of Iran by globin chain analysis on HPLC.

    PubMed

    Khatami, Shohreh; Najmabadi, Hossein; Rouhi, Soghra; Mirzazadeh, Roghieh; Bayat, Parastoo; Sadeghi, Sedigheh

    2013-12-01

    Many abnormal α-chain hemoglobins (Hbs) are caused by single nucleotide mutations in α1- or α2-goblin genes. One of these Hbs is Hb Q-Iran which is resulted from a point mutation at codon 75 of the α1-globin gene (Asp→His). The identification of Hb Q-Iran was observed in two members of a family from the Central Province of Iran. In this study, Globin chain analysis on high performance liquid chromatography (HPLC) and DNA sequencing were applied. An unusual Hb variant, like HbS on alkaline pH electrophoresis was identified from samples of a father and his son from Arak city in the Central Province of Iran. The variant was further characterized by globin chain analysis and DNA sequencing methods. Globin chain analysis revealed an unknown globin chain peak after α-globin chain peak with a different retention time from βs-globin chain, as the control in both samples. Genetic analysis led to the identification of an unknown Hb variant, Hb Q-Iran. Globin chain analysis showed the presence of an unknown globin chain, and likewise DNA sequencing revealed HbQ-Iran. In other words, Globin chain analysis procedure could preliminarily detect an unknown globin chain.

  2. Zscan4 transiently reactivates early embryonic genes during the generation of induced pluripotent stem cells.

    PubMed

    Hirata, Tetsuya; Amano, Tomokazu; Nakatake, Yuhki; Amano, Misa; Piao, Yulan; Hoang, Hien G; Ko, Minoru S H

    2012-01-01

    The generation of induced pluripotent stem cells (iPSCs) by the forced expression of defined transcription factors in somatic cells holds great promise for the future of regenerative medicine. However, the initial reprogramming mechanism is still poorly understood. Here we show that Zscan4, expressed transiently in2-cell embryos and embryonic stem cells (ESCs), efficiently produces iPSCs from mouse embryo fibroblasts when coexpressed with Klf4, Oct4, and Sox2. Interestingly, the forced expression of Zscan4 is required onlyfor the first few days of iPSC formation. Microarray analysis revealed transient and early induction of preimplantation-specific genes in a Zscan4-dependent manner. Our work indicates that Zscan4 is a previously unidentified potent natural factor that facilitates the reprogramming process and reactivates early embryonic genes.

  3. DNA context represents transcription regulation of the gene in mouse embryonic stem cells

    PubMed Central

    Ha, Misook; Hong, Soondo

    2016-01-01

    Understanding gene regulatory information in DNA remains a significant challenge in biomedical research. This study presents a computational approach to infer gene regulatory programs from primary DNA sequences. Using DNA around transcription start sites as attributes, our model predicts gene regulation in the gene. We find that H3K27ac around TSS is an informative descriptor of the transcription program in mouse embryonic stem cells. We build a computational model inferring the cell-type-specific H3K27ac signatures in the DNA around TSS. A comparison of embryonic stem cell and liver cell-specific H3K27ac signatures in DNA shows that the H3K27ac signatures in DNA around TSS efficiently distinguish the cell-type specific H3K27ac peaks and the gene regulation. The arrangement of the H3K27ac signatures inferred from the DNA represents the transcription regulation of the gene in mESC. We show that the DNA around transcription start sites is associated with the gene regulatory program by specific interaction with H3K27ac. PMID:27075878

  4. DNA context represents transcription regulation of the gene in mouse embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Ha, Misook; Hong, Soondo

    2016-04-01

    Understanding gene regulatory information in DNA remains a significant challenge in biomedical research. This study presents a computational approach to infer gene regulatory programs from primary DNA sequences. Using DNA around transcription start sites as attributes, our model predicts gene regulation in the gene. We find that H3K27ac around TSS is an informative descriptor of the transcription program in mouse embryonic stem cells. We build a computational model inferring the cell-type-specific H3K27ac signatures in the DNA around TSS. A comparison of embryonic stem cell and liver cell-specific H3K27ac signatures in DNA shows that the H3K27ac signatures in DNA around TSS efficiently distinguish the cell-type specific H3K27ac peaks and the gene regulation. The arrangement of the H3K27ac signatures inferred from the DNA represents the transcription regulation of the gene in mESC. We show that the DNA around transcription start sites is associated with the gene regulatory program by specific interaction with H3K27ac.

  5. Developmental analysis of tropomyosin gene expression in embryonic stem cells and mouse embryos.

    PubMed Central

    Muthuchamy, M; Pajak, L; Howles, P; Doetschman, T; Wieczorek, D F

    1993-01-01

    Tropomyosins (TMs) comprise a family of actin-binding proteins which play an important role in the regulation of contractility in muscle (cardiac, skeletal, and smooth) and nonmuscle cells. Although they are present in all cells, different isoforms are characteristic of specific cell types. In vertebrates, there are four different TM genes (alpha-TM, beta-TM, TM30, and TM4), three of which generate alternatively spliced isoforms. This study defines the expression patterns of these isoforms during murine embryogenesis, using both in vivo and in vitro conditions. The embryonic stem cell culture system, which has been shown to mimic different stages of mouse embryonic development, including the differentiation of primitive organ systems such as the myocardium, is used for our in vitro analysis. Our results demonstrate that several TM isoforms are expressed in specific developmental patterns, often correlated with the differentiation of particular tissues or organs. Surprisingly, other TMs, such as the striated muscle beta-TM and smooth muscle alpha-TM, are expressed constitutively. This study also demonstrates that there is an excellent correlation between the expression patterns of the TM isoforms observed in developing embryonic stem cells and mouse embryos. In addition, a quantitative molecular analysis of TM isoforms was conducted in embryonic, neonatal, and adult cardiac tissue. Our results show for the first time that the alpha- and beta-TM striated muscle transcripts are present in the earliest functional stages of the heart, and these TM isoforms are identical to those present throughout cardiac development. Images PMID:7684495

  6. Beta Thalassemia: mutations which affect processing of the beta-Globin mRNA precursor.

    PubMed

    Kantor, J A; Turner, P H; Nienhuis, A W

    1980-08-01

    To define the molecular lesion which causes decreased beta-globin synthesis in beta+ thalessemia, four patients of diverse ethnic origin were studied. Each had a 2--3 fold higher concentration of beta-globin mRNA precursor than that found in control bone marrow cells from patients with sickle cell anemia. Globin RNA metabolism was analyzed in two of these patients. Transcription of the beta-globin gene appeared to be normal, since analysis of nuclear RNA indicated that beta-globin mRNA synthesis exceeded that of alpha in a 2 hr pulse but the cytoplasm contained a relative deficiency of labeled beta-globin mRNA. An abnormal RNA species approximately 650 nucleotides in length, which contained sequences transcribed from both the large intron and coding portions of the beta-globin gene, was found in one patient's bone marrow cells. The second patient's cells contained a significant amount of a 1320 nucleotide RNA species, not initially evident in normal cells, from which part but not all of the large intervening sequence had been removed. Our data thus indicate that mutations which affect RNA processing cause beta thalessemia.

  7. 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.

  8. Discovery of a novel imprinted gene by transcriptional analysis of parthenogenetic embryonic stem cells

    PubMed Central

    Sritanaudomchai, Hathaitip; Ma, Hong; Clepper, Lisa; Gokhale, Sumita; Bogan, Randy; Hennebold, Jon; Wolf, Don; Mitalipov, Shoukhrat

    2010-01-01

    BACKGROUND Parthenogenetic embryonic stem cells (PESCs) may have future utilities in cell replacement therapies since they are closely related to the female from which the activated oocyte was obtained. Furthermore, the avoidance of parthenogenetic development in mammals provides the most compelling rationale for the evolution of genomic imprinting, and the biological process of parthenogenesis raises complex issues regarding differential gene expression. METHODS AND RESULTS We describe here homozygous rhesus monkey PESCs derived from a spontaneously duplicated, haploid oocyte genome. Since the effect of homozygosity on PESCs pluripotency and differentiation potential is unknown, we assessed the similarities and differences in pluripotency markers and developmental potential by in vitro and in vivo differentiation of homozygous and heterozygous PESCs. To understand the differences in gene expression regulation between parthenogenetic and biparental embryonic stem cells (ESCs), we conducted microarray analysis of genome-wide mRNA profiles of primate PESCs and ESCs derived from fertilized embryos using the Affymetrix Rhesus Macaque Genome array. Several known paternally imprinted genes were in the highly down-regulated group in PESCs compared with ESCs. Furthermore, allele-specific expression analysis of other genes whose expression is also down-regulated in PESCs, led to the identification of one novel imprinted gene, inositol polyphosphate-5-phosphatase F (INPP5F), which was exclusively expressed from a paternal allele. CONCLUSION Our findings suggest that PESCs could be used as a model for studying genomic imprinting, and in the discovery of novel imprinted genes. PMID:20522441

  9. Differential Loss and Retention of Cytoglobin, Myoglobin, and Globin-E during the Radiation of Vertebrates

    PubMed Central

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

    2011-01-01

    If rates of postduplication gene retention are positively correlated with levels of functional constraint, then gene duplicates that have been retained in a restricted number of taxonomic lineages would be expected to exhibit relatively low levels of sequence conservation. Paradoxical patterns are presented by gene duplicates that have been retained in a small number of taxa but which are nonetheless subject to strong purifying selection relative to paralogous members of the same multigene family. This pattern suggests that such genes may have been co-opted for novel, lineage-specific functions. One possible example involves the enigmatic globin-E gene (GbE), which appears to be exclusively restricted to birds. Available data indicate that this gene is expressed exclusively in the avian eye, but its physiological function remains a mystery. In contrast to the highly restricted phyletic distribution of GbE, the overwhelming majority of jawed vertebrates (gnathostomes) possess copies of the related cytoglobin (Cygb) and myoglobin (Mb) genes. The purpose of the present study was 1) to assess the phyletic distribution of the Cygb, Mb, and GbE genes among vertebrates, 2) to elucidate the duplicative origins and evolutionary histories of these three genes, and 3) to evaluate the relative levels of functional constraint of these genes based on comparative sequence analysis. To accomplish these objectives, we conducted a combined phylogenetic and comparative genomic analysis involving taxa that represent each of the major lineages of gnathostome vertebrates. Results of synteny comparisons and phylogenetic topology tests revealed that GbE is clearly not the product of a recent, bird-specific duplication event. Instead, GbE originated via duplication of a proto-Mb gene in the stem lineage of gnathostomes. Unlike the Mb gene, which has been retained in all major gnathostome lineages other than amphibians, the GbE gene has been retained only in the lineage leading to modern

  10. Time-Series Interactions of Gene Expression, Vascular Growth and Hemodynamics during Early Embryonic Arterial Development

    PubMed Central

    Goktas, Selda; Uslu, Fazil E.; Kowalski, William J.; Ermek, Erhan; Keller, Bradley B.

    2016-01-01

    The role of hemodynamic forces within the embryo as biomechanical regulators for cardiovascular morphogenesis, growth, and remodeling is well supported through the experimental studies. Furthermore, clinical experience suggests that perturbed flow disrupts the normal vascular growth process as one etiology for congenital heart diseases (CHD) and for fetal adaptation to CHD. However, the relationships between hemodynamics, gene expression and embryonic vascular growth are poorly defined due to the lack of concurrent, sequential in vivo data. In this study, a long-term, time-lapse optical coherence tomography (OCT) imaging campaign was conducted to acquire simultaneous blood velocity, pulsatile micro-pressure and morphometric data for 3 consecutive early embryonic stages in the chick embryo. In conjunction with the in vivo growth and hemodynamics data, in vitro reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to track changes in transcript expression relevant to histogenesis and remodeling of the embryonic arterial wall. Our non-invasive extended OCT imaging technique for the microstructural data showed continuous vessel growth. OCT data coupled with the PIV technique revealed significant but intermitted increases in wall shear stress (WSS) between first and second assigned stages and a noticeable decrease afterwards. Growth rate, however, did not vary significantly throughout the embryonic period. Among all the genes studied, only the MMP-2 and CASP-3 expression levels remained unchanged during the time course. Concurrent relationships were obtained among the transcriptional modulation of the genes, vascular growth and hemodynamics-related changes. Further studies are indicated to determine cause and effect relationships and reversibility between mechanical and molecular regulation of vasculogenesis. PMID:27552150

  11. Time-Series Interactions of Gene Expression, Vascular Growth and Hemodynamics during Early Embryonic Arterial Development.

    PubMed

    Goktas, Selda; Uslu, Fazil E; Kowalski, William J; Ermek, Erhan; Keller, Bradley B; Pekkan, Kerem

    2016-01-01

    The role of hemodynamic forces within the embryo as biomechanical regulators for cardiovascular morphogenesis, growth, and remodeling is well supported through the experimental studies. Furthermore, clinical experience suggests that perturbed flow disrupts the normal vascular growth process as one etiology for congenital heart diseases (CHD) and for fetal adaptation to CHD. However, the relationships between hemodynamics, gene expression and embryonic vascular growth are poorly defined due to the lack of concurrent, sequential in vivo data. In this study, a long-term, time-lapse optical coherence tomography (OCT) imaging campaign was conducted to acquire simultaneous blood velocity, pulsatile micro-pressure and morphometric data for 3 consecutive early embryonic stages in the chick embryo. In conjunction with the in vivo growth and hemodynamics data, in vitro reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to track changes in transcript expression relevant to histogenesis and remodeling of the embryonic arterial wall. Our non-invasive extended OCT imaging technique for the microstructural data showed continuous vessel growth. OCT data coupled with the PIV technique revealed significant but intermitted increases in wall shear stress (WSS) between first and second assigned stages and a noticeable decrease afterwards. Growth rate, however, did not vary significantly throughout the embryonic period. Among all the genes studied, only the MMP-2 and CASP-3 expression levels remained unchanged during the time course. Concurrent relationships were obtained among the transcriptional modulation of the genes, vascular growth and hemodynamics-related changes. Further studies are indicated to determine cause and effect relationships and reversibility between mechanical and molecular regulation of vasculogenesis. PMID:27552150

  12. Direct demonstration that the A gamma T globin gene is linked to the 4 bp promoter deletion in the beta A chromosome of sickle cell traits.

    PubMed

    Gilman, J G; Josifovska, O; Erlingsson, S; Milner, P F; Nagel, R L

    1993-08-01

    In beta zero-thalassemia and sickle cell patients, a 4 bp deletion at -222 to -225 of the A gamma globin promoter was associated with low expression of the A gamma T variant (threonine at codon 75 of A gamma), whereas A gamma I (isoleucine at 75) had the normal A gamma promoter and higher expression. However, it has been reported that the beta A chromosomes of sickle cell trait cases have the 4 bp deletion as a common polymorphism unlinked to the A gamma T allele. We now present data demonstrating the association of the A gamma T allele with the 4 bp deletion in beta A chromosomes of sickle cell traits.

  13. Tbx18 targets dermal condensates for labeling, isolation, and gene ablation during embryonic hair follicle formation.

    PubMed

    Grisanti, Laura; Clavel, Carlos; Cai, Xiaoqiang; Rezza, Amelie; Tsai, Su-Yi; Sennett, Rachel; Mumau, Melanie; Cai, Chen-Leng; Rendl, Michael

    2013-02-01

    How cell fate decisions of stem and progenitor cells are regulated by their microenvironment or niche is a central question in stem cell and regenerative biology. Although functional analysis of hair follicle epithelial stem cells by gene targeting is well established, the molecular and genetic characterization of the dermal counterpart during embryonic morphogenesis has been lacking because of the absence of cell type-specific drivers. Here, we report that T-box transcription factor Tbx18 specifically marks dermal papilla (DP) precursor cells during embryonic hair follicle morphogenesis. With Tbx18(LacZ), Tbx18(H2BGFP), and Tbx18(Cre) knock-in mouse models, we demonstrate LacZ and H2BGFP (nuclear green fluorescent protein) expression and Cre activity in dermal condensates of nascent first-wave hair follicles at E14.5. As Tbx18 expression becomes more widespread throughout the dermis at later developmental stages, we use tamoxifen-inducible Cre-expressing mice, Tbx18(MerCreMer), to exclusively target DP precursor cells and their progeny. Finally, we ablate Tbx18 in full knockout mice, but find no perturbations in hair follicle formation, suggesting that Tbx18 is dispensable for normal DP function. In summary, our study establishes Tbx18 as a genetic driver to target for the first time embryonic DP precursors for labeling, isolation, and gene ablation that will greatly enhance investigations into their molecular functions during hair follicle morphogenesis. PMID:22992803

  14. Tbx18 targets dermal condensates for labeling, isolation, and gene ablation during embryonic hair follicle formation.

    PubMed

    Grisanti, Laura; Clavel, Carlos; Cai, Xiaoqiang; Rezza, Amelie; Tsai, Su-Yi; Sennett, Rachel; Mumau, Melanie; Cai, Chen-Leng; Rendl, Michael

    2013-02-01

    How cell fate decisions of stem and progenitor cells are regulated by their microenvironment or niche is a central question in stem cell and regenerative biology. Although functional analysis of hair follicle epithelial stem cells by gene targeting is well established, the molecular and genetic characterization of the dermal counterpart during embryonic morphogenesis has been lacking because of the absence of cell type-specific drivers. Here, we report that T-box transcription factor Tbx18 specifically marks dermal papilla (DP) precursor cells during embryonic hair follicle morphogenesis. With Tbx18(LacZ), Tbx18(H2BGFP), and Tbx18(Cre) knock-in mouse models, we demonstrate LacZ and H2BGFP (nuclear green fluorescent protein) expression and Cre activity in dermal condensates of nascent first-wave hair follicles at E14.5. As Tbx18 expression becomes more widespread throughout the dermis at later developmental stages, we use tamoxifen-inducible Cre-expressing mice, Tbx18(MerCreMer), to exclusively target DP precursor cells and their progeny. Finally, we ablate Tbx18 in full knockout mice, but find no perturbations in hair follicle formation, suggesting that Tbx18 is dispensable for normal DP function. In summary, our study establishes Tbx18 as a genetic driver to target for the first time embryonic DP precursors for labeling, isolation, and gene ablation that will greatly enhance investigations into their molecular functions during hair follicle morphogenesis.

  15. Prediction of developmental chemical toxicity based on gene networks of human embryonic stem cells.

    PubMed

    Yamane, Junko; Aburatani, Sachiyo; Imanishi, Satoshi; Akanuma, Hiromi; Nagano, Reiko; Kato, Tsuyoshi; Sone, Hideko; Ohsako, Seiichiroh; Fujibuchi, Wataru

    2016-07-01

    Predictive toxicology using stem cells or their derived tissues has gained increasing importance in biomedical and pharmaceutical research. Here, we show that toxicity category prediction by support vector machines (SVMs), which uses qRT-PCR data from 20 categorized chemicals based on a human embryonic stem cell (hESC) system, is improved by the adoption of gene networks, in which network edge weights are added as feature vectors when noisy qRT-PCR data fail to make accurate predictions. The accuracies of our system were 97.5-100% for three toxicity categories: neurotoxins (NTs), genotoxic carcinogens (GCs) and non-genotoxic carcinogens (NGCs). For two uncategorized chemicals, bisphenol-A and permethrin, our system yielded reasonable results: bisphenol-A was categorized as an NGC, and permethrin was categorized as an NT; both predictions were supported by recently published papers. Our study has two important features: (i) as the first study to employ gene networks without using conventional quantitative structure-activity relationships (QSARs) as input data for SVMs to analyze toxicogenomics data in an hESC validation system, it uses additional information of gene-to-gene interactions to significantly increase prediction accuracies for noisy gene expression data; and (ii) using only undifferentiated hESCs, our study has considerable potential to predict late-onset chemical toxicities, including abnormalities that occur during embryonic development. PMID:27207879

  16. Early embryonic failure: Expression and imprinted status of candidate genes on human chromosome 21

    SciTech Connect

    Sherman, L.S.; Bennett, P.R.; Moore, G.E.

    1994-09-01

    Two cases of maternal uniparental (hetero)disomy for human chromosome 21 (mUPD21) have been identified in a systematic search for UPD in 23 cases of early embryonic failure (EEF). Bi-parental origin of the other chromosome pairs was confirmed using specific VNTR probes or dinucleotide repeat analysis. Both maternally and paternally derived isochromosomes 21q have previously been identified in two individuals with normal phenotypes. Full UPD21 has a different mechanism of origin than uniparental isochromosome 21q and its effect on imprinted genes and phenotypic outcome will therefore not necessarily be the same. EEF associated with mUPD21 suggests that developmentally important genes on HSA 21 may be imprinted such that they are only expressed from either the maternally or paternally derived alleles. We have searched for monoallelic expression of candidate genes on HSA 21 in human pregnancy (CBS, IFNAR, COL6A1) using intragenic DNA polymorphisms. These genes were chosen either because their murine homologues lie in imprinted regions or because they are potentially important in embryogenesis. Once imprinted candidate genes have been identified, their methylation status and expression in normal, early embryonic failure and uniparental disomy 21 pregnancies will be studied. At the same time, a larger number of cases of EEF are being examined to further investigate the incidence of UPD21 in this group.

  17. Prediction of developmental chemical toxicity based on gene networks of human embryonic stem cells

    PubMed Central

    Yamane, Junko; Aburatani, Sachiyo; Imanishi, Satoshi; Akanuma, Hiromi; Nagano, Reiko; Kato, Tsuyoshi; Sone, Hideko; Ohsako, Seiichiroh; Fujibuchi, Wataru

    2016-01-01

    Predictive toxicology using stem cells or their derived tissues has gained increasing importance in biomedical and pharmaceutical research. Here, we show that toxicity category prediction by support vector machines (SVMs), which uses qRT-PCR data from 20 categorized chemicals based on a human embryonic stem cell (hESC) system, is improved by the adoption of gene networks, in which network edge weights are added as feature vectors when noisy qRT-PCR data fail to make accurate predictions. The accuracies of our system were 97.5–100% for three toxicity categories: neurotoxins (NTs), genotoxic carcinogens (GCs) and non-genotoxic carcinogens (NGCs). For two uncategorized chemicals, bisphenol-A and permethrin, our system yielded reasonable results: bisphenol-A was categorized as an NGC, and permethrin was categorized as an NT; both predictions were supported by recently published papers. Our study has two important features: (i) as the first study to employ gene networks without using conventional quantitative structure-activity relationships (QSARs) as input data for SVMs to analyze toxicogenomics data in an hESC validation system, it uses additional information of gene-to-gene interactions to significantly increase prediction accuracies for noisy gene expression data; and (ii) using only undifferentiated hESCs, our study has considerable potential to predict late-onset chemical toxicities, including abnormalities that occur during embryonic development. PMID:27207879

  18. The genes and mRNA coding for the heavy chains of chick embryonic skeletal myosin.

    PubMed

    Patrinou-Georgoulas, M; John, H A

    1977-10-01

    A size class of polysomes was isolated from chick embryonic leg skeletal muscle which synthesized almost exclusively a polypeptide chain with a molecular weight identical to the myosin heavy chain. The mRNA purified from these polysomes was shown to synthesize the 200,000 dalton polypeptide in the wheat germ cell-free translation system. At least 90% of the polypeptide had properties similar to the myosin heavy chain. Isoelectric focusing indicated that the myosin heavy chain synthesized in vitro contained two chains in equal amounts, as did purified embryonic leg skeletal muscle myosin. The kinetics of hybridization of the complementary DNA with an excess of the myosin heavy chain mRNA (MHC mRNA) indicated the presence of two different mRNA sequences. Reassociation of the cDNA to an excess of the DNA of the genome suggest that there is little, if any, reiteration of the myosin heavy chain genes.

  19. Erythroid-Specific Expression of β-globin from Sleeping Beauty-Transduced Human Hematopoietic Progenitor Cells

    PubMed Central

    Sjeklocha, Lucas M.; Park, Chang-Won; Wong, Phillip Y-P; Roney, Mark J.; Belcher, John D.; Kaufman, Dan S.; Vercellotti, Gregory M.; Hebbel, Robert P.; Steer, Clifford J.

    2011-01-01

    Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34+ cells with DsRed and a hybrid IHK–β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p = 0.05), indicating expression of β-globin from the integrated SB transgene. IHK–β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK–β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK–β-globin transgene for gene therapy of sickle cell disease. PMID:22216176

  20. Erythroid-specific expression of β-globin from Sleeping Beauty-transduced human hematopoietic progenitor cells.

    PubMed

    Sjeklocha, Lucas M; Park, Chang-Won; Wong, Phillip Y-P; Roney, Mark J; Belcher, John D; Kaufman, Dan S; Vercellotti, Gregory M; Hebbel, Robert P; Steer, Clifford J

    2011-01-01

    Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34(+) cells with DsRed and a hybrid IHK-β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p=0.05), indicating expression of β-globin from the integrated SB transgene. IHK-β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK-β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK-β-globin transgene for gene therapy of sickle cell disease.

  1. Efficient and specific repair of sickle beta-globin RNA by trans-splicing ribozymes.

    PubMed

    Byun, Jonghoe; Lan, Ning; Long, Meredith; Sullenger, Bruce A

    2003-10-01

    Previously we demonstrated that a group I ribozyme can perform trans-splicing to repair sickle beta-globin transcripts upon transfection of in vitro transcribed ribozyme into mammalian cells. Here, we sought to develop expression cassettes that would yield high levels of active ribozyme after gene transfer. Our initial expression constructs were designed to generate trans-slicing ribozymes identical to those used in our previous RNA transfection studies with ribozymes containing 6-nucleotide long internal guide sequences. The ribozymes expressed from these cassettes, however, were found to be unable to repair sickle beta-globin RNAs. Further experiments revealed that two additional structural elements are important for ribozyme-mediate RNA repair: the P10 interaction formed between the 5' end of the ribozyme and the beginning of the 3' exon and an additional base-pairing interaction formed between an extended guide sequence and the substrate RNA. These optimized expression cassettes yield ribozymes that are able to amend 10%-50% of the sickle beta-globin RNAs in transfected mammalian cells. Finally, a ribozyme with a 5-bp extended guide sequence preferentially reacts with sickle beta-globin RNAs over wild-type beta-globin RNAs, although the wild-type beta-globin transcript forms only a single mismatch with the ribozyme. These results demonstrate that trans-splicing ribozyme expression cassettes can be generated to yield ribozymes that can repair a clinically relevant fraction of sickle beta-globin RNAs in mammalian cells with greatly improved specificity.

  2. Integrated protein quality-control pathways regulate free α-globin in murine β-thalassemia

    PubMed Central

    Khandros, Eugene; Thom, Christopher S.; D'Souza, Janine

    2012-01-01

    Cells remove unstable polypeptides through protein quality-control (PQC) pathways such as ubiquitin-mediated proteolysis and autophagy. In the present study, we investigated how these pathways are used in β-thalassemia, a common hemoglobinopathy in which β-globin gene mutations cause the accumulation and precipitation of cytotoxic α-globin subunits. In β-thalassemic erythrocyte precursors, free α-globin was polyubiquitinated and degraded by the proteasome. These cells exhibited enhanced proteasome activity, and transcriptional profiling revealed coordinated induction of most proteasome subunits that was mediated by the stress-response transcription factor Nrf1. In isolated thalassemic cells, short-term proteasome inhibition blocked the degradation of free α-globin. In contrast, prolonged in vivo treatment of β-thalassemic mice with the proteasome inhibitor bortezomib did not enhance the accumulation of free α-globin. Rather, systemic proteasome inhibition activated compensatory proteotoxic stress-response mechanisms, including autophagy, which cooperated with ubiquitin-mediated proteolysis to degrade free α-globin in erythroid cells. Our findings show that multiple interregulated PQC responses degrade excess α-globin. Therefore, β-thalassemia fits into the broader framework of protein-aggregation disorders that use PQC pathways as cell-protective mechanisms. PMID:22427201

  3. 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

  4. High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells.

    PubMed

    Carlson-Stevermer, Jared; Goedland, Madelyn; Steyer, Benjamin; Movaghar, Arezoo; Lou, Meng; Kohlenberg, Lucille; Prestil, Ryan; Saha, Krishanu

    2016-01-12

    CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.

  5. High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells

    PubMed Central

    Carlson-Stevermer, Jared; Goedland, Madelyn; Steyer, Benjamin; Movaghar, Arezoo; Lou, Meng; Kohlenberg, Lucille; Prestil, Ryan; Saha, Krishanu

    2016-01-01

    Summary CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing. PMID:26771356

  6. The nucleoporin Nup153 regulates embryonic stem cell pluripotency through gene silencing

    PubMed Central

    Jacinto, Filipe V.; Benner, Chris; Hetzer, Martin W.

    2015-01-01

    Nucleoporins (Nups) are a family of proteins best known as the constituent building blocks of nuclear pore complexes (NPCs), membrane-embedded channels that mediate nuclear transport across the nuclear envelope. Recent evidence suggests that several Nups have additional roles in controlling the activation and silencing of developmental genes; however, the mechanistic details of these functions remain poorly understood. Here, we show that depletion of Nup153 in mouse embryonic stem cells (mESCs) causes the derepression of developmental genes and induction of early differentiation. This loss of stem cell identity is not associated with defects in the nuclear import of key pluripotency factors. Rather, Nup153 binds around the transcriptional start site (TSS) of developmental genes and mediates the recruitment of the polycomb-repressive complex 1 (PRC1) to a subset of its target loci. Our results demonstrate a chromatin-associated role of Nup153 in maintaining stem cell pluripotency by functioning in mammalian epigenetic gene silencing. PMID:26080816

  7. Variation in RNA-Seq Transcriptome Profiles of Peripheral Whole Blood from Healthy Individuals with and without Globin Depletion

    PubMed Central

    Fishbane, Nick; Ruan, Jian; Zhou, Mi; Balshaw, Robert; Wilson-McManus, Janet E.; Ng, Raymond T.; McManus, Bruce M.; Tebbutt, Scott J.

    2014-01-01

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

  8. MicroRNA and gene expression patterns in the differentiation of human embryonic stem cells

    PubMed Central

    Ren, Jiaqiang; Jin, Ping; Wang, Ena; Marincola, Francesco M; Stroncek, David F

    2009-01-01

    Background The unique features of human embryonic stem (hES) cells make them the best candidate resource for both cell replacement therapy and development research. However, the molecular mechanisms responsible for the simultaneous maintenance of their self-renewal properties and undifferentiated state remain unclear. Non-coding microRNAs (miRNA) which regulate mRNA cleavage and inhibit encoded protein translation exhibit temporal or tissue-specific expression patterns and they play an important role in development timing. Results In this study, we analyzed miRNA and gene expression profiles among samples from 3 hES cell lines (H9, I6 and BG01v), differentiated embryoid bodies (EB) derived from H9 cells at different time points, and 5 adult cell types including Human Microvascular Endothelial Cells (HMVEC), Human Umbilical Vein Endothelial Cells (HUVEC), Umbilical Artery Smooth Muscle Cells (UASMC), Normal Human Astrocytes (NHA), and Lung Fibroblasts (LFB). This analysis rendered 104 miRNAs and 776 genes differentially expressed among the three cell types. Selected differentially expressed miRNAs and genes were further validated and confirmed by quantitative real-time-PCR (qRT-PCR). Especially, members of the miR-302 cluster on chromosome 4 and miR-520 cluster on chromosome 19 were highly expressed in undifferentiated hES cells. MiRNAs in these two clusters displayed similar expression levels. The members of these two clusters share a consensus 7-mer seed sequence and their targeted genes had overlapping functions. Among the targeted genes, genes with chromatin structure modification function are enriched suggesting a role in the maintenance of chromatin structure. We also found that the expression level of members of the two clusters, miR-520b and miR-302c, were negatively correlated with their targeted genes based on gene expression analysis Conclusion We identified the expression patterns of miRNAs and gene transcripts in the undifferentiation of human embryonic

  9. A mouse embryonic stem cell bank for inducible overexpression of human chromosome 21 genes

    PubMed Central

    2010-01-01

    Background Dosage imbalance is responsible for several genetic diseases, among which Down syndrome is caused by the trisomy of human chromosome 21. Results To elucidate the extent to which the dosage imbalance of specific human chromosome 21 genes perturb distinct molecular pathways, we developed the first mouse embryonic stem (ES) cell bank of human chromosome 21 genes. The human chromosome 21-mouse ES cell bank includes, in triplicate clones, 32 human chromosome 21 genes, which can be overexpressed in an inducible manner. Each clone was transcriptionally profiled in inducing versus non-inducing conditions. Analysis of the transcriptional response yielded results that were consistent with the perturbed gene's known function. Comparison between mouse ES cells containing the whole human chromosome 21 (trisomic mouse ES cells) and mouse ES cells overexpressing single human chromosome 21 genes allowed us to evaluate the contribution of single genes to the trisomic mouse ES cell transcriptome. In addition, for the clones overexpressing the Runx1 gene, we compared the transcriptome changes with the corresponding protein changes by mass spectroscopy analysis. Conclusions We determined that only a subset of genes produces a strong transcriptional response when overexpressed in mouse ES cells and that this effect can be predicted taking into account the basal gene expression level and the protein secondary structure. We showed that the human chromosome 21-mouse ES cell bank is an important resource, which may be instrumental towards a better understanding of Down syndrome and other human aneuploidy disorders. PMID:20569505

  10. [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

  11. A new role for muscle segment homeobox genes in mammalian embryonic diapause

    PubMed Central

    Cha, Jeeyeon; Sun, Xiaofei; Bartos, Amanda; Fenelon, Jane; Lefèvre, Pavine; Daikoku, Takiko; Shaw, Geoff; Maxson, Robert; Murphy, Bruce D.; Renfree, Marilyn B.; Dey, Sudhansu K.

    2013-01-01

    Mammalian embryonic diapause is a phenomenon defined by the temporary arrest in blastocyst growth and metabolic activity within the uterus which synchronously becomes quiescent to blastocyst activation and implantation. This reproductive strategy temporally uncouples conception from parturition until environmental or maternal conditions are favourable for the survival of the mother and newborn. The underlying molecular mechanism by which the uterus and embryo temporarily achieve quiescence, maintain blastocyst survival and then resume blastocyst activation with subsequent implantation remains unknown. Here, we show that uterine expression of Msx1 or Msx2, members of an ancient, highly conserved homeobox gene family, persists in three unrelated mammalian species during diapause, followed by rapid downregulation with blastocyst activation and implantation. Mice with uterine inactivation of Msx1 and Msx2 fail to achieve diapause and reactivation. Remarkably, the North American mink and Australian tammar wallaby share similar expression patterns of MSX1 or MSX2 as in mice—it persists during diapause and is rapidly downregulated upon blastocyst activation and implantation. Evidence from mouse studies suggests that the effects of Msx genes in diapause are mediated through Wnt5a, a known transcriptional target of uterine Msx. These studies provide strong evidence that the Msx gene family constitutes a common conserved molecular mediator in the uterus during embryonic diapause to improve female reproductive fitness. PMID:23615030

  12. A new role for muscle segment homeobox genes in mammalian embryonic diapause.

    PubMed

    Cha, Jeeyeon; Sun, Xiaofei; Bartos, Amanda; Fenelon, Jane; Lefèvre, Pavine; Daikoku, Takiko; Shaw, Geoff; Maxson, Robert; Murphy, Bruce D; Renfree, Marilyn B; Dey, Sudhansu K

    2013-04-01

    Mammalian embryonic diapause is a phenomenon defined by the temporary arrest in blastocyst growth and metabolic activity within the uterus which synchronously becomes quiescent to blastocyst activation and implantation. This reproductive strategy temporally uncouples conception from parturition until environmental or maternal conditions are favourable for the survival of the mother and newborn. The underlying molecular mechanism by which the uterus and embryo temporarily achieve quiescence, maintain blastocyst survival and then resume blastocyst activation with subsequent implantation remains unknown. Here, we show that uterine expression of Msx1 or Msx2, members of an ancient, highly conserved homeobox gene family, persists in three unrelated mammalian species during diapause, followed by rapid downregulation with blastocyst activation and implantation. Mice with uterine inactivation of Msx1 and Msx2 fail to achieve diapause and reactivation. Remarkably, the North American mink and Australian tammar wallaby share similar expression patterns of MSX1 or MSX2 as in mice-it persists during diapause and is rapidly downregulated upon blastocyst activation and implantation. Evidence from mouse studies suggests that the effects of Msx genes in diapause are mediated through Wnt5a, a known transcriptional target of uterine Msx. These studies provide strong evidence that the Msx gene family constitutes a common conserved molecular mediator in the uterus during embryonic diapause to improve female reproductive fitness. PMID:23615030

  13. Gene expression analysis of mouse embryonic stem cells following levitation in an ultrasound standing wave trap.

    PubMed

    Bazou, Despina; Kearney, Roisin; Mansergh, Fiona; Bourdon, Celine; Farrar, Jane; Wride, Michael

    2011-02-01

    In the present paper, gene expression analysis of mouse embryonic stem (ES) cells levitated in a novel ultrasound standing wave trap (USWT) (Bazou et al. 2005a) at variable acoustic pressures (0.08-0.85 MPa) and times (5-60 min) was performed. Our results showed that levitation of ES cells at the highest employed acoustic pressure for 60 min does not modify gene expression and cells maintain their pluripotency. Embryoid bodies (EBs) also expressed the early and late neural differentiation markers, which were also unaffected by the acoustic field. Our results suggest that the ultrasound trap microenvironment is minimally invasive as the biologic consequences of ES cell replication and EB differentiation proceed without significantly affecting gene expression. The technique holds great promise in safe cell manipulation techniques for a variety of applications including tissue engineering and regenerative medicine. PMID:21208732

  14. Gene Expression Analysis of Mouse Embryonic Stem Cells Following Levitation in an Ultrasound Standing Wave Trap

    PubMed Central

    Bazou, Despina; Kearney, Roisin; Mansergh, Fiona; Bourdon, Celine; Farrar, Jane; Wride, Michael

    2011-01-01

    In the present paper, gene expression analysis of mouse embryonic stem (ES) cells levitated in a novel ultrasound standing wave trap (USWT) (Bazou et al. 2005a) at variable acoustic pressures (0.08–0.85 MPa) and times (5–60 min) was performed. Our results showed that levitation of ES cells at the highest employed acoustic pressure for 60 min does not modify gene expression and cells maintain their pluripotency. Embryoid bodies (EBs) also expressed the early and late neural differentiation markers, which were also unaffected by the acoustic field. Our results suggest that the ultrasound trap microenvironment is minimally invasive as the biologic consequences of ES cell replication and EB differentiation proceed without significantly affecting gene expression. The technique holds great promise in safe cell manipulation techniques for a variety of applications including tissue engineering and regenerative medicine. (E-mail: Bazoud@tcd.ie) PMID:21208732

  15. Dual promoter activation by the human beta-globin locus control region.

    PubMed Central

    Bresnick, E H; Felsenfeld, G

    1994-01-01

    The human beta-globin locus control region (LCR) is necessary for high-level and position-independent expression of globin genes in erythroid cells. A variety of mechanisms have been proposed for the cis-activation of individual members of the beta-globin gene family by the LCR located 10-50 kilobases upstream. It is not known, however, whether a given LCR can activate all developmentally appropriate globin family members on its chromosome or whether, within a given chromosome, the LCR must be committed to activating only a single gene. We have devised an experiment to distinguish between these possibilities. This experiment takes advantage of the fact that if two genes in a cluster are transcriptionally active and their promoters, therefore, are in a conformation hypersensitive to nucleases, restriction enzymes that cleave the promoters will excise the intervening chromatin fragment. The Apa I sites on human fetal G gamma- and A gamma-globin gene promoters are accessible to cleavage in nuclei from the human erythroleukemia cell line K562, which expresses these genes, but not in HeLa cells. We find that Apa I digestion leads to excision in high yield of the fragment spanning these promoters, showing that a LCR element is capable of sharing its activating function among members of a gene cluster on a single chromosome. Images PMID:8108408

  16. Comparative analysis of temporal gene expression patterns in the developing ovary of the embryonic chicken

    PubMed Central

    YU, Minli; XU, Yali; YU, Defu; YU, Debing; DU, Wenxing

    2015-01-01

    Many genes participate in the process of ovarian germ cell development, while the combined action mechanisms of these molecular regulators still need clarification. The present study was focused on determination of differentially expressed genes and gene functions at four critical time points in chicken ovarian development. Comparative transcriptional profiling of ovaries from embryonic day 5.5 (E5.5), E12.5, E15.5 and E18.5 was performed using an Affymetrix GeneChip chicken genome microarray. Differential expression patterns for genes specifically depleted and enriched in each stage were identified. The results showed that most of the up- and downregulated genes were involved in the metabolism of retinoic acid (RA) and synthesis of hormones. Among them, a higher number of up- and downregulated genes in the E15.5 ovary were identified as being involved in steroid biosynthesis and retinol metabolism, respectively. To validate gene changes, expressions of twelve candidate genes related to germ cell development were examined by real-time PCR and found to be consistent with the of GeneChip data. Moreover, the immunostaining results suggested that ovarian development during different stages was regulated by different genes. Furthermore, a Raldh2 knockdown chicken model was produced to investigate the fundamental role of Raldh2 in meiosis initiation. It was found that meiosis occurred abnormally in Raldh2 knockdown ovaries, but the inhibitory effect on meiosis was reversed by the addition of exogenous RA. This study offers insights into the profile of gene expression and mechanisms regulating ovarian development, especially the notable role of Raldh2 in meiosis initiation in the chicken. PMID:25736178

  17. Functional Genomics Screening Utilizing Mutant Mouse Embryonic Stem Cells Identifies Novel Radiation-Response Genes

    PubMed Central

    Loesch, Kimberly; Galaviz, Stacy; Hamoui, Zaher; Clanton, Ryan; Akabani, Gamal; Deveau, Michael; DeJesus, Michael; Ioerger, Thomas; Sacchettini, James C.; Wallis, Deeann

    2015-01-01

    Elucidating the genetic determinants of radiation response is crucial to optimizing and individualizing radiotherapy for cancer patients. In order to identify genes that are involved in enhanced sensitivity or resistance to radiation, a library of stable mutant murine embryonic stem cells (ESCs), each with a defined mutation, was screened for cell viability and gene expression in response to radiation exposure. We focused on a cancer-relevant subset of over 500 mutant ESC lines. We identified 13 genes; 7 genes that have been previously implicated in radiation response and 6 other genes that have never been implicated in radiation response. After screening, proteomic analysis showed enrichment for genes involved in cellular component disassembly (e.g. Dstn and Pex14) and regulation of growth (e.g. Adnp2, Epc1, and Ing4). Overall, the best targets with the highest potential for sensitizing cancer cells to radiation were Dstn and Map2k6, and the best targets for enhancing resistance to radiation were Iqgap and Vcan. Hence, we provide compelling evidence that screening mutant ESCs is a powerful approach to identify genes that alter radiation response. Ultimately, this knowledge can be used to define genetic variants or therapeutic targets that will enhance clinical therapy. PMID:25853515

  18. DNA demethylation reactivates a subset of imprinted genes in uniparental mouse embryonic fibroblasts.

    PubMed

    El Kharroubi, A; Piras, G; Stewart, C L

    2001-03-23

    Although most imprinted genes show allelic differences in DNA methylation, it is not clear whether methylation regulates the expression of some or all imprinted genes in somatic cells. To examine the mechanisms of silencing of imprinted alleles, we generated novel uniparental mouse embryonic fibroblasts exclusively containing either the paternal or the maternal genome. These fibroblasts retain parent-of-origin allele-specific expression of 12 imprinted genes examined for more than 30 cell generations. We show that p57(Kip2) (cyclin-dependent kinase inhibitor protein 2) and Igf2 (insulin-like growth factor 2) are induced by inhibiting histone deacetylases; however, their activated state is reversed quickly by withdrawal of trichostatin A. In contrast, DNA demethylation results in the heritable expression of a subset of imprinted genes including H19 (H19 fetal liver mRNA), p57(Kip2), Peg3/Pw1 (paternally expressed gene 3), and Zac1 (zinc finger-binding protein regulating apoptosis and cell cycle arrest). Other imprinted genes such as Grb10 (growth factor receptor-bound protein 10), Peg1/Mest (paternally expressed gene 1/mesoderm-specific transcript), Sgce (epsilon-sarcoglycan), Snrpn (small nuclear ribonucleoprotein polypeptide N), and U2af1 (U2 small nuclear ribonucleoprotein auxiliary factor), remain inactive, despite their exposure to inhibitors of histone deacetylases and DNA methylation. These results demonstrate that changes in DNA methylation but not histone acetylation create a heritable epigenetic state at some imprinted loci in somatic cells. PMID:11124954

  19. MicroRNA-10 modulates Hox genes expression during Nile tilapia embryonic development.

    PubMed

    Giusti, Juliana; Pinhal, Danillo; Moxon, Simon; Campos, Camila Lovaglio; Münsterberg, Andrea; Martins, Cesar

    2016-05-01

    Hox gene clusters encode a family of transcription factors that govern anterior-posterior axis patterning during embryogenesis in all bilaterian animals. The time and place of Hox gene expression are largely determined by the relative position of each gene within its cluster. Furthermore, Hox genes were shown to have their expression fine-tuned by regulatory microRNAs (miRNAs). However, the mechanisms of miRNA-mediated regulation of these transcription factors during fish early development remain largely unknown. Here we have profiled three highly expressed miR-10 family members of Nile tilapia at early embryonic development, determined their genomic organization as well as performed functional experiments for validation of target genes. Quantitative analysis during developmental stages showed miR-10 family expression negatively correlates with the expression of HoxA3a, HoxB3a and HoxD10a genes, as expected for bona fide miRNA-mRNA interactions. Moreover, luciferase assays demonstrated that HoxB3a and HoxD10a are targeted by miR-10b-5p. Overall, our data indicate that the miR-10 family directly regulates members of the Hox gene family during Nile tilapia embryogenesis. PMID:26980108

  20. Gene Targeting Using Homologous Recombination in Embryonic Stem Cells: The Future for Behavior Genetics?

    PubMed Central

    Gerlai, Robert

    2016-01-01

    Gene targeting with homologous recombination in embryonic stem cells created a revolution in the analysis of the function of genes in behavioral brain research. The technology allowed unprecedented precision with which one could manipulate genes and study the effect of this manipulation on the central nervous system. With gene targeting, the uncertainty inherent in psychopharmacology regarding whether a particular compound would act only through a specific target was removed. Thus, gene targeting became highly popular. However, with this popularity came the realization that like other methods, gene targeting also suffered from some technical and principal problems. For example, two decades ago, issues about compensatory changes and about genetic linkage were raised. Since then, the technology developed, and its utility has been better delineated. This review will discuss the pros and cons of the technique along with these advancements from the perspective of the neuroscientist user. It will also compare and contrast methods that may represent novel alternatives to the homologous recombination based gene targeting approach, including the TALEN and the CRISPR/Cas9 systems. The goal of the review is not to provide detailed recipes, but to attempt to present a short summary of these approaches a behavioral geneticist or neuroscientist may consider for the analysis of brain function and behavior. PMID:27148349

  1. Systematic identification of gene family regulators in mouse and human embryonic stem cells

    PubMed Central

    Aaronson, Yair; Livyatan, Ilana; Gokhman, David; Meshorer, Eran

    2016-01-01

    Pluripotent self-renewing embryonic stem cells (ESCs) have been the focus of a growing number of high-throughput experiments, revealing the genome-wide locations of hundreds of transcription factors and histone modifications. While most of these datasets were used in a specific context, all datasets combined offer a comprehensive view of chromatin characteristics and regulatory elements that govern cell states. Here, using hundreds of datasets in ESCs, we generated colocalization maps of chromatin proteins and modifications, and built a discovery pipeline for regulatory proteins of gene families. By comparing genome-wide binding data with over-expression and knockdown analysis of hundreds of genes, we discovered that the pluripotency-related factor NR5A2 separates mitochondrial from cytosolic ribosomal genes, regulating their expression. We further show that genes with a common chromatin profile are enriched for distinct Gene Ontology (GO) categories. Our approach can be generalized to reveal common regulators of any gene group; discover novel gene families, and identify common genomic elements based on shared chromatin features. PMID:27084933

  2. Gene Targeting Using Homologous Recombination in Embryonic Stem Cells: The Future for Behavior Genetics?

    PubMed

    Gerlai, Robert

    2016-01-01

    Gene targeting with homologous recombination in embryonic stem cells created a revolution in the analysis of the function of genes in behavioral brain research. The technology allowed unprecedented precision with which one could manipulate genes and study the effect of this manipulation on the central nervous system. With gene targeting, the uncertainty inherent in psychopharmacology regarding whether a particular compound would act only through a specific target was removed. Thus, gene targeting became highly popular. However, with this popularity came the realization that like other methods, gene targeting also suffered from some technical and principal problems. For example, two decades ago, issues about compensatory changes and about genetic linkage were raised. Since then, the technology developed, and its utility has been better delineated. This review will discuss the pros and cons of the technique along with these advancements from the perspective of the neuroscientist user. It will also compare and contrast methods that may represent novel alternatives to the homologous recombination based gene targeting approach, including the TALEN and the CRISPR/Cas9 systems. The goal of the review is not to provide detailed recipes, but to attempt to present a short summary of these approaches a behavioral geneticist or neuroscientist may consider for the analysis of brain function and behavior.

  3. The oct3 gene, a gene for an embryonic transcription factor, is controlled by a retinoic acid repressible enhancer.

    PubMed Central

    Okazawa, H; Okamoto, K; Ishino, F; Ishino-Kaneko, T; Takeda, S; Toyoda, Y; Muramatsu, M; Hamada, H

    1991-01-01

    Oct3 is an embryonic octamer-binding transcription factor, whose expression is rapidly repressed by retinoic acid (RA). In this report, we have determined the transcriptional control region of the oct3 gene and studied the mechanism of the RA-mediated repression. The chromosomal oct3 gene consists of five exons. Three subdomains of the POU region and transactivating domain are located in separate exons. Transcription initiates at multiple sites in the GC-rich region lacking a typical TATA box. The upstream 2 kb region can confer the cell type-specific expression and RA-mediated repression. Analysis of the upstream region by deletion mutagenesis locates a cis element (RARE1) which functions as a stem cell-specific, yet RA-repressible, enhancer. Footprint and gel-retardation assays show that RARE1 is composed of two domains, each of which is recognized by distinct factors. Microinjection of oct3-lacZ constructs into fertilized eggs indicates that RARE1 can function in early embryos. We suggest that RARE1 is a critical cis element for oct3 gene expression in embryonic stem cells and for the RA-mediated repression. Images PMID:1915274

  4. Myogenic Differentiation of Mouse Embryonic Stem Cells That Lack a Functional Pax7 Gene

    PubMed Central

    Czerwinska, Areta M.; Grabowska, Iwona; Archacka, Karolina; Bem, Joanna; Swierczek, Barbara; Helinska, Anita; Streminska, Wladyslawa; Fogtman, Anna; Iwanicka-Nowicka, Roksana; Koblowska, Marta

    2016-01-01

    The transcription factor Pax7 plays a key role during embryonic myogenesis and sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Overexpression of Pax7 has been shown to promote the myogenic differentiation of pluripotent stem cells. However, the effects of the absence of functional Pax7 in differentiating embryonic stem cells (ESCs) have not yet been directly tested. Herein, we studied mouse stem cells that lacked a functional Pax7 gene and characterized the differentiation of these stem cells under conditions that promoted the derivation of myoblasts in vitro. We analyzed the expression of myogenic factors, such as myogenic regulatory factors and muscle-specific microRNAs, in wild-type and mutant cells. Finally, we compared the transcriptome of both types of cells and did not find substantial differences in the expression of genes related to the regulation of myogenesis. As a result, we showed that the absence of functional Pax7 does not prevent the in vitro myogenic differentiation of ESCs. PMID:26649785

  5. Characterization of tweety gene (ttyh1-3) expression in Xenopus laevis during embryonic development

    PubMed Central

    Rabe, Brian A.; Huyck, Ryan W.; Williams, Cheyenne C.; Saha, Margaret S.

    2015-01-01

    The tweety family of genes encodes large-conductance chloride channels and has been implicated in a wide array of cellular processes including cell division, cell adhesion, regulation of calcium activity, and tumorigenesis, particularly in neuronal cells. However, their expression patterns during early development remain largely unknown. Here, we describe the spatial and temporal patterning of ttyh1, ttyh2, and ttyh3 in Xenopus laevis during early embryonic development. Ttyh1 and ttyh3 are initially expressed at the late neurula stage are and primarily localized to the developing nervous system; however ttyh1 and ttyh3 both show transient expression in the somites. By swimming tadpole stages, all three genes are expressed in the brain, spinal cord, eye, and cranial ganglia. While ttyh1 is restricted to proliferative, ventricular zones, ttyh3 is primarily localized to postmitotic regions of the developing nervous system. Ttyh2, however, is strongly expressed in cranial ganglia V, VII, IX and X. The differing temporal and spatial expression patterns of ttyh1, ttyh2, and ttyh3 suggest that they may play distinct roles throughout embryonic development. PMID:25541457

  6. MIWI2 as an Effector of DNA Methylation and Gene Silencing in Embryonic Male Germ Cells.

    PubMed

    Kojima-Kita, Kanako; Kuramochi-Miyagawa, Satomi; Nagamori, Ippei; Ogonuki, Narumi; Ogura, Atsuo; Hasuwa, Hidetoshi; Akazawa, Takashi; Inoue, Norimitsu; Nakano, Toru

    2016-09-13

    During the development of mammalian embryonic germ cells, global demethylation and de novo DNA methylation take place. In mouse embryonic germ cells, two PIWI family proteins, MILI and MIWI2, are essential for the de novo DNA methylation of retrotransposons, presumably through PIWI-interacting RNAs (piRNAs). Although piRNA-associated MIWI2 has been reported to play critical roles in the process, its molecular mechanisms have remained unclear. To identify the mechanism, transgenic mice were produced; they contained a fusion protein of MIWI2 and a zinc finger (ZF) that recognized the promoter region of a type A LINE-1 gene. The ZF-MIWI2 fusion protein brought about DNA methylation, suppression of the type A LINE-1 gene, and a partial rescue of the impaired spermatogenesis of MILI-null mice. In addition, ZF-MIWI2 was associated with the proteins involved in DNA methylation. These data indicate that MIWI2 functions as an effector of de novo DNA methylation of the retrotransposon. PMID:27626653

  7. Expression dynamics of WOX genes mark cell fate decisions during early embryonic patterning in Arabidopsis thaliana.

    PubMed

    Haecker, Achim; Gross-Hardt, Rita; Geiges, Bernd; Sarkar, Ananda; Breuninger, Holger; Herrmann, Marita; Laux, Thomas

    2004-02-01

    During embryonic pattern formation, the main body axes are established and cells of different developmental fates are specified from a single-cell zygote. Despite the fundamental importance of this process, in plants, the underlying mechanisms are largely unknown. We show that expression dynamics of novel WOX (WUSCHEL related homeobox) gene family members reveal early embryonic patterning events in Arabidopsis. WOX2 and WOX8 are co-expressed in the egg cell and zygote and become confined to the apical and basal daughter cells of the zygote, respectively, by its asymmetric division. WOX2 not only marks apical descendants of the zygote, but is also functionally required for their correct development, suggesting that the asymmetric division of the plant zygote separates determinants of apical and basal cell fates. WOX9 expression is initiated in the basal daughter cell of the zygote and subsequently shifts into the descendants of the apical daughter apparently in response to signaling from the embryo proper. Expression of WOX5 shows that identity of the quiescent center is initiated very early in the hypophyseal cell, and highlights molecular and developmental similarities between the stem cell niches of root and shoot meristems. Together, our data suggest that during plant embryogenesis region-specific transcription programs are initiated very early in single precursor cells and that WOX genes play an important role in this process.

  8. Disruption of Murine mp29/Syf2/Ntc31 Gene Results in Embryonic Lethality with Aberrant Checkpoint Response

    PubMed Central

    Lee, Liekyeow; Lien, Huang-Wei; Lin, Tse-Ling; Fan, Chi-Chen; Chi, Peter; Huang, Chang-Jen; Chang, Mau-Sun

    2012-01-01

    Human p29 is a putative component of spliceosomes, but its role in pre-mRNA is elusive. By siRNA knockdown and stable overexpression, we demonstrated that human p29 is involved in DNA damage response and Fanconi anemia pathway in cultured cells. In this study, we generated p29 knockout mice (mp29GT/GT) using the mp29 gene trap embryonic stem cells to study the role of mp29 in DNA damage response in vivo. Interruption of mp29 at both alleles resulted in embryonic lethality. Embryonic abnormality occurred as early as E6.5 in mp29GT/GT mice accompanied with decreased mRNA levels of α-tubulin and Chk1. The reduction of α-tubulin and Chk1 mRNAs is likely due to an impaired post-transcriptional event. An aberrant G2/M checkpoint was found in mp29 gene trap embryos when exposed to aphidicolin and UV light. This embryonic lethality was rescued by crossing with mp29 transgenic mice. Additionally, the knockdown of zfp29 in zebrafish resulted in embryonic death at 72 hours of development postfertilization (hpf). A lower level of acetylated α-tubulin was also observed in zfp29 morphants. Together, these results illustrate an indispensable role of mp29 in DNA checkpoint response during embryonic development. PMID:22448250

  9. Differential and stage-related expression in embryonic tissues of a new human homoeobox gene.

    PubMed

    Mavilio, F; Simeone, A; Giampaolo, A; Faiella, A; Zappavigna, V; Acampora, D; Poiana, G; Russo, G; Peschle, C; Boncinelli, E

    The homoeobox is a 183 base-pair (bp) DNA sequence conserved in several Drosophila genes controlling segmentation and segment identity. Homoeobox sequences have been detected in the genome of species ranging from insects and anellids to vertebrates and homoeobox containing genes have been cloned from Xenopus, mouse and man. We recently isolated human homoeobox containing complementary DNA clones, that represent transcripts from four different human genes. One clone (HHO.c10) is selectively expressed in a 2.1 kilobase (kb) polyadenylated transcript in the spinal cord of human embryos and fetuses 5-10 weeks after fertilization. We report the characterization of a second cDNA clone, termed HHO.c13, that represents a new homoeobox gene. This clone encodes a protein of 255 amino-acid residues, which includes a pentapeptide, upstream of the homoeo domain, conserved in other Drosophila, Xenopus, murine and human homoeobox genes. By Northern analysis HHO.c13 detects multiple embryonic transcripts, which are differentially expressed in spinal cord, brain, backbone rudiments, limb buds and heart in 5-9-week-old human embryos and fetuses, in a striking organ- and stage-specific pattern. These observations suggest that in early mammalian development homoeobox genes may exert a wide spectrum of control functions in a variety of organs and body parts, in addition to the spinal cord. PMID:2879245

  10. Inference of the Xenopus tropicalis embryonic regulatory network and spatial gene expression patterns

    PubMed Central

    2014-01-01

    Background During embryogenesis, signaling molecules produced by one cell population direct gene regulatory changes in neighboring cells and influence their developmental fates and spatial organization. One of the earliest events in the development of the vertebrate embryo is the establishment of three germ layers, consisting of the ectoderm, mesoderm and endoderm. Attempts to measure gene expression in vivo in different germ layers and cell types are typically complicated by the heterogeneity of cell types within biological samples (i.e., embryos), as the responses of individual cell types are intermingled into an aggregate observation of heterogeneous cell types. Here, we propose a novel method to elucidate gene regulatory circuits from these aggregate measurements in embryos of the frog Xenopus tropicalis using gene network inference algorithms and then test the ability of the inferred networks to predict spatial gene expression patterns. Results We use two inference models with different underlying assumptions that incorporate existing network information, an ODE model for steady-state data and a Markov model for time series data, and contrast the performance of the two models. We apply our method to both control and knockdown embryos at multiple time points to reconstruct the core mesoderm and endoderm regulatory circuits. Those inferred networks are then used in combination with known dorsal-ventral spatial expression patterns of a subset of genes to predict spatial expression patterns for other genes. Both models are able to predict spatial expression patterns for some of the core mesoderm and endoderm genes, but interestingly of different gene subsets, suggesting that neither model is sufficient to recapitulate all of the spatial patterns, yet they are complementary for the patterns that they do capture. Conclusion The presented methodology of gene network inference combined with spatial pattern prediction provides an additional layer of validation to

  11. Dissecting the heterogeneity of gene expressions in mouse embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Zou, Ling-Nan; Thomson, Matt; Liu, S. John; Ramanathan, Sharad

    2011-03-01

    A population of genetically identical cells, of the same nominal cell type, and cultured in the same petri dish, will nevertheless often exhibit varying patterns of gene expression. Taking mouse embryonic stem (ES) cells as a model system, we use immunofluorescence and flow cytometry to examine in detail the distribution of expression levels for various transcription factors key to the maintenance of the ES cell identity. We find the population-level distribution of many proteins, once rescaled by the average expression level, have very similar shapes. This suggest the largest component of observed heterogeneity comes from a single source. More subtly, we find the expression many of genes appears to modulate with the cell cycle. This may suggest that the program for maintaining ES cell identity is tightly coupled to the cell cycle machinery. This work is supported by the Harvard Stem Cell Institute and the Jane Coffin Childs Memorial Fund for Medical Research.

  12. Divergent RNA Localisation Patterns of Maternal Genes Regulating Embryonic Patterning in the Butterfly Pararge aegeria.

    PubMed

    Carter, Jean-Michel; Gibbs, Melanie; Breuker, Casper J

    2015-01-01

    The maternal effect genes responsible for patterning the embryo along the antero-posterior (AP) axis are broadly conserved in insects. The precise function of these maternal effect genes is the result of the localisation of their mRNA in the oocyte. The main developmental mechanisms involved have been elucidated in Drosophila melanogaster, but recent studies have shown that other insect orders often diverge in RNA localisation patterns. A recent study has shown that in the butterfly Pararge aegeria the distinction between blastodermal embryonic (i.e. germ band) and extra-embryonic tissue (i.e. serosa) is already specified in the oocyte during oogenesis in the ovariole, long before blastoderm cellularisation. To examine the extent by which a female butterfly specifies and patterns the AP axis within the region fated to be the germ band, and whether she specifies a germ plasm, we performed in situ hybridisation experiments on oocytes in P. aegeria ovarioles and on early embryos. RNA localisation of the following key maternal effect genes were investigated: caudal (cad), orthodenticle (otd), hunchback (hb) and four nanos (nos) paralogs, as well as TDRD7 a gene containing a key functional domain (OST-HTH/LOTUS) shared with oskar. TDRD7 was mainly confined to the follicle cells, whilst hb was exclusively zygotically transcribed. RNA of some of the nos paralogs, otd and cad revealed complex localisation patterns within the cortical region prefiguring the germ band (i.e. germ cortex). Rather interestingly, otd was localised within and outside the anterior of the germ cortex. Transcripts of nos-O formed a distinct granular ring in the middle of the germ cortex possibly prefiguring the region where germline stem cells form. These butterfly RNA localisation patterns are highly divergent with respect to other insects, highlighting the diverse ways in which different insect orders maternally regulate early embryogenesis of their offspring.

  13. Divergent RNA Localisation Patterns of Maternal Genes Regulating Embryonic Patterning in the Butterfly Pararge aegeria.

    PubMed

    Carter, Jean-Michel; Gibbs, Melanie; Breuker, Casper J

    2015-01-01

    The maternal effect genes responsible for patterning the embryo along the antero-posterior (AP) axis are broadly conserved in insects. The precise function of these maternal effect genes is the result of the localisation of their mRNA in the oocyte. The main developmental mechanisms involved have been elucidated in Drosophila melanogaster, but recent studies have shown that other insect orders often diverge in RNA localisation patterns. A recent study has shown that in the butterfly Pararge aegeria the distinction between blastodermal embryonic (i.e. germ band) and extra-embryonic tissue (i.e. serosa) is already specified in the oocyte during oogenesis in the ovariole, long before blastoderm cellularisation. To examine the extent by which a female butterfly specifies and patterns the AP axis within the region fated to be the germ band, and whether she specifies a germ plasm, we performed in situ hybridisation experiments on oocytes in P. aegeria ovarioles and on early embryos. RNA localisation of the following key maternal effect genes were investigated: caudal (cad), orthodenticle (otd), hunchback (hb) and four nanos (nos) paralogs, as well as TDRD7 a gene containing a key functional domain (OST-HTH/LOTUS) shared with oskar. TDRD7 was mainly confined to the follicle cells, whilst hb was exclusively zygotically transcribed. RNA of some of the nos paralogs, otd and cad revealed complex localisation patterns within the cortical region prefiguring the germ band (i.e. germ cortex). Rather interestingly, otd was localised within and outside the anterior of the germ cortex. Transcripts of nos-O formed a distinct granular ring in the middle of the germ cortex possibly prefiguring the region where germline stem cells form. These butterfly RNA localisation patterns are highly divergent with respect to other insects, highlighting the diverse ways in which different insect orders maternally regulate early embryogenesis of their offspring. PMID:26633019

  14. Divergent RNA Localisation Patterns of Maternal Genes Regulating Embryonic Patterning in the Butterfly Pararge aegeria

    PubMed Central

    Carter, Jean-Michel; Gibbs, Melanie; Breuker, Casper J.

    2015-01-01

    The maternal effect genes responsible for patterning the embryo along the antero-posterior (AP) axis are broadly conserved in insects. The precise function of these maternal effect genes is the result of the localisation of their mRNA in the oocyte. The main developmental mechanisms involved have been elucidated in Drosophila melanogaster, but recent studies have shown that other insect orders often diverge in RNA localisation patterns. A recent study has shown that in the butterfly Pararge aegeria the distinction between blastodermal embryonic (i.e. germ band) and extra-embryonic tissue (i.e. serosa) is already specified in the oocyte during oogenesis in the ovariole, long before blastoderm cellularisation. To examine the extent by which a female butterfly specifies and patterns the AP axis within the region fated to be the germ band, and whether she specifies a germ plasm, we performed in situ hybridisation experiments on oocytes in P. aegeria ovarioles and on early embryos. RNA localisation of the following key maternal effect genes were investigated: caudal (cad), orthodenticle (otd), hunchback (hb) and four nanos (nos) paralogs, as well as TDRD7 a gene containing a key functional domain (OST-HTH/LOTUS) shared with oskar. TDRD7 was mainly confined to the follicle cells, whilst hb was exclusively zygotically transcribed. RNA of some of the nos paralogs, otd and cad revealed complex localisation patterns within the cortical region prefiguring the germ band (i.e. germ cortex). Rather interestingly, otd was localised within and outside the anterior of the germ cortex. Transcripts of nos-O formed a distinct granular ring in the middle of the germ cortex possibly prefiguring the region where germline stem cells form. These butterfly RNA localisation patterns are highly divergent with respect to other insects, highlighting the diverse ways in which different insect orders maternally regulate early embryogenesis of their offspring. PMID:26633019

  15. The Production and Characteristics of a Mouse's Embryonic Stem Cell Lineage, Transfected by the Glia Neurotrophic Factor and Gene Fused with the Green Fluorescent Protein Gene

    PubMed Central

    Arsenieva, E. L.; Kuzmin, I. V.; Manuilova, E. S.; Novosadova, E. V.; Murkin, E. V.; Pavlova, G. V.; Tarantul, V. Z.

    2009-01-01

    The influence that the expression of the human (glial-derived neurotrophic factor (GDNF)) neurotrophic factor has on the morphology and proliferative activity of embryonic stem cells (SC) of a mouse with R1 lineage, as well as their ability to form embroid bodies (EB), has been studied. Before that, using a PCR (polymerase chain reaction) coupled with reverse transcription, it was shown that, in this very lineage of the embryonic SC, the expression of the receptors' genes is being fulfilled for the neurotropfic RET and GFRα1 glia factor. The mouse's embryonic SC lineage has been obtained, transfected by the human GDNF gene, and has been fused with the "green" fluorescent protein (GFP) gene. The presence of the expression of the human GDNF gene in the cells was shown by northern hybridization and the synthesis of its albuminous product by immunocitochemical coloration with the use of specific antibodies. The reliable slowing-down of the embriod-body formation by the embryonic SC transfected by the GDNF gene has been shown. No significant influence of the expression of the GDNF gene on the morphology and the proliferative activity of the transfected embryonic SCs has been found when compared with the control ones. PMID:22649595

  16. Expression profiles of Wnt genes during neural differentiation of mouse embryonic stem cells.

    PubMed

    Nordin, Norshariza; Li, Meng; Mason, John O

    2008-03-01

    The Wnt family of secreted signaling proteins regulates many aspects of animal development and the behavior of several types of stem cells, including embryonic stem (ES) cells. Activation of canonical Wnt signaling has been shown to either inhibit or promote the differentiation of ES cells into neurons, depending on the stage of differentiation. Here, we describe the expression of all 19 mouse Wnt genes during this process. Using the well-established retinoic acid induction protocol we found that all Wnt genes except Wnt8b are expressed as ES cells differentiate into neurons, many of them in dynamic patterns. The expression pattern of 12 Wnt genes was analyzed quantitatively at 2-day intervals throughout neural differentiation, showing that multiple Wnt genes are expressed at each stage. A large proportion of these, including both canonical and noncanonical Wnts, are expressed at highest levels during later stages of differentiation. The complexity of the patterns observed indicates that disentangling specific roles for individual Wnt genes in the differentiation process will be a significant challenge.

  17. Selection-Independent Generation of Gene Knockout Mouse Embryonic Stem Cells Using Zinc-Finger Nucleases

    PubMed Central

    Osiak, Anna; Radecke, Frank; Guhl, Eva; Radecke, Sarah; Dannemann, Nadine; Lütge, Fabienne; Glage, Silke; Rudolph, Cornelia; Cantz, Tobias; Schwarz, Klaus; Heilbronn, Regine; Cathomen, Toni

    2011-01-01

    Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10−6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells. PMID:22194948

  18. Ethanol upregulates NMDA receptor subunit gene expression in human embryonic stem cell-derived cortical neurons.

    PubMed

    Xiang, Yangfei; Kim, Kun-Yong; Gelernter, Joel; Park, In-Hyun; Zhang, Huiping

    2015-01-01

    Chronic alcohol consumption may result in sustained gene expression alterations in the brain, leading to alcohol abuse or dependence. Because of ethical concerns of using live human brain cells in research, this hypothesis cannot be tested directly in live human brains. In the present study, we used human embryonic stem cell (hESC)-derived cortical neurons as in vitro cellular models to investigate alcohol-induced expression changes of genes involved in alcohol metabolism (ALDH2), anti-apoptosis (BCL2 and CCND2), neurotransmission (NMDA receptor subunit genes: GRIN1, GRIN2A, GRIN2B, and GRIN2D), calcium channel activity (ITPR2), or transcriptional repression (JARID2). hESCs were differentiated into cortical neurons, which were characterized by immunostaining using antibodies against cortical neuron-specific biomarkers. Ethanol-induced gene expression changes were determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). After a 7-day ethanol (50 mM) exposure followed by a 24-hour ethanol withdrawal treatment, five of the above nine genes (including all four NMDA receptor subunit genes) were highly upregulated (GRIN1: 1.93-fold, P = 0.003; GRIN2A: 1.40-fold, P = 0.003; GRIN2B: 1.75-fold, P = 0.002; GRIN2D: 1.86-fold, P = 0.048; BCL2: 1.34-fold, P = 0.031), and the results of GRIN1, GRIN2A, and GRIN2B survived multiple comparison correction. Our findings suggest that alcohol responsive genes, particularly NMDA receptor genes, play an important role in regulating neuronal function and mediating chronic alcohol consumption-induced neuroadaptations.

  19. Chibby functions in Xenopus ciliary assembly, embryonic development, and the regulation of gene expression

    PubMed Central

    Shi, Jianli; Zhao, Ying; Galati, Domenico; Winey, Mark; Klymkowsky, Michael W.

    2015-01-01

    Wnt signaling and ciliogenesis are core features of embryonic development in a range of metazoans. Chibby (Cby), a basal-body associated protein, regulates β-catenin-mediated Wnt signaling in the mouse but not Drosophila. Here we present an analysis of Cby’s embryonic expression and morphant phenotypes in Xenopus laevis. Cby RNA is supplied maternally, negatively regulated by Snail2 but not Twist1, preferentially expressed in the neuroectoderm, and regulates β-catenin-mediated gene expression. Reducing Cby levels reduced the density of multiciliated cells, the number of basal bodies per multiciliated cell, and the numbers of neural tube primary cilia; it also led to abnormal development of the neural crest, central nervous system, and pronephros, all defects that were rescued by a Cby-GFP chimera. Reduction of Cby led to an increase in Wnt8a and decreases in Gli2, Gli3, and Shh RNA levels. Many, but not all, morphant phenotypes were significantly reversed by the Wnt inhibitor SFRP2. These observations extend our understanding of Cby’s role in mediating the network of interactions between ciliogenesis, signaling systems and tissue patterning. PMID:25220153

  20. Haploinsufficiency screen highlights two distinct groups of ribosomal protein genes essential for embryonic stem cell fate

    PubMed Central

    Fortier, Simon; MacRae, Tara; Bilodeau, Mélanie; Sargeant, Tobias; Sauvageau, Guy

    2015-01-01

    In a functional genomics screen of mouse embryonic stem cells (ESCs) with nested hemizygous chromosomal deletions, we reveal that ribosomal protein (RP) genes are the most significant haploinsufficient determinants for embryoid body (EB) formation. Hemizygocity for three RP genes (Rps5, Rps14, or Rps28), distinguished by the proximity of their corresponding protein to the ribosome's mRNA exit site, is associated with the most profound phenotype. This EB phenotype was fully rescued by BAC or cDNA complementation but not by the reduction of p53 levels, although such reduction was effective with most other RP-deleted clones corresponding to non-mRNA exit-site proteins. RNA-sequencing studies further revealed that undifferentiated ESCs hemizygous for Rps5 showed reduced expression levels of several mesoderm-specific genes as compared with wild-type counterparts. Together, these results reveal that RP gene dosage limits the differentiation, not the self-renewal, of mouse ESCs. They also highlight two separate mechanisms underlying this process, one of which is p53 independent. PMID:25646475

  1. A gene expression signature shared by human mature oocytes and embryonic stem cells

    PubMed Central

    Assou, Said; Cerecedo, Doris; Tondeur, Sylvie; Pantesco, Véronique; Hovatta, Outi; Klein, Bernard; Hamamah, Samir; De Vos, John

    2009-01-01

    Background The first week of human pre-embryo development is characterized by the induction of totipotency and then pluripotency. The understanding of this delicate process will have far reaching implication for in vitro fertilization and regenerative medicine. Human mature MII oocytes and embryonic stem (ES) cells are both able to achieve the feat of cell reprogramming towards pluripotency, either by somatic cell nuclear transfer or by cell fusion, respectively. Comparison of the transcriptome of these two cell types may highlight genes that are involved in pluripotency initiation. Results Based on a microarray compendium of 205 samples, we compared the gene expression profile of mature MII oocytes and human ES cells (hESC) to that of somatic tissues. We identified a common oocyte/hESC gene expression profile, which included a strong cell cycle signature, genes associated with pluripotency such as LIN28 and TDGF1, a large chromatin remodelling network (TOP2A, DNMT3B, JARID2, SMARCA5, CBX1, CBX5), 18 different zinc finger transcription factors, including ZNF84, and several still poorly annotated genes such as KLHL7, MRS2, or the Selenophosphate synthetase 1 (SEPHS1). Interestingly, a large set of genes was also found to code for proteins involved in the ubiquitination and proteasome pathway. Upon hESC differentiation into embryoid bodies, the transcription of this pathway declined. In vitro, we observed a selective sensitivity of hESC to the inhibition of the activity of the proteasome. Conclusion These results shed light on the gene networks that are concurrently overexpressed by the two human cell types with somatic cell reprogramming properties. PMID:19128516

  2. 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

  3. Muscle segment homeobox genes direct embryonic diapause by limiting inflammation in the uterus

    SciTech Connect

    Cha, Jeeyeon; Burnum-Johnson, Kristin E.; Bartos, Amanda; Li, Yingju; Baker, Erin Shammel; Tilton, Susan C.; Webb-Robertson, Bobbie-Jo M.; Piehowski, Paul D.; Monroe, Matthew E.; Jegga, Anil; Murata, Shigeo; Hirota, Yasushi; Dey, Sudhansu K.

    2015-06-11

    Embryonic diapause (delayed implantation) is a reproductive strategy widespread in the animal kingdom. Under this condition, embryos at the blastocyst stage become dormant simultaneously with uterine quiescence until environmental or physiological conditions are favorable for the survival of the mother and newborn. Under favorable conditions, activation of the blastocyst and uterus ensues with implantation and progression of pregnancy. Although endocrine factors are known to participate in this process, the underlying molecular mechanism coordinating this phenomenon is not clearly understood. We recently found that uterine muscle segment homeobox (Msx) transcription factors are critical for the initiation and maintenance of delayed implantation in mice. To better understand why Msx genes are critical for delayed implantation, we compared uterine proteomics profiles between littermate floxed (Msx1/Msx2f/f) mice and mice with uterine deletion of Msx genes (Msx1/Msx2d/d) under delayed conditions. In Msx1/Msx2d/d uteri, pathways including protein translation, ubiquitin-proteasome system, inflammation, chaperone-mediated protein folding, and endoplasmic reticulum (ER) stress were enriched, and computational modeling showed intersection of these pathways on inflammatory responses. Indeed, increases in the ubiquitin-proteasome system and inflammation conformed to proteotoxic and ER stress in Msx1/Msx2d/d uteri under delayed conditions. Interestingly, treatment with a proteasome inhibitor bortezomib further exacerbated ER stress in Msx1/Msx2d/d uteri with aggravated inflammatory response, deteriorating rate of blastocyst recovery and failure to sustain delayed implantation. This study highlights a previously unrecognized role for Msx in preventing proteotoxic stress and inflammatory responses to coordinate embryo dormancy and uterine quiescence during embryonic diapause.

  4. Interactions between the otitis media gene, Fbxo11, and p53 in the mouse embryonic lung.

    PubMed

    Tateossian, Hilda; Morse, Susan; Simon, Michelle M; Dean, Charlotte H; Brown, Steve D M

    2015-12-01

    Otitis media with effusion (OME) is the most common cause of hearing loss in children, and tympanostomy (ear tube insertion) to alleviate the condition remains the commonest surgical intervention in children in the developed world. Chronic and recurrent forms of otitis media (OM) are known to have a very substantial genetic component; however, until recently, little was known of the underlying genes involved. The Jeff mouse mutant carries a mutation in the Fbxo11 gene, a member of the F-box family, and develops deafness due to a chronic proliferative OM. We previously reported that Fbxo11 is involved in the regulation of transforming growth factor beta (TGF-β) signalling by regulating the levels of phospho-Smad2 in the epithelial cells of palatal shelves, eyelids and airways of the lungs. It has been proposed that FBXO11 regulates the cell's response to TGF-β through the ubiquitination of CDT2. Additional substrates for FBXO11 have been identified, including p53. Here, we have studied both the genetic and biochemical interactions between FBXO11 and p53 in order to better understand the function of FBXO11 in epithelial development and its potential role in OM. In mice, we show that p53 (also known as Tp53) homozygous mutants and double heterozygous mutants (Jf/+ p53/+) exhibit similar epithelial developmental defects to Fbxo11 homozygotes. FBXO11 and p53 interact in the embryonic lung, and mutation in Fbxo11 prevents the interaction with p53. Both p53 and double mutants show raised levels of pSMAD2, recapitulating that seen in Fbxo11 homozygotes. Overall, our results support the conclusion that FBXO11 regulates the TGF-β pathway in the embryonic lung via cross-talk with p53.

  5. Concentration-dependent gene expression responses to flusilazole in embryonic stem cell differentiation cultures

    SciTech Connect

    Dartel, Dorien A.M. van; Pennings, Jeroen L.A.; Fonteyne, Liset J.J. de la; Brauers, Karen J.J.; Claessen, Sandra; Delft, Joost H. van; Kleinjans, Jos C.S.; Piersma, Aldert H.

    2011-03-01

    The murine embryonic stem cell test (EST) is designed to evaluate developmental toxicity based on compound-induced inhibition of embryonic stem cell (ESC) differentiation into cardiomyocytes. The addition of transcriptomic evaluation within the EST may result in enhanced predictability and improved characterization of the applicability domain, therefore improving usage of the EST for regulatory testing strategies. Transcriptomic analyses assessing factors critical for risk assessment (i.e. dose) are needed to determine the value of transcriptomic evaluation in the EST. Here, using the developmentally toxic compound, flusilazole, we investigated the effect of compound concentration on gene expression regulation and toxicity prediction in ESC differentiation cultures. Cultures were exposed for 24 h to multiple concentrations of flusilazole (0.54-54 {mu}M) and RNA was isolated. In addition, we sampled control cultures 0, 24, and 48 h to evaluate the transcriptomic status of the cultures across differentiation. Transcriptomic profiling identified a higher sensitivity of development-related processes as compared to cell division-related processes in flusilazole-exposed differentiation cultures. Furthermore, the sterol synthesis-related mode of action of flusilazole toxicity was detected. Principal component analysis using gene sets related to normal ESC differentiation was used to describe the dynamics of ESC differentiation, defined as the 'differentiation track'. The concentration-dependent effects on development were reflected in the significance of deviation of flusilazole-exposed cultures from this transcriptomic-based differentiation track. Thus, the detection of developmental toxicity in EST using transcriptomics was shown to be compound concentration-dependent. This study provides further insight into the possible application of transcriptomics in the EST as an improved alternative model system for developmental toxicity testing.

  6. Meta-analysis of differentiating mouse embryonic stem cell gene expression kinetics reveals early change of a small gene set.

    PubMed

    Glover, Clive H; Marin, Michael; Eaves, Connie J; Helgason, Cheryl D; Piret, James M; Bryan, Jennifer

    2006-11-24

    Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC) lines obtained at specific time points during the course of their differentiation into various lineages. We developed methods to identify genes with expression changes that correlated with the altered frequency of functionally defined, undifferentiated ESC in culture. In each dataset, we computed a novel statistical confidence measure for every gene which captured the certainty that a particular gene exhibited an expression pattern of interest within that dataset. This permitted a joint analysis of the datasets, despite the different experimental designs. Using a ranking scheme that favored genes exhibiting patterns of interest, we focused on the top 88 genes whose expression was consistently changed when ESC were induced to differentiate. Seven of these (103728_at, 8430410A17Rik, Klf2, Nr0b1, Sox2, Tcl1, and Zfp42) showed a rapid decrease in expression concurrent with a decrease in frequency of undifferentiated cells and remained predictive when evaluated in additional maintenance and differentiating protocols. Through a novel meta-analysis, this study identifies a small set of genes whose expression is useful for identifying changes in stem cell frequencies in cultures of mouse ESC. The methods and findings have broader applicability to understanding the regulation of self-renewal of other stem cell types.

  7. Association of milk protein genes with fertilization rate and early embryonic development in Holstein dairy cattle.

    PubMed

    Peñagaricano, Francisco; Khatib, Hasan

    2012-02-01

    Concomitant with intensive selection for increased milk yield, reproductive performance of dairy cows has declined in the last decades, in part due to an unfavourable genetic relationship between these traits. Given that the six main milk protein genes (i.e. whey proteins and caseins) are directly involved in milk production and hence have been a target of the strong selection aimed at improving milk yield in dairy cattle, we hypothesized that these genes could show selection footprints associated with fertility traits. In this study, we used an in-vitro fertilization (IVF) system to test genetic association between 66 single nucleotide polymorphisms (SNPs) in the four caseins (αS1-casein, αS2-casein, β-casein and κ-casein) and the two whey protein genes (α-lactalbumin and β-lactoglobulin) with fertilization rate and early embryonic development in the Holstein breed. A total of 6893 in-vitro fertilizations were performed and a total of 4661 IVF embryos were produced using oocytes from 399 ovaries and semen samples from 12 bulls. Associations between SNPs and fertility traits were analysed using a mixed linear model with genotype as fixed effect and ovary and bull as random effects. A multiple testing correction approach was used to account for the correlation between SNPs due to linkage disequilibrium. After correction, polymorphisms in the LALBA and LGB genes showed significant associations with fertilization success and blastocyst rate. No significant associations were detected between SNPs located in the casein region and IVF fertility traits. Although the molecular mechanisms underlying the association between whey protein genes and fertility have not yet been characterized, this study provides the first evidence of association between these genes and fertility traits. Furthermore, these results could shed light on the antagonistic relationship that exists between milk yield and fertility in dairy cattle.

  8. The embryonic leaf identity gene FUSCA3 regulates vegetative phase transitions by negatively modulating ethylene-regulated gene expression in Arabidopsis

    PubMed Central

    2012-01-01

    Background The embryonic temporal regulator FUSCA3 (FUS3) plays major roles in the establishment of embryonic leaf identity and the regulation of developmental timing. Loss-of-function mutations of this B3 domain transcription factor result in replacement of cotyledons with leaves and precocious germination, whereas constitutive misexpression causes the conversion of leaves into cotyledon-like organs and delays vegetative and reproductive phase transitions. Results Herein we show that activation of FUS3 after germination dampens the expression of genes involved in the biosynthesis and response to the plant hormone ethylene, whereas a loss-of-function fus3 mutant shows many phenotypes consistent with increased ethylene signaling. This FUS3-dependent regulation of ethylene signaling also impinges on timing functions outside embryogenesis. Loss of FUS3 function results in accelerated vegetative phase change, and this is again partially dependent on functional ethylene signaling. This alteration in vegetative phase transition is dependent on both embryonic and vegetative FUS3 function, suggesting that this important transcriptional regulator controls both embryonic and vegetative developmental timing. Conclusion The results of this study indicate that the embryonic regulator FUS3 not only controls the embryonic-to-vegetative phase transition through hormonal (ABA/GA) regulation but also functions postembryonically to delay vegetative phase transitions by negatively modulating ethylene-regulated gene expression. PMID:22348746

  9. Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

    SciTech Connect

    Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith , E-Mail: Judith.hall@ncl.ac.uk

    2005-09-16

    In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of the TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian.

  10. The Regulation of rRNA Gene Transcription during Directed Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Liu, Zhong; Zhao, Rui; Giles, Keith E.

    2016-01-01

    It has become increasingly clear that proper cellular control of pluripotency and differentiation is related to the regulation of rRNA synthesis. To further our understanding of the role that the regulation of rRNA synthesis has in pluripotency we monitored rRNA synthesis during the directed differentiation of human embryonic stem cells (hESCs). We discovered that the rRNA synthesis rate is reduced ~50% within 6 hours of ACTIVIN A treatment. This precedes reductions in expression of specific stem cell markers and increases in expression of specific germ layer markers. The reduction in rRNA synthesis is concomitant with dissociation of the Pol I transcription factor, UBTF, from the rRNA gene promoter and precedes any increase to heterochromatin throughout the rRNA gene. To directly investigate the role of rRNA synthesis in pluripotency, hESCs were treated with the Pol I inhibitor, CX-5461. The direct reduction of rRNA synthesis by CX-5461 induces the expression of markers for all three germ layers, reduces the expression of pluripotency markers, and is overall similar to the ACTIVIN A induced changes. This work indicates that the dissociation of UBTF from the rRNA gene, and corresponding reduction in transcription, represent early regulatory events during the directed differentiation of pluripotent stem cells. PMID:27299313

  11. Gene-modified embryonic stem cell test to characterize chemical risks.

    PubMed

    Kitada, Kohei; Kizu, Akane; Teramura, Takeshi; Takehara, Toshiyuki; Hayashi, Masami; Tachibana, Daisuke; Wanibuchi, Hideki; Fukushima, Shoji; Koyama, Masayasu; Yoshida, Kayo; Morita, Takashi

    2015-11-01

    A high-throughput test of cell growth inhibition was performed using mouse embryonic stem (ES) cells to assess chemical toxicities. We herein demonstrated using a 96-well culture plate approach and the MTT assay that this method was suitable for prioritization of chemicals for their cytotoxic properties. In order to categorize chemicals, we used p53 gene-modified mouse ES cells as well as wild-type ES cells. The p53 gene is a well-known tumor suppressor and controls programmed cell death (apoptosis) and cellular senescence that is triggered by DNA-damaging agents such as alkylating agents and radiation. In the present study, p53-deficient ES cells were found to be more resistant to a tumor initiator, diethylnitrosamine (DEN), than wild-type ES cells, suggesting the inhibition of apoptosis or senescence by a dysfunction in p53. Chromosome aberrations were more frequently detected in p53-deficient ES cells than in wild-type cells, indicating genomic instability due to the deletion of p53. Other tumor initiators, methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (NMU), did not reveal apparent differences in cytotoxicity between wild-type and p53-deficient ES cells. Thus, ES test system using gene-modified ES cells may be used to categorize chemicals by detecting their characteristic effects on apoptosis, genotoxic potentials as well as general cytotoxicity.

  12. Cell-Free Transcription of Mammalian Chromatin: Transcription of Globin Messenger RNA Sequences from Bone-Marrow Chromatin with Mammalian RNA Polymerase

    PubMed Central

    Steggles, A. W.; Wilson, G. N.; Kantor, J. A.; Picciano, D. J.; Falvey, A. K.; Anderson, W. F.

    1974-01-01

    A mammalian cell-free transcriptional system was developed in which mammalian RNA polymerase synthesizes globin messenger RNA sequences from bone-marrow chromatin. The messenger RNA sequences are detected by measurement of the ability of the transcribed RNA to hybridize with globin complementary DNA. The globin complementary DNA is synthesized by the enzyme from avian myeloblastosis virus, RNA-directed DNA polymerase, with purified globin messenger RNA as template. The specificity of the globin complementary DNA in annealing reactions was verified by preparing DNA complementary to liver messenger RNA and showing that the globin and liver complementary DNAs are specific for their own messenger RNAs. Both DNA-dependent RNA polymerase II from sheep liver and RNA polymerase from Escherichia coli can transcribe globin messenger RNA sequences from rabbit bone-marrow chromatin; however, the mammalian enzyme appears to be more specific in that globin gene sequences represent a higher proportion of the RNA synthesized. Neither polymerase can transcribe globin messenger RNA sequences from rabbit-liver chromatin. This cell-free assay system should be useful in searching for mammalian transcriptional regulatory factors. PMID:4364529

  13. Impact of thermal stress during incubation on gene expression in embryonic muscle of Peking ducks (Anasplatyrhynchos domestica).

    PubMed

    Liu, Hehe; Liu, Junying; Yan, Xiping; Li, Qingqing; Zhao, Yangmei; Wang, Yan; Zhang, Rongping; Wang, Guosong; Wang, Haohan; Li, Xinxin; Yang, Chao; Li, Liang; Han, Chunchun; Wang, Jiwen

    2015-10-01

    Changes in temperature will influence poultry embryonic muscle development. However, little is known about the changes in molecular processes impacted by incubation temperature in avians. In this study, we investigated the effects of increasing the incubation temperature by 1°C from day 11-20 on the embryonic and posthatch skeletal muscle development of the Peking duck, and identified the differentially expressed genes using RNA-seq of leg muscle tissues. The results showed that altering the incubation temperature had immediate and long-lasting effects on phenotypic changes in the embryonic and post-hatching muscle development. It was shown that expression levels of total 1370 genes were altered in muscle tissues by the thermal treatments. The gene ontology (GO) analyses indicated that cellular processes including metabolism, cell cycle, catalytic activity, and enzyme regulatory activity may have involved in the muscle mass impacted by thermal manipulation. TGF-beta and insulin pathways as two classical muscle development related pathways may also involve in regulating muscle mass. These data may be helpful for understanding the physiological and biochemical processes of muscle development under environmental treatments in embryonic avians.

  14. 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

  15. High expression of hTERT and stemness genes in BORIS/CTCFL positive cells isolated from embryonic cancer cells.

    PubMed

    Alberti, Loredana; Renaud, Stéphanie; Losi, Lorena; Leyvraz, Serge; Benhattar, Jean

    2014-01-01

    BORIS/CTCFL is a member of cancer testis antigen family normally expressed in germ cells. In tumors, it is aberrantly expressed although its functions are not completely well-defined. To better understand the functions of BORIS in cancer, we selected the embryonic cancer cells as a model. Using a molecular beacon, which specifically targets BORIS mRNA, we demonstrated that BORIS positive cells are a small subpopulation of tumor cells (3-5% of total). The BORIS-positive cells isolated using BORIS-molecular beacon, expressed higher telomerase hTERT, stem cell (NANOG, OCT4, SOX2) and cancer stem cell marker genes (CD44 and ALDH1) compared to the BORIS-negative tumor cells. In order to define the functional role of BORIS, stable BORIS-depleted embryonic cancer cells were generated. BORIS silencing strongly down-regulated the expression of hTERT, stem cell and cancer stem cell marker genes. Moreover, the BORIS knockdown increased cellular senescence in embryonic cancer cells, revealing a putative role of BORIS in the senescence biological program. Our data indicate an association of BORIS expressing cells subpopulation with the expression of stemness genes, highlighting the critical role played by BORIS in embryonic neoplastic disease.

  16. Analysis of enhancer function of the HS-40 core sequence of the human alpha-globin cluster.

    PubMed Central

    Chen, H; Lowrey, C H; Stamatoyannopoulos, G

    1997-01-01

    HS-40 is the major regulatory element of the human alpha-globin locus, located 40 kb upstream of the zeta-globin gene. To test for potential interactions between HS-40 and the beta- or the gamma-globin gene promoters in stable transfection assays, the HS-40 core sequence was cloned upstream of either the beta promoter or the gamma promoter driving the neomycin phosphotransferase gene and enhancer activity was measured using a colony assay. In K562 or in MEL cells, enhancer activity of HS-40 was higher than that of the individual core sequences of the DNase I hypersensitive sites (HS) of the beta-globin locus control region (LCR), and approximately 60% of the enhancer activity of a 2.5 kb microLCR, which contains the core elements of DNase I hypersensitive sites 1-4. In contrast to the synergistic interaction between the DNase I hypersensitive sites of beta locus LCR, combination of HS-40 with these DNase I hypersensitive sites failed to display cooperativity in K562 cells and inhibited enhancer function in MEL cells. Inhibition of enhancer function was also observed when two copies of the HS-40 were arranged tandemly. We conclude that the core element of HS-40 (i) is a powerful enhancer of gamma- and beta-globin gene expression, (ii) in contrast to other classical enhancers, acts best as a single copy, (iii) does not cooperate with the regulatory elements of the beta-globin locus control region. PMID:9207043

  17. Polymorphic core promoter GA-repeats alter gene expression of the early embryonic developmental genes.

    PubMed

    Valipour, E; Kowsari, A; Bayat, H; Banan, M; Kazeminasab, S; Mohammadparast, S; Ohadi, M

    2013-12-01

    Protein complexes that bind to 'GAGA' DNA elements are necessary to replace nucleosomes to create a local chromatin environment that facilitates a variety of site-specific regulatory responses. Three to four elements are required for the disruption of a preassembled nucleosome. We have previously identified human protein-coding gene core promoters that are composed of exceptionally long GA-repeats. The functional implication of those GA-repeats is beginning to emerge in the core promoter of the human SOX5 gene, which is involved in multiple developmental processes. In the current study, we analyze the functional implication of GA-repeats in the core promoter of two additional genes, MECOM and GABRA3, whose expression is largely limited to embryogenesis. We report a significant difference in gene expression as a result of different alleles across those core promoters in the HEK-293 cell line. Across-species homology check for the GABRA3 GA-repeats revealed that those repeats are evolutionary conserved in mouse and primates (p<1 × 10(-8)). The MECOM core promoter GA-repeats are also conserved in numerous species, of which human has the longest repeat and complexity. We propose a novel role for GA-repeat core promoters to regulate gene expression in the genes involved in development and evolution.

  18. Erythropoietic differentiation of a human embryonic stem cell line harbouring the sickle cell anaemia mutation

    PubMed Central

    Pryzhkova, Marina V; Peters, Ann; Zambidis, Elias T

    2012-01-01

    Herein is reported efficient erythropoietic differentiation of a human embryonic stem cell (ESC) line derived from a preimplantation genetic diagnosis (PGD)-screened embryo that harbours the homozygous sickle cell disease (SCD) haemoglobinopathy mutation. This human ESC line possesses typical pluripotency characteristics and forms multilineage teratomas in vivo. SCD-human ESC efficiently differentiated to the haematopoietic lineage under serum-free and stromal co-culture conditions and gave rise to robust primitive and definitive erythrocytes. Expression of embryonic, fetal and adult sickle globin genes in SCD PGD-derived human ESC-derived erythrocytes was confirmed by quantitative real-time PCR, intracytoplasmic fluorescence-activated cell sorting and insitu immunostaining of PGD-derived human ESC teratoma sections. These data introduce important methodologies and paradigms for using patient-specific human ESC to generate normal and haemoglobinopathic erythroid progenitors for biomedical research. PMID:20541472

  19. Gene expression signatures affected by alcohol-induced DNA methylomic deregulation in human embryonic stem cells

    PubMed Central

    Kim, Hyun-Sung; Hoang, Michael; Tu, Thanh G.; Elie, Omid; Lee, Connie; Vu, Catherine; Horvath, Steve; Spigelman, Igor; Kim, Yong

    2014-01-01

    Stem cells, especially human embryonic stem cells (hESCs), are useful models to study molecular mechanisms of human disorders that originate during gestation. Alcohol (ethanol, EtOH) consumption during pregnancy causes a variety of prenatal and postnatal disorders collectively referred to as fetal alcohol spectrum disorders (FASDs). To better understand the molecular events leading to FASDs, we performed a genome-wide analysis of EtOH's effects on the maintenance and differentiation of hESCs in culture. Gene Co-expression Network Analysis showed significant alterations in gene profiles of EtOH-treated differentiated or undifferentiated hESCs, particularly those associated with molecular pathways for metabolic processes, oxidative stress, and neuronal properties of stem cells. A genome-wide DNA methylome analysis revealed widespread EtOH-induced alterations with significant hypermethylation of many regions of chromosomes. Undifferentiated hESCs were more vulnerable to EtOH's effect than their differentiated counterparts, with methylation on the promoter regions of chromosomes 2, 16 and 18 in undifferentiated hESCs most affected by EtOH exposure. Combined transcriptomic and DNA methylomic analysis produced a list of differentiation-related genes dysregulated by EtOH-induced DNA methylation changes, which likely play a role in EtOH-induced decreases in hESC pluripotency. DNA sequence motif analysis of genes epigenetically altered by EtOH identified major motifs representing potential binding sites for transcription factors. These findings should help in deciphering the precise mechanisms of alcohol-induced teratogenesis. PMID:24751885

  20. Kisspeptin Activates Ankrd 26 Gene Expression in Migrating Embryonic GnRH Neurons.

    PubMed

    Soga, Tomoko; Lim, Wei Ling; Khoo, Alan Soo-Beng; Parhar, Ishwar S

    2016-01-01

    Kisspeptin, a newly discovered neuropeptide, regulates gonadotropin-releasing hormone (GnRH). Kisspeptins are a large RF-amide family of peptides. The kisspeptin coded by KiSS-1 gene is a 145-amino acid protein that is cleaved to C-terminal peptide kisspeptin-10. G-protein-coupled receptor 54 (GPR54) has been identified as a kisspeptin receptor, and it is expressed in GnRH neurons and in a variety of cancer cells. In this study, enhanced green fluorescent protein (EGFP) labeled GnRH cells with migratory properties, which express GPR54, served as a model to study the effects of kisspeptin on cell migration. We monitored EGFP-GnRH neuronal migration in brain slide culture of embryonic day 14 transgenic rat by live cell imaging system and studied the effects of kisspeptin-10 (1 nM) treatment for 36 h on GnRH migration. Furthermore, to determine kisspeptin-induced molecular pathways related with apoptosis and cytoskeletal changes during neuronal migration, we studied the expression levels of candidate genes in laser-captured EGFP-GnRH neurons by real-time PCR. We found that there was no change in the expression level of genes related to cell proliferation and apoptosis. The expression of ankyrin repeat domain-containing protein (ankrd) 26 in EGFP-GnRH neurons was upregulated by the exposure to kisspeptin. These studies suggest that ankrd 26 gene plays an unidentified role in regulating neuronal movement mediated by kisspeptin-GPR54 signaling, which could be a potential pathway to suppress cell migration. PMID:26973595

  1. Kisspeptin Activates Ankrd 26 Gene Expression in Migrating Embryonic GnRH Neurons

    PubMed Central

    Soga, Tomoko; Lim, Wei Ling; Khoo, Alan Soo-Beng; Parhar, Ishwar S.

    2016-01-01

    Kisspeptin, a newly discovered neuropeptide, regulates gonadotropin-releasing hormone (GnRH). Kisspeptins are a large RF-amide family of peptides. The kisspeptin coded by KiSS-1 gene is a 145-amino acid protein that is cleaved to C-terminal peptide kisspeptin-10. G-protein-coupled receptor 54 (GPR54) has been identified as a kisspeptin receptor, and it is expressed in GnRH neurons and in a variety of cancer cells. In this study, enhanced green fluorescent protein (EGFP) labeled GnRH cells with migratory properties, which express GPR54, served as a model to study the effects of kisspeptin on cell migration. We monitored EGFP–GnRH neuronal migration in brain slide culture of embryonic day 14 transgenic rat by live cell imaging system and studied the effects of kisspeptin-10 (1 nM) treatment for 36 h on GnRH migration. Furthermore, to determine kisspeptin-induced molecular pathways related with apoptosis and cytoskeletal changes during neuronal migration, we studied the expression levels of candidate genes in laser-captured EGFP–GnRH neurons by real-time PCR. We found that there was no change in the expression level of genes related to cell proliferation and apoptosis. The expression of ankyrin repeat domain-containing protein (ankrd) 26 in EGFP–GnRH neurons was upregulated by the exposure to kisspeptin. These studies suggest that ankrd 26 gene plays an unidentified role in regulating neuronal movement mediated by kisspeptin–GPR54 signaling, which could be a potential pathway to suppress cell migration. PMID:26973595

  2. Differential gene expression in mouse spermatogonial stem cells and embryonic stem cells

    PubMed Central

    Bai, Yinshan; Feng, Meiying; Liu, Shanshan; Wei, Hengxi; Li, Li; Zhang, Xianwei; Shen, Chao; Zhang, Shouquan; Ma, Ningfang

    2016-01-01

    Mouse spermatogonial stem cells (mSSCs) may be reprogrammed to become pluripotent stem cells under in vitro culture conditions, due to epigenetic modifications, which are closely associated with the expression of transcription factors and epigenetic factors. Thus, this study was conducted to compare the gene expression of transcription factors and epigenetic factors in mSSCs and mouse embryonic stem cells (mESCs). Firstly, the freshly isolated mSSCs [mSSCs (f)] were enriched by magnetic-activated cell sorting with Thy1.2 (CD90.2) microbeads, and the typical morphological characteristics were maintained under in vitro culture conditions for over 5 months to form long-term propagated mSSCs [mSSCs (l)]. These mSSCs (l) expressed pluripotency-associated genes and were induced to differentiate into sperm. Our findings indicated that the mSSCs (l) expressed high levels of the transcription factors, Lin28 and Prmt5, and the epigenetic factors, Tet3, Parp1, Max, Tert and Trf1, in comparison with the mESCs, with the levels of Prmt5, Tet3, Parp1 and Tert significantly higher than those in the mESCs. There was no significant difference in Kdm2b expression between mSSCs (l) and mESCs. Furthermore, the gene expression of N-Myc, Dppa2, Tbx3, Nr5a2, Prmt5, Tet3, Parp1, Max, Tert and Trf1 in the mSSCs (l) was markedly higher in comparison to that in the mSSCs (f). Collectively, our results suggest that the mSSCs and the mESCs displayed differential gene expression profiles, and the mSSCs possessed the potential to acquire pluripotency based on the high expression of transcription factors and epigenetic factors. These data may provide novel insights into the reprogramming mechanism of mSSCs. PMID:27353491

  3. Embryonic expression and evolutionary analysis of the amphioxus Dickkopf and Kremen family genes.

    PubMed

    Zhang, Yujun; Mao, Bingyu

    2010-09-01

    The secreted Wnt signaling inhibitor Dickkopf1 (Dkk1) plays key role in vertebrate head induction. Its receptor Kremen synergizes with Dkk1 in Wnt inhibition. Here we have carried out expression and functional studies of the Dkk and Kremen genes in amphioxus (Branchiostoma belcheri). During embryonic and larval development, BbDkk1/2/4 is expressed in the posterior mesoendoderm, anterior somatic mesoderm and the pharyngeal regions. Its expression becomes restricted to the pharyngeal region on the left side at larval stages. In 45 h larvae, BbDkk1/2/4 is expressed specifically in the cerebral vesicle. BbDkk3 was only detected at larval stages in the mid-intestine region. Seven Kremen related genes were identified in the genome of the Florida amphioxus (Branchiostoma floridae), clustered in 4 scaffolds, and are designated Kremen1-4 and Kremen-like 1-3, respectively. In B. belcheri, Kremen1 is strongly expressed in the mesoendoderm during early development and Kremen3 is expressed asymmetrically in spots in the larval pharyngeal region. In luciferase reporter assays, BbDkk1/2/4 can strongly inhibit Wnt signaling, while BbDkk3, BbKremen1 and BbKremen3 can not. No co-operative effect was observed between amphioxus Dkk1/2/4 and Kremens, suggesting that the interaction between Dkk and Kremen likely originated later during evolution.

  4. RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

    PubMed Central

    Vieceli, Felipe M.; Yan, C.Y. Irene

    2014-01-01

    In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development. PMID:25406837

  5. Shortening and Improving the Embryonic Stem Cell Test through the Use of Gene Biomarkers of Differentiation

    PubMed Central

    Romero, Andrea C.; Vilanova, Eugenio; Sogorb, Miguel A.

    2011-01-01

    The embryonic Stem cell Test (EST) is a validated assay for testing embryotoxicity in vitro. The total duration of this protocol is 10 days, and its main end-point is based on histological determinations. It is suggested that improvements on EST must be focused toward molecular end-points and, if possible, to reduce the total assay duration. Five days of exposure of D3 cells in monolayers under spontaneous differentiation to 50 ng/mL of the strong embryotoxic 5-fluorouracil or to 75 μg/mL of the weak embryotoxic 5,5-diphenylhydeantoin caused between 20 and 74% of reductions in the expression of the following genes: Pnpla6, Afp, Hdac7, Vegfa, and Nes. The exposure to 1 mg/mL of nonembryotoxic saccharin only caused statistically significant reductions in the expression of Nes. These exposures reduced cell viability of D3 cells by 15, 28, and 34%. We applied these records to the mathematical discriminating function of the EST method to find that this approach is able to correctly predict the embryotoxicity of all three above-mentioned chemicals. Therefore, this work proposes the possibility of improve EST by reducing its total duration and by introducing gene expression as biomarker of differentiation, which might be very interesting for in vitro risk assessment embryotoxicity. PMID:21876691

  6. EFFECTS OF CYTOSINE ARABINOSIDE ON DIFFERENTIAL GENE EXPRESSION IN EMBRYONIC NEURAL RETINA

    PubMed Central

    Jones, R. E.; Moscona, A. A.

    1974-01-01

    The analogue of cytidine, cytosine arabinoside (Ara-C), elicited a significant increase in the level of glutamine synthetase (GS) in embryonic chick neural retina in the absence of the steroid inducer of the enzyme. The increase was due to de novo synthesis of GS and was mediated by RNA which accumulated in the presence of the effective concentration of Ara-C. Accumulation of GS did not result from the inhibition of DNA synthesis for which Ara-C is best known. This new effect of Ara-C involves differential suppression of macromolecular synthesis in this system: the concentration of Ara-C which caused maximum GS accumulation suppressed overall protein and RNA syntheses 65–75% without inhibiting the transcription and translation of templates essential for GS synthesis. Withdrawal of Ara-C resulted in restoration of RNA synthesis and cessation of GS accumulation, even though preformed templates for the enzyme were present; however, if all RNA synthesis was arrested with actinomycin D at the time of Ara-C withdrawal, GS continued to accumulate. The results are consistent with the hypothesis that Ara-C differentially affects the activity of structural and regulatory genes involved in the regulation of GS levels in the retina: Ara-C allows transcription of the enzyme-specific templates, but reversibly inhibits the expression of regulatory genes which limit the accumulation of GS. PMID:4151790

  7. Gene expression signatures defining fundamental biological processes in pluripotent, early, and late differentiated embryonic stem cells.

    PubMed

    Gaspar, John Antonydas; Doss, Michael Xavier; Winkler, Johannes; Wagh, Vilas; Hescheler, Jürgen; Kolde, Raivo; Vilo, Jaak; Schulz, Herbert; Sachinidis, Agapios

    2012-09-01

    Investigating the molecular mechanisms controlling the in vivo developmental program postembryogenesis is challenging and time consuming. However, the developmental program can be partly recapitulated in vitro by the use of cultured embryonic stem cells (ESCs). Similar to the totipotent cells of the inner cell mass, gene expression and morphological changes in cultured ESCs occur hierarchically during their differentiation, with epiblast cells developing first, followed by germ layers and finally somatic cells. Combination of high throughput -omics technologies with murine ESCs offers an alternative approach for studying developmental processes toward organ-specific cell phenotypes. We have made an attempt to understand differentiation networks controlling embryogenesis in vivo using a time kinetic, by identifying molecules defining fundamental biological processes in the pluripotent state as well as in early and the late differentiation stages of ESCs. Our microarray data of the differentiation of the ESCs clearly demonstrate that the most critical early differentiation processes occur at days 2 and 3 of differentiation. Besides monitoring well-annotated markers pertinent to both self-renewal and potency (capacity to differentiate to different cell lineage), we have identified candidate molecules for relevant signaling pathways. These molecules can be further investigated in gain and loss-of-function studies to elucidate their role for pluripotency and differentiation. As an example, siRNA knockdown of MageB16, a gene highly expressed in the pluripotent state, has proven its influence in inducing differentiation when its function is repressed.

  8. Generation and gene expression profiling of 48 transcription-factor-inducible mouse embryonic stem cell lines

    PubMed Central

    Yamamizu, Kohei; Sharov, Alexei A.; Piao, Yulan; Amano, Misa; Yu, Hong; Nishiyama, Akira; Dudekula, Dawood B.; Schlessinger, David; Ko, Minoru S. H.

    2016-01-01

    Mouse embryonic stem cells (ESCs) can differentiate into a wide range – and possibly all cell types in vitro, and thus provide an ideal platform to study systematically the action of transcription factors (TFs) in cell differentiation. Previously, we have generated and analyzed 137 TF-inducible mouse ESC lines. As an extension of this “NIA Mouse ESC Bank,” we generated and characterized 48 additional mouse ESC lines, in which single TFs in each line could be induced in a doxycycline-controllable manner. Together, with the previous ESC lines, the bank now comprises 185 TF-manipulable ESC lines (>10% of all mouse TFs). Global gene expression (transcriptome) profiling revealed that the induction of individual TFs in mouse ESCs for 48 hours shifts their transcriptomes toward specific differentiation fates (e.g., neural lineages by Myt1 Isl1, and St18; mesodermal lineages by Pitx1, Pitx2, Barhl2, and Lmx1a; white blood cells by Myb, Etv2, and Tbx6, and ovary by Pitx1, Pitx2, and Dmrtc2). These data also provide and lists of inferred target genes of each TF and possible functions of these TFs. The results demonstrate the utility of mouse ESC lines and their transcriptome data for understanding the mechanism of cell differentiation and the function of TFs. PMID:27150017

  9. Modulation of chromatin modifying factors' gene expression in embryonic and induced pluripotent stem cells.

    PubMed

    Luzzani, Carlos; Solari, Claudia; Losino, Noelia; Ariel, Waisman; Romorini, Leonardo; Bluguermann, Carolina; Sevlever, Gustavo; Barañao, Lino; Miriuka, Santiago; Guberman, Alejandra

    2011-07-15

    Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are a promising source of cells for regenerative medicine because of their potential of self renew and differentiation. Multiple evidences highlight the relationship of chromatin remodeling with stem cell properties, differentiation programs and reprogramming for iPSC obtention. With the purpose of finding chromatin modifying factors relevant to these processes, and based on ChIP on chip studies, we selected several genes that could be modulated by Oct4, Sox2 and Nanog, critical transcription factors in stem cells, and studied their expression profile along the differentiation in mouse and human ESCs, and in mouse iPSCs. In this work, we analyzed the expression of Gcn5l2, GTF3C3, TAF15, ATF7IP, Myst2, HDAC2, HDAC3, HDAC5, HDAC10, SUV39H2, Jarid2, and Bmi-1. We found some genes from different functional groups that were highly modulated, suggesting that they could be relevant both in the undifferentiated state and during differentiation. These findings could contribute to the comprehension of molecular mechanisms involved in pluripotency, early differentiation and reprogramming. We believe that a deeper knowledge of the epigenetic regulation of ESC will allow improving somatic cell reprogramming for iPSC obtention and differentiation protocols optimization.

  10. Nuclear matrix association of the human beta-globin locus utilizing a novel approach to quantitative real-time PCR.

    PubMed

    Ostermeier, G Charles; Liu, Zhandong; Martins, Rui Pires; Bharadwaj, Rikki R; Ellis, James; Draghici, Sorin; Krawetz, Stephen A

    2003-06-15

    The human beta-globin locus is home to five genes that are regulated in a tissue-specific and developmental stage-specific manner. While the exact mode of expression remains somewhat enigmatic, a significant effort has been focused at the locus control region (LCR). The LCR is marked by five DNase I-hypersensitive sites (HS) approximately 15 kb upstream of the epsilon-globin gene. Nuclear matrix-associated regions (MARs) organize chromatin into functional domains and at least one of the HS appears bound to the nuclear matrix. We have employed an in vivo based PCR MAR assay to investigate the role of MAR-mediated regulation of the beta-globin locus. This was facilitated with a novel reaction efficiency based quantitative real-time PCR analysis software tool, Target Analysis Quantification. Using a log-linear regression strategy, discordances were eliminated. This allowed us to reliably estimate the relative amount of initial template associated with the nuclear matrix at 15 unique regions spanning the beta-globin locus in both non-expressing and expressing cell lines. A dynamic association dependent on expression status was revealed both at the LCR/5'HS region and within the second intron of the beta-globin gene. These results provide the first evidence that nuclear matrix association dynamically mediates the looping of the beta-globin locus to achieve transcriptional control.

  11. 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

  12. Exploiting the power of LINE-1 retrotransposon mutagenesis for identification of genes involved in embryonic stem cell differentiation.

    PubMed

    Lenka, Nibedita; Krishnan, Shruthi; Board, Philip; Rangasamy, Danny

    2014-06-01

    Identifying the genes or epigenetic factors that control the self-renewal and differentiation of stem cells is critical to understanding the molecular basis of cell commitment. Although a number of insertional mutagenesis vectors have been developed for identifying gene functions in animal models, the L1 retrotransposition system offers additional advantages as a tool to disrupt genes in embryonic stem cells in order to identify their functions and the phenotypes associated with them. Recent advances in producing synthetic versions of L1 retrotransposon vector system and the optimization of techniques to accurately identify retrotransposon integration sites have increased their utility for gene discovery applications. We have developed a novel episomal, nonviral L1 retrotransposon vector using scaffold/matrix attachment regions that provides stable, sustained levels of retrotransposition in cell cultures without being affected by epigenetic silencing or from some of the common problems of vector integration. This modified vector contains a GFP marker whose expression occurs only after successful gene disruption events and thus the cells with disrupted genes can be easily picked for functional analysis. Here we present a method to disrupt gene function in embryonic stem cells that aid in the identification of genes involved in stem cell differentiation processes. The methods presented here can be easily adapted to the study of other types of cancer stem cells or induced pluripotent stem cells using the L1 retrotransposon as an insertional mutagen.

  13. Microarray analysis of gene expression in mouse (strain 129) embryonic stem cells after typical synthetic musk exposure.

    PubMed

    Shi, Jiachen; Li, Ming; Jiao, Zhihao; Zhang, Jing; Feng, Yixing; Shao, Bing

    2013-01-01

    Synthetic musks are widely used in personal-care products and can readily accumulate in the adipose tissue, breast milk, and blood of humans. In this study, the Affymetrix Mouse Genome GeneChip was used to identify alterations in gene expression of embryonic stem cells from the 129 strain of the laboratory mouse after treatment with the synthetic musk tonalide (AHTN). Among the 45,037 transcripts in the microarray, 2,879 genes were differentially expressed. According to the microarray analysis, the potential influence of AHTN on the development to embryo should be of concern, and the toxicological effects of it and related musk compounds should be studied further.

  14. c-Rel Regulates Inscuteable Gene Expression during Mouse Embryonic Stem Cell Differentiation.

    PubMed

    Ishibashi, Riki; Kozuki, Satoshi; Kamakura, Sachiko; Sumimoto, Hideki; Toyoshima, Fumiko

    2016-02-12

    Inscuteable (Insc) regulates cell fate decisions in several types of stem cells. Although it is recognized that the expression levels of mouse INSC govern the balance between symmetric and asymmetric stem cell division, regulation of mouse Insc gene expression remains poorly understood. Here, we showed that mouse Insc expression transiently increases at an early stage of differentiation, when mouse embryonic stem (mES) cells differentiate into bipotent mesendoderm capable of producing both endoderm and mesoderm in defined culture conditions. We identified the minimum transcriptional regulatory element (354 bases) that drives mouse Insc transcription in mES cells within a region >5 kb upstream of the mouse Insc transcription start site. We found that the transcription factor reticuloendotheliosis oncogene (c-Rel) bound to the minimum element and promoted mouse Insc expression in mES cells. In addition, short interfering RNA-mediated knockdown of either mouse INSC or c-Rel protein decreased mesodermal cell populations without affecting differentiation into the mesendoderm or endoderm. Furthermore, overexpression of mouse INSC rescued the mesoderm-reduced phenotype induced by knockdown of c-Rel. We propose that regulation of mouse Insc expression by c-Rel modulates cell fate decisions during mES cell differentiation.

  15. Genome-wide gene expression analysis of mouse embryonic stem cells exposed to p-dichlorobenzene.

    PubMed

    Tani, Hidenori; Takeshita, Jun-Ichi; Aoki, Hiroshi; Abe, Ryosuke; Toyoda, Akinobu; Endo, Yasunori; Miyamoto, Sadaaki; Gamo, Masashi; Torimura, Masaki

    2016-09-01

    Because of the limitations of whole animal testing approaches for toxicological assessment, new cell-based assay systems have been widely studied. In this study, we focused on two biological products for toxicological assessment: mouse embryonic stem cells (mESCs) and long noncoding RNAs (lncRNAs). mESCs possess the abilities of self-renewal and differentiation into multiple cell types. LlncRNAs are an important class of pervasive non-protein-coding transcripts involved in the molecular mechanisms associated with responses to chemicals. We exposed mESCs to p-dichlorobenzene (p-DCB) for 1 or 28 days (daily dose), extracted total RNA, and performed deep sequencing analyses. The genome-wide gene expression analysis indicated that mechanisms modulating proteins occurred following acute and chronic exposures, and mechanisms modulating genomic DNA occurred following chronic exposure. Moreover, our results indicate that three novel lncRNAs (Snora41, Gm19947, and Scarna3a) in mESCs respond to p-DCB exposure. We propose that these lncRNAs have the potential to be surrogate indicators of p-DCB responses in mESCs. PMID:26975756

  16. Directed neuronal differentiation of mouse embryonic and induced pluripotent stem cells and their gene expression profiles.

    PubMed

    Chen, Xuesong; Gu, Qi; Wang, Xiang; Ma, Qingwen; Tang, Huixiang; Yan, Xiaoshuang; Guo, Xinbing; Yan, Hao; Hao, Jie; Zeng, Fanyi

    2013-07-01

    Embryonic stem cells (ESCs) may be useful as a therapeutic source of cells for the production of healthy tissue; however, they are associated with certain challenges including immunorejection as well as ethical issues. Induced pluripotent stem cells (iPSCs) are a promising substitute since a patient's own adult cells would serve as tissue precursors. Ethical concerns prevent a full evaluation of the developmental potency of human ESCs and iPSCs, therefore, mouse iPSC models are required for protocol development and safety assessments. We used a modified culturing protocol to differentiate pluripotent cells from a mouse iPS cell line and two mouse ES cell lines into neurons. Our results indicated that all three pluripotent stem cell lines underwent nearly the same differentiation process when induced to form neurons in vitro. Genomic expression microarray profiling and single-cell RT-qPCR were used to analyze the neural lineage differentiation process, and more than one thousand differentially expressed genes involved in multiple molecular processes relevant to neural development were identified.

  17. Identification of positional candidates for bovine placental genes responsible for early embryonic death during cloning-attempted pregnancy.

    PubMed

    Yamada, Takahisa; Muramatsu, Youji; Taniguchi, Yukio; Sasaki, Yoshiyuki

    Our previous study detected 291 and 77 genes showing early embryonic death-associated elevation and reduction of expression, respectively, in the fetal placenta of the cow carrying somatic nuclear transfer-derived cloned embryo. In this study, we mapped the 10 genes showing the elevation and the 10 genes doing the reduction most significantly, using somatic cell hybrid and bovine draft genome sequence. We then compared the mapped positions for these genes with the genomic locations of bovine quantitative trait loci for still-birth and/or abortion. Among the mapped genes, peptidylglycine alpha-amidating monooxygenase (PAM), spectrin, beta, nonerythrocytic 1 (SPTBNI), and an unknown novel gene containing AU277832 expressed sequence tag were intriguing, in that the mapped positions were consistent with the genomic locations of bovine still-birth and/or abortion quantitative trait loci, and thus identified as positional candidates for bovine placental genes responsible for the early embryonic death during the pregnancy attempted by somatic nuclear transfer-derived cloning.

  18. Generation of KCL035 research grade human embryonic stem cell line carrying a mutation in HBB gene.

    PubMed

    Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-03-01

    The KCL035 human embryonic stem cell line was derived from an embryo donated for research that carried a mutation in the HBB gene, which is linked to the β-thalassemia syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345970

  19. Generation of KCL035 research grade human embryonic stem cell line carrying a mutation in HBB gene

    PubMed Central

    Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL035 human embryonic stem cell line was derived from an embryo donated for research that carried a mutation in the HBB gene, which is linked to the β-thalassemia syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345970

  20. Cationic star copolymers based on β-cyclodextrins for efficient gene delivery to mouse embryonic stem cell colonies.

    PubMed

    Loh, Xian Jun; Wu, Yun-Long

    2015-07-11

    A cationic star copolymer with a β-cyclodextrin core was developed for nonviral gene transfer to mouse embryonic stem cells (mESCs). The copolymer comprises poly(2-dimethyl aminoethyl methacrylate) as the cationic component and poly(2-hydroxyethyl methacrylate) as the non-toxic stealth component. These materials have very low toxicity and show highly efficient transfection to mESC colonies. PMID:26040469

  1. 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.

  2. An amphioxus netrin gene is expressed in midline structures during embryonic and larval development.

    PubMed

    Shimeld, S

    2000-07-01

    Members of the netrin gene family have been identified in vertebrates, Drosophila and Caenorhabditis elegans and found to encode secreted molecules involved in axon guidance. Here I use the conserved function of netrins in triploblasts, coupled with the phylogenetic position of amphioxus (the closest living relative of the vertebrates), to investigate the evolution of an axon guidance cue in chordates. A single amphioxus netrin gene was isolated by PCR and cDNA library screening and named AmphiNetrin. The predicted AmphiNetrin protein showed high identity to other netrin family members but differed in that the third of three EGF repeats found in other netrins was absent. Molecular phylogene-tic analysis showed that despite the absent EGF repeat AmphiNetrin is most closely related to the vertebrate netrins. AmphiNetrin expression was identified in embryonic notochord and floor plate, a pattern similar to that of vertebrate netrin-1 expression. AmphiNetrin expression was also identified more widely in the posterior larval brain, and in the anterior extension of the notochord that underlies the anterior of the amphioxus brain. All of these areas of expression are correlated with developing axon trajectories: The floor plate with ventrally projecting somatic motor neurons and Rohde cell projections, the posterior brain with the ventral commissure and primary motor centre and the anterior extension of the notochord with ventrally projecting neurons associated with the median eye. Amphioxus is naturally cyclopaedic and also lacks the ventral brain cells that the induction of which results in the splitting of the vertebrate eye field and, when missing, result in cyclopaedia. These cells normally express netrins required for developing axon tracts in the brain, and the expression of AmphiNetrin in the anterior extension of the notochord underlying the brain may explain how amphioxus is able to maintain ventral guidance cues while lacking these cells.

  3. Functional equivalence of Hox gene products in the specification of the tritocerebrum during embryonic brain development of Drosophila.

    PubMed

    Hirth, F; Loop, T; Egger, B; Miller, D F; Kaufman, T C; Reichert, H

    2001-12-01

    Hox genes encode evolutionarily conserved transcription factors involved in the specification of segmental identity during embryonic development. This specification of identity is thought to be directed by differential Hox gene action, based on differential spatiotemporal expression patterns, protein sequence differences, interactions with co-factors and regulation of specific downstream genes. During embryonic development of the Drosophila brain, the Hox gene labial is required for the regionalized specification of the tritocerebral neuromere; in the absence of labial, the cells in this brain region do not acquire a neuronal identity and major axonal pathfinding deficits result. We have used genetic rescue experiments to investigate the functional equivalence of the Drosophila Hox gene products in the specification of the tritocerebral neuromere. Using the Gal4-UAS system, we first demonstrate that the labial mutant brain phenotype can be rescued by targeted expression of the Labial protein under the control of CNS-specific labial regulatory elements. We then show that under the control of these CNS-specific regulatory elements, all other Drosophila Hox gene products, except Abdominal-B, are able to efficiently replace Labial in the specification of the tritocerebral neuromere. We also observe a correlation between the rescue efficiency of the Hox proteins and the chromosomal arrangement of their encoding loci. Our results indicate that, despite considerably diverged sequences, most Hox proteins are functionally equivalent in their ability to replace Labial in the specification of neuronal identity. This suggests that in embryonic brain development, differences in Hox gene action rely mainly on cis-acting regulatory elements and not on Hox protein specificity. PMID:11731458

  4. Embryonic and tissue-specific regulation of myostatin-1 and -2 gene expression in zebrafish

    PubMed Central

    Helterline, Deri L.I.; Garikipati, Dilip; Stenkamp, Deborah L.; Rodgers, Buel D.

    2008-01-01

    Myostatin is a member of the TGF-β superfamily and a potent negative regulator of muscle growth and development in mammals. Its expression is limited primarily to skeletal muscle in mammals, but occurs in many different fish tissues, although quantitative measurements of the embryonic and tissue-specific expression profiles are lacking. A recent phylogenetic analysis of all known myostatin genes identified a novel paralogue in zebrafish, zfMSTN-2, and prompted the reclassification of the entire subfamily to include MSTN-1 and -2 sister clades in the bony fishes. The differential expression profiles of both genes were therefore determined using custom RNA panels generated from pooled (100–150/sampling) embryos at different stages of development and from individual adult tissues. High levels of both transcripts were transiently present at the blastula stage, but were undetectable throughout gastrulation (7 hpf). Levels of zfMSTN-2 peaked during early somitogenesis (11 hpf), returned to basal levels during late somitogenesis and did not begin to rise again until hatching (72 hpf). By contrast, zfMSTN-1 mRNA levels peaked during late somitogenesis (15.5–19 hpf), returned to baseline at 21.5 hpf and eventually rose 25-fold by 72 hpf. In adults, both transcripts were present in a wide variety of tissues, including some not previously known to express myostatin. Expression of zfMSTN-1 was highest in brain, muscle, heart and testes and was 1–3 log orders above that in other tissues. It was also greater than zfMSTN-2 expression in most tissues, nevertheless, levels of both transcripts increased almost 600-fold in spleens of fish subjected to stocking stress. Myostatin expression was also detected in mouse spleens, suggesting that myostatin may influence immune cell development in mammals as well as fish. These studies indicate that zfMSTN-1 and -2 gene expression is differentially regulated in developing fish embryos and in adult tissues. The increased expression of

  5. 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

  6. 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.

  7. Similarities in Gene Expression Profiles during In Vitro Aging of Primary Human Embryonic Lung and Foreskin Fibroblasts

    PubMed Central

    Marthandan, Shiva; Priebe, Steffen; Baumgart, Mario; Groth, Marco; Cellerino, Alessandro; Guthke, Reinhard; Hemmerich, Peter; Diekmann, Stephan

    2015-01-01

    Replicative senescence is of fundamental importance for the process of cellular aging, since it is a property of most of our somatic cells. Here, we elucidated this process by comparing gene expression changes, measured by RNA-seq, in fibroblasts originating from two different tissues, embryonic lung (MRC-5) and foreskin (HFF), at five different time points during their transition into senescence. Although the expression patterns of both fibroblast cell lines can be clearly distinguished, the similar differential expression of an ensemble of genes was found to correlate well with their transition into senescence, with only a minority of genes being cell line specific. Clustering-based approaches further revealed common signatures between the cell lines. Investigation of the mRNA expression levels at various time points during the lifespan of either of the fibroblasts resulted in a number of monotonically up- and downregulated genes which clearly showed a novel strong link to aging and senescence related processes which might be functional. In terms of expression profiles of differentially expressed genes with age, common genes identified here have the potential to rule the transition into senescence of embryonic lung and foreskin fibroblasts irrespective of their different cellular origin. PMID:26339636

  8. 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

  9. Generation of KCL026 research grade human embryonic stem cell line carrying a mutation in SMN1 gene

    PubMed Central

    Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL026 human embryonic stem cell line was derived from an embryo donated for research that carried a mutation in the SMN1 gene encoding survival of motor neuron 1, telomeric (exons 7 and 8 deletion). Mutations in this gene are associated with spinal muscular atrophy. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345977

  10. Generation of KCL025 research grade human embryonic stem cell line carrying a mutation in NF1 gene

    PubMed Central

    Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL025 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation in the NF1 gene encoding neurofibromin (c.3739–3742 ΔTTTG). Mutations in this gene have been linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345978

  11. Generation of KCL024 research grade human embryonic stem cell line carrying a mutation in NF1 gene

    PubMed Central

    Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL024 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation in the NF1 gene encoding neurofibromin (c.3739–3742 ∆ TTTG). Mutations in this gene have been linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345975

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    1995-05-25

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

  14. 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

  15. 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.

  16. Targeting of the Human Coagulation Factor IX Gene at rDNA Locus of Human Embryonic Stem Cells

    PubMed Central

    Yang, Junlin; Xue, Jinfeng; Hu, Youjin; Feng, Mai; Niu, Wenbin; Yang, Qiurui; Lei, Ming; Xia, Jiahui; Wu, Lingqian; Liang, Desheng

    2012-01-01

    Background Genetic modification is a prerequisite to realizing the full potential of human embryonic stem cells (hESCs) in human genetic research and regenerative medicine. Unfortunately, the random integration methods that have been the primary techniques used keep creating problems, and the primary alternative method, gene targeting, has been effective in manipulating mouse embryonic stem cells (mESCs) but poorly in hESCs. Methodology/Principal Findings Human ribosomal DNA (rDNA) repeats are clustered on the short arm of acrocentric chromosomes. They consist of approximately 400 copies of the 45S pre-RNA (rRNA) gene per haploid. In the present study, we targeted a physiological gene, human coagulation factor IX, into the rDNA locus of hESCs via homologous recombination. The relative gene targeting efficiency (>50%) and homologous recombination frequency (>10−5) were more than 10-fold higher than those of loci targeted in previous reports. Meanwhile, the targeted clones retained both a normal karyotype and the main characteristics of ES cells. The transgene was found to be stably and ectopically expressed in targeted hESCs. Conclusion/Significance This is the first targeting of a human physiological gene at a defined locus on the hESC genome. Our findings indicate that the rDNA locus may serve as an ideal harbor for transgenes in hESCs. PMID:22615895

  17. C. elegans SoxB genes are dispensable for embryonic neurogenesis but required for terminal differentiation of specific neuron types

    PubMed Central

    Vidal, Berta; Santella, Anthony; Serrano-Saiz, Esther; Bao, Zhirong; Chuang, Chiou-Fen; Hobert, Oliver

    2015-01-01

    Neurogenesis involves deeply conserved patterning molecules, such as the proneural basic helix-loop-helix transcription factors. Sox proteins and specifically members of the SoxB and SoxC groups are another class of conserved transcription factors with an important role in neuronal fate commitment and differentiation in various species. In this study, we examine the expression of all five Sox genes of the nematode C. elegans and analyze the effect of null mutant alleles of all members of the SoxB and SoxC groups on nervous system development. Surprisingly, we find that, unlike in other systems, neither of the two C. elegans SoxB genes sox-2 (SoxB1) and sox-3 (SoxB2), nor the sole C. elegans SoxC gene sem-2, is broadly expressed throughout the embryonic or adult nervous system and that all three genes are mostly dispensable for embryonic neurogenesis. Instead, sox-2 is required to maintain the developmental potential of blast cells that are generated in the embryo but divide only postembryonically to give rise to differentiated neuronal cell types. Moreover, sox-2 and sox-3 have selective roles in the terminal differentiation of specific neuronal cell types. Our findings suggest that the common themes of SoxB gene function across phylogeny lie in specifying developmental potential and, later on, in selectively controlling terminal differentiation programs of specific neuron types, but not in broadly controlling neurogenesis. PMID:26153233

  18. TGFβ-1 and Wnt-3a interact to induce unique gene expression profiles in murine embryonic palate mesenchymal cells

    PubMed Central

    Warner, Dennis R.; Mukhopadhyay, Partha; Brock, Guy N.; Pihur, Vasyl; Pisano, M. Michele; Greene, Robert M.

    2011-01-01

    Development of the secondary palate in mammals is a complex process under the control of numerous growth and differentiation factors that regulate key processes such as cell proliferation, synthesis of extracellular matrix molecules, and epithelial-mesenchymal transdifferentiation. Alterations in any one of these processes either through genetic mutation or environmental insult have the potential to lead to clefts of the secondary palate. Members of the TGFβ family of cytokines are crucial mediators of these processes and emerging evidence supports a pivotal role for members of the Wnt family of secreted growth and differentiation factors. Previous work in this laboratory demonstrated cross-talk between the Wnt and TGFβ signaling pathways in cultured mouse embryonic palate mesenchymal cells. In the current study we tested the hypothesis that unique gene expression profiles are induced in murine embryonic palate mesenchymal cells as a result of this cross-talk between the TGFβ and Wnt signal transduction pathways. PMID:20955781

  19. Toward the Beginning of Time: Circadian Rhythms in Metabolism Precede Rhythms in Clock Gene Expression in Mouse Embryonic Stem Cells

    PubMed Central

    Paulose, Jiffin K.; Rucker, Edmund B.; Cassone, Vincent M.

    2012-01-01

    The appearance, progression, and potential role for circadian rhythms during early development have previously focused mainly on the suprachiasmatic nucleus (SCN) and peri- and postnatal expression of canonical clock genes. More recently, gene expression studies in embryonic stem cells have shown that some clock genes are expressed in undifferentiated cells; however rhythmicity was only established when cells are directed toward a neural fate. These studies also concluded that a functional clock is not present in ESCs, based solely on their gene expression. The null hypothesis underlying the present study is that embryonic stem cells become rhythmic in both clock gene expression and glucose utilization only when allowed to spontaneously differentiate. Undifferentiated stem cells (ESCs, n = 6 cultures/timepoint for all experiments) were either maintained in their pluripotent state or released into differentiation (dESCs, n = 6 cultures/timepoint for all experiments). Glucose utilization was assayed through 2-deoxyglucose uptake measurement, and clock gene and glucose transporter expression was assayed every 4 hours for 2 days in ESCs and dESCs by quantitative PCR (qPCR) in the same cell lysates. Undifferentiated stem cells expressed a self-sustained rhythm in glucose uptake that was not coincident with rhythmic expression of clock genes. This physiological rhythm was paralleled by glucose transporter mRNA expression. Upon differentiation, circadian patterns of some but not all clock genes were expressed, and the amplitude of the glucose utilization rhythm was enhanced in dESCs. These data provide the earliest evidence of a functional circadian clock, in addition to further challenging the idea that rhythmic transcription of clock genes are necessary for rhythmic physiological output and suggest a role for a clock-controlled physiology in the earliest stages of development. PMID:23155474

  20. Determinants of specific RNA interference-mediated silencing of human beta-globin alleles differing by a single nucleotide polymorphism.

    PubMed

    Dykxhoorn, Derek M; Schlehuber, Lisa D; London, Irving M; Lieberman, Judy

    2006-04-11

    A single nucleotide polymorphism (SNP) in the sickle beta-globin gene (beta(S)) leads to sickle cell anemia. Sickling increases sharply with deoxy sickle Hb concentration and decreases with increasing fetal gamma-globin concentration. Measures that decrease sickle Hb concentration should have an antisickling effect. RNA interference (RNAi) uses small interfering (si)RNAs for sequence-specific gene silencing. A beta(S) siRNA with position 10 of the guide strand designed to align with the targeted beta(S) SNP specifically silences beta(S) gene expression without affecting the expression of the gamma-globin or normal beta-globin (beta(A)) genes. Silencing is increased by altering the 5' end of the siRNA antisense (guide) strand to enhance its binding to the RNA-induced silencing complex (RISC). Specific beta(S) silencing was demonstrated by using a luciferase reporter and full-length beta(S) cDNA transfected into HeLa cells and mouse erythroleukemia cells, where it was expressed in the context of the endogenous beta-globin gene promoter and the locus control region enhancers. When this strategy was used to target beta(E), silencing was not limited to the mutant gene but also targeted the normal beta(A) gene. siRNAs, mismatched with their target at position 10, guided mRNA cleavage in all cases except when two bulky purines were aligned. The specific silencing of the beta(S)-globin gene, as compared with beta(E), as well as studies of silencing SNP mutants in other diseases, indicates that siRNAs developed to target a disease-causing SNP will be specific if the mutant residue is a pyrimidine and the normal residue is a purine.

  1. Control of mouse U1 snRNA gene expression during in vitro differentiation of mouse embryonic stem cells.

    PubMed Central

    Cheng, Y; Lund, E; Kahan, B W; Dahlberg, J E

    1997-01-01

    Early in mouse development, two classes of U1 RNAs, mU1a and mU1b, are synthesized, but as development proceeds, transcription of the embryo-specific mU1b genes is selectively down-regulated to a barely detectable level. We show here that during in vitro differentiation of mouse embryonic stem (ES) cells, both exogenously introduced and endogenous U1b genes are subject to normal developmental regulation. Thus, ES cells represent a convenient isogenic system for studying the control of expression of developmentally regulated snRNA genes. Using this system, we have identified a region in the proximal 5'flanking region, located outside the PSE element, that is responsible for differential transcription of the mU1a and mU1b genes in both developing cells and transiently transfected NIH 3T3 cells. PMID:9153321

  2. Serum-based culture conditions provoke gene expression variability in mouse embryonic stem cells as revealed by single cell analysis

    PubMed Central

    Guo, Guoji; Pinello, Luca; Han, Xiaoping; Lai, Shujing; Shen, Li; Lin, Ta-Wei; Zou, Keyong; Yuan, Guo-Cheng; Orkin, Stuart H.

    2015-01-01

    Summary Variation in gene expression is an important feature of mouse embryonic stem cells (ESCs). However, the mechanisms responsible for global gene expression variation in ESCs are not fully understood. We performed single cell mRNA-seq analysis of mouse ESCs and uncovered significant heterogeneity in ESCs cultured in serum. We define highly variable gene clusters with distinct chromatin states; and show that bivalent genes are prone to expression variation. At the same time, we identify an ESC priming pathway that initiates the exit from the naïve ESC state. Finally, we provide evidence that a large proportion of intracellular network variability is due to the extracellular culture environment. Serum free culture reduces cellular heterogeneity and transcriptome variation in ESCs. PMID:26804902

  3. Compound-specific effects of diverse neurodevelopmental toxicants on global gene expression in the neural embryonic stem cell test (ESTn).

    PubMed

    Theunissen, P T; Robinson, J F; Pennings, J L A; van Herwijnen, M H; Kleinjans, J C S; Piersma, A H

    2012-08-01

    Alternative assays for developmental toxicity testing are needed to reduce animal use in regulatory toxicology. The in vitro murine neural embryonic stem cell test (ESTn) was designed as an alternative for neurodevelopmental toxicity testing. The integration of toxicogenomic-based approaches may further increase predictivity as well as provide insight into underlying mechanisms of developmental toxicity. In the present study, we investigated concentration-dependent effects of six mechanistically diverse compounds, acetaldehyde (ACE), carbamazepine (CBZ), flusilazole (FLU), monoethylhexyl phthalate (MEHP), penicillin G (PENG) and phenytoin (PHE), on the transcriptome and neural differentiation in the ESTn. All compounds with the exception of PENG altered ESTn morphology (cytotoxicity and neural differentiation) in a concentration-dependent manner. Compound induced gene expression changes and corresponding enriched gene ontology biological processes (GO-BP) were identified after 24h exposure at equipotent differentiation-inhibiting concentrations of the compounds. Both compound-specific and common gene expression changes were observed between subsets of tested compounds, in terms of significance, magnitude of regulation and functionality. For example, ACE, CBZ and FLU induced robust changes in number of significantly altered genes (≥ 687 genes) as well as a variety of GO-BP, as compared to MEHP, PHE and PENG (≤ 55 genes with no significant changes in GO-BP observed). Genes associated with developmentally related processes (embryonic morphogenesis, neuron differentiation, and Wnt signaling) showed diverse regulation after exposure to ACE, CBZ and FLU. In addition, gene expression and GO-BP enrichment showed concentration dependence, allowing discrimination of non-toxic versus toxic concentrations on the basis of transcriptomics. This information may be used to define adaptive versus toxic responses at the transcriptome level.

  4. Compound-specific effects of diverse neurodevelopmental toxicants on global gene expression in the neural embryonic stem cell test (ESTn)

    SciTech Connect

    Theunissen, P.T.; Robinson, J.F.; Pennings, J.L.A.; Herwijnen, M.H. van; Kleinjans, J.C.S.; Piersma, A.H.

    2012-08-01

    Alternative assays for developmental toxicity testing are needed to reduce animal use in regulatory toxicology. The in vitro murine neural embryonic stem cell test (ESTn) was designed as an alternative for neurodevelopmental toxicity testing. The integration of toxicogenomic-based approaches may further increase predictivity as well as provide insight into underlying mechanisms of developmental toxicity. In the present study, we investigated concentration-dependent effects of six mechanistically diverse compounds, acetaldehyde (ACE), carbamazepine (CBZ), flusilazole (FLU), monoethylhexyl phthalate (MEHP), penicillin G (PENG) and phenytoin (PHE), on the transcriptome and neural differentiation in the ESTn. All compounds with the exception of PENG altered ESTn morphology (cytotoxicity and neural differentiation) in a concentration-dependent manner. Compound induced gene expression changes and corresponding enriched gene ontology biological processes (GO–BP) were identified after 24 h exposure at equipotent differentiation-inhibiting concentrations of the compounds. Both compound-specific and common gene expression changes were observed between subsets of tested compounds, in terms of significance, magnitude of regulation and functionality. For example, ACE, CBZ and FLU induced robust changes in number of significantly altered genes (≥ 687 genes) as well as a variety of GO–BP, as compared to MEHP, PHE and PENG (≤ 55 genes with no significant changes in GO–BP observed). Genes associated with developmentally related processes (embryonic morphogenesis, neuron differentiation, and Wnt signaling) showed diverse regulation after exposure to ACE, CBZ and FLU. In addition, gene expression and GO–BP enrichment showed concentration dependence, allowing discrimination of non-toxic versus toxic concentrations on the basis of transcriptomics. This information may be used to define adaptive versus toxic responses at the transcriptome level.

  5. Beta-globin active chromatin Hub formation in differentiating erythroid cells and in p45 NF-E2 knock-out mice.

    PubMed

    Kooren, Jurgen; Palstra, Robert-Jan; Klous, Petra; Splinter, Erik; von Lindern, Marieke; Grosveld, Frank; de Laat, Wouter

    2007-06-01

    Expression of the beta-globin genes proceeds from basal to exceptionally high levels during erythroid differentiation in vivo. High expression is dependent on the locus control region (LCR) and coincides with more frequent LCR-gene contacts. These contacts are established in the context of an active chromatin hub (ACH), a spatial chromatin configuration in which the LCR, together with other regulatory sequences, loops toward the active beta-globin-like genes. Here, we used recently established I/11 cells as a model system that faithfully recapitulates the in vivo erythroid differentiation program to study the molecular events that accompany and underlie ACH formation. Upon I/11 cell induction, histone modifications changed, the ACH was formed, and the beta-globin-like genes were transcribed at rates similar to those observed in vivo. The establishment of frequent LCR-gene contacts coincided with a more efficient loading of polymerase onto the beta-globin promoter. Binding of the transcription factors GATA-1 and EKLF to the locus, although previously shown to be required, was not sufficient for ACH formation. Moreover, we used knock-out mice to show that the erythroid transcription factor p45 NF-E2, which has been implicated in beta-globin gene regulation, is dispensable for beta-globin ACH formation.

  6. 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

  7. Differential regulation of osteogenic marker gene expression by Wnt-3a in embryonic mesenchymal multipotential progenitor cells.

    PubMed

    Derfoul, Assia; Carlberg, Alyssa L; Tuan, Rocky S; Hall, David J

    2004-06-01

    The Wnt family of secreted glycoproteins plays an integral role in embryonic development and differentiation. To explore the role of Wnt's in one aspect of differentiation, namely osteogenesis, we employed a retroviral gene transfer approach to express Wnt-3a in the multipotent murine embryonic mesenchymal cell line C3H10T1/2. We found that expression of Wnt-3a in these cells had a significant, positive effect on cell growth in serum-containing medium, in that the cells grew to very high densities compared to the control cells. Additionally, apoptosis was markedly inhibited by Wnt-3a. However, when the cells were grown in serum-deficient medium, the Wnt-3a-expressing cells arrested efficiently in G1 phase, indicating that serum growth factors were needed in addition to Wnt-3a for enhanced proliferation. Wnt-3a-expressing cells exhibited high levels of alkaline phosphatase gene expression and enzymatic activity, but did not show any matrix mineralization. Unexpectedly, basal expression of bone sialoprotein, osteocalcin, and osteopontin were markedly inhibited by Wnt-3a, as were other known target genes of Wnt-3a, such as Brachyury, FGF-10, and Cdx1. When Wnt-3a-expressing cells were treated with osteogenic supplements in the presence of BMP-2, alkaline phosphatase gene expression and activity were further elevated. Additionally, BMP-2 was able to reverse the inhibitory effect of Wnt-3a on osteocalcin and osteopontin gene expression. These results indicate that while Wnt-3a represses basal expression of some osteogenic genes, this repression can be partially reversed by BMP-2. Finally, the enhanced gene expression of alkaline phosphatase induced by Wnt-3a could be effectively suppressed by the combined action of dexamethasone and 1,25-dihydroxyvitamin D(3). These data show for the first time that Wnt-3a has an unusual effect on multipotential embryonic cells, in that it enhances cellular proliferation and expression of alkaline phosphatase, while it represses most

  8. New frontiers in gene targeting and cloning: success, application and challenges in domestic animals and human embryonic stem cells.

    PubMed

    Denning, Chris; Priddle, Helen

    2003-07-01

    Until recently, precise modification of the animal genome by gene targeting was restricted to the mouse because germline competent embryonic stem cells are not available in any other mammalian species. Nuclear transfer (NT) technology now provides an alternative route for cell-based transgenesis in domestic species, offering new opportunities in genetic modification. Livestock that produce human therapeutic proteins in their milk, have organs suitable for xenotransplantation, or that could provide resistance to diseases such as spongiform encephalopathies have been produced by NT from engineered, cultured somatic cells. However, improvements in the efficiency of somatic cell gene targeting and a greater understanding of the reprogramming events that occur during NT are required for the routine application of what is currently an inefficient process. The ability to reprogramme and genetically manipulate cells will also be crucial for full exploitation of human embryonic stem (hES) cells, which offer unparalleled opportunities in human health and biotechnology. Particularly pertinent are directed differentiation of hES lines to specific cell lineages, production of cells that evade the patient's immune system and ensuring the safety of ensuing transplants. This review will discuss some of the successes, applications and challenges facing gene targeting in livestock and hES cells.

  9. Distinct gene expression responses of two anticonvulsant drugs in a novel human embryonic stem cell based neural differentiation assay protocol.

    PubMed

    Schulpen, Sjors H W; de Jong, Esther; de la Fonteyne, Liset J J; de Klerk, Arja; Piersma, Aldert H

    2015-04-01

    Hazard assessment of chemicals and pharmaceuticals is increasingly gaining from knowledge about molecular mechanisms of toxic action acquired in dedicated in vitro assays. We have developed an efficient human embryonic stem cell neural differentiation test (hESTn) that allows the study of the molecular interaction of compounds with the neural differentiation process. Within the 11-day differentiation protocol of the assay, embryonic stem cells lost their pluripotency, evidenced by the reduced expression of stem cell markers Pou5F1 and Nanog. Moreover, stem cells differentiated into neural cells, with morphologically visible neural structures together with increased expression of neural differentiation-related genes such as βIII-tubulin, Map2, Neurogin1, Mapt and Reelin. Valproic acid (VPA) and carbamazepine (CBZ) exposure during hESTn differentiation led to concentration-dependent reduced expression of βIII-tubulin, Neurogin1 and Reelin. In parallel VPA caused an increased gene expression of Map2 and Mapt which is possibly related to the neural protective effect of VPA. These findings illustrate the added value of gene expression analysis for detecting compound specific effects in hESTn. Our findings were in line with and could explain effects observed in animal studies. This study demonstrates the potential of this assay protocol for mechanistic analysis of specific compound-induced inhibition of human neural cell differentiation.

  10. Generation of KCL018 research grade human embryonic stem cell line carrying a mutation in the DMPK gene

    PubMed Central

    Miere, Cristian; Hewitson, Heema; Devito, Liani; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL018 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the DMPK gene encoding the dystrophia myotonica protein kinase (2200 trinucleotide repeats; 14 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27346000

  11. Generation of KCL027 research grade human embryonic stem cell line carrying a mutation in the HTT gene

    PubMed Central

    Jacquet, Laureen; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Hobbs, Carl; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL027 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (43 trinucleotide repeats; 21 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays. PMID:27345982

  12. Generation of KCL017 research grade human embryonic stem cell line carrying a mutation in VHL gene

    PubMed Central

    Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL017 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting splicing site of the VHL gene encoding von Hippel–Lindau tumor suppressor E3 ubiquitin protein ligase (676 + 3 A > T). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345980

  13. Generation of KCL013 research grade human embryonic stem cell line carrying a mutation in the HTT gene

    PubMed Central

    Jacquet, Laureen; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL013 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (42 trinucleotide repeats; 17 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345987

  14. Generation of KCL016 research grade human embryonic stem cell line carrying a mutation in VHL gene

    PubMed Central

    Miere, Cristian; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL016 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting splicing site of the VHL gene encoding von Hippel–Lindau tumor suppressor E3 ubiquitin protein ligase (676 + 3A > T). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345783

  15. Generation of KCL012 research grade human embryonic stem cell line carrying a mutation in the HTT gene

    PubMed Central

    Jacquet, Laureen; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Hobbs, Carl; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL012 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (46 trinucleotide repeats; 17 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays. PMID:27345979

  16. Generation of KCL028 research grade human embryonic stem cell line carrying a mutation in the HTT gene

    PubMed Central

    Jacquet, Laureen; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Hobbs, Carl; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL028 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (43 trinucleotide repeats; 21 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays. PMID:27345983

  17. Generation of KCL036 research grade human embryonic stem cell line carrying a mutation in the HTT gene

    PubMed Central

    Jacquet, Laureen; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Hobbs, Carl; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL036 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (38 trinucleotide repeats; 14 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays. PMID:27346001

  18. Generation of KCL017 research grade human embryonic stem cell line carrying a mutation in VHL gene.

    PubMed

    Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-03-01

    The KCL017 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting splicing site of the VHL gene encoding von Hippel-Lindau tumor suppressor E3 ubiquitin protein ligase (676+3A>T). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345980

  19. Generation of KCL016 research grade human embryonic stem cell line carrying a mutation in VHL gene.

    PubMed

    Miere, Cristian; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL016 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting splicing site of the VHL gene encoding von Hippel-Lindau tumor suppressor E3 ubiquitin protein ligase (676+3A>T). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345783

  20. A Limited Number of Caenorhabditis Elegans Genes Are Readily Mutable to Dominant, Temperature-Sensitive Maternal-Effect Embryonic Lethality

    PubMed Central

    Mitenko, N. L.; Eisner, J. R.; Swiston, J. R.; Mains, P. E.

    1997-01-01

    Dominant gain-of-function mutations can give unique insights into the study of gene function. In addition, gain-of-function mutations, unlike loss-of-function alleles, are not biased against the identification of genetically redundant loci. To identify novel genetic functions active during Caenorhabditis elegans embryogenesis, we have collected a set of dominant temperature-sensitive maternal-effect embryonic lethal mutations. In a previous screen, we isolated eight such mutations, distributed among six genes. In the present study, we describe eight new dominant mutations that identify only three additional genes, yielding a total of 16 dominant mutations found in nine genes. Therefore, it appears that a limited number of C. elegans genes mutate to this phenotype at appreciable frequencies. Five of the genes that we identified by dominant mutations have loss-of-function alleles. Two of these genes may lack loss-of-function phenotypes, indicating that they are nonessential and so may represent redundant loci. Loss-of-function mutations of three other genes are associated with recessive lethality, indicating nonredundancy. PMID:9409829

  1. Identification of Estrogen Target Genes during Zebrafish Embryonic Development through Transcriptomic Analysis

    EPA Science Inventory

    Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio) as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 μM 17β-estradiol (E2) or vehicle from 3 hours to 4 days post...

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

    PubMed Central

    Boosalis, Michael S.; Sangerman, Jose I.; White, Gary L.; Wolf, Roman F.; Shen, Ling; Dai, Yan; White, Emily; Makala, Levi H.; Li, Biaoru; Pace, Betty S.; Nouraie, Mehdi; Faller, Douglas V.; Perrine, Susan P.

    2015-01-01

    High-level fetal (γ) globin expression ameliorates clinical severity of the beta (β) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies. PMID:26713848

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

    PubMed

    Boosalis, Michael S; Sangerman, Jose I; White, Gary L; Wolf, Roman F; Shen, Ling; Dai, Yan; White, Emily; Makala, Levi H; Li, Biaoru; Pace, Betty S; Nouraie, Mehdi; Faller, Douglas V; Perrine, Susan P

    2015-01-01

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

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

    PubMed Central

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

    2001-01-01

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

  5. Complete TCR-α gene locus control region activity in T cells derived in vitro from embryonic stem cells.

    PubMed

    Lahiji, Armin; Kucerová-Levisohn, Martina; Lovett, Jordana; Holmes, Roxanne; Zúñiga-Pflücker, Juan Carlos; Ortiz, Benjamin D

    2013-07-01

    Locus control regions (LCRs) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin's gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage-expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. In this study, we report the manifestation of all key features of mouse TCR-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells. High-level, copy number-related TCR-α LCR-linked reporter gene expression levels are cell type restricted in this system, and upregulated during the expected stage transition of T cell development. We also report that de novo introduction of TCR-α LCR-linked transgenes into existing T cell lines yields incomplete LCR activity. These data indicate that establishing full TCR-α LCR activity requires critical molecular events occurring prior to final T lineage determination. This study also validates a novel, tractable, and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering.

  6. An optimized gene set for transcriptomics based neurodevelopmental toxicity prediction in the neural embryonic stem cell test.

    PubMed

    Pennings, Jeroen L A; Theunissen, Peter T; Piersma, Aldert H

    2012-10-28

    The murine neural embryonic stem cell test (ESTn) is an in vitro model for neurodevelopmental toxicity testing. Recent studies have shown that application of transcriptomics analyses in the ESTn is useful for obtaining more accurate predictions as well as mechanistic insights. Gene expression responses due to stem cell neural differentiation versus toxicant exposure could be distinguished using the Principal Component Analysis based differentiation track algorithm. In this study, we performed a de novo analysis on combined raw data (10 compounds, 19 exposures) from three previous transcriptomics studies to identify an optimized gene set for neurodevelopmental toxicity prediction in the ESTn. By evaluating predictions of 200,000 randomly selected gene sets, we identified genes which significantly contributed to the prediction reliability. A set of 100 genes was obtained, predominantly involved in (neural) development. Further stringency restrictions resulted in a set of 29 genes that allowed for 84% prediction accuracy (area under the curve 94%). We anticipate these gene sets will contribute to further improve ESTn transcriptomics studies aimed at compound risk assessment.

  7. Multiple elements in human beta-globin locus control region 5' HS 2 are involved in enhancer activity and position-independent, transgene expression.

    PubMed Central

    Caterina, J J; Ciavatta, D J; Donze, D; Behringer, R R; Townes, T M

    1994-01-01

    The human beta-globin Locus Control Region (LCR) has two important activities. First, the LCR opens a 200 kb chromosomal domain containing the human epsilon-, gamma- and beta-globin genes and, secondly, these sequences function as a powerful enhancer of epsilon-, gamma- and beta-globin gene expression. Erythroid-specific, DNase I hypersensitive sites (HS) mark sequences that are critical for LCR activity. Previous experiments demonstrated that a 1.9 kb fragment containing the 5' HS 2 site confers position-independent expression in transgenic mice and enhances human beta-globin gene expression 100-fold. Further analysis of this region demonstrates that multiple sequences are required for maximal enhancer activity; deletion of SP1, NF-E2, GATA-1 or USF binding sites significantly decrease beta-globin gene expression. In contrast, no single site is required for position-independent transgene expression; all mice with site-specific mutations in 5' HS 2 express human beta-globin mRNA regardless of the site of transgene integration. Apparently, multiple combinations of protein binding sites in 5' HS 2 are sufficient to prevent chromosomal position effects that inhibit transgene expression. PMID:8152905

  8. Paternal poly (ADP-ribose) metabolism modulates retention of inheritable sperm histones and early embryonic gene expression.

    PubMed

    Ihara, Motomasa; Meyer-Ficca, Mirella L; Leu, N Adrian; Rao, Shilpa; Li, Fan; Gregory, Brian D; Zalenskaya, Irina A; Schultz, Richard M; Meyer, Ralph G

    2014-05-01

    To achieve the extreme nuclear condensation necessary for sperm function, most histones are replaced with protamines during spermiogenesis in mammals. Mature sperm retain only a small fraction of nucleosomes, which are, in part, enriched on gene regulatory sequences, and recent findings suggest that these retained histones provide epigenetic information that regulates expression of a subset of genes involved in embryo development after fertilization. We addressed this tantalizing hypothesis by analyzing two mouse models exhibiting abnormal histone positioning in mature sperm due to impaired poly(ADP-ribose) (PAR) metabolism during spermiogenesis and identified altered sperm histone retention in specific gene loci genome-wide using MNase digestion-based enrichment of mononucleosomal DNA. We then set out to determine the extent to which expression of these genes was altered in embryos generated with these sperm. For control sperm, most genes showed some degree of histone association, unexpectedly suggesting that histone retention in sperm genes is not an all-or-none phenomenon and that a small number of histones may remain associated with genes throughout the genome. The amount of retained histones, however, was altered in many loci when PAR metabolism was impaired. To ascertain whether sperm histone association and embryonic gene expression are linked, the transcriptome of individual 2-cell embryos derived from such sperm was determined using microarrays and RNA sequencing. Strikingly, a moderate but statistically significant portion of the genes that were differentially expressed in these embryos also showed different histone retention in the corresponding gene loci in sperm of their fathers. These findings provide new evidence for the existence of a linkage between sperm histone retention and gene expression in the embryo.

  9. Compensatory embryonic response to allele-specific inactivation of the murine X-linked gene Hcfc1.

    PubMed

    Minocha, Shilpi; Sung, Tzu-Ling; Villeneuve, Dominic; Lammers, Fabienne; Herr, Winship

    2016-04-01

    Early in female mammalian embryonic development, cells randomly inactivate one of the two X chromosomes to achieve overall equal inactivation of parental X-linked alleles. Hcfc1 is a highly conserved X-linked mouse gene that encodes HCF-1 - a transcriptional co-regulator implicated in cell proliferation in tissue culture cells. By generating a Cre-recombinase inducible Hcfc1 knock-out (Hcfc1(lox)) allele in mice, we have probed the role of HCF-1 in actively proliferating embryonic cells and in cell-cycle re-entry of resting differentiated adult cells using a liver regeneration model. HCF-1 function is required for both extraembryonic and embryonic development. In heterozygous Hcfc1(lox/+) female embryos, however, embryonic epiblast-specific Cre-induced Hcfc1 deletion (creating an Hcfc1(epiKO) allele) around E5.5 is well tolerated; it leads to a mixture of HCF-1-positive and -negative epiblast cells owing to random X-chromosome inactivation of the wild-type or Hcfc1(epiKO) mutant allele. At E6.5 and E7.5, both HCF-1-positive and -negative epiblast cells proliferate, but gradually by E8.5, HCF-1-negative cells disappear owing to cell-cycle exit and apoptosis. Although generating a temporary developmental retardation, the loss of HCF-1-negative cells is tolerated, leading to viable heterozygous offspring with 100% skewed inactivation of the X-linked Hcfc1(epiKO) allele. In resting adult liver cells, the requirement for HCF-1 in cell proliferation was more evident as hepatocytes lacking HCF-1 fail to re-enter the cell cycle and thus to proliferate during liver regeneration. The survival of the heterozygous Hcfc1(epiKO/+) female embryos, even with half the cells genetically compromised, illustrates the developmental plasticity of the post-implantation mouse embryo - in this instance, permitting survival of females heterozygous for an X-linked embryonic lethal allele.

  10. Transcriptional Profiling Identifies Location-Specific and Breed-Specific Differentially Expressed Genes in Embryonic Myogenesis in Anas Platyrhynchos

    PubMed Central

    Zhang, Rong-Ping; Liu, He-He; Liu, Jun-Ying; Hu, Ji-Wei; Yan, Xi-Ping; Wang, Ding-Min-Cheng; Li, Liang; Wang, Ji-Wen

    2015-01-01

    Skeletal muscle growth and development are highly orchestrated processes involving significant changes in gene expressions. Differences in the location-specific and breed-specific genes and pathways involved have important implications for meat productions and meat quality. Here, RNA-Seq was performed to identify differences in the muscle deposition between two muscle locations and two duck breeds for functional genomics studies. To achieve those goals, skeletal muscle samples were collected from the leg muscle (LM) and the pectoral muscle (PM) of two genetically different duck breeds, Heiwu duck (H) and Peking duck (P), at embryonic 15 days. Functional genomics studies were performed in two experiments: Experiment 1 directly compared the location-specific genes between PM and LM, and Experiment 2 compared the two breeds (H and P) at the same developmental stage (embryonic 15 days). Almost 13 million clean reads were generated using Illumina technology (Novogene, Beijing, China) on each library, and more than 70% of the reads mapped to the Peking duck (Anas platyrhynchos) genome. A total of 168 genes were differentially expressed between the two locations analyzed in Experiment 1, whereas only 8 genes were differentially expressed when comparing the same location between two breeds in Experiment 2. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) were used to functionally annotate DEGs (differentially expression genes). The DEGs identified in Experiment 1 were mainly involved in focal adhesion, the PI3K-Akt signaling pathway and ECM-receptor interaction pathways (corrected P-value<0.05). In Experiment 2, the DEGs were associated with only the ribosome signaling pathway (corrected P-value<0.05). In addition, quantitative real-time PCR was used to confirm 15 of the differentially expressed genes originally detected by RNA-Seq. A comparative transcript analysis of the leg and pectoral muscles of two duck breeds not only improves our

  11. Maternal Experience with Predation Risk Influences Genome-Wide Embryonic Gene Expression in Threespined Sticklebacks (Gasterosteus aculeatus)

    PubMed Central

    Mommer, Brett C.; Bell, Alison M.

    2014-01-01

    There is growing evidence for nongenetic effects of maternal experience on offspring. For example, previous studies have shown that female threespined stickleback fish (Gasterosteus aculeatus) exposed to predation risk produce offspring with altered behavior, metabolism and stress physiology. Here, we investigate the effect of maternal exposure to predation risk on the embryonic transcriptome in sticklebacks. Using RNA-sequencing we compared genome-wide transcription in three day post-fertilization embryos of predator-exposed and control mothers. There were hundreds of differentially expressed transcripts between embryos of predator-exposed mothers and embryos of control mothers including several non-coding RNAs. Gene Ontology analysis revealed biological pathways involved in metabolism, epigenetic inheritance, and neural proliferation and differentiation that differed between treatments. Interestingly, predation risk is associated with an accelerated life history in many vertebrates, and several of the genes and biological pathways that were identified in this study suggest that maternal exposure to predation risk accelerates the timing of embryonic development. Consistent with this hypothesis, embryos of predator-exposed mothers were larger than embryos of control mothers. These findings point to some of the molecular mechanisms that might underlie maternal effects. PMID:24887438

  12. Novel Genes Implicated in Embryonal, Alveolar, and Pleomorphic Rhabdomyosarcoma: A Cytogenetic and Molecular Analysis of Primary Tumors1

    PubMed Central

    Goldstein, Myriam; Meller, Isaac; Issakov, Josephine; Orr-Urtreger, Avi

    2006-01-01

    Abstract Rhabdomyosarcoma, the most common pediatric soft tissue sarcoma, likely results from deregulation of the skeletal myogenesis program. Although associations between PAX3, PAX7, FOXO1A, and RMS tumorigenesis are well recognized, the entire spectrum of genetic factors underlying RMS development and progression is unclear. Using a combined approach of spectral karyotyping, array-based comparative genomic hybridization (CGH), and expression analysis, we examined 10 primary RMS tumors, including embryonal, alveolar, and the rare adult pleomorphic variant, to explore the involvement of different genes and genetic pathways in RMS tumorigenesis. A complete karyotype established for each tumor revealed a high aneuploidy level, mostly tetraploidy, with double minutes and additional structural aberrations. Quantitative expression analysis detected the overexpression of the AURKA gene in all tumors tested, suggesting a role for this mitotic regulator in the aneuploidy and chromosomal instability observed in RMS. Array-based CGH analysis in primary RMS tumors detected copy number changes of genes involved in multiple genetic pathways, including transcription factors such as MYC-related gene from lung cancer and the cytoskeleton and cell adhesion-encoding genes laminin γ-2 and p21-activated kinase-1. Our data suggest the involvement of genes encoding cell adhesion, cytoskeletal signaling, and transcriptional and cell cycle components in RMS tumorigenesis. PMID:16790082

  13. An epigenetic switch regulates de novo DNA methylation at a subset of pluripotency gene enhancers during embryonic stem cell differentiation

    PubMed Central

    Petell, Christopher J.; Alabdi, Lama; He, Ming; San Miguel, Phillip; Rose, Richard; Gowher, Humaira

    2016-01-01

    Coordinated regulation of gene expression that involves activation of lineage specific genes and repression of pluripotency genes drives differentiation of embryonic stem cells (ESC). For complete repression of pluripotency genes during ESC differentiation, chromatin at their enhancers is silenced by the activity of the Lsd1-Mi2/NuRD complex. The mechanism/s that regulate DNA methylation at these enhancers are largely unknown. Here, we investigated the affect of the Lsd1-Mi2/NuRD complex on the dynamic regulatory switch that induces the local interaction of histone tails with the Dnmt3 ATRX-DNMT3-DNMT3L (ADD) domain, thus promoting DNA methylation at the enhancers of a subset of pluripotency genes. This is supported by previous structural studies showing a specific interaction between Dnmt3-ADD domain with H3K4 unmethylated histone tails that is disrupted by histone H3K4 methylation and histone acetylation. Our data suggest that Dnmt3a activity is triggered by Lsd1-Mi2/NuRD-mediated histone deacetylation and demethylation at these pluripotency gene enhancers when they are inactivated during mouse ESC differentiation. Using Dnmt3 knockout ESCs and the inhibitors of Lsd1 and p300 histone modifying enzymes during differentiation of E14Tg2A and ZHBTc4 ESCs, our study systematically reveals this mechanism and establishes that Dnmt3a is both reader and effector of the epigenetic state at these target sites. PMID:27179026

  14. Detecting the developmental toxicity of bFGF in the embryonic stem cell test using differential gene expression of differentiation-related genes.

    PubMed

    Deng, Shu-Qin; Xu, Hua; He, Qing; Jiang, Hai-Xiang; Su, Ben-Jin; Zhang, Qi-Hao

    2014-06-01

    Basic fibroblast growth factor (bFGF) is a mitogenic cytokine that can stimulate mesoderm-and neuroectoderm-originated cell proliferation. This study was performed to investigate the effects of bFGF on cell differentiation and the expression of specific markers at different embryonic developmental stages. We firstly evaluated the embryotoxic potential of bFGF in vitro using a modified EST protocol. Sequentially, we further investigated how bFGF impact the different tissue-special genes and proteins expressions during the differentiation of murine ES cells in vitro and attempt to reveal the effects of bFGF on differentiation processes. This analysis was focused on key tissue- and stage-specific genes involved in ectodermal, mesodermal, and endodermal differentiation, including ectodermal-specific gene Nestin, Oligo2 and Syn, mesodermal-specific gene MHC and MyoD, and endodermal-specific gene GATA6, TTR and ALB, as well as undifferentiated gene Sox-2 and Oct-4. The results demonstrate that bFGF could promote expression of ectodermal-specific genes and protein, but suppress the expressions of endoderm-specific and some mesoderm-specific gene and protein. A conclusion can be drawn that bFGF exhibits weak embryotoxicity and mainly promotes ES cell differentiation towards the ectodermal lineages but suppress differentiation into endoderm lineages. These opposing effects of bFGF on the embryonic development of the three germ layers may be related to its weak embryotoxic potential. More specifically, inhibition of expression of the endodermal-specific markers transthyretin (TTR), and albumin (ALB) by bFGF may be of more value in detecting the embryotoxic potential of bFGF.

  15. Generation of KCL029 research grade human embryonic stem cell line carrying a mutation in WAS gene

    PubMed Central

    Miere, Cristian; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL029 human embryonic stem cell line was derived from an embryo donated for research that carried a c.814 T > C mutation in the WAS gene, which is linked to the Wiskott-Aldrich syndrome, a rare, inherited, X-linked, recessive disease characterized by immune dysregulation and microthrombocytopenia. The line is also carrier for a mutation p.N1152H in the gene encoding the cystic fibrosis transmembrane conductance regulator CFTR. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345811

  16. Generation of KCL029 research grade human embryonic stem cell line carrying a mutation in WAS gene.

    PubMed

    Miere, Cristian; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL029 human embryonic stem cell line was derived from an embryo donated for research that carried a c.814T>C mutation in the WAS gene, which is linked to the Wiskott-Aldrich syndrome, a rare, inherited, X-linked, recessive disease characterized by immune dysregulation and microthrombocytopenia. The line is also carrier for a mutation p.N1152H in the gene encoding the cystic fibrosis transmembrane conductance regulator CFTR. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345811

  17. High-Sensitivity Mass Spectrometry for Probing Gene Translation in Single Embryonic Cells in the Early Frog (Xenopus) Embryo

    PubMed Central

    Lombard-Banek, Camille; Moody, Sally A.; Nemes, Peter

    2016-01-01

    Direct measurement of protein expression with single-cell resolution promises to deepen the understanding of the basic molecular processes during normal and impaired development. High-resolution mass spectrometry provides detailed coverage of the proteomic composition of large numbers of cells. Here we discuss recent mass spectrometry developments based on single-cell capillary electrophoresis that extend discovery proteomics to sufficient sensitivity to enable the measurement of proteins in single cells. The single-cell mass spectrometry system is used to detect a large number of proteins in single embryonic cells in the 16-cell embryo of the South African clawed frog (Xenopus laevis) that give rise to distinct tissue types. Single-cell measurements of protein expression provide complementary information on gene transcription during early development of the vertebrate embryo, raising a potential to understand how differential gene expression coordinates normal cell heterogeneity during development. PMID:27761436

  18. Embryonic Stem Cell-Derived Microvesicles Induce Gene Expression Changes in Müller Cells of the Retina

    PubMed Central

    Katsman, Diana; Stackpole, Emma J.; Domin, Daniel R.; Farber, Debora B.

    2012-01-01

    Cell-derived microvesicles (MVs), recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs) have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC) mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation. PMID:23226281

  19. Gene Expression Profiling of Shoot-Derived Calli from Adult Radiata Pine and Zygotic Embryo-Derived Embryonal Masses

    PubMed Central

    Garcia-Mendiguren, O.; Montalbán, I. A.; Stewart, D.; Moncaleán, P.; Klimaszewska, K.; Rutledge, R. G.

    2015-01-01

    Background Although somatic embryogenesis has an unprecedented potential for large-scale clonal propagation of conifers, the ability to efficiently induce the embryonal cultures required for somatic embryo production has long been a challenge. Furthermore, because early stage zygotic embryos remain the only responsive explants for pines, it is not possible to clone individual trees from vegetative explants at a commercial scale. This is of particular interest for adult trees because many elite characteristics only become apparent following sexual maturation. Findings Shoot explants collected from adult radiata pine trees were cultured on four induction media differing in plant growth regulator composition, either directly after collection or from in vitro-generated axillary shoots. Six callus lines were selected for microscopic examination, which failed to reveal any embryonal masses (EM). qPCR expression profiling of five of these lines indicated that explant type influenced the absolute level of gene expression, but not the type of genes that were expressed. The analysis, which also included three EM lines induced from immature zygotic embryos, encompassed five categories of genes reflective of metabolic, mitotic and meristematic activity, along with putative markers of embryogenicity. Culture medium was found to have no significant impact on gene expression, although differences specific to the explant’s origin were apparent. Expression of transcriptional factors associated with vegetative meristems further suggested that all of the callus lines possessed a substantive vegetative character. Most notable, however, was that they all also expressed a putative embryogenic marker (LEC1). Conclusions While limited in scope, these results illustrate the utility of expression profiling for characterizing tissues in culture. For example, although the biological significance of LEC1 expression is unclear, it does present the possibility that these callus lines possess

  20. Early embryonic death-associated changes in genome-wide gene expression profiles in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo.

    PubMed

    Oishi, Masahito; Gohma, Hiroshi; Hashizume, Kazuyoshi; Taniguchi, Yukio; Yasue, Hiroshi; Takahashi, Seiya; Yamada, Takahisa; Sasaki, Yoshiyuki

    2006-04-01

    Successful somatic nuclear transfer-derived cloning has been reported in cattle; however, the cloned embryo is highly susceptible to death around day 60 of gestation leading to early embryonic loss. The early embryonic death is postulated to possibly arise in part from an atypical placentation. We have performed cDNA macroarray analysis using 3,353 of the previously cataloged 4,165 genes, in order to characterize the early embryonic death-associated changes in genome-wide gene expression profiles in the fetal placenta of the cow carrying somatic nuclear transfer-derived cloned embryo. A more marked difference in the expression profiles was observed between the fetal placentas of the cows with the cloned immotile embryo (CD) and with the cloned motile embryo (CL) or artificial insemination-derived motile embryo (AI), as compared to between the CL and AI placentas, suggesting an aberration of the expression profile in the CD placenta among the three placentas. Further, 291 and 77 genes showed more than twofold elevation and less than 50% reduction, respectively, in either or both of two CD (CD1 and CD2) placentas in comparison with the CL placenta, but no differential expression between the CL and AI placentas. The expression patterns of six genes in the AI, CL, and CD placentas were confirmed in an experiment with an additional sample for each of the three placentas. Among the placental genes showing the early embryonic death-associated changes of expression in the cow with the cloned embryo, IGF2 (elevated gene), and HBA1, HBA2, SPTB, and SPTBN1 genes (reduced gene) are intriguing in that the changes of expression in these genes were observed in an additional sample of CD placenta as well as the CD1 and CD2 placentas, and in that overexpression (for IGF2) and dysfunction or deficiency (for HBA1, HBA2, SPTB, and SPTBN1) result in embryonic lethality.

  1. 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

  2. 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.

  3. Embryonic Lethality in Homozygous Human Her-2 Transgenic Mice Due to Disruption of the Pds5b Gene

    PubMed Central

    Yong, Carmen S. M.; Sharkey, Janelle; Duscio, Belinda; Venville, Ben; Wei, Wei-Zen; Jones, Richard F.; Slaney, Clare Y.; Mir Arnau, Gisela; Papenfuss, Anthony T.

    2015-01-01

    The development of antigen-targeted therapeutics is dependent on the preferential expression of tumor-associated antigens (TAA) at targetable levels on the tumor. Tumor-associated antigens can be generated de novo or can arise from altered expression of normal basal proteins, such as the up-regulation of human epidermal growth factor receptor 2 (Her2/ErbB2). To properly assess the development of Her2 therapeutics in an immune tolerant model, we previously generated a transgenic mouse model in which expression of the human Her2 protein was present in both the brain and mammary tissue. This mouse model has facilitated the development of Her2 targeted therapies in a clinically relevant and suitable model. While heterozygous Her2+/- mice appear to develop in a similar manner to wild type mice (Her2-/-), it has proven difficult to generate homozygous Her2+/+ mice, potentially due to embryonic lethality. In this study, we performed whole genome sequencing to determine if the integration site of the Her2 transgene was responsible for this lethality. Indeed, we report that the Her2 transgene had integrated into the Pds5b (precocious dissociation of sisters) gene on chromosome 5, as a 162 copy concatemer. Furthermore, our findings demonstrate that Her2+/+ mice, similar to Pds5b-/- mice, are embryonic lethal and confirm the necessity for Pds5b in embryonic development. This study confirms the value of whole genome sequencing in determining the integration site of transgenes to gain insight into associated phenotypes. PMID:26334628

  4. P-Element Insertion Alleles of Essential Genes on the Third Chromosome of Drosophila Melanogaster: Mutations Affecting Embryonic Pns Development

    PubMed Central

    Salzberg, A.; Prokopenko, S. N.; He, Y.; Tsai, P.; Pal, M.; Maroy, P.; Glover, D. M.; Deak, P.; Bellen, H. J.

    1997-01-01

    To identify novel genes and to isolate tagged mutations in known genes that are required for the development of the peripheral nervous system (PNS), we have screened a novel collection of 2460 strains carrying lethal or semilethal P-element insertions on the third chromosome. Monoclonal antibody 22C10 was used as a marker to visualize the embryonic PNS. We identified 109 mutant strains that exhibited reproducible phenotypes in the PNS. Cytological and genetic analyses of these strains indicated that 87 mutations affect previously identified genes: tramtrack (n = 18 alleles), string (n = 15), cyclin A (n = 13), single-minded (n = 13), Delta (n = 9), neuralized (n = 4), pointed (n = 4), extra macrochaetae (n = 4), prospero (n = 3), tartan (n = 2), and pebble (n = 2). In addition, 13 mutations affect genes that we identified recently in a chemical mutagenesis screen designed to isolate similar mutants: hearty (n = 3), dorsotonals (n = 2), pavarotti (n = 2), sanpodo (n = 2), dalmatian (n = 1), missensed (n = 1), senseless (n = 1), and sticky ch1 (n = 1). The remaining nine mutations define seven novel complementation groups. The data presented here demonstrate that this collection of P elements will be useful for the identification and cloning of novel genes on the third chromosome, since >70% of mutations identified in the screen are caused by the insertion of a P element. A comparison between this screen and a chemical mutagenesis screen undertaken earlier highlights the complementarity of the two types of genetic screens. PMID:9409832

  5. Gene expression classification using epigenetic features and DNA sequence composition in the human embryonic stem cell line H1.

    PubMed

    Su, Wen-Xia; Li, Qian-Zhong; Zhang, Lu-Qiang; Fan, Guo-Liang; Wu, Cheng-Yan; Yan, Zhen-He; Zuo, Yong-Chun

    2016-10-30

    Epigenetic factors are known to correlate with gene expression in the existing studies. However, quantitative models that accurately classify the highly and lowly expressed genes based on epigenetic factors are currently lacking. In this study, a new machine learning method combines histone modifications, DNA methylation, DNA accessibility, transcription factors, and trinucleotide composition with support vector machines (SVM) is developed in the context of human embryonic stem cell line (H1). The results indicate that the predictive accuracy will be markedly improved when the epigenetic features are considered. The predictive accuracy and Matthews correlation coefficient of the best model are as high as 95.96% and 0.92 for 10-fold cross-validation test, and 95.58% and 0.92 for independent dataset test, respectively. Our model provides a good way to judge a gene is either highly or lowly expressed gene by using genetic and epigenetic data, when the expression data of the gene is lacking. And a web-server GECES for our analysis method is established at http://202.207.14.87:8032/fuwu/GECES/index.asp, so that other scientists can easily get their desired results by our web-server, without going through the mathematical details.

  6. Gene expression classification using epigenetic features and DNA sequence composition in the human embryonic stem cell line H1.

    PubMed

    Su, Wen-Xia; Li, Qian-Zhong; Zhang, Lu-Qiang; Fan, Guo-Liang; Wu, Cheng-Yan; Yan, Zhen-He; Zuo, Yong-Chun

    2016-10-30

    Epigenetic factors are known to correlate with gene expression in the existing studies. However, quantitative models that accurately classify the highly and lowly expressed genes based on epigenetic factors are currently lacking. In this study, a new machine learning method combines histone modifications, DNA methylation, DNA accessibility, transcription factors, and trinucleotide composition with support vector machines (SVM) is developed in the context of human embryonic stem cell line (H1). The results indicate that the predictive accuracy will be markedly improved when the epigenetic features are considered. The predictive accuracy and Matthews correlation coefficient of the best model are as high as 95.96% and 0.92 for 10-fold cross-validation test, and 95.58% and 0.92 for independent dataset test, respectively. Our model provides a good way to judge a gene is either highly or lowly expressed gene by using genetic and epigenetic data, when the expression data of the gene is lacking. And a web-server GECES for our analysis method is established at http://202.207.14.87:8032/fuwu/GECES/index.asp, so that other scientists can easily get their desired results by our web-server, without going through the mathematical details. PMID:27468948

  7. Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment.

    PubMed

    Sekinaka, Tamotsu; Hayashi, Yohei; Noce, Toshiaki; Niwa, Hitoshi; Matsui, Yasuhisa

    2016-09-09

    Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs.

  8. PDX1 binds and represses hepatic genes to ensure robust pancreatic commitment in differentiating human embryonic stem cells.

    PubMed

    Teo, Adrian Kee Keong; Tsuneyoshi, Norihiro; Hoon, Shawn; Tan, Ee Kim; Stanton, Lawrence W; Wright, Christopher V E; Dunn, N Ray

    2015-04-14

    Inactivation of the Pancreatic and Duodenal Homeobox 1 (PDX1) gene causes pancreatic agenesis, which places PDX1 high atop the regulatory network controlling development of this indispensable organ. However, little is known about the identity of PDX1 transcriptional targets. We simulated pancreatic development by differentiating human embryonic stem cells (hESCs) into early pancreatic progenitors and subjected this cell population to PDX1 chromatin immunoprecipitation sequencing (ChIP-seq). We identified more than 350 genes bound by PDX1, whose expression was upregulated on day 17 of differentiation. This group included known PDX1 targets and many genes not previously linked to pancreatic development. ChIP-seq also revealed PDX1 occupancy at hepatic genes. We hypothesized that simultaneous PDX1-driven activation of pancreatic and repression of hepatic programs underlie early divergence between pancreas and liver. In HepG2 cells and differentiating hESCs, we found that PDX1 binds and suppresses expression of endogenous liver genes. These findings rebrand PDX1 as a context-dependent transcriptional repressor and activator within the same cell type. PMID:25843046

  9. Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment

    PubMed Central

    Sekinaka, Tamotsu; Hayashi, Yohei; Noce, Toshiaki; Niwa, Hitoshi; Matsui, Yasuhisa

    2016-01-01

    Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs. PMID:27608931

  10. Thin-film assembly of diethanolamine-based lipidic material as potential gene carrier in mouse embryonic neural stem cells.

    PubMed

    Kusumoto, Ken-Ichi; Yamashita, Satoko; Nagata, Takahiro; Ido, Takeshi; Hamachi, Itaru; Akao, Tetsuyuki

    2009-10-01

    Understanding of lipidic materials used for gene delivery system is essential for the effective design and development of potential applications in basic and therapeutic research. This study aimed to evaluate the biological activity of totally synthesized ditetradecylacetyldiethanolaminetrimethylammonium (TMA-C2-DEA-C14) as gene carriers for neural stem cells. The transfer abilities were estimated by expressing green fluorescent protein (GFP) in mouse embryonic neural stem cells. Here, we demonstrate that lipidic assembly of TMA-C2-DEA-C14, which was self-organized by incubation in water for a month at 25 degrees C, can provide an efficient gene delivery with low cytotoxicity ( approximately 40% of GFP-expressed neural stem cells). However, when dispersed by ultrasonication, TMA-C2-DEA-C14 showed low effect ( approximately 4%). Moreover, electron microscopic analysis showed that TMA-C2-DEA-C14 assembly is characterized by thin-film structures with polygonal shapes ( approximately 2.7 mum), and after association with DNA, their structures dramatically changes to form liposome complexes that can effectively deliver DNA into the cellular cytoplasm of neural stem cells. Thus, TMA-C2-DEA-C14 assembly identified in this study was determined to have an effective activity as gene carriers for primary neural stem cells. Our findings suggest that this approach can serve as a novel model for the development of lipidic materials on nonviral gene delivery system.

  11. Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment

    NASA Astrophysics Data System (ADS)

    Sekinaka, Tamotsu; Hayashi, Yohei; Noce, Toshiaki; Niwa, Hitoshi; Matsui, Yasuhisa

    2016-09-01

    Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs.

  12. Analysis of RNA Interference Lines Identifies New Functions of Maternally-Expressed Genes Involved in Embryonic Patterning in Drosophila melanogaster.

    PubMed

    Liu, Niankun; Lasko, Paul

    2015-03-31

    Embryonic patterning in Drosophila melanogaster is initially established through the activity of a number of maternally expressed genes that are expressed during oogenesis. mRNAs from some of these genes accumulate in the posterior pole plasm of the oocyte and early embryo and localize further into RNA islands, which are transient ring-like structures that form around the nuclei of future primordial germ cells (pole cells) at stage 3 of embryogenesis. As mRNAs from several genes with known functions in anterior-posterior patterning and/or germ cell specification accumulate in RNA islands, we hypothesized that some other mRNAs that localize in this manner might also function in these developmental processes. To test this, we investigated the developmental functions of 51 genes whose mRNAs accumulate in RNA islands by abrogating their activity in the female germline using RNA interference. This analysis revealed requirements for ttk, pbl, Hip14, eIF5, eIF4G, and CG9977 for progression through early oogenesis. We observed dorsal appendage defects in a proportion of eggs produced by females expressing double-stranded RNA targeting Mkrn1 or jvl, implicating these two genes in dorsal-ventral patterning. In addition, posterior patterning defects and a reduction in pole cell number were seen in the progeny of Mkrn1 females. Because the mammalian ortholog of Mkrn1 acts as an E3 ubiquitin ligase, these results suggest an additional link between protein ubiquitination and pole plasm activity.

  13. Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment.

    PubMed

    Sekinaka, Tamotsu; Hayashi, Yohei; Noce, Toshiaki; Niwa, Hitoshi; Matsui, Yasuhisa

    2016-01-01

    Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs. PMID:27608931

  14. Antagonistic roles of the ERK and p38 MAPK signalling pathways in globin expression, haem biosynthesis and iron uptake.

    PubMed

    Mardini, Louay; Gasiorek, Jadwiga; Derjuga, Anna; Carrière, Lucie; Schranzhofer, Matthias; Paw, Barry H; Ponka, Prem; Blank, Volker

    2010-11-15

    Late-stage erythroid cells synthesize large quantities of haemoglobin, a process requiring the co-ordinated regulation of globin and haem synthesis as well as iron uptake. In the present study, we investigated the role of the ERK (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) signalling pathways in MEL (mouse erythroleukaemia) cell differentiation. We found that treatment of HMBA (hexamethylene bisacetamide)-induced MEL cells with the ERK pathway inhibitor UO126 results in an increase in intracellular haem and haemoglobin levels. The transcript levels of the genes coding for β(major)-globin, the haem biosynthesis enzyme 5-aminolevulinate synthase 2 and the mitochondrial iron transporter mitoferrin 1 are up-regulated. We also showed enhanced expression of globin and transferrin receptor 1 proteins upon UO126 treatment. With respect to iron uptake, we found that ERK inhibitor treatment led to an increase in both haem-bound and total iron. In contrast, treatment of MEL cells with the p38 MAPK pathway inhibitor SB202190 had the opposite effect, resulting in decreased globin expression, haem synthesis and iron uptake. Reporter assays showed that globin promoter and HS2 enhancer-mediated transcription was under the control of MAPKs, as inhibition of the ERK and p38 MAPK pathways led to increased and decreased gene activity respectively. Our present results suggest that the ERK1/2 and p38α/β MAPKs play antagonistic roles in HMBA-induced globin gene expression and erythroid differentiation. These results provide a novel link between MAPK signalling and the regulation of haem biosynthesis and iron uptake in erythroid cells.

  15. Contrasting effects of alpha and beta globin regulatory elements on chromatin structure may be related to their different chromosomal environments.

    PubMed Central

    Craddock, C F; Vyas, P; Sharpe, J A; Ayyub, H; Wood, W G; Higgs, D R

    1995-01-01

    Expression of the human alpha and beta globin gene clusters is regulated by remote sequences, referred to as HS -40 and the beta-locus control region (beta-LCR) that lie 5-40 kb upstream of the genes they activate. Because of their common ancestry, similar organization and coordinate expression it has often been assumed that regulation of the globin gene clusters by HS -40 and the beta-LCR occurs via similar mechanisms. Using interspecific hybrids containing chromosomes with naturally occurring deletions of HS -40 we have shown that, in contrast to the beta-LCR, this element exerts no discernible effect on long-range chromatin structure and in addition does not influence formation of DNase I hypersensitive sites at the alpha globin promoters. These differences in the behaviour of HS -40 and the beta-LCR may reflect their contrasting influence on gene expression in transgenic mice and may result from the differing requirements of these elements in their radically different, natural chromosomal environments; the alpha cluster lying within a region of constitutively 'open' chromatin and the beta cluster in a segment of chromatin which opens in a tissue-specific manner. Differences in the hierarchical control of the alpha and beta globin clusters may exemplify more general differences in the regulation of eukaryotic genes which lie in similar open or closed chromosomal regions. Images PMID:7737123

  16. 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.

  17. 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

  18. A 66-bp deletion in growth hormone releasing hormone gene 5'-flanking region with largemouth bass recessive embryonic lethal.

    PubMed

    Ma, D M; Han, L Q; Bai, J J; Li, S J; Fan, J J; Yu, L Y; Quan, Y C

    2014-06-01

    Growth hormone releasing hormone (GHRH) regulates the secretion of growth hormone (GH) in the pituitary gland. A 66-bp deletion (c.-923_-858del) was detected in the 5'-flanking sequence of the largemouth bass (Micropterus salmoides) GHRH gene. In two cultured random populations of adult individuals (A: n = 170 and B: n = 150), the genotype ratios of +/+:+/- were 2.5:1 and 2.8:1 respectively. Only one -/- fish was detected. A Largemouth bass family was constructed with two heterozygous individuals (+/-) as parents. The genotype ratio of +/+:+/-:-/- in the filial generation embryos was 1:1.6:0.1 at the neurula and 1:2:0 at hatched larvae stages. This indicated that the 66-bp deletion was a recessive lethal site and that homozygous individuals (-/-) died off in embryonic development. The growth traits (body weight, body length and body depth) were measured, and the GHRH mRNA expression levels in brain tissue were detected using real-time PCR. The effects of genotype (+/-) on growth traits and GHRH mRNA expression were not significant. Although the cause of death was not clear, the results hint that the 66-bp deletion site in GHRH 5'-flanking sequence significantly affects the livability in largemouth bass embryonic development. PMID:24697798

  19. A 66-bp deletion in growth hormone releasing hormone gene 5'-flanking region with largemouth bass recessive embryonic lethal.

    PubMed

    Ma, D M; Han, L Q; Bai, J J; Li, S J; Fan, J J; Yu, L Y; Quan, Y C

    2014-06-01

    Growth hormone releasing hormone (GHRH) regulates the secretion of growth hormone (GH) in the pituitary gland. A 66-bp deletion (c.-923_-858del) was detected in the 5'-flanking sequence of the largemouth bass (Micropterus salmoides) GHRH gene. In two cultured random populations of adult individuals (A: n = 170 and B: n = 150), the genotype ratios of +/+:+/- were 2.5:1 and 2.8:1 respectively. Only one -/- fish was detected. A Largemouth bass family was constructed with two heterozygous individuals (+/-) as parents. The genotype ratio of +/+:+/-:-/- in the filial generation embryos was 1:1.6:0.1 at the neurula and 1:2:0 at hatched larvae stages. This indicated that the 66-bp deletion was a recessive lethal site and that homozygous individuals (-/-) died off in embryonic development. The growth traits (body weight, body length and body depth) were measured, and the GHRH mRNA expression levels in brain tissue were detected using real-time PCR. The effects of genotype (+/-) on growth traits and GHRH mRNA expression were not significant. Although the cause of death was not clear, the results hint that the 66-bp deletion site in GHRH 5'-flanking sequence significantly affects the livability in largemouth bass embryonic development.

  20. Brachyury and SMAD signalling collaboratively orchestrate distinct mesoderm and endoderm gene regulatory networks in differentiating human embryonic stem cells.

    PubMed

    Faial, Tiago; Bernardo, Andreia S; Mendjan, Sasha; Diamanti, Evangelia; Ortmann, Daniel; Gentsch, George E; Mascetti, Victoria L; Trotter, Matthew W B; Smith, James C; Pedersen, Roger A

    2015-06-15

    The transcription factor brachyury (T, BRA) is one of the first markers of gastrulation and lineage specification in vertebrates. Despite its wide use and importance in stem cell and developmental biology, its functional genomic targets in human cells are largely unknown. Here, we use differentiating human embryonic stem cells to study the role of BRA in activin A-induced endoderm and BMP4-induced mesoderm progenitors. We show that BRA has distinct genome-wide binding landscapes in these two cell populations, and that BRA interacts and collaborates with SMAD1 or SMAD2/3 signalling to regulate the expression of its target genes in a cell-specific manner. Importantly, by manipulating the levels of BRA in cells exposed to different signalling environments, we demonstrate that BRA is essential for mesoderm but not for endoderm formation. Together, our data illuminate the function of BRA in the context of human embryonic development and show that the regulatory role of BRA is context dependent. Our study reinforces the importance of analysing the functions of a transcription factor in different cellular and signalling environments.

  1. Brachyury and SMAD signalling collaboratively orchestrate distinct mesoderm and endoderm gene regulatory networks in differentiating human embryonic stem cells

    PubMed Central

    Faial, Tiago; Bernardo, Andreia S.; Mendjan, Sasha; Diamanti, Evangelia; Ortmann, Daniel; Gentsch, George E.; Mascetti, Victoria L.; Trotter, Matthew W. B.; Smith, James C.; Pedersen, Roger A.

    2015-01-01

    The transcription factor brachyury (T, BRA) is one of the first markers of gastrulation and lineage specification in vertebrates. Despite its wide use and importance in stem cell and developmental biology, its functional genomic targets in human cells are largely unknown. Here, we use differentiating human embryonic stem cells to study the role of BRA in activin A-induced endoderm and BMP4-induced mesoderm progenitors. We show that BRA has distinct genome-wide binding landscapes in these two cell populations, and that BRA interacts and collaborates with SMAD1 or SMAD2/3 signalling to regulate the expression of its target genes in a cell-specific manner. Importantly, by manipulating the levels of BRA in cells exposed to different signalling environments, we demonstrate that BRA is essential for mesoderm but not for endoderm formation. Together, our data illuminate the function of BRA in the context of human embryonic development and show that the regulatory role of BRA is context dependent. Our study reinforces the importance of analysing the functions of a transcription factor in different cellular and signalling environments. PMID:26015544

  2. Inactivation of HDAC1 or HDAC2 induces gamma globin expression without altering cell cycle or proliferation.

    PubMed

    Esrick, Erica B; McConkey, Marie; Lin, Katherine; Frisbee, Alyse; Ebert, Benjamin L

    2015-07-01

    Other than hydroxyurea, no pharmacologic agents are clinically available for fetal hemoglobin (HbF) induction in sickle cell disease (SCD). An optimal candidate would induce HbF without causing cell cycle inhibition and would act independently of hydroxyurea in order to yield additional HbF induction when combined. We explored whether inhibition of histone deacetylase (HDAC) 1 or HDAC2 could achieve these goals. In human erythroid progenitor cells, shRNA knockdown of the HDAC1 or HDAC2 genes induced gamma globin, without altering cellular proliferation in vitro, and without altering cell cycle phase. Treatment with hydroxyurea in combination with HDAC2 knockdown yielded a further increase in gamma globin expression. Additionally, when CD34+ cells were treated with both hydroxyurea and MS-275 (an inhibitor of HDAC 1, 2, and 3), an additive induction of relative gamma globin expression was achieved. Our findings support further clinical investigation of HDAC inhibitors in combination with hydroxyurea in SCD patients.

  3. Genetics of Hemoglobin in the Deer Mouse, PEROMYSCUS MANICULATUS . I. Multiple α- and β-Globin Structural Loci

    PubMed Central

    Snyder, Lee R. G.

    1978-01-01

    Genetic data, together with molecular structure studies, have demonstrated that the complex hemoglobin phenotypes in four subspecies of P. maniculatus are generated by at least four, and probably five, globin structural loci. The Hba and Hbc loci apparently arose by duplication of an ancestral α-type locus, while Hbb, Hbd, and Hbe apparently were derived from a β-type locus. The Hba and Hbb structural loci are electrophoretically monomorphic, while Hbc, Hbd, and Hbe are each polymorphic for at least two electrophoretic alleles. Alleles at Hbc and Hbd segregate independently. The globin products of Hbc and Hbd are electrophoretically indistinguishable; therefore, it is impossible to enumerate true gene frequencies in population surveys. Combined evidence from Peromyscus, Mus and Rattus indicates a remarkable similarity in the numbers of duplicated globin structural loci and in their linkage relationships to coat color loci. PMID:669255

  4. Muscle Segment Homeobox Genes Direct Embryonic Diapause by Limiting Inflammation in the Uterus.

    PubMed

    Cha, Jeeyeon; Burnum-Johnson, Kristin E; Bartos, Amanda; Li, Yingju; Baker, Erin S; Tilton, Susan C; Webb-Robertson, Bobbie-Jo M; Piehowski, Paul D; Monroe, Matthew E; Jegga, Anil G; Murata, Shigeo; Hirota, Yasushi; Dey, Sudhansu K

    2015-06-12

    Embryonic diapause is a reproductive strategy widespread in the animal kingdom. This phenomenon is defined by a temporary arrest in blastocyst growth and metabolic activity within a quiescent uterus without implantation until the environmental and maternal milieu become favorable for pregnancy to progress. We found that uterine Msx expression persists during diapause across species; their inactivation in the mouse uterus results in termination of diapause with the development of implantation-like responses ("pseudoimplantation") that ultimately succumbed to resorption. To understand the cause of this failure, we compared proteome profiles between floxed and Msx-deleted uteri. In deleted uteri, several functional networks, including transcription/translation, ubiquitin-proteasome, inflammation, and endoplasmic reticulum stress, were dysregulated. Computational modeling predicted intersection of these pathways on an enhanced inflammatory signature. Further studies showed that this signature was reflected in increased phosphorylated IκB levels and nuclear NFκB in deleted uteri. This was associated with enhanced proteasome activity and endoplasmic reticulum stress. Interestingly, treatment with anti-inflammatory glucocorticoid (dexamethasone) reduced the inflammatory signature with improvement of the diapause phenotype. These findings highlight an unexpected role of uterine Msx in limiting aberrant inflammatory responses to maintain embryonic diapause.

  5. The Amotl2 Gene Inhibits Wnt/β-Catenin Signaling and Regulates Embryonic Development in Zebrafish*

    PubMed Central

    Li, Zhiqiang; Wang, Yeqi; Zhang, Min; Xu, Pengfei; Huang, Huizhe; Wu, Di; Meng, Anming

    2012-01-01

    The Motin family proteins can regulate cell polarity, cell mobility, and proliferation during embryonic development by controlling distinct signaling pathways. In this study, we demonstrate that amotl2 knockdown in zebrafish wild-type embryos results in embryonic dorsalization, and this effect can be antagonized by co-knockdown of the dorsal inducer β-catenin2. Overexpression of amotl2 in masterblind (mbl) homozygous embryos, in which canonical Wnt signaling is up-regulated due to an axin1 mutation, transforms eyeless phenotype into smaller eyes, whereas co-knockdown of amot, amotl1, and amotl2 leads to development of smaller eyes in mbl heterozygotes. In cultured mammalian cells, Motin family members all possess the ability to attenuate Wnt/β-catenin signaling. Focusing on Amotl2, we show that Amotl2 can associate with and trap β-catenin in the Rab11-positive recycling endosomes, and as a result, the amount of β-catenin in the cytosol and nucleus is reduced. Thus, our findings provide novel insights into functions of Motin family members and regulation of Wnt/β-catenin signaling. PMID:22362771

  6. Muscle Segment Homeobox Genes Direct Embryonic Diapause by Limiting Inflammation in the Uterus*

    PubMed Central

    Cha, Jeeyeon; Burnum-Johnson, Kristin E.; Bartos, Amanda; Li, Yingju; Baker, Erin S.; Tilton, Susan C.; Webb-Robertson, Bobbie-Jo M.; Piehowski, Paul D.; Monroe, Matthew E.; Jegga, Anil G.; Murata, Shigeo; Hirota, Yasushi; Dey, Sudhansu K.

    2015-01-01

    Embryonic diapause is a reproductive strategy widespread in the animal kingdom. This phenomenon is defined by a temporary arrest in blastocyst growth and metabolic activity within a quiescent uterus without implantation until the environmental and maternal milieu become favorable for pregnancy to progress. We found that uterine Msx expression persists during diapause across species; their inactivation in the mouse uterus results in termination of diapause with the development of implantation-like responses (“pseudoimplantation”) that ultimately succumbed to resorption. To understand the cause of this failure, we compared proteome profiles between floxed and Msx-deleted uteri. In deleted uteri, several functional networks, including transcription/translation, ubiquitin-proteasome, inflammation, and endoplasmic reticulum stress, were dysregulated. Computational modeling predicted intersection of these pathways on an enhanced inflammatory signature. Further studies showed that this signature was reflected in increased phosphorylated IκB levels and nuclear NFκB in deleted uteri. This was associated with enhanced proteasome activity and endoplasmic reticulum stress. Interestingly, treatment with anti-inflammatory glucocorticoid (dexamethasone) reduced the inflammatory signature with improvement of the diapause phenotype. These findings highlight an unexpected role of uterine Msx in limiting aberrant inflammatory responses to maintain embryonic diapause. PMID:25931120

  7. Dose response analysis of monophthalates in the murine embryonic stem cell test assessed by cardiomyocyte differentiation and gene expression.

    PubMed

    Schulpen, Sjors H W; Robinson, Joshua F; Pennings, Jeroen L A; van Dartel, Dorien A M; Piersma, Aldert H

    2013-01-01

    The embryonic stem cell test (EST) is based on compound-induced inhibition of cardiomyocyte differentiation of pluripotent stem cells. We examined the use of transcriptomics to assess concentration-effect relationships and performed potency ranking within a chemical class. Three embryotoxic phthalate monoesters, monobutyl phthalate (MBuP), monobenzyl phthalate (MBzP) and mono-(2-ethylhexyl) phthalate (MEHP) and the non-embryotoxic monomethyl phthalate (MMP) were studied for their effects on gene expression. Effects on gene expression were observed at concentrations that did not inhibit cardiomyocyte differentiation or induce cytotoxicity. The embryotoxic phthalate monoesters altered the expression of 668 commonly expressed genes in a concentration-dependent fashion. The same potency ranking was observed for morphology and gene expression (MEHP>MBzP>MBuP>MMP). These results indicate that integrating transcriptomics provides a sensitive method to measure the dose-dependent effects of phthalate monoester exposure and enables potency ranking based on a common mode of action within a class of compounds. Transcriptomic approaches may improve the applicability of the EST, in terms of sensitivity and specificity.

  8. Generation of gene-targeted mice using embryonic stem cells derived from a transgenic mouse model of Alzheimer's disease.

    PubMed

    Yamamoto, Satoshi; Ooshima, Yuki; Nakata, Mitsugu; Yano, Takashi; Matsuoka, Kunio; Watanabe, Sayuri; Maeda, Ryouta; Takahashi, Hideki; Takeyama, Michiyasu; Matsumoto, Yoshio; Hashimoto, Tadatoshi

    2013-06-01

    Gene-targeting technology using mouse embryonic stem (ES) cells has become the "gold standard" for analyzing gene functions and producing disease models. Recently, genetically modified mice with multiple mutations have increasingly been produced to study the interaction between proteins and polygenic diseases. However, introduction of an additional mutation into mice already harboring several mutations by conventional natural crossbreeding is an extremely time- and labor-intensive process. Moreover, to do so in mice with a complex genetic background, several years may be required if the genetic background is to be retained. Establishing ES cells from multiple-mutant mice, or disease-model mice with a complex genetic background, would offer a possible solution. Here, we report the establishment and characterization of novel ES cell lines from a mouse model of Alzheimer's disease (3xTg-AD mouse, Oddo et al. in Neuron 39:409-421, 2003) harboring 3 mutated genes (APPswe, TauP301L, and PS1M146V) and a complex genetic background. Thirty blastocysts were cultured and 15 stable ES cell lines (male: 11; female: 4) obtained. By injecting these ES cells into diploid or tetraploid blastocysts, we generated germline-competent chimeras. Subsequently, we confirmed that F1 mice derived from these animals showed similar biochemical and behavioral characteristics to the original 3xTg-AD mice. Furthermore, we introduced a gene-targeting vector into the ES cells and successfully obtained gene-targeted ES cells, which were then used to generate knockout mice for the targeted gene. These results suggest that the present methodology is effective for introducing an additional mutation into mice already harboring multiple mutated genes and/or a complex genetic background.

  9. Differential display system with vertebrate-common degenerate oligonucleotide primers: uncovering genes responsive to dioxin in avian embryonic liver.

    PubMed

    Teraoka, Hiroki; Ito, Shino; Ikeda, Haruki; Kubota, Akira; Abou Elmagd, M M; Kitazawa, Takio; Kim, Eun-Young; Iwata, Hisato; Endoh, Daiji

    2012-01-01

    To assess possible impacts of environmental pollutants on gene expression profiles in a variety of organisms, we developed a novel differential display system with primer sets that are common in seven vertebrate species, based on degenerate oligonucleotide-primed PCR (DOP-PCR). An 8-mer inverse repeat motif was found in most transcripts from the seven vertebrates including fish to primates with detailed transcriptome information; more than 10,000 motifs were recognized in common in the transcripts of the seven species. Among them, we selected 275 common motifs that cover about 40-70% of transcripts throughout these species, and designed 275 DOP-PCR primers that were common to seven vertebrate species (common DOP-PCR primers). To detect genes responsive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in developing embryos, differential display with common DOP-PCR primers was applied to embryonic liver of two avian species, the chicken (Gallus gallus) and the common cormorant (Phalacrocorax carbo), which were exposed in ovo to TCDD. The cDNA bands that showed differences between the control and TCDD-treated groups were sequenced and the mRNA expression levels were confirmed by real-time RT-PCR. This approach succeeded in isolating novel dioxin-responsive genes that include 10 coding genes in the chicken, and 1 coding gene and 1 unknown transcript in the cormorant, together with cytochrome P450 1As that have already been well established as dioxin markers. These results highlighted the usefulness of systematically designed novel differential display systems to search genes responsive to chemicals in vertebrates, including wild species, for which transcriptome information is not available. PMID:21786751

  10. Expression of the mouse corticotropin-releasing hormone gene in vivo and targeted inactivation in embryonic stem cells.

    PubMed Central

    Muglia, L J; Jenkins, N A; Gilbert, D J; Copeland, N G; Majzoub, J A

    1994-01-01

    Corticotropin-releasing hormone (CRH), one of the primary regulators of the hypothalamic-pituitary-adrenal (HPA) axis, exhibits abnormal regulation in pathologic states such as depression and anorexia nervosa. Analysis of the role of CRH in regulation of the HPA axis would be facilitated by the creation of animal models in which CRH gene structure and function could be manipulated. We have determined the DNA sequence of the mouse CRH gene. Using a highly sensitive reverse transcription-polymerase chain reaction method, we have found expression of CRH mRNA in adrenal, ovary, testis, gut, heart, anterior pituitary, lung, and spleen, in addition to cerebral cortex and hypothalamus. Within the spleen, CRH mRNA is localized specifically to T-lymphocytes. We mapped the chromosomal location of mouse CRH via interspecific mouse backcrosses to chromosome 3, which is not the site of any naturally occurring mutations consistent with CRH deficiency. Because of this, we inactivated a CRH allele in mouse embryonic stem (ES) cells by homologous recombination with a mutant mouse CRH gene lacking the entire coding region of preproCRH. Mice chimeric for each of two ES clones with an inactivated CRH allele are being used to generate animals with complete CRH deficiency. Images PMID:8182138

  11. Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells.

    PubMed

    Yeo, Seungeun; Jeong, Sangkyun; Kim, Janghwan; Han, Jee-Soo; Han, Yong-Mahn; Kang, Yong-Kook

    2007-08-01

    Pluripotent human embryonic stem cells (hESCs) have the distinguishing feature of innate capacity to allow indefinite self-renewal. This attribute continues until specific constraints or restrictions, such as DNA methylation, are imposed on the genome, usually accompanied by differentiation. With the aim of utilizing DNA methylation as a sign of early differentiation, we probed the genomic regions of hESCs, particularly focusing on stem cell marker (SCM) genes to identify regulatory sequences that display differentiation-sensitive alterations in DNA methylation. We show that the promoter regions of OCT4 and NANOG, but not SOX2, REX1 and FOXD3, undergo significant methylation during hESCs differentiation in which SCM genes are substantially repressed. Thus, following exposure to differentiation stimuli, OCT4 and NANOG gene loci are modified relatively rapidly by DNA methylation. Accordingly, we propose that the DNA methylation states of OCT4 and NANOG sequences may be utilized as barometers to determine the extent of hESC differentiation.

  12. Neuronal developmental gene and miRNA signatures induced by histone deacetylase inhibitors in human embryonic stem cells

    PubMed Central

    Meganathan, K; Jagtap, S; Srinivasan, S P; Wagh, V; Hescheler, J; Hengstler, J; Leist, M; Sachinidis, A

    2015-01-01

    Human embryonic stem cells (hESCs) may be applied to develop human-relevant sensitive in vitro test systems for monitoring developmental toxicants. The aim of this study was to identify potential developmental toxicity mechanisms of the histone deacetylase inhibitors (HDAC) valproic acid (VPA), suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) relevant to the in vivo condition using a hESC model in combination with specific differentiation protocols and genome-wide gene expression and microRNA profiling. Analysis of the gene expression data showed that VPA repressed neural tube and dorsal forebrain (OTX2, ISL1, EMX2 and SOX10)-related transcripts. In addition, VPA upregulates axonogenesis and ventral forebrain-associated genes, such as SLIT1, SEMA3A, DLX2/4 and GAD2. HDACi-induced expression of miR-378 and knockdown of miR-378 increases the expression of OTX2 and EMX2, which supports our hypothesis that HDACi targets forebrain markers through miR-378. In conclusion, multilineage differentiation in vitro test system is very sensitive for monitoring molecular activities relevant to in vivo neuronal developmental toxicity. Moreover, miR-378 seems to repress the expression of the OTX2 and EMX2 and therefore could be a regulator of the development of neural tube and dorsal forebrain neurons. PMID:25950486

  13. Activated Notch1 Target Genes during Embryonic Cell Differentiation Depend on the Cellular Context and Include Lineage Determinants and Inhibitors

    PubMed Central

    Meier-Stiegen, Franziska; Schwanbeck, Ralf; Bernoth, Kristina; Martini, Simone; Hieronymus, Thomas; Ruau, David; Zenke, Martin; Just, Ursula

    2010-01-01

    Background Notch receptor signaling controls developmental cell fates in a cell-context dependent manner. Although Notch signaling directly regulates transcription via the RBP-J/CSL DNA binding protein, little is known about the target genes that are directly activated by Notch in the respective tissues. Methodology/Principal Findings To analyze how Notch signaling mediates its context dependent function(s), we utilized a Tamoxifen-inducible system to activate Notch1 in murine embryonic stem cells at different stages of mesodermal differentiation and performed global transcriptional analyses. We find that the majority of genes regulated by Notch1 are unique for the cell type and vary widely dependent on other signals. We further show that Notch1 signaling regulates expression of genes playing key roles in cell differentiation, cell cycle control and apoptosis in a context dependent manner. In addition to the known Notch1 targets of the Hes and Hey families of transcriptional repressors, Notch1 activates the expression of regulatory transcription factors such as Sox9, Pax6, Runx1, Myf5 and Id proteins that are critically involved in lineage decisions in the absence of protein synthesis. Conclusion/Significance We suggest that Notch signaling determines lineage decisions and expansion of stem cells by directly activating both key lineage specific transcription factors and their repressors (Id and Hes/Hey proteins) and propose a model by which Notch signaling regulates cell fate commitment and self renewal in dependence of the intrinsic and extrinsic cellular context. PMID:20628604

  14. Parental RNA interference of genes involved in embryonic development of the western corn rootworm, Diabrotica virgifera virgifera LeConte.

    PubMed

    Khajuria, Chitvan; Vélez, Ana M; Rangasamy, Murugesan; Wang, Haichuan; Fishilevich, Elane; Frey, Meghan L F; Carneiro, Newton Portilho; Gandra, Premchand; Narva, Kenneth E; Siegfried, Blair D

    2015-08-01

    RNA interference (RNAi) is being developed as a potential tool for insect pest management and one of the most likely target pest species for transgenic plants that express double stranded RNA (dsRNA) is the western corn rootworm. Thus far, most genes proposed as targets for RNAi in rootworm cause lethality in the larval stage. In this study, we describe RNAi-mediated knockdown of two developmental genes, hunchback (hb) and brahma (brm), in the western corn rootworm delivered via dsRNA fed to adult females. dsRNA feeding caused a significant decrease in hb and brm transcripts in the adult females. Although total oviposition was not significantly affected, there was almost complete absence of hatching in the eggs collected from females exposed to dsRNA for either gene. These results confirm that RNAi is systemic in nature for western corn rootworms. These results also indicate that hunchback and brahma play important roles in rootworm embryonic development and could provide useful RNAi targets in adult rootworms to prevent crop injury by impacting the population of larval progeny of exposed adults. The ability to deliver dsRNA in a trans-generational manner by feeding to adult rootworms may offer an additional approach to utilizing RNAi for rootworm pest management. The potential to develop parental RNAi technology targeting progeny of adult rootworms in combination with Bt proteins or dsRNA lethal to larvae may increase opportunities to develop sustainable approaches to rootworm management involving RNAi technologies for rootworm control.

  15. Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells.

    PubMed

    Yeo, Seungeun; Jeong, Sangkyun; Kim, Janghwan; Han, Jee-Soo; Han, Yong-Mahn; Kang, Yong-Kook

    2007-08-01

    Pluripotent human embryonic stem cells (hESCs) have the distinguishing feature of innate capacity to allow indefinite self-renewal. This attribute continues until specific constraints or restrictions, such as DNA methylation, are imposed on the genome, usually accompanied by differentiation. With the aim of utilizing DNA methylation as a sign of early differentiation, we probed the genomic regions of hESCs, particularly focusing on stem cell marker (SCM) genes to identify regulatory sequences that display differentiation-sensitive alterations in DNA methylation. We show that the promoter regions of OCT4 and NANOG, but not SOX2, REX1 and FOXD3, undergo significant methylation during hESCs differentiation in which SCM genes are substantially repressed. Thus, following exposure to differentiation stimuli, OCT4 and NANOG gene loci are modified relatively rapidly by DNA methylation. Accordingly, we propose that the DNA methylation states of OCT4 and NANOG sequences may be utilized as barometers to determine the extent of hESC differentiation. PMID:17548060

  16. 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

  17. 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

  18. 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

  19. Targeting of c-myc and beta-globin coding sequences to cytoskeletal-bound polysomes by c-myc 3' untranslated region.

    PubMed Central

    Hesketh, J; Campbell, G; Piechaczyk, M; Blanchard, J M

    1994-01-01

    The influence of the 3' untranslated region on mRNA localization was investigated by measuring the distribution of myc, beta-globin and hybrid myc-globin mRNAs between free, cytoskeletal-bound and membrane-bound polysomes in cells transfected with either control or chimeric gene constructs. c-myc sequences and beta-globin-coding sequences linked to the myc 3' untranslated region were present at greatest enrichment in cytoskeletal-bound polysomes. beta-Globin mRNA and myc-coding sequences linked to the beta-globin 3' untranslated region were recovered largely in the free polysomes. In situ hybridization confirmed that replacement of the c-myc 3' untranslated region by that of globin caused a relocalization of the mRNA. The results suggest that mRNA localization in differentiated eukaryotic cells depends on a mechanism that involves directional information in the 3' untranslated region of mRNAs. Images Figure 2 Figure 3 PMID:8129712

  20. 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.

  1. 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

  2. Differential expression of hoxa2a and hoxa2b genes during striped bass embryonic development.

    PubMed

    Scemama, Jean-Luc; Vernon, Jamie L; Stellwag, Edmund J

    2006-10-01

    Here, we report the cloning and expression analysis of two previously uncharacterized paralogs group 2 Hox genes, striped bass hoxa2a and hoxa2b, and the developmental regulatory gene egr2. We demonstrate that both Hox genes are expressed in the rhombomeres of the developing hindbrain and the pharyngeal arches albeit with different spatio-temporal distributions relative to one another. While both hoxa2a and hoxa2b share the r1/r2 anterior boundary of expression characteristic of the hoxa2 paralog genes of other species, hoxa2a gene expression extends throughout the hindbrain, whereas hoxa2b gene expression is restricted to the r2-r5 region. Egr2, which is used in this study as an early developmental marker of rhombomeres 3 and 5, is expressed in two distinct bands with a location and spacing typical for these two rhombomeres in other species. Within the pharyngeal arches, hoxa2a is expressed at higher levels in the second pharyngeal arch, while hoxa2b is more strongly expressed in the posterior arches. Further, hoxa2b expression within the arches becomes undetectable at 60hpf, while hoxa2a expression is maintained at least up until the beginning of chondrogenesis. Comparison of the striped bass HoxA cluster paralog group 2 (PG2) genes to their orthologs and trans-orthologs shows that the striped bass hoxa2a gene expression pattern is similar to the overall expression pattern described for the hoxa2 genes in the lobe-finned fish lineage and for the hoxa2b gene from zebrafish. It is notable that the pharyngeal arch expression pattern of the striped bass hoxa2a gene is more divergent from its sister paralog, hoxa2b, than from the zebrafish hoxa2b gene. Overall, our results suggest that differences in the Hox PG2 gene complement of striped bass and zebrafish affects both their rhombomeric and pharyngeal arch expression patterns and may account for the similarities in pharyngeal arch expression between striped bass hoxa2a and zebrafish hoxa2b.

  3. Preliminary evidence of phenytoin-induced alterations in embryonic gene expression in a mouse model.

    PubMed

    Musselman, A C; Bennett, G D; Greer, K A; Eberwine, J H; Finnell, R H

    1994-01-01

    SWV mouse embryos collected on gestational days (GD) 9:12 and 10:00 following chronic in utero exposure to teratogenic concentrations of phenytoin were utilized for in situ transcription studies of gene expression. The substrate cDNA obtained from the frozen embryo sections was amplified into radiolabelled antisense RNA (RT/aRNA) and used as a probe to screen a panel of 20 cDNA clones representing genes that are important regulators of craniofacial and neural development. The magnitude of alteration in gene expression following phenytoin treatment was determined densitometrically by changes in the hybridization intensity of the aRNA probes to the cDNA clones immobilized to the slot blots. We found that both Wnt-1 and the calcium channel gene were developmentally regulated, as their level of expression decreased significantly between the two collection times. Phenytoin treatment produced a significant downregulation in the level of expression for 25% of the genes examined in the GD 9:12 embryos, including the growth factors TGF-beta and NT3, the proto-oncogene Wnt-1, the nicotinic receptor, and the voltage sensitive calcium channel gene. Additional changes in the coordinate expression of several of the growth and transcription factors were observed at both gestational timepoints. The application of RT/aRNA technology has extended our appreciation of the normal patterns of gene expression during craniofacial and neural development, and provided the first demonstration of multiple coordinate changes in transcription patterns following teratogenic insult.

  4. Transcriptional regulatory program in wild-type and retinoblastoma gene-deficient mouse embryonic fibroblasts during adipocyte differentiation

    PubMed Central

    2011-01-01

    Background Although many molecular regulators of adipogenesis have been identified a comprehensive catalogue of components is still missing. Recent studies showed that the retinoblastoma protein (pRb) was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs), and retinoblastoma gene-deficient MEFs (Rb-/- MEFs). Findings Comparative analysis of the expression profiles of 3T3-L1 cells and wild-type MEFs revealed genes involved specifically in early regulation of the adipocyte differentiation as well as secreted factors and signaling molecules regulating the later phase of differentiation. In an attempt to identify transcription factors regulating adipogenesis, bioinformatics analysis of the promoters of coordinately and highly expressed genes was performed. We were able to identify a number of high-confidence target genes for follow-up experimental studies. Additionally, combination of experimental data and computational analyses pinpointed a feedback-loop between Pparg and Foxo1. To analyze the effects of the retinoblastoma protein at the transcriptional level we chose a perturbated system (Rb-/- MEFs) for comparison to the transcriptional program of wild-type MEFs. Gene ontology analysis of 64 deregulated genes showed that the Rb-/- MEF model exhibits a brown(-like) adipocyte phenotype. Additionally, the analysis results indicate a different or additional role for pRb family member involvement in the lineage commitment. Conclusion In this study a number of commonly modulated genes during adipogenesis in 3T3-L1 cells and MEFs, potential transcriptional regulation mechanisms, and differentially regulated targets during adipocyte differentiation of Rb

  5. Selective repression of retinoic acid target genes by RIP140 during induced tumor cell differentiation of pluripotent human embryonal carcinoma cells

    PubMed Central

    Heim, Kelly C; White, Kristina A; Deng, Dexin; Tomlinson, Craig R; Moore, Jason H; Freemantle, Sarah J; Spinella, Michael J

    2007-01-01

    Background The use of retinoids as anti-cancer agents has been limited due to resistance and low efficacy. The dynamics of nuclear receptor coregulation are incompletely understood. Cell-and context-specific activities of nuclear receptors may be in part due to distinct coregulator complexes recruited to distinct subsets of target genes. RIP140 (also called NRIP1) is a ligand-dependent corepressor that is inducible with retinoic acid (RA). We had previously shown that RIP140 limits RA induced tumor cell differentiation of embryonal carcinoma; the pluriopotent stem cells of testicular germ cell tumors. This implies that RIP140 represses key genes required for RA-mediated tumor cell differentiation. Identification of these genes would be of considerable interest. Results To begin to address this issue, microarray technology was employed to elucidate in a de novo fashion the global role of RIP140 in RA target gene regulation of embryonal carcinoma. Subclasses of genes were affected by RIP140 in distinct manners. Interestingly, approximately half of the RA-dependent genes were unaffected by RIP140. Hence, RIP140 appears to discriminate between different classes of RA target genes. In general, RIP140-dependent gene expression was consistent with RIP140 functioning to limit RA signaling and tumor cell differentiation. Few if any genes were regulated in a manner to support a role for RIP140 in "active repression". We also demonstrated that RIP140 silencing sensitizes embryonal carcinoma cells to low doses of RA. Conclusion Together the data demonstrates that RIP140 has profound effects on RA-mediated gene expression in this cancer stem cell model. The RIP140-dependent RA target genes identified here may be particularly important in mediating RA-induced tumor cell differentiation and the findings suggest that RIP140 may be an attractive target to sensitize tumor cells to retinoid-based differentiation therapy. We discuss these data in the context of proposed models of RIP

  6. 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.

  7. Conditional-ready mouse embryonic stem cell derived macrophages enable the study of essential genes in macrophage function

    PubMed Central

    Yeung, A. T. Y.; Hale, C.; Xia, J.; Tate, P. H.; Goulding, D.; Keane, J. A.; Mukhopadhyay, S.; Forrester, L.; Billker, O.; Skarnes, W. C.; Hancock, R. E. W.; Dougan, G.

    2015-01-01

    The ability to differentiate genetically modified mouse embryonic stem (ES) cells into functional macrophages provides a potentially attractive resource to study host-pathogen interactions without the need for animal experimentation. This is particularly useful in instances where the gene of interest is essential and a knockout mouse is not available. Here we differentiated mouse ES cells into macrophages in vitro and showed, through a combination of flow cytometry, microscopic imaging, and RNA-Seq, that ES cell-derived macrophages responded to S. Typhimurium, in a comparable manner to mouse bone marrow derived macrophages. We constructed a homozygous mutant mouse ES cell line in the Traf2 gene that is known to play a role in tumour necrosis factor-α signalling but has not been studied for its role in infections or response to Toll-like receptor agonists. Interestingly, traf2-deficient macrophages produced reduced levels of inflammatory cytokines in response to lipopolysaccharide (LPS) or flagellin stimulation and exhibited increased susceptibility to S. Typhimurium infection. PMID:25752829

  8. Gene Editing of Human Embryonic Stem Cells via an Engineered Baculoviral Vector Carrying Zinc-finger Nucleases

    PubMed Central

    Lei, Yuning; Lee, Chi-Lin; Joo, Kye-Il; Zarzar, Jonathan; Liu, Yarong; Dai, Bingbing; Fox, Victoria; Wang, Pin

    2011-01-01

    Human embryonic stem (hES) cells are renewable cell sources that have potential applications in regenerative medicine. The development of technologies to produce permanent and site-specific genome modifications is in demand to achieve future medical implementation of hES cells. We report herein that a baculoviral vector (BV) system carrying zinc-finger nucleases (ZFNs) can successfully modify the hES cell genome. BV-mediated transient expression of ZFNs specifically disrupted the CCR5 locus in transduced cells and the modified cells exhibited resistance to HIV-1 transduction. To convert the BV to a gene targeting vector, a DNA donor template and ZFNs were incorporated into the vector. These hybrid vectors yielded permanent site-specific gene addition in both immortalized human cell lines (10%) and hES cells (5%). Modified hES cells were both karyotypically normal and pluripotent. These results suggest that this baculoviral delivery system can be engineered for site-specific genetic manipulation in hES cells. PMID:21326219

  9. Development of retrovirus vectors useful for expressing genes in cultured murine embryonal cells and hematopoietic cells in vivo.

    PubMed Central

    Guild, B C; Finer, M H; Housman, D E; Mulligan, R C

    1988-01-01

    A series of retrovirus vectors were constructed in which cellular promoter elements derived from the chicken beta-actin and human histone H4 genes were introduced within the proviral transcriptional unit of Moloney murine leukemia virus in order to promote expression of inserted sequences. Each of these vectors gave rise to high titer of virus capable of transferring the expected proviral structure to cells. Inclusion of normal 5' splice sequences or a portion of viral gag sequences in these constructions resulted in significant increases in virus titer. Each construction was transcriptionally active in NIH 3T3 cells and in undifferentiated F9 cells. One of the vectors, HSG-neo, which contained the human histone H4 promoter, was shown to be transcriptionally active in hematopoietic cells derived from long-term reconstituted bone marrow transplant recipients engrafted with transduced stem cells. These vectors should be of general use for obtaining efficient gene expression in embryonal and hematopoietic cells. Images PMID:3418785

  10. The embryonic RNA helicase gene (ERH): a new member of the DEAD box family of RNA helicases.

    PubMed Central

    Sowden, J; Putt, W; Morrison, K; Beddington, R; Edwards, Y

    1995-01-01

    DEAD box proteins share several highly conserved motifs including the characteristic Asp-Glu-Ala-Asp (D-E-A-D in the amino acid single-letter code) motif and have established or putative ATP-dependent RNA helicase activity. These proteins are implicated in a range of cellular processes that involve regulation of RNA function, including translation initiation, RNA splicing and ribosome assembly. Here we describe the isolation and characterization of an embryonic RNA helicase gene, ERH, which maps to mouse chromosome 1 and encodes a new member of the DEAD box family of proteins. The predicted ERH protein shows high sequence similarity to the testes-specific mouse PL10 and to the maternally acting Xenopus An3 helicase proteins. The ERH expression profile is similar, to that of An3, which localizes to the animal hemisphere of oocytes and is abundantly expressed in the embryo. ERH is expressed in oocytes and is a ubiquitous mRNA in the 9 days-post-conception embryo, and at later stages of development shows a more restricted pattern of expression in brain and kidney. The similarities in sequence and in expression profile suggest that ERH is the murine equivalent of the Xenopus An3 gene, and we propose that ERH plays a role in translational activation of mRNA in the oocyte and early embryo. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8948440

  11. The Hand1 and Hand2 transcription factors regulate expansion of the embryonic cardiac ventricles in a gene dosage-dependent manner.

    PubMed

    McFadden, David G; Barbosa, Ana C; Richardson, James A; Schneider, Michael D; Srivastava, Deepak; Olson, Eric N

    2005-01-01

    The basic helix-loop-helix transcription factors Hand1 and Hand2 display dynamic and spatially restricted expression patterns in the developing heart. Mice that lack Hand2 die at embryonic day 10.5 from right ventricular hypoplasia and vascular defects, whereas mice that lack Hand1 die at embryonic day 8.5 from placental and extra-embryonic abnormalities that preclude analysis of its potential role in later stages of heart development. To determine the cardiac functions of Hand1, we generated mice harboring a conditional Hand1-null allele and excised the gene by cardiac-specific expression of Cre recombinase. Embryos homozygous for the cardiac Hand1 gene deletion displayed defects in the left ventricle and endocardial cushions, and exhibited dysregulated ventricular gene expression. However, these embryos survived until the perinatal period when they died from a spectrum of cardiac abnormalities. Creation of Hand1/2 double mutant mice revealed gene dose-sensitive functions of Hand transcription factors in the control of cardiac morphogenesis and ventricular gene expression. These findings demonstrate that Hand factors play pivotal and partially redundant roles in cardiac morphogenesis, cardiomyocyte differentiation and cardiac-specific transcription.

  12. Changes in WNT signaling-related gene expression associated with development and cloning in bovine extra-embryonic and endometrial tissues during the peri-implantation period.

    PubMed

    Biase, Fernando H; Rabel, Chanaka; Guillomot, Michel; Sandra, Olivier; Andropolis, Kalista; Olmstead, Colleen; Oliveira, Rosane; Wallace, Richard; Le Bourhis, Daniel; Richard, Christophe; Campion, Evelyne; Chaulot-Talmon, Aurélie; Giraud-Delville, Corinne; Taghouti, Géraldine; Jammes, Hélène; Hue, Isabelle; Renard, Jean Paul; Lewin, Harris A

    2013-12-01

    We determined if somatic cell nuclear transfer (SCNT) cloning is associated with WNT-related gene expression in cattle development, and if the expression of genes in the WNT pathway changes during the peri-implantation period. Extra-embryonic and endometrial tissues were collected at gestation days 18 and 34 (d18, d34). WNT5A, FZD4, FZD5, LRP5, CTNNB1, GNAI2, KDM1A, BCL2L1, and SFRP1 transcripts were localized in extra-embryonic tissue, whereas SFRP1 and DKK1 were localized in the endometrium. There were no differences in the localization of these transcripts in extra-embryonic tissue or endometrium from SCNT or artificial insemination (AI) pregnancies. Expression levels of WNT5A were 11-fold greater in the allantois of SCNT than AI samples. In the trophoblast, expression of WNT5A, FZD5, CTNNB1, and DKK1 increased significantly from d18 to d34, whereas expression of KDM1A and SFRP1 decreased, indicating that implantation is associated with major changes in WNT signaling. SCNT was associated with altered WNT5A expression in trophoblasts, with levels increasing 2.3-fold more in AI than SCNT conceptuses from d18 to d34. In the allantois, expression of WNT5A increased 6.3-fold more in SCNT than AI conceptuses from d18 to d34. Endometrial tissue expression levels of the genes tested did not differ between AI or SCNT pregnancies, although expression of individual genes showed variation across developmental stages. Our results demonstrate that SCNT is associated with altered expression of specific WNT-related genes in extra-embryonic tissue in a time- and tissue-specific manner. The pattern of gene expression in the WNT pathway suggests that noncanonical WNT signal transduction is important for implantation of cattle conceptuses.

  13. Graded Smad2/3 Activation Is Converted Directly into Levels of Target Gene Expression in Embryonic Stem Cells

    PubMed Central

    Mavrakis, Konstantinos J.; Goggolidou, Paraskevi; Norris, Dominic P.; Episkopou, Vasso

    2009-01-01

    The Transforming Growth Factor (TGF) β signalling family includes morphogens, such as Nodal and Activin, with important functions in vertebrate development. The concentration of the morphogen is critical for fate decisions in the responding cells. Smad2 and Smad3 are effectors of the Nodal/Activin branch of TGFβ signalling: they are activated by receptors, enter the nucleus and directly transcribe target genes. However, there have been no studies correlating levels of Smad2/3 activation with expression patterns of endogenous target genes in a developmental context over time. We used mouse Embryonic Stem (ES) cells to create a system whereby levels of activated Smad2/3 can be manipulated by an inducible constitutively active receptor (Alk4*) and an inhibitor (SB-431542) that blocks specifically Smad2/3 activation. The transcriptional responses were analysed by microarrays at different time points during activation and repression. We identified several genes that follow faithfully and reproducibly the Smad2/3 activation profile. Twenty-seven of these were novel and expressed in the early embryo downstream of Smad2/3 signalling. As they responded to Smad2/3 activation in the absence of protein synthesis, they were considered direct. These immediate responsive genes included negative intracellular feedback factors, like SnoN and I-Smad7, which inhibit the transcriptional activity of Smad2/3. However, their activation did not lead to subsequent repression of target genes over time, suggesting that this type of feedback is inefficient in ES cells or it is counteracted by mechanisms such as ubiquitin-mediated degradation by Arkadia. Here we present an ES cell system along with a database containing the expression profile of thousands of genes downstream of Smad2/3 activation patterns, in the presence or absence of protein synthesis. Furthermore, we identify primary target genes that follow proportionately and with high sensitivity changes in Smad2/3 levels over 15–30

  14. 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

  15. Regulation of the Nanog Gene by Both Positive and Negative cis-Regulatory Elements in Embryonal Carcinoma Cells and Embryonic Stem Cells

    PubMed Central

    Boer, Brian; Cox, Jesse L.; Claassen, David; Mallanna, Sunil Kumar; Desler, Michelle; Rizzino, Angie

    2008-01-01

    The transcription factor Nanog is essential for mammalian embryogenesis, as well as the pluripotency of embryonic stem (ES) cells. Work with ES cells and embryonal carcinoma (EC) cells previously identified positive and negative cis-regulatory elements that influence the activity of the Nanog promoter, including adjacent cis-regulatory elements that bind Sox2 and Oct-3/4. Given the importance of Nanog during mammalian development, we examined the cis-regulatory elements required for Nanog promoter activity more closely. In this study, we demonstrate that two positive cis-regulatory elements previously shown to be active in F9 EC cells are also active in ES cells. We also identify a novel negative regulatory region that is located in close proximity to two other positive Nanog cis-regulatory elements. Although this negative regulatory region is active in F9 EC cells and ES cells, it is inactive in P19 EC cells. Furthermore, we demonstrate that one of the positive cis-regulatory elements active in F9 EC cells and ES cells is inactive in P19 EC cells. Together, these and other studies suggest that Nanog transcription is regulated by the interplay of positive and negative cis-regulatory elements. Given that P19 appears to be more closely related to a later developmental stage of mammalian development than F9 and ES cells, differential utilization of cis-regulatory elements may reflect mechanisms used during development to achieve the correct level of Nanog expression as embryogenesis unfolds. PMID:18537119

  16. Neuroglobins, pivotal proteins associated with emerging neural systems and precursors of metazoan globin diversity.

    PubMed

    Lechauve, Christophe; Jager, Muriel; Laguerre, Laurent; Kiger, Laurent; Correc, Gaëlle; Leroux, Cédric; Vinogradov, Serge; Czjzek, Mirjam; Marden, Michael C; Bailly, Xavier

    2013-03-01

    Neuroglobins, previously thought to be restricted to vertebrate neurons, were detected in the brain of a photosymbiotic acoel, Symsagittifera roscoffensis, and in neurosensory cells of the jellyfish Clytia hemisphaerica. For the neuroglobin of S. roscoffensis, a member of a lineage that originated either at the base of the bilateria or of the deuterostome clade, we report the ligand binding properties, crystal structure at 2.3 Å, and brain immunocytochemical pattern. We also describe in situ hybridizations of two neuroglobins specifically expressed in differentiating nematocytes (neurosensory cells) and in statocytes (ciliated mechanosensory cells) of C. hemisphaerica, a member of the early branching animal phylum cnidaria. In silico searches using these neuroglobins as queries revealed the presence of previously unidentified neuroglobin-like sequences in most metazoan lineages. Because neural systems are almost ubiquitous in metazoa, the constitutive expression of neuroglobin-like proteins strongly supports the notion of an intimate association of neuroglobins with the evolution of animal neural systems and hints at the preservation of a vitally important function. Neuroglobins were probably recruited in the first protoneurons in early metazoans from globin precursors. Neuroglobins were identified in choanoflagellates, sponges, and placozoans and were conserved during nervous system evolution. Because the origin of neuroglobins predates the other metazoan globins, it is likely that neuroglobin gene duplication followed by co-option and subfunctionalization led to the emergence of globin families in protostomes and deuterostomes (i.e. convergent evolution).

  17. Reactive oxygen species and nuclear factor-kappa B pathway mediate high glucose-induced Pax-2 gene expression in mouse embryonic mesenchymal epithelial cells and kidney explants.

    PubMed

    Chen, Y-W; Liu, F; Tran, S; Zhu, Y; Hébert, M-J; Ingelfinger, J R; Zhang, S-L

    2006-11-01

    Diabetic mellitus confers a major risk of congenital malformations, and is associated with diabetic embryopathy, affecting multiple organs including the kidney. The DNA paired box-2 (Pax-2) gene is essential in nephrogenesis. We investigated whether high glucose alters Pax-2 gene expression and aimed to delineate its underlying mechanism(s) of action using both in vitro (mouse embryonic mesenchymal epithelial cells (MK4) and ex vivo (kidney explant from Hoxb7-green florescent protein (GFP) mice) approaches. Pax-2 gene expression was determined by reverse transcriptase-polymerase chain reaction, Western blotting, and immunofluorescent staining. A fusion gene containing the full-length 5'-flanking region of the human Pax-2 promoter linked to a luciferase reporter gene, pGL-2/hPax-2, was transfected into MK4 cells with or without dominant negative IkappaBalpha (DN IkappaBalpha) cotransfection. Fusion gene expression level was quantified by cellular luciferase activity. Reactive oxygen species (ROS) generation was measured by lucigenin assay. Embryonic kidneys from Hoxb7-GFP mice were cultured ex vivo. High D(+) glucose (25 mM), compared to normal glucose (5 mM), specifically induced Pax-2 gene expression in MK4 cells and kidney explants. High glucose-induced Pax-2 gene expression is mediated, at least in part, via ROS generation and activation of the nuclear factor kappa B signaling pathway, but not via protein kinase C, p38 mitogen-activated protein kinase (MAPK), and p44/42 MAPK signaling.

  18. 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 ...

  19. Paternal H3K4 methylation is required for minor zygotic gene activation and early mouse embryonic development

    PubMed Central

    Aoshima, Keisuke; Inoue, Erina; Sawa, Hirofumi; Okada, Yuki

    2015-01-01

    Epigenetic modifications, such as DNA methylation and histone modifications, are dynamically altered predominantly in paternal pronuclei soon after fertilization. To identify which histone modifications are required for early embryonic development, we utilized histone K-M mutants, which prevent endogenous histone methylation at the mutated site. We prepared four single K-M mutants for histone H3.3, K4M, K9M, K27M, and K36M, and demonstrate that overexpression of H3.3 K4M in embryos before fertilization results in developmental arrest, whereas overexpression after fertilization does not affect the development. Furthermore, loss of H3K4 methylation decreases the level of minor zygotic gene activation (ZGA) predominantly in the paternal pronucleus, and we obtained similar results from knockdown of the H3K4 methyltransferase Mll3/4. We therefore conclude that H3K4 methylation, likely established by Mll3/4 at the early pronuclear stage, is essential for the onset of minor ZGA in the paternal pronucleus, which is necessary for subsequent preimplantation development in mice. PMID:25925669

  20. Paternal H3K4 methylation is required for minor zygotic gene activation and early mouse embryonic development.

    PubMed

    Aoshima, Keisuke; Inoue, Erina; Sawa, Hirofumi; Okada, Yuki

    2015-07-01

    Epigenetic modifications, such as DNA methylation and histone modifications, are dynamically altered predominantly in paternal pronuclei soon after fertilization. To identify which histone modifications are required for early embryonic development, we utilized histone K-M mutants, which prevent endogenous histone methylation at the mutated site. We prepared four single K-M mutants for histone H3.3, K4M, K9M, K27M, and K36M, and demonstrate that overexpression of H3.3 K4M in embryos before fertilization results in developmental arrest, whereas overexpression after fertilization does not affect the development. Furthermore, loss of H3K4 methylation decreases the level of minor zygotic gene activation (ZGA) predominantly in the paternal pronucleus, and we obtained similar results from knockdown of the H3K4 methyltransferase Mll3/4. We therefore conclude that H3K4 methylation, likely established by Mll3/4 at the early pronuclear stage, is essential for the onset of minor ZGA in the paternal pronucleus, which is necessary for subsequent preimplantation development in mice. PMID:25925669

  1. Cloning and molecular characterization of an ornithine decarboxylase gene and its expression during embryonic development of the housefly, Musca domestica.

    PubMed

    Toutges, Michelle J; Santoso, Adi

    2011-10-01

    We are interested in identifying targets that may be used to develop new control products for the common housefly, Musca domestica, a vector of disease for many vertebrates. One such target, ornithine decarboxylase (ODC), is an embryonic enzyme involved in the regulation of polyamines and is a critical enzyme during M. domestica development. In this study, the cDNA for ODC from M. domestica was cloned, sequenced, and characterized. The full-length cDNA was 1,337-bp, consistent with a single band of approximately 1.35 kb obtained by northern analysis. The open-reading frame contains 1,191 bp, yielding a deduced polypeptide of 396 amino acid residues with a predicted mass of 44,618 Da. The deduced M. domestica ODC protein was homologous to other ODC proteins. mRNA expression profiles analyzed by real-time PCR indicated that the ODC transcript is temporally regulated throughout embryogenesis. Sequence data and Southern blot analysis suggests that there were likely only one or two closely linked copies of the M. domestica ODC gene.

  2. Cloning and molecular characterization of an ornithine decarboxylase gene and its expression during embryonic development of the housefly, Musca domestica.

    PubMed

    Toutges, Michelle J; Santoso, Adi

    2011-10-01

    We are interested in identifying targets that may be used to develop new control products for the common housefly, Musca domestica, a vector of disease for many vertebrates. One such target, ornithine decarboxylase (ODC), is an embryonic enzyme involved in the regulation of polyamines and is a critical enzyme during M. domestica development. In this study, the cDNA for ODC from M. domestica was cloned, sequenced, and characterized. The full-length cDNA was 1,337-bp, consistent with a single band of approximately 1.35 kb obtained by northern analysis. The open-reading frame contains 1,191 bp, yielding a deduced polypeptide of 396 amino acid residues with a predicted mass of 44,618 Da. The deduced M. domestica ODC protein was homologous to other ODC proteins. mRNA expression profiles analyzed by real-time PCR indicated that the ODC transcript is temporally regulated throughout embryogenesis. Sequence data and Southern blot analysis suggests that there were likely only one or two closely linked copies of the M. domestica ODC gene. PMID:21928394

  3. Gas1 is a pleiotropic regulator of cellular functions: from embryonic development to molecular actions in cancer gene therapy.

    PubMed

    Segovia, José; Zarco, Natanael

    2014-01-01

    Cellular homeostasis is governed by a precise regulation of the molecular mechanisms of action of several proteins in a given time. There is a group of proteins that have a particular role depending on the cellular context in which they are present and are known as pleiotropic proteins. The Gas1 (Growth Arrest Specific 1) gene was isolated from a subtraction library from serum arrested versus growing NIH3T3 mouse fibroblast. Gas1 is a member of the alpha receptors (GFRα) for the family of GDNF ligands (GFL), we have previously shown that Gas1 acts as a negative modulator of the GDNF-induced intracellular signaling and induces cell arrest and apoptosis. This modulating activity is the cause of the capacity of Gas1 to act as a tumor suppressor. On the other hand, several studies have shown the interaction between Gas1 and Hh (Hedgehog) proteins to potentiate the positive regulation of this pathway, which is involved in the development of the nervous system, and in both the origin and progression of different tumors. This review summarizes our current understanding of the structure of Gas1 and the molecular mechanism of action in different cellular functions, both during embryonic development, in the adult and its effects inhibiting cell growth and inducing apoptosis of cancer cells.

  4. Aire regulates the expression of differentiation-associated genes and self-renewal of embryonic stem cells.

    PubMed

    Gu, Bin; Zhang, Jiarong; Chen, Qi; Tao, Bo; Wang, Wei; Zhou, Yang; Chen, Liangbiao; Liu, Yusen; Zhang, Ming

    2010-04-01

    Embryonic stem cells (ESCs) are pluripotent stem cells from early embryos. It has been well recognized that ESC genomes are maintained in a globally transcriptional hyperactive state, which genetically poised ESCs to the high differentiation potential. However, the transcription factors regulating the global transcription activities in ESCs are not well defined. We show here that mouse and human ESCs express two transcription factors, Aire and Deaf1. Previously known to function in the thymus stromal cells and peripheral lymphoid organs respectively, Aire and Deaf1 help regulate the ectopic expression of diverse tissue-specific antigens to establish self-immune tolerance. Differentiation of ESCs greatly reduced Aire and Deaf1 expression, in a pattern similar to the pluripotent factors, Oct4 and Nanog. Knockdown of Aire in mouse ESCs resulted in significantly decreased clone-forming efficiency as well as attenuated cell cycle, suggesting Aire plays a role in ESC self-renewal. In addition, some differentiation-associated genes that are sporadically expressed in ESCs were reduced in expression upon Aire knockdown. These results suggest that transcription factors such as Aire and Deaf1, which exert global transcriptional regulatory functions, may play important roles in self-renewal of ESCs and maintaining ESC in a transcriptionally hyperactive state.

  5. Generation of KCL021 research grade human embryonic stem cell line carrying a ΔF508 mutation in the CFTR gene

    PubMed Central

    Miere, Cristian; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL021 human embryonic stem cell line was derived from an embryo donated for research that carried a ΔF508 mutation affecting the CFTR gene encoding the cystic fibrosis transmembrane conductance regulator. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345808

  6. Generation of KCL021 research grade human embryonic stem cell line carrying a ΔF508 mutation in the CFTR gene.

    PubMed

    Miere, Cristian; Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL021 human embryonic stem cell line was derived from an embryo donated for research that carried a ΔF508 mutation affecting the CFTR gene encoding the cystic fibrosis transmembrane conductance regulator. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345808

  7. Impact of supplementary royal jelly on in vitro maturation of sheep oocytes: genes involved in apoptosis and embryonic development.

    PubMed

    Valiollahpoor Amiri, Mohammad; Deldar, Hamid; Ansari Pirsaraei, Zarbakht

    2016-01-01

    Optimizing culture conditions lead to the improvement of oocyte developmental competence and additives with anti-oxidative activity in culture media improved embryonic development. Royal jelly (RJ) is a product from the cephalic glands of nurse bees that has considerable health effects. The aim of this study was to investigate the effect of different concentrations of RJ on the maturation, cleavage, and blastocyst rates and gene expression in the oocyte and cumulus cells during in vitro maturation (IVM) of sheep oocyte. IVM of oocyte was performed in the presence of control (RJ0), 2.5 (RJ2.5), 5 (RJ5), 10 (RJ10), 20 (RJ20), and 40 (RJ40) mg/mL of RJ. Following the maturation period, parthenogenetic activation was carried out in two treatment groups (RJ0 and RJ10) and embryonic development was examined three and eight days thereafter. Moreover, the relative expression of BCL2 and BAX in oocyte as well as BCL2, BAX, HAS2, PTGS2, and STAR in cumulus cells were assessed. The results indicated that the addition of 10 mg/mL of RJ (90 ± 4.51%) to the maturation medium linearly increased the oocyte maturation rate compared to the control group (57 ± 2.42%), then it remained constant to the RJ40 (93 ± 3.10%) group. The higher RJ concentrations were associated with increased (p < 0.01) cleavage (53.3 ± 1.55% to 82.3 ± 2.82%) and blastocyst rate (15.5 ± 1.16% to 33.8 ± 3.09%) from the RJ0 to the RJ10 group. The relative mRNA expression of BCL2 and BAX in the oocyte was higher at RJ10. In cumulus cells, the expression of BCL2 was not affected, but that of BAX decreased, and expression of HAS2, PTGS2, and STAR were increased following the addition of RJ to the maturation media. In conclusion, the addition of 10 mg/mL of RJ to maturation medium improved blastocyst formation and decreased the apoptotic incidence in sheep cumulus cells and the oocyte during the in vitro development.

  8. Embryonic stem cells lacking a functional inhibitory G-protein subunit (alpha i2) produced by gene targeting of both alleles.

    PubMed Central

    Mortensen, R M; Zubiaur, M; Neer, E J; Seidman, J G

    1991-01-01

    The alpha i2 subunit of the inhibitory heterotrimeric guanine nucleotide-binding proteins is highly conserved in mammals and is expressed in all cell types, but its exact function is not yet defined. We have investigated the role of this protein by producing embryonic stem (ES) cells lacking a functional alpha i2 gene. These alpha i2-null cell lines regulate adenylyl cyclase and grow and differentiate in vitro the same as wild-type ES cells. Homologous recombination was used to sequentially inactivate both copies of the alpha i2 gene. The first allele was inactivated by insertion of a neomycin-resistance gene. We modified the hygromycin B-resistance gene for improved expression in ES cells and used this gene to inactivate the remaining normal allele. The techniques used should be generally applicable to other genes whether or not they are expressed in ES cells. Images PMID:1908087

  9. Human alpha2-globin nonsense-mediated mRNA decay induced by a novel alpha-thalassaemia frameshift mutation at codon 22.

    PubMed

    Pereira, Francisco J C; do Céu Silva, Maria; Picanço, Isabel; Seixas, Maria T; Ferrão, Anabela; Faustino, Paula; Romão, Luísa

    2006-04-01

    We describe a novel alpha-thalassaemia determinant in a 3-year-old girl presenting a mild microcytic and hypochromic anaemia, and normal haemoglobin A2 level. Molecular studies revealed heterozygosity for a novel microdeletion (-C) at codon 22 of the alpha2-globin gene. As the frameshift mutation generates a premature translation termination codon at position 48/49, we investigated the effect of the nonsense codon on the alpha2-globin gene expression. Although it does not affect RNA splicing, the premature nonsense codon induces accelerated mRNA degradation. To our knowledge, this is the first time the nonsense-mediated mRNA decay has been reported to occur in human alpha-globin mRNA.

  10. Tributyltin alters osteocalcin, matrix metalloproteinase 20 and dentin sialophosphoprotein gene expression in mineralizing mouse embryonic tooth in vitro.

    PubMed

    Salmela, Eija; Alaluusua, Satu; Sahlberg, Carin; Lukinmaa, Pirjo-Liisa

    2012-01-01

    We showed in a previous in vitro study that tributyltin (TBT) arrests dentin mineralization and enamel formation in developing mouse tooth. The present aim was to investigate the effect of TBT on the expression of genes associated with mineralization of dental hard tissues. Embryonic day 18 mouse mandibular first molars were cultured for 3, 5 or 7 days and exposed to 1.0 μM TBT and studied by real-time quantitative polymerase chain reaction (RT-QPCR) for the expressions of osteocalcin (Ocn), alkaline phosphatase (Alpl), dentin matrix protein 1 (Dmp1), dentin sialophosphoprotein (Dspp) and matrix metalloproteinase 20 (Mmp-20).Ocn, Mmp-20 and Dspp, whose expressions showed changes in RT- QPCR, were further analyzed by in situ hybridization of tissue sections. In situ hybridization showed that TBT decreased Ocn expression in odontoblasts but increased the expression in the epithelial tooth compartment. In QPCR assays, the net effect in the whole tooth was increased expression. TBT also reduced Mmp-20 expression in ameloblasts and odontoblasts. Dspp expression varied but both QPCR assays and in situ hybridization showed a decreasing trend. TBT exposure had no clear effect on Alpl and Dmp1 expressions. Increased Ocn expression by epithelial enamel organ may inhibit dentin mineralization and enamel formation. Decreased Ocn, Mmp-20 and Dspp expressions in odontoblasts may indicate delayed cell differentiation, or TBT may specifically decrease the expression of genes involved in dentin mineralization. While decreased Mmp-20 expression by TBT in ameloblasts may impair enamel mineralization, the coincident reduction in Mmp-20 and Dspp expressions in odontoblasts may potentiate the delay of dentin mineralization.

  11. RING1B O-GlcNAcylation regulates gene targeting of polycomb repressive complex 1 in human embryonic stem cells.

    PubMed

    Maury, Julien Jean Pierre; El Farran, Chadi A; Ng, Daniel; Loh, Yuin-Han; Bi, Xuezhi; Bardor, Muriel; Choo, Andre Boon-Hwa

    2015-07-01

    O-linked-N-acetylglucosamine (O-GlcNAc) post-translationally modifies and regulates thousands of proteins involved in various cellular mechanisms. Recently, O-GlcNAc has been linked to human embryonic stem cells (hESC) differentiation, however the identity and function of O-GlcNAc proteins regulating hESC remain unknown. Here, we firstly identified O-GlcNAc modified human stem cell regulators such as hnRNP K, HP1γ, and especially RING1B/RNF2. Thereafter, we focused our work on RING1B which is the catalytic subunit of the polycomb repressive complex 1 (PRC1) a major epigenetic repressor essential for pluripotency maintenance and differentiation. By point-mutation, we show that T(250)/S(251) and S(278) RING1B residues are bearing O-GlcNAc, and that T(250)/S(251) O-GlcNAcylation decreases during differentiation. O-GlcNAc seems to regulate RING1B-DNA binding as suggested by our ChIP-sequencing results. Non-O-GlcNAcylated RING1B is found to be enriched near cell cycle genes whereas O-GlcNAcylated RING1B seems preferentially enriched near neuronal genes. Our data suggest that during hESC differentiation, the decrease of RING1B O-GlcNAcylation might enable PRC1 to switch its target to induce neuron differentiation. Overall, we demonstrate that O-GlcNAc modifies and regulates an essential epigenetic tool, RING1B, which may contribute to hESC pluripotency maintenance and differentiation. PMID:26100231

  12. Abscisic acid regulation of DC8, a carrot embryonic gene. [Daucus carota

    SciTech Connect

    Hatzopoulos, P.; Fong, F.; Sung, Z.R. Texas A M Univ., College Station )

    1990-10-01

    DC8 encodes a hydrophylic 66 kilodalton protein located in the cytoplasm and cell walls of carrot (Daucus carota) embryo and endosperm. During somatic embryogenesis, the levels of DC8 mRNA and protein begin to increase 5 days after removal of auxin. To study the role of abscisic acid (ABA) in the regulation of DC8 gene, fluridone, 1-methyl-3-phenyl,-5(3-trifluoro-methyl-phenyl)-4(1H)-pyridinone, was used to inhibit the endogenous ABA content of the embryos. Fluridone, 50 micrograms per milliliter, effectively inhibits the accumulation of ABA in globular-tage embryos. Western and Northern analysis show that when fluridone is added to the culture medium DC8 protein and mRNA decrease to very low levels. ABA added to fluridone supplemented culture media restores the DC8 protein and mRNA to control levels. Globular-stage embryos contain 0.9 to 1.4 {times} 10{sup {minus}7} molar ABA while 10{sup {minus}6} molar exogenously supplied ABA is the optimal concentration for restoration of DC8 protein accumulation in fluridone-treated embryos. The mRNA level is increased after 15 minutes of ABA addition and reaches maximal levels by 60 minutes. Evidence is presented that, unlike other ABA-regulated genes, DC8 is not induced in nonembryonic tissues via desiccation nor addition of ABA.

  13. Expression of Immune-Related Genes during Loach (Misgurnus anguillicaudatus) Embryonic and Early Larval Development

    PubMed Central

    Lee, Jang Wook; Kim, Jung Eun; Goo, In Bon; Hwang, Ju-Ae; Im, Jea Hyun; Choi, Hye-Sung; Lee, Jeong-Ho

    2015-01-01

    Early life stage mortality in fish is one of the problems faced by loach aquaculture. However, our understanding of immune system in early life stage fish is still incomplete, and the information available is restricted to a few fish species. In the present work, we investigated the expression of immune-related transcripts in loach during early development. In fishes, recombination-activating gene 1 (RAG-1) and sacsin (SACS) have been considered as immunological function. In this study, the expression of the both genes was assessed throughout the early developmental stages of loach using real-time PCR method. maRAG-1 mRNA was first detected in 0 dph, observed the increased mostly until 40 dph. Significant expression of maRAG-1 was detected in 0 to 40 dph. These patterns of expression may suggest that the loach start to develop its function after hatching. On the other hand, maSACS was detected in unfertilized oocyte to molura stages and 0 to 40 dph. maSACS mRNA transcripts were detected in unfertilized oocytes, suggesting that they are maternally transferred. PMID:26973969

  14. Gene-Specific Assessment of Guanine Oxidation as an Epigenetic Modulator for Cardiac Specification of Mouse Embryonic Stem Cells.

    PubMed

    Park, Joonghoon; Park, Jong Woo; Oh, Hawmok; Maria, Fernanda S; Kang, Jaeku; Tian, Xiuchun

    2016-01-01

    Epigenetics have essential roles in development and human diseases. Compared to the complex histone modifications, epigenetic changes on mammalian DNA are as simple as methylation on cytosine. Guanine, however, can be oxidized as an epigenetic change which can undergo base-pair transversion, causing a genetic difference. Accumulating evidence indicates that reactive oxygen species (ROS) are important signaling molecules for embryonic stem cell (ESC) differentiation, possibly through transient changes on genomic DNA such as 7,8-dihydro-8-oxoguanine (8-oxoG). Technical limitations on detecting such DNA modifications, however, restrict the investigation of the role of 8-oxoG in ESC differentiation. Here, we developed a Hoogsteen base pairing-mediated PCR-sequencing assay to detect 8-oxoG lesions that can subsequently cause G to T transversions during PCR. We then used this assay to assess the epigenetic and transient 8-oxoG formation in the Tbx5 gene of R1 mouse ESCs subjected to oxidative stress by removing 2-mercaptoethanol (2ME) from the culture media. To our surprise, significantly higher numbers of 8-oxoG-mediated G∙C to C∙G transversion, not G∙C to T∙A, were detected at 7th and 9th base position from the transcription start site of exon 1 of Tbx5 in ESCs in the (-)2ME than (+)2ME group (p < 0.05). This was consistent with the decrease in the amount of amplifiable of DNA harboring the 8-oxoG lesions at the Tbx5 promoter region in the oxidative stressed ESCs. The ESCs responded to oxidative stress, possibly through the epigenetic effects of guanine oxidation with decreased proliferation (p < 0.05) and increased formation of beating embryoid bodies (EBs; p < 0.001). Additionally, the epigenetic changes of guanine induced up-regulation of Ogg1 and PolB, two base excision repairing genes for 8-oxoG, in ESCs treated with (-)2ME (p < 0.01). Together, we developed a gene-specific and direct quantification assay for guanine oxidation. Using oxidative stressed

  15. Gene-Specific Assessment of Guanine Oxidation as an Epigenetic Modulator for Cardiac Specification of Mouse Embryonic Stem Cells

    PubMed Central

    Park, Joonghoon; Park, Jong Woo; Oh, Hawmok; Maria, Fernanda S.; Kang, Jaeku; Tian, Xiuchun

    2016-01-01

    Epigenetics have essential roles in development and human diseases. Compared to the complex histone modifications, epigenetic changes on mammalian DNA are as simple as methylation on cytosine. Guanine, however, can be oxidized as an epigenetic change which can undergo base-pair transversion, causing a genetic difference. Accumulating evidence indicates that reactive oxygen species (ROS) are important signaling molecules for embryonic stem cell (ESC) differentiation, possibly through transient changes on genomic DNA such as 7,8-dihydro-8-oxoguanine (8-oxoG). Technical limitations on detecting such DNA modifications, however, restrict the investigation of the role of 8-oxoG in ESC differentiation. Here, we developed a Hoogsteen base pairing-mediated PCR-sequencing assay to detect 8-oxoG lesions that can subsequently cause G to T transversions during PCR. We then used this assay to assess the epigenetic and transient 8-oxoG formation in the Tbx5 gene of R1 mouse ESCs subjected to oxidative stress by removing 2-mercaptoethanol (2ME) from the culture media. To our surprise, significantly higher numbers of 8-oxoG-mediated G∙C to C∙G transversion, not G∙C to T∙A, were detected at 7th and 9th base position from the transcription start site of exon 1 of Tbx5 in ESCs in the (-)2ME than (+)2ME group (p < 0.05). This was consistent with the decrease in the amount of amplifiable of DNA harboring the 8-oxoG lesions at the Tbx5 promoter region in the oxidative stressed ESCs. The ESCs responded to oxidative stress, possibly through the epigenetic effects of guanine oxidation with decreased proliferation (p < 0.05) and increased formation of beating embryoid bodies (EBs; p < 0.001). Additionally, the epigenetic changes of guanine induced up-regulation of Ogg1 and PolB, two base excision repairing genes for 8-oxoG, in ESCs treated with (-)2ME (p < 0.01). Together, we developed a gene-specific and direct quantification assay for guanine oxidation. Using oxidative stressed

  16. Early Exposure of Murine Embryonic Stem Cells to Hematopoietic Cytokines Differentially Directs Definitive Erythropoiesis and Cardiomyogenesis in Alginate Hydrogel Three-Dimensional Cultures

    PubMed Central

    Fauzi, Iliana

    2014-01-01

    HepG2-conditioned medium (CM) facilitates early differentiation of murine embryonic stem cells (mESCs) into hematopoietic cells in two-dimensional cultures through formation of embryoid-like colonies (ELCs), bypassing embryoid body (EB) formation. We now demonstrate that three-dimensional (3D) cultures of alginate-encapsulated mESCs cultured in a rotating wall vessel bioreactor can be differentially driven toward definitive erythropoiesis and cardiomyogenesis in the absence of ELC formation. Three groups were evaluated: mESCs in maintenance medium with leukemia inhibitory factor (LIF, control) and mESCs cultured with HepG2 CM (CM1 and CM2). Control and CM1 groups were cultivated for 8 days in early differentiation medium with murine stem cell factor (mSCF) followed by 10 days in hematopoietic differentiation medium (HDM) containing human erythropoietin, m-interleukin (mIL)-3, and mSCF. CM2 cells were cultured for 18 days in HDM, bypassing early differentiation. In CM1, a fivefold expansion of hematopoietic colonies was observed at day 14, with enhancement of erythroid progenitors, hematopoietic genes (Gata-2 and SCL), erythroid genes (EKLF and β-major globin), and proteins (Gata-1 and β-globin), although ζ-globin was not expressed. In contrast, CM2 primarily produced beating colonies in standard hematopoietic colony assay and expressed early cardiomyogenic markers, anti-sarcomeric α-actinin and Gata-4. In conclusion, a scalable, automatable, integrated, 3D bioprocess for the differentiation of mESC toward definitive erythroblasts has been established. Interestingly, cardiomyogenesis was also directed in a specific protocol with HepG2 CM and hematopoietic cytokines making this platform a useful tool for the study of erythroid and cardiomyogenic development. PMID:24926614

  17. Metaxin, a gene contiguous to both thrombospondin 3 and glucocerebrosidase, is required for embryonic development in the mouse: implications for Gaucher disease.

    PubMed Central

    Bornstein, P; McKinney, C E; LaMarca, M E; Winfield, S; Shingu, T; Devarayalu, S; Vos, H L; Ginns, E I

    1995-01-01

    We have identified a murine gene, metaxin, that spans the 6-kb interval separating the glucocerebrosidase gene (GC) from the thrombospondin 3 gene on chromosome 3E3-F1. Metaxin and GC are transcribed convergently; their major polyadenylylation sites are only 431 bp apart. On the other hand, metaxin and the thrombospondin 3 gene are transcribed divergently and share a common promoter sequence. The cDNA for metaxin encodes a 317-aa protein, without either a signal sequence or consensus for N-linked glycosylation. Metaxin protein is expressed ubiquitously in tissues of the young adult mouse, but no close homologues have been found in the DNA or protein data bases. A targeted mutation (A-->G in exon 9) was introduced into GC by homologous recombination in embryonic stem cells to establish a mouse model for a mild form of Gaucher disease. A phosphoglycerate kinase-neomycin gene cassette was also inserted into the 3'-flanking region of GC as a selectable marker, at a site later identified as the terminal exon of metaxin. Mice homozygous for the combined mutations die early in gestation. Since the same amino acid mutation in humans is associated with mild type 1 Gaucher disease, we suggest that metaxin protein is likely to be essential for embryonic development in mice. Clearly, the contiguous gene organization at this locus limits targeting strategies for the production of murine models of Gaucher disease. Images Fig. 3 Fig. 4 PMID:7753840

  18. Profiles of mRNA expression of related genes in the duck hypothalamus-pituitary growth axis during embryonic and early post-hatch development.

    PubMed

    Hu, Yan; Liu, Hongxiang; Song, Chi; Xu, Wenjuan; Ji, Gaige; Zhu, Chunhong; Shu, Jingting; Li, Huifang

    2015-03-15

    In this study, the ontogeny of body and liver weight and the pattern of related gene mRNA expression in the hypothalamus-pituitary growth axis (HPGA) of two different duck breeds (Anas platyrhynchos domestica) were compared during embryonic and post-hatch development. Duck hypothalamic growth hormone release hormone (GHRH), somatostatin (SS), pituitary growth hormone (GH), liver growth hormone receptor (GHR) and insulin-like growth factor-I (IGF-1) mRNA were first detected on the 13th embryonic day. During early duck development, SS maintained a lower expression status, whereas the other four genes exhibited highly significant variations in an age-specific manner. Highly significant breed specificity was observed with respect to hepatic IGF-1 mRNA expression, which showed a significant breed-age interaction effect. Compared with previous studies on chickens, significant species differences were observed regarding the mRNA expression of bird embryonic HPGA-related genes. During early development, highly significant breed and age specificity were observed with respect to developmental changes in body and liver weight, and varying degrees of significant linear correlation were found between these performances and the mRNA expression of HPGA-related genes in the duck HPGA. These results suggest that different genetic backgrounds may lead to differences in duck growth and HPGA-related gene mRNA expression, and the differential mRNA expression of related genes in the duck HPGA may be particularly important in the early growth of ducks. Furthermore, hepatic IGF-1 mRNA expression presented highly significant breed specificity, and evidence suggests the involvement of hepatic IGF-1 in mediating genetic effects on embryo and offspring growth in ducks.

  19. Expression Pattern of Early Growth Response Gene 1 during Olive Flounder (Paralichthys olivaceus) Embryonic Development

    PubMed Central

    Yang, Hyun; Lee, Jeong-Ho; Noh, Jae Koo; Kim, Hyun Chul; Park, Choul-Ji; Park, Jong-Won; Kim, Kyung-Kil

    2014-01-01

    The early growth response protein 1 (Egr-1) is a widely reported zinc finger protein and a well known transcription factor encoded by the Egr-1 gene, which plays key roles in many aspects of vertebrate embryogenesis and in adult vertebrates. The Egr-1 expression is important in the formation of the gill vascular system in flounders, which develops during the post-hatching phase and is essential for survival during the juvenile period. However, the complete details of Egr-1 expression during embryo development in olive flounder are not available. We assessed the expression patterns of Egr-1 during the early development of olive flounders by using reverse transcription polymerase chain reaction (RT-PCR) analysis. Microscopic observations showed that gill filament formation corresponded with the Egr-1 expression. Thus, we showed that Egr-1 plays a vital role in angiogenesis in the gill filaments during embryogenesis. Further, Egr-1 expression was found to be strong at 5 days after hatching (DAH), in the development of the gill vascular system, and this strong expression level was maintained throughout all the development stages. Our findings have important implications with respect to the biological role of Egr-1 and evolution of the first respiratory blood vessels in the gills of olive flounder. Further studies are required to elucidate the Egr-1-mediated stress response and to decipher the functional role of Egr-1 in developmental stages. PMID:25949193

  20. MicroRNA-323-3p Regulates the Activity of Polycomb Repressive Complex 2 (PRC2) via Targeting the mRNA of Embryonic Ectoderm Development (Eed) Gene in Mouse Embryonic Stem Cells*

    PubMed Central

    Zhang, Ying; Teng, Fei; Luo, Guan-Zheng; Wang, Meng; Tong, Man; Zhao, Xiaoyang; Wang, Liu; Wang, Xiu-Jie; Zhou, Qi

    2013-01-01

    PRC2 (Polycomb repressive complex 2) mediates epigenetic gene silencing by catalyzing the triple methylation of histone H3 Lys-27 (H3K27me3) to establish a repressive epigenetic state. PRC2 is involved in the regulation of many fundamental biological processes and is especially essential for embryonic stem cells. However, how the formation and function of PRC2 are regulated is largely unknown. Here, we show that a microRNA encoded by the imprinted Dlk1-Dio3 region of mouse chromosome 12, miR-323-3p, targets Eed (embryonic ectoderm development) mRNA, which encodes one of the core components of PRC2, the EED protein. Binding of miR-323-3p to Eed mRNA resulted in reduced EED protein abundance and cellular H3K27me3 levels, indicating decreased PRC2 activity. Such regulation seems to be conserved among mammals, at least between mice and humans. We demonstrate that induced pluripotent stem cells with varied developmental abilities had different miR-323-3p as well as EED and H3K27me3 levels, indicating that miR-323-3p may be involved in the regulation of stem cell pluripotency through affecting PRC2 activity. Mouse embryonic fibroblast cells had much higher miR-323-3p expression and nearly undetectable H3K27me3 levels. These findings identify miR-323-3p as a new regulator for PRC2 and provide a new approach for regulating PRC2 activity via microRNAs. PMID:23821546

  1. PID1 (NYGGF4), a new growth-inhibitory gene in embryonal brain tumors and gliomas

    PubMed Central

    Erdreich-Epstein, Anat; Robison, Nathan; Ren, Xiuhai; Zhou, Hong; Xu, Jingying; Davidson, Tom B.; Schur, Mathew; Gilles, Floyd H.; Ji, Lingyun; Malvar, Jemily; Shackleford, Gregory M.; Margol, Ashley S.; Krieger, Mark D.; Judkins, Alexander R.; Jones, David T.W.; Pfister, Stefan; Kool, Marcel; Sposto, Richard; Asgharazadeh, Shahab

    2014-01-01

    Purpose We present here the first report of PID1 (Phosphotyrosine Interaction Domain containing 1; NYGGF4) in cancer. PID1 was identified in 2006 as a gene that modulates insulin signaling and mitochondrial function in adipocytes and muscle cells. Experimental Design and Results Using four independent medulloblastoma datasets, we show that mean PID1 mRNA levels were lower in unfavorable medulloblastomas (Groups 3 and 4, and anaplastic histology) compared with favorable medulloblastomas (SHH and WNT groups, and desmoplastic/nodular histology) and with fetal cerebellum. In two large independent glioma datasets PID1 mRNA was lower in glioblastomas (GBMs), the most malignant gliomas, compared to other astrocytomas, oligodendrogliomas and non-tumor brains. Neural and proneural GBM subtypes had higher PID1 mRNA compared to classical and mesenchymal GBM. Importantly, overall survival and radiation-free progression-free survival were longer in medulloblastoma patients with higher PID1 mRNA (univariate and multivariate analyses). Higher PID1 mRNA also correlated with longer overall survival in glioma and GBM patients. In cell culture, overexpression of PID1 inhibited colony formation in medulloblastoma, atypical teratoid rhabdoid tumor (ATRT) and GBM cell lines. Increasing PID1 also increased cell death and apoptosis, inhibited proliferation, induced mitochondrial depolarization, and decreased serum-mediated phosphorylation of AKT and ERK in medulloblastoma, ATRT and/or GBM cell lines, whereas siRNA to PID1 diminished mitochondrial depolarization. Conclusions These data are the first to link PID1 to cancer and suggest that PID1 may have a tumor inhibitory function in these pediatric and adult brain tumors. PMID:24300787

  2. Quantitative expression of pluripotency-related genes in parthenogenetically produced buffalo (Bubalus bubalis) embryos and in putative embryonic stem cells derived from them.

    PubMed

    Singh, K P; Kaushik, R; Mohapatra, S K; Garg, V; Rameshbabu, K; Singh, M K; Palta, P; Manik, R S; Singla, S K; Chauhan, M S

    2014-09-01

    Parthenogenetically produced embryos and embryonic stem (ES) cells derived from them offer a unique model for investigating the role of transcription factors in embryonic genome activation (EGA), pluripotent lineage specification and in pluripotency and self-renewal of ES cells because of the unique nature of these embryos. There is little information on the quantitative expression of important genes in parthenogenetically produced embryos and in ES cells derived from them. The present study examined the quantitative expression of some important genes in parthenogenetically produced buffalo embryos and in putative parthenogenetic ES cells (pES) cells. The quantitative expression of OCT-4, SOX-2, NANOG, REX-1, FOXD-3 and NUCLEOSTEMIN, which is very low in immature and mature oocytes, and in embryos at 2-, 4- and 8- to 16-cell stage, increases significantly at morula and blastocyst stage. The expression level of TELOMERASE, c-MYC and STAT-3, which is high in immature oocytes decreases during embryonic development followed by either an increase at the morula stage (TELOMERASE) or a low expression level maintained throughout development till blastocyst stage (c-MYC and STAT-3). There is a progressive decline in the expression level of OCT-4, SOX-2, c-MYC, REX-1, NUCLEOSTEMIN, TELOMERASE and STAT-3 during long term culture of pES cells. PMID:25077841

  3. Hydrogen gas attenuates embryonic gene expression and prevents left ventricular remodeling induced by intermittent hypoxia in cardiomyopathic hamsters.

    PubMed

    Kato, Ryuji; Nomura, Atsuo; Sakamoto, Aiji; Yasuda, Yuki; Amatani, Koyuha; Nagai, Sayuri; Sen, Yoko; Ijiri, Yoshio; Okada, Yoshikatsu; Yamaguchi, Takehiro; Izumi, Yasukatsu; Yoshiyama, Minoru; Tanaka, Kazuhiko; Hayashi, Tetsuya

    2014-12-01

    The prevalence of sleep apnea is very high in patients with heart failure (HF). The aims of this study were to investigate the influence of intermittent hypoxia (IH) on the failing heart and to evaluate the antioxidant effect of hydrogen gas. Normal male Syrian hamsters (n = 22) and cardiomyopathic (CM) hamsters (n = 33) were exposed to IH (repeated cycles of 1.5 min of 5% oxygen and 5 min of 21% oxygen for 8 h during the daytime) or normoxia for 14 days. Hydrogen gas (3.05 vol/100 vol) was inhaled by some CM hamsters during hypoxia. IH increased the ratio of early diastolic mitral inflow velocity to mitral annulus velocity (E/e', 21.8 vs. 16.9) but did not affect the LV ejection fraction (EF) in normal Syrian hamsters. However, IH increased E/e' (29.4 vs. 21.5) and significantly decreased the EF (37.2 vs. 47.2%) in CM hamsters. IH also increased the cardiomyocyte cross-sectional area (672 vs. 443 μm(2)) and interstitial fibrosis (29.9 vs. 9.6%), along with elevation of oxidative stress and superoxide production in the left ventricular (LV) myocardium. Furthermore, IH significantly increased the expression of brain natriuretic peptide, β-myosin heavy chain, c-fos, and c-jun mRNA in CM hamsters. Hydrogen gas inhalation significantly decreased both oxidative stress and embryonic gene expression, thus preserving cardiac function in CM hamsters. In conclusion, IH accelerated LV remodeling in CM hamsters, at least partly by increasing oxidative stress in the failing heart. These findings might explain the poor prognosis of patients with HF and sleep apnea.

  4. A statistical approach towards the derivation of predictive gene sets for potency ranking of chemicals in the mouse embryonic stem cell test.

    PubMed

    Schulpen, Sjors H W; Pennings, Jeroen L A; Tonk, Elisa C M; Piersma, Aldert H

    2014-03-21

    The embryonic stem cell test (EST) is applied as a model system for detection of embryotoxicants. The application of transcriptomics allows a more detailed effect assessment compared to the morphological endpoint. Genes involved in cell differentiation, modulated by chemical exposures, may be useful as biomarkers of developmental toxicity. We describe a statistical approach to obtain a predictive gene set for toxicity potency ranking of compounds within one class. This resulted in a gene set based on differential gene expression across concentration-response series of phthalatic monoesters. We determined the concentration at which gene expression was changed at least 1.5-fold. Genes responding with the same potency ranking in vitro and in vivo embryotoxicity were selected. A leave-one-out cross-validation showed that the relative potency of each phthalate was always predicted correctly. The classical morphological 50% effect level (ID50) in EST was similar to the predicted concentration using gene set expression responses. A general down-regulation of development-related genes and up-regulation of cell-cycle related genes was observed, reminiscent of the differentiation inhibition in EST. This study illustrates the feasibility of applying dedicated gene set selections as biomarkers for developmental toxicity potency ranking on the basis of in vitro testing in the EST.

  5. Surface functionalization of inorganic nano-crystals with fibronectin and E-cadherin chimera synergistically accelerates trans-gene delivery into embryonic stem cells.

    PubMed

    Kutsuzawa, K; Chowdhury, E H; Nagaoka, M; Maruyama, K; Akiyama, Y; Akaike, T

    2006-11-24

    Stem cells holding great promises in regenerative medicine have the potential to be differentiated to a specific cell type through genetic manipulation. However, conventional ways of gene transfer to such progenitor cells suffer from a number of disadvantages particularly involving safety and efficacy issues. Here, we report on the development of a bio-functionalized inorganic nano-carrier of DNA by embedding fibronectin and E-cadherin chimera on the carrier, leading to its high affinity interactions with embryonic stem cell surface and accelerated trans-gene delivery for subsequent expression. While only apatite nano-particles were very inefficient in transfecting embryonic stem cells, fibronectin-anchored particles and to a more significant extent, fibronectin and E-cadherin-Fc-associated particles dramatically enhanced trans-gene delivery with a value notably higher than that of commercially available lipofection system. The involvement of both cell surface integrin and E-cadherin in mediating intracellular localization of the hybrid carrier was verified by blocking integrin binding site with excess free fibronectin and up-regulating both integrin and E-cadherin through PKC activation. Thus, the new establishment of a bio-functional hybrid gene-carrier would promote and facilitate development of stem cell-based therapy in regenerative medicine.

  6. Surface functionalization of inorganic nano-crystals with fibronectin and E-cadherin chimera synergistically accelerates trans-gene delivery into embryonic stem cells

    SciTech Connect

    Kutsuzawa, K.; Chowdhury, E.H.; Nagaoka, M.; Maruyama, K.; Akiyama, Y.; Akaike, T. . E-mail: takaike@bio.titech.ac.jp

    2006-11-24

    Stem cells holding great promises in regenerative medicine have the potential to be differentiated to a specific cell type through genetic manipulation. However, conventional ways of gene transfer to such progenitor cells suffer from a number of disadvantages particularly involving safety and efficacy issues. Here, we report on the development of a bio-functionalized inorganic nano-carrier of DNA by embedding fibronectin and E-cadherin chimera on the carrier, leading to its high affinity interactions with embryonic stem cell surface and accelerated trans-gene delivery for subsequent expression. While only apatite nano-particles were very inefficient in transfecting embryonic stem cells, fibronectin-anchored particles and to a more significant extent, fibronectin and E-cadherin-Fc-associated particles dramatically enhanced trans-gene delivery with a value notably higher than that of commercially available lipofection system. The involvement of both cell surface integrin and E-cadherin in mediating intracellular localization of the hybrid carrier was verified by blocking integrin binding site with excess free fibronectin and up-regulating both integrin and E-cadherin through PKC activation. Thus, the new establishment of a bio-functional hybrid gene-carrier would promote and facilitate development of stem cell-based therapy in regenerative medicine.

  7. 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

  8. 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

  9. Expression of insulin-like growth factor system genes in liver tissue during embryonic and early post-hatch development in duck (Anas platyrhynchos Domestica).

    PubMed

    Jianmin, Zou; Jingting, Shu; Yanju, Shan; Yan, Hu; Chi, Song; Wenqi, Zhu

    2014-04-01

    The IGF system is one of the most important endocrine and paracrine growth factor systems that regulate fetal and placental growth, whereas the liver is the principal source of circulation IGF-I. In the present study, expression of IGF-I, IGF type-I receptor (IGF-IR), and IGF binding protein (IGFBP)-3 genes was quantified by RT-PCR in the liver tissue on days 13, 17, 21, 25, and 27 of embryonic development, as well as at 7 days post-hatching (PH) in meat-type Gaoyou ducks and egg-type Jinding ducks. The results showed that IGF-I mRNA could be detected as early as on E 13d, but the expression level was low throughout embryonic development before increasing dramatically by E 27d and 7 days PH in both duck breeds. However, Gaoyou ducks exhibited higher IGF-I mRNA level than Jinding ducks, and the differences were significant on E 13d, E 21d, and at 7 days PH. Expression of IGF-IR in liver increased gradually in the former stages of the embryonic development, reaching its highest point on E 21d, and then declined up until 7 days PH. The expression pattern of IGFBP-3 gene was similar to that of IGF-IR gene, increasing significantly from E 17d. The expression peak appeared on E 25d, then declined significantly just prior to hatching (day 27) and was followed by an increase at 7 days PH. In general, the expression level of IGF-IR and IGFBP-3 genes in Jinding ducks was higher than that in Gaoyou ducks. Inverse relationships were observed for the expression of IGF-I and IGF-IR, and IGF-I and IGFBP-3, whereas a positive relationship was observed for the expression of IGF-IR and IGFBP-3. Our data indicate a differential expression of selected genes that comprise the IGF system in the duck liver tissue during embryonic and early PH growth and development.

  10. Inducible and reversible suppression of Npm1 gene expression using stably integrated small interfering RNA vector in mouse embryonic stem cells

    SciTech Connect

    Wang Beibei; Lu Rui; Wang Weicheng; Jin Ying . E-mail: yjin@sibs.ac.cn

    2006-09-08

    The tetracycline (Tc)-inducible small interference RNA (siRNA) is a powerful tool for studying gene function in mammalian cells. However, the system is infrequently utilized in embryonic stem (ES) cells. Here, we present First application of the Tc-inducible, stably integrated plasmid-based siRNA system in mouse ES cells to down-regulate expression of Npm1, an essential gene for embryonic development. The physiological role of Npm1 in ES cells has not been defined. Our data show that the knock-down of Npm1 expression by this siRNA system was not only highly efficient, but also Tc- dose- and induction time-dependent. Particularly, the down-regulation of Npm1 expression was reversible. Importantly, suppression of Npm1 expression in ES cells resulted in reduced cell proliferation. Taken together, this system allows for studying gene function in a highly controlled manner, otherwise difficult to achieve in ES cells. Moreover, our results demonstrate that Npm1 is essential for ES cell proliferation.

  11. Expression of biomarker genes of differentiation in D3 mouse embryonic stem cells after exposure to different embryotoxicant and non-embryotoxicant model chemicals

    PubMed Central

    Romero, Andrea C.; del Río, Eva; Vilanova, Eugenio; Sogorb, Miguel A.

    2015-01-01

    There is a necessity to develop in vitro methods for testing embryotoxicity (Romero et al., 2015) [1]. We studied the progress of D3 mouse embryonic stem cells differentiation exposed to model embryotoxicants and non-embryotoxicants chemicals through the expression of biomarker genes. We studied a set of 16 different genes biomarkers of general cellular processes (Cdk1, Myc, Jun, Mixl, Cer and Wnt3), ectoderm formation (Nrcam, Nes, Shh and Pnpla6), mesoderm formation (Mesp1, Vegfa, Myo1e and Hdac7) and endoderm formation (Flk1 and Afp). We offer dose response in order to derive the concentration causing either 50% or 200% of expression of the biomarker gene. These records revealed to be a valuable end-point to predict in vitro the embryotoxicity of chemicals (Romero et al., 2015) [1]. PMID:26568980