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Sample records for alpha gene transfer

  1. Gene transfer mediated by alpha2-macroglobulin.

    PubMed Central

    Schneider, H; Huse, K; Birkenmeier, G; Otto, A; Scholz, G H

    1996-01-01

    alpha2-Macroglobulin covalently linked to poly(L)-lysine can be used as a vehicle for receptor-mediated gene transfer. This modified alpha2-macroglobulin maintains its ability to bind to the alpha2-macroglobulin receptor, and was shown to introduce a luciferase reporter gene plasmid into HepG2 human hepatoma cells in vitro. The alpha2-macroglobulin receptor is a very large and multifunctional cell surface receptor, whose rapid and efficient internalization rate makes it attractive for gene therapy, e.g. for hepatic gene targeting via injection into the portal vein. PMID:8871570

  2. Modulation of ozone-sensitive genes in alpha-tocopherol transfer protein null mice

    PubMed Central

    Vasu, Vihas T.; Oommen, Saji; Lim, Yunsook; Valacchi, Giuseppe; Hobson, Brad; Eiserich, Jason P.; Leonard, Scott W.; Traber, Maret G.; Cross, Carroll E.; Gohil, Kishorchandra

    2009-01-01

    Alpha-tocopherol transfer protein (ATTP) null mice (ATTP−/−) have a systemic alpha-tocopherol (AT) deficiency, with their lung AT levels being < 10% of those in AT-replete ATTP+/+ mice when fed a standard rodent chow diet. ATTP+/+ and ATTP−/− mice (4 wk old male mice, n = 16 per group) were fed a standard diet (35 IU AT/kg diet) for 8–12 wk, exposed 6 h/day for 3 days to either to O3 (0.5 ppm) or filtered air, then sacrificed. No significant differences in plasma or lung AT concentrations were observed in response to this level of O3 exposure. Lung genomic responses of the lungs to O3 were determined using Affymetrix 430A 2.0 arrays containing over 22,600 probe sets representing 14,000 well-characterized mouse genes. As compared with filtered air exposure, O3 exposure resulted in 99 genes being differentially expressed in ATTP−/− mice, as compared to 52 differentially expressed genes in ATTP+/+ mice. The data revealed an O3-induced upregulation of genes related to cell proliferation/DNA repair and inflammatory-immune responses in both ATTP+/+ and ATTP−/− mice, with the expression of 22 genes being common to both, whereas 30 and 77 genes were unique to ATTP+/+ and ATTP−/− mice, respectively. The expressions of O3 sensitive genes—Timp1, Areg, Birc5 and Tnc—were seen to be further modulated by AT status. The present study reveals AT modulation of adaptive response of lung genome to O3 exposure. PMID:19555225

  3. Amelioration of laminin-alpha2-deficient congenital muscular dystrophy by somatic gene transfer of miniagrin.

    PubMed

    Qiao, Chunping; Li, Jianbin; Zhu, Tong; Draviam, Romesh; Watkins, Simon; Ye, Xiaojing; Chen, Chunlian; Li, Juan; Xiao, Xiao

    2005-08-23

    Congenital muscular dystrophy (CMD) is characterized by severe muscle wasting, premature death in early childhood, and lack of effective treatment. Most of the CMD cases are caused by genetic mutations of laminin-alpha2, which is essential for the structural integrity of muscle extracellular matrix. Here, we report that somatic gene delivery of a structurally unrelated protein, a miniature version of agrin, functionally compensates for laminin-alpha2 deficiency in the murine models of CMD. Adeno-associated virus-mediated overexpression of miniagrin restored the structural integrity of myofiber basal lamina, inhibited interstitial fibrosis, and ameliorated dystrophic pathology. Furthermore, systemic gene delivery of miniagrin into multiple vital muscles significantly improved whole body growth and motility and quadrupled the lifespan (50% survival) of the dystrophic mice. Thus, our study demonstrated the efficacy of somatic gene therapy in a mouse model of CMD. PMID:16103356

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

    PubMed

    Helder, J; Deisseroth, A

    1987-04-01

    The loss of alpha-globin gene transcriptional activity rarely occurs as an acquired abnormality during the evolution of myeloproliferative disease or preleukemia. To test whether the mutation responsible for the loss of alpha-globin gene expression (hemoglobin H disease) in these patients is linked with the alpha-globin genes on chromosome 16, we transferred chromosome 16 from preleukemic patients with acquired hemoglobin H disease to mouse erythroleukemia cells and measured the transcriptional activity of the human alpha-globin genes. After transfer to mouse erythroleukemia cells, the expression of human alpha-globin genes from the peripheral blood or marrow cells of preleukemic patients with acquired hemoglobin H disease was similar to that of human alpha-globin genes transferred to mouse erythroleukemia cells from normal donors. These data showed that factor(s) in the mouse erythroleukemia cell can genetically complement the alpha-globin gene defect in these preleukemia patients with acquired hemoglobin H disease and suggest that altered expression of a gene in trans to the alpha-globin gene may be responsible for the acquisition of hemoglobin H disease in these patients. PMID:3031681

  5. Retroviral interleukin 1alpha gene transfer in bone marrow stromal cells in a primate model: induction of myelopoiesis stimulation.

    PubMed

    de Revel, Thierry; Becard, Nicolas; Sorg, Tania; Rousseau, Sandrine; Spano, Jean Philippe; Thiebot, Hugues; Methali, Magid; Gras, Gabriel; Le Grand, Roger; Dormont, Dominique

    2002-09-01

    Effects of interleukin 1-alpha (IL-1alpha), a proinflammatory cytokine with pleiotropic activity, in the myelopoietic setting, is mainly linked to its ability to increase haematopoietic growth factor production by bone marrow stromal cells. In order to minimize systemic effects of IL-1alpha therapy, we proposed a model of retroviral IL-1alpha gene transfer within bone marrow stromal cells in the macaque cynomolgus. Invitro, 10-15% of bone marrow stromal cells was effectively transduced by retroviral vector (murine Moloney leukaemia virus-derived) expressing IL-1alpha/LacZ, or LacZ alone as control marker, as assessed by betaGal staining. IL-1alpha gene expression was upregulated [semiquantitative reverse transcription polymerase chain reaction (RT-PCR)] within the transduced cells and the cell supernatant showed an increased production of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage (GM)-CSF (enzyme-linked immunosorbent assay) and an increased clonogenic activity (colony-forming cell assay). Ex vivo autologous expanded IL-1alpha/LacZ transduced bone marrow stromal cells were reinfused in two macaques (and two control animals for LacZ alone as controls), without clinical systemic toxicity; LacZ expression by RT-PCR was detected in one animal of each group between d 4 and 9. A slight increase of the peripheral blood leucocyte counts (both polymorphonuclear cells and monocytes) of the two animals transduced with IL-1alpha/LacZ was observed within 10 d, indicating stimulation of myelopoiesis. PMID:12181061

  6. Gene transfer of master autophagy regulator TFEB results in clearance of toxic protein and correction of hepatic disease in alpha-1-anti-trypsin deficiency

    PubMed Central

    Pastore, Nunzia; Blomenkamp, Keith; Annunziata, Fabio; Piccolo, Pasquale; Mithbaokar, Pratibha; Maria Sepe, Rosa; Vetrini, Francesco; Palmer, Donna; Ng, Philip; Polishchuk, Elena; Iacobacci, Simona; Polishchuk, Roman; Teckman, Jeffrey; Ballabio, Andrea; Brunetti-Pierri, Nicola

    2013-01-01

    Alpha-1-anti-trypsin deficiency is the most common genetic cause of liver disease in children and liver transplantation is currently the only available treatment. Enhancement of liver autophagy increases degradation of mutant, hepatotoxic alpha-1-anti-trypsin (ATZ). We investigated the therapeutic potential of liver-directed gene transfer of transcription factor EB (TFEB), a master gene that regulates lysosomal function and autophagy, in PiZ transgenic mice, recapitulating the human hepatic disease. Hepatocyte TFEB gene transfer resulted in dramatic reduction of hepatic ATZ, liver apoptosis and fibrosis, which are key features of alpha-1-anti-trypsin deficiency. Correction of the liver phenotype resulted from increased ATZ polymer degradation mediated by enhancement of autophagy flux and reduced ATZ monomer by decreased hepatic NFκB activation and IL-6 that drives ATZ gene expression. In conclusion, TFEB gene transfer is a novel strategy for treatment of liver disease of alpha-1-anti-trypsin deficiency. This study may pave the way towards applications of TFEB gene transfer for treatment of a wide spectrum of human disorders due to intracellular accumulation of toxic proteins. PMID:23381957

  7. Differential effects of angiostatin, endostatin and interferon-alpha(1) gene transfer on in vivo growth of human breast cancer cells.

    PubMed

    Indraccolo, S; Gola, E; Rosato, A; Minuzzo, S; Habeler, W; Tisato, V; Roni, V; Esposito, G; Morini, M; Albini, A; Noonan, D M; Ferrantini, M; Amadori, A; Chieco-Bianchi, L

    2002-07-01

    The administration of different angiogenesis inhibitors by gene transfer has been shown to result in inhibition of tumor growth in animal tumor models, but the potency of these genes has been only partially evaluated in comparative studies to date. To identify the most effective anti-angiogenic molecule for delivery by retroviral vectors, we investigated the effects of angiostatin, endostatin and interferon(IFN)-alpha(1) gene transfer in in vivo models of breast cancer induced neovascularization and tumor growth. Moloney leukemia virus-based retroviral vectors for expression of murine angiostatin, endostatin and IFN-alpha(1) were generated, characterized, and used to transduce human breast cancer cell lines (MCF7 and MDA-MB435). Secretion of the recombinant proteins was confirmed by biological and Western blotting assays. Their production did not impair in vitro growth of these breast cancer cells nor their viability, and did not interfere with the expression of angiogenic factors. However, primary endothelial cell proliferation and migration in vitro were inhibited by supernatants of the transduced cells containing angiostatin, endostatin, and IFN-alpha(1). Stable gene transfer of the IFN-alpha(1) cDNA by retroviral vectors in both MCF7 and MDA-MB435 cells resulted in a marked and long-lasting inhibition of tumor growth in nude mice that was associated with reduced vascularization. Endostatin reduced the in vivo growth of MDA-MB435, but not MCF7 cells, despite similar levels of in vivo production, and angiostatin did not impair the in vivo growth of either cell line. These findings indicate heterogeneity in the therapeutic efficacy of angiostatic molecules delivered by viral vectors and suggest that gene therapy with IFN-alpha(1) and endostatin might be useful for treatment of breast cancer. PMID:12080381

  8. Mutational analysis of essential IncP alpha plasmid transfer genes traF and traG and involvement of traF in phage sensitivity.

    PubMed Central

    Waters, V L; Strack, B; Pansegrau, W; Lanka, E; Guiney, D G

    1992-01-01

    Although the broad-host-range IncP plasmids can vegetatively replicate in diverse gram-negative bacteria, the development of shuttle vector systems has established that the host range for IncP plasmid conjugative transfer is greater than the range of bacteria that sustain IncP replicons. Towards understanding IncP plasmid conjugation and the connection between IncP conjugation and Agrobacterium tumefaciens T-DNA transfer to plants, two sets of mutants were generated in the larger transfer region (Tra1) of the IncP alpha plasmid RK2. Mutagenesis strategies were chosen to minimize transcriptional polar effects. Mutant Tra1 clones were mapped, sequenced, and processed to reconstruct 49.5-kb Tra2-containing plasmid derivatives in order to assay for transfer activity and IncP plasmid-specific phage sensitivity. Focusing on the activities of the gene products of traF and traG in Escherichia coli, we found that mutations in traF abolished transfer activity and rendered the host cells phage resistant and mutations in traG abolished transfer activity but had no effect on phage sensitivity. Complementation of these mutant derivatives with corresponding trans-acting clones carrying traF or traG restored transfer activity and, in the case of the traF mutant, the phage sensitivity of the host cell. We conclude that in E. coli, both TraF and TraG are essential for IncP plasmid transfer and that TraF is necessary (but not sufficient) for donor-specific phage sensitivity, and sequencing data suggest that both TraF and TraG are membrane spanning. Images PMID:1400217

  9. Expression of the Alpha Tocopherol Transfer Protein gene is regulated by Oxidative Stress and Common Single Nucleotide Polymorphisms

    PubMed Central

    Ulatowski, Lynn; Dreussi, Cara; Noy, Noa; Barnholtz-Sloan, Jill; Klein, Eric; Manor, Danny

    2012-01-01

    Vitamin E (α-tocopherol) is the major lipid soluble antioxidant in most animal species. By controlling the secretion of vitamin E from the liver, the α-tocopherol transfer protein (αTTP) regulates whole-body distribution and levels of this vital nutrient. However, the mechanism(s) that regulate the expression of this protein are poorly understood. Here we report that transcription of the TTPA gene in immortalized human hepatocytes (IHH) is induced by oxidative stress and by hypoxia, by agonists of the nuclear receptors PPARα and RXR, and by increased cAMP levels. The data show further that induction of TTPA transcription by oxidative stress is mediated by an already-present transcription factor, and does not require de novo protein synthesis. Silencing of the cAMP response element binding (CREB) transcription factor attenuated transcriptional responses of the TTPA gene to added peroxide, suggesting that CREB mediates responses of this gene to oxidative stress. Using a 1.9 Kb proximal segment of the human TTPA promoter together with site-directed mutagenesis approach, we found that single nucleotide polymorphisms (SNPs) that are commonly found in healthy humans dramatically affect promoter activity. These observations suggest that oxidative stress and individual genetic makeup contribute to vitamin E homeostasis in humans. These findings may explain the variable responses to vitamin E supplementation observed in human clinical trials. PMID:23079030

  10. Rescue of type I collagen-deficient phenotype by retroviral-vector-mediated transfer of human pro alpha 1(I) collagen gene into Mov-13 cells.

    PubMed Central

    Stacey, A; Mulligan, R; Jaenisch, R

    1987-01-01

    A full-length cDNA clone corresponding to the human pro alpha 1(I) collagen gene was isolated and inserted into a retrovirus vector. Cell lines were obtained which produced recombinant viruses transducing the collagen cDNA (HUC virus). To test whether the transduced cDNA was functional, Mov-13 mouse cells were infected with the virus. These cells do not produce any type I collagen due to an insertional mutation of the pro alpha 1(I) gene which blocks transcription. While normal amounts of pro alpha 2(I) RNA were synthesized, no alpha 2(I) collagen chains were detectable in the mutant Mov-13 cells. Infection with HUC virus, however, resulted in the production of stable type I collagen, which was secreted into the medium. Analysis of pepsin-resistant proteins indicated that interspecies heterotrimers consisting of human alpha 1(I) and mouse alpha 2(I) collagen chains were secreted by the infected Mov-13 cells. Our results show that pro alpha (I) collagen chains from species as distant as human and mouse can associate to form stable type I collagen. The availability of a retrovirus vector transducing a functional pro alpha 1(I) collagen gene combined with the Mov-13 mutant system should enable us to study the effect of specific mutations on the synthesis, assembly, and function of type I collagen, not only in tissue culture but also in the animal. Images PMID:3599181

  11. Pedigree Analysis and Exclusion of Alpha-Tocopherol Transfer Protein (TTPA) as a Candidate Gene for Neuroaxonal Dystrophy in the American Quarter Horse

    PubMed Central

    Finno, C.J.; Famula, T.; Aleman, M.; Higgins, R.J.; Madigan, J.E.; Bannasch, D.L.

    2015-01-01

    Background Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (NAD/EDM) is a neurodegenerative disorder affecting young horses of various breeds that resembles ataxia with vitamin E deficiency in humans, an inherited disorder caused by mutations in the alpha-tocopherol transfer protein gene (TTPA). To evaluate variants found upon sequencing TTPA in the horse, the mode of inheritance for NAD/EDM had to be established. Hypothesis NAD/EDM in the American Quarter Horse (QH) is caused by a mutation in TTPA. Animals 88 clinically phenotyped (35 affected [ataxia score ≥2], 53 unaffected) QHs with a diagnosis of NAD/EDM with 6 affected and 4 unaffected cases confirmed at postmortem examination. Procedures Pedigrees and genotypes across 54,000 single nucleotide polymorphism (SNP) markers were assessed to determine heritability and mode of inheritance of NAD/EDM. TTPA sequence of exon/intron boundaries was evaluated in 2 affected and 2 control horses. An association analysis was performed by 71 SNPs surrounding TTPA and 8 SNPs within TTPA that were discovered by sequencing. RT-PCR for TTPA was performed on mRNA from the liver of 4 affected and 4 control horses. Results Equine NAD/EDM appears to be inherited as a polygenic trait and, within this family of QHs, demonstrates high heritability. Sequencing of TTPA identified 12 variants. No significant association was found using the 79 available variants in and surrounding TTPA. RT-PCR yielded PCR products of equivalent sizes between affected cases and controls. Conclusions and Clinical Importance NAD/EDM demonstrates heritability in this family of QHs. Variants in TTPA are not responsible for NAD/EDM in this study population. PMID:23186252

  12. L-alpha-glycerylphosphorylcholine inhibits the transfer function of phosphatidylinositol transfer protein alpha.

    PubMed

    Komatsu, Hiroaki; Westerman, Jan; Snoek, Gerry T; Taraschi, Theodore F; Janes, Nathan

    2003-12-30

    Phosphatidylinositol transfer protein alpha (PITP-alpha) is a bifunctional phospholipid transfer protein that is highly selective for phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho). Polar lipid metabolites, including L-alpha-glycerylphosphorylcholine (GroPCho), increasingly have been linked to changes in cellular function and to disease. In this study, polar lipid metabolites of PtdIns and PtdCho were tested for their ability to influence PITP-alpha activity. GroPCho inhibited the ability of PITP-alpha to transfer PtdIns or PtdCho between liposomes. The IC(50) of both processes was dependent on membrane composition. D-myo-inositol 1-phosphate and glycerylphosphorylinositol modestly enhanced PITP-alpha-mediated phospholipid transfer. Choline, phosphorylcholine (PCho), CDP-choline, glyceryl-3-phosphate, myo-inositol and D-myo-inositol 1,4,5-trisphosphate had little effect. Membrane surface charge was a strong determinant of the GroPCho inhibition with the inhibition being greatest for highly anionic membranes. GroPCho was shown to enhance the binding of PITP-alpha to anionic vesicles. In membranes of low surface charge, phosphatidylethanolamine (PtdEtn) was a determinant enabling the GroPCho inhibition. Anionic charge and PtdEtn content appeared to increase the strength of PITP-alpha-membrane interactions. The GroPCho-enhanced PITP-alpha-membrane binding was sufficient to cause inhibition, but not sufficient to account for the extent of inhibition observed. Processes associated with strengthened PITP-alpha-membrane binding in the presence of GroPCho appeared to impair the phospholipid insertion/extraction process. PMID:14729069

  13. Divergence of human [alpha]-chain constant region gene sequences: A novel recombinant [alpha]2 gene

    SciTech Connect

    Chintalacharuvu, K. R.; Morrison, S.L. ); Raines, M. )

    1994-06-01

    IgA is the major Ig synthesized in humans and provides the first line of defense at the mucosal surfaces. The constant region of IgA heavy chain is encoded by the [alpha] gene on chromosome 14. Previous studies have indicated the presence of two [alpha] genes, [alpha]1 and [alpha]2 existing in two allotypic forms, [alpha]2 m(1) and [alpha]2 m(2). Here the authors report the cloning and complete nucleotide sequence determination of a novel human [alpha] gene. Nucleotide sequence comparison with the published [alpha] sequences suggests that the gene arose as a consequence of recombination or gene conversion between the two [alpha]2 alleles. The authors have expressed the gene as a chimeric protein in myeloma cells indicating that it encodes a functional protein. The novel IgA resembles IgA2 m(2) in that disulfide bonds link H and L chains. This novel recombinant gene provides insights into the mechanisms of generation of different constant regions and suggests that within human populations, multiple alleles of [alpha] may be present providing IgAs of different structures.

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

    PubMed

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

    1983-04-01

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

  15. Challenges and Prospects for Alpha-1 Antitrypsin Deficiency Gene Therapy.

    PubMed

    Wozniak, Joanna; Wandtke, Tomasz; Kopinski, Piotr; Chorostowska-Wynimko, Joanna

    2015-11-01

    Alpha-1 antitrypsin (AAT) is a protease inhibitor belonging to the serpin family. A number of identified mutations in the SERPINA1 gene encoding this protein result in alpha-1 antitrypsin deficiency (AATD). A decrease in AAT serum concentration or reduced biological activity causes considerable risk of chronic respiratory and liver disorders. As a monogenic disease, AATD appears to be an attractive target for gene therapy, particularly for patients with pulmonary dysfunction, where augmentation of functional AAT levels in plasma might slow down respiratory disease development. The short AAT coding sequence and its activity in the extracellular matrix would enable an increase in systemic serum AAT production by cellular secretion. In vitro and in vivo experimental AAT gene transfer with gamma-retroviral, lentiviral, adenoviral, and adeno-associated viral (AAV) vectors has resulted in enhanced AAT serum levels and a promising safety profile. Human clinical trials using intramuscular viral transfer with AAV1 and AAV2 vectors of the AAT gene demonstrated its safety, but did not achieve a protective level of AAT >11 μM in serum. This review provides an in-depth critical analysis of current progress in AATD gene therapy based on viral gene transfer. The factors affecting transgene expression levels, such as site of administration, dose and type of vector, and activity of the immune system, are discussed further as crucial variables for optimizing the clinical effectiveness of gene therapy in AATD subjects. PMID:26413996

  16. Lateral gene transfer in eukaryotes.

    PubMed

    Andersson, J O

    2005-06-01

    Lateral gene transfer -- the transfer of genetic material between species -- has been acknowledged as a major mechanism in prokaryotic genome evolution for some time. Recently accumulating data indicate that the process also occurs in the evolution of eukaryotic genomes. However, there are large rate variations between groups of eukaryotes; animals and fungi seem to be largely unaffected, with a few exceptions, while lateral gene transfer frequently occurs in protists with phagotrophic lifestyles, possibly with rates comparable to prokaryotic organisms. Gene transfers often facilitate the acquisition of functions encoded in prokaryotic genomes by eukaryotic organisms, which may enable them to colonize new environments. Transfers between eukaryotes also occur, mainly into larger phagotrophic eukaryotes that ingest eukaryotic cells, but also between plant lineages. These findings have implications for eukaryotic genomic research in general, and studies of the origin and phylogeny of eukaryotes in particular. PMID:15761667

  17. Gene Transfer into Cardiac Myocytes

    PubMed Central

    Lang, Sarah E.; Westfall, Margaret V.

    2016-01-01

    Traditional methods for DNA transfection are often inefficient and toxic for terminally differentiated cells, such as cardiac myocytes. Vector-based gene transfer is an efficient approach for introducing exogenous cDNA into these types of primary cell cultures. In this chapter, separate protocols for adult rat cardiac myocyte isolation and gene transfer with recombinant adenovirus are provided and are routinely utilized for studying the effects of sarcomeric proteins on myofilament function. PMID:25836585

  18. In vivo transfer of soluble TNF-alpha receptor 1 gene improves cardiac function and reduces infarct size after myocardial infarction in rats.

    PubMed

    Sugano, Masahiro; Tsuchida, Keiko; Hata, Tomoji; Makino, Naoki

    2004-05-01

    Increased circulating and cardiac TNF-alpha levels during myocardial ischemia have been found in both experimental animals and patients with ischemic heart disease and advanced heart failure. Soluble TNF-alpha receptor 1 (sTNFR1) is an antagonist to TNF-alpha. In the present study, we examined whether sTNFR1 improves cardiac function in rats after myocardial infarction. Male Wistar rats were subjected to left coronary artery (LCA) ligation. Immediately after the ligation, a total of 200 microg of either the sTNFR1 or LacZ plasmid was injected into three different sites in the left ventricular wall. From 1 to 21 days after LCA ligation, TNF-alpha bioactivity in the heart was higher in rats receiving LacZ plasmid than in sham-operated rats, whereas sTNFR1 plasmid significantly suppressed the increase. The LV diastolic dimension was significantly lower, and the fractional shortening was significantly higher in rats treated with the sTNFR1 plasmid than in those treated with the LacZ plasmid. At 21 days after LCA ligation, the LV end-diastolic pressure was also significantly lower in the rats treated with the sTNFR1 plasmid. In addition, the sTNFR1 expression plasmid had significantly reduced the infarct size. In conclusion, TNF-alpha bioactivity in the heart increased during the early stage of infarction and remained elevated. This elevation seemed partially responsible for the impairment of LV function and the increased infarct size. Suppression of TNF-alpha bioactivity from the early stage of infarction with the sTNFR1 plasmid improved cardiac function and reduced infarct size. PMID:15117889

  19. Gene transfer for erythropoiesis enhancement.

    PubMed

    Naffakh, N; Danos, O

    1996-08-01

    The spectrum of anemias treated with recombinant human erythropoietin is rapidly broadening. Lifelong treatment with very high doses is now under evaluation for beta-thalassemia and sickle cell anemia. These indications make it worthwhile to search for methods that will allow a permanent systemic delivery of the hormone. Here, we review experimental gene-transfer-based procedures for erythropoietin delivery in vivo. In mice, both ex vivo and direct in vivo approaches for gene transfer have resulted in the long-term production of therapeutic levels of the hormone. Gene transfer of erythropoietin could become a viable alternative to the injection of the purified recombinant protein once reliable procedures for controlling transgene expression are available. PMID:8796920

  20. Characterization of the bovine C alpha gene.

    PubMed Central

    Brown, W R; Rabbani, H; Butler, J E; Hammarström, L

    1997-01-01

    The complete genomic sequence of a bovine C alpha gene is reported here. The genomic sequence was obtained from a C alpha phage clone that had been cloned from a genomic EMBL4 phage vector library. The C alpha sequence had previously been expressed as a chimeric antibody and identified as IgA using IgA-specific antibodies. Intron/exon boundaries were determined by comparison of the genomic sequence with an expressed bovine C alpha sequence obtained from spleen by reverse transcription-polymerase chain reaction (RT-PCR). Analysis of 50 Swedish bovine genomic DNA samples using genomic blots and five different restriction enzymes failed to detect evidence of polymorphism. However, PstI digests of Brown Swiss DNA showed a restriction fragment length polymorphism (RFLP), suggesting that at least two allelic variants of bovine IgA exist. Comparison of the deduced amino acid sequence of bovine IgA with sequences available for other species indicated that the highest homology was with that of swine, another artiodactyl. This was the highest homology observed for all mammalian IgA compared except for that between IgA1 and IgA2 in humans. Bovine IgA shares with rabbit IgA3 and IgA4, an additional N-linked glycosylation site at position 282. However, the collective data indicate that cattle are like swine and rodents and unlike rabbits in having a single locus of the gene encoding IgA of this species. Images Figure 4 PMID:9203958

  1. Gene transfer: transduction.

    PubMed

    Frangipani, Emanuela

    2014-01-01

    Bacteriophages able to propagate on Pseudomonas strains are very common and can be easily isolated from natural environments or lysogenic strains. The development of transducing systems has allowed bacterial geneticists to perform chromosome analyses and mutation mapping. Moreover, these systems have also been proved to be a successful tool for molecular microbiologists to introduce a foreign gene or a mutation into the chromosome of a bacterial cell. This chapter provides a description of the phage methodology illustrated by Adams in 1959 and applicable to strain PAO1 derivatives. PMID:24818891

  2. Gene transfer in intact animals

    NASA Astrophysics Data System (ADS)

    Cline, M. J.; Stang, H.; Mercola, K.; Morse, L.; Ruprecht, R.; Browne, J.; Salser, W.

    1980-04-01

    Resistance to methotrexate was induced in bone marrow cells of mice by transformation in vitro with DNA from a drug-resistant cell line. Transformed cells were injected in vivo and haematopoietic cells expressing resistance were selected by drug treatment of recipients. Transformed cells had elevated levels of dihydrofolate reductase and demonstrated a proliferative advantage over untransformed cells, indicating successful gene transfer.

  3. Horizontal gene transfer in plants.

    PubMed

    Gao, Caihua; Ren, Xiaodong; Mason, Annaliese S; Liu, Honglei; Xiao, Meili; Li, Jiana; Fu, Donghui

    2014-03-01

    Horizontal gene transfer (HGT) describes the transmission of genetic material across species boundaries. HGT often occurs in microbic and eukaryotic genomes. However, the pathways by which HGTs occur in multicellular eukaryotes, especially in plants, are not well understood. We systematically summarized more than ten possible pathways for HGT. The intimate contact which frequently occurs in parasitism, symbiosis, pathogen, epiphyte, entophyte, and grafting interactions could promote HGTs between two species. Besides these direct transfer methods, genes can be exchanged with a vector as a bridge: possible vectors include pollen, fungi, bacteria, viruses, viroids, plasmids, transposons, and insects. HGT, especially when involving horizontal transfer of transposable elements, is recognized as a significant force propelling genomic variation and biological innovation, playing an important functional and evolutionary role in both eukaryotic and prokaryotic genomes. We proposed possible mechanisms by which HGTs can occur, which is useful in understanding the genetic information exchange among distant species or distant cellular components. PMID:24132513

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

    PubMed

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

    2002-12-01

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

  5. Classification and evolution of alpha-amylase genes in plants.

    PubMed

    Huang, N; Stebbins, G L; Rodriguez, R L

    1992-08-15

    The DNA sequences for 17 plant genes for alpha-amylase (EC 3.2.1.1) were analyzed to determine their phylogenetic relationship. A phylogeny for these genes was obtained using two separate approaches, one based on molecular clock assumptions and the other based on a comparison of sequence polymorphisms (i.e., small and localized insertions) in the alpha-amylase genes. These polymorphisms are called "alpha-amylase signatures" because they are diagnostic of the gene subfamily to which a particular alpha-amylase gene belongs. Results indicate that the cereal alpha-amylase genes fall into two major classes: AmyA and AmyB. The AmyA class is subdivided into the Amy1 and Amy2 subfamilies previously used to classify alpha-amylase genes in barley and wheat. The AmyB class includes the Amy3 subfamily to which most of the alpha-amylase genes of rice belong. Using polymerase chain reaction and oligonucleotide primers that flank one of the two signature regions, we show that the AmyA and AmyB gene classes are present in approximately equal amounts in all grass species examined except barley. The AmyB (Amy3 subfamily) genes in the latter case are comparatively underrepresented. Additional evidence suggests that the AmyA genes appeared recently and may be confined to the grass family. PMID:1502164

  6. Fibrinogen {alpha} genes: Conservation of bipartite transcripts and carboxy-terminal-extended {alpha} subunits in vertebrates

    SciTech Connect

    Fu, Y.; Cao, Y.; Hertzberg, K.M.; Grieninger, G.

    1995-11-01

    All three well-studied subunits of the clotting protein fibrinogen ({alpha}, {beta}, {gamma}) share N-terminal structural homologies, but until recently only the {beta} and {gamma} chains were recognized as having similar globular C-termini. With the discovery of an extra exon in the human fibrinogen {alpha} gene (exon VI), a minor form of the {alpha} subunit ({alpha}{sub E}) with an extended {beta}- and {gamma}-like C-terminus has been identified. In the present study, the polymerase chain reaction has been used to identify sequences that encode counterparts to {alpha}{sub E} in chicken, rabbit, rat, and baboon. The basic six-exon structure of the fibrinogen {alpha} genes is shown to be conserved among mammals and birds, as are the intron positions. Bipartite transcripts - still bearing an intron prior to the last exon - are found among the products of the various vertebrate fibrinogen {alpha} genes. The last exon represents the largest conserved segment of the gene and, in each species examined, encodes exactly 236 amino acids. The C-termini of these {alpha}{sub E} chains align without a single gap and are between 76 and 99% identical. Since the exon VI-encoded domain of {alpha}{sub E} is as well conserved as the corresponding regions of the {beta} and {gamma} chains, it follows that it is equally important and that {alpha}{sub E}-fibrinogen plays a vital, if as-yet unrecognized physiological role. 21 refs., 7 figs., 1 tab.

  7. Population of mixed-symmetry states via {alpha} transfer reactions

    SciTech Connect

    Alonso, C. E.; Arias, J. M.; Fortunato, L.; Vitturi, A.; Pietralla, N.

    2008-07-15

    Within the neutron-proton interacting boson model we study the population of mixed-symmetry states via {alpha} transfer processes. Closed expressions are deduced in the case of the limiting U{sub {pi}}{sub +{nu}}(5) and SU{sub {pi}}{sub +{nu}}(3). We find that the population of the lowest mixed-symmetry 2{sup +} state, vanishing along the N{sub {pi}}=N{sub {nu}} line, depends on the number of active bosons and is normally smaller than that of the lowest full symmetric 2{sup +} state. In particular, for deformed nuclei where the number of bosons is normally large, the relative population of the mixed-symmetry 2{sup +} state is of the order of a few percent. More favorable cases can be found near shell closures, as in the case of {alpha} transfer leading to {sup 140}Ba.

  8. Structure of the mouse calcitonin/calcitonin gene-related peptide alpha and beta genes.

    PubMed

    Thomas, P M; Nasonkin, I; Zhang, H; Gagel, R F; Cote, G J

    2001-01-01

    We report the cloning, genomic organization and sequence of the mouse alpha-CALC and beta-CALC genes. The two genes share extensive sequence homology. The transcription units of both genes contain 6 exons. Transcripts of the alpha-CALC gene were found to alternatively include exon 4 or exons 5 and 6. For the beta-CALC gene exon 4 was not detected in transcripts derived from this gene. The predicted mouse alpha-CGRP was found to be identical to rat alpha-CGRP, however, beta-CGRP predicted amino acid sequences revealed three amino acid differences suggesting these residues are not critical to CGRP function. PMID:11761712

  9. Characterization of the human rod transducin alpha-subunit gene.

    PubMed Central

    Fong, S L

    1992-01-01

    The human rod transducin alpha subunit (Tr alpha) gene has been cloned. A cDNA clone, HG14, contained a 1.1 kb insertion when compared with the human Tr alpha cDNA published by Van Dop et al. (1). Based on two overlapping clones isolated from a human genomic library, the human Tr alpha gene is 4.9 kb in length and consists of nine exons interrupted by eight introns. Northern blots of human retina total RNA showed that the gene is transcribed by rod photoreceptors into two species of mRNA, 1.3 kb and 2.4 kb in size. Apparently, this is the result of alternative splicing. Two putative transcription initiation sites were determined by primer extension and S1 nuclease protection assays. The putative promoter regions of the human and mouse Tr alpha genes have an identity of 78.1%. As found in the mouse gene (2), no TATA consensus sequence is present in the human gene. Images PMID:1614872

  10. RADIATIVE TRANSFER MODELING OF Ly{alpha} EMITTERS. I. STATISTICS OF SPECTRA AND LUMINOSITY

    SciTech Connect

    Zheng Zheng; Cen Renyue; Trac, Hy; Miralda-Escude, Jordi

    2010-06-10

    We combine a cosmological reionization simulation with box size of 100 h {sup -1} Mpc on a side and a Monte Carlo Ly{alpha} radiative transfer code to model Ly{alpha} Emitters (LAEs) at z {approx} 5.7. The model introduces Ly{alpha} radiative transfer as the single factor for transforming the intrinsic Ly{alpha} emission properties into the observed ones. Spatial diffusion of Ly{alpha} photons from radiative transfer results in extended Ly{alpha} emission and only the central part with high surface brightness can be observed. Because of radiative transfer, the appearance of LAEs depends on density and velocity structures in circumgalactic and intergalactic media as well as the viewing angle, which leads to a broad distribution of apparent (observed) Ly{alpha} luminosity for a given intrinsic Ly{alpha} luminosity. Radiative transfer also causes frequency diffusion of Ly{alpha} photons. The resultant Ly{alpha} line is asymmetric with a red tail. The peak of the Ly{alpha} line shifts toward longer wavelength and the shift is anti-correlated with the apparent-to-intrinsic Ly{alpha} luminosity ratio. The simple radiative transfer model provides a new framework for studying LAEs. It is able to explain an array of observed properties of z {approx} 5.7 LAEs in Ouchi et al., producing Ly{alpha} spectra, morphology, and apparent Ly{alpha} luminosity function (LF) similar to those seen in observation. The broad distribution of apparent Ly{alpha} luminosity at fixed UV luminosity provides a natural explanation for the observed UV LF, especially the turnover toward the low luminosity end. The model also reproduces the observed distribution of Ly{alpha} equivalent width (EW) and explains the deficit of UV bright, high EW sources. Because of the broad distribution of the apparent-to-intrinsic Ly{alpha} luminosity ratio, the model predicts effective duty cycles and Ly{alpha} escape fractions for LAEs.

  11. Organization and sequence of the human alpha-lactalbumin gene.

    PubMed Central

    Hall, L; Emery, D C; Davies, M S; Parker, D; Craig, R K

    1987-01-01

    A recombinant bacteriophage containing the entire alpha-lactalbumin gene was isolated from a human genomic library constructed in bacteriophage lambda L47. Within this recombinant the 2.5 kb alpha-lactalbumin gene is flanked by about 5 kb of sequence on either side. The complete nucleotide sequence of the gene and its immediate flanking sequences were determined and compared with those of the rat alpha-lactalbumin gene. These studies showed that the size, organization and sequence of the exons have been highly conserved, whereas the introns have diverged considerably. In particular, the first intron of the human gene was found to contain an Alu repetitive sequence not present in the rat. A high degree of homology (67%) was also observed in the 5' flanking regions, extending as far as 655 nucleotide residues upstream of the transcriptional initiation site. Comparison of the 5' flanking sequences of these two alpha-lactalbumin genes with those of five casein genes has revealed the presence of a highly conserved region [consensus sequence: RGAAGRAAA(N)TGGACAGAAATCAA(CG)TTTCTA], extending from position -140 to -110 in all seven sequences examined, suggesting a possible regulatory role in the hormonal control or tissue-specific expression of milk protein genes in the mammary gland. Images Fig. 1. PMID:2954544

  12. Regulation of alpha o expression by the 5'-flanking region of the alpha o gene.

    PubMed

    Li, Y; Mortensen, R; Neer, E J

    1994-11-01

    Many responses of cells to external signals require activation of the heterotrimeric G proteins. These responses depend on the type and amount of G proteins that are expressed. Each cell has a characteristic complement of G protein subunits. For example, the alpha o subunit is very abundant in neural tissues. Very little is known about the mechanisms that determine cellular levels of G proteins. In the present study, we have isolated a genomic clone for mouse alpha o gene and identified the promoter region. There are multiple transcription initiation sites located about 750 base pairs upstream of the translational start site. The promoter region is GC-rich and contains neither a TATA-box nor a CAAT box. Transient expression assays using a series of constructs containing various lengths of the 5'-flanking region of the alpha o promoter demonstrated that the region 300-700 base pairs upstream of the transcription initiation sites is responsible for the basic promoter activity. The relative activity of alpha o promoter is 8-12-fold higher in cells expressing alpha o than in cells lacking alpha o. The level of alpha o in cells may also be regulated at the level of protein translation because deletions in the 5'-noncoding region of alpha o gene increase reporter enzyme expression without a corresponding increase in reporter enzyme mRNA level. Our results suggest that both transcriptional and post-transcriptional mechanisms are involved in regulating the expression of alpha o in vivo. Transcriptional regulation probably is important for control of tissue-specific expression, while posttranscriptional mechanisms may be used to regulate the alpha o level in cells. PMID:7961675

  13. Identification of noncollagenous sites encoding specific interactions and quaternary assembly of alpha 3 alpha 4 alpha 5(IV) collagen: implications for Alport gene therapy.

    PubMed

    Kang, Jeong Suk; Colon, Selene; Hellmark, Thomas; Sado, Yoshikazu; Hudson, Billy G; Borza, Dorin-Bogdan

    2008-12-12

    Defective assembly of alpha 3 alpha 4 alpha 5(IV) collagen in the glomerular basement membrane causes Alport syndrome, a hereditary glomerulonephritis progressing to end-stage kidney failure. Assembly of collagen IV chains into heterotrimeric molecules and networks is driven by their noncollagenous (NC1) domains, but the sites encoding the specificity of these interactions are not known. To identify the sites directing quaternary assembly of alpha 3 alpha 4 alpha 5(IV) collagen, correctly folded NC1 chimeras were produced, and their interactions with other NC1 monomers were evaluated. All alpha1/alpha 5 chimeras containing alpha 5 NC1 residues 188-227 replicated the ability of alpha 5 NC1 to bind to alpha3NC1 and co-assemble into NC1 hexamers. Conversely, substitution of alpha 5 NC1 residues 188-227 by alpha1NC1 abolished these quaternary interactions. The amino-terminal 58 residues of alpha3NC1 encoded binding to alpha 5 NC1, but this interaction was not sufficient for hexamer co-assembly. Because alpha 5 NC1 residues 188-227 are necessary and sufficient for assembly into alpha 3 alpha 4 alpha 5 NC1 hexamers, whereas the immunodominant alloantigenic sites of alpha 5 NC1 do not encode specific quaternary interactions, the findings provide a basis for the rational design of less immunogenic alpha 5(IV) collagen constructs for the gene therapy of X-linked Alport patients. PMID:18930919

  14. Lateral Gene Transfer from the Dead

    PubMed Central

    Szöllősi, Gergely J.; Tannier, Eric; Lartillot, Nicolas; Daubin, Vincent

    2013-01-01

    In phylogenetic studies, the evolution of molecular sequences is assumed to have taken place along the phylogeny traced by the ancestors of extant species. In the presence of lateral gene transfer, however, this may not be the case, because the species lineage from which a gene was transferred may have gone extinct or not have been sampled. Because it is not feasible to specify or reconstruct the complete phylogeny of all species, we must describe the evolution of genes outside the represented phylogeny by modeling the speciation dynamics that gave rise to the complete phylogeny. We demonstrate that if the number of sampled species is small compared with the total number of existing species, the overwhelming majority of gene transfers involve speciation to and evolution along extinct or unsampled lineages. We show that the evolution of genes along extinct or unsampled lineages can to good approximation be treated as those of independently evolving lineages described by a few global parameters. Using this result, we derive an algorithm to calculate the probability of a gene tree and recover the maximum-likelihood reconciliation given the phylogeny of the sampled species. Examining 473 near-universal gene families from 36 cyanobacteria, we find that nearly a third of transfer events (28%) appear to have topological signatures of evolution along extinct species, but only approximately 6% of transfers trace their ancestry to before the common ancestor of the sampled cyanobacteria. [Gene tree reconciliation; lateral gene transfer; macroevolution; phylogeny.] PMID:23355531

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

    SciTech Connect

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

    1989-01-01

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

  16. Alpha-amylase gene transcription in tissues of normal dog.

    PubMed

    Mocharla, H; Mocharla, R; Hodes, M E

    1990-02-25

    We studied the distribution of alpha-amylase mRNA in normal dog tissues by northern blotting (NB) and reverse transcription-polymerase chain reaction (RT-PCR) with human pancreatic (AMY2) and salivary (AMY1) alpha-amylase cDNA-specific primers. Analysis of poly(A+) RNA from various normal tissues by NB indicated the presence of detectable levels of alpha-amylase mRNA transcripts only in pancreas. Dot-blot analysis of DNA amplified with primers common to both (human) isoamylase mRNAs showed presence of alpha-amylase gene transcripts not only in pancreas but also in liver, small intestine, large intestine and fallopian tube. Traces of amylase gene transcripts were also observed in ovary, uterus and lung. Interestingly, amylase transcripts were not detectable in the parotid gland by NB or RT-PCR. We have also localized alpha-amylase mRNA transcripts to dog pancreas by in situ transcription and in situ hybridization. Our results suggest that there is high degree of homology between the alpha-amylase mRNA sequences in dog and human at least in the exon 3-4 regions of the human gene. PMID:2315015

  17. Differential expressions of essential and nonessential alpha-tubulin genes in Schizosaccharomyces pombe.

    PubMed

    Adachi, Y; Toda, T; Niwa, O; Yanagida, M

    1986-06-01

    The fission yeast Schizosaccharomyces pombe has two alpha-tubulin genes and one beta-tubulin gene. Gene disruption experiments showed that the alpha 1-tubulin gene (NDA2) is essential whereas the alpha 2 gene is dispensable. The alpha 2-disrupted cells missing alpha 2 transcript and alpha 2 polypeptide could grow and sporulate normally. The alpha 2 gene, however, was expressed in the wild type and the alpha 1 mutant. Alpha 2-Tubulin was distinguished as an electrophoretic band and was assembled into microtubules. The alpha 2-disrupted cells had an increased sensitivity to an antimicrotubule drug thiabendazole, and the alpha 1(cold-sensitive [cs]) alpha 2 (disrupted) cells became not only cs but also temperature sensitive. Northern blot experiments indicated that alpha 2 transcription was minor and constitutive whereas alpha 1 transcription was major and modulated, depending on the gene copy number of the alpha 2 gene. The amounts of alpha 1 and alpha 2 polypeptides estimated by beta-galactosidase activities of the lacZ-fused genes integrated on the chromosome and by intensities of the electrophoretic bands in crude tubulin fractions, however, were comparable, indicating that alpha 2 tubulin is not a minor subtype. We assume that the cells of Schizosaccharomyces pombe have no excess tubulin pool. alpha 1 mutants would then be blocked in the cell cycle because only half the amount of functional alpha-tubulin required for growth can be produced by the alpha 2 gene. On the other hand, the alpha 2-disrupted cells became viable because the synthesis of alpha 1 tubulin was increased by transcriptional or translational modulation or both. The real cause for essential alpha 1 and dispensable alpha 2 genes seems to be in their regulatory sequences instead of the coding sequences. PMID:3785193

  18. Horizontal gene transfer between bacteria and animals.

    PubMed

    Dunning Hotopp, Julie C

    2011-04-01

    Horizontal gene transfer is increasingly described between bacteria and animals. Such transfers that are vertically inherited have the potential to influence the evolution of animals. One classic example is the transfer of DNA from mitochondria and chloroplasts to the nucleus after the acquisition of these organelles by eukaryotes. Even today, many of the described instances of bacteria-to-animal transfer occur as part of intimate relationships such as those of endosymbionts and their invertebrate hosts, particularly insects and nematodes, while numerous transfers are also found in asexual animals. Both of these observations are consistent with modern evolutionary theory, in particular the serial endosymbiotic theory and Muller's ratchet. Although it is tempting to suggest that these particular lifestyles promote horizontal gene transfer, it is difficult to ascertain given the nonrandom sampling of animal genome sequencing projects and the lack of a systematic analysis of animal genomes for such transfers. PMID:21334091

  19. High expression hampers horizontal gene transfer.

    PubMed

    Park, Chungoo; Zhang, Jianzhi

    2012-01-01

    Horizontal gene transfer (HGT), the movement of genetic material from one species to another, is a common phenomenon in prokaryotic evolution. Although the rate of HGT is known to vary among genes, our understanding of the cause of this variation, currently summarized by two rules, is far from complete. The first rule states that informational genes, which are involved in DNA replication, transcription, and translation, have lower transferabilities than operational genes. The second rule asserts that protein interactivity negatively impacts gene transferability. Here, we hypothesize that high expression hampers HGT, because the fitness cost of an HGT to the recipient, arising from the 1) energy expenditure in transcription and translation, 2) cytotoxic protein misfolding, 3) reduction in cellular translational efficiency, 4) detrimental protein misinteraction, and 5) disturbance of the optimal protein concentration or cell physiology, increases with the expression level of the transferred gene. To test this hypothesis, we examined laboratory and natural HGTs to Escherichia coli. We observed lower transferabilities of more highly expressed genes, even after controlling the confounding factors from the two established rules and the genic GC content. Furthermore, expression level predicts gene transferability better than all other factors examined. We also confirmed the significant negative impact of gene expression on the rate of HGTs to 127 of 133 genomes of eubacteria and archaebacteria. Together, these findings establish the gene expression level as a major determinant of horizontal gene transferability. They also suggest that most successful HGTs are initially slightly deleterious, fixed because of their negligibly low costs rather than high benefits to the recipient. PMID:22436996

  20. Horizontal gene transfer, genome innovation and evolution.

    PubMed

    Gogarten, J Peter; Townsend, Jeffrey P

    2005-09-01

    To what extent is the tree of life the best representation of the evolutionary history of microorganisms? Recent work has shown that, among sets of prokaryotic genomes in which most homologous genes show extremely low sequence divergence, gene content can vary enormously, implying that those genes that are variably present or absent are frequently horizontally transferred. Traditionally, successful horizontal gene transfer was assumed to provide a selective advantage to either the host or the gene itself, but could horizontally transferred genes be neutral or nearly neutral? We suggest that for many prokaryotes, the boundaries between species are fuzzy, and therefore the principles of population genetics must be broadened so that they can be applied to higher taxonomic categories. PMID:16138096

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

    PubMed Central

    Whitelaw, E; Proudfoot, N J

    1983-01-01

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

  2. Gene Transfers Between Distantly Related Organisms

    NASA Technical Reports Server (NTRS)

    Doolittle, Russell F.

    2003-01-01

    With the completion of numerous microbial genome sequences, reports of individual gene transfers between distantly related prokaryotes have become commonplace. On the other hand, transfers between prokaryotes and eukaryotes still excite the imagination. Many of these claims may be premature, but some are certainly valid. In this chapter, the kinds of supporting data needed to propose transfers between distantly related organisms and cite some interesting examples are considered.

  3. Identification of the human ApoAV gene as a novel ROR{alpha} target gene

    SciTech Connect

    Lind, Ulrika; Nilsson, Tina; McPheat, Jane; Stroemstedt, Per-Erik; Bamberg, Krister; Balendran, Clare; Kang, Daiwu . E-mail: Daiwu.Kang@astrazeneca.com

    2005-04-29

    Retinoic acid receptor-related orphan receptor-{alpha} (ROR{alpha}) (NR1F1) is an orphan nuclear receptor with a potential role in metabolism. Previous studies have shown that ROR{alpha} regulates transcription of the murine Apolipoprotein AI gene and human Apolipoprotein CIII genes. In the present study, we present evidence that ROR{alpha} also induces transcription of the human Apolipoprotein AV gene, a recently identified apolipoprotein associated with triglyceride levels. Adenovirus-mediated overexpression of ROR{alpha} increased the endogenous expression of ApoAV in HepG2 cells and ROR{alpha} also enhanced the activity of an ApoAV promoter construct in transiently transfected HepG2 cells. Deletion and mutation studies identified three AGGTCA motifs in the ApoAV promoter that mediate ROR{alpha} transactivation, one of which overlaps with a previously identified binding site for PPAR{alpha}. Together, these results suggest a novel mechanism whereby ROR{alpha} modulates lipid metabolism and implies ROR{alpha} as a potential target for the treatment of dyslipidemia and atherosclerosis.

  4. Horizontal gene transfer of stress resistance genes through plasmid transport.

    PubMed

    Shoeb, Erum; Badar, Uzma; Akhter, Jameela; Shams, Hina; Sultana, Maria; Ansari, Maqsood A

    2012-03-01

    The horizontal gene transfer of plasmid-determined stress tolerance was achieved under lab conditions. Bacterial isolates, Enterobacter cloacae (DGE50) and Escherichia coli (DGE57) were used throughout the study. Samples were collected from contaminated marine water and soil to isolate bacterial strains having tolerance against heavy metals and antimicrobial agents. We have demonstrated plasmid transfer, from Amp(+)Cu(+)Zn(-) strain (DGE50) to Amp(-)Cu(-)Zn(+) strain (DGE57), producing Amp(+)Cu(+)Zn(+) transconjugants (DGE(TC50→57)) and Amp(+)Cu(-)Zn(+) transformants (DGE(TF50→57)). DGE57 did not carry any plasmid, therefore, it can be speculated that zinc tolerance gene in DGE57 is located on chromosome. DGE50 was found to carry three plasmids, out of which two were transferred through conjugation into DGE57, and only one was transferred through transformation. Plasmid transferred through transformation was one out of the two transferred through conjugation. Through the results of transformation it was revealed that the genes of copper and ampicillin tolerance in DGE50 were located on separate plasmids, since only ampicillin tolerance genes were transferred through transformation as a result of one plasmid transfer. By showing transfer of plasmids under lab conditions and monitoring retention of respective phenotype via conjugation and transformation, it is very well demonstrated how multiple stress tolerant strains are generated in nature. PMID:22805823

  5. Characterization of the L. manihotivorans alpha-amylase gene.

    PubMed

    Morlon-Guyot, J; Mucciolo-Roux, F; Rodriguez Sanoja, R; Guyot, J P

    2001-07-01

    Primers and probes were established from the sequences of the alpha-amylase genes (amyA) of L. amylovorus CIP 102989 and of L. plantarum A6 (Giraud and Cuny 1997). They were successfully used for the detection of the amyA gene in L. manihotivorans strain LMG 18010T and a 2842 bp region, containing the entire gene (2706 bp) with its putative promoter has been sequenced. More than 98% nucleotide sequence identities was found with L. amylovorus and L. plantarum amyA genes. The deduced amino acid sequence shares more than 96% amino acid sequence identities with L. amylovorus and L. plantarum alpha-amylases, and also 65% and 59% identities with the alpha-amylases of B. subtilis and S. bovis, respectively. The 3' terminal part of L. manihotivorans LMG 18010T amyA gene contained four repeated sequences (SRU). The amyA genes of the three lactobacilli species differed mainly in the number of SRU and in the size of the flanking regions of the SRU. PMID:11697143

  6. In vitro gene transfer by electrosonoporation.

    PubMed

    Escoffre, J M; Kaddur, K; Rols, M P; Bouakaz, A

    2010-10-01

    Among the nonviral methods for gene delivery in vitro, electroporation is simple, inexpensive and safe. To upregulate the expression level of transfected gene, we investigated the applicability of electrosonoporation. This approach consists of a combination of electric pulses and ultrasound assisted with gas microbubbles. Cells were first electroporated with plasmid DNA encoding-enhanced green fluorescent protein and then sonoporated in presence of contrast microbubbles. Twenty-four hours later, cells that received electrosonoporation demonstrated a four-fold increase in transfection level and a six-fold increase in transfection efficiency compared with cells having undergone electroporation alone. Although electroporation induced the formation of DNA aggregates into the cell membrane, sonoporation induced its direct propulsion into the cytoplasm. Sonoporation can improve the transfer of electro-induced DNA aggregates by allowing its free and rapid entrance into the cells. These results demonstrated that in vitro gene transfer by electrosonoporation could provide a new potent method for gene transfer. PMID:20850028

  7. Human gene transfer: Characterization of human tumor-infiltrating lymphocytes as vehicles for retroviral-mediated gene transfer in man

    SciTech Connect

    Kasid, A.; Morecki, S.; Aebersold, P.; Cornetta, K.; Culver, K.; Freeman, S.; Director, E.; Lotze, M.T.; Blaese, R.M.; Anderson, W.F.; Rosenberg, S.A. )

    1990-01-01

    Tumor-infiltrating lymphocytes (TILs) are cells generated from tumor suspensions cultured in interleukin 2 that can mediate cancer regression when adoptively transferred into mice or humans. Since TILs proliferate rapidly in vitro, recirculate, and preferentially localize at the tumor site in vivo, they provide an attractive model for delivery of exogenous genetic material into man. To determine whether efficient gene transfer into TILs is feasible. The authors transduced human TILs with the bacterial gene for neomycin-resistance (Neo{sup R}) using the retroviral vector N2. The transduced TIL populations were stable and polyclonal with respect to the intact Neo{sup R} gene integration and expressed high levels of neomycin phosphotransferase activity. The Neo{sup R} gene insertion did not alter the in vitro growth pattern and interleukin 2 dependence of the transduced TILs. Analyses of T-cell receptor gene rearrangement for {beta}- and {gamma}-chain genes revealed the oligoclonal nature of the TIL populations with no major change in the DNA rearrangement patterns or the levels of mRNA expression of the {beta} and {gamma} chains following transduction and selection of TILs in the neomycin analog G418. Human TILs expressed mRNA for tumor necrosis factors ({alpha} and {beta}) and interleukin 2 receptor P55. This pattern of cytokine-mRNA expression was not significantly altered following the transduction of TILs. The studies demonstrate the feasibility of TILs as suitable cellular vehicles for the introduction of therapeutic genes into patients receiving autologous TILs.

  8. Interleukin-2 gene transfer potentiates the alpha-galactosylceramide-stimulated antitumor effect by the induction of TRAIL in NKT and NK cells in mouse models of subcutaneous and metastatic carcinoma.

    PubMed

    Nishihori, Yoshiki; Kato, Kazunori; Tanaka, Maki; Okamoto, Tetsuro; Hagiwara, Seiya; Araki, Naoko; Kogawa, Katsuhisa; Kuribayashi, Kageaki; Nakamura, Kiminori; Niitsu, Yoshiro

    2009-09-01

    Alpha-Galactosylceramide (alpha-GalCer) is a potent CD1d ligand that activates natural killer like T-cells (NKT), leading to the production of helper T (Th) 1 and Th2 cytokines that mediate various immunemodulatory and antitumor effects. Here, we determined whether the administration of adenovirus-vector-encoding mouse interleukin-2 (AdmIL-2) can augment the antitumor effect of alpha-GalCer on subcutaneous and metastatic tumors in mice. Mice were intraperitoneally injected with alpha-GalCer on days 7, 10 and 13 after tumor inoculation, with or without intratumoral injection of AdmIL-2 on day 7. alpha-GalCer treatment increased the serum levels of interferon-gamma, while intratumoral injection of AdmIL-2 elevated serum IL-2 levels. A combination of alpha-GalCer and AdmIL-2 (alpha-GalCer/AdmIL-2) inhibited the in vivo tumor growth and improved the survival of tumor-bearing mice, as compared to the use of a single agent. Experiments on spontaneous metastasis models revealed that alpha-GalCer/AdmIL-2 reduced lung metastasis and prolonged survival, as compared to control groups. In addition, the splenic and liver mononuclear cells from mice treated with alpha-GalCer/AdmIL-2 showed enhanced cytolytic activity against NK-sensitive YAC-1 and NK-resistant 3LL tumors. Moreover, alpha-GalCer/AdmIL-2 treatment expanded the absolute numbers of lung and liver NK, NKT and T-cells as well as the TNF-related apoptosis-inducing ligand (TRAIL) expression of these cells. This study shows the efficacy of alpha-GalCer/AdmIL-2 immunomodulatory therapy, and provides a cellular mechanism on how it exerts the antitumor effects. PMID:19901518

  9. Tumour necrosis factor-alpha gene polymorphisms and Alzheimer's disease.

    PubMed

    Culpan, Doris; MacGowan, Sian H; Ford, Julia M; Nicoll, James A R; Griffin, W Sue; Dewar, Deborah; Cairns, Nigel J; Hughes, Anthony; Kehoe, Patrick G; Wilcock, Gordon K

    2003-10-16

    Recent findings suggest that production of pro-inflammatory cytokines, such as tumour necrosis factor-alpha (TNF-alpha), is increased in the brains of people with Alzheimer's disease (AD). We used direct sequencing methods on a section of the enhancer/promoter region and on a smaller fragment located 10.5 kb upstream of the TNF-alpha gene to respectively examine TNF-alpha polymorphisms and TNF-a and -b microsatellite alleles in a cohort of 235 post-mortem confirmed AD and 130 control cases. None of the TNF-alpha point mutations or microsatellite alleles investigated proved to be independent risk factors for AD. However, when -308/A, -238/G and TNF-a2 were examined as a 2-1-2 haplotype, we observed that the absence of that haplotype was significantly associated with AD (P = 0.014, Fisher's exact test) suggesting that the 2-1-2 haplotype may be protective against AD. PMID:12962917

  10. Genetic analysis of the Drosophila Gs(alpha) gene.

    PubMed Central

    Wolfgang, W J; Hoskote, A; Roberts, I J; Jackson, S; Forte, M

    2001-01-01

    One of the best understood signal transduction pathways activated by receptors containing seven transmembrane domains involves activation of heterotrimeric G-protein complexes containing Gs(alpha), the subsequent stimulation of adenylyl cyclase, production of cAMP, activation of protein kinase A (PKA), and the phosphorylation of substrates that control a wide variety of cellular responses. Here, we report the identification of "loss-of-function" mutations in the Drosophila Gs(alpha) gene (dgs). Seven mutants have been identified that are either complemented by transgenes representing the wild-type dgs gene or contain nucleotide sequence changes resulting in the production of altered Gs(alpha) protein. Examination of mutant alleles representing loss-of-Gs(alpha) function indicates that the phenotypes generated do not mimic those created by mutational elimination of PKA. These results are consistent with the conclusion reached in previous studies that activation of PKA, at least in these developmental contexts, does not depend on receptor-mediated increases in intracellular cAMP, in contrast to the predictions of models developed primarily on the basis of studies in cultured cells. PMID:11454767

  11. Viral Vectors for in Vivo Gene Transfer

    NASA Astrophysics Data System (ADS)

    Thévenot, E.; Dufour, N.; Déglon, N.

    The transfer of DNA into the nucleus of a eukaryotic cell (gene transfer) is a central theme of modern biology. The transfer is said to be somatic when it refers to non-germline organs of a developed individual, and germline when it concerns gametes or the fertilised egg of an animal, with the aim of transmitting the relevant genetic modification to its descendents [1]. The efficient introduction of genetic material into a somatic or germline cell and the control of its expression over time have led to major advances in understanding how genes work in vivo, i.e., in living organisms (functional genomics), but also to the development of innovative therapeutic methods (gene therapy). The efficiency of gene transfer is conditioned by the vehicle used, called the vector. Desirable features for a vector are as follows: Easy to produce high titer stocks of the vector in a reproducible way. Absence of toxicity related to transduction (transfer of genetic material into the target cell, and its expression there) and no immune reaction of the organism against the vector and/or therapeutic protein. Stability in the expression of the relevant gene over time, and the possibility of regulation, e.g., to control expression of the therapeutic protein on the physiological level, or to end expression at the end of treatment. Transduction of quiescent cells should be as efficient as transduction of dividing cells. Vectors currently used fall into two categories: non-viral and viral vectors. In non-viral vectors, the DNA is complexed with polymers, lipids, or cationic detergents (described in Chap. 3). These vectors have a low risk of toxicity and immune reaction. However, they are less efficient in vivo than viral vectors when it comes to the number of cells transduced and long-term transgene expression. (Naked DNA transfer or electroporation is rather inefficient in the organism. This type of gene transfer will not be discussed here, and the interested reader is referred to the

  12. Unsupervised learning in detection of gene transfer.

    PubMed

    Hamel, L; Nahar, N; Poptsova, M S; Zhaxybayeva, O; Gogarten, J P

    2008-01-01

    The tree representation as a model for organismal evolution has been in use since before Darwin. However, with the recent unprecedented access to biomolecular data, it has been discovered that, especially in the microbial world, individual genes making up the genome of an organism give rise to different and sometimes conflicting evolutionary tree topologies. This discovery calls into question the notion of a single evolutionary tree for an organism and gives rise to the notion of an evolutionary consensus tree based on the evolutionary patterns of the majority of genes in a genome embedded in a network of gene histories. Here, we discuss an approach to the analysis of genomic data of multiple genomes using bipartition spectral analysis and unsupervised learning. An interesting observation is that genes within genomes that have evolutionary tree topologies, which are in substantial conflict with the evolutionary consensus tree of an organism, point to possible horizontal gene transfer events which often delineate significant evolutionary events. PMID:18509479

  13. Lateral gene transfer in the subsurface

    SciTech Connect

    Barkay, Tamar; Sobecky, Patricia

    2007-08-27

    Lateral gene transfer (LGT) is an important adaptive mechanism among prokaryotic organisms. This mechanism is particularly important for the response of microorganisms to changing environmental conditions because it facilitates the transfer of a large number of genes and their rapid expression. Together the transferred genes promote rapid genetic and metabolic changes that may enhance survival to newly established and sometimes hostile environmental conditions. The goal of our project was to examine if and how LGT enhances microbial adaptation to toxic heavy metals in subsurface environments that had been contaminated by mixed wastes due to activities associated with the production of nuclear energy and weapons. This task has been accomplished by dividing the project to several sub-tasks. Thus, we: (1) Determined the level of resistance of subsurface bacterial isolates to several toxic metals, all identified as pollutants of concern in subsurface environments; (2) Designed, tested, and applied, a molecular approach that determined whether metal resistance genes had evolved by LGT among subsurface bacteria; and (3) Developed a DNA hybridization array for the identification of broad host range plasmids and of metal resistance plasmids. The results are briefly summarized below with references to published papers and manuscripts in preparation where details about our research can be found. Additional information may be found in copies of our published manuscripts and conference proceedings, and our yearly reports that were submitted through the RIMS system.

  14. Gene Therapy for Alpha-1 Antitrypsin Deficiency Lung Disease.

    PubMed

    Chiuchiolo, Maria J; Crystal, Ronald G

    2016-08-01

    Alpha-1 antitrypsin (AAT) deficiency, characterized by low plasma levels of the serine protease inhibitor AAT, is associated with emphysema secondary to insufficient protection of the lung from neutrophil proteases. Although AAT augmentation therapy with purified AAT protein is efficacious, it requires weekly to monthly intravenous infusion of AAT purified from pooled human plasma, has the risk of viral contamination and allergic reactions, and is costly. As an alternative, gene therapy offers the advantage of single administration, eliminating the burden of protein infusion, and reduced risks and costs. The focus of this review is to describe the various strategies for AAT gene therapy for the pulmonary manifestations of AAT deficiency and the state of the art in bringing AAT gene therapy to the bedside. PMID:27564673

  15. Human hTM. cap alpha. gene: Expression in muscle and nonmuscle tissue

    SciTech Connect

    MacLeod, A.R.; Gooding, C.

    1988-01-01

    The authors isolated a cDNA clone from a human skeletal muscle library which contains the complete protein-coding sequence of a skeletal muscle ..cap alpha..-tropomyosin. This cDNA sequence defines a fourth human tropomyosin gene, the hTM..cap alpha.. gene, which is distinct from the hTM/sub nm/ gene encoding a closely related isoform of skeletal muscle ..cap alpha..-tropomyosin. In cultured human fibroblasts, the hTM..cap alpha.. gene encodes both skeletal-muscle- and smooth-muscle-type ..cap alpha..-tropomyosins by using an alternative mRNA-splicing mechanism.

  16. Clinical Applications Involving CNS Gene Transfer

    PubMed Central

    Kantor, Boris; McCown, Thomas; Leone, Paola; Gray, Steven J.

    2015-01-01

    Diseases of the central nervous system (CNS) have traditionally been the most difficult to treat by traditional pharmacological methods, due mostly to the blood–brain barrier and the difficulties associated with repeated drug administration targeting the CNS. Viral vector gene transfer represents a way to permanently provide a therapeutic protein within the nervous system after a single administration, whether this be a gene replacement strategy for an inherited disorder or a disease-modifying protein for a disease such as Parkinson's. Gene therapy approaches for CNS disorders has evolved considerably over the last two decades. Although a breakthrough treatment has remained elusive, current strategies are now considerably safer and potentially much more effective. This chapter will explore the past, current, and future status of CNS gene therapy, focusing on clinical trials utilizing adeno-associated virus and lentiviral vectors. PMID:25311921

  17. Perinatal Gene Transfer to the Liver

    PubMed Central

    McKay, Tristan R; Rahim, Ahad A; Buckley, Suzanne M.K; Ward, Natalie J; Chan, Jerry K.Y; Howe, Steven J; Waddington, Simon N

    2011-01-01

    The liver acts as a host to many functions hence raising the possibility that any one may be compromised by a single gene defect. Inherited or de novo mutations in these genes may result in relatively mild diseases or be so devastating that death within the first weeks or months of life is inevitable. Some diseases can be managed using conventional medicines whereas others are, as yet, untreatable. In this review we consider the application of early intervention gene therapy in neonatal and fetal preclinical studies. We appraise the tools of this technology, including lentivirus, adenovirus and adeno-associated virus (AAV)-based vectors. We highlight the application of these for a range of diseases including hemophilia, urea cycle disorders such as ornithine transcarbamylase deficiency, organic acidemias, lysosomal storage diseases including mucopolysaccharidoses, glycogen storage diseases and bile metabolism. We conclude by assessing the advantages and disadvantages associated with fetal and neonatal liver gene transfer. PMID:21774770

  18. The human HLA class II alpha chain gene DZ alpha is distinct from genes in the DP, DQ and DR subregions.

    PubMed Central

    Trowsdale, J; Kelly, A

    1985-01-01

    A new human HLA class II alpha gene DZ alpha was sequenced. The structure and organisation of the gene was similar to other alpha chain genes except for a particularly small intron (95 bp) after the exon encoding the alpha 2 domain, and the position of the stop codon, which was on a different exon to that encoding the cytoplasmic portion of the molecule. Comparison of the DZ alpha sequence with other class II genes showed that the gene is about as distantly related to alpha chain genes in the DP, DQ and DR subregions as they are to each other. The DZ alpha gene results in an unusually large mRNA transcript of greater than 3.0 kb, detected on Northern blots of B cell lines. From the sequence, there are no obvious features that would render DZ alpha a pseudogene, except for an unusual poly(A)+ addition signal, ACTAAA. Analysis of Northern blots shows that sequences downstream (3') of this signal are present in mature mRNA. The large transcripts are probably due to defects in the signals for processing of the mRNA transcript at the 3' end. Images Fig. 4. PMID:3000765

  19. Association of tumor necrosis factor-alpha(TNF-alpha)gene promoter polymorphisms with TNF-alpha response to endotoxin (LPS)in calves.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Attenuation of TNF-alpha gene expression is a NF-'B-mediated regulatory process essential to avoid deleterious effects of excessive or prolonged synthesis of TNF-alpha, especially in response to gram-negative bacterial infection or LPS. An uncommon G to A transition polymorphism in the promoter regi...

  20. Modulation of gene expression by alpha-tocopherol and alpha-tocopheryl phosphate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The naturally occurring vitamin E analogue, alpha-tocopheryl phosphate (alphaTP), has been reported to be more potent in reducing cell proliferation and the expression of the CD36 scavenger receptor than the un-phosphorylated alpha-tocopherol (alpha T). We have now assessed the effects of alpha T an...

  1. cap alpha. -skeletal and. cap alpha. -cardiac actin genes are coexpressed in adult human skeletal muscle and heart

    SciTech Connect

    Gunning, P.; Ponte, P.; Blau, H.; Kedes, L.

    1983-11-01

    The authors determined the actin isotypes encoded by 30 actin cDNA clones previously isolated from an adult human muscle cDNA library. Using 3' untranslated region probes, derived from ..cap alpha.. skeletal, ..beta..- and ..gamma..-actin cDNAs and from an ..cap alpha..-cardiac actin genomic clone, they showed that 28 of the cDNAs correspond to ..cap alpha..-skeletal actin transcripts. Unexpectedly, however, the remaining two cDNA clones proved to derive from ..cap alpha..-cardiac actin mRNA. Sequence analysis confirmed that the two skeletal muscle ..cap alpha..-cardiac actin cDNAs are derived from transcripts of the cloned ..cap alpha..-cardiac actin gene. Comparison of total actin mRNA levels in adult skeletal muscle and adult heart revealed that the steady-state levels in skeletal muscle are about twofold greater, per microgram of total cellular RNA, than those in heart. Thus, in skeletal muscle and in heart, both of the sarcomeric actin mRNA isotypes are quite abundant transcripts. They conclude that ..cap alpha..-skeletal and ..cap alpha..-cardiac actin genes are coexpressed as an actin pair in human adult striated muscles. Since the smooth-muscle actins (aortic and stomach) and the cytoplasmic actins (..beta.. and ..gamma..) are known to be coexpressed in smooth muscle and nonmuscle cells, respectively, they postulate that coexpression of actin pairs may be a common feature of mammalian actin gene expression in all tissues.

  2. Foamy virus vectors for gene transfer

    PubMed Central

    Trobridge, Grant D.

    2009-01-01

    Foamy virus (FV) vectors are efficient gene delivery vehicles that have shown great promise for gene therapy in preclinical animal models. FVs or spumaretroviruses are not endemic in humans, but are prevalent in nonhuman primates and in other mammals. They have evolved means for efficient horizontal transmission in their host species without pathology. FV vectors have several unique properties that make them well-suited for therapeutic gene transfer including a desirable safety profile, a broad tropism, a large transgene capacity, and the ability to persist in quiescent cells. They mediate efficient and stable gene transfer to hematopoietic stem cells (HSCs) in mouse models, and in the canine large animal model. Analysis of FV vector integration sites in vitro and in hematopoietic repopulating cells shows they have a unique integration profile, and suggests they may be safer than gammaretroviruses or lentiviral vectors. Here properties of FVs relevant to the safety and efficacy of FV vectors are discussed. The development of FV vector systems is described, and studies evaluating their potential in vitro, and in small and large animal models is reviewed. PMID:19743892

  3. Horizontal Gene Transfer, Dispersal and Haloarchaeal Speciation

    PubMed Central

    Papke, R. Thane; Corral, Paulina; Ram-Mohan, Nikhil; de la Haba, Rafael R.; Sánchez-Porro, Cristina; Makkay, Andrea; Ventosa, Antonio

    2015-01-01

    The Halobacteria are a well-studied archaeal class and numerous investigations are showing how their diversity is distributed amongst genomes and geographic locations. Evidence indicates that recombination between species continuously facilitates the arrival of new genes, and within species, it is frequent enough to spread acquired genes amongst all individuals in the population. To create permanent independent diversity and generate new species, barriers to recombination are probably required. The data support an interpretation that rates of evolution (e.g., horizontal gene transfer and mutation) are faster at creating geographically localized variation than dispersal and invasion are at homogenizing genetic differences between locations. Therefore, we suggest that recurrent episodes of dispersal followed by variable periods of endemism break the homogenizing forces of intrapopulation recombination and that this process might be the principal stimulus leading to divergence and speciation in Halobacteria. PMID:25997110

  4. RADIATIVE TRANSFER MODELING OF Ly{alpha} EMITTERS. II. NEW EFFECTS ON GALAXY CLUSTERING

    SciTech Connect

    Zheng Zheng; Cen Renyue; Trac, Hy; Miralda-Escude, Jordi

    2011-01-01

    We study the clustering properties of z {approx} 5.7 Ly{alpha} emitters (LAEs) in a cosmological reionization simulation with a full Ly{alpha} radiative transfer calculation. Ly{alpha} radiative transfer substantially modifies the intrinsic Ly{alpha} emission properties, compared to observed ones, depending on the density and velocity structure environment around the Ly{alpha}-emitting galaxy. This environment-dependent Ly{alpha} selection introduces new features in LAE clustering, suppressing (enhancing) the line-of-sight (transverse) density fluctuations and giving rise to scale-dependent galaxy bias. In real space, the contours of the three-dimensional two-point correlation function of LAEs appear to be prominently elongated along the line of sight on large scales, an effect that is opposite to and much stronger than the linear redshift-space distortion effect. The projected two-point correlation function is greatly enhanced in amplitude by a factor of up to a few, compared to the case without the environment-dependent selection effect. The new features in LAE clustering can be understood with a simple, physically motivated model, where Ly{alpha} selection depends on matter density, velocity, and their gradients. We discuss the implications and consequences of the effects on galaxy clustering from Ly{alpha} selection in interpreting clustering measurements and in constraining cosmology and reionization from LAEs.

  5. Probable new type of reaction mechanism: Double. cap alpha. direct transfer process

    SciTech Connect

    Xu Shu-wei; Wu Guo-hua; Miao Rong-zhi; Han Fei

    1983-10-01

    It is assumed that /sup 8/Be consists of two ..cap alpha.. particles which are close to each other in configuration space. A spectroscopic density of /sup 8/Be cluster in the residue nuclei is then obtained, which is proportional to the square of the preformation probability of ..cap alpha.. particle at nuclear surface. Using the improved method of parametrization of EFR-DWBA overlap integral,/sup 1//sup en-dash//sup 2/ we calculate the double differential energy spectra and angular distributions of ..cap alpha.. particles for the reactions /sup 209/Bi (/sup 12/C, ..cap alpha..) /sup 217/Fr and extract the preformation probability of ..cap alpha.. particle at the surface of /sup 217/Fr nuclei from fitting the experimental data. The agreement within the range of calculation error between the preformation probabilities extracted from transfer reactions and ..cap alpha.. decay suggests that the reaction /sup 209/Bi(/sup 12/C, ..cap alpha..) /sup 217/Fr may be explained as a double ..cap alpha.. direct transfer process.

  6. Polymorphism in the interferon-alpha gene family.

    PubMed Central

    Golovleva, I.; Kandefer-Szerszen, M.; Beckman, L.; Lundgren, E.

    1996-01-01

    A pronounced genetic polymorphism of the interferon type I gene family has been assumed on the basis of RFLP analysis of the genomic region as well as the large number of sequences published compared to the number of loci. However, IFNA2 is the only locus that has been carefully analyzed concerning gene frequency, and only naturally occurring rare alleles have been found. We have extended the studies on a variation of expressed sequences by studying the IFNA1, IFNA2, IFNA10, IFNA13, IFNA14, and IFNA17 genes. Genomic white-blood-cell DNA from a population sample of blood donors and from a family material were screened by single-nucleotide primer extension (allele-specific primer extension) of PCR fragments. Because of sequence similarities, in some cases "nested" PCR was used, and, when applicable, restriction analysis or control sequencing was performed. All individuals carried the interferon-alpha 1 and interferon-alpha 13 variants but not the LeIF D variant. At the IFNA2 and IFNA14 loci only one sequence variant was found, while in the IFNA10 and IFNA17 groups two alleles were detected in each group. The IFNA10 and IFNA17 alleles segregated in families and showed a close fit to the Hardy-Weinberg equilibrium. There was a significant linkage disequilibrium between IFNA10 and IFNA17 alleles. The fact that the extent of genetic polymorphism was lower than expected suggests that a majority of the previously described gene sequences represent nonpolymorphic rare mutants that may have arisen in tumor cell lines. Images Figure 1 Figure 2 Figure 3 PMID:8751858

  7. Molecular genetic and expression analysis of alpha-tocopherol transfer protein mRNA in German shepherd dogs with degenerative myelopathy.

    PubMed

    Fechner, Henry; Johnston, Pamela E; Sharp, Nicholas J; Montague, Paul; Griffiths, Ian R; Wang, Xiaomin; Olby, Natasha; Looman, Alfred C; Poller, Wolfgang; Flegel, Thomas

    2003-01-01

    Degenerative Myelopathy (DM) is a progressive neurological disorder of the spinal cord preferentially occurring in German shepherd dogs. The pathogenesis of the disease is unknown. However, there are indications that vitamin E deficiency may be involved in the pathogenesis of DM. Therefore, we analyzed the expression and the nucleotide sequence of the canine alpha-tocopherol transfer protein (alpha Ttp) of German shepherd dogs with DM in order to determine whether a deficiency or a defect of the alpha Ttp could be a primary factor in the pathogenesis of DM, as found in human patients with Ataxia with vitamin E deficiency (AVED). The cDNA of the coding region of the canine alpha Ttp-mRNA was generated from total liver RNA using RT-PCR and 5' RACE technique. We determined the sequence of 707 out of 834 base pairs or 84.8% of the canine alpha Ttp coding region. Sequence comparison of canine alpha Ttp between affected and control dogs revealed no differences in either nucleotide or predicted amino acid sequence. Using Northern blot analysis alpha Ttp-mRNA expression was solely found in the liver of the dogs, rats and humans, while various other organs showed no alpha Ttp-mRNA expression. No significant differences in expression levels of canine alpha Ttp mRNA were found between DM and control dogs. Our data suggest that the canine alpha Ttp gene is unlikely to be involved in the pathogenesis of DM in German shepherd dogs. PMID:12592926

  8. Simple rapid method for gene transfer

    SciTech Connect

    Cockburn, A.F.; Meier, H.

    1990-01-30

    The object of the present invention is to provide methods for gene transfer that reduce or eliminate cellular pretreatment steps, e.g., the removal of cell wall by chemical or enzymatic methods, is rapid and can be practiced without the need of additional expensive equipment. Cells, embryos or tissues selected for genetic manipulation are suspended in an Eppendorf tube in an aliquot of the desired genetic material to be transferred to which the resulting mixture is added and is agitated by vortexing from about 30 to about 90 seconds. The cells, embryos or tissue are sedimented and the DNA supernatant removed. After sedimentation, the injected material is resuspended in or on a growth medium to assay for expression.

  9. Gene Transfer between Salmonella enterica Serovar Typhimurium inside Epithelial Cells

    PubMed Central

    Ferguson, Gayle C.; Heinemann, Jack A.; Kennedy, Martin A.

    2002-01-01

    Virulence and antibiotic resistance genes transfer between bacteria by bacterial conjugation. Conjugation also mediates gene transfer from bacteria to eukaryotic organisms, including yeast and human cells. Predicting when and where genes transfer by conjugation could enhance our understanding of the risks involved in the release of genetically modified organisms, including those being developed for use as vaccines. We report here that Salmonella enterica serovar Typhimurium conjugated inside cultured human cells. The DNA transfer from donor to recipient bacteria was proportional to the probability that the two types of bacteria occupied the same cell, which was dependent on viable and invasive bacteria and on plasmid tra genes. Based on the high frequencies of gene transfer between bacteria inside human cells, we suggest that such gene transfers occur in situ. The implications of gene transfer between bacteria inside human cells, particularly in the context of antibiotic resistance, are discussed. PMID:11914355

  10. Effect of long-term exposure to mobile phone radiation on alpha-Int1 gene sequence of Candida albicans

    PubMed Central

    Shahin-jafari, Ariyo; Bayat, Mansour; Shahhosseiny, Mohammad Hassan; Tajik, Parviz; Roudbar-mohammadi, Shahla

    2015-01-01

    Over the last decade, communication industries have witnessed a tremendous expansion, while, the biological effects of electromagnetic waves have not been fully elucidated. Current study aimed at evaluating the mutagenic effect of long-term exposure to 900-MHz radiation on alpha-Int1 gene sequences of Candida albicans. A standard 900 MHz radiation generator was used for radiation. 10 ml volumes from a stock suspension of C. albicans were transferred into 10 polystyrene tubes. Five tubes were exposed at 4 °C to a fixed magnitude of radiation with different time periods of 10, 70, 210, 350 and 490 h. The other 5 tubes were kept far enough from radiation. The samples underwent genomic DNA extraction. PCR amplification of alpha-Int1 gene sequence was done using one set of primers. PCR products were resolved using agarose gel electrophoresis and the nucleotide sequences were determined. All samples showed a clear electrophoretic band around 441 bp and further sequencing revealed the amplified DNA segments are related to alpha-Int1 gene of the yeast. No mutations in the gene were seen in radiation exposed samples. Long-term exposure of the yeast to mobile phone radiation under the above mentioned conditions had no mutagenic effect on alpha-Int1 gene sequence. PMID:27081370

  11. Effect of long-term exposure to mobile phone radiation on alpha-Int1 gene sequence of Candida albicans.

    PubMed

    Shahin-Jafari, Ariyo; Bayat, Mansour; Shahhosseiny, Mohammad Hassan; Tajik, Parviz; Roudbar-Mohammadi, Shahla

    2016-05-01

    Over the last decade, communication industries have witnessed a tremendous expansion, while, the biological effects of electromagnetic waves have not been fully elucidated. Current study aimed at evaluating the mutagenic effect of long-term exposure to 900-MHz radiation on alpha-Int1 gene sequences of Candida albicans. A standard 900 MHz radiation generator was used for radiation. 10 ml volumes from a stock suspension of C. albicans were transferred into 10 polystyrene tubes. Five tubes were exposed at 4 °C to a fixed magnitude of radiation with different time periods of 10, 70, 210, 350 and 490 h. The other 5 tubes were kept far enough from radiation. The samples underwent genomic DNA extraction. PCR amplification of alpha-Int1 gene sequence was done using one set of primers. PCR products were resolved using agarose gel electrophoresis and the nucleotide sequences were determined. All samples showed a clear electrophoretic band around 441 bp and further sequencing revealed the amplified DNA segments are related to alpha-Int1 gene of the yeast. No mutations in the gene were seen in radiation exposed samples. Long-term exposure of the yeast to mobile phone radiation under the above mentioned conditions had no mutagenic effect on alpha-Int1 gene sequence. PMID:27081370

  12. Non random usage of T cell receptor alpha gene expression in atopy using anchored PCR.

    PubMed

    Mansur, A H; Gelder, C M; Holland, D; Campell, D A; Griffin, A; Cunliffe, W; Markham, A F; Morrison, J F

    1996-01-01

    The T cell receptor (TCR) alpha beta heterodimer recognises antigenic peptide fragments presented by Class II MHC. This interaction initiates T cell activation and cytokine release with subsequent recruitment of inflammatory cells. Previous work from our group suggests a qualitative difference in variable alpha gene expression in atopy as compared to non atopic controls. In this study we examine TCR alpha repertoire using anchored PCR to provide a quantitative assessment of the V alpha and J alpha repertoire. One atopic (DRB1*0701,DRB1*15: DRB4*0101, DRB5*01: DQB1* 0303, DQB1*601/2) and one non-atopic (DRB1*0701,DRB1*03011/2: DRB4*01, DRB3*0x: DQB1* 0303, DQB1*0201/2) control were studied. Variable gene usage was markedly limited in the atopic individual. V alpha 1, 3, 8 accounted for 60% and J alpha 12, 31 30% of the gene usage. There was evidence of preferential V alpha-J alpha gene pairing and clonal expansion. We conclude that there is a marked non random TCR alpha gene distribution in atopy using both V alpha family and anchored PCR. This may be due in part to antigen driven clonal expansion. PMID:9095269

  13. The human alpha 2(IV) collagen gene, COL4A2, is syntenic with the alpha 1(IV) gene, COL4A1, on chromosome 13.

    PubMed

    Solomon, E; Hall, V; Kurkinen, M

    1987-05-01

    We have previously assigned the gene for the alpha 1 chain of type IV collagen to chromosome 13. In this report we show that the gene coding for the second chain of this heterotrimer is on the same chromosome. This is the first example of the genes for both chains of one collagen molecule being syntenic. PMID:3674752

  14. Reducible cationic lipids for gene transfer.

    PubMed Central

    Wetzer, B; Byk, G; Frederic, M; Airiau, M; Blanche, F; Pitard, B; Scherman, D

    2001-01-01

    One of the main challenges of gene therapy remains the increase of gene delivery into eukaryotic cells. We tested whether intracellular DNA release, an essential step for gene transfer, could be facilitated by using reducible cationic DNA-delivery vectors. For this purpose, plasmid DNA was complexed with cationic lipids bearing a disulphide bond. This reduction-sensitive linker is expected to be reduced and cleaved in the reducing milieu of the cytoplasm, thus potentially improving DNA release and consequently transfection. The DNA--disulphide-lipid complexation was monitored by ethidium bromide exclusion, and the size of complexes was determined by dynamic light scattering. It was found that the reduction kinetics of disulphide groups in DNA--lipid complexes depended on the position of the disulphide linker within the lipid molecule. Furthermore, the internal structure of DNA--lipid particles was examined by small-angle X-ray scattering before and after lipid reduction. DNA release from lipid complexes was observed after the reduction of disulphide bonds of several lipids. Cell-transfection experiments suggested that complexes formed with selected reducible lipids resulted in up to 1000-fold higher reporter-gene activity, when compared with their analogues without disulphide bonds. In conclusion, reduction-sensitive groups introduced into cationic lipid backbones potentially allow enhanced DNA release from DNA--lipid complexes after intracellular reduction and represent a tool for improved vectorization. PMID:11389682

  15. Horizontal gene transfer from Agrobacterium to plants

    PubMed Central

    Matveeva, Tatiana V.; Lutova, Ludmila A.

    2014-01-01

    Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A. rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named “cellular T-DNA” (cT-DNA). It represents an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14) and an opine synthesis gene (Ngmis). A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologs of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role. PMID:25157257

  16. Genome analysis of DNA repair genes in the alpha proteobacterium Caulobacter crescentus

    PubMed Central

    Martins-Pinheiro, Marinalva; Marques, Regina CP; Menck, Carlos FM

    2007-01-01

    Background The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria. Results In this study, we employed a bioinformatic approach, to analyse and describe the open reading frames potentially related to DNA repair from the genome of the alpha-proteobacterium Caulobacter crescentus. This was performed by comparison with known DNA repair related genes found in public databases. As expected, although C. crescentus and E. coli bacteria belong to separate phylogenetic groups, many of their DNA repair genes are very similar. However, some important DNA repair genes are absent in the C. crescentus genome and other interesting functionally related gene duplications are present, which do not occur in E. coli. These include DNA ligases, exonuclease III (xthA), endonuclease III (nth), O6-methylguanine-DNA methyltransferase (ada gene), photolyase-like genes, and uracil-DNA-glycosylases. On the other hand, the genes imuA and imuB, which are involved in DNA damage induced mutagenesis, have recently been described in C. crescentus, but are absent in E. coli. Particularly interesting are the potential atypical phylogeny of one of the photolyase genes in alpha-proteobacteria, indicating an origin by horizontal transfer, and the duplication of the Ada orthologs, which have diverse structural configurations, including one that is still unique for C. crescentus. Conclusion The absence and the presence of certain genes are discussed and predictions are made considering the particular aspects of the C. crescentus

  17. Gene therapy: Biological pacemaker created by gene transfer

    NASA Astrophysics Data System (ADS)

    Miake, Junichiro; Marbán, Eduardo; Nuss, H. Bradley

    2002-09-01

    The pacemaker cells of the heart initiate the heartbeat, sustain the circulation, and dictate the rate and rhythm of cardiac contraction. Circulatory collapse ensues when these specialized cells are damaged by disease, a situation that currently necessitates the implantation of an electronic pacemaker. Here we report the use of viral gene transfer to convert quiescent heart-muscle cells into pacemaker cells, and the successful generation of spontaneous, rhythmic electrical activity in the ventricle in vivo. Our results indicate that genetically engineered pacemakers could be developed as a possible alternative to implantable electronic devices.

  18. Tumor necrosis factor-alpha inhibits albumin gene expression in a murine model of cachexia.

    PubMed Central

    Brenner, D A; Buck, M; Feitelberg, S P; Chojkier, M

    1990-01-01

    The mechanisms responsible for decreased serum albumin levels in patients with cachexia-associated infection, inflammation, and cancer are unknown. Since tumor necrosis factor-alpha (TNF alpha) is elevated in cachexia-associated diseases, and chronic administration of TNF alpha induces cachexia in animal models, we assessed the regulation of albumin gene expression by TNF alpha in vivo. In this animal model of cachexia, Chinese hamster ovary cells transfected with the functional gene for human TNF alpha were inoculated into nude mice (TNF alpha mice). TNF alpha mice became cachectic and manifested decreased serum albumin levels, albumin synthesis, and albumin mRNA levels. However, even before the TNF alpha mice lost weight, their albumin mRNA steady-state levels were decreased approximately 90%, and in situ hybridization revealed a low level of albumin gene expression throughout the hepatic lobule. The mRNA levels of several other genes were unchanged. Hepatic nuclei from TNF alpha mice before the onset of weight loss were markedly less active in transcribing the albumin gene than hepatic nuclei from control mice. Therefore, TNF alpha selectively inhibits the genetic expression of albumin in this model before weight loss. Images PMID:2295699

  19. Technology Transfer and Outreach for SNL/Rochester ALPHA Project.

    SciTech Connect

    Sinars, Daniel

    2016-01-01

    This report describes the next stage goals and resource needs for the joint Sandia and University of Rochester ARPA-E project. A key portion of this project is Technology Transfer and Outreach, with the goal being to help ensure that this project develops a credible method or tool that the magneto-inertial fusion (MIF) research community can use to broaden the advocacy base, to pursue a viable path to commercial fusion energy, and to develop other commercial opportunities for the associated technology. This report describes an analysis of next stage goals and resource needs as requested by Milestone 5.1.1.

  20. Sequence heterogeneity and differential expression of the alpha-Amy2 gene family in wheat.

    PubMed

    Huttly, A K; Martienssen, R A; Baulcombe, D C

    1988-10-01

    The alpha-Amy2 genes of wheat are a multigene family which is expressed in the aleurone cells of germinating grain under control of the plant hormone gibberellin. A subset of the genes are also expressed in developing grain. Comparison of five genomic clones containing alpha-Amy2 genes, using DNA sequence analysis and Southern hybridisation, showed that the extent of similarity between genes differed. Two of the most heterogeneous genes compared were located to the same group 7 chromosome while the most similar genes alpha-Amy2/54 and alpha-Amy2/8 were located to different ones; hence sequence variation could not be correlated to the ancestry of the alpha-Amy2 genes during the separate existence of the constituent genomes of hexaploid wheat. Expression of the cloned genes was measured using an S1 nuclease protection assay and this identified alpha-Amy2/54 and alpha-Amy2/8 as part of the subset of alpha-Amy2 genes expressed in both the developing grain and in aleurone cells. Comparison of the 5' upstream regions of all five genes showed high similarity, with the exception of one gene, up to -280 nucleotides from the transcriptional start, while similarity between alpha-Amy2/54 and alpha-Amy2/8 extended a further 90 bp upstream of this point. It is suggested that regulatory elements responsible for tissue specificity and gibberellin regulation may be located within these regions of similarity. PMID:2467183

  1. Localization of an alpha-amanitin resistance mutation in the gene encoding the largest subunit of mouse RNA polymerase II.

    PubMed Central

    Bartolomei, M S; Corden, J L

    1987-01-01

    RNA polymerase II is inhibited by the mushroom toxin alpha-amanitin. A mouse BALB/c 3T3 cell line was selected for resistance to alpha-amanitin and characterized in detail. This cell line, designated A21, was heterozygous, possessing both amanitin-sensitive and -resistant forms of RNA polymerase II; the mutant form was 500 times more resistant to alpha-amanitin than the sensitive form. By using the wild-type mouse RNA polymerase II largest subunit (RPII215) gene (J.A. Ahearn, M.S. Bartolomei, M. L. West, and J. L. Corden, submitted for publication) as the probe, RPII215 genes were isolated from an A21 genomic DNA library. The mutant allele was identified by its ability to transfer amanitin resistance in a transfection assay. Genomic reconstructions between mutant and wild-type alleles localized the mutation to a 450-base-pair fragment that included parts of exons 14 and 15. This fragment was sequenced and compared with the wild-type sequence; a single AT-to-GC transition was detected at nucleotide 6819, corresponding to an asparagine-to-aspartate substitution at amino acid 793 of the predicted protein sequence. Knowledge of the position of the A21 mutation should facilitate the study of the mechanism of alpha-amanitin resistance. Furthermore, the A21 gene will be useful for studying the phenotype of site-directed mutations in the RPII215 gene. Images PMID:3821724

  2. Gene duplication and transfer events in plant mitochondria genome

    SciTech Connect

    Xiong Aisheng Peng Rihe; Zhuang Jing; Gao Feng; Zhu Bo; Fu Xiaoyan; Xue Yong; Jin Xiaofen; Tian Yongsheng; Zhao Wei; Yao Quanhong

    2008-11-07

    Gene or genome duplication events increase the amount of genetic material available to increase the genomic, and thereby phenotypic, complexity of organisms during evolution. Gene duplication and transfer events have been important to molecular evolution in all three domains of life, and may be the first step in the emergence of new gene functions. Gene transfer events have been proposed as another accelerator of evolution. The duplicated gene or genome, mainly nuclear, has been the subject of several recent reviews. In addition to the nuclear genome, organisms have organelle genomes, including mitochondrial genome. In this review, we briefly summarize gene duplication and transfer events in the plant mitochondrial genome.

  3. Kidney-specific Sonoporation-mediated Gene Transfer

    PubMed Central

    Ishida, Ryo; Kami, Daisuke; Kusaba, Tetsuro; Kirita, Yuhei; Kishida, Tsunao; Mazda, Osam; Adachi, Takaomi; Gojo, Satoshi

    2016-01-01

    Sonoporation can deliver agents to target local organs by systemic administration, while decreasing the associated risk of adverse effects. Sonoporation has been used for a variety of materials and in a variety of organs. Herein, we demonstrated that local sonoporation to the kidney can offer highly efficient transfer of oligonucleotides, which were systemically administrated to the tubular epithelium with high specificity. Ultrasonic wave irradiation to the kidney collapsed the microbubbles and transiently affected the glomerular filtration barrier and increased glomerular permeability. Oligonucleotides were passed through the barrier all at once and were absorbed throughout the tubular epithelium. Tumor necrosis factor alpha (TNFα), which plays a central role in renal ischemia–reperfusion injury, was targeted using small interfering RNA (siRNA) with renal sonoporation in a murine model. The reduction of TNFα expression after single gene transfer significantly inhibited the expression of kidney injury markers, suggesting that systemic administration of siRNA under temporary and local sonoporation could be applicable in the clinical setting of ischemic acute kidney injury. PMID:26419704

  4. Lactation is disrupted by alpha-lactalbumin deficiency and can be restored by human alpha-lactalbumin gene replacement in mice.

    PubMed Central

    Stacey, A; Schnieke, A; Kerr, M; Scott, A; McKee, C; Cottingham, I; Binas, B; Wilde, C; Colman, A

    1995-01-01

    Mice carrying either a deletion of the murine alpha-lactalbumin (alpha-lac) gene (null allele) or its replacement by the human alpha-lac gene (humanized allele) have been generated by gene targeting. Homozygous null females are alpha-lac-deficient, produce reduced amounts of thickened milk containing little or no lactose, and cannot sustain their offspring. This provides definitive evidence that alpha-lac is required for lactose synthesis and that lactose is important for milk production. Females homozygous for the humanized allele lactate normally, indicating that human alpha-lac can replace murine alpha-lac. Mouse and human alpha-lac expression was compared in mice heterozygous for the humanized allele. The human gene expressed approximately 15-fold greater mRNA and approximately 14-fold greater protein than the mouse, indicating that the major determinants of human alpha-lac expression are close to, or within, the human gene and that the mouse locus does not exert a negative influence on alpha-lac expression. Variations in alpha-lac expression levels in nondeficient mice did not affect milk lactose concentration, but the volume of milk increased slightly in mice homozygous for the humanized allele. These variations demonstrated that alpha-lac expression in mice is gene dosage dependent. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7708733

  5. Targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies.

    PubMed Central

    Wickham, T J; Segal, D M; Roelvink, P W; Carrion, M E; Lizonova, A; Lee, G M; Kovesdi, I

    1996-01-01

    A major hurdle to adenovirus (Ad)-mediated gene transfer is that the target issue lacks sufficient levels of receptors to mediate vector attachment via its fiber coat protein. Endothelial and smooth muscle cells are primary targets in gene therapy approaches to prevent restenosis following angioplasty or to promote or inhibit angiogenesis. However, Ad poorly binds and transduces these cells because of their low or undetectable levels of functional Ad fiber receptor. The Ad-binding deficiency of these cells was overcome by targeting Ad binding to alpha v integrin receptors that are sufficiently expressed by these cells. In order to target alpha v integrins, a bispecific antibody (bsAb) that comprised a monoclonal Ab to the FLAG peptide epitope, DYKDDDDK, and a monoclonal Ab to alpha v integrins was constructed. In conjunction with the bsAb, a new vector, AdFLAG, which incorporated the FLAG peptide epitope into its penton base protein was constructed. Complexing AdFLAG with the bsAb increased the beta-glucuronidase transduction of human venule endothelial cells and human intestinal smooth muscle cells by seven- to ninefold compared with transduction by AdFLAG alone. The increased transduction efficiency was shown to occur through the specific interaction of the complex with alpha v integrins. These results demonstrate that bsAbs can be successfully used to target Ad to a specific cellular receptor and thereby increase the efficiency of gene transfer. PMID:8794324

  6. The neuronal nicotinic acetylcholine receptor {alpha}7 subunit gene: Cloning, mapping, structure, and targeting in mouse

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1994-09-01

    The neuronal nicotinic acetylcholine receptor {alpha}7 subunit is a member of a family of ligand-gated ion channels, and is the only subunit know to bind {alpha}-bungarotoxin in mammalian brain. {alpha}-Bungarotoxin binding sites are known to be more abundant in the hippocampus of mouse strains that are particularly sensitive to nicotine-induced seizures. The {alpha}7 receptor is highly permeable to calcium, which could suggest a role in synaptic plasticity in the nervous system. Auditory gating deficiency, an abnormal response to a second auditory stimulus, is characteristic of schizophrenia. Mouse strains that exhibit a similar gating deficit have reduced hippocampal expression of the {alpha}7 subunit. We have cloned and sequenced the full length cDNA for the mouse {alpha}7 gene (Acra-7) and characterized its gene structure. The murine {alpha}7 shares amino acid identity of 99% and 93% with the rat and human {alpha}7 subunits, respectively. Using an interspecies backcross panel, the murine gene was mapped to chromosome 7 near the p locus, a region syntenic with human chromosome 15; the human gene (CHRNA7) was confirmed to map to 15q13-q14 by FISH. To generate a mouse {alpha}7 mutant by homologous recombination, we have constructed a replacement vector which will delete transmembrane domains II-IV and the cytoplasmic domain from the gene product. Recombinant embryonic stem (ES) cell clones were selected and used to develop mouse chimeras that are currently being bred to obtain germline transmission.

  7. Optical gene transfer by femtosecond laser pulses

    NASA Astrophysics Data System (ADS)

    Konig, Karsten; Riemann, Iris; Tirlapur, Uday K.

    2003-07-01

    Targeted transfection of cells is an important technique for gene therapy and related biomedical applications. We delineate how high-intensity (1012 W/cm2) near-infrared (NIR) 80 MHz nanojoule femtosecond laser pulses can create highly localised membrane perforations within a minute focal volume, enabling non-invasive direct transfection of mammalian cells with DNA. We suspended Chinese hamster ovarian (CHO), rat kangaroo kidney epithelial (PtK2) and rat fibroblast cells in 0.5 ml culture medium in a sterile miniaturized cell chamber (JenLab GmbH, Jena, Germany) containing 0.2 μg plasmid DNA vector pEGFP-N1 (4.7 kb), which codes for green fluorescent protein (GFP). The NIR laser beam was introduced into a femtosecond laser scanning microscope (JenLab GmbH, Jena, Germany; focussed on the edge of the cell membrane of a target cell for 16 ms. The integration and expression efficiency of EGFP were assessed in situ by two-photon fluorescence-lifetime imaging using time-correlated single photon counting. The unique capability to transfer foreign DNA safely and efficiently into specific cell types (including stem cells), circumventing mechanical, electrical or chemical means, will have many applications, such as targeted gene therapy and DNA vaccination.

  8. Lentiviral vector gene transfer to porcine airways.

    PubMed

    Sinn, Patrick L; Cooney, Ashley L; Oakland, Mayumi; Dylla, Douglas E; Wallen, Tanner J; Pezzulo, Alejandro A; Chang, Eugene H; McCray, Paul B

    2012-01-01

    In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE) and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE). Interestingly, feline immunodeficiency virus (FIV)-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1-based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF).Molecular Therapy - Nucleic Acids (2012) 1, e56; doi:10.1038/mtna.2012.47; published online 27 November 2012. PMID:23187455

  9. Plant transformation via pollen tube-mediated gene transfer

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic transformation using foreign genes and the subsequent development of transgenic plants has been employed to develop enhanced elite germplasm. Although some skepticism exits regarding pollen tube-mediated gene transfer (PTT), reports demonstrating improved transformation efficiency with PTT ...

  10. Gene Transfer & Hybridization Studies in Hyperthermophilic Species

    SciTech Connect

    Nelson, Karen E.

    2005-10-14

    A. ABSTRACT The importance of lateral gene transfer (LGT) in the evolution of microbial species has become increasingly evident with each completed microbial genome sequence. Most significantly, the genome of Thermotoga maritima MSB8, a hyperthermophilic bacterium isolated by Karl Stetter and workers from Vulcano Italy in 1986, and sequenced at The Institute for Genomic Research (TIGR) in Rockville Maryland in 1999, revealed extensive LGT between % . this bacterium and members of the archaeal domain (in particular Archaeoglobus fulgidus, and Pyracoccus frcriosus species). Based on whole genome comparisons, it was estimated that 24% of the genetic information in this organism was acquired by genetic exchange with archaeal species, Independent analyses including periodicity analysis of the T. maritimu genomic DNA sequence, phylogenetic reconstruction based on genes that appear archaeal-like, and codon and amino acid usage, have provided additional evidence for LGT between T. maritima and the archaea. More recently, DiRuggiero and workers have identified a very recent LGT event between two genera of hyperthermophilic archaea, where a nearly identical DNA fragment of 16 kb in length flanked by insertion sequence (IS) elements, exists. Undoubtedly, additional examples of LGT will be identified as more microbial genomes are completed. For the present moment however, the genome sequence of T. maritima and other hyperthermophiles including P. furiosus, Pyrococcus horikoshii, Pyrococcus abyssi, A. fulgidus, and Aquifex aeolicus, have significantly increased out awareness of evolution being a web of life rather than a tree of life, as suggested by single gene phylogenies. In this proposal, we will aim to determine the extent of LGT across the hyperthemophiles, employing iY maritima as the model organism. A variety of biochemical techniques and phylogenetic reconstructions will allow for a detailed and thorough characterization of the extent of LGT in this species. The

  11. Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.

    PubMed Central

    Wickham, T J; Tzeng, E; Shears, L L; Roelvink, P W; Li, Y; Lee, G M; Brough, D E; Lizonova, A; Kovesdi, I

    1997-01-01

    Alteration of the natural tropism of adenovirus (Ad) will permit gene transfer into specific cell types and thereby greatly broaden the scope of target diseases that can be treated by using Ad. We have constructed two Ad vectors which contain modifications to the Ad fiber coat protein that redirect virus binding to either alpha(v) integrin [AdZ.F(RGD)] or heparan sulfate [AdZ.F(pK7)] cellular receptors. These vectors were constructed by a novel method involving E4 rescue of an E4-deficient Ad with a transfer vector containing both the E4 region and the modified fiber gene. AdZ.F(RGD) increased gene delivery to endothelial and smooth muscle cells expressing alpha(v) integrins. Likewise, AdZ.F(pK7) increased transduction 5- to 500-fold in multiple cell types lacking high levels of Ad fiber receptor, including macrophage, endothelial, smooth muscle, fibroblast, and T cells. In addition, AdZ.F(pK7) significantly increased gene transfer in vivo to vascular smooth muscle cells of the porcine iliac artery following balloon angioplasty. These vectors may therefore be useful in gene therapy for vascular restenosis or for targeting endothelial cells in tumors. Although binding to the fiber receptor still occurs with these vectors, they demonstrate the feasibility of tissue-specific receptor targeting in cells which express low levels of Ad fiber receptor. PMID:9343173

  12. Methods for Gene Transfer to the Central Nervous System

    PubMed Central

    Kantor, Boris; Bailey, Rachel M.; Wimberly, Keon; Kalburgi, Sahana N.; Gray, Steven J.

    2015-01-01

    Gene transfer is an increasingly utilized approach for research and clinical applications involving the central nervous system (CNS). Vectors for gene transfer can be as simple as an unmodified plasmid, but more commonly involve complex modifications to viruses to make them suitable gene delivery vehicles. This chapter will explain how tools for CNS gene transfer have been derived from naturally occurring viruses. The current capabilities of plasmid, retroviral, adeno-associated virus, adenovirus, and herpes simplex virus vectors for CNS gene delivery will be described. These include both focal and global CNS gene transfer strategies, with short- or long-term gene expression. As is described in this chapter, an important aspect of any vector is the cis-acting regulatory elements incorporated into the vector genome that control when, where, and how the transgene is expressed. PMID:25311922

  13. Gene transfer system for Rhodopseudomonas viridis.

    PubMed Central

    Lang, F S; Oesterhelt, D

    1989-01-01

    A gene transfer system for Rhodopseudomonas viridis was established which uses conjugation with Escherichia coli S17-I as the donor and mobilizable plasmids as vectors. Initially, plasmids of the incompatibility group P1 (pRK290 and pRK404) were used. The more effective shuttle vectors between E. coli and R. viridis, pKV1 and pKVS1, were derived from plasmid pBR322 and showed the highest conjugation frequency (10(-2] thus far demonstrated in purple bacteria. It was also demonstrated that Rhizobium meliloti can be used as a donor for conjugation with R. viridis. From a genomic cosmid library of R. viridis constructed in the vector pHC79, clones that coded for subunits H (puh operon), L, M and cytochrome c (puf operon) of the photosynthetic reaction center were isolated and characterized. For linkage of the two operons on the genome, cosmids that overlapped with the operon-carrying clones were identified. The relative positions of the two operons could not be determined, but the operons must be more than 100 kilobase pairs apart. Thus, the genomic organization of the reaction center in R. viridis is different from that of Rhodobacter capsulatus, for which a distance of about 39 kilobase pairs was determined. From a spontaneous mutant of R. viridis that is resistant to the herbicide terbutryn, the puf operon was cloned in pKVS1 and transferred by conjugation into R. viridis wild-type cells. The resulting exconjugants were resistant to the herbicide, which demonstrated that the puf operon on pKVS1 constructions was functionally expressed in R. viridis. Images PMID:2666398

  14. EFFECT OF DUST ON Ly{alpha} PHOTON TRANSFER IN AN OPTICALLY THICK HALO

    SciTech Connect

    Yang Yang; Shu Chiwang; Roy, Ishani; Fang Lizhi

    2011-10-01

    We investigate the effects of dust on Ly{alpha} photons emergent from an optically thick medium by solving the integro-differential equation of radiative transfer of resonant photons. To solve the differential equations numerically, we use the weighted essentially non-oscillatory method. Although the effects of dust on radiative transfer are well known, the resonant scattering of Ly{alpha} photons makes the problem non-trivial. For instance, if the medium has an optical depth of dust absorption and scattering of {tau}{sub a} >> 1, {tau} >> 1, and {tau} >> {tau}{sub a}, the effective absorption optical depth in a random walk scenario would be equal to {radical}({tau}{sub a}({tau}{sub a}+{tau})). We show, however, that for a resonant scattering at frequency {nu}{sub 0}, the effective absorption optical depth would be even larger than {tau}({nu}{sub 0}). If the cross section of dust scattering and absorption is frequency-independent, the double-peaked structure of the frequency profile given by the resonant scattering is basically dust-independent. That is, dust causes neither narrowing nor widening of the width of the double-peaked profile. One more result is that the timescales of the Ly{alpha} photon transfer in an optically thick halo are also basically independent of the dust scattering, even when the scattering is anisotropic. This is because those timescales are mainly determined by the transfer in the frequency space, while dust scattering, either isotropic or anisotropic, does not affect the behavior of the transfer in the frequency space when the cross section of scattering is wavelength-independent. This result does not support the speculation that dust will lead to the smoothing of the brightness distribution of a Ly{alpha} photon source with an optically thick halo.

  15. Transcriptional regulation of human retinoic acid receptor-alpha (RAR-{alpha}) by Wilms` tumour gene product

    SciTech Connect

    Goodyer, P.R.; Torban, E.; Dehbi, M.

    1994-09-01

    The Wilms` tumor gene encodes a 47-49 kDa transcription factor expressed in kidney, gonads and mesothelium during embryogenesis. Inherited mutations of WT1 lead to aberrant urogenital development and Wilms` tumor, but the role of WT1 in development is not fully understood. Since the human RAR-{alpha} gene contains a potential WT1 binding site at its 5{prime} end, we studied the effect of WT1 co-transfection on expression of an RAR-{alpha} promoter/CAT reporter construct in COS cells. COS cells were plated at 5X10{sup 5} cells/dish in DMEM with 10% FBS and transfected by the Ca/PO4 method with an expression plasmid containing the full-length WT1 (-/-) cDNA under the control of the CMV promoter, plasmid containing the RAR-{alpha} promoter (-519 to +36)/CAT reporter and TK/growth hormone plasmid to control for efficiency of transfection. CAT/GH activity at 48 hours was inhibited by co-transfection with increasing amounts of WT1 (-/-); maximum inhibition = 5% of control. WT1 co-transfection did not affect expression of TKGH, nor of a CMV-CAT vector. Expression of WT1 protein in tranfected COS cells was demonstrated by Western blotting. Minimal inhibiton of RAR-{alpha}/CAT activity was seen when cells were co-transfected with vectors containing WT1 deletion mutants, alternate WT1 splicing variants, or WT1 (-/-) cDNA bearing a mutation identified in a patient with Drash syndrome. Gel shift assays indicated binding of WT1 to RAR-{alpha} cDNA but not to an RAR-{alpha} deletion mutant lacking the GCGGGGGGCG site. These observations suggest that WT1 may function to regulate RAR-{alpha} expression during normal development.

  16. Problems associated with gene transfer and opportunities for microgravity environments

    NASA Astrophysics Data System (ADS)

    Tennessen, Daniel J.

    1997-01-01

    The method of crop improvement by gene transfer is becoming increasingly routine with transgenic foods and ornamental crops now being marketed to consumers. However, biological processes of plants, and the physical barriers of current protocols continue to limit the application of gene transfer in many commercial crops. The goal of this paper is to outline the current limitations of gene transfer and to hypothesize possible opportunities for use of microgravity to overcome such limitations. The limitations detailed in this paper include host-range specificity of Agrobacterium mediated transformation, probability of gene insertion, position effects of the inserted genes, gene copy number, stability of foreign gene expression in host plants, and regeneration of recalcitrant plant species. Microgravity offers an opportunity for gene transfer where cell growth kinetics, DNA synthesis, and genetic recombination rates can be altered. Such biological conditions may enhance the ability for recombination of reporter genes and other genes of interest to agriculture. Proposed studies would be useful for understanding instability of foreign gene expression and may lead to stable transformed plants. Other aspects of gene transfer in microgravity are discussed.

  17. Estrogen-related receptor {alpha} modulates the expression of adipogenesis-related genes during adipocyte differentiation

    SciTech Connect

    Ijichi, Nobuhiro; Ikeda, Kazuhiro; Horie-Inoue, Kuniko; Yagi, Ken; Okazaki, Yasushi; Inoue, Satoshi . E-mail: INOUE-GER@h.u-tokyo.ac.jp

    2007-07-06

    Estrogen-related receptor {alpha} (ERR{alpha}) is an orphan nuclear receptor that regulates cellular energy metabolism by modulating gene expression involved in fatty acid oxidation and mitochondrial biogenesis in brown adipose tissue. However, the physiological role of ERR{alpha} in adipogenesis and white adipose tissue development has not been well studied. Here, we show that ERR{alpha} and ERR{alpha}-related transcriptional coactivators, peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) coactivator-1{alpha} (PGC-1{alpha}) and PGC-1{beta}, can be up-regulated in 3T3-L1 preadipocytes at mRNA levels under the adipogenic differentiation condition including the inducer of cAMP, glucocorticoid, and insulin. Gene knockdown by ERR{alpha}-specific siRNA results in mRNA down-regulation of fatty acid binding protein 4, PPAR{gamma}, and PGC-1{alpha} in 3T3-L1 cells in the adipogenesis medium. ERR{alpha} and PGC-1{beta} mRNA expression can be also up-regulated in another preadipocyte lineage DFAT-D1 cells and a pluripotent mesenchymal cell line C3H10T1/2 under the differentiation condition. Furthermore, stable expression of ERR{alpha} in 3T3-L1 cells up-regulates adipogenic marker genes and promotes triglyceride accumulation during 3T3-L1 differentiation. These results suggest that ERR{alpha} may play a critical role in adipocyte differentiation by modulating the expression of various adipogenesis-related genes.

  18. Binding of alpha-bungarotoxin to proteolytic fragments of the alpha subunit of Torpedo acetylcholine receptor analyzed by protein transfer on positively charged membrane filters.

    PubMed Central

    Wilson, P T; Gershoni, J M; Hawrot, E; Lentz, T L

    1984-01-01

    Proteolytic fragments of the alpha subunit of the acetylcholine receptor retain the ability to bind alpha-bungarotoxin following resolution by polyacrylamide gel electrophoresis and immobilization on protein transfers. The alpha subunit of the acetylcholine receptor of Torpedo electric organ was digested with four proteases: Staphylococcus aureus V-8 protease, papain, bromelain, and proteinase K. The proteolytic fragments resolved on 15% polyacrylamide gels were electrophoretically transferred onto positively charged nylon membrane filters. When incubated with 0.3 nM 125I-labeled alpha-bungarotoxin and autoradiographed, the transfers yielded patterns of labeled bands characteristic for each protease. The molecular masses of the fragments binding toxin ranged from 7 to 34 kDa, with major groupings in the 8-, 18-, and 28-kDa ranges. The apparent affinity of the fragments for alpha-bungarotoxin as determined from the IC50 value was 6.7 X 10(-8) M. The labeling of fragments with alpha-bungarotoxin could be inhibited by prior affinity alkylation of receptor-containing membranes with 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide. These findings demonstrate that immobilized proteolytic fragments as small as 1/5 the size of the alpha subunit retain the structural characteristics necessary for binding alpha-bungarotoxin, although the toxin is bound to the fragments with lower affinity than to the native receptor. The effect of affinity ligand alkylation demonstrates that the alpha-bungarotoxin binding site detected on the proteolytic fragments is the same as the affinity-labeled acetylcholine binding site on the intact acetylcholine receptor. Images PMID:6371817

  19. Identification of Core Alpha 1,3-Fucosyltransferase Gene From Silkworm: An Insect Popularly Used to Express Mammalian Proteins

    PubMed Central

    Minagawa, Sachi; Sekiguchi, Satoshi; Nakaso, Yuzuru; Tomita, Masahiro; Takahisa, Manabu; Yasuda, Hideyo

    2015-01-01

    Silkworm has great potential as production system of recombinant mammalian proteins. When the protein products are used for medical purpose, it is required to reduce the risk of an allergy, the content of core alpha 1,3-fucosyl residue attached to the N-glycan of proteins, for example. We isolated the gene of an enzyme responsible for the transfer of core alpha 1,3-fucosyl residue, core alpha 1,3-fucosyltransferase (Fuc-T C3), from silkworm. A candidate cDNA for silkworm Fuc-T C3 was isolated as a homolog of the fruit fly enzyme gene fucTA. The gene was located on chromosome 7 of the silkworm genome and was composed of seven exons, which spanned approximately 10 kb on the genome. The coding region of the gene was 1,350 bp and encoded a 450-amino acid protein with a molecular mass of 52.2 kDa. Deduced amino acid sequence of the coding region showed one transmembrane domain in its N-terminal and typical motifs common to fucosyltransferases including Fuc-T C3s of other organisms in its C-terminal. The extract of CHO cells transfected with the cDNA showed Fuc-T C3 activity using GDP-fucose and DABS-GnGn peptide as substrates. These results showed this cDNA clone actually encodes silkworm Fuc-T C3. PMID:26223947

  20. Expression pattern of the RAR alpha-PML fusion gene in acute promyelocytic leukemia.

    PubMed

    Alcalay, M; Zangrilli, D; Fagioli, M; Pandolfi, P P; Mencarelli, A; Lo Coco, F; Biondi, A; Grignani, F; Pelicci, P G

    1992-06-01

    Two chimeric genes, PML-RAR alpha and RAR alpha-PML, are formed as a consequence of the acute promyelocytic leukemia (APL)-specific reciprocal translocation of chromosomes 15 and 17 [t(15;17)]. PML-RAR alpha is expressed as a fusion protein. We investigated the organization and expression pattern of the RAR alpha-PML gene in a series of APL patients representative of the molecular heterogeneity of the t(15;17) and found (i) two types of RAR alpha-PML mRNA junctions (RAR alpha exon 2/PML exon 4 or RAR alpha exon 2/PML exon 7) that maintain the RAR alpha and PML longest open reading frames aligned and are the result of chromosome 15 breaking at two different sites; and (ii) 10 different RAR alpha-PML fusion transcripts that differ for the assembly of their PML coding exons. A RAR alpha-PML transcript was present in most, but not all, APL patients. PMID:1317574

  1. Ly{alpha} RADIATIVE TRANSFER IN COSMOLOGICAL SIMULATIONS USING ADAPTIVE MESH REFINEMENT

    SciTech Connect

    Laursen, Peter; Razoumov, Alexei O.; Sommer-Larsen, Jesper E-mail: razoumov@ap.smu.ca

    2009-05-01

    A numerical code for solving various Ly{alpha} radiative transfer (RT) problems is presented. The code is suitable for an arbitrary, three-dimensional distribution of Ly{alpha} emissivity, gas temperature, density, and velocity field. Capable of handling Ly{alpha} RT in an adaptively refined grid-based structure, it enables detailed investigation of the effects of clumpiness of the interstellar (or intergalactic) medium. The code is tested against various geometrically and physically idealized configurations for which analytical solutions exist, and subsequently applied to three different simulated high-resolution 'Lyman-break galaxies', extracted from high-resolution cosmological simulations at redshift z = 3.6. Proper treatment of the Ly{alpha} scattering reveals a diversity of surface brightness (SB) and line profiles. Specifically, for a given galaxy the maximum observed SB can vary by an order of magnitude, and the total flux by a factor of 3-6, depending on the viewing angle. This may provide an explanation for differences in observed properties of high-redshift galaxies, and in particular a possible physical link between Lyman-break galaxies and regular Ly{alpha} emitters.

  2. Comparison of nonerythroid alpha-spectrin genes reveals strict homology among diverse species.

    PubMed Central

    Leto, T L; Fortugno-Erikson, D; Barton, D; Yang-Feng, T L; Francke, U; Harris, A S; Morrow, J S; Marchesi, V T; Benz, E J

    1988-01-01

    The spectrins are a family of widely distributed filamentous proteins. In association with actin, spectrins form a supporting and organizing scaffold for cell membranes. Using antibodies specific for human brain alpha-spectrin (alpha-fodrin), we have cloned a rat brain alpha-spectrin cDNA from an expression library. Several closely related human clones were also isolated by hybridization. Comparison of sequences of these and other overlapping nonerythroid and erythroid alpha-spectrin genes demonstrated that the nonerythroid genes are strictly conserved across species, while the mammalian erythroid genes have diverged rapidly. Peptide sequences deduced from these cDNAs revealed that the nonerythroid alpha-spectrin chain, like the erythroid spectrin, is composed of multiple 106-amino-acid repeating units, with the characteristic invariant tryptophan as well as other charged and hydrophobic residues in conserved locations. However, the carboxy-terminal sequence varies markedly from this internal repeat pattern and may represent a specialized functional site. The nonerythroid alpha-spectrin gene was mapped to human chromosome 9, in contrast to the erythroid alpha-spectrin gene, which has previously been assigned to a locus on chromosome 1. Images PMID:3336352

  3. Partial activation of gene activity and chromatin remodeling of the human 14q32.1 serpin gene cluster by HNF-1 alpha and HNF-4 in fibroblast microcell hybrids.

    PubMed

    Rollini, P; Xu, L; Fournier, R E

    1999-07-01

    The genes encoding alpha 1-antitrypsin (alpha 1AT, gene symbol P I) and corticosteroid-binding globulin (CBG) are part of a cluster of serine protease inhibitor (serpin) genes on human chromosome 14q32.1. Both genes are highly expressed in the liver and in cultured hepatoma cells, and the approximately 100-kb region around these genes contains an extensive array of expression-associated DNase I-hypersensitive sites (DHSs). Activation of human alpha 1AT and CBG transcription occurred when human chromosome 14 was transferred from nonexpressing cells to rat hepatoma cells. This activation event was accompanied by long-range chromatin reorganization of the entire region and the de novo formation of 17 expression-associated DHSs. Both gene activation and chromatin remodeling in hepatic cells required the liver-enriched transactivators hepatocyte nuclear factors-1 alpha and -4 (HNF-1 alpha and HNF-4). In this study, we tested whether ectopic expression of HNF-1 alpha and HNF-4 in nonexpressing cells could activate alpha 1AT and/or CBG transcription, and we monitored the chromatin structure of the locus in stably transfected fibroblasts. We report that both alpha 1AT and CBG mRNAs were expressed in fibroblast transfectants that stably expressed HNF-1 alpha and HNF-4, but expression was only approximately 1-10% of that observed in hepatic cells. Gene activation in these cells was accompanied by partial chromatin remodeling, as 6 of 17 expression-associated DHSs were formed. The potential implications of these results are discussed. PMID:11586788

  4. A recently transferred cluster of bacterial genes in Trichomonas vaginalis - lateral gene transfer and the fate of acquired genes

    PubMed Central

    2014-01-01

    Background Lateral Gene Transfer (LGT) has recently gained recognition as an important contributor to some eukaryote proteomes, but the mechanisms of acquisition and fixation in eukaryotic genomes are still uncertain. A previously defined norm for LGTs in microbial eukaryotes states that the majority are genes involved in metabolism, the LGTs are typically localized one by one, surrounded by vertically inherited genes on the chromosome, and phylogenetics shows that a broad collection of bacterial lineages have contributed to the transferome. Results A unique 34 kbp long fragment with 27 clustered genes (TvLF) of prokaryote origin was identified in the sequenced genome of the protozoan parasite Trichomonas vaginalis. Using a PCR based approach we confirmed the presence of the orthologous fragment in four additional T. vaginalis strains. Detailed sequence analyses unambiguously suggest that TvLF is the result of one single, recent LGT event. The proposed donor is a close relative to the firmicute bacterium Peptoniphilus harei. High nucleotide sequence similarity between T. vaginalis strains, as well as to P. harei, and the absence of homologs in other Trichomonas species, suggests that the transfer event took place after the radiation of the genus Trichomonas. Some genes have undergone pseudogenization and degradation, indicating that they may not be retained in the future. Functional annotations reveal that genes involved in informational processes are particularly prone to degradation. Conclusions We conclude that, although the majority of eukaryote LGTs are single gene occurrences, they may be acquired in clusters of several genes that are subsequently cleansed of evolutionarily less advantageous genes. PMID:24898731

  5. Cotransfer of linked eukaryotic genes and efficient transfer of hypoxanthine phosphoribosyltransferase by DNA-mediated gene transfer.

    PubMed Central

    Peterson, J L; McBride, O W

    1980-01-01

    The efficiency of DNA-mediated transfer of the gene (hprt) for hypoxanthine phosphoribosyltransferase (HPRT; IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) is dependent upon the recipient cell used. hprt has been transferred into mouse TG8 or Chinese hamster CHTG49 cells at a high frequency, similar to the frequency of the gene (tk) for thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) transfer into mouse LMTK- cells (i.e., 10(-6)). In contrast, the frequency of transfer of hprt into mouse A9 cells was about two orders of magnitude less. The identification of efficient recipient cells for hprt transfer permits the use of DNA-mediated transfer as a bioassay for the gene. Cotransfer of the linked tk gene and the gene (galk) for galactokinase (ATP: D-galactose 1-phosphotransferase, EC 2.7.1.6) to LMTK- cells has been detected once among 87 tk transferrents. This suggests that the distance between the tk and galk genes in the Chinese hamster genome may be smaller than was previously thought. Significant differences between chromosome-mediated and DNA-mediated gene transfer were observed with respect to both the size of the transferred functional genetic fragment and the recipient cell specificity. Images PMID:6929511

  6. Gene Transfer Strategies to Promote Chondrogenesis and Cartilage Regeneration.

    PubMed

    Im, Gun-Il

    2016-04-01

    Gene transfer has been used experimentally to promote chondrogenesis and cartilage regeneration. While it is controversial to apply gene therapy for nonlethal conditions such as cartilage defect, there is a possibility that the transfer of therapeutic transgenes may dramatically increase the effectiveness of cell therapy and reduce the quantity of cells that are needed to regenerate cartilage. Single or combination of growth factors and transcription factors has been transferred to mesenchymal stem cells or articular chondrocytes using both nonviral and viral approaches. The current challenge for the clinical applications of genetically modified cells is ensuring the safety of gene therapy while guaranteeing effectiveness. Viral gene delivery methods have been mainstays currently with enhanced safety features being recently refined. On the other hand, efficiency has been greatly improved in nonviral delivery. This review summarizes the history and recent update on the gene transfer to enhance chondrogenesis from stem cells or articular chondrocytes. PMID:26414246

  7. Electron-transfer photochemistry of. cap alpha. -silylamine-cyclohexenone systems. Medium effects on reaction pathways followed

    SciTech Connect

    Yoon, U.C.; Kim, J.U.; Hasegawa, E.; Mariano, P.S.

    1987-07-08

    Previously, the authors demonstrated how photostimulated electron-transfer (SET) processes of ..cap alpha..-trialkylsilyl-substituted electron donors can be employed to generate free radical systems. Initial efforts focused on SET processes of iminium salts. Recently, the authors expanded this methodology to include arenecarbonitriles. In this communication they report on the SET photochemistry of ..cap alpha..,..beta..-unsaturated cyclohexenones with an ..cap alpha..-silyl tertiary amine donor.

  8. Tumor necrosis factor-alpha gene is not associated with obsessive-compulsive disorder.

    PubMed

    Zai, Gwyneth; Arnold, Paul D; Burroughs, Eliza; Richter, Margaret A; Kennedy, James L

    2006-02-01

    Dysregulation of the immune system has been suggested to play a role in the complex etiology of obsessive-compulsive disorder. In this context, tumor necrosis factor-alpha is considered an interesting candidate for genetic studies as overproduction of tumor necrosis factor-alpha, which may be genetically modulated, can exert neurotoxic effects and influence neural cell growth and proliferation. Moreover, the tumor necrosis factor-alpha gene is located on chromosome 6p21.3, a region that has been found to be weakly associated with obsessive-compulsive disorder in linkage studies. One functional polymorphism, G-308A, has been found within the gene. PMID:16395130

  9. Reaction kinetics in dehydrochlorination of. cap alpha. -isomer of hexachlorocyclohexane in the presence of a phase transfer catalyst

    SciTech Connect

    Sirovskii, F.S.; Chimishkyan, A.L.; Panova, M.V.; Treger, Y.A.; Velichko, S.M.

    1986-01-01

    This paper presents a kinetic study of the reaction of aqueous caustic dehydrochlorination of the alpha-isomer of hexachlorocyclohexane (alpha-HCCH) with catalysis by tetrabutyl-ammonium hydroxide. It has been shown that the process is described by a scheme of phase transfer catalysis.

  10. Direct transfer of IL-12 gene into growing Renca tumors.

    PubMed

    Budryk, M; Wilczyńska, U; Szary, J; Szala, S

    2000-01-01

    We investigated the feasibility of transferring naked plasmid DNA containing a therapeutic gene (IL-12) into mice harboring growing Renca tumors. We found that naked DNA transferred into growing Renca and B16(F10) tumors gives higher expression level of reporter gene than complexes of DNA with DDAB/DOPE or DC-Chol/DOPE. Transfer of naked DNA carrying the IL-12 gene into growing Renca tumors causes a distinct therapeutic effect that depends on the time span between inoculation of mice with cancer cells and the beginning of the therapy. Therapy started on day 3 resulted in total cure (100%) of mice. PMID:11051203

  11. LATERAL GENE TRANSFER AND THE HISTORY OF BACTERIAL GENOMES

    SciTech Connect

    Howard Ochman

    2006-02-22

    The aims of this research were to elucidate the role and extent of lateral transfer in the differentiation of bacterial strains and species, and to assess the impact of gene transfer on the evolution of bacterial genomes. The ultimate goal of the project is to examine the dynamics of a core set of protein-coding genes (i.e., those that are distributed universally among Bacteria) by developing conserved primers that would allow their amplification and sequencing in any bacterial taxa. In addition, we adopted a bioinformatic approach to elucidate the extent of lateral gene transfer in sequenced genome.

  12. Thiazolidinediones inhibit REG I{alpha} gene transcription in gastrointestinal cancer cells

    SciTech Connect

    Yamauchi, Akiyo; Takahashi, Iwao; Takasawa, Shin; Nata, Koji; Noguchi, Naoya; Ikeda, Takayuki; Yoshikawa, Takeo; Shervani, Nausheen J.; Suzuki, Iwao; Uruno, Akira; Unno, Michiaki; Okamoto, Hiroshi Sugawara, Akira

    2009-02-13

    REG (Regenerating gene) I{alpha} protein functions as a growth factor for gastrointestinal cancer cells, and its mRNA expression is strongly associated with a poor prognosis in gastrointestinal cancer patients. We here demonstrated that PPAR{gamma}-agonist thiazolidinediones (TZDs) inhibited cell proliferation and REG I{alpha} protein/mRNA expression in gastrointestinal cancer cells. TZDs inhibited the REG I{alpha} gene promoter activity, via its cis-acting element which lacked PPAR response element and could not bind to PPAR{gamma}, in PPAR{gamma}-expressing gastrointestinal cancer cells. The inhibition was reversed by co-treatment with a specific PPAR{gamma}-antagonist GW9662. Although TZDs did not inhibit the REG I{alpha} gene promoter activity in PPAR{gamma}-non-expressing cells, PPAR{gamma} overexpression in the cells recovered their inhibitory effect. Taken together, TZDs inhibit REG I{alpha} gene transcription through a PPAR{gamma}-dependent pathway. The TZD-induced REG I{alpha} mRNA reduction was abolished by cycloheximide, indicating the necessity of novel protein(s) synthesis. TZDs may therefore be a candidate for novel anti-cancer drugs for patients with gastrointestinal cancer expressing both REG I{alpha} and PPAR{gamma}.

  13. Intracellular gene transfer: Reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase

    PubMed Central

    Daley, Daniel O.; Clifton, Rachel; Whelan, James

    2002-01-01

    Subunit 2 of cytochrome c oxidase (Cox2) in legumes offers a rare opportunity to investigate factors necessary for successful gene transfer of a hydrophobic protein that is usually mitochondrial-encoded. We found that changes in local hydrophobicity were necessary to allow import of this nuclear-encoded protein into mitochondria. All legume species containing both a mitochondrial and nuclear encoded Cox2 displayed a similar pattern, with a large decrease in hydrophobicity evident in the first transmembrane region of the nuclear encoded protein compared with the organelle-encoded protein. Mitochondrial-encoded Cox2 could not be imported into mitochondria under the direction of the mitochondrial targeting sequence that readily supports the import of nuclear encoded Cox2. Removal of the first transmembrane region promotes import ability of the mitochondrial-encoded Cox2. Changing just two amino acids in the first transmembrane region of mitochondrial-encoded Cox2 to the corresponding amino acids in the nuclear encoded Cox2 also promotes import ability, whereas changing the same two amino acids in the nuclear encoded Cox2 to what they are in the mitochondrial-encoded copy prevents import. Therefore, changes in amino acids in the mature protein were necessary and sufficient for gene transfer to allow import under the direction of an appropriate signal to achieve the functional topology of Cox2. PMID:12142462

  14. Mapping of the {alpha}{sub 4} subunit gene (GABRA4) to human chromosome 4 defines an {alpha}{sub 2}-{alpha}{sub 4}-{beta}{sub 1}-{gamma}{sub 1} gene cluster: Further evidence that modern GABA{sub a} receptor gene clusters are derived from an ancestral cluster

    SciTech Connect

    McLean, P.J.; Farb, D.H.; Russek, S.J.

    1995-04-10

    We demonstrated previously that an {alpha}{sub 1}-{beta}{sub 2}-{gamma}{sub 2} gene cluster of the {gamma}-aminobutyric acid (GABA{sub A}) receptor is located on human chromosome 5q34-q35 and that an ancestral {alpha}-{beta}-{gamma} gene cluster probably spawned clusters on chromosomes 4, 5, and 15. Here, we report that the {alpha}{sub 4} gene (GABRA4) maps to human chromosome 4p14-q12, defining a cluster comprising the {alpha}{sub 2}, {alpha}{sub 4}, {beta}{sub 1}, and {gamma}{sub 1} genes. The existence of an {alpha}{sub 2}-{alpha}{sub 4}-{beta}{sub 1}-{gamma}{sub 2} cluster on chromosome 4 and an {alpha}{sub 1}-{alpha}{sub 6}-{beta}{sub 2}-{gamma}{sub 2} cluster on chromosome 5 provides further evidence that the number of ancestral GABA{sub A} receptor subunit genes has been expanded by duplication within an ancestral gene cluster. Moreover, if duplication of the {alpha} gene occurred before duplication of the ancestral gene cluster, then a heretofore undiscovered subtype of a subunit should be located on human chromosome 15q11-q13 within an {alpha}{sub 5}-{alpha}{sub x}-{beta}{sub 3}-{gamma}{sub 3} gene cluster at the locus for Angelman and Prader-Willi syndromes. 34 refs., 6 figs., 1 tab.

  15. RAmy2A; a novel alpha-amylase-encoding gene in rice.

    PubMed

    Huang, N; Reinl, S J; Rodriguez, R L

    1992-02-15

    The structure and expression of the alpha-amylase-encoding gene, RAmy2A, are described. This only representative of the Amy2 subfamily in rice differs from other cereal alpha-amylase-encoding genes in several respects. It contains the largest introns of all the cereal alpha-amylase-encoding genes examined to date. Moreover, the second of three introns in this gene contains a long inverted repeat sequence that can potentially form a large and stable stem-loop structure in the unspliced RNA transcript. Finally, RAmy2A is constitutively expressed at very low levels in germinated seeds, root, etiolated leaves, immature seeds and callus. This is in marked contrast to the Amy2 genes of wheat and barley which are highly expressed in the aleurone layer of the germinated seeds. PMID:1541400

  16. Differential expression of rice alpha-amylase genes during seedling development under anoxia.

    PubMed

    Hwang, Y S; Thomas, B R; Rodriguez, R L

    1999-08-01

    The unique capability of rice (Oryza sativa L.) seedlings to grow under anoxic conditions may result in part from their ability to express alpha-amylase and maintain the supply of sugar needed for energy metabolism. Previous studies have demonstrated that under aerobic conditions the Amy1 and Amy2 subfamily genes are regulated primarily by phytohormones while the Amy3 subfamily genes are induced during sugar starvation. The expression patterns for these alpha-amylase genes were considerably different in anoxic vs. aerobic rice seedlings. The level of total alpha-amylase mRNA under anoxic conditions was decreased in aleurone layers while it increased in the embryo. Anoxic conditions greatly diminished the expression of the Amy1A gene in aleurone. Conversely, expression of many Amy3 subfamily genes was up-regulated and prolonged in embryo tissues under anoxic conditions. PMID:10527416

  17. Modulation of gene expression by alpha-tocopherol and alpha-tocopheryl phosphate in thp-1 monocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The naturally occurring vitamin E analogue, alpha-tocopheryl phosphate (alphaTP), has been reported to be more potent than the un-phosphorylated alpha alpha-tocopherol (alphaT). We have now measured plasma levels of alphaTP and compared the cellular effects of alphaTP and gamma-tocopheryl phosphate ...

  18. Human tumor necrosis factor alpha gene regulation by virus and lipopolysaccharide.

    PubMed Central

    Goldfeld, A E; Doyle, C; Maniatis, T

    1990-01-01

    We have identified a region of the human tumor necrosis factor alpha (TNF-alpha) gene promoter that is necessary for maximal constitutive, virus-induced, and lipopolysaccharide (LPS)-induced transcription. This region contains three sites that match an NF-kappa B binding-site consensus sequence. We show that these three sites specifically bind NF-kappa B in vitro, yet each of these sites can be deleted from the TNF-alpha promoter with little effect on the induction of the gene by virus or LPS. Moreover, when multimers of these three sites are placed upstream from a truncated TNF-alpha promoter, or a heterologous promoter, an increase in the basal level of transcription is observed that is influenced by sequence context and cell type. However, these multimers are not sufficient for virus or LPS induction of either promoter. Thus, unlike other virus- and LPS-inducible promoters that contain NF-kappa B binding sites, these sites from the TNF-alpha promoter are neither required nor sufficient for virus or LPS induction. Comparison of the sequence requirements of virus induction of the human TNF-alpha gene in mouse L929 and P388D1 cells reveals significant differences, indicating that the sequence requirements for virus induction of the gene are cell type-specific. However, the sequences required for virus and LPS induction of the gene in a single cell type, P388D1, overlap. Images PMID:2263628

  19. Fibrin-mediated lentivirus gene transfer: implications for lentivirus microarrays

    PubMed Central

    Raut, Shruti; Lei, Pedro; Padmashali, Roshan; Andreadis, Stelios T.

    2010-01-01

    We employed fibrin hydrogel as bioactive matrix for lentivirus mediated gene transfer. Fibrin-mediated gene transfer was highly efficient and exhibited strong dependence on fibrinogen concentration. Efficient gene transfer was achieved with fibrinogen concentration between 3.75 – 7.5 mg/mL. Lower fibrinogen concentrations resulted in diffusion of virus out of the gel while higher concentrations led to ineffective fibrin degradation by target cells. Addition of fibrinolytic inhibitors decreased gene transfer in a dose-dependent manner suggesting that fibrin degradation by target cells may be necessary for successful gene delivery. Under these conditions transduction may be limited only to cells interacting with the matrix thereby providing a method for spatially localized gene delivery. Indeed, when lentivirus-containing fibrin microgels were spotted in an array format gene transfer was confined to virus-containing fibrin spots with minimal cross-contamination between neighboring sites. Collectively, our data suggest that fibrin may provide an effective matrix for spatially-localized gene delivery with potential applications in high-throughput lentiviral microarrays and in regenerative medicine. PMID:20153386

  20. Gene expression profile of human lymphocytes exposed to (211)At alpha particles.

    PubMed

    Turtoi, A; Brown, I; Schläger, M; Schneeweiss, F H A

    2010-08-01

    In this study, the Whole Human Genome 44K DNA microarray assay was used for the first time to obtain gene expression profiles in human peripheral blood lymphocytes 2 h after exposure (in suspension) to 6.78 MeV mean energy alpha particles from extracellular (211)At. Lymphocytes were exposed to fluences of 0.3-9.6 x 10(6) alpha particles/cm(2) [corresponding to mean absorbed alpha-particle doses (D(alpha)) of 0.05-1.60 Gy] over 30 min. Significantly modulated expression was identified in 338 early-response genes. Up-regulated expression was evident in 183 early-response genes, while the remaining 155 were down-regulated. Over half of the up-regulated genes and 40% of the down-regulated genes had a known biological process related primarily to cell growth and maintenance and cell communication. Genes associated with cell death were found only in the up-regulated genes and those with development only in the down-regulated genes. Eight selected early-response genes that displayed a sustained up- or down-regulation (CD36, HSPA2, MS4A6A, NFIL3, IL1F9, IRX5, RASL11B and SULT1B1) were further validated in alpha-particle-irradiated lymphocytes of two human individuals using the TaqMan(R) RT-qPCR technique. The results confirmed the observed microarray gene expression patterns. The expression modulation profiles of IL1F9, IRX5, RASL11B and SULT1B1 genes demonstrated similar trends in the two individuals studied. However, no significant linear correlation between increasing relative gene expression and the alpha-particle dose was evident. The results suggest the possibility that a panel of genes that react to alpha-particle radiation does exist and that they merit further study in a greater number of individuals to determine their possible value regarding alpha-particle biodosimetry. PMID:20681779

  1. Tobacco plants transformed with the bean. alpha. ai gene express an inhibitor of insect. alpha. -amylase in their seeds. [Nicotiana tabacum; Tenebrio molitor

    SciTech Connect

    Altabella, T.; Chrispeels, M.J. )

    1990-06-01

    Bean (Phaseolus vulgaris L.) seeds contain a putative plant defense protein that inhibits insect and mammalian but not plant {alpha}-amylases. We recently presented strong circumstantial evidence that this {alpha}-amylase inhibitor ({alpha}Al) is encoded by an already-identified lectin gene whose product is referred to as lectin-like-protein (LLP). We have now made a chimeric gene consisting of the coding sequence of the lectin gene that encodes LLP and the 5{prime} and 3{prime} flanking sequences of the lectin gene that encodes phytohemagglutinin-L. When this chimeric gene was expressed in transgenic tobacco (Nicotiana tabacum), we observed in the seeds a series of polypeptides (M{sub r} 10,000-18,000) that cross-react with antibodies to the bean {alpha}-amylase inhibitor. Most of these polypeptides bind to a pig pancreas {alpha}-amylase affinity column. An extract of the seeds of the transformed tobacco plants inhibits pig pancreas {alpha}-amylase activity as well as the {alpha}-amylase present in the midgut of Tenebrio molitor. We suggest that introduction of this lectin gene (to be called {alpha}ai) into other leguminous plants may be a strategy to protect the seeds from the seed-eating larvae of Coleoptera.

  2. Phytol directly activates peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) and regulates gene expression involved in lipid metabolism in PPAR{alpha}-expressing HepG2 hepatocytes

    SciTech Connect

    Goto, Tsuyoshi; Takahashi, Nobuyuki; Kato, Sota; Egawa, Kahori; Ebisu, Shogo; Moriyama, Tatsuya; Fushiki, Tohru; Kawada, Teruo . E-mail: fat@kais.kyoto-u.ac.jp

    2005-11-18

    The peroxisome proliferator-activated receptor (PPAR) is one of the indispensable transcription factors for regulating lipid metabolism in various tissues. In our screening for natural compounds that activate PPAR using luciferase assays, a branched-carbon-chain alcohol (a component of chlorophylls), phytol, has been identified as a PPAR{alpha}-specific activator. Phytol induced the increase in PPAR{alpha}-dependent luciferase activity and the degree of in vitro binding of a coactivator, SRC-1, to GST-PPAR{alpha}. Moreover, the addition of phytol upregulated the expression of PPAR{alpha}-target genes at both mRNA and protein levels in PPAR{alpha}-expressing HepG2 hepatocytes. These findings indicate that phytol is functional as a PPAR{alpha} ligand and that it stimulates the expression of PPAR{alpha}-target genes in intact cells. Because PPAR{alpha} activation enhances circulating lipid clearance, phytol may be important in managing abnormalities in lipid metabolism.

  3. ''Pulling'' Nanoparticles into Water: Phase Transfer of Oleic Acid Stabilized Monodisperse Nanoparticles into Aqueous Solutions of alpha-Cyclodextrin

    SciTech Connect

    Wang, Y.; Wong, J.F.; Teng, X.; Lin, X.Z.; Yang, H.

    2003-10-18

    (B204)This paper describes a general method to drastically improve the disparity of oleic acid stabilized nanoparticles in aqueous solutions. We use oleic acid stabilized monodisperse nanoparticles of iron oxides and silver as model systems, and have modified the surface properties of these nanoparticles through the formation of an inclusion complex between surface-bound surfactant molecules and alpha-cyclodextrin (alpha-CD). After the modification, the nanoparticles of both iron oxide and Ag can transfer from hydrophobic solvents, such as hexane, to alpha-CD aqueous phase. The efficiency of the phase transfer to the aqueous solutions depend son the initial alpha-CD concentration. The alpha-CD/oleic acid complex stabilized nanoparticles can be stable for long periods of time in aqueous phase under ambient atmospheric conditions. Transmission electron microscopy (TME), ultraviolet-visible (UV-vis) spectroscopy, Fourier transform-infrared (FT-IR) spectroscopy, and colorimetric methods have been used in the characterization of these nanoparticles.

  4. Molecular evolution of serpins: homologous structure of the human. cap alpha. /sub 1/-antichymotrypsin and. cap alpha. /sub 1/-antitrypsin genes

    SciTech Connect

    Bao, J.; Sifers, R.N.; Kidd, V.J.; Ledley, F.D.; Woo, S.L.C.

    1987-12-01

    ..cap alpha../sub 1/-Antichymotrypsin belongs to a supergene family that includes ..cap alpha../sub 1/-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. The human chromosomal ..cap alpha../sub 1/-antichymotrypsin gene has been cloned and its molecular structure established. The gene is approximately 12 kb in length and contains five exons and four introns. The locations of the introns within the ..cap alpha../sub 1/-antichymotrypsin gene are identical with those of the human ..cap alpha../sub 1/-antitrypsin and angiotensinogen genes. Other members of this supergene family contain introns located at nonhomologous positions of the genes. The homologous organization of the ..cap alpha../sub 1/-antichymotrypsin and ..cap alpha../sub 1/-antitrypsin genes corresponds with the high degree of homology between their protein sequences and suggest that these loci arose by recent gene duplication. A model is presented for the evolution of both the genomic structure and the protein sequences of the serine protease inhibitor superfamily.

  5. Assessment of fecal bacteria with bile acid 7 alpha-dehydroxylating activity for the presence of bai-like genes.

    PubMed Central

    Doerner, K C; Takamine, F; LaVoie, C P; Mallonee, D H; Hylemon, P B

    1997-01-01

    Eubacterium sp. strain VPI 12708 has several bile acid-inducible (bai) genes which encode enzymes in the bile acid 7 alpha-dehydroxylation (7 alpha DeOH) pathway. Twelve 7 alpha DeOH-positive intestinal bacterial strains were assayed for 7 alpha DeOH activity, and 13 strains were tested for hybridization with bai genes. Cholic acid 7 alpha DeOH activity varied greatly (> 100-fold) among these strains. Southern blot experiments showed that DNA prepared from 7 of 13 strains hybridized with at least one of the bai genes from Eubacterium sp. strain VPI 12708. PMID:9055436

  6. Global Analysis of Horizontal Gene Transfer in Fusarium verticillioides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The co-occurrence of microbes within plants and other specialized niches may facilitate horizontal gene transfer (HGT) affecting host-pathogen interactions. We recently identified fungal-to-fungal HGTs involving metabolic gene clusters. For a global analysis of HGTs in the maize pathogen Fusarium ve...

  7. Tumor necrosis factor-alpha modulates monocyte/macrophage apoprotein E gene expression.

    PubMed Central

    Duan, H; Li, Z; Mazzone, T

    1995-01-01

    apo E has been shown to modulate cholesterol balance in arterial wall cells. Production of apo E by macrophages in atherosclerotic plaques could thereby influence the development of the plaque lesion. Cytokines, including TNF alpha, have been identified in human lesions, therefore, we undertook a series of studies to evaluate the effect of TNF alpha on monocyte/macrophage apo E production. The addition of TNF alpha to freshly isolated human monocytes led to a four- to fivefold increase of apo E mRNA abundance. The addition of TNF alpha to fully differentiated macrophages either had no effect or modestly inhibited apo E mRNA expression. THP1 human monocytic cells also responded to TNF alpha in a phenotype-specific manner. Treatment of these cells with TNF alpha produced a dose- and time-dependent increase in apo E mRNA. This increase was reflected in apo E synthesis and was associated with inhibition of DNA synthesis, and with induction of c-fos and ICAM-1 gene expression. Cell-permanent analogues of ceramide did not reproduce TNF alpha effect on apo E, but antagonists of protein kinase C did inhibit its effect. TNF alpha induction of apo E mRNA abundance was associated with stimulation of apo E promoter-dependent gene transcription. In summary, TNF alpha stimulates apo E gene transcription, mRNA abundance, and protein synthesis in the monocyte/macrophage in a phenotype-specific manner. Such regulation could significantly modify the amount of apo E present in vessel wall lesions. Images PMID:7635986

  8. Evolution of and horizontal gene transfer in the Endornavirus genus.

    PubMed

    Song, Dami; Cho, Won Kyong; Park, Sang-Ho; Jo, Yeonhwa; Kim, Kook-Hyung

    2013-01-01

    The transfer of genetic information between unrelated species is referred to as horizontal gene transfer. Previous studies have demonstrated that both retroviral and non-retroviral sequences have been integrated into eukaryotic genomes. Recently, we identified many non-retroviral sequences in plant genomes. In this study, we investigated the evolutionary origin and gene transfer of domains present in endornaviruses which are double-stranded RNA viruses. Using the available sequences for endornaviruses, we found that Bell pepper endornavirus-like sequences homologous to the glycosyltransferase 28 domain are present in plants, fungi, and bacteria. The phylogenetic analysis revealed the glycosyltransferase 28 domain of Bell pepper endornavirus may have originated from bacteria. In addition, two domains of Oryza sativa endornavirus, a glycosyltransferase sugar-binding domain and a capsular polysaccharide synthesis protein, also exhibited high similarity to those of bacteria. We found evidence that at least four independent horizontal gene transfer events for the glycosyltransferase 28 domain have occurred among plants, fungi, and bacteria. The glycosyltransferase sugar-binding domains of two proteobacteria may have been horizontally transferred to the genome of Thalassiosira pseudonana. Our study is the first to show that three glycome-related viral genes in the genus Endornavirus have been acquired from marine bacteria by horizontal gene transfer. PMID:23667703

  9. Evolution of and Horizontal Gene Transfer in the Endornavirus Genus

    PubMed Central

    Park, Sang-Ho; Jo, Yeonhwa; Kim, Kook-Hyung

    2013-01-01

    The transfer of genetic information between unrelated species is referred to as horizontal gene transfer. Previous studies have demonstrated that both retroviral and non-retroviral sequences have been integrated into eukaryotic genomes. Recently, we identified many non-retroviral sequences in plant genomes. In this study, we investigated the evolutionary origin and gene transfer of domains present in endornaviruses which are double-stranded RNA viruses. Using the available sequences for endornaviruses, we found that Bell pepper endornavirus-like sequences homologous to the glycosyltransferase 28 domain are present in plants, fungi, and bacteria. The phylogenetic analysis revealed the glycosyltransferase 28 domain of Bell pepper endornavirus may have originated from bacteria. In addition, two domains of Oryza sativa endornavirus, a glycosyltransferase sugar-binding domain and a capsular polysaccharide synthesis protein, also exhibited high similarity to those of bacteria. We found evidence that at least four independent horizontal gene transfer events for the glycosyltransferase 28 domain have occurred among plants, fungi, and bacteria. The glycosyltransferase sugar-binding domains of two proteobacteria may have been horizontally transferred to the genome of Thalassiosira pseudonana. Our study is the first to show that three glycome-related viral genes in the genus Endornavirus have been acquired from marine bacteria by horizontal gene transfer. PMID:23667703

  10. Electrical characterization of a Space Station Freedom alpha utility transfer assembly

    NASA Technical Reports Server (NTRS)

    Yenni, Edward J.

    1994-01-01

    Electrical power, command signals and data are transferred across the Space Station Freedom solar alpha rotary joint by roll rings, which are incorporated within the Utility Transfer Assembly (UTA) designed and manufactured by Honeywell Space Systems Operations. A developmental Model of the UTA was tested at the NASA Lewis Research Center using the Power Management and Distribution DC test bed. The objectives of these tests were to obtain data for calibrating system models and to support final design of qualification and flight units. This testing marked the first time the UTA was operated at high power levels and exposed to electrical conditions similar to that which it will encounter on the actual Space Station. Satisfactory UTA system performance was demonstrated within the scope of this testing.

  11. [Cloning the alpha-amylase gene of Streptococcus bovis and its expression in Bacillus subtilis cells].

    PubMed

    Iakorski, P; Kuntsova, M M; Loseva, E F; Khasanov, F K

    1991-06-01

    The gene coding for alpha-amylase from the ruminant bacterium Streptococcus bovis was cloned on the plasmid pMX39 in Bacillus subtilis cells. An alpha-amylase positive colony was isolated in the initial screening of 3900 colonies on the medium containing insoluble starch. The size of the insert was approximately 2.8 kb. The recombinant plasmid was stably maintained in Bacillus subtilis cells under the nonselective conditions. PMID:1944323

  12. Sequence of the dog immunoglobulin alpha and epsilon constant region genes

    SciTech Connect

    Patel, M.; Selinger, D.; Mark, G.E.; Hollis, G.F.; Hickey, G.J.

    1995-03-01

    The immunoglobulin alpha (IGHAC) and epsilon (IGHEC) germline constant region genes were isolated from a dog liver genomic DNA library. Sequence analysis indicates that the dog IGHEC gene is encoded by four exons spread out over 1.7 kilobases (kb). The IGHAC sequence encompasses 1.5 kb and includes all three constant region coding exons. The complete exon/intron sequence of these genes is described. 28 refs., 2 figs., 2 tabs.

  13. Genes for the dimerization cofactor of hepatocyte nuclear factor-1[alpha] (DCOH) are on human and murine chromsomes 10

    SciTech Connect

    Milatovich, A.; Mendel, D.B.; Crabtree, G.R.; Francke, U. )

    1993-04-01

    Hepatocyte nuclear factor-1[alpha] (HNF-1[alpha]; gene symbol, TCF1) forms dimers with itself as well as with HNF-1[beta] and regulates the expression of several liver-specific genes. Recently, a dimerization cofactor of hepatocyte nuclear factor-1[alpha], called DCOH, has been identified. Here, the authors report the chromosomal localization of the genes for this cofactor to chromosomes 10 in both humans and mice by Southern blot analyses of somatic cell hybrids. 25 refs., 1 fig., 2 tabs.

  14. Estrogen-related receptor {alpha} is essential for the expression of antioxidant protection genes and mitochondrial function

    SciTech Connect

    Rangwala, Shamina M. . E-mail: shamina.rangwala@novartis.com; Li, Xiaoyan; Lindsley, Loren; Wang, Xiaomei; Shaughnessy, Stacey; Daniels, Thomas G.; Szustakowski, Joseph; Nirmala, N.R.; Wu, Zhidan; Stevenson, Susan C.

    2007-05-25

    Estrogen-related receptor {alpha} (ERR{alpha}) is an important mediator of mitochondrial biogenesis and function. To investigate the transcriptional network controlling these phenomena, we investigated mitochondrial gene expression in embryonic fibroblasts isolated from ERR{alpha} null mice. Peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) stimulated mitochondrial gene expression program in control cells, but not in the ERR{alpha} null cells. Interestingly, the induction of levels of mitochondrial oxidative stress protection genes in response to increased PGC-1{alpha} levels was dependent on ERR{alpha}. Furthermore, we found that the PGC-1{alpha}-mediated induction of estrogen-related receptor {gamma} and nuclear respiratory factor 2 (NRF-2), was dependent on the presence of ERR{alpha}. Basal levels of NRF-2 were decreased in the absence of ERR{alpha}. The absence of ERR{alpha} resulted in a decrease in citrate synthase enzyme activity in response to PGC-1{alpha} overexpression. Our results indicate an essential role for ERR{alpha} as a key regulator of oxidative metabolism.

  15. Characterization of alpha-gliadin genes from diploid wheats and the comparative analysis with those from polyploid wheats.

    PubMed

    Ma, Z C; Wei, Y M; Yan, Z H; Zheng, Y L

    2007-11-01

    To carry out the comparative analysis of alpha-gliadin genes on A genomes of diploid and polyploid wheats, 8 full-length alpha-gliadin genes, including 3 functional genes and 5 pseudogenes, were obtained from diploid wheats, among which 2, 2 and 4 alpha-gliadin genes were isolated from T. urartu, T. monococcum and T. boeoticum, respectively. The results indicated that higher number of alpha-gliadin pseudogenes have been present in diploid wheats before the formation of polyploid wheats. Amino acid sequence comparative analysis among 26 alpha-gliadin genes, including 16 functional genes and 10 pseudogenes, from diploid and polyploid wheats was conducted. The results indicated that all alpha-gliadins contained four coeliac toxic peptide sequences (i.e., PSQQ, QQQP, QQPY and QPYP). The polyglutamine domains are highly variable, and the second polyglutamine stretch is usually disrupted by the lysine or arginine residue at the fourth position. The unique domain I is the most conserved domain. There are 4 and 2 conserved cysteine residues in the unique domains I and II, respectively. Comparative analysis indicated that the functional alpha-gliadin genes from A genome are highly conserved, whereas the identity of pseudogenes in diploid wheats are higher than those in hexaploid wheats. Phylogenetic analysis indicated that all the analyzed functional alpha-gliadin genes could be clustered into two major groups, among which one group could be further divided into 5 subgroups. The origin of alpha-gliadin pseudogene and functional genes were also discussed. PMID:18186192

  16. Neuronal-type alpha-bungarotoxin receptors and the alpha 5-nicotinic receptor subunit gene are expressed in neuronal and nonneuronal human cell lines.

    PubMed Central

    Chini, B; Clementi, F; Hukovic, N; Sher, E

    1992-01-01

    alpha-Bungarotoxin (alpha Bgtx) is a toxin known to interact with muscle nicotinic receptors and with some neuronal nicotinic receptors. We show that alpha Bgtx binding sites are also expressed in nonmuscle and nonneuronal human cells, including small cell lung carcinoma and several epithelial cell lines. These receptors are immunologically related to the alpha Bgtx receptors of unknown function described in the nervous system and in the IMR32 neuroblastoma cell line and are distinct from muscle nicotinic receptors. We have also cloned from IMR32 cells the human alpha 5-nicotinic receptor subunit, which is supposed to participate in the formation of alpha Bgtx receptors. Transcripts corresponding to the alpha 5-subunit gene were found not only in neuroblastoma cells but also in all the cell lines expressing alpha Bgtx receptors, with the exception of the TE671 cell line, whose nicotinic receptor subunits are of the muscle type. We conclude that both alpha Bgtx receptors and the alpha 5-nicotinic subunit gene are not neuron-specific, as previously thought, but are expressed in a number of human cell lines of various origin. Images PMID:1542648

  17. Structure and expression of the guinea-pig alpha-lactalbumin gene.

    PubMed Central

    Laird, J E; Jack, L; Hall, L; Boulton, A P; Parker, D; Craig, R K

    1988-01-01

    The entire guinea-pig alpha-lactalbumin gene was isolated from a genomic DNA library constructed in the bacteriophage lambda L47. The complete nucleotide sequence of the gene and its immediate 5' and 3' flanking sequences were determined and compared with those of the human and rat alpha-lactalbumin genes. This demonstrates that the size, organization and sequence of the exons is highly conserved between species, and reveals the presence of the highly conserved potential regulatory 'milk box' consensus sequence [RGAAGRAAA(N)TGGACAGAAATCAA(CG)TTTCTA] between positions -140 and -110 relative to the transcriptional start point. A guinea-pig LINE repeat sequence was also present in the 5' flanking region between positions -520 and -1195. Transfection of the alpha-lactalbumin gene cloned in a bovine papilloma virus vector into the mouse C127 and human MCF-7 mammary tumour cell-lines gave rise to stable but seemingly constitutive expression of alpha-lactalbumin. Expression was from the correct transcriptional start point, resulting in the accumulation of correctly processed mRNA and the secretion of alpha-lactalbumin into the culture medium. Images Fig. 3. Fig. 5. Fig. 6. Fig. 7. PMID:2845947

  18. Sequence heterogeneity, multiplicity, and genomic organization of. cap alpha. - and. beta. -tubulin genes in Sea Urchins

    SciTech Connect

    Alexandraki, D.; Ruderman, J.V.

    1981-12-01

    The authors analyzed the multiplicity, heterogeneity, and organization of the genes encoding the ..cap alpha.. and ..beta.. tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. ..cap alpha.. and ..beta..-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The ..cap alpha.. cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of ..cap alpha.. tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The ..beta.. cDNA insertion contains the coding sequence for the 100 C-terminal amino acids of ..beta.. tubulin and 83 base pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous ..cap alpha..- and ..beta..-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of ..cap alpha..-tubulin genes with ..beta..-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered.

  19. A lectin gene encodes the alpha-amylase inhibitor of the common bean.

    PubMed Central

    Moreno, J; Chrispeels, M J

    1989-01-01

    An alpha-amylase inhibitor that inhibits insect and mammalian alpha-amylases but not plant alpha-amylases, is present in seeds of the common bean (Phaseolus vulgaris). We have purified the alpha-amylase inhibitor by using a selective heat treatment in acidic medium and affinity chromatography with porcine pancreas alpha-amylase coupled to agarose. Under sodium dodecyl sulfate gel electrophoresis, the purified inhibitor gave rise to five bands with mobilities corresponding to molecular masses ranging from 14 to 19 kDa. N-terminal sequencing (up to 15 amino acids) of the polypeptides obtained from these bands resulted in only two different sequences matching two stretches of the amino acid sequence deduced from an already described lectin gene [Hoffman, L. M. (1984) J. Mol. Appl. Gen. 2,447-453]. This gene is different from but closely related to the genes that code for phytohemagglutinin, the major lectin of bean. Further evidence based on amino acid composition, identification of a precursor, and recognition of the product of the gene (expressed in Escherichia coli) by an anti-alpha-amylase inhibitor serum confirms that the inhibitor is encoded by this or a closely related lectin gene. This finding assigns a biological function, which has been described at the molecular level, to a plant lectin gene product and supports the defense role postulated for seed lectins. The lack of homology with other families of enzyme inhibitors suggests that this may be the first member of a new family of plant enzyme inhibitors. Images PMID:2682631

  20. Horizontal functional gene transfer from bacteria to fishes

    PubMed Central

    Sun, Bao-Fa; Li, Tong; Xiao, Jin-Hua; Jia, Ling-Yi; Liu, Li; Zhang, Peng; Murphy, Robert W.; He, Shun-Min; Huang, Da-Wei

    2015-01-01

    Invertebrates can acquire functional genes via horizontal gene transfer (HGT) from bacteria but fishes are not known to do so. We provide the first reliable evidence of one HGT event from marine bacteria to fishes. The HGT appears to have occurred after emergence of the teleosts. The transferred gene is expressed and regulated developmentally. Its successful integration and expression may change the genetic and metabolic repertoire of fishes. In addition, this gene contains conserved domains and similar tertiary structures in fishes and their putative donor bacteria. Thus, it may function similarly in both groups. Evolutionary analyses indicate that it evolved under purifying selection, further indicating its conserved function. We document the first likely case of HGT of functional gene from prokaryote to fishes. This discovery certifies that HGT can influence vertebrate evolution. PMID:26691285

  1. Advancements in gene transfer-based therapy for hemophilia A

    PubMed Central

    Doering, Christopher B; Spencer, H Trent

    2010-01-01

    Gene therapy has promised clinical benefit to those suffering with hemophilia A, but this benefit has not yet been realized. However, during the past two decades, basic and applied gene therapy research has progressed and the goal of gene therapy for hemophilia A is once again in our sights. The hemophilia A patient population suffers from a disease that requires invasive, lifelong management, is exorbitantly expensive to treat, has geographically limited treatment access and can become untreatable due to immune reactions to the treatment product. Subsequent to the cloning of the factor VIII gene and cDNA in the early 1980s, academic and commercial research laboratories began to pursue gene transfer-based therapies to supplement or supplant the available protein replacement therapy. However, to date, clinical trials for gene therapy of hemophilia A have been unsuccessful. Three trials have been conducted with each having tested a different gene-transfer strategy and each demonstrating that there is a considerable barrier to achieving sustained expression of therapeutic amounts of factor VIII. Recent progress has been made in gene-transfer technology and, relevant to hemophilia A, towards increasing the biosynthetic efficiency of factor VIII. These advances are now being combined to develop novel strategies to treat and possibly cure hemophilia A. PMID:20577574

  2. MHC evolution in three salmonid species: a comparison between class II alpha and beta genes.

    PubMed

    Gómez, Daniela; Conejeros, Pablo; Marshall, Sergio H; Consuegra, Sofia

    2010-08-01

    The genes of the major histocompatibility complex (MHC) are amongst the most variable in vertebrates and represent some of the best candidates to study processes of adaptive evolution. However, despite the number of studies available, most of the information on the structure and function of these genes come from studies in mammals and birds in which the MHC class I and II genes are tightly linked and class II alpha exhibits low variability in many cases. Teleost fishes are among the most primitive vertebrates with MHC and represent good organisms for the study of MHC evolution because their class I and class II loci are not physically linked, allowing for independent evolution of both classes of genes. We have compared the diversity and molecular mechanisms of evolution of classical MH class II alpha and class II beta loci in farm populations of three salmonid species: Oncorhynchus kisutch, Oncorhynchus mykiss and Salmo salar. We found single classical class II loci and high polymorphism at both class II alpha and beta genes in the three species. Mechanisms of evolution were common for both class II genes, with recombination and point mutation involved in generating diversity and positive selection acting on the peptide-binding residues. These results suggest that the maintenance of variability at the class IIalpha gene could be a mechanism to increase diversity in the MHC class II in salmonids in order to compensate for the expression of one single classical locus and to respond to a wider array of parasites. PMID:20521040

  3. Inhibition of DNA topoisomerase II alpha gene expression by the p53 tumor suppressor.

    PubMed Central

    Wang, Q; Zambetti, G P; Suttle, D P

    1997-01-01

    DNA topoisomerase II (topo II) is an essential nuclear enzyme involved in major cellular functions such as DNA replication, transcription, recombination, and mitosis. While an elevated level of topo II alpha is associated with cell proliferation, wild-type (wt) p53 inhibits the expression of various growth-stimulatory genes. To determine if p53 downregulates topo II alpha gene expression, a murine cell line, (10)1val, that expresses a temperature-sensitive p53 was utilized. The (10)1val cells had significantly lower levels of topo II alpha mRNA and protein following incubation for 24 h at 32 degrees C (p53 with wt conformation) than at 39 degrees C (p53 with mutant conformation). The effect of p53 on the human topo II alpha gene promoter was determined by using luciferase reporter plasmids containing varying lengths of the topo II alpha promoter transiently cotransfected into p53-deficient (10)1 cells together with wt or mutant p53 expression plasmids. Transcription from the full-length (bp -557 to +90) topo II alpha promoter was decreased 15-fold by wt p53 in a concentration-dependent manner, whereas mutant p53 exerted much weaker inhibition. Consecutive deletion of the five inverted CCAAT elements (ICEs) from the topo II alpha promoter reduced both the basal promoter activity and wt p53-induced suppression. Transcription of the minimal promoter (-32 to +90), which contains no ICE, was slightly stimulated by wt or mutant p53 expression. When point mutations were introduced into the most proximal ICE (-68), the inhibitory effect of wt p53 was alleviated and stimulation of topo II alpha expression resulted. Our study suggests that wt p53 functions as a transcriptional repressor of topo II alpha gene expression, possibly through a functional interaction with specific ICEs. Inactivation of wt p53 may reduce normal regulatory suppression of topo II alpha and contribute to abortive cell cycle checkpoints, accelerated cell proliferation, and alterations in genomic

  4. Structural organization of the human cardiac [alpha]-myosin heavy chain gene (MYH6)

    SciTech Connect

    Epp, T.A.; Dixon, I.M.C.; Wang, H.Y.; Sole, M.J.; Liew, C.C. )

    1993-12-01

    The human myocardium expresses two cardiac myosin heavy chain (MyHC) isoforms, [alpha] and [beta], that exist in tandem array on chromosome 14q12. The authors have previously sequenced the entire human cardiac [beta]-MyHC gene and now report the complete nucleotide sequence of the human cardiac [alpha]-MyHC, encompassing 26,159 bp as well as the entire 4484-bp 5'-flanking intergenic region. The gene (MYH6) consists of 39 exons, 37 of which contain coding information. The 5'-untranslated region is split into 3 exons, with the third exon containing the AUG translocation initiation codon. With the exception of the 13th intron of the human cardiac [beta]-MyHC, which is not present within the [alpha]-isogene, all exon/intron boundaries are conserved. Conspicuous sequence motifs contained within the [alpha]-MyHC gene include four Alu repeats, a single (GT)[sub n] element, and a homopurine-homopyrimidine tract containing 23 GAA repeating units followed by 10 GAG repeating units. Comparison of the encoded amino acid sequence with a previously reported human [alpha]-MyHC cDNA sequence reveals several potential polymorphisms. 29 refs., 1 fig., 1 tab.

  5. The mouse dead-end gene isoform alpha is necessary for germ cell and embryonic viability.

    PubMed

    Bhattacharya, Chitralekha; Aggarwal, Sita; Zhu, Rui; Kumar, Madhu; Zhao, Ming; Meistrich, Marvin L; Matin, Angabin

    2007-03-30

    Inactivation of the dead-end (Dnd1) gene in the Ter mouse strain results in depletion of primordial germ cells (PGCs) so that mice become sterile. However, on the 129 mouse strain background, loss of Dnd1 also increases testicular germ cell tumor incidence in parallel to PGC depletion. We report that inactivation of Dnd1 also affects embryonic viability in the 129 strain. Mouse Dnd1 encodes two protein isoforms, DND1-isoform alpha (DND1-alpha) and DND1-isoform beta (DND1-beta). Using isoform-specific antibodies, we determined DND1-alpha is expressed in embryos and embryonic gonads whereas DND1-beta expression is restricted to germ cells of the adult testis. Our data implicate DND1-alpha isoform to be necessary for germ cell viability and therefore its loss in Ter mice results in PGC depletion, germ cell tumor development and partial embryonic lethality in the 129 strain. PMID:17291453

  6. Horizontal gene transfer in the human gastrointestinal tract: potential spread of antibiotic resistance genes

    PubMed Central

    Huddleston, Jennifer R

    2014-01-01

    Bacterial infections are becoming increasingly difficult to treat due to widespread antibiotic resistance among pathogens. This review aims to give an overview of the major horizontal transfer mechanisms and their evolution and then demonstrate the human lower gastrointestinal tract as an environment in which horizontal gene transfer of resistance determinants occurs. Finally, implications for antibiotic usage and the development of resistant infections and persistence of antibiotic resistance genes in populations as a result of horizontal gene transfer in the large intestine will be discussed. PMID:25018641

  7. Experiments on gene transferring to primary hematopoietic cells by liposome.

    PubMed

    Hu, L; Zhang, B

    2000-01-01

    Liposomes have showed many advantages in mediating exogenous gene into many cell types in vitro and in vivo. But few data are available concerning gene transfer into hematopoietic cells. In this report, we described two-marker genes (Neo R and Lac Z) co-transferred into hematopoietic cells of human and mouse by using liposome in vitro. The efficiency of gene transfer was tested by X-gal staining and observation of colony formation. The X-gal blue staining rate of transduced cells was about (13.33 +/- 2.68)% in human and about (16.28 +/- 2.95)% in mouse without G418 selection. After G418 selection, the blue cell rate was (46.06 +/- 3.47)% in human and (43.45 +/- 4.1)% in mouse, which were markedly higher than those before selection, suggesting that high-efficiency gene transfer and expression could be attained in primary hematopoietic cells using this easy and harmless transduction protocol. At the same time, this protocol provided experimental data for clinicians to investigate the biology of marrow reconstitution and trace the origin of relapse after autologous bone marrow transplantation for the patients with leukemia. PMID:12840913

  8. Estimating the Frequency of Horizontal Gene Transfer Using Phylogenetic Models of Gene Gain and Loss.

    PubMed

    Zamani-Dahaj, Seyed Alireza; Okasha, Mohamed; Kosakowski, Jakub; Higgs, Paul G

    2016-07-01

    We analyze patterns of gene presence and absence in a maximum likelihood framework with rate parameters for gene gain and loss. Standard methods allow independent gains and losses in different parts of a tree. While losses of the same gene are likely to be frequent, multiple gains need to be considered carefully. A gene gain could occur by horizontal transfer or by origin of a gene within the lineage being studied. If a gene is gained more than once, then at least one of these gains must be a horizontal transfer. A key parameter is the ratio of gain to loss rates, a/v We consider the limiting case known as the infinitely many genes model, where a/v tends to zero and a gene cannot be gained more than once. The infinitely many genes model is used as a null model in comparison to models that allow multiple gains. Using genome data from cyanobacteria and archaea, it is found that the likelihood is significantly improved by allowing for multiple gains, but the average a/v is very small. The fraction of genes whose presence/absence pattern is best explained by multiple gains is only 15% in the cyanobacteria and 20% and 39% in two data sets of archaea. The distribution of rates of gene loss is very broad, which explains why many genes follow a treelike pattern of vertical inheritance, despite the presence of a significant minority of genes that undergo horizontal transfer. PMID:27189546

  9. Repression by ARP-1 sensitizes apolipoprotein AI gene responsiveness to RXR alpha and retinoic acid.

    PubMed Central

    Widom, R L; Rhee, M; Karathanasis, S K

    1992-01-01

    The gene coding for apolipoprotein AI (apoAI), a lipid binding protein involved in the transport of cholesterol and other lipids in the plasma, is expressed in mammals predominantly in the liver and the intestine. Liver-specific expression is controlled by synergistic interactions between transcription factors bound to three separate sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within a powerful liver-specific enhancer located between nucleotides -222 and -110 upstream of the apoAI gene transcription start site (+1). Previous studies in our laboratory have shown that ARP-1, a member of the nuclear receptor superfamily whose ligand is unknown (orphan receptor), binds to site A and represses transcription of the apoAI gene in liver cells. In a more recent series of experiments, we found that site A is a retinoic acid (RA) response element that responds preferentially to the recently identified RA-responsive receptor RXR alpha over the previously characterized RA receptors RAR alpha and RAR beta. In this study we investigated the combined effects of ARP-1 and RXR alpha on apoAI gene expression in liver cells. Transient transfection assays showed that site A is necessary and sufficient for RXR alpha-mediated transactivation of the apoAI gene basal promoter in human hepatoma HepG2 cells in the presence of RA and that this transactivation is abolished by increasing amounts of cotransfected ARP-1. Electrophoretic mobility shift assays and subsequent Scatchard analysis of the data revealed that ARP-1 and RXR alpha bind to site A with similar affinities. These assays also revealed that ARP-1 and RXR alpha bind to site A as heterodimers with an affinity approximately 10 times greater than that of either ARP-1 or RXR alpha alone. Further transfection assays in HepG2 cells, using as a reporter a construct containing the apoAI gene basal promoter and its upstream regulatory elements (including site A) in their natural context, revealed that RXR alpha

  10. Cholesteryl ester hydroperoxides increase macrophage CD36 gene expression via PPAR{alpha}

    SciTech Connect

    Jedidi, Iness; Couturier, Martine; Therond, Patrice; Gardes-Albert, Monique; Legrand, Alain; Barouki, Robert; Bonnefont-Rousselot, Dominique; Aggerbeck, Martine . E-mail: Martine.Aggerbeck@univ-paris5.fr

    2006-12-22

    The uptake of oxidized LDL by macrophages is a key event in the development of atherosclerosis. The scavenger receptor CD36 is one major receptor that internalizes oxidized LDL. In differentiated human macrophages, we compared the regulation of CD36 expression by copper-oxidized LDL or their products. Only oxidized derivatives of cholesteryl ester (CEOOH) increased the amount of CD36 mRNA (2.5-fold). Both oxidized LDL and CEOOH treatment increased two to fourfold the transcription of promoters containing peroxisome-proliferator-activated-receptor responsive elements (PPRE) in the presence of PPAR{alpha} or {gamma}. Electrophoretic-mobility-shift-assays with nuclear extracts prepared from macrophages treated by either oxidized LDL or CEOOH showed increased binding of PPAR{alpha} to the CD36 gene promoter PPRE. In conclusion, CEOOH present in oxidized LDL increase CD36 gene expression in a pathway involving PPAR{alpha}.

  11. The PPAR alpha gene is associated with triglyceride, low-density cholesterol and inflammation marker response to fenofibrate intervention: the GOLDN study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As a peroxisome proliferator-activated receptor alpha (PPAR Alpha) agonist, fenofibrate favorably modulates dyslipidemia and inflammation markers, which are associated with cardiovascular risk. To determine whether variation in the PPAR Alpha receptor gene was associated with lipid and inflammatory ...

  12. Horizontal gene transfer in eukaryotes: The weak-link model

    PubMed Central

    Huang, Jinling

    2013-01-01

    The significance of horizontal gene transfer (HGT) in eukaryotic evolution remains controversial. Although many eukaryotic genes are of bacterial origin, they are often interpreted as being derived from mitochondria or plastids. Because of their fixed gene pool and gene loss, however, mitochondria and plastids alone cannot adequately explain the presence of all, or even the majority, of bacterial genes in eukaryotes. Available data indicate that no insurmountable barrier to HGT exists, even in complex multicellular eukaryotes. In addition, the discovery of both recent and ancient HGT events in all major eukaryotic groups suggests that HGT has been a regular occurrence throughout the history of eukaryotic evolution. A model of HGT is proposed that suggests both unicellular and early developmental stages as likely entry points for foreign genes into multicellular eukaryotes. PMID:24037739

  13. Interkingdom Gene Transfer May Contribute to the Evolution of Phytopathogenicity in Botrytis Cinerea

    PubMed Central

    Zhu, Bo; Zhou, Qing; Xie, Guanlin; Zhang, Guoqing; Zhang, Xiaowei; Wang, Yanli; Sun, Gunchang; Li, Bin; Jin, Gulei

    2012-01-01

    The ascomycete Botrytis cinerea is a phytopathogenic fungus infecting and causing significant yield losses in a number of crops. The genome of B. cinerea has been fully sequenced while the importance of horizontal gene transfer (HGT) to extend the host range in plant pathogenic fungi has been recently appreciated. However, recent data confirm that the B. cinerea fungus shares conserved virulence factors with other fungal plant pathogens with narrow host range. Therefore, interkingdom HGT may contribute to the evolution of phytopathogenicity in B. cinerea. In this study, a stringent genome comparison pipeline was used to identify potential genes that have been obtained by B. cinerea but not by other fungi through interkingdom HGT. This search led to the identification of four genes: a UDP-glucosyltransferase (UGT), a lipoprotein and two alpha/beta hydrolase fold proteins. Phylogenetic analysis of the four genes suggests that B. cinerea acquired UGT from plants and the other 3 genes from bacteria. Based on the known gene functions and literature searching, a correlation between gene acquision and the evolution of pathogenicity in B. cinerea can be postulated. PMID:22346340

  14. Lifetime and g-factor measurements of excited states using Coulomb excitation and alpha transfer reactions

    NASA Astrophysics Data System (ADS)

    Guevara, Z. E.; Torres, D. A.

    2016-07-01

    In this contribution the challenges in the use of a setup to simultaneously measure lifetimes and g-factor values will be presented. The simultaneous use of the transient field technique and the Doppler Shift Attenuation Method, to measure magnetic moments and lifetimes respectively, allows to obtain a complete characterization of the currents of nucleons and the deformation in excited states close to the ground state. The technique is at the moment limited to Coulomb excitation and alpha-transfer reactions, what opens an interesting perspective to consider this type of experiments with radioactive beams. The use of deep-inelastic and fusion-evaporation reactions will be discussed. An example of a setup that makes use of a beam of 106Cd to study excited states of 110Sn and the beam nuclei itself will be presented.

  15. Hepatocyte nuclear factor-4alpha and bile acids regulate human concentrative nucleoside transporter-1 gene expression.

    PubMed

    Klein, Kerstin; Kullak-Ublick, Gerd A; Wagner, Martin; Trauner, Michael; Eloranta, Jyrki J

    2009-04-01

    The concentrative nucleoside transporter-1 (CNT1) is a member of the solute carrier 28 (SLC28) gene family and is expressed in the liver, intestine, and kidneys. CNT1 mediates the uptake of naturally occurring pyrimidine nucleosides, but also nucleoside analogs used in anticancer and antiviral therapy. Thus expression levels of CNT1 may affect the pharmacokinetics of these drugs and the outcome of drug therapy. Because little is known about the transcriptional regulation of human CNT1 gene expression, we have characterized the CNT1 promoter with respect to DNA response elements and their binding factors. The transcriptional start site of the CNT1 gene was determined by 5'-RACE. In silico analysis revealed the existence of three putative binding sites for the nuclear receptor hepatocyte nuclear factor-4alpha (HNF-4alpha) within the CNT1 promoter. A luciferase reporter gene construct containing the CNT1 promoter region was transactivated by HNF-4alpha in human cell lines derived from the liver, intestine, and kidneys. Consistent with this, we showed in electromobility shift assays that HNF-4alpha specifically binds to two conserved direct repeat-1 motifs within the proximal CNT1 promoter. In cotransfection experiments, the transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1alpha further increased, whereas the bile acid-inducible corepressor small heterodimer partner reduced, HNF-4alpha-dependent CNT1 promoter activity. Consistent with the latter phenomenon, CNT1 mRNA expression levels were suppressed in primary human hepatocytes upon bile acid treatment. Supporting the physiological relevance and species conservation of this effect, ileal Cnt1 mRNA expression was decreased upon bile acid feeding and increased upon bile duct ligation in mice. PMID:19228884

  16. CHARACTERIZATION AND GENE EXPRESSION OF BABESIA BOVIS ELONGATION FACTOR-1ALPHA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Elongation factor 1 alpha (EF-1') is a constitutively expressed, abundant protein that is a key element in eukaryotic protein translation. Because of its high level of transcription, the EF-1''promoter has been utilized to drive exogenous gene expression in transfected cells. In this study, we ident...

  17. Viral mediated gene transfer to sprouting blood vessels during angiogenesis.

    PubMed

    Alian, Akram; Eldor, Amiram; Falk, Haya; Panet, Amos

    2002-08-01

    Several experimental systems have been applied to investigate the development of new blood vessels. Angiogenesis can be followed ex-vivo by culturing explants of rat aorta 'rings' in biomatrix gels. This angiogenesis system was modified for the study of viral vector mediated gene transfer, using adenovirus, vaccinia- and retroviral vectors. Two modifications were introduced to the model in order to facilitate efficient viral mediated gene transfer, (i) placing the aorta ring on top of a thin layer of collagen such that the angiogenic tissue will be accessible to the viral vector; and (ii) infection of the aorta rings prior to embedding them into the collagen matrix. While adenovirus and vaccinia vectors infected efficiently the aorta rings they induced cell death. Subsequent gene transfer experiments were, therefore, carried with retroviral vectors containing vascular endothelial growth factor (VEGF) and the beta-interferon (IFN) genes. Overexpression of VEGF enhanced significantly microvessel sprouting, while overexpression of IFN-beta induced an antiviral effect. The experimental system described in this study can facilitate the application of other viral vectors to the study of genes that may regulate the complex angiogenic process and thereby open new avenues for vascular gene therapy. PMID:12176137

  18. Gene Transfer in Mycobacterium tuberculosis: Shuttle Phasmids to Enlightenment

    PubMed Central

    JACOBS, WILLIAM R.

    2016-01-01

    Infectious diseases have plagued humankind throughout history and have posed serious public health problems. Yet vaccines have eradicated smallpox and antibiotics have drastically decreased the mortality rate of many infectious agents. These remarkable successes in the control of infections came from knowing the causative agents of the diseases, followed by serendipitous discoveries of attenuated viruses and antibiotics. The discovery of DNA as genetic material and the understanding of how this information translates into specific phenotypes have changed the paradigm for developing new vaccines, drugs, and diagnostic tests. Knowledge of the mechanisms of immunity and mechanisms of action of drugs has led to new vaccines and new antimicrobial agents. The key to the acquisition of the knowledge of these mechanisms has been identifying the elemental causes (i.e., genes and their products) that mediate immunity and drug resistance. The identification of these genes is made possible by being able to transfer the genes or mutated forms of the genes into causative agents or surrogate hosts. Such an approach was limited in Mycobacterium tuberculosis by the difficulty of transferring genes or alleles into M. tuberculosis or a suitable surrogate mycobacterial host. The construction of shuttle phasmids—chimeric molecules that replicate in Escherichia coli as plasmids and in mycobacteria as mycobacteriophages—was instrumental in developing gene transfer systems for M. tuberculosis. This review will discuss M. tuberculosis genetic systems and their impact on tuberculosis research. “I had to know my enemy in order to prevail against him.”Nelson Mandela PMID:26105819

  19. Structure-function of human 3 alpha-hydroxysteroid dehydrogenases: genes and proteins.

    PubMed

    Penning, T M; Jin, Y; Steckelbroeck, S; Lanisnik Rizner, T; Lewis, M

    2004-02-27

    Four soluble human 3 alpha-hydroxysteroid dehydrogenase (HSD) isoforms exist which are aldo-keto reductase (AKR) superfamily members. They share 86% sequence identity and correspond to: AKR1C1 (20 alpha(3 alpha)-HSD); AKR1C2 (type 3 3 alpha-HSD and bile-acid binding protein); AKR1C3 (type 2 3 alpha-HSD and type 5 17 beta-HSD); and AKR1C4 (type 1 3 alpha-HSD). Each of the homogeneous recombinant enzymes are plastic and display 3-, 17- and 20-ketosteroid reductase and 3 alpha- 17 beta- and 20 alpha-hydroxysteroid oxidase activities with different k(cat)/K(m) ratios in vitro. The crystal structure of the AKR1C2.NADP(+).ursodeoxycholate complex provides an explanation for this functional plasticity. Ursodeoxycholate is bound backwards (D-ring in the A-ring position) and upside down (beta-face of steroid inverted) relative to the position of 3-ketosteroids in the related rat liver 3 alpha-HSD (AKR1C9) structure. Transient transfection indicates that in COS-1 cells, AKR1C enzymes function as ketosteroid reductases due to potent inhibition of their oxidase activity by NADPH. By acting as ketosteroid reductases they may regulate the occupancy of the androgen, estrogen and progesterone receptors. RT-PCR showed that AKRs are discretely localized. AKR1C4 is virtually liver specific, while AKR1C2 and AKR1C3 are dominantly expressed in prostate and mammary gland. AKR1C genes are highly conserved in structure and may be transcriptionally regulated by steroid hormones and stress. PMID:15026176

  20. Cloning, characterization, and expression of two alpha-amylase genes from Aspergillus niger var. awamori.

    PubMed

    Korman, D R; Bayliss, F T; Barnett, C C; Carmona, C L; Kodama, K H; Royer, T J; Thompson, S A; Ward, M; Wilson, L J; Berka, R M

    1990-03-01

    Using synthetic oligonucleotide probes, we cloned genomic DNA sequences encoding an alpha-amylase gene from Aspergillus niger var. awamori (A. awamori) on a 5.8 kb EcoRI fragment. Hybridization experiments, using a portion of this cloned fragment to probe DNA from A. awamori, suggested the presence of two alpha-amylase gene copies which were subsequently cloned as 7 kb (designated as amyA) and 4 kb (amyB) HindIII fragments. DNA sequence analysis of the amyA and amyB genes revealed the following: (1) Both genes are arranged as nine exons and eight introns; (2) The nucleotide sequences of amyA and amyB are identical throughout all but the last few nucleotides of their respective coding regions; (3) The amyA and amyB genes from A. awamori share extensive homology (greater than or equal to 98% identity) with the genes encoding Taka-amylase from A. oryzae. In order to test whether both amyA and amyB were functional in the genome, we constructed vectors containing gene fusions of either amyA and amyB to bovine prochymosin cDNA and used these vectors to transform A. awamori. Transformants which contained either the amyA- or amyB-prochymosin gene fusions produced extracellular chymosin, suggesting that both genes are functional. PMID:2340591

  1. Detecting Horizontal Gene Transfer between Closely Related Taxa.

    PubMed

    Adato, Orit; Ninyo, Noga; Gophna, Uri; Snir, Sagi

    2015-10-01

    Horizontal gene transfer (HGT), the transfer of genetic material between organisms, is crucial for genetic innovation and the evolution of genome architecture. Existing HGT detection algorithms rely on a strong phylogenetic signal distinguishing the transferred sequence from ancestral (vertically derived) genes in its recipient genome. Detecting HGT between closely related species or strains is challenging, as the phylogenetic signal is usually weak and the nucleotide composition is normally nearly identical. Nevertheless, there is a great importance in detecting HGT between congeneric species or strains, especially in clinical microbiology, where understanding the emergence of new virulent and drug-resistant strains is crucial, and often time-sensitive. We developed a novel, self-contained technique named Near HGT, based on the synteny index, to measure the divergence of a gene from its native genomic environment and used it to identify candidate HGT events between closely related strains. The method confirms candidate transferred genes based on the constant relative mutability (CRM). Using CRM, the algorithm assigns a confidence score based on "unusual" sequence divergence. A gene exhibiting exceptional deviations according to both synteny and mutability criteria, is considered a validated HGT product. We first employed the technique to a set of three E. coli strains and detected several highly probable horizontally acquired genes. We then compared the method to existing HGT detection tools using a larger strain data set. When combined with additional approaches our new algorithm provides richer picture and brings us closer to the goal of detecting all newly acquired genes in a particular strain. PMID:26439115

  2. Detecting Horizontal Gene Transfer between Closely Related Taxa

    PubMed Central

    Adato, Orit; Ninyo, Noga; Gophna, Uri; Snir, Sagi

    2015-01-01

    Horizontal gene transfer (HGT), the transfer of genetic material between organisms, is crucial for genetic innovation and the evolution of genome architecture. Existing HGT detection algorithms rely on a strong phylogenetic signal distinguishing the transferred sequence from ancestral (vertically derived) genes in its recipient genome. Detecting HGT between closely related species or strains is challenging, as the phylogenetic signal is usually weak and the nucleotide composition is normally nearly identical. Nevertheless, there is a great importance in detecting HGT between congeneric species or strains, especially in clinical microbiology, where understanding the emergence of new virulent and drug-resistant strains is crucial, and often time-sensitive. We developed a novel, self-contained technique named Near HGT, based on the synteny index, to measure the divergence of a gene from its native genomic environment and used it to identify candidate HGT events between closely related strains. The method confirms candidate transferred genes based on the constant relative mutability (CRM). Using CRM, the algorithm assigns a confidence score based on “unusual” sequence divergence. A gene exhibiting exceptional deviations according to both synteny and mutability criteria, is considered a validated HGT product. We first employed the technique to a set of three E. coli strains and detected several highly probable horizontally acquired genes. We then compared the method to existing HGT detection tools using a larger strain data set. When combined with additional approaches our new algorithm provides richer picture and brings us closer to the goal of detecting all newly acquired genes in a particular strain. PMID:26439115

  3. Specific Gene Repression by CRISPRi System Transferred through Bacterial Conjugation

    PubMed Central

    2014-01-01

    In microbial communities, bacterial populations are commonly controlled using indiscriminate, broad range antibiotics. There are few ways to target specific strains effectively without disrupting the entire microbiome and local environment. Here, we use conjugation, a natural DNA horizontal transfer process among bacterial species, to deliver an engineered CRISPR interference (CRISPRi) system for targeting specific genes in recipient Escherichia coli cells. We show that delivery of the CRISPRi system is successful and can specifically repress a reporter gene in recipient cells, thereby establishing a new tool for gene regulation across bacterial cells and potentially for bacterial population control. PMID:25409531

  4. Antibiotics and gene transfer in swine gut bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mammalian gastrointestinal (GI) tract hosts a diverse collection bacteria, most of which are beneficial for host health. This bacterial community also supports a community of viruses that infect bacteria (called bacteriophages or phages). Phages transfer genes between bacteria, and phage-media...

  5. Localisation of cis elements in the promoter of a wheat alpha-Amy2 gene.

    PubMed

    Huttly, A K; Phillips, A L; Tregear, J W

    1992-09-01

    A functional analysis of the promoter from the wheat alpha-amylase gene alpha-Amy2/54 is described. Mutant alpha-Amy2/54 promoters containing replacements or deletions were constructed and their ability to direct expression of the reporter gene beta-glucuronidase (GUS) in gibberellin-responsive oat aleurone protoplasts analysed. Chimaeric promoters using regions of the cauliflower mosaic virus (CaMV) 35S and alpha-Amy2/54 promoters were also analysed. The results suggest that at least three regions within the alpha-Amy2/54 promoter contain cis elements that are necessary for high-level gibberellin-regulated transcription. Fusion of 1.8 kb of promoter sequence upstream from -117 bp to a minimal (-55 CaMV 35S) promoter gave rise to hormone-independent expression implying that the region 3' to -117 bp contains an element which represses transcription in the absence of gibberellin or presence of abscisic acid. PMID:1511136

  6. Kidney development and gene expression in the HIF2alpha knockout mouse.

    PubMed

    Steenhard, Brooke M; Freeburg, Paul B; Isom, Kathryn; Stroganova, Larysa; Borza, Dorin-Bogdan; Hudson, Billy G; St John, Patricia L; Zelenchuk, Adrian; Abrahamson, Dale R

    2007-04-01

    The hypoxia-inducible transcription factor-2 (HIF2), a heterodimer composed of HIF2alpha and HIF1beta subunits, drives expression of genes essential for vascularization, including vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2, Flk-1). Here, we used a HIF2alpha/LacZ transgenic mouse to define patterns of HIF2alpha transcription during kidney development and maturation. Our results from embryonic heterozygotes showed HIF2alpha/LacZ expression by apparently all renal endothelial cells. At 4 weeks of age, glomerular mesangial and vascular smooth muscle cells were also positive together with endothelial cells. These expression patterns were confirmed by electron microscopy using Bluo-gal as a beta-galactosidase substrate. Small numbers of glomerular and tubular epithelial cells were also positive at all stages examined. Light and electron microscopic examination of kidneys from HIF2alpha null embryos showed no defects in renal vascular development or nephrogenesis. Similarly, the same amounts of Flk-1 protein were seen on Western blots of kidney extracts from homozygous and heterozygous HIF2alpha mutants. To examine responsiveness of HIF2alpha null kidneys to hypoxia, embryonic day 13.5 metanephroi were cultured in room air or in mild (5% O(2)) hypoxia. For both heterozygous and null samples, VEGF mRNA levels doubled when metanephroi were cultured in mild hypoxia. Anterior chamber grafts of embryonic HIF2alpha knockouts were morphologically indistinguishable from heterozygous grafts. Endothelial markers, platelet endothelial cell adhesion molecule and BsLB4, as well as glomerular epithelial markers, GLEPP1 and WT-1, were all expressed appropriately. Finally, we undertook quantitative real-time polymerase chain reaction of kidneys from HIF2alpha null embryos and wild-type siblings and found no compensatory up-regulation of HIF1alpha or -3alpha. Our results show that, although HIF2alpha was widely transcribed by kidney endothelium and vascular

  7. Transferred interbacterial antagonism genes augment eukaryotic innate immune function

    PubMed Central

    Chou, Seemay; Daugherty, Matthew D.; Peterson, S. Brook; Biboy, Jacob; Yang, Youyun; Jutras, Brandon L.; Fritz-Laylin, Lillian K.; Ferrin, Michael A.; Harding, Brittany N.; Jacobs-Wagner, Christine; Yang, X. Frank; Vollmer, Waldemar; Malik, Harmit S.

    2015-01-01

    Horizontal gene transfer (HGT) allows organisms to rapidly acquire adaptive traits1. Though documented instances of HGT from bacteria to eukaryotes remain rare, bacteria represent a rich source of new functions potentially available for co-option2. One benefit that genes of bacterial origin could provide to eukaryotes is the capacity to produce anti-bacterials, which have evolved in prokaryotes as the result of eons of interbacterial competition. The type VI secretion amidase effector (Tae) proteins are potent bacteriocidal enzymes that degrade the cell wall when delivered into competing bacterial cells by the type VI secretion system (T6SS)3. Here we show that tae genes have been transferred to eukaryotes on at least six occasions, and that the resulting domesticated amidase effector (dae) genes have been preserved for hundreds of millions of years via purifying selection. We show that the dae genes acquired eukaryotic secretion signals, are expressed within recipient organisms, and encode active antibacterial toxins that possess substrate specificity matching extant Tae proteins of the same lineage. Finally, we show that a dae gene in the deer tick Ixodes scapularis limits proliferation of Borrelia burgdorferi, the etiologic agent of Lyme disease. Our work demonstrates that a family of horizontally acquired toxins honed to mediate interbacterial antagonism confers previously undescribed antibacterial capacity to eukaryotes. We speculate that the selective pressure imposed by competition between bacteria has produced a reservoir of genes encoding diverse antimicrobial functions that are tailored for facile co-option by eukaryotic innate immune systems. PMID:25470067

  8. Concomitant alpha- and gamma-sarcoglycan deficiencies in a Turkish boy with a novel deletion in the alpha-sarcoglycan gene.

    PubMed

    Diniz, Gulden; Tosun Yildirim, Hulya; Gokben, Sarenur; Serdaroglu, Gul; Hazan, Filiz; Yararbas, Kanay; Tukun, Ajlan

    2014-01-01

    Limb-girdle muscular dystrophy type 2D (LGMD-2D) is caused by autosomal recessive defects in the alpha-sarcoglycan gene located on chromosome 17q21. In this study, we present a child with alpha-sarcoglycanopathy and describe a novel deletion in the alpha-sarcoglycan gene. A 5-year-old boy had a very high serum creatinine phosphokinase level, which was determined incidentally, and a negative molecular test for the dystrophin gene. Muscle biopsy showed dystrophic features. Immunohistochemistry showed that there was diminished expression of alpha- and gamma-sarcoglycans. DNA analysis revealed a novel 7 bp homozygous deletion in exon 3 of the alpha-sarcoglycan gene. His parents were consanguineous heterozygous carriers of the same deletion. We believe this is the first confirmed case of primary alpha-sarcoglycanopathy with a novel deletion in Turkey. In addition, this study demonstrated that both muscle biopsy and DNA analysis remain important methods for the differential diagnosis of muscular dystrophies because dystrophinopathies and sarcoglycanopathies are so similar. PMID:25050186

  9. Detection of horizontal gene transfers from phylogenetic comparisons.

    PubMed

    Pylro, Victor Satler; Vespoli, Luciano de Souza; Duarte, Gabriela Frois; Yotoko, Karla Suemy Clemente

    2012-01-01

    Bacterial phylogenies have become one of the most important challenges for microbial ecology. This field started in the mid-1970s with the aim of using the sequence of the small subunit ribosomal RNA (16S) tool to infer bacterial phylogenies. Phylogenetic hypotheses based on other sequences usually give conflicting topologies that reveal different evolutionary histories, which in some cases may be the result of horizontal gene transfer events. Currently, one of the major goals of molecular biology is to understand the role that horizontal gene transfer plays in species adaptation and evolution. In this work, we compared the phylogenetic tree based on 16S with the tree based on dszC, a gene involved in the cleavage of carbon-sulfur bonds. Bacteria of several genera perform this survival task when living in environments lacking free mineral sulfur. The biochemical pathway of the desulphurization process was extensively studied due to its economic importance, since this step is expensive and indispensable in fuel production. Our results clearly show that horizontal gene transfer events could be detected using common phylogenetic methods with gene sequences obtained from public sequence databases. PMID:22675653

  10. Rescuing the Failing Heart by Targeted Gene Transfer

    PubMed Central

    Kawase, Yoshiaki; Ladage, Dennis; Hajjar, Roger J.

    2011-01-01

    Congestive heart failure is a major cause of morbidity and mortality in the US. While progress in conventional treatments is making steady and incremental gains to reduce heart failure mortality, there is a critical need to explore new therapeutic approaches. Gene therapy was initially applied in the clinical setting for inherited monogenic disorders. It is now apparent that gene therapy has broader potential that also includes acquired polygenic diseases, such as congestive heart failure. Recent advances in understanding of the molecular basis of myocardial dysfunction, together with the evolution of increasingly efficient gene transfer technology, has placed heart failure within reach of gene-based therapy. Furthermore, the recent successful and safe completion of a phase 2 trial targeting the sarcoplasmic reticulum calcium ATPase pump (SERCA2a) along with the start of more recent phase 1 trials usher a new era for gene therapy for the treatment of heart failure. PMID:21371634

  11. Analyses of alpha/beta-type gliadin genes from diploid and hexaploid wheats.

    PubMed

    Reeves, C D; Okita, T W

    1987-01-01

    The alpha/beta-gliadin genes isolated from both hexaploid wheat (cv. Yamhill) and the diploid A genome progenitor Triticum urartu had remarkably similar sequences and differ by only a few point mutations. Primer extension analysis indicated that the transcriptional start points for individual genes in the family cluster within a few nucleotides. Comparison of the promoter region of several alpha/beta-gliadin and B-hordein genes reveals two conserved regions at about -130 and -250 bp. DNA from the hexaploid cultivars, Cheyenne and Chinese Spring, and the diploid progenitors T. urartu and Aegilops squarrosa was analysed by Southern blotting. Restriction fragment lengths of the alpha/beta-gliadin genes varied only slightly between the various wheats, although the overall copy number varied significantly. A region between approx. -1700 and -700 bp upstream from the TATA box was highly repeated in all three wheat genomes. For the hexaploid-derived gene, over 1700 bp of sequence upstream from the TATA box was determined, revealing an additional open reading frame between approx. -1550 and -1250 bp relative to the gliadin TATA box. Northern blot analysis indicated that RNA homologous to this repeated sequence family was present only in developing seed and accumulated to a maximum at late stages of maturation. PMID:3038689

  12. Multiple octamer binding sites in the promoter region of the bovine alpha s2-casein gene.

    PubMed Central

    Groenen, M A; Dijkhof, R J; van der Poel, J J; van Diggelen, R; Verstege, E

    1992-01-01

    Using a set of overlapping oligonucleotides from the promoter region of the bovine alpha s2-casein gene we have identified two nuclear factors which probably are involved in expression of this gene and the related calcium sensitive alpha s1- and beta-casein genes. One of these factors which was present in extracts of all tissues that have been tested including Hela cells turned out to be the octamer binding protein OCT-1. Oct-1 binds with different affinity to 4 sites at positions centred around -480, -260, -210 and -50. The strongest of these 4 binding sites, the one around position -50, is highly conserved in all calcium sensitive caseins of mouse, rat, rabbit and cattle. The other nuclear factor (MGF, mammary gland factor) which is specifically expressed in the mammary gland, binds to a site around position -90. This binding site is also highly conserved in all calcium sensitive caseins of mouse, rat, rabbit and cattle. Images PMID:1508722

  13. AAV-mediated gene transfer to the mouse CNS

    PubMed Central

    Stoica, Lorelei; Ahmed, Seemin S.

    2013-01-01

    Recombinant adeno associated virus (rAAV) vectors are great tools for gene transfer due to their ability to mediate long-term gene expression. Recombinant AAVs have been used at various ages of development with no apparent toxicity. There are multiple ways of delivering AAV vectors to the CNS, depending on the stage of development of the mouse. In neonates, intravascular injections into the facial vein are often used. In adults, direct injections into target regions of the brain are achieved with great spatiotemporal control through stereotaxic surgeries. Recently, discoveries of new AAV vectors with the ability to cross the blood brain barrier have made it possible to also target the adult CNS by intravascular injections. rAAVs have been successfully used as gene transfer vehicles in multiple animal models of CNS disorders, and several clinical trials are currently underway. PMID:23686825

  14. The bean. alpha. -amylase inhibitor is encoded by a lectin gene

    SciTech Connect

    Moreno, J.; Altabella, T.; Chrispeels, M.J. )

    1989-04-01

    The common bean, Phaseolus vulgaris, contains an inhibitor of insect and mammalian {alpha}-amylases that does not inhibit plant {alpha}-amylase. This inhibitor functions as an anti-feedant or seed-defense protein. We purified this inhibitor by affinity chromatography and found that it consists of a series of glycoforms of two polypeptides (Mr 14,000-19,000). Partial amino acid sequencing was carried out, and the sequences obtained are identical with portions of the derived amino acid sequence of a lectin-like gene. This lectin gene encodes a polypeptide of MW 28,000, and the primary in vitro translation product identified by antibodies to the {alpha}-amylase inhibitor has the same size. Co- and posttranslational processing of this polypeptide results in glycosylated polypeptides of 14-19 kDa. Our interpretation of these results is that the bean lectins constitute a gene family that encodes diverse plant defense proteins, including phytohemagglutinin, arcelin and {alpha}-amylase inhibitor.

  15. A functional polymorphism of the TNF-{alpha} gene that is associated with type 2 DM

    SciTech Connect

    Susa, Shinji; Daimon, Makoto Sakabe, Jun-Ichi; Sato, Hidenori; Oizumi, Toshihide; Karasawa, Shigeru; Wada, Kiriko; Jimbu, Yumi; Kameda, Wataru; Emi, Mitsuru; Muramatsu, Masaaki; Kato, Takeo

    2008-05-09

    To examine the association of the tumor necrosis factor-{alpha} (TNF-{alpha}) gene region with type 2 diabetes (DM), 11 single-nucleotide polymorphisms (SNPs) of the region were analyzed. The initial study using a sample set (148 cases vs. 227 controls) showed a significant association of the SNP IVS1G + 123A of the TNF-{alpha} gene with DM (p = 0.0056). Multiple logistic regression analysis using an enlarged sample set (225 vs. 716) revealed the significant association of the SNP with DM independently of any clinical traits examined (OR: 1.49, p = 0.014). The functional relevance of the SNP were examined by the electrophoretic mobility shift assays using nuclear extracts from the U937 and NIH3T3 cells and luciferase assays in these cells with Simian virus 40 promoter- and TNF-{alpha} promoter-reporter gene constructs. The functional analyses showed that YY1 transcription factor bound allele-specifically to the SNP region and, the IVS1 + 123A allele had an increase in luciferase expression compared with the G allele.

  16. Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes

    PubMed Central

    Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F.

    2015-01-01

    Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners—the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)—and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic—and plant and algal—lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller’s ratchet—the origin of eukaryotic recombination, or sex—might have required surprisingly little evolutionary innovation. PMID:25733873

  17. Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes.

    PubMed

    Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F

    2015-08-18

    Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners--the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)--and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic--and plant and algal--lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller's ratchet--the origin of eukaryotic recombination, or sex--might have required surprisingly little evolutionary innovation. PMID:25733873

  18. Integration and expression of alpha-amylase and endoglucanase genes in the Lactobacillus plantarum chromosome.

    PubMed Central

    Scheirlinck, T; Mahillon, J; Joos, H; Dhaese, P; Michiels, F

    1989-01-01

    A commercial grass silage starter strain of Lactobacillus plantarum was transformed by high-frequency electroporation with plasmids containing an alpha-amylase gene from Bacillus stearothermophilus and an endoglucanase gene from Clostridium thermocellum. Both genes were expressed from their native regulatory signals, and active enzymes were found in the supernatant. However, the segregational stability of the transforming plasmids was rather low. Therefore, the transforming genes were inserted in the L. plantarum chromosome by means of single homologous recombination. In the majority of the transformants, this led to extremely stable segregation and expression of the transforming genes, without generating secondary mutations in the host. Increased selective pressure led to tandem amplification of the transforming DNA. The transformed strains demonstrated the ability of L. plantarum to express heterologous gene products; they can be used to detect the inoculum in silage ecology studies; and they demonstrate the feasibility of engineering truly cellulolytic silage starter bacteria. Images PMID:2679379

  19. Do HLA genes play a prominent role in determining T cell receptor V{alpha} segment usage in humans?

    SciTech Connect

    Gulwani-Akolkar, B.; Shi, B.; Akolkar, P.N.

    1995-04-15

    Previous studies in humans have demonstrated that HLA genes can profoundly influence the TCR V{beta} repertoire. To similarly assess the influence of HLA genes on the TCR V{alpha} segment repertoire, the V{alpha} repertoires of 12 individuals from three unrelated families were determined by quantitative PCR. Each family contained at least one pair of HLA-identical and -nonidentical siblings. Repertoire analysis was performed on purified CD4{sup +} and CD8{sup +} cells by using V{alpha}-specific primers. We were unable to demonstrate more similar V{alpha} repertoires between HLA-identical siblings than between HLA-nonidentical siblings. In contrast, when a similar analysis was performed on the same individuals for the V{beta} repertoire, HLA-identical siblings were found to have significantly more similar repertoires than HLA-nonidentical siblings. Furthermore, both the V{alpha} and V{beta} repertoires of monozygotic twins showed striking similarity. Despite our inability to shown an influence of HLA genes on the V{alpha} repertoire, we did observe a very strong skewing in terms of preferential expression on CD4{sup +} or CD8{sup +} cells of several V{alpha} segments, notably TCRAV1, -2, -5, -6, -7, -11, -12, and -13. These studies suggest that HLA genes play less of a role in determining V{alpha} segment usage than V{beta}. Nevertheless, the pronounced skewing of V{alpha} segment expression in the CD4{sup +} or CD8{sup +} populations suggests some role for HLA genes in determining the V{alpha} TCR repertoire. Furthermore, the striking similarity of V{alpha} repertoires of identical twins suggests a major role for non-HLA genes in determining the V{alpha} repertoire. 35 refs., 8 figs., 3 tabs.

  20. Growth-related gene product {alpha}: A chemotactic cytokine for neutrophils in rheumatoid arthritis

    SciTech Connect

    Koch, A.E.; Pope, R.M. |; Shah, M.R.; Hosaka, S.

    1995-10-01

    Leukocyte recruitment is critical in the inflammation seen in rheumatoid arthritis (RA). To determine whether the chemokine growth-related gene product {alpha} (gro{alpha}) plays a role in this process, we examined synovial tissue (ST), synovial fluid (SF), and plasma samples from 102 patients with arthritis. RA SF contained more antigenic gro{alpha} (mean 5.3 {+-} 1.9 ng/ml) than did SFs from either osteoarthritis (OA) or other forms of arthritis (mean 0.1 ng/ml) (p < 0.05). RA plasma contained more gro{alpha} (mean 4.3 {+-} 1.8 ng/ml) than normal plasma (mean 0.1 ng/ml) (p < 0.05). RA ST fibroblasts (1.2 x 10{sup 5}/cells/ml RPMI 1640/24 h) produced antigenic gro{alpha} (mean 0.2 {+-} 0.1 ng/ml), and this production was increased significantly upon incubation with TNF-{alpha} (mean 1.3 {+-} 0.3 ng/ml) or IL-1{beta} (mean 2.3 {+-} 0.6 ng/ml) (p < 0.05). Cells from RA SF also produced gro{alpha}: neutrophils (PMNs) (10{sup 7} cells/ml/24 h) produced 3.7 {+-} 0.7 ng/ml. RA SF mononuclear cells produced gro{alpha}, particularly upon incubation with LPS or PHA. Immunoreactive ST gro{alpha} was found in greater numbers of RA compared with either OA or normal lining cells, as well as in RA compared with OA subsynovial macrophages (p < 0.05). IL-8 accounted for a mean of 36% of the RA SF chemotactic activity for PMNs, while epithelial neutrophil-activating peptide-78 accounted for 34%, and gro{alpha} for 28%, of this activity. Combined neutralization of all three chemokines in RA SFs resulted in a mean decrease of 50% of the chemotactic activity for PMNs present in the RA SFs. These results indicate that gro{alpha} plays an important role in the ingress of PMNs into the RA joint. 54 refs., 6 figs., 1 tab.

  1. Immunotherapy of Malignancy by in vivo Gene Transfer into Tumors

    NASA Astrophysics Data System (ADS)

    Plautz, Gregory E.; Yang, Zhi-Yong; Wu, Bei-Yue; Gao, Xiang; Huang, Leaf; Nabel, Gary J.

    1993-05-01

    The immune system confers protection against a variety of pathogens and contributes to the surveillance and destruction of neoplastic cells. Several cell types participate in the recognition and lysis of tumors, and appropriate immune stimulation provides therapeutic effects in malignancy. Foreign major histocompatibility complex (MHC) proteins also serve as a potent stimulus to the immune system. In this report, a foreign MHC gene was introduced directly into malignant tumors in vivo in an effort to stimulate tumor rejection. In contrast to previous attempts to induce tumor immunity by cell-mediated gene transfer, the recombinant gene was introduced directly into tumors in vivo. Expression of the murine class I H-2K^s gene within the CT26 mouse colon adenocarcinoma (H-2K^d) or the MCA 106 fibrosarcoma (H-2K^b) induced a cytotoxic T-cell response to H-2K^s and, more importantly, to other antigens present on unmodified tumor cells. This immune response attenuated tumor growth and caused complete tumor regression in many cases. Direct gene transfer in vivo can therefore induce cell-mediated immunity against specific gene products, which provides an immunotherapeutic effect for malignancy, and potentially can be applied to the treatment of cancer and infectious diseases in man.

  2. Three NF-kappa B sites in the I kappa B-alpha promoter are required for induction of gene expression by TNF alpha.

    PubMed Central

    Ito, C Y; Kazantsev, A G; Baldwin, A S

    1994-01-01

    NF-kappa B was first identified as a postive regulator which bound to a 10 bp sequence in the first intron of the Ig kappa light chain gene. Further characterization of this transcription factor has revealed that NF-kappa B is kept from binding to its consensus sequence by its inhibitor, IkB-alpha, which retains NF-kappa B in the cytoplasm. Upon receiving various extra- and intracellular signals, I kappa B-alpha is rapidly degraded and NF-kappa B is induced to translocate into the nucleus. This process precedes the rapid induction of I kappa B-alpha mRNA and protein. To understand how I kappa B-alpha is replenished, we have cloned and sequenced the 5' flanking region of the I kappa B-alpha gene and have identified the transcription start site and three NF-kappa B sites in this region. Further characterization of these NF-kappa B sites show that they have different affinities for three specific protein complexes which we identify here to consist of various members of the Rel family. In transient assays, cotransfection with a p65 expression vector is able to activate an I kappa B-alpha promoter-CAT reporter construct and all three NF-kappa B sites are required for full activation of the I kappa B-alpha gene following stimulation with TNF-alpha. Our data confirm a transcriptional autoregulatory loop involved in maintaining appropriate NF-kappa B and I kappa B-alpha levels in the cell. Images PMID:7937093

  3. Cloning and characterization of a third type of human alpha-amylase gene, AMY2B.

    PubMed

    Yokouchi, H; Horii, A; Emi, M; Tomita, N; Doi, S; Ogawa, M; Mori, T; Matsubara, K

    1990-06-15

    We have previously reported concerning the existence of a third type of human alpha-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229-235; Tomita et al., Gene 76 (1989) 11-18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human alpha-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively. Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197-1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5' region; hence the promoter lies far upstream relative to the other two AMY genes. PMID:2401405

  4. [Experimental study of vascular gene transfer using soluble stent].

    PubMed

    Huang, Z; Gou, J; Li, X

    1997-05-01

    We assessed the possibility of gene transfer into anastomotic arteries in vivo using soluble stent containing Adv5-CMV/LacZ. After being soaked in a high concentration solution of glucose containing Adv5-CMV (control group) or Adv5-CMV/LacZ (treatment group) for 30 minutes, we inserted soluble stents into the lumina of cut rat carotid arteries, and end-to-end anastomoses of cut rat carotid were performed with standard microvascular surgical technique. 16 rats were killed after two weeks, the segments of anastomotic carotid arteries were prepared for assessing beta-galactosidase activity and histochemical staining. In the control group, the anastomotic arteries did not have detectable level of beta-galactosidase expression. In the treatment group, the amount of beta-galactosidase expression was 9.80 x 10-3 u/g tissue. Microscopic examination of histochemically stained arteries demonstrated that gene transfered not only to endothelial cells but also to smooth muscle cells, and all anastomotic arteries were transfered in the treatment group, but none of arteries revealed blue in the control group. The results of this experimental study suggested that soluble stent be a new method of direct gene transfer into arteries in vivo. PMID:10374573

  5. Rapid evolution and diversification of mammalian alpha-defensins as revealed by comparative analysis of rodent and primate genes.

    PubMed

    Patil, Amar; Hughes, Austin L; Zhang, Guolong

    2004-12-15

    Mammalian alpha-defensins constitute a family of cysteine-rich, cationic antimicrobial peptides produced by phagocytes and intestinal Paneth cells, playing an important role in innate host defense. Following comprehensive computational searches, here we report the discovery of complete repertoires of the alpha-defensin gene family in the human, chimpanzee, rat, and mouse with new genes identified in each species. The human genome was found to encode a cluster of 10 distinct alpha-defensin genes and pseudogenes expanding 132 kb continuously on chromosome 8p23. Such alpha-defensin loci are also conserved in the syntenic chromosomal regions of chimpanzee, rat, and mouse. Phylogenetic analyses showed formation of two distinct clusters with primate alpha-defensins forming one cluster and rodent enteric alpha-defensins forming the other cluster. Species-specific clustering of genes is evident in nonprimate species but not in the primates. Phylogenetically distinct subsets of alpha-defensins also exist in each species, with most subsets containing multiple members. In addition, natural selection appears to have acted to diversify the functionally active mature defensin region but not signal or prosegment sequences. We concluded that mammalian alpha-defensin genes may have evolved from two separate ancestors originated from beta-defensins. The current repertoires of the alpha-defensin gene family in each species are primarily a result of repeated gene duplication and positive diversifying selection after divergence of mammalian species from each other, except for the primate genes, which were evolved prior to the separation of the primate species. We argue that the presence of multiple, divergent subsets of alpha-defensins in each species may help animals to better cope with different microbial challenges in the ecological niches which they inhabit. PMID:15494476

  6. Functions of AAV-CMV-F.IX And AAV-EF1alpha-F.IX in gene therapy for hemophilia B.

    PubMed

    Wiwanitkit, Viroj

    2007-02-01

    There has been substantial progress in using gene therapy to treat animals with hemophilia. Adeno-associated viral (AAV) gene transfer of coagulation factor IX to skeletal muscle and liver of murine and canine models of hemophilia has resulted in sustained systemic expression and, in several studies, in complete cure of the bleeding disorder. Two AAV vectors widely used at present are AAV-CMV-F.IX and AAV-EF1alpha-F.IX. This work compares the predicted molecular functions of AAV-CMV-F.IX and AAV-EF1alpha -F.IX by sequence docking and gene ontology. It is shown that both AAV-CMV-F.IX and AAV-EF1alpha -F.IX induce coagulation factor IXa activity; however, AAV-CMV-F.IX administration also yields coagulation factor XIa activity and AAV-EF1alpha -F.IX treatment results in coagulation factor Xa activity. Therefore, AAV-CMV-F.IX might be useful for factor XI deficiency. AAV-CMV-F.IX has several additional molecular functions and processes compared with AAV-CMV-F.IX. PMID:17266422

  7. Promoter activity of the 5'-flanking regions of medaka fish soluble guanylate cyclase alpha1 and beta1 subunit genes.

    PubMed Central

    Yamamoto, Takehiro; Suzuki, Norio

    2002-01-01

    We examined the spatial expression pattern of medaka fish (Oryzias latipes) soluble guanylate cyclase alpha(1) and beta(1) subunit genes, OlGCS-alpha(1) and OlGCS-beta(1), and characterized the 5'-flanking region required for expression of both genes by introducing various promoter-luciferase fusion-gene constructs into COS-1 cells and medaka fish embryos. The OlGCS-alpha(1) and OlGCS-beta(1) gene transcripts were detected in whole brain and kidney in 7-day and 9-day embryos. Primer-extension analysis demonstrated that there were no differences among various adult organs (brain, eye, kidney, ovary and testis) in the transcription start site of the OlGCS-alpha(1) and OlGCS-beta(1) genes. Neither gene contained the functional TATA box within its 5'-flanking region, and the basal promoter activity was found between nucleotides +33 and +42 in the OlGCS-alpha(1) gene and between nucleotides +146 and +155 in the OlGCS-beta(1) gene. In the assay of medaka fish embryos, the 5'-flanking region of the OlGCS-beta(1) gene exhibited lower promoter activity than that of the OlGCS-alpha(1) gene. In the experiments on dual-luciferase fusion-gene constructs, the 5'-flanking region of the OlGCS-alpha(1) gene connected to the 5'-flanking region of the OlGCS-beta(1) gene was introduced into medaka fish embryos, and the 5'-flanking regions of both subunit genes were shown to mutually influence each other's promoter activity. PMID:11772405

  8. Nano-Sized Sunflower Polycations As Effective Gene Transfer Vehicles.

    PubMed

    Cheng, Yilong; Wei, Hua; Tan, James-Kevin Y; Peeler, David J; Maris, Don O; Sellers, Drew L; Horner, Philip J; Pun, Suzie H

    2016-05-01

    The architecture of polycations plays an important role in both gene transfection efficiency and cytotoxicity. In this work, a new polymer, sunflower poly(2-dimethyl amino)ethyl methacrylate) (pDMAEMA), is prepared by atom transfer radical polymerization and employed as nucleic acid carriers compared to linear pDMAEMA homopolymer and comb pDMAEMA. The sunflower pDMAEMAs show higher IC50 , greater buffering capacity, and stronger binding capacity toward plasmid DNA than their linear and comb counterparts. In vitro transfection studies demonstrate that sunflower pDMAEMAs exhibit high transfection efficiency as well as relatively low cytotoxicity in complete growth medium. In vivo gene delivery by intraventricular injection to the brain shows that sunflower polymer delivers plasmid DNA more effectively than comb polymer. This study provides a new insight into the relationship between polymeric architecture and gene delivery capability, and as well as a useful means to design potent vectors for successful gene delivery. PMID:27061622

  9. Six mouse alpha-tubulin mRNAs encode five distinct isotypes: testis-specific expression of two sister genes.

    PubMed Central

    Villasante, A; Wang, D; Dobner, P; Dolph, P; Lewis, S A; Cowan, N J

    1986-01-01

    Five mouse alpha-tubulin isotypes are described, each distinguished by the presence of unique amino acid substitutions within the coding region. Most, though not all of these isotype-specific amino acids, are clustered at the carboxy terminus. One of the alpha-tubulin isotypes described is expressed exclusively in testis and is encoded by two closely related genes (M alpha 3 and M alpha 7) which have homologous 3' untranslated regions but which differ at multiple third codon positions and in their 5' untranslated regions. We show that a subfamily of alpha-tubulin genes encoding the same testis-specific isotype also exists in humans. Thus, we conclude that the duplication event leading to a pair of genes encoding a testis-specific alpha-tubulin isotype predated the mammalian radiation, and both members of the duplicated sequence have been maintained since species divergence. A second alpha-tubulin gene, M alpha 6, is expressed ubiquitously at a low level, whereas a third gene, M alpha 4, is unique in that it does not encode a carboxy-terminal tyrosine residue. This gene yields two transcripts: a 1.8-kilobase (kb) mRNA that is abundant in muscle and a 2.4-kb mRNA that is abundant in testis. Whereas the 1.8-kb mRNA encodes a distinct alpha-tubulin isotype, the 2.4-kb mRNA is defective in that the methionine residue required for translational initiation is missing. Patterns of developmental expression of the various alpha-tubulin isotypes are presented. Our data support the view that individual tubulin isotypes are capable of conferring functional specificity on different kinds of microtubules. Images PMID:3785200

  10. Comparative gene expression profiles induced by PPAR{gamma} and PPAR{alpha}/{gamma} agonists in rat hepatocytes

    SciTech Connect

    Rogue, Alexandra; Renaud, Marie Pierre; Claude, Nancy; Guillouzo, Andre; Spire, Catherine

    2011-07-01

    Species-differential toxic effects have been described with PPAR{alpha} and PPAR{gamma} agonists between rodent and human liver. PPAR{alpha} agonists (fibrates) are potent hypocholesterolemic agents in humans while they induce peroxisome proliferation and tumors in rodent liver. By contrast, PPAR{gamma} agonists (glitazones) and even dual PPAR{alpha}/{gamma} agonists (glitazars) have caused idiosyncratic hepatic and nonhepatic toxicities in human without evidence of any damage in rodent during preclinical studies. The mechanisms involved in such differences remain largely unknown. Several studies have identified the major target genes of PPAR{alpha} agonists in rodent liver while no comprehensive analysis has been performed on gene expression changes induced by PPAR{gamma} and dual PPAR{alpha}/{gamma} agonists. Here, we investigated transcriptomes of rat hepatocytes after 24 h treatment with two PPAR{gamma} (troglitazone and rosiglitazone) and two PPAR{alpha}/{gamma} (muraglitazar and tesaglitazar) agonists. Although, hierarchical clustering revealed a gene expression profile characteristic of each PPAR agonist class, only a limited number of genes was specifically deregulated by glitazars. Functional analyses showed that many genes known as PPAR{alpha} targets were also modulated by both PPAR{gamma} and PPAR{alpha}/{gamma} agonists and quantitative differences in gene expression profiles were observed between these two classes. Moreover, most major genes modulated in rat hepatocytes were also found to be deregulated in rat liver after tesaglitazar treatment. Taken altogether, these results support the conclusion that differential toxic effects of PPAR{alpha} and PPAR{gamma} agonists in rodent liver do not result from transcriptional deregulation of major PPAR target genes but rather from qualitative and/or quantitative differential responses of a small subset of genes.

  11. Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

    PubMed

    Bilbao, R; Reay, D P; Hughes, T; Biermann, V; Volpers, C; Goldberg, L; Bergelson, J; Kochanek, S; Clemens, P R

    2003-10-01

    High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle. PMID:12960972

  12. Detection of Clostridium perfringens alpha toxin gene in lambs by loop mediated isothermal amplification

    PubMed Central

    Radhika, B.; Kumar, N. Vinod; Sreenivasulu, D.

    2016-01-01

    Aim: The loop mediated isothermal amplification (LAMP) was standardized for rapid detection of Clostridium perfringens. Materials and Methods: A total of 120 fecal samples were collected from enterotoxemia suspected lambs were used for screening of C. perfringens cpa gene by LAMP. The specificity of the LAMP amplified products was tested by digesting with restriction enzyme XmnI for alpha toxin gene. Results: Out of 120 samples screened 112 (93.3%) samples were positive by both LAMP and polymerase chain reaction (PCR) for detection of cpa gene which indicated the equal sensitivity of both the tests. The enzyme produced single cut in 162 base pair amplified product of alpha toxin gene at 81 base pair resulting in a single band in gel electrophoresis. Conclusion: Both LAMP and PCR for detection of cpa gene indicated the equal sensitivity of both the tests. Standardization of LAMP reaction for amplification of epsilon and beta toxin genes will help to identify the C. perfringens toxin types from the clinical samples. The test could be a suitable alternative to the PCR in detection of toxin types without the help of sophisticated machinery like thermal cycler. Considering its simplicity in operation and high sensitivity, there is the potential use of this technique in clinical diagnosis and surveillance of infectious diseases. PMID:27051186

  13. Electroporation-Mediated Gene Transfer Directly to the Swine Heart

    PubMed Central

    Hargrave, Barbara; Downey, Harre; Strange, Robert; Murray, Len; Cinnamond, Cade; Lundberg, Cathryn; Israel, Annelise; Chen, Yeong-Jer; Marshall, William; Heller, Richard

    2012-01-01

    In vivo gene transfer to the ischemic heart via electroporation holds promise as a potential therapeutic approach for the treatment of heart disease. In the current study, we investigated the use of in vivo electroporation for gene transfer using 3 different penetrating electrodes and one non-penetrating electrode. The hearts of adult male swine were exposed through a sternotomy. Eight electric pulses synchronized to the rising phase of the R wave of the ECG were administered at varying pulse widths and field strengths following an injection of either a plasmid encoding luciferase or one encoding green fluorescent protein. Four sites on the anterior wall of the left ventricle were treated. Animals were euthanized 48 hours after injection and electroporation and gene expression was determined. Results were compared to sites in the heart that received plasmid injection but no electric pulses or were not treated. Gene expression was higher in all electroporated sites when compared to injection only sites demonstrating the robustness of this approach. Our results provide evidence that in vivo electroporation can be a safe and effective non-viral method for delivering genes to the heart, in vivo. PMID:22456328

  14. Characterization of an Ancient Lepidopteran Lateral Gene Transfer

    PubMed Central

    Wheeler, David; Redding, Amanda J.; Werren, John H.

    2013-01-01

    Bacteria to eukaryote lateral gene transfers (LGT) are an important potential source of material for the evolution of novel genetic traits. The explosion in the number of newly sequenced genomes provides opportunities to identify and characterize examples of these lateral gene transfer events, and to assess their role in the evolution of new genes. In this paper, we describe an ancient lepidopteran LGT of a glycosyl hydrolase family 31 gene (GH31) from an Enterococcus bacteria. PCR amplification between the LGT and a flanking insect gene confirmed that the GH31 was integrated into the Bombyx mori genome and was not a result of an assembly error. Database searches in combination with degenerate PCR on a panel of 7 lepidopteran families confirmed that the GH31 LGT event occurred deep within the Order approximately 65–145 million years ago. The most basal species in which the LGT was found is Plutella xylostella (superfamily: Yponomeutoidea). Array data from Bombyx mori shows that GH31 is expressed, and low dN/dS ratios indicates the LGT coding sequence is under strong stabilizing selection. These findings provide further support for the proposition that bacterial LGTs are relatively common in insects and likely to be an underappreciated source of adaptive genetic material. PMID:23533610

  15. alpha. -Amylase of Clostridium thermosulfurogenes EM1: Nucleotide sequence of the gene, processing of the enzyme, and comparison to other. alpha. -amylases

    SciTech Connect

    Bahl, H.; Burchhardt, G.; Spreinat, A.; Haeckel, K.; Wienecke, A.; Antranikian, G.; Schmidt, B. )

    1991-05-01

    The nucleotide sequence of the {alpha}-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes Em1 suggested that the {alpha}-amylase is translated form mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature {alpha}-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 {alpha}-amylase with those from other bacterial and eukaryotic {alpha}-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca{sup 2+}-binding site (consensus region I) of this Ca{sub 2+}-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the {alpha}-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the {beta}-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.

  16. PPAR{alpha} gene expression is up-regulated by LXR and PXR activators in the small intestine

    SciTech Connect

    Inoue, Jun; Satoh, Shin-ichi; Kita, Mariko; Nakahara, Mayuko; Hachimura, Satoshi; Miyata, Masaaki; Nishimaki-Mogami, Tomoko; Sato, Ryuichiro

    2008-07-11

    LXR, PXR, and PPAR{alpha} are members of a nuclear receptor family which regulate the expression of genes involved in lipid metabolism. Here, we show the administration of T0901317 stimulates PPAR{alpha} gene expression in the small intestine but not in the liver of both normal and FXR-null mice. The administration of LXR specific ligand GW3965, or PXR specific ligand PCN has the same effect, indicating that ligand-dependent activation of LXR and PXR, but not FXR, is responsible for the increased gene expression of PPAR{alpha} in the mouse small intestine.

  17. The alpha-helical domain of liver fatty acid binding protein is responsible for the diffusion-mediated transfer of fatty acids to phospholipid membranes.

    PubMed

    Córsico, Betina; Liou, Heng Ling; Storch, Judith

    2004-03-30

    Intestinal fatty acid binding protein (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms for the transfer of fatty acids (FAs) to acceptor membranes. Transfer from IFABP occurs during protein-membrane collisional interactions, while for LFABP, transfer occurs by diffusion through the aqueous phase. Earlier, we had shown that the helical domain of IFABP is critical in determining its collisional FA transfer mechanism. In the study presented here, we have engineered a pair of chimeric proteins, one with the "body" (ligand binding domain) of IFABP and the alpha-helical region of LFABP (alphaLbetaIFABP) and the other with the ligand binding pocket of LFABP and the helical domain of IFABP (alphaIbetaLFABP). The objective of this work was to determine whether the change in the alpha-helical domain of each FABP would alter the rate and mechanism of transfer of FA from the chimeric proteins in comparison with those of the wild-type proteins. The fatty acid transfer properties of the FABP chimeras were examined using a fluorescence resonance transfer assay. The results showed a significant modification of the absolute rate of FA transfer from the chimeric proteins compared to that of the wild type, indicating that the slower rate of FA transfer observed for wild-type LFABP relative to that of wild-type IFABP is, in part, determined by the helical domain of the proteins. In addition to these quantitative changes, it was of great interest to observe that the apparent mechanism of FA transfer also changed when the alpha-helical domain was exchanged, with transfer from alphaLbetaIFABP occurring by aqueous diffusion and transfer from alphaIbetaLFABP occurring via protein-membrane collisional interactions. These results demonstrate that the alpha-helical region of LFABP is responsible for its diffusional mechanism of fatty acid transfer to membranes. PMID:15035630

  18. Adenovirus serotype 5 hexon mediates liver gene transfer.

    PubMed

    Waddington, Simon N; McVey, John H; Bhella, David; Parker, Alan L; Barker, Kristeen; Atoda, Hideko; Pink, Rebecca; Buckley, Suzanne M K; Greig, Jenny A; Denby, Laura; Custers, Jerome; Morita, Takashi; Francischetti, Ivo M B; Monteiro, Robson Q; Barouch, Dan H; van Rooijen, Nico; Napoli, Claudio; Havenga, Menzo J E; Nicklin, Stuart A; Baker, Andrew H

    2008-02-01

    Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo. PMID:18267072

  19. Gene transfer from a parasitic flowering plant to a fern

    PubMed Central

    Davis, Charles C; Anderson, William R; Wurdack, Kenneth J

    2005-01-01

    The rattlesnake fern (Botrychium virginianum (L.) Sw.) is obligately mycotrophic and widely distributed across the northern hemisphere. Three mitochondrial gene regions place this species with other ferns in Ophioglossaceae, while two regions place it as a member of the largely parasitic angiosperm order Santalales (sandalwoods and mistletoes). These discordant phylogenetic placements suggest that part of the genome in B. virginianum was acquired by horizontal gene transfer (HGT), perhaps from root-parasitic Loranthaceae. These transgenes are restricted to B. virginianum and occur across the range of the species. Molecular and life-history traits indicate that the transfer preceded the global expansion of B. virginianum, and that the latter may have happened very rapidly. This is the first report of HGT from an angiosperm to a fern, through either direct parasitism or the mediation of interconnecting fungal symbionts. PMID:16191635

  20. A Rice Stowaway MITE for Gene Transfer in Yeast

    PubMed Central

    Fattash, Isam; Bhardwaj, Priyanka; Hui, Caleb; Yang, Guojun

    2013-01-01

    Miniature inverted repeat transposable elements (MITEs) lack protein coding capacity and often share very limited sequence similarity with potential autonomous elements. Their capability of efficient transposition and dramatic amplification led to the proposition that MITEs are an untapped rich source of materials for transposable element (TE) based genetic tools. To test the concept of using MITE sequence in gene transfer, a rice Stowaway MITE previously shown to excise efficiently in yeast was engineered to carry cargo genes (neo and gfp) for delivery into the budding yeast genome. Efficient excision of the cargo gene cassettes was observed even though the excision frequency generally decreases with the increase of the cargo sizes. Excised elements insert into new genomic loci efficiently, with about 65% of the obtained insertion sites located in genes. Elements at the primary insertion sites can be remobilized, frequently resulting in copy number increase of the element. Surprisingly, the orientation of a cargo gene (neo) on a construct bearing dual reporter genes (gfp and neo) was found to have a dramatic effect on transposition frequency. These results demonstrated the concept that MITE sequences can be useful in engineering genetic tools to deliver cargo genes into eukaryotic genomes. PMID:23704977

  1. Detection of homologous horizontal gene transfer in SNP data

    Energy Science and Technology Software Center (ESTSC)

    2012-07-23

    We study the detection of mutations, sequencing errors, and homologous horizontal gene transfers (HGT) in a set of closely related microbial genomes. We base the model on single nucleotide polymorphisms (SNP's) and break the genomes into blocks to handle the rearrangement problem. Then we apply a synamic programming algorithm to model whether changes within each block are likely a result of mutations, sequencing errors, or HGT.

  2. Organization of the V gene segments in mouse T-cell antigen receptor [alpha]/[delta] locus

    SciTech Connect

    Wang, K.; Klotz, J.L.; Kiser, G.; Bristol, G.; Hays, E.; Lai, E.; Gese, E.; Kronenberg, M.; Hood, L. )

    1994-04-01

    The mouse T-cell receptor (TCR) [alpha]/[delta] was mapped using 17 V[alpha] and 4 V[delta] subfamily-specific probes. Four complementary methods were used: (1) an estimate of the V gene repertoire by Southern blot analysis of genomic DNA with subfamily-specific probes; (2) an analysis of V gene segments deleted by TCR gene rearrangements from a panel of T-cell tumors and hybridomas; (3) an analysis of overlapping clusters of cosmid clones; and (4) an analysis of large DNA fragments separated by field-inversion gel electrophoresis. The [alpha]/[delta] locus spans about 1 Mb. The distance between the 3[prime]-most V gene segments (V[delta]1) and the [delta] constant gene (C[delta]) is no more than 150 kb. Sixty-six V gene segments have been mapped physically on cosmids. The members of individual V[alpha] gene segments subfamilies are dispersed throughout the locus. In contrast, the V[delta] gene segments V[delta]1 to 5 are clustered at the 3[prime] end of the V gene segments cluster. At least two DNA segment duplications, 45 to 80 kb in length, are present in the locus. These data provide information on the evolution of the [alpha]/[delta] locus and on organizational features that might influence the expression of specific V gene segments in [gamma][delta] cells. 35 refs., 5 figs., 2 tabs.

  3. Characterization of the 5'-flanking region of the gene for the alpha chain of human fibrinogen.

    PubMed

    Hu, C H; Harris, J E; Davie, E W; Chung, D W

    1995-11-24

    The 5'-flanking region of the gene coding for the alpha chain of human fibrinogen was isolated, sequenced, and characterized. The principal site of transcription initiation was determined by primer extension analysis and the RNase protection assay and shown to be at an adenine residue located 55 nucleotides upstream from the initiator methionine codon, or 13,399 nucleotides down-stream from the polyadenylation site of the gene coding for the gamma chain. Transient expression of constructs containing sequentially deleted 5'-flanking sequences of the alpha chain gene fused to the chloramphenicol acetyltransferase reporter gene showed that the promoter was liver-specific and inducible by interleukin 6 (IL-6). The shortest DNA fragment with significant promoter activity and full response to IL-6 stimulation encompassed the region from -217 to +1 base pairs (bp). Although six potential IL-6 responsive sequences homologous to the type II IL-6 responsive element were present, a single sequence of CTGGGA localized from -122 to -127 bp was shown to be a functional element in IL-6 induction. A hepatocyte nuclear factor 1 (HNF-1) binding site, present from -47 to -59 bp, in combination with other upstream elements, was essential for liver-specific expression of the gene. A functional CCAAT/enhancer binding protein site (C/EBP, -134 to -142 bp) was also identified within 217 bp from the transcription initiation site. An additional positive element (-1393 to -1133 bp) and a negative element (-1133 to -749 bp) were also found in the upstream region of the alpha-fibrinogen gene. PMID:7499335

  4. Cloning and sequencing of the gene for alpha antigen from Mycobacterium avium and mapping of B-cell epitopes.

    PubMed Central

    Ohara, N; Matsuo, K; Yamaguchi, R; Yamazaki, A; Tasaka, H; Yamada, T

    1993-01-01

    The complete nucleotide sequence of alpha antigen secreted from Mycobacterium avium (A-alpha) was determined. The gene encodes 330 amino acids, including 40 amino acids for the signal peptide, followed by 290 amino acids for the mature protein with a molecular mass of 30,811 Da. This is the first sequence of A-alpha. Comparisons between A-alpha and alpha antigens of Mycobacterium leprae, Mycobacterium bovis BCG, and Mycobacterium kansasii showed highly homologous regions which suggested a conserved functional domain and two less-homologous regions. Serological analysis of recombinant A-alpha, expressed by a series of deletion constructs, indicated the possibility that A-alpha carries at least six B-cell epitopes. The three antigenic determinants were common to Mycobacterium tuberculosis, M. kansasii, and M. avium. The results also suggested the possibility that there are three species-specific epitopes. Images PMID:7681039

  5. The integrity of the alpha-helical domain of intestinal fatty acid binding protein is essential for the collision-mediated transfer of fatty acids to phospholipid membranes.

    PubMed

    Franchini, G R; Storch, J; Corsico, B

    2008-04-01

    Intestinal FABP (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms of fatty acid transfer to acceptor model membranes. Transfer from IFABP occurs during protein-membrane collisional interactions, while for LFABP transfer occurs by diffusion through the aqueous phase. In addition, transfer from IFABP is markedly faster than from LFABP. The overall goal of this study was to further explore the structural differences between IFABP and LFABP which underlie their large functional differences in ligand transport. In particular, we addressed the role of the alphaI-helix domain in the unique transport properties of intestinal FABP. A chimeric protein was engineered with the 'body' (ligand binding domain) of IFABP and the alphaI-helix of LFABP (alpha(I)LbetaIFABP), and the fatty acid transfer properties of the chimeric FABP were examined using a fluorescence resonance energy transfer assay. The results showed a significant decrease in the absolute rate of FA transfer from alpha(I)LbetaIFABP compared to IFABP. The results indicate that the alphaI-helix is crucial for IFABP collisional FA transfer, and further indicate the participation of the alphaII-helix in the formation of a protein-membrane "collisional complex". Photo-crosslinking experiments with a photoactivable reagent demonstrated the direct interaction of IFABP with membranes and further support the importance of the alphaI helix of IFABP in its physical interaction with membranes. PMID:18284926

  6. A Theoretical Investigation of the Charge Transfer System TCNQ-F4 and Alpha-Sexithiophene

    NASA Astrophysics Data System (ADS)

    Braun, Kai-Felix

    2005-03-01

    The electronic and geometrical structures of the charge-transfer system of alpha-sexihiophene and tetrafluorotetracyanoquinodimethane are calculated self-consistently from first principles. By means of density functional theory (DFT) methods several configurations of the free molecules are calculated within LDA and B3LYP employing a plane wave basis and different atomic orbital sets. The combined system exhibits preferential binding of the center of the TCNQ-F4 on top of a c-c bond of the sexithiophene, thereby the central configuration having the lowest energy. As opposed to the periodic arrangement in a crystal of the related system dimethylquaterthiophene and TCNQ-F4, the free system exhibits a strong interaction going along with a substantial polarization of both molecules. For comparison with scanning tunneling spectroscopy results, the molecules were adsorbed in a parallel geometry on a Au(111) slab. To take into account the voltage applied to the STM tip the system was finally calculated within an electric field. This work is financially supported by the US-DOE grant no. DE-FG02-02ER46012.

  7. Reduction and Methyl Transfer Kinetics of the Alpha Subunit from Acetyl-Coenzyme A Synthase

    SciTech Connect

    Xiangshi Tan; Christopher Sewell; Qingwu Yang; Paul A. Lindahl

    2003-01-15

    OAK-B135 Stopped-flow was used to evaluate the methylation and reduction kinetics of the isolated alpha subunit of acetyl-Coenzyme A synthase from Moorella thermoacetica. This catalytically active subunit contains a novel Ni-X-Fe4S4 cluster and a putative unidentified n =2 redox site called D. The D-site must be reduced for a methyl group to transfer from a corrinoid-iron-sulfur protein, a key step in the catalytic synthesis of acetyl-CoA. The Fe4S4 component of this cluster is also redox active, raising the possibility that it is the D-site or a portion thereof. Results presented demonstrate that the D-site reduces far faster than the Fe4S4 component, effectively eliminating this possibility. Rather, this component may alter catalytically important properties of the Ni center. The D-site is reduced through a pathway that probably does not involve the Fe4S4 component of this active-site cluster.

  8. Selective Gene Transfer to the Retina Using Intravitreal Ultrasound Irradiation

    PubMed Central

    Sonoda, Shozo; Tachibana, Katsuro; Yamashita, Toshifumi; Shirasawa, Makoto; Terasaki, Hiroto; Uchino, Eisuke; Suzuki, Ryo; Maruyama, Kazuo; Sakamoto, Taiji

    2012-01-01

    This paper aims to evaluate the efficacy of intravitreal ultrasound (US) irradiation for green fluorescent protein (GFP) plasmid transfer into the rabbit retina using a miniature US transducer. Intravitreal US irradiation was performed by a slight modification of the transconjunctival sutureless vitrectomy system utilizing a small probe. After vitrectomy, the US probe was inserted through a scleral incision. A mixture of GFP plasmid (50 μL) and bubble liposomes (BLs; 50 μL) was injected into the vitreous cavity, and US was generated to the retina using a SonoPore 4000. The control group was not exposed to US. After 72 h, the gene-transfer efficiency was quantified by counting the number of GFP-positive cells. The retinas that received plasmid, BL, and US showed a significant increase in the number (average ± SEM) of GFP-positive cells (32 ± 4.9; n = 7; P < 0.01 ). No GFP-positive cells were observed in the control eyes (n = 7). Intravitreal retinal US irradiation can transfer the GFP plasmid into the retina without causing any apparent damage. This procedure could be used to transfer genes and drugs directly to the retina and therefore has potential therapeutic value. PMID:22518277

  9. 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

  10. Dynamic monitoring of horizontal gene transfer in soil

    NASA Astrophysics Data System (ADS)

    Cheng, H. Y.; Masiello, C. A.; Silberg, J. J.; Bennett, G. N.

    2015-12-01

    Soil microbial gene expression underlies microbial behaviors (phenotypes) central to many aspects of C, N, and H2O cycling. However, continuous monitoring of microbial gene expression in soils is challenging because genetically-encoded reporter proteins widely used in the lab are difficult to deploy in soil matrices: for example, green fluorescent protein cannot be easily visualized in soils, even in the lab. To address this problem we have developed a reporter protein that releases small volatile gases. Here, we applied this gas reporter in a proof-of-concept soil experiment, monitoring horizontal gene transfer, a microbial activity that alters microbial genotypes and phenotypes. Horizontal gene transfer is central to bacterial evolution and adaptation and is relevant to problems such as the spread of antibiotic resistance, increasing metal tolerance in superfund sites, and bioremediation capability of bacterial consortia. This process is likely to be impacted by a number of matrix properties not well-represented in the petri dish, such as microscale variations in water, nutrients, and O2, making petri-dish experiments a poor proxy for environmental processes. We built a conjugation system using synthetic biology to demonstrate the use of gas-reporting biosensors in safe, lab-based biogeochemistry experiments, and here we report the use of these sensors to monitor horizontal gene transfer in soils. Our system is based on the F-plasmid conjugation in Escherichia coli. We have found that the gas signal reports on the number of cells that acquire F-plasmids (transconjugants) in a loamy Alfisol collected from Kellogg Biological Station. We will report how a gas signal generated by transconjugants varies with the number of F-plasmid donor and acceptor cells seeded in a soil, soil moisture, and soil O2 levels.

  11. [Synthesis of new gene-loaded microbubbles serve as gene delivery vehicle applied in reporter gene transfer into cardiac myocytes].

    PubMed

    Wang, Guozhong; Hu, Shenjiang; Zheng, Zhelan; Sun, Jian; Zheng, Xia; Zhu, Zhaohui; Li, Jiang; Yao, Yumei

    2006-08-01

    To improve the stability and gene-carried capability of gene-attached microbubbles, the method for manufacture of albumin microbubbles was modified and new gene-loaded microbubbles were synthesized by incorporated gene-PEI complex into the shell of microbubbles. Agarose gel electrophoresis and bacteria transformation showed that PEI had the ability to provide the protection of plasmid DNA from ultrasonic degradation. The new gene-loaded microbubbles exhibited excellent acoustical and hemorheological properties. Moreover, they could carry more plasmid DNA than gene-attached microbubbles. beta-galactosidase plasmid transfection into cardiac myocytes was performed by using ultrasound targeted destruction of new gene-loaded microbubbles or gene-attached microbubbles. Gene expression in cardiac myocytes was detected by beta-galactosidase in situ staining and quantitive assay. It was shown that beta-galactosidase activity in cardiac myocytes was enhanced 107-fold by ultrasonic destruction of gene-loaded microbubbles compared with naked plasmid transfection and new gene-loaded microbubbles resulted in 6.85-fold increase in beta-galactosidase activity compared with optimal transfection mediated by gene-attached microbubbles. These results suggested that ultrasonic destruction of the gene-loaded microbubbles can enhance the cardiac myocytes exogenous gene transfer efficiency significantly and new gene-loaded microbubbles is an efficient and safe gene delivery vehicle. PMID:17002125

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

    PubMed Central

    Borgio, J. Francis

    2015-01-01

    Alpha-thalassemia (α-thal) is a disorder caused by the deletion of single or double α-globin genes, and/or point mutations in the α-globin genes. There are 2 common types of α-globin genes; HBA2 and HBA1. Recently, it has been discovered that the HBA2 gene is replaced by a unique HBA12 gene convert in 5.7% of the Saudi population. The α-globin genes have been emerging as a molecular target for the treatment of β-thalassemia (β-thal). Hence, it is essential to understand the molecular nature of α-globin genes to treat the most prevalent hemoglobin disorders, such as sickle cell disease, α-thal, and β-thal prevalent in the Kingdom of Saudi Arabia. Thirty-two different α-globin genotypes have been observed in the Saudi population. This review outlines the classification of the α-globin genes on the basis of their molecular nature and complex combinations of α-globin genes, and their variants predominant in Saudis. PMID:26593158

  13. Nucleotide sequence and expression of alpha-glucosidase-encoding gene (agdA) from Aspergillus oryzae.

    PubMed

    Minetoki, T; Gomi, K; Kitamoto, K; Kumagai, C; Tamura, G

    1995-08-01

    We have isolated an alpha-glucosidase(AGL)-encoding gene (agdA) from Aspergillus oryzae by heterologous hybridization using the corresponding Aspergillus niger gene as a probe. Southern hybridization analysis showed that the agdA gene is on a 5.0-kb ScaI fragment and there is a single copy in the A. oryzae chromosome. Comparison with the A. niger agdA gene indicated that the agdA gene contains three putative introns from 52 to 59 nucleotides long, and that it encodes 985 amino acid residues. The deduced amino acid sequence of A. oryzae AGL is 78% homologous with the A. niger AGL. The high degree of homology with the amino acid sequence bordering the putative catalytic residue of a number of AGL enzymes, and this enzyme suggests that Asp492 is a catalytic residue of A. oryzae AGL. The cloned gene was functional. Transformants of A. oryzae containing multiple copies of the cloned agdA gene showed a 6-16 fold increase in AGL activity. Like the Taka-amylase A and glucoamylase genes of A. oryzae, expression of the agdA gene was induced when maltose was provided as a carbon source, but expression was not induced by glucose. This result suggested that cis-element(s) involved in maltose induction may be also present in the agdA promoter region. PMID:7549103

  14. Effect of TNF{alpha} on activities of different promoters of human apolipoprotein A-I gene

    SciTech Connect

    Orlov, Sergey V.; Mogilenko, Denis A.; Shavva, Vladimir S.; Dizhe, Ella B.; Ignatovich, Irina A.; Perevozchikov, Andrej P.

    2010-07-23

    Research highlights: {yields} TNF{alpha} stimulates the distal alternative promoter of human apoA-I gene. {yields} TNF{alpha} acts by weakening of promoter competition within apoA-I gene (promoter switching). {yields} MEK1/2 and nuclear receptors PPAR{alpha} and LXRs take part in apoA-I promoter switching. -- Abstract: Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoproteins. The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1{beta} and TNF{alpha}. Recently, two novel additional (alternative) promoters for human apoA-I gene have been identified. Nothing is known about the role of alternative promoters in TNF{alpha}-mediated downregulation of apoA-I gene. In this article we report for the first time about the different effects of TNF{alpha} on two alternative promoters of human apoA-I gene. Stimulation of HepG2 cells by TNF{alpha} leads to activation of the distal alternative apoA-I promoter and downregulation of the proximal alternative and the canonical apoA-I promoters. This effect is mediated by weakening of the promoter competition within human apoA-I 5'-regulatory region (apoA-I promoter switching) in the cells treated by TNF{alpha}. The MEK1/2-ERK1/2 cascade and nuclear receptors PPAR{alpha} and LXRs are important for TNF{alpha}-mediated apoA-I promoter switching.

  15. Horizontal Gene Transfer Contributes to the Evolution of Arthropod Herbivory

    PubMed Central

    Wybouw, Nicky; Pauchet, Yannick; Heckel, David G.; Van Leeuwen, Thomas

    2016-01-01

    Within animals, evolutionary transition toward herbivory is severely limited by the hostile characteristics of plants. Arthropods have nonetheless counteracted many nutritional and defensive barriers imposed by plants and are currently considered as the most successful animal herbivores in terrestrial ecosystems. We gather a body of evidence showing that genomes of various plant feeding insects and mites possess genes whose presence can only be explained by horizontal gene transfer (HGT). HGT is the asexual transmission of genetic information between reproductively isolated species. Although HGT is known to have great adaptive significance in prokaryotes, its impact on eukaryotic evolution remains obscure. Here, we show that laterally transferred genes into arthropods underpin many adaptations to phytophagy, including efficient assimilation and detoxification of plant produced metabolites. Horizontally acquired genes and the traits they encode often functionally diversify within arthropod recipients, enabling the colonization of more host plant species and organs. We demonstrate that HGT can drive metazoan evolution by uncovering its prominent role in the adaptations of arthropods to exploit plants. PMID:27307274

  16. Evidence of horizontal gene transfer between obligate leaf nodule symbionts.

    PubMed

    Pinto-Carbó, Marta; Sieber, Simon; Dessein, Steven; Wicker, Thomas; Verstraete, Brecht; Gademann, Karl; Eberl, Leo; Carlier, Aurelien

    2016-09-01

    Bacteria of the genus Burkholderia establish an obligate symbiosis with plant species of the Rubiaceae and Primulaceae families. The bacteria, housed within the leaves, are transmitted hereditarily and have not yet been cultured. We have sequenced and compared the genomes of eight bacterial leaf nodule symbionts of the Rubiaceae plant family. All of the genomes exhibit features consistent with genome erosion. Genes potentially involved in the biosynthesis of kirkamide, an insecticidal C7N aminocyclitol, are conserved in most Rubiaceae symbionts. However, some have partially lost the kirkamide pathway due to genome erosion and are unable to synthesize the compound. Kirkamide synthesis is therefore not responsible for the obligate nature of the symbiosis. More importantly, we find evidence of intra-clade horizontal gene transfer (HGT) events affecting genes of the secondary metabolism. This indicates that substantial gene flow can occur at the early stages following host restriction in leaf nodule symbioses. We propose that host-switching events and plasmid conjugative transfers could have promoted these HGTs. This genomic analysis of leaf nodule symbionts gives, for the first time, new insights in the genome evolution of obligate symbionts in their early stages of the association with plants. PMID:26978165

  17. Horizontal Gene Transfer Contributes to the Evolution of Arthropod Herbivory.

    PubMed

    Wybouw, Nicky; Pauchet, Yannick; Heckel, David G; Van Leeuwen, Thomas

    2016-01-01

    Within animals, evolutionary transition toward herbivory is severely limited by the hostile characteristics of plants. Arthropods have nonetheless counteracted many nutritional and defensive barriers imposed by plants and are currently considered as the most successful animal herbivores in terrestrial ecosystems. We gather a body of evidence showing that genomes of various plant feeding insects and mites possess genes whose presence can only be explained by horizontal gene transfer (HGT). HGT is the asexual transmission of genetic information between reproductively isolated species. Although HGT is known to have great adaptive significance in prokaryotes, its impact on eukaryotic evolution remains obscure. Here, we show that laterally transferred genes into arthropods underpin many adaptations to phytophagy, including efficient assimilation and detoxification of plant produced metabolites. Horizontally acquired genes and the traits they encode often functionally diversify within arthropod recipients, enabling the colonization of more host plant species and organs. We demonstrate that HGT can drive metazoan evolution by uncovering its prominent role in the adaptations of arthropods to exploit plants. PMID:27307274

  18. PEGylated Cationic Serum Albumin for Boosting Retroviral Gene Transfer.

    PubMed

    Palesch, David; Boldt, Felix; Müller, Janis A; Eisele, Klaus; Stürzel, Christina M; Wu, Yuzhou; Münch, Jan; Weil, Tanja

    2016-08-17

    Retroviral vectors are common tools for introducing genes into the genome of a cell. However, low transduction rates are a major limitation in retroviral gene transfer, especially in clinical applications. We generated cationic human serum albumin (cHSA) protected by a shell of poly(ethylene glycol) (PEG); this significantly enhanced retroviral gene transduction with potentially attractive pharmacokinetics and low immunogenicity. By screening a panel of chemically optimized HSA compounds, we identified a very potent enhancer that boosted the transduction rates of viral vectors. Confocal microscopy revealed a drastically increased number of viral particles attached to the surfaces of target cells. In accordance with the positive net charge of cationic and PEGylated HSA, this suggests a mechanism of action in which the repulsion of the negatively charged cellular and viral vector membranes is neutralized, thereby promoting attachment and ultimately transduction. Importantly, the transduction-enhancing PEGylated HSA derivative evaded recognition by HSA-specific antibodies and macrophage activation. Our findings hold great promise for facilitating improved retroviral gene transfer. PMID:27239020

  19. Modular organization and development activity of an Arabidopsis thaliana EF-1 alpha gene promoter.

    PubMed

    Curie, C; Axelos, M; Bardet, C; Atanassova, R; Chaubet, N; Lescure, B

    1993-04-01

    The activity of the Arabidopsis thalana A1 EF-1 alpha gene promoter was analyzed in transgenic Arabidopsis plants. The 5' upstream sequence of the A1 gene and several promoter deletions were fused to the beta-glucuronidase (GUS) coding region. Promoter activity was monitored by quantitative and histochemical assays of GUS activity. The results show that the A1 promoter exhibits a modular organization. Sequences both upstream and downstream relative to the transcription initiation site are involved in quantitative and tissue-specific expression during vegetative growth. One upstream element may be involved in the activation of expression in meristematic tissues; the downstream region, corresponding to an intron within the 5' non-coding region (5'IVS), is important for expression in roots; both upstream and downstream sequences are required for expression in leaves, suggesting combinatorial properties of EF-1 alpha cis-regulatory elements. This notion of specific combinatorial regulation is reinforced by the results of transient expression experiments in transfected Arabidopsis protoplasts. The deletion of the 5'IVS has much more effect on expression when the promoter activity is under the control of A1 EF-1 alpha upstream sequences than when these upstream sequences were replaced by the 35S enhancer. Similarly, a synthetic oligonucleotide corresponding to an A1 EF-1 alpha upstream cis-acting element (the TEF1 box), is able to restore partially the original activity when fused to a TEF1-less EF1-alpha promoter but has no significant effect when fused to an enhancer-less 35S promoter. PMID:8492811

  20. HGTree: database of horizontally transferred genes determined by tree reconciliation

    PubMed Central

    Jeong, Hyeonsoo; Sung, Samsun; Kwon, Taehyung; Seo, Minseok; Caetano-Anollés, Kelsey; Choi, Sang Ho; Cho, Seoae; Nasir, Arshan; Kim, Heebal

    2016-01-01

    The HGTree database provides putative genome-wide horizontal gene transfer (HGT) information for 2472 completely sequenced prokaryotic genomes. This task is accomplished by reconstructing approximate maximum likelihood phylogenetic trees for each orthologous gene and corresponding 16S rRNA reference species sets and then reconciling the two trees under parsimony framework. The tree reconciliation method is generally considered to be a reliable way to detect HGT events but its practical use has remained limited because the method is computationally intensive and conceptually challenging. In this regard, HGTree (http://hgtree.snu.ac.kr) represents a useful addition to the biological community and enables quick and easy retrieval of information for HGT-acquired genes to better understand microbial taxonomy and evolution. The database is freely available and can be easily scaled and updated to keep pace with the rapid rise in genomic information. PMID:26578597

  1. PCR-based detection of gene transfer vectors: application to gene doping surveillance.

    PubMed

    Perez, Irene C; Le Guiner, Caroline; Ni, Weiyi; Lyles, Jennifer; Moullier, Philippe; Snyder, Richard O

    2013-12-01

    Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR. PMID:23912835

  2. Indications for Acquisition of Reductive Dehalogenase Genes through Horizontal Gene Transfer by Dehalococcoides ethenogenes Strain 195

    PubMed Central

    Regeard, Christophe; Maillard, Julien; Dufraigne, Christine; Deschavanne, Patrick; Holliger, Christof

    2005-01-01

    The genome of Dehalococcoides ethenogenes strain 195, an anaerobic dehalorespiring bacterium, contains 18 copies of putative reductive dehalogenase genes, including the well-characterized tceA gene, whose gene product functions as the key enzyme in the environmentally important dehalorespiration process. The genome of D. ethenogenes was analyzed using a bioinformatic tool based on the frequency of oligonucleotides. The results in the form of a genomic signature revealed several local disruptions of the host signature along the genome sequence. These fractures represent DNA segments of potentially foreign origin, so-called atypical regions, which may have been acquired by an ancestor through horizontal gene transfer. Most interestingly, 15 of the 18 reductive dehalogenase genes, including the tceA gene, were found to be located in these regions, strongly indicating the foreign nature of the dehalorespiration activity. The GC content and the presence of recombinase genes within some of these regions corroborate this hypothesis. A hierarchical classification of the atypical regions containing the reductive dehalogenase genes indicated that these regions were probably acquired by several gene transfer events. PMID:15932990

  3. Interindividual concordance of methylation profiles in human genes for tumor necrosis factors alpha and beta.

    PubMed Central

    Kochanek, S; Toth, M; Dehmel, A; Renz, D; Doerfler, W

    1990-01-01

    The DNA in mammalian genomes is characterized by complex patterns of DNA methylation that reflect the states of all genetic activities of that genome. The modified nucleotide 5-methyldeoxycytidine (5mdC) can affect the interactions of specific proteins with DNA sequence motifs. The most extensively studied effect of sequence-specific methylations is that of the long-term silencing of eukaryotic (mammalian) promoters. We have initiated studies on the methylation status of parts of the human genome to view patterns of DNA methylation as indicators for genetic activities. In this report, analyses using both restriction enzyme--Southern blotting and the very precise genomic sequencing technique have been done. The genes for tumor necrosis factors (TNF) alpha and beta--in particular, their 5'-upstream and promoter regions--have been investigated in DNA isolated from human lymphocytes, granulocytes, and sperm. The results are characterized by a remarkable interindividual concordance of DNA methylation in specific human cell types. The patterns are identical in the DNA from one cell type for different individuals even of different genetic origins but different in the DNA from different cell types. As an example, in the DNA from human granulocytes of 15 different individuals (ages 20-48 yr, both sexes), 5mdC residues have been localized by the genomic sequencing technique in three identical sequence positions in the 5'-upstream region and in one downstream position of the gene encoding TNF-alpha. The promoter of this gene is free of 5mdC, and TNF-alpha is expressed in human granulocytes. The TNF-beta promoter is methylated in granulocytes from 9 different individuals, and TNF-beta is not expressed. In human lymphocytes, the main source of TNF-beta, the TNF-beta promoter is free of 5mdC residues. All 5'-CG-3' sites studied in the TNF-alpha and -beta genes are methylated in DNA from human sperm. In human cell lines HL-60, Jurkat, and RPMI 1788, the extent of DNA methylation

  4. Tumor necrosis factor alpha negatively regulates hepatitis B virus gene expression in transgenic mice.

    PubMed Central

    Gilles, P N; Fey, G; Chisari, F V

    1992-01-01

    It is well known that several inflammatory cytokines can modulate hepatocellular gene expression in a complex physiological process known as the hepatic acute-phase response. Since hepatitis B virus (HBV) characteristically induces a vigorous lymphomononuclear inflammatory response in the liver during acute and chronic hepatitis, it is possible that hepatocellular HBV gene expression may also be modulated by one or more of the cytokines produced by these cells. Using bacterial lipopolysaccharide (LPS) as a surrogate inducer of inflammatory cytokines in vivo, we have tested this hypothesis in a transgenic mouse model system. In experiments with two independent transgenic mouse lineages that express the HBV envelope region under the control of either HBV or cellular promoters, we observed a 50 to 80% reduction in the hepatic steady-state content of a 2.1-kb HBV mRNA following administration of a single intraperitoneal dose of LPS. The regulatory influence of several inflammatory cytokines known to be induced by LPS was also examined in this system. The negative regulatory effect of LPS was consistently reproduced by the administration of a single nontoxic dose of tumor necrosis factor alpha, and it was occasionally observed following the administration of high doses of alpha interferon and interleukin-6, while no effect was detectable in response to high-dose interleukin-1 alpha or to gamma interferon. These observations suggest that tumor necrosis factor alpha and perhaps other cytokines may activate a heretofore unsuspected intracellular pathway that negatively regulates HBV gene expression. The intracellular mechanism(s) responsible for this effect and its pathophysiologic relevance remain to be elucidated. Images PMID:1583737

  5. Identification of gene-based responses in human blood cells exposed to alpha particle radiation

    PubMed Central

    2014-01-01

    Background The threat of a terrorist-precipitated nuclear event places humans at danger for radiological exposures. Isotopes which emit alpha (α)-particle radiation pose the highest risk. Currently, gene expression signatures are being developed for radiation biodosimetry and triage with respect to ionizing photon radiation. This study was designed to determine if similar gene expression profiles are obtained after exposures involving α-particles. Methods Peripheral blood mononuclear cells (PBMCs) were used to identify sensitive and robust gene-based biomarkers of α-particle radiation exposure. Cells were isolated from healthy individuals and were irradiated at doses ranging from 0-1.5 Gy. Microarray technology was employed to identify transcripts that were differentially expressed relative to unirradiated cells 24 hours post-exposure. Statistical analysis identified modulated genes at each of the individual doses. Results Twenty-nine genes were common to all doses with expression levels ranging from 2-10 fold relative to control treatment group. This subset of genes was further assessed in independent complete white blood cell (WBC) populations exposed to either α-particles or X-rays using quantitative real-time PCR. This 29 gene panel was responsive in the α-particle exposed WBCs and was shown to exhibit differential fold-changes compared to X-irradiated cells, though no α-particle specific transcripts were identified. Conclusion Current gene panels for photon radiation may also be applicable for use in α-particle radiation biodosimetry. PMID:25017500

  6. Noninvasive monitoring of therapeutic gene transfer in animal models of muscular dystrophies.

    PubMed

    Bartoli, M; Poupiot, J; Goyenvalle, A; Perez, N; Garcia, L; Danos, O; Richard, I

    2006-01-01

    Muscular dystrophies are a genetically and phenotypically heterogeneous group of degenerative muscle diseases. A subset of them are due to genetic deficiencies in proteins which form the dystrophin-associated complex at the membrane of the myofibers. In this report, we utilized recombinant adeno-associated virus containing a U7 cassette carrying an antisense sequence aimed at inducing exon skipping of the dystrophin gene or containing the alpha-sarcoglycan gene to alleviate the dystrophic phenotype of the mdx and Sgca-null mice, respectively. As these diseases are characterized by cycle of degeneration/regeneration, we postulated that a reporter gene coadministered at the time of the treatment would make it possible to follow the extent of muscle repair. We observed that the murine secreted alkaline phosphatase (muSeAP) level was very much lower in these animal models than in normal mice. Upon treatment of the dystrophic muscle by gene transfer, the level of muSeAP was restored and correlated with the expression of the therapeutic transgene and with the level of muscle improvement. The system described here provides a simple and noninvasive procedure for monitoring the outcome of a therapeutic strategy involving cell survival. PMID:16107863

  7. Bacterial gene transfer by natural genetic transformation in the environment.

    PubMed Central

    Lorenz, M G; Wackernagel, W

    1994-01-01

    Natural genetic transformation is the active uptake of free DNA by bacterial cells and the heritable incorporation of its genetic information. Since the famous discovery of transformation in Streptococcus pneumoniae by Griffith in 1928 and the demonstration of DNA as the transforming principle by Avery and coworkers in 1944, cellular processes involved in transformation have been studied extensively by in vitro experimentation with a few transformable species. Only more recently has it been considered that transformation may be a powerful mechanism of horizontal gene transfer in natural bacterial populations. In this review the current understanding of the biology of transformation is summarized to provide the platform on which aspects of bacterial transformation in water, soil, and sediments and the habitat of pathogens are discussed. Direct and indirect evidence for gene transfer routes by transformation within species and between different species will be presented, along with data suggesting that plasmids as well as chromosomal DNA are subject to genetic exchange via transformation. Experiments exploring the prerequisites for transformation in the environment, including the production and persistence of free DNA and factors important for the uptake of DNA by cells, will be compiled, as well as possible natural barriers to transformation. The efficiency of gene transfer by transformation in bacterial habitats is possibly genetically adjusted to submaximal levels. The fact that natural transformation has been detected among bacteria from all trophic and taxonomic groups including archaebacteria suggests that transformability evolved early in phylogeny. Probable functions of DNA uptake other than gene acquisition will be discussed. The body of information presently available suggests that transformation has a great impact on bacterial population dynamics as well as on bacterial evolution and speciation. PMID:7968924

  8. Genes encoding tumor necrosis factor alpha and granzyme A are expressed during development of autoimmune diabetes.

    PubMed Central

    Held, W; MacDonald, H R; Weissman, I L; Hess, M W; Mueller, C

    1990-01-01

    Progressive destruction of the insulin-producing beta cells in nonobese diabetic mice is observed after infiltration of the pancreas with lymphocytes [Makino, S., Kunimoto, K., Muraoka, Y., Mizushima, Y., Katagiri, K. & Tochino, Y. (1980) Exp. Anim. (Tokyo) 29, 1-13]. We show that the genes for tumor necrosis factor alpha and granzyme A, a serine protease associated with cytoplasmic granules of cytotoxic cells, are expressed during the development of spontaneous diabetes mellitus in the nonobese diabetic mouse. Granzyme A-positive cells are found both in and surrounding the islets, implying induction prior to islet infiltration. Tumor necrosis factor alpha expression is exclusively observed in the intra-islet infiltrate, predominantly in lymphocytes adjacent to insulin-producing beta cells, the targets of the autoimmune destruction, implying that tumor necrosis factor alpha expression is induced locally--i.e., in the islet. A considerable portion of cells expressing tumor necrosis factor alpha appear to be CD4+ T cells. This T-cell subset was previously shown to be necessary for development of the disease. Thus, these findings may be important for understanding the pathogenesis of autoimmune diabetes mellitus and potentially also for that of other T-cell-mediated autoimmune diseases. Images PMID:2179951

  9. Matrix metalloproteinase-12 gene regulation by a PPAR alpha agonist in human monocyte-derived macrophages

    SciTech Connect

    Souissi, Imen Jguirim; Billiet, Ludivine; Cuaz-Perolin, Clarisse; Rouis, Mustapha

    2008-11-01

    MMP-12, a macrophage-specific matrix metalloproteinase with large substrate specificity, has been reported to be highly expressed in mice, rabbits and human atherosclerotic lesions. Increased MMP-12 from inflammatory macrophages is associated with several degenerative diseases such as atherosclerosis. In this manuscript, we show that IL-1{beta}, a proinflammatory cytokine found in atherosclerotic plaques, increases both mRNA and protein levels of MMP-12 in human monocyte-derived macrophages (HMDM). Since peroxisome proliferator-activated receptors (PPARs), such as PPAR{alpha} and PPAR{gamma}, are expressed in macrophages and because PPAR activation exerts an anti-inflammatory effect on vascular cells, we have investigated the effect of PPAR{alpha} and {gamma} isoforms on MMP-12 regulation in HMDM. Our results show that MMP-12 expression (mRNA and protein) is down regulated in IL-1{beta}-treated macrophages only in the presence of a specific PPAR{alpha} agonist, GW647, in a dose-dependent manner. In contrast, this inhibitory effect was abolished in IL-1{beta}-stimulated peritoneal macrophages isolated from PPAR{alpha}{sup -/-} mice and treated with the PPAR{alpha} agonist, GW647. Moreover, reporter gene transfection experiments using different MMP-12 promoter constructs showed a reduction of the promoter activities by {approx} 50% in IL-1{beta}-stimulated PPAR{alpha}-pre-treated cells. However, MMP-12 promoter analysis did not reveal the presence of a PPRE response element. The IL-1{beta} effect is known to be mediated through the AP-1 binding site. Mutation of the AP-1 site, located at - 81 in the MMP-12 promoter region relative to the transcription start site, followed by transfection analysis, gel shift and ChIP experiments revealed that the inhibitory effect was the consequence of the protein-protein interaction between GW 647-activated PPAR{alpha} and c-Fos or c-Jun transcription factors, leading to inhibition of their binding to the AP-1 motif. These studies

  10. [Association of TNF-alpha and IL-13 genes polymorphisms with bronchial asthma].

    PubMed

    Liang, Wenhua; Zhou, Zhaoshan; Ji, Zhongqiang; Wang, Yanqing; Xue, Weilin; Zhang, Xiaoyan

    2015-10-01

    OBJECTIVE To assess the association of polymorphisms of TNF-alpha gene (rs1799724, rs1800630, rs1799964 and rs769178) and IL-13 gene (rs2158177 and rs1295687) with susceptibility to asthma among ethnic Chinese in Qingdao region. METHODS For 400 asthma patients and 200 healthy subjects, above polymorphisms were detected with a SNaPshot method. RESULTS For rs2158177, the frequency of genotype of GG in the asthma group was significantly lower than the control group (2.8% vs. 5%, OR = 0.31, 95%CI: 0.12-0.82, P = 0.021). No significant difference was detected in the genotypic frequencies for the remaining 5 polymorphisms between the two groups (All P > 0.05). CONCLUSION The study has indicated that rs2158177 polymorphism of the IL-13 gene is associated with asthma in ethnic Han Chinese from Qingdao. No association has been found between polymorphisms of TNF-alpha gene with susceptibility to asthma. PMID:26418997

  11. In vivo Cytokine Gene Transfer by Gene Gun Reduces Tumor Growth in Mice

    NASA Astrophysics Data System (ADS)

    Sun, Wenn H.; Burkholder, Joseph K.; Sun, Jian; Culp, Jerilyn; Turner, Joel; Lu, Xing G.; Pugh, Thomas D.; Ershler, William B.; Yang, Ning-Sun

    1995-03-01

    Implantation of tumor cells modified by in vitro cytokine gene transfer has been shown by many investigators to result in potent in vivo antitumor activities in mice. Here we describe an approach to tumor immunotherapy utilizing direct transfection of cytokine genes into tumorbearing animals by particle-mediated gene transfer. In vivo transfection of the human interleukin 6 gene into the tumor site reduced methylcholanthrene-induced fibrosarcoma growth, and a combination of murine tumor necrosis factor α and interferon γ genes inhibited growth of a renal carcinoma tumor model (Renca). In addition, treatment with murine interleukin 2 and interferon γ genes prolonged the survival of Renca tumor-bearing mice and resulted in tumor eradication in 25% of the test animals. Transgene expression was demonstrated in treated tissues by ELISA and immunohistochemical analysis. Significant serum levels of interleukin 6 and interferon γ were detected, demonstrating effective secretion of transgenic proteins from treated skin into the bloodstream. This in vivo cytokine gene therapy approach provides a system for evaluating the antitumor properties of various cytokines in different tumor models and has potential utility for human cancer gene therapy.

  12. Association of ER-alpha gene polymorphism with metabolic phenotypes in Chinese Hans.

    PubMed

    Chen, Yan; Jiang, Xiao-yan; Xu, Li; Li, Xia; Cao, Fei-fei; Li, Lei; Lu, Ming; Jin, Li; Wang, Xiao-feng

    2009-08-01

    Recently, two polymorphisms (rs1884052 and rs3778099) of estrogen receptor alpha (ER-alpha) gene were identified as being associated with primary quantitative bone mineral density (BMD) in a genome-wide association (GWA) study in Framingham cohorts. In this study we aimed at investigating the association of rs1884052 and rs3778099, and another polymorphism (rs2234693) located at intron 1 of the ER-alpha gene with BMD, body mass index (BMI), glucose, triglyceride, and total cholesterol (CHO) levels in Chinese Hans. We recruited 425 consecutive adult volunteers who had a physical examination in the Jinan Maternity and Child Care Hospital. We did not observe significant association of rs1884052 and rs3778099 with BMD, BMI, glucose, triglyceride, and total cholesterol (CHO) levels. For rs2234693, increased levels of BMD for hip, spine or whole-body regions were consistently observed in TT/TC genotype carriers than in CC genotype carriers, although the board line significance diminished after adjusting for age and gender. However, significant association of rs2234693 with glucose and CHO levels were observed in our sample. Subjects with TC/CC genotypes were associated with an increased level of glucose (p = 0.013) and CHO (p = 0.032) levels than subjects with TT genotypes. In conclusion, we did not confirm the association of rs1884052 and rs3778099 with BMD originally discovered in a GWA study; however, we made novel discoveries that rs2234693 was associated with glucose and CHO levels in Chinese Hans. PMID:19578917

  13. Molecular cloning and characterization of a Candida tsukubaensis alpha-glucosidase gene in the yeast Saccharomyces cerevisiae.

    PubMed

    Kinsella, B T; Larkin, A; Bolton, M; Cantwell, B A

    1991-07-01

    The molecular cloning of an alpha-glucosidase gene isolated from a Candida tsukubaensis (CBS 6389) genomic library in Saccharomyces cervisiae is reported. The cloned gene is contained within a 6.2 kb Sau3A DNA fragment and directs the synthesis and secretion of an amylolytic enzyme into the extracellular medium of the recombinant host, S. cerevisiae. The cloned enzyme was found to have an unusually broad substrate specificity and is capable of hydrolysing alpha-1,2, alpha-1,3, alpha-1,4 and alpha-1,6 linked, as well as aryl and alkyl, D-glucosides. On the basis of its substrate specificity profile, the cloned enzyme was classified as an alpha-glucosidase (E.C. 3.2.1.20). It has a pH optimum in the range 4.2-4.6, a temperature optimum of 58 degrees C and is readily inactivated at pasteurization temperature (60 degrees C). Southern blot analysis failed to reveal any homology between the cloned gene and genomic DNA isolated from other well characterized amylolytic yeasts. A rapid plate-assay, based on the utilization of a chromogenic substrate X-alpha-D-glucoside to detect the expression of the cloned alpha-glucosidase in S. cerevisiae transformants, was developed. PMID:1934116

  14. Adaptive Horizontal Gene Transfers between Multiple Cheese-Associated Fungi.

    PubMed

    Ropars, Jeanne; Rodríguez de la Vega, Ricardo C; López-Villavicencio, Manuela; Gouzy, Jérôme; Sallet, Erika; Dumas, Émilie; Lacoste, Sandrine; Debuchy, Robert; Dupont, Joëlle; Branca, Antoine; Giraud, Tatiana

    2015-10-01

    Domestication is an excellent model for studies of adaptation because it involves recent and strong selection on a few, identified traits [1-5]. Few studies have focused on the domestication of fungi, with notable exceptions [6-11], despite their importance to bioindustry [12] and to a general understanding of adaptation in eukaryotes [5]. Penicillium fungi are ubiquitous molds among which two distantly related species have been independently selected for cheese making-P. roqueforti for blue cheeses like Roquefort and P. camemberti for soft cheeses like Camembert. The selected traits include morphology, aromatic profile, lipolytic and proteolytic activities, and ability to grow at low temperatures, in a matrix containing bacterial and fungal competitors [13-15]. By comparing the genomes of ten Penicillium species, we show that adaptation to cheese was associated with multiple recent horizontal transfers of large genomic regions carrying crucial metabolic genes. We identified seven horizontally transferred regions (HTRs) spanning more than 10 kb each, flanked by specific transposable elements, and displaying nearly 100% identity between distant Penicillium species. Two HTRs carried genes with functions involved in the utilization of cheese nutrients or competition and were found nearly identical in multiple strains and species of cheese-associated Penicillium fungi, indicating recent selective sweeps; they were experimentally associated with faster growth and greater competitiveness on cheese and contained genes highly expressed in the early stage of cheese maturation. These findings have industrial and food safety implications and improve our understanding of the processes of adaptation to rapid environmental changes. PMID:26412136

  15. Ocular gene transfer of active TGF-beta induces changes in anterior segment morphology and elevated IOP in rats.

    PubMed

    Robertson, Jennifer V; Golesic, Elizabeth; Gauldie, Jack; West-Mays, Judith A

    2010-01-01

    Purpose. Transforming growth factor beta (TGF-beta) is known to play a crucial role in wound healing and fibrotic tissue remodeling. A large body of evidence suggests a role for this cytokine in the pathogenesis of glaucoma; however, the mechanisms by which it affects anterior segment morphology are not well understood. Therefore, the purpose of this study was to examine the effects of TGF-beta overexpression on anterior segment morphology and subsequent effects on intraocular pressure. Methods. Adenoviral gene transfer was used to deliver active TGF-beta1 to the rat eye. Measurements of intraocular pressure were taken with a tonometer on days 0, 14, 21, and 29. Histologic analysis was undertaken to examine anterior segment morphology, and markers of matrix deposition and fibrosis were used. Results. Gene transfer of TGF-beta in the anterior segment resulted in the formation of peripheral anterior synechiae (PAS), which consisted of a fibroproliferative region of corneal endothelial cells, matrix accumulation, and decrease in trabecular meshwork expression of alpha-smooth muscle actin. These features were accompanied by ocular hypertension. Conclusions. Gene transfer of TGF-beta into the anterior segment induces aberrant PAS associated with the transition of corneal endothelial cells and subsequent matrix deposition. These features are highly reminiscent of human iridocorneal endothelial (ICE) syndrome. Gene transfer of TGF-beta can, therefore, be used to induce anatomic changes in the anterior segment in a rodent model that result in ocular hypertension. PMID:19696167

  16. High tumor necrosis factor alpha (TNF-alpha) production in Trypanosoma cruzi-infected pregnant mice and increased TNF-alpha gene transcription in their offspring.

    PubMed Central

    Rivera, M T; Marques de Araujo, S; Lucas, R; Deman, J; Truyens, C; Defresne, M P; de Baetselier, P; Carlier, Y

    1995-01-01

    Since tumor necrosis factor alpha (TNF-alpha) is known to be involved in the feto-maternal relationship, this cytokine was studied in Trypanosoma cruzi-infected pregnant BALB/c mice and their fetuses and offspring. Pregnant chronically infected mice displayed significantly higher levels of circulating TNF-alpha than animals either only infected or only pregnant. TNF-alpha was undetectable in sera of uninfected and nonpregnant mice as well as in breast milk obtained from infected and uninfected animals. Fetuses from infected mice exhibited significantly more cells containing TNF-alpha mRNA in their thymus than fetuses from uninfected mothers. When infected 2 months after birth, offspring born to infected and uninfected mothers displayed similar amounts of circulating TNF-alpha during chronic infection, whereas this cytokine was only weakly detectable during the acute phase of the disease. An intravenous injection of lipopolysaccharide during acute infection strongly increased the production of TNF-alpha in offspring born to infected mothers to levels higher than those in progeny from uninfected mice. These results suggest that TNF-alpha is an important cytokine in the feto-maternal relationship during T. cruzi infection and that fetuses and offspring of infected mothers are primed to produce elevated levels of TNF-alpha. PMID:7822027

  17. Effects of 12 metal ions on iron regulatory protein 1 (IRP-1) and hypoxia-inducible factor-1 alpha (HIF-1{alpha}) and HIF-regulated genes

    SciTech Connect

    Li Qin; Chen Haobin; Huang Xi; Costa, Max . E-mail: costam@env.med.nyu.edu

    2006-06-15

    Several metal ions that are carcinogenic affect cellular iron homeostasis by competing with iron transporters or iron-regulated enzymes. Some metal ions can mimic a hypoxia response in cells under normal oxygen tension, and induce expression of HIF-1{alpha}-regulated genes. This study investigated whether 12 metal ions altered iron homeostasis in human lung carcinoma A549 cells as measured by an activation of IRP-1 and ferritin level. We also studied hypoxia signaling by measuring HIF-1{alpha} protein levels, hypoxia response element (HRE)-driven luciferase reporter activity, and Cap43 protein level (an HIF-1{alpha} responsive gene). Our results show the following: (i) Ni(II), Co(II), V(V), Mn(II), and to a lesser extent As(III) and Cu(II) activated the binding of IRP-1 to IRE after 24 h, while the other metal ions had no effect; (ii) 10 of 12 metal ions induced HIF-1{alpha} protein but to strikingly different degrees. Two of these metal ions, Al(III) and Cd(II), did not induce HIF-1{alpha} protein; however, as indicated below, only Ni(II), Co (II), and to lesser extent Mn(II) and V(V) activated HIF-1{alpha}-dependent transcription. The combined effects of both [Ni(II) + As(III)] and [Ni(II) + Cr(VI)] on HIF-1{alpha} protein were synergistic; (iii) Addition of Fe(II) with Ni(II), Co(II), and Cr(VI) attenuated the induction of HIF-1{alpha} after 4 h treatment; (iv) Ni(II), Co(II), and Mn(II) significantly decrease ferritin level after 24 h exposure; (v) Ni(II), Co(II), V(V), and Mn(II) activated HRE reporter gene after 20 h treatment; (vi) Ni(II), Co(II), V(V), and Mn(II) increased the HIF-1-dependent Cap43 protein level after 24 h treatment. In conclusion, only Ni (II), Co (II), and to a lesser extent Mn(II) and V(V) significantly stabilized HIF-1{alpha} protein, activated IRP, decreased the levels of ferritin, induced the transcription of HIF-dependent reporter, and increased the expression of Cap43 protein levels (HIF-dependent gene). The mechanism for the

  18. Emergence of new alleles of the MSP-3alpha gene in Plasmodium vivax isolates from Korea.

    PubMed

    Nam, Deok Hwa; Oh, Jun Seo; Nam, Myoung Hyun; Park, Hae Chul; Lim, Chae Seung; Lee, Won Ja; Sattabongkot, Jetsumon; Klein, Terry A; Ayala, Francisco J

    2010-04-01

    Nucleotide sequence analysis of the Plasmodium vivax PvMSP-3alpha gene was conducted on blood from 143 malaria patients admitted to Korea University Medical Center from 1996 to 2007 in the Republic of Korea (ROK). From 1996 to 2002, the PvMSP-3alpha alleles were of two types, SKOR-67 (2.53 kb) and SKOR-69 (1.78 kb), which differed in length and amino acid sequence. Two new variants with similar size to SKOR-67 were first observed in 2002 and in 2006-2007 accounted for nearly 50% (25/51) of the sampled isolates. The new variants had the same amino acid sequence as SKOR-69 in the N-terminal region, but in Blocks I and II and in the C-terminal region, they were similar to previously reported isolates from Thailand, Papua New Guinea, India, Brazil, and Ecuador strains. PMID:20348492

  19. Expression of a bacterial alpha-amylase gene in transgenic rice seeds.

    PubMed

    Xu, Xiaoli; Fang, Jun; Wang, Wei; Guo, Jianli; Chen, Pinnan; Cheng, Jiaan; Shen, Zhicheng

    2008-08-01

    An alpha-amylase gene from Bacillus stearothermophilus under the control of the promoter of a major rice-seed storage protein was introduced into rice. The transgenic line with the highest alpha-amylase activity reached about 15,000 U/g of seeds (one unit is defined as the amount of enzyme that produces 1 mumol of reducing sugar in 1 min at 70 degrees C). The enzyme produced in the seeds had an optimum pH of 5.0-5.5 and optimum temperature of 60-70 degrees C. Without extraction or purification, the power of transgenic rice seeds was able to liquify 100 times its weight of corn powder in 2 h. Thus, the transgenic rice could be used for industrial starch liquefaction. PMID:17926139

  20. The alpha/beta fold family of proteins database and the cholinesterase gene server ESTHER.

    PubMed Central

    Cousin, X; Hotelier, T; Giles, K; Lievin, P; Toutant, J P; Chatonnet, A

    1997-01-01

    ESTHER (for esterases, alpha/betahydrolase enzyme and relatives) is a database of sequences phylogenetically related to cholinesterases. These sequences define a homogeneous group of enzymes (carboxylesterases, lipases and hormone-sensitive lipases) sharing a similar structure of a central beta-sheet surrounded by alpha-helices. Among these proteins a wide range of functions can be found (hydrolases, adhesion molecules, hormone precursors). The purpose of ESTHER is to help comparison of structures and functions of members of the family. Since the last release, new features have been added to the server. A BLAST comparison tool allows sequence homology searches within the database sequences. New sections are available: kinetics and inhibitors of cholinesterases, fasciculin-acetylcholinesterase interaction and a gene structure review. The mutation analysis compilation has been improved with three-dimensional images. A mailing list has been created. PMID:9016525

  1. Three alpha-amylase genes of Aspergillus oryzae exhibit identical intron-exon organization.

    PubMed

    Wirsel, S; Lachmund, A; Wildhardt, G; Ruttkowski, E

    1989-01-01

    We have cloned three genes (amy1, amy2 and amy3) encoding alpha-amylase in the filamentous fungus Aspergillus oryzae. The established overall sequences have a very high degree of homology, showing divergences mainly in the 3'-untranslated regions. The positions and the sequences of the eight introns were found to be absolutely identical in the three genes. The sequence analysis of the 5'-regions revealed presumptive TATA, CAAT and GC boxes. Primer extension analysis was performed to determine the transcription start. We were able to detect mRNAs from amy1 and amy3 but not from amy2 with gene-specific oligonucleotide probes complementary to the 3'-noncoding regions. PMID:2785629

  2. The Drosophila melanogaster importin alpha3 locus encodes an essential gene required for the development of both larval and adult tissues.

    PubMed Central

    Mason, D Adam; Máthé, Endre; Fleming, Robert J; Goldfarb, David S

    2003-01-01

    The nuclear transport of classical nuclear localization signal (cNLS)-containing proteins is mediated by the cNLS receptor importin alpha. The conventional importin alpha gene family in metazoan animals is composed of three clades that are conserved between flies and mammals and are referred to here as alpha1, alpha2, and alpha3. In contrast, plants and fungi contain only alpha1 genes. In this study we report that Drosophila importin alpha3 is required for the development of both larval and adult tissues. Importin alpha3 mutant flies die around the transition from first to second instar larvae, and homozygous importin alpha3 mutant eyes are defective. The transition to second instar larvae was rescued with importin alpha1, alpha2, or alpha3 transgenes, indicating that Importin alpha3 is normally required at this stage for an activity shared by all three importin alpha's. In contrast, an alpha3-specific biochemical activity(s) of Importin alpha3 is probably required for development to adults and photoreceptor cell development, since only an importin alpha3 transgene rescued these processes. These results are consistent with the view that the importin alpha's have both overlapping and distinct functions and that their role in animal development involves the spatial and temporal control of their expression. PMID:14704178

  3. Interaction between Conjugative and Retrotransposable Elements in Horizontal Gene Transfer

    PubMed Central

    Novikova, Olga; Smith, Dorie; Hahn, Ingrid; Beauregard, Arthur; Belfort, Marlene

    2014-01-01

    Mobile genetic elements either encode their own mobilization machineries or hijack them from other mobile elements. Multiple classes of mobile elements often coexist within genomes and it is unclear whether they have the capacity to functionally interact and even collaborate. We investigate the possibility that molecular machineries of disparate mobile elements may functionally interact, using the example of a retrotransposon, in the form of a mobile group II intron, found on a conjugative plasmid pRS01 in Lactococcus lactis. This intron resides within the pRS01 ltrB gene encoding relaxase, the enzyme required for nicking the transfer origin (oriT) for conjugal transmission of the plasmid into a recipient cell. Here, we show that relaxase stimulates both the frequency and diversity of retrotransposition events using a retromobility indicator gene (RIG), and by developing a high-throughput genomic retrotransposition detection system called RIG-Seq. We demonstrate that LtrB relaxase not only nicks ssDNA of its cognate oriT in a sequence- and strand-specific manner, but also possesses weak off-target activity. Together, the data support a model in which the two different mobile elements, one using an RNA-based mechanism, the other using DNA-based transfer, do functionally interact. Intron splicing facilitates relaxase expression required for conjugation, whereas relaxase introduces spurious nicks in recipient DNA that stimulate both the frequency of intron mobility and the density of events. We hypothesize that this functional interaction between the mobile elements would promote horizontal conjugal gene transfer while stimulating intron dissemination in the donor and recipient cells. PMID:25474706

  4. Gene Transfer and Molecular Cloning of the Human NGF Receptor

    NASA Astrophysics Data System (ADS)

    Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

    1986-04-01

    Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

  5. Site-Specific Gene Expression in Vivo by Direct Gene Transfer into the Arterial Wall

    NASA Astrophysics Data System (ADS)

    Nabel, Elizabeth G.; Plautz, Gregory; Nabel, Gary J.

    1990-09-01

    A recombinant β-galactosidase gene has been expressed in a specific arterial segment in vivo by direct infection with a murine amphotropic retroviral vector or by DNA transfection with the use of liposomes. Several cell types in the vessel wall were transduced, including endothelial and vascular smooth muscle cells. After retroviral infection, a recombinant reporter gene was expressed for at least 5 months, and no helper virus was detected. Recombinant gene expression achieved by direct retroviral infection or liposome-mediated DNA transfection was limited to the site of infection and was absent from liver, lung, kidney, and spleen. These results demonstrate that site-specific gene expression can be achieved by direct gene transfer in vivo and could be applied to the treatment of such human diseases as atherosclerosis or cancer.

  6. Laterally Transferred Gene Recruited as a Venom in Parasitoid Wasps.

    PubMed

    Martinson, Ellen O; Martinson, Vincent G; Edwards, Rachel; Mrinalini; Werren, John H

    2016-04-01

    Parasitoid wasps use venom to manipulate the immunity and metabolism of their host insects in a variety of ways to provide resources for their offspring. Yet, how genes are recruited and evolve to perform venom functions remain open questions. A recently recognized source of eukaryotic genome innovation is lateral gene transfer (LGT). Glycoside hydrolase family 19 (GH19) chitinases are widespread in bacteria, microsporidia, and plants where they are used in nutrient acquisition or defense, but have previously not been known in metazoans. In this study, a GH19 chitinase LGT is described from the unicellular microsporidia/Rozella clade into parasitoid wasps of the superfamily Chalcidoidea, where it has become recruited as a venom protein. The GH19 chitinase is present in 15 species of chalcidoid wasps representing four families, and phylogenetic analysis indicates that it was laterally transferred near or before the origin of Chalcidoidea (∼95 Ma). The GH19 chitinase gene is highly expressed in the venom gland of at least seven species, indicating a role in the complex host manipulations performed by parasitoid wasp venom. RNAi knockdown in the model parasitoid Nasonia vitripennis reveals that-following envenomation-the GH19 chitinase induces fly hosts to upregulate genes involved in an immune response to fungi. A second, independent LGT of GH19 chitinase from microsporidia into mosquitoes was also found, also supported by phylogenetic reconstructions. Besides these two LGT events, GH19 chitinase is not found in any other sequenced animal genome, or in any fungi outside the microsporidia/Rozella clade. PMID:26715630

  7. CXCR4 gene transfer prevents pressure overload induced heart failure

    PubMed Central

    LaRocca, Thomas J.; Jeong, Dongtak; Kohlbrenner, Erik; Lee, Ahyoung; Chen, JiQiu; Hajjar, Roger J.; Tarzami, Sima T.

    2012-01-01

    Stem cell and gene therapies are being pursued as strategies for repairing damaged cardiac tissue following myocardial infarction in an attempt to prevent heart failure. The chemokine receptor-4 (CXCR4) and its ligand, CXCL12, play a critical role in stem cell recruitment post-acute myocardial infarction. Whereas progenitor cell migration via the CXCL12/CXCR4 axis is well characterized, little is known about the molecular mechanisms of CXCR4 mediated modulation of cardiac hypertrophy and failure. We used gene therapy to test the effects of CXCR4 gene delivery on adverse ventricular remodeling due to pressure overload. We assessed the effect of cardiac overexpression of CXCR4 during trans-aortic constriction (TAC) using a cardiotropic adeno-associated viral vector (AAV9) carrying the CXCR4 gene. Cardiac overexpression of CXCR4 in mice with pressure overload prevented ventricular remodeling, preserved capillary density and maintained function as determined by echocardiography and in vivo hemodynamics. In isolated adult rat cardiac myocytes, CXCL12 treatment prevented isoproterenol induced hypertrophy and interrupted the calcineurin/NFAT pathway. Finally, a complex involving the L-type calcium channel, β2-adenoreceptor, and CXCR4 (Cav1.2/β2AR/CXCR4) was identified in healthy cardiac myocytes and was shown to dissociate as a consequence of heart failure. CXCR4 administered to the heart via gene transfer prevents pressure overload induced heart failure. The identification of CXCR4 participation in a Cav1.2-β2AR regulatory complex provides further insight into the mechanism by which CXCR4 modulates calcium homeostasis and chronic pressure overload responses in the cardiac myocyte. Together these results suggest AAV9.CXCR4 gene therapy is a potential therapeutic approach for congestive heart failure. PMID:22668785

  8. Variation in salivary and pancreatic alpha-amylase genes in Italian horse breeds.

    PubMed

    Coizet, Beatrice; Nicoloso, Letizia; Marletta, Donata; Tamiozzo-Calligarich, Alessandra; Pagnacco, Giulio; Crepaldi, Paola

    2014-01-01

    The dietary demand of the modern horse relies on high-cereal feeding and limited forage compared with natural grazing conditions, predisposing the horse to several important diseases. Salivary and pancreatic alpha-amylases (coded by AMY1 and AMY2 genes, respectively) play a crucial role in carbohydrate digestion in nonruminants, but little is known about these 2 genes in the horse. Aim of this work has been to distinguish genomic sequences of horse AMY1 and AMY2 genes and to analyze any polymorphisms in breeds historically characterized by marked differences in nutritional management. A single nucleotide polymorphism detection was performed and 7 novel single nucleotide polymorphisms were found. Three single nucleotide polymorphisms are in exons and were genotyped in 112 horses belonging to 6 breeds. One single nucleotide polymorphism in AMY1 gene distinguished Haflinger and the Italian native Murgese from the other breeds, whereas both the single nucleotide polymorphisms in AMY2 gene showed different allelic frequencies in Friesian compared with the other breeds. These differences are confirmed by quite high fixation index (Fst) values for these 2 nonsynonymous single nucleotide polymorphisms. These preliminary results highlight marked divergences in allele frequencies of AMY1 and AMY2 genes, involved in starch digestion, between horse breeds characterized by different histories of selection, thus providing first indications of possible relations between genetics and nutritional management. PMID:24558100

  9. c-Ha-ras down regulates the alpha-fetoprotein gene but not the albumin gene in human hepatoma cells.

    PubMed Central

    Nakao, K; Lawless, D; Ohe, Y; Miyao, Y; Nakabayashi, H; Kamiya, H; Miura, K; Ohtsuka, E; Tamaoki, T

    1990-01-01

    We studied the effects of transfection of the normal c-Ha-ras gene, rasGly-12, and its oncogenic mutant, rasVal-12, on expression of the alpha-fetoprotein (AFP) and albumin genes in a human hepatoma cell line, HuH-7. The mutant and, to a lesser extent, the normal ras gene caused reduction of the AFP mRNA but not the albumin mRNA level in transfected HuH-7 cells. Cotransfection experiments with a rasVal-12 expression plasmid and a chloramphenicol acetyltransferase reporter gene fused to AFP regulatory sequences showed that rasVal-12 suppressed the activity of enhancer and promoter regions containing A + T-rich sequences (AT motif). In contrast, rasVal-12 did not affect the promoter activity of the albumin and human hepatitis B virus pre-S1 genes even though these promoters contain homologous A + T-rich elements. ras transfection appeared to induce phosphorylation of nuclear proteins that interact with the AFP AT motif, since gel mobility analysis revealed the formation of slow-moving complexes which was reversed by phosphatase treatment. However, similar changes in complex formation were observed with the albumin and hepatitis B surface antigen pre-S1 promoters. Therefore, this effect alone cannot explain the specific down regulation of the AFP promoter and enhancer activity. ras-mediated suppression of the AFP gene may reflect the process of developmental gene regulation in which AFP gene transcription is controlled by a G-protein-linked signal transduction cascade triggered by external growth stimuli. Images PMID:1690841

  10. Expression of modified gene encoding functional human alpha-1-antitrypsin protein in transgenic tomato plants.

    PubMed

    Agarwal, Saurabh; Singh, Rahul; Sanyal, Indraneel; Amla, D V

    2008-10-01

    Transgenic plants offer promising alternative for large scale, sustainable production of safe, functional, recombinant proteins of therapeutic and industrial importance. Here, we report the expression of biologically active human alpha-1-antitrypsin in transgenic tomato plants. The 1,182 bp cDNA sequence of human AAT was strategically designed, modified and synthesized to adopt codon usage pattern of dicot plants, elimination of mRNA destabilizing sequences and modifications around 5' and 3' flanking regions of the gene to achieve high-level regulated expression in dicot plants. The native signal peptide sequence was substituted with modified signal peptide sequence of tobacco (Nicotiana tabacum) pathogenesis related protein PR1a, sweet potato (Ipomoea batatas) sporamineA and with dicot-preferred native signal peptide sequence of AAT gene. A dicot preferred translation initiation context sequence, 38 bp alfalfa mosaic virus untranslated region were incorporated at 5' while an endoplasmic reticulum retention signal (KDEL) was incorporated at 3' end of the gene. The modified gene was synthesized by PCR based method using overlapping oligonucleotides. Tomato plants were genetically engineered by nuclear transformation with Agrobacterium tumefaciens harbouring three different constructs pPAK, pSAK and pNAK having modified AAT gene with different signal peptide sequences under the control of CaMV35S duplicated enhancer promoter. Promising transgenic plants expressing recombinant AAT protein upto 1.55% of total soluble leaf protein has been developed and characterized. Plant-expressed recombinant AAT protein with molecular mass of around approximately 50 kDa was biologically active, showing high specific activity and efficient inhibition of elastase activity. The enzymatic deglycosylation established proper glycosylation of the plant-expressed recombinant AAT protein in contrast to unglycosylated rAAT expressed in E. coli ( approximately 45 kDa). Our results demonstrate

  11. Resistance Gene Transfer during Treatments for Experimental Avian Colibacillosis

    PubMed Central

    Dheilly, Alexandra; Le Devendec, Laëtitia; Mourand, Gwenaëlle; Bouder, Axelle; Jouy, Eric

    2012-01-01

    An experiment was conducted in animal facilities to compare the impacts of four avian colibacillosis treatments—oxytetracycline (OTC), trimethoprim-sulfadimethoxine (SXT), amoxicillin (AMX), or enrofloxacin (ENR)—on the susceptibility of Escherichia coli in broiler intestinal tracts. Birds were first orally inoculated with rifampin-resistant E. coli strains bearing plasmid genes conferring resistance to fluoroquinolones (qnr), cephalosporins (blaCTX-M or blaFOX), trimethoprim-sulfonamides, aminoglycosides, or tetracyclines. Feces samples were collected before, during, and after antimicrobial treatments. The susceptibilities of E. coli strains were studied, and resistance gene transfer was analyzed. An increase in the tetracycline-resistant E. coli population was observed only in OTC-treated birds, whereas multiresistant E. coli was detected in the dominant E. coli populations of SXT-, AMX-, or ENR-treated birds. Most multiresistant E. coli strains were susceptible to rifampin and exhibited various pulsed-field gel electrophoresis profiles, suggesting the transfer of one of the multiresistance plasmids from the inoculated strains to other E. coli strains in the intestinal tract. In conclusion, this study clearly illustrates how, in E. coli, “old” antimicrobials may coselect antimicrobial resistance to recent and critical molecules. PMID:21986830

  12. Lateral gene transfers have polished animal genomes: lessons from nematodes

    PubMed Central

    Danchin, Etienne G. J.; Rosso, Marie-Noëlle

    2012-01-01

    It is now accepted that lateral gene transfers (LGT), have significantly contributed to the composition of bacterial genomes. The amplitude of the phenomenon is considered so high in prokaryotes that it challenges the traditional view of a binary hierarchical tree of life to correctly represent the evolutionary history of species. Given the plethora of transfers between prokaryotes, it is currently impossible to infer the last common ancestral gene set for any extant species. For this ensemble of reasons, it has been proposed that the Darwinian binary tree of life may be inappropriate to correctly reflect the actual relations between species, at least in prokaryotes. In contrast, the contribution of LGT to the composition of animal genomes is less documented. In the light of recent analyses that reported series of LGT events in nematodes, we discuss the importance of this phenomenon in the evolutionary history and in the current composition of an animal genome. Far from being neutral, it appears that besides having contributed to nematode genome contents, LGT have favored the emergence of important traits such as plant-parasitism. PMID:22919619

  13. Synthetic Fatty Acids Prevent Plasmid-Mediated Horizontal Gene Transfer

    PubMed Central

    Getino, María; Sanabria-Ríos, David J.; Fernández-López, Raúl; Campos-Gómez, Javier; Sánchez-López, José M.; Fernández, Antonio; Carballeira, Néstor M.

    2015-01-01

    ABSTRACT Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic resistance genes among human pathogens. Antibiotic resistance spread could be halted or diminished by molecules that interfere with the conjugation process. In this work, synthetic 2-alkynoic fatty acids were identified as a novel class of conjugation inhibitors. Their chemical properties were investigated by using the prototype 2-hexadecynoic acid and its derivatives. Essential features of effective inhibitors were the carboxylic group, an optimal long aliphatic chain of 16 carbon atoms, and one unsaturation. Chemical modification of these groups led to inactive or less-active derivatives. Conjugation inhibitors were found to act on the donor cell, affecting a wide number of pathogenic bacterial hosts, including Escherichia, Salmonella, Pseudomonas, and Acinetobacter spp. Conjugation inhibitors were active in inhibiting transfer of IncF, IncW, and IncH plasmids, moderately active against IncI, IncL/M, and IncX plasmids, and inactive against IncP and IncN plasmids. Importantly, the use of 2-hexadecynoic acid avoided the spread of a derepressed IncF plasmid into a recipient population, demonstrating the feasibility of abolishing the dissemination of antimicrobial resistances by blocking bacterial conjugation. PMID:26330514

  14. Molecular cloning and expression of two alpha-amylase genes from Streptococcus bovis 148 in Escherichia coli.

    PubMed Central

    Satoh, E; Niimura, Y; Uchimura, T; Kozaki, M; Komagata, K

    1993-01-01

    The alpha-amylase genes of Streptococcus bovis 148 were cloned in Escherichia coli MC1061, using pBR322. The recombinant plasmids were classified into two groups on the basis of their restriction maps. Southern blot analysis did not show homology between the two types of alpha-amylase genes, and the two alpha-amylase genes existed on the chromosomal DNA of S. bovis 148. The enzymatic properties and N-terminal amino acid sequences of the two purified enzymes produced by the cloned E. coli strains were quite different from each other. Particularly, one alpha-amylase (Amy I) was adsorbed on raw corn starch and hydrolyzed raw corn starch, and another (Amy II) was not adsorbed on raw corn starch and did not hydrolyze raw corn starch. Amy I was considered to be the same as the extracellular alpha-amylase of S. bovis 148 in raw starch absorbability, ability to hydrolyze raw corn starch, enzymatic characteristics, N-terminal amino acid sequence, and mode of action on soluble starch. Amy II showed a unique pattern of oligosaccharide production from soluble starch compared with the extracellular alpha-amylase of S. bovis 148. Amy II was suggested to be an intracellular alpha-amylase of S. bovis 148. Images PMID:8285674

  15. Molecular cloning and expression of two alpha-amylase genes from Streptococcus bovis 148 in Escherichia coli.

    PubMed

    Satoh, E; Niimura, Y; Uchimura, T; Kozaki, M; Komagata, K

    1993-11-01

    The alpha-amylase genes of Streptococcus bovis 148 were cloned in Escherichia coli MC1061, using pBR322. The recombinant plasmids were classified into two groups on the basis of their restriction maps. Southern blot analysis did not show homology between the two types of alpha-amylase genes, and the two alpha-amylase genes existed on the chromosomal DNA of S. bovis 148. The enzymatic properties and N-terminal amino acid sequences of the two purified enzymes produced by the cloned E. coli strains were quite different from each other. Particularly, one alpha-amylase (Amy I) was adsorbed on raw corn starch and hydrolyzed raw corn starch, and another (Amy II) was not adsorbed on raw corn starch and did not hydrolyze raw corn starch. Amy I was considered to be the same as the extracellular alpha-amylase of S. bovis 148 in raw starch absorbability, ability to hydrolyze raw corn starch, enzymatic characteristics, N-terminal amino acid sequence, and mode of action on soluble starch. Amy II showed a unique pattern of oligosaccharide production from soluble starch compared with the extracellular alpha-amylase of S. bovis 148. Amy II was suggested to be an intracellular alpha-amylase of S. bovis 148. PMID:8285674

  16. Estrogen Receptor beta binds Sp1 and recruits a Corepressor Complex to the Estrogen Receptor alpha Gene Promoter

    PubMed Central

    Bartella, V; Rizza, P; Barone, I; Zito, D; Giordano, F; Giordano, C; Catalano, S; Mauro, L; Sisci, D; Panno, ML; Fuqua, SA; Andò, Sebastiano

    2015-01-01

    Human estrogen receptors (ERs) alpha and beta are crucially involved in the regulation of mammary growth and development. Normal breast tissues display a prevalently expression of ER beta than ER alpha, which drastically increases during breast tumorogenesis. So, it is reasonable to assume how a dysregulation of the two estrogen receptor subtypes may induce breast cancer development. However, the molecular mechanism underlying the opposite role played by the two estrogen receptors on tumor cell growth remains to be elucidated. In the present study, we have demonstrated that ER beta overexpression in breast cancer cells decreases cell proliferation and down-regulates ER alpha mRNA and protein content along with a concomitant repression of estrogen-regulated genes. Transient transfection experiments, using a vector containing the human ER alpha promoter region, showed that elevated levels of the ER beta down-regulated basal ER alpha promoter activity. Furthermore, side-directed mutagenesis and deletion analysis have revealed that the proximal GC-rich motifs at −223 and −214 is crucial for the ER beta-induced ER alpha down-regulation in breast cancer cells. This occurred through ER beta-Sp1 protein-protein interaction within the ER alpha promoter region and the recruitment of a corepressor complex containing NCoR/SMRT (nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid hormone receptor), accompanied by hypoacetylation of histone H4 and displacement of RNA polymerase II. Silencing of NCoR gene expression by RNA interference reversed the down-regulatory effect of ER beta on ER alpha gene expression and cell proliferation. Our results provide evidence for a novel mechanism by which overexpression of ER beta through NCoR is able to down regulate ER alpha gene expression, thus inhibiting ER alpha’s driving role on breast cancer cell growth. PMID:22622808

  17. 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

  18. Genome-wide experimental determination of barriers to horizontal gene transfer.

    PubMed

    Sorek, Rotem; Zhu, Yiwen; Creevey, Christopher J; Francino, M Pilar; Bork, Peer; Rubin, Edward M

    2007-11-30

    Horizontal gene transfer, in which genetic material is transferred from the genome of one organism to that of another, has been investigated in microbial species mainly through computational sequence analyses. To address the lack of experimental data, we studied the attempted movement of 246,045 genes from 79 prokaryotic genomes into Escherichia coli and identified genes that consistently fail to transfer. We studied the mechanisms underlying transfer inhibition by placing coding regions from different species under the control of inducible promoters. Our data suggest that toxicity to the host inhibited transfer regardless of the species of origin and that increased gene dosage and associated increased expression may be a predominant cause for transfer failure. Although these experimental studies examined transfer solely into E. coli, a computational analysis of gene-transfer rates across available bacterial and archaeal genomes supports that the barriers observed in our study are general across the tree of life. PMID:17947550

  19. Genome-wide experimental determination of barriers to horizontal gene transfer

    SciTech Connect

    Rubin, Edward; Sorek, Rotem; Zhu, Yiwen; Creevey, Christopher J.; Francino, M. Pilar; Bork, Peer; Rubin, Edward M.

    2007-09-24

    Horizontal gene transfer, in which genetic material is transferred from the genome of one organism to another, has been investigated in microbial species mainly through computational sequence analyses. To address the lack of experimental data, we studied the attempted movement of 246,045 genes from 79 prokaryotic genomes into E. coli and identified genes that consistently fail to transfer. We studied the mechanisms underlying transfer inhibition by placing coding regions from different species under the control of inducible promoters. Their toxicity to the host inhibited transfer regardless of the species of origin and our data suggest that increased gene dosage and associated increased expression is a predominant cause for transfer failure. While these experimental studies examined transfer solely into E. coli, a computational analysis of gene transfer rates across available bacterial and archaeal genomes indicates that the barriers observed in our study are general across the tree of life.

  20. Particle size and interfacial effects on heat transfer characteristics of water and {alpha}-SiC nanofluids.

    SciTech Connect

    Timofeeva, E.; Smith, D. S.; Yu, W.; France, D. M.; Singh, D.; Routbort, J. L.

    2010-01-01

    The effect of average particle sizes on basic macroscopic properties and heat transfer performance of {alpha}-SiC/water nanofluids was investigated. The average particle sizes, calculated from the specific surface area of nanoparticles, were varied from 16 to 90 nm. Nanofluids with larger particles of the same material and volume concentration provide higher thermal conductivity and lower viscosity increases than those with smaller particles because of the smaller solid/liquid interfacial area of larger particles. It was also demonstrated that the viscosity of water-based nanofluids can be significantly decreased by pH of the suspension independently from the thermal conductivity. Heat transfer coefficients were measured and compared to the performance of base fluids as well as to nanofluids reported in the literature. Criteria for evaluation of the heat transfer performance of nanofluids are discussed and optimum directions in nanofluid development are suggested.

  1. The palladium assisted transfer reduction of. alpha. ,. beta. -unsaturated nitroalkenes to oximes using ammonium formate

    SciTech Connect

    Kabalka, G.W.; Pace, R.D.; Wadgaonkar, P.P. )

    1990-01-01

    {alpha},{beta}-Unsaturated nitroalkenes are readily reduced to the corresponding oximes in good yields using ammonium formate in the presence of palladium. The reactions occur rapidly at room temperature in a solvent system of methanol and tetrahydrofuran.

  2. [Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

    PubMed

    Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki

    2007-01-01

    Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity. PMID:18154441

  3. Analysis and expression of the alpha-expansin and beta-expansin gene families in maize

    NASA Technical Reports Server (NTRS)

    Wu, Y.; Meeley, R. B.; Cosgrove, D. J.

    2001-01-01

    Expansins comprise a multigene family of proteins in maize (Zea mays). We isolated and characterized 13 different maize expansin cDNAs, five of which are alpha-expansins and eight of which are beta-expansins. This paper presents an analysis of these 13 expansins, as well as an expression analysis by northern blotting with materials from young and mature maize plants. Some expansins were expressed in restricted regions, such as the beta-expansins ExpB1 (specifically expressed in maize pollen) and ExpB4 (expressed principally in young husks). Other expansins such as alpha-expansin Exp1 and beta-expansin ExpB2 were expressed in several organs. The expression of yet a third group was not detected in the selected organs and tissues. An analysis of expansin sequences from the maize expressed sequence tag collection is also presented. Our results indicate that expansin genes may have general, overlapping expression in some instances, whereas in other cases the expression may be highly specific and limited to a single organ or cell type. In contrast to the situation in Arabidopsis, beta-expansins in maize seem to be more numerous and more highly expressed than are alpha-expansins. The results support the concept that beta-expansins multiplied and evolved special functions in the grasses.

  4. 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

  5. Long-chain fatty acids regulate liver carnitine palmitoyltransferase I gene (L-CPT I) expression through a peroxisome-proliferator-activated receptor alpha (PPARalpha)-independent pathway.

    PubMed Central

    Louet, J F; Chatelain, F; Decaux, J F; Park, E A; Kohl, C; Pineau, T; Girard, J; Pegorier, J P

    2001-01-01

    Liver carnitine palmitoyltransferase I (L-CPT I) catalyses the transfer of long-chain fatty acid (LCFA) for translocation across the mitochondrial membrane. Expression of the L-CPT I gene is induced by LCFAs as well as by lipid-lowering compounds such as clofibrate. Previous studies have suggested that the peroxisome-proliferator-activated receptor alpha (PPARalpha) is a common mediator of the transcriptional effects of LCFA and clofibrate. We found that free LCFAs rather than acyl-CoA esters are the signal metabolites responsible for the stimulation of L-CPT I gene expression. Using primary culture of hepatocytes we found that LCFAs failed to stimulate L-CPT I gene expression both in wild-type and PPARalpha-null mice. These results suggest that the PPARalpha-knockout mouse does not represent a suitable model for the regulation of L-CPT I gene expression by LCFAs in the liver. Finally, we determined that clofibrate stimulates L-CPT I through a classical direct repeat 1 (DR1) motif in the promoter of the L-CPT I gene while LCFAs induce L-CPT I via elements in the first intron of the gene. Our results demonstrate that LCFAs can regulate gene expression through PPARalpha-independent pathways and suggest that the regulation of gene expression by dietary lipids is more complex than previously proposed. PMID:11171094

  6. Perilipin, a critical regulator of fat storage and breakdown, is a target gene of estrogen receptor-related receptor {alpha}

    SciTech Connect

    Akter, Mst. Hasina; Yamaguchi, Tomohiro; Hirose, Fumiko; Osumi, Takashi

    2008-04-11

    Perilipin is a protein localized on lipid droplet surfaces in adipocytes and steroidogenic cells, playing a central role in regulated lipolysis. Expression of the perilipin gene is markedly induced during adipogenesis. We found that transcription from the perilipin gene promoter is activated by an orphan nuclear receptor, estrogen receptor-related receptor (ERR){alpha}. A response element to this receptor was identified in the promoter region by a gene reporter assay, the electrophoretic-gel mobility-shift assay and the chromatin immunoprecipitation assay. Peroxisome proliferator-activated receptor {gamma} coactivator (PGC)-1{alpha} enhanced, whereas small heterodimer partner (SHP) repressed, the transactivating function of ERR{alpha} on the promoter. Thus, the perilipin gene expression is regulated by a transcriptional network controlling energy metabolism, substantiating the functional importance of perilipin in the maintenance of body energy balance.

  7. Repeated, recent and diverse transfers of a mitochondrial gene to the nucleus in flowering plants.

    PubMed

    Adams, K L; Daley, D O; Qiu, Y L; Whelan, J; Palmer, J D

    2000-11-16

    A central component of the endosymbiotic theory for the bacterial origin of the mitochondrion is that many of its genes were transferred to the nucleus. Most of this transfer occurred early in mitochondrial evolution; functional transfer of mitochondrial genes has ceased in animals. Although mitochondrial gene transfer continues to occur in plants, no comprehensive study of the frequency and timing of transfers during plant evolution has been conducted. Here we report frequent loss (26 times) and transfer to the nucleus of the mitochondrial gene rps10 among 277 diverse angiosperms. Characterization of nuclear rps10 genes from 16 out of 26 loss lineages implies that many independent, RNA-mediated rps10 transfers occurred during recent angiosperm evolution; each of the genes may represent a separate functional gene transfer. Thus, rps10 has been transferred to the nucleus at a surprisingly high rate during angiosperm evolution. The structures of several nuclear rps10 genes reveal diverse mechanisms by which transferred genes become activated, including parasitism of pre-existing nuclear genes for mitochondrial or cytoplasmic proteins, and activation without gain of a mitochondrial targeting sequence. PMID:11099041

  8. Tumor necrosis factor alpha -308 gene locus promoter polymorphism: an analysis of association with health and disease.

    PubMed

    Elahi, Maqsood M; Asotra, Kamlesh; Matata, Bashir M; Mastana, Sarabjit S

    2009-03-01

    Tumor necrosis factor-alpha (TNF-alpha) is a potent immunomediator and proinflammatory cytokine that has been implicated in the pathogenesis of a large number of human diseases. The location of its gene within major histocompatibility complex and biological activities has raised the possibility that polymorphisms within this locus may contribute to the pathogenesis of wide range of autoimmune and infectious diseases. For example, a bi-allelic single nucleotide substitution of G (TNFA1 allele) with A (TNFA2 allele) polymorphism at -308 nucleotides upstream from the transcription initiation site in the TNF-alpha promoter is associated with elevated TNF-alpha levels and disease susceptibilities. However, it is still unclear whether TNF-alpha -308 polymorphism plays a part in the disease process, in particular whether it could affect transcription factor binding and in turn influence TNF-alpha transcription and synthesis. Several studies have suggested that TNFA2 allele is significantly linked with the high TNF-alpha-producing autoimmune MHC haplotype HLA-A1, B8, DR3, with elevated serum TNF-alpha levels and a more severe outcome in diseases. This review discusses the genetics of the TNF-alpha -308 polymorphism in selected major diseases and evaluates its common role in health and disease. PMID:19708125

  9. Double gene deletion reveals the lack of cooperation between PPAR{alpha} and PPAR{beta} in skeletal muscle

    SciTech Connect

    Bedu, E.; Desplanches, D.; Pequignot, J.; Bordier, B.; Desvergne, B. . E-mail: beatrice.desvergne@unil.ch

    2007-06-15

    The peroxisome proliferator-activated receptors (PPARs) are involved in the regulation of most of the pathways linked to lipid metabolism. PPAR{alpha} and PPAR{beta} isotypes are known to regulate muscle fatty acid oxidation and a reciprocal compensation of their function has been proposed. Herein, we investigated muscle contractile and metabolic phenotypes in PPAR{alpha}-/-, PPAR{beta}-/-, and double PPAR{alpha}-/- {beta}-/- mice. Heart and soleus muscle analyses show that the deletion of PPAR{alpha} induces a decrease of the HAD activity ({beta}-oxidation) while soleus contractile phenotype remains unchanged. A PPAR{beta} deletion alone has no effect. However, these mild phenotypes are not due to a reciprocal compensation of PPAR{beta} and PPAR{alpha} functions since double gene deletion PPAR{alpha}-PPAR{beta} mostly reproduces the null PPAR{alpha}-mediated reduced {beta}-oxidation, in addition to a shift from fast to slow fibers. In conclusion, PPAR{beta} is not required for maintaining skeletal muscle metabolic activity and does not compensate the lack of PPAR{alpha} in PPAR{alpha} null mice.

  10. Molecular study of the 5 {alpha}-reductase type 2 gene in three European families with 5 {alpha}-reductase deficiency

    SciTech Connect

    Boudon, C.; Lumbroso, S.; Lobaccaro, J.M.

    1995-07-01

    The molecular basis of 5{alpha}-reductase (5{alpha}R) deficiency was investigated in four patients from three European families. In the French family, the first patient was raised as a female, and gonadectomy was performed before puberty. The second sibling, also raised as female, differed in that gonadal removal was performed after the onset of pubertal masculinization. The other two patients, both from Polish families, developed masculinization of external genitalia during puberty. All patients developed a female sexual identity. In all cases, no known consanguinity or family history of 5{alpha}R deficiency was reported. The genomic DNAs of the patients were sequenced after polymerase chain reaction amplification of the five exons of the 5{alpha}R type 2 gene. We found two homozygous mutations responsible for gutamine to arginine and histidine to arginine substitution in families 1 and 3, respectively. In family 2, we found a heterozygous mutation responsible for an asparagine to serine substitution at position 193. The glutamine/arginine 126 mutation in the French family was previously reported in a Creole ethnic group, and the Polish histidine/arginine 231 mutation was previously reported in a patient from Chicago, Moreover, all of the mutations created new restriction sites, which were used to determine the kindred carrier status in the three families. Because 5{alpha}R deficiency is known to be heterogenous disease in terms of clinical and biochemical expression, our data suggest that molecular biology analysis of the type 2 gene could be an essential step in diagnosing 5{alpha}R deficiency. 22 refs., 3 figs., 1 tab.

  11. Adaptive Horizontal Gene Transfers between Multiple Cheese-Associated Fungi

    PubMed Central

    Ropars, Jeanne; Rodríguez de la Vega, Ricardo C.; López-Villavicencio, Manuela; Gouzy, Jérôme; Sallet, Erika; Dumas, Émilie; Lacoste, Sandrine; Debuchy, Robert; Dupont, Joëlle; Branca, Antoine; Giraud, Tatiana

    2015-01-01

    Summary Domestication is an excellent model for studies of adaptation because it involves recent and strong selection on a few, identified traits [1–5]. Few studies have focused on the domestication of fungi, with notable exceptions [6–11], despite their importance to bioindustry [12] and to a general understanding of adaptation in eukaryotes [5]. Penicillium fungi are ubiquitous molds among which two distantly related species have been independently selected for cheese making—P. roqueforti for blue cheeses like Roquefort and P. camemberti for soft cheeses like Camembert. The selected traits include morphology, aromatic profile, lipolytic and proteolytic activities, and ability to grow at low temperatures, in a matrix containing bacterial and fungal competitors [13–15]. By comparing the genomes of ten Penicillium species, we show that adaptation to cheese was associated with multiple recent horizontal transfers of large genomic regions carrying crucial metabolic genes. We identified seven horizontally transferred regions (HTRs) spanning more than 10 kb each, flanked by specific transposable elements, and displaying nearly 100% identity between distant Penicillium species. Two HTRs carried genes with functions involved in the utilization of cheese nutrients or competition and were found nearly identical in multiple strains and species of cheese-associated Penicillium fungi, indicating recent selective sweeps; they were experimentally associated with faster growth and greater competitiveness on cheese and contained genes highly expressed in the early stage of cheese maturation. These findings have industrial and food safety implications and improve our understanding of the processes of adaptation to rapid environmental changes. PMID:26412136

  12. PU.1 (Spi-1) and C/EBP alpha regulate expression of the granulocyte-macrophage colony-stimulating factor receptor alpha gene.

    PubMed Central

    Hohaus, S; Petrovick, M S; Voso, M T; Sun, Z; Zhang, D E; Tenen, D G

    1995-01-01

    Growth factor receptors play an important role in hematopoiesis. In order to further understand the mechanisms directing the expression of these key regulators of hematopoiesis, we initiated a study investigating the transcription factors activating the expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha gene. Here, we demonstrate that the human GM-CSF receptor alpha promoter directs reporter gene activity in a tissue-specific fashion in myelomonocytic cells, which correlates with its expression pattern as analyzed by reverse transcription PCR. The GM-CSF receptor alpha promoter contains an important functional site between positions -53 and -41 as identified by deletion analysis of reporter constructs. We show that the myeloid and B cell transcription factor PU.1 binds specifically to this site. Furthermore, we demonstrate that a CCAAT site located upstream of the PU.1 site between positions -70 and -54 is involved in positive-negative regulation of the GM-CSF receptor alpha promoter activity. C/EBP alpha is the major CCAAT/enhancer-binding protein (C/EBP) form binding to this site in nuclear extracts of U937 cells. Point mutations of either the PU.1 site or the C/EBP site that abolish the binding of the respective factors result in a significant decrease of GM-CSF receptor alpha promoter activity in myelomonocytic cells only. Furthermore, we demonstrate that in myeloid and B cell extracts, PU.1 forms a novel, specific, more slowly migrating complex (PU-SF) when binding the GM-CSF receptor alpha promoter PU.1 site. This is the first demonstration of a specific interaction with PU.1 on a myeloid PU.1 binding site. The novel complex is distinct from that described previously as binding to B cell enhancer sites and can be formed by addition of PU.1 to extracts from certain nonmyeloid cell types which do not express PU.1, including T cells and epithelial cells, but not from erythroid cells. Furthermore, we demonstrate that the PU

  13. Alpha-fetoprotein-targeted reporter gene expression imaging in hepatocellular carcinoma.

    PubMed

    Kim, Kwang Il; Chung, Hye Kyung; Park, Ju Hui; Lee, Yong Jin; Kang, Joo Hyun

    2016-07-21

    Hepatocellular carcinoma (HCC) is one of the most common cancers in Eastern Asia, and its incidence is increasing globally. Numerous experimental models have been developed to better our understanding of the pathogenic mechanism of HCC and to evaluate novel therapeutic approaches. Molecular imaging is a convenient and up-to-date biomedical tool that enables the visualization, characterization and quantification of biologic processes in a living subject. Molecular imaging based on reporter gene expression, in particular, can elucidate tumor-specific events or processes by acquiring images of a reporter gene's expression driven by tumor-specific enhancers/promoters. In this review, we discuss the advantages and disadvantages of various experimental HCC mouse models and we present in vivo images of tumor-specific reporter gene expression driven by an alpha-fetoprotein (AFP) enhancer/promoter system in a mouse model of HCC. The current mouse models of HCC development are established by xenograft, carcinogen induction and genetic engineering, representing the spectrum of tumor-inducing factors and tumor locations. The imaging analysis approach of reporter genes driven by AFP enhancer/promoter is presented for these different HCC mouse models. Such molecular imaging can provide longitudinal information about carcinogenesis and tumor progression. We expect that clinical application of AFP-targeted reporter gene expression imaging systems will be useful for the detection of AFP-expressing HCC tumors and screening of increased/decreased AFP levels due to disease or drug treatment. PMID:27468205

  14. Differential Effects of Alpha-Particle Radiation and X-Irradiation on Genes Associated with Apoptosis

    PubMed Central

    Chauhan, Vinita; Howland, Matthew; Chen, Jeremy; Kutzner, Barbara; Wilkins, Ruth C.

    2011-01-01

    This study examined differential effects of alpha-(α-) particle radiation and X-rays on apoptosis and associated changes in gene expression. Human monocytic cells were exposed to α-particle radiation and X-rays from 0 to 1.5 Gy. Four days postexposure, cell death was measured by flow cytometry and 84 genes related to apoptosis were analyzed using real-time PCR. On average, 33% of the cells were apoptotic at 1.5 Gy of α-particle radiation. Transcript profiling showed statistical expression of 15 genes at all three doses tested. Cells exposed to X-rays were <5% apoptotic at ~1.5 Gy and induced less than a 2-fold expression in 6 apoptotic genes at the higher doses of radiation. Among these 6 genes, Fas and TNF-α were common to the α-irradiated cells. This data suggests that α-particle radiation initiates cell death by TNF-α and Fas activation and through intermediate signalling mediators that are distinct from X-irradiated cells. PMID:22091383

  15. Preferential expression of an alpha-tubulin gene of Arabidopsis in pollen.

    PubMed Central

    Carpenter, J L; Ploense, S E; Snustad, D P; Silflow, C D

    1992-01-01

    The pool of tubulin protein in tissues of Arabidopsis is provided by the expression of multiple alpha-tubulin (TUA) and beta-tubulin genes. Whereas most tubulin genes are expressed in many tissues, previous evidence suggested that the TUA1 gene might be expressed primarily in pollen. We now report a detailed analysis of TUA1 expression during Arabidopsis development. In RNA from tissues of dissected flowers, TUA1 transcripts were detected only in stamens and mature pollen. Chimeric genes containing TUA1 5' flanking DNA fused to the beta-glucuronidase (GUS) coding region were used to create transgenic Arabidopsis plants. Plants containing a chimeric gene with 533 bp of 5' flanking sequence were analyzed by histochemical assay to localize GUS expression within the plant. The blue product of GUS enzyme activity accumulated very rapidly in postmitotic pollen grains. Much lower levels of GUS activity were detected in anthers with uninucleate pollen grains, in flower receptacles, and in a few vegetative tissues. Analysis of 5' deletions of the TUA1 promoter suggested that 97 bp of 5' flanking DNA is sufficient to drive GUS expression in pollen and young anthers, whereas at least 380 bp is required to detect GUS expression in the receptacle. Examination of the TUA1 promoter sequence revealed several motifs that are repeated within the TUA1 promoter and are similar to sequences in other pollen-specific promoters. PMID:1498610

  16. Asymmetrical transfer of alpha-aminoisobutyric acid (AIB), leucine and lysine across the in vitro perfused human placenta.

    PubMed

    Schneider, H; Proegler, M; Sodha, R; Dancis, J

    1987-01-01

    The mechanism for establishing transplacental gradients for leucine, lysine and alpha-aminoisobutyric acid (AIB) has been investigated in the perfused human placenta. Experiments were done with either the maternal or the fetal circulation closed and the donor circulation open. Transfer of the amino acids towards the fetal side was more rapid than it was in the reverse direction. When the maternal perfusate was recirculated, the amino acid concentrations were maintained at a considerably lower level in the maternal circulation than in the open fetal circuit. When the fetal circuit was closed, the concentrations approached or slightly exceeded those in the maternal perfusate over a period of three hours. Within the placenta, higher concentrations were established during the experiments with transfer towards the fetal side than in the reverse direction. Of the three amino acids, leucine was transferred most rapidly across the placenta while AIB reached the highest concentrations in the placental tissue. The asymmetry of the transplacental amino acid flux is favoured by rapid uptake from the maternal circulation and transfer towards the fetus. Both rates exceed those observed in the reverse direction. The transfer rate of D-leucine was 1.7 times that of L-glucose. For in vitro studies of the transfer rate of physiological compounds a correction for diffusion is required. The results may differ considerably depending on which marker is used as the basis. PMID:3112761

  17. Aleurone nuclear proteins bind to similar elements in the promoter regions of two gibberellin-regulated alpha-amylase genes.

    PubMed

    Rushton, P J; Hooley, R; Lazarus, C M

    1992-09-01

    Binding of nuclear proteins from wild oat aleurone protoplasts to the promoter regions of two gibberellin-regulated wheat alpha-amylase genes (alpha-Amy1/18 and alpha-Amy2/54) has been studied by gel retardation and DNase 1 footprinting. Gel retardation studies using 300-430 bp fragments of the promoters showed similar binding characteristics with nuclear extracts from both gibberellin A1-treated and untreated protoplasts. DNase 1 footprints localised binding of nuclear proteins from gibberellin A1-treated aleurone protoplasts to regions in both promoters. Similar sequence elements in the promoter regions of both genes were protected from digestion although the location and number of footprints in each promoter region were different. Each footprint contained either a sequence similar to the cAMP and/or phorbol ester response elements, or a hyphenated palindrome sequence. The presence of cAMP and/or phorbol ester response element-like sequences in the footprints suggests that transcription factors of the bZIP type may be involved in the expression of alpha-amylase genes in aleurone cells. Footprints containing hyphenated palindrome sequences, found in the promoter regions of both genes, suggest the possible involvement of other classes of transcription factor. The conserved alpha-amylase promoter sequence TAA-CAGA was also shown to bind nuclear protein in the alpha-Amy2/54 promoter. These observations are discussed in relation to alpha-amylase gene expression in aleurone and to functional data concerning these genes. PMID:1511135

  18. Extensive Intra-Kingdom Horizontal Gene Transfer Converging on a Fungal Fructose Transporter Gene

    PubMed Central

    Coelho, Marco A.; Gonçalves, Carla; Sampaio, José Paulo; Gonçalves, Paula

    2013-01-01

    Comparative genomics revealed in the last decade a scenario of rampant horizontal gene transfer (HGT) among prokaryotes, but for fungi a clearly dominant pattern of vertical inheritance still stands, punctuated however by an increasing number of exceptions. In the present work, we studied the phylogenetic distribution and pattern of inheritance of a fungal gene encoding a fructose transporter (FSY1) with unique substrate selectivity. 109 FSY1 homologues were identified in two sub-phyla of the Ascomycota, in a survey that included 241 available fungal genomes. At least 10 independent inter-species instances of horizontal gene transfer (HGT) involving FSY1 were identified, supported by strong phylogenetic evidence and synteny analyses. The acquisition of FSY1 through HGT was sometimes suggestive of xenolog gene displacement, but several cases of pseudoparalogy were also uncovered. Moreover, evidence was found for successive HGT events, possibly including those responsible for transmission of the gene among yeast lineages. These occurrences do not seem to be driven by functional diversification of the Fsy1 proteins because Fsy1 homologues from widely distant lineages, including at least one acquired by HGT, appear to have similar biochemical properties. In summary, retracing the evolutionary path of the FSY1 gene brought to light an unparalleled number of independent HGT events involving a single fungal gene. We propose that the turbulent evolutionary history of the gene may be linked to the unique biochemical properties of the encoded transporter, whose predictable effect on fitness may be highly variable. In general, our results support the most recent views suggesting that inter-species HGT may have contributed much more substantially to shape fungal genomes than heretofore assumed. PMID:23818872

  19. Nemaline myopathy caused by mutations in the muscle alpha-skeletal-actin gene.

    PubMed

    Ilkovski, B; Cooper, S T; Nowak, K; Ryan, M M; Yang, N; Schnell, C; Durling, H J; Roddick, L G; Wilkinson, I; Kornberg, A J; Collins, K J; Wallace, G; Gunning, P; Hardeman, E C; Laing, N G; North, K N

    2001-06-01

    Nemaline myopathy (NM) is a clinically and genetically heterogeneous disorder characterized by muscle weakness and the presence of nemaline bodies (rods) in skeletal muscle. Disease-causing mutations have been reported in five genes, each encoding a protein component of the sarcomeric thin filament. Recently, we identified mutations in the muscle alpha-skeletal-actin gene (ACTA1) in a subset of patients with NM. In the present study, we evaluated a new series of 35 patients with NM. We identified five novel missense mutations in ACTA1, which suggested that mutations in muscle alpha-skeletal actin account for the disease in approximately 15% of patients with NM. The mutations appeared de novo and represent new dominant mutations. One proband subsequently had two affected children, a result consistent with autosomal dominant transmission. The seven patients exhibited marked clinical variability, ranging from severe congenital-onset weakness, with death from respiratory failure during the 1st year of life, to a mild childhood-onset myopathy, with survival into adulthood. There was marked variation in both age at onset and clinical severity in the three affected members of one family. Common pathological features included abnormal fiber type differentiation, glycogen accumulation, myofibrillar disruption, and "whorling" of actin thin filaments. The percentage of fibers with rods did not correlate with clinical severity; however, the severe, lethal phenotype was associated with both severe, generalized disorganization of sarcomeric structure and abnormal localization of sarcomeric actin. The marked variability, in clinical phenotype, among patients with different mutations in ACTA1 suggests that both the site of the mutation and the nature of the amino acid change have differential effects on thin-filament formation and protein-protein interactions. The intrafamilial variability suggests that alpha-actin genotype is not the sole determinant of phenotype. PMID:11333380

  20. Glucocorticoid regulation of branched-chain alpha-ketoacid dehydrogenase E2 subunit gene expression.

    PubMed Central

    Costeas, P A; Chinsky, J M

    2000-01-01

    Regulation of the mammalian branched-chain alpha-ketoacid dehydrogenase complex (BCKAD) occurs under a variety of stressful conditions associated with changes in circulating glucocorticoids. Multiple levels of regulation in hepatocytes, including alteration of the levels of the structural subunits available for assembly (E1, alpha-ketoacid decarboxylase; E2, dihydrolipoamide acyltransferase; and E3, dihydrolipoamide dehydrogenase), as well as BCKAD kinase, which serves to phosphorylate the E1alpha subunit and inactivate complex activity, have been proposed. The direct role of glucocorticoids in regulating the expression of the murine gene encoding the major BCKAD subunit E2, upon which the other BCKAD subunits assemble, was therefore examined. Deletion analysis of the 5' proximal 7.0 kb of the murine E2 promoter sequence, using E2 promoter/luciferase expression minigene plasmids introduced into the hepatic H4IIEC3 cell line, suggested a promoter proximal region responsive to glucocorticoid regulation. Linker-scanning mutagenesis combined with deletion analysis established this functional glucocorticoid-responsive unit (GRU) to be located near the murine E2 proximal promoter site at -140 to -70 bp upstream from the transcription initiation site. The presence of this region in plasmid minigenes, containing varying amounts of the murine genomic sequence 5' upstream from proximal E2 promoter sequences, conferred 2-10 fold increases in luciferase reporter gene expression in H4IIEC3 cells, whether introduced by transient transfection or following co-selection for stable transfectants. The GRU region itself appeared to contain multiple interacting elements that combine to regulate overall E2 promoter activity in response to changing physiological conditions associated with varying concentrations of glucocorticoids and likely other hormonal effectors. PMID:10749674

  1. Molecular analysis of a Clostridium butyricum NCIMB 7423 gene encoding 4-alpha-glucanotransferase and characterization of the recombinant enzyme produced in Escherichia coli.

    PubMed

    Goda, S K; Eissa, O; Akhtar, M; Minton, N P

    1997-10-01

    An Escherichia coli clone was detected in a Clostridium butyricum NCIMB 7423 plasmid library capable of degrading soluble amylose. Deletion subcloning of its recombinant plasmid indicated that the gene(s) responsible for amylose degradation was localized on a 1.8 kb NspHI-Scal fragment. This region was sequenced in its entirety and shown to encompass a large ORF capable of encoding a protein with a calculated molecular mass of 57,184 Da. Although the deduced amino acid sequence showed only weak similarity with known amylases, significant sequences identity was apparent with the 4-alpha-glucano-transferase enzymes of Streptococcus pneumoniae (46.9%), potato (42.9%) and E. coli (16.2%). The clostridial gene (designated maIQ) was followed by a second ORF which, through its homology to the equivalent enzymes of E. coli and S. pneumoniae, was deduced to encode maltodextrin phosphorylase (MaIP). The translation stop codon of MaIQ overlapped the translation start codon of the putative maIP gene, suggesting that the two genes may be both transcriptionally and translationally coupled. 4-alpha-Glucanotransferase catalyses a disproportionation reaction in which single or multiple glucose units from oligosaccharides are transferred to the 4-hydroxyl group of acceptor sugars. Characterization of the recombinant C. butyricum enzyme demonstrated that glucose, maltose and maltotriose could act as acceptor, whereas of the three only maltotriose could act as donor. The enzyme therefore shares properties with the E. coli MaIQ protein, but differs significantly from the glucanotransferase of Thermotoga maritima, which is unable to use maltotriose as donor or glucose as acceptor. Physiologically, the concerted action of 4-alpha-glucanotransferase and maltodextrin phosphorylase provides C. butyricum with a mechanism of utilizing amylose/maltodextrins with little drain on cellular ATP reserves. PMID:9353929

  2. Molecular analysis of the 5 alpha-steroid reductase type 2 gene in a family with deficiency of the enzyme.

    PubMed

    Vilchis, F; Canto, P; Chávez, B; Ulloa-Aguirre, A; Méndez, J P

    1997-03-01

    This report describes the identification of a point mutation in the 5 alpha-reductase type 2 (5 alpha-SR2) gene from a family in which both sibs (6 and 3 years old) have steroid 5 alpha-reductase 2 deficiency. The five exons of the gene were individually amplified by the polymerase chain reaction (PCR) and analysed for single-strand conformation polymorphisms (SSCP) to detect mutations. Direct sequencing of the mutant PCR products demonstrated a single C-->T mutation, within exon 4, changing codon 227 from CGA (Arg) to TGA (premature termination signal). Both patients were homozygous for the mutation, but their parents were heterozygous. These results suggest that the mutation at codon 227 impairs normal 5 alpha-SR2 function, thus leading to the phenotypical expression of this rare enzymatic defect. PMID:9066886

  3. Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and. alpha. 1-antitrypsin genes

    SciTech Connect

    Costa, R.H. ); Grayson, D.R. ); Darnell, J.E. Jr. )

    1989-04-01

    Transthyretin (TTR) and {alpha}1-antitrypsin ({alpha}1-AT) are expressed at high levels in the liver and also in at least one other cell type. The authors report here a detailed analysis of the proximal regulatory region of the TTR gene, which has uncovered two new DNA-binding factors that are present mainly (or only) in hepatocytes. One of these new factors, hepatocyte nuclear factor 3 (HNF-3), binds to two sites that are crucial in TTR expression as well as to two additional sites in the {alpha}1-AT proximal enhancer region. The second new factor, HNF-4, binds to two sites in TTR that are required for gene activity. The authors had previously identified binding sites for another hepatocyte-enriched DNA-binding protein (C/EBP or a relative thereof), and additional promoter-proximal sites for that protein in both TTR and {alpha}1-AT are also reported here. From these results it seems clear that cell-specific expression is not simply the result of a single cell-specific factor for each gene but the results of a combination of such factors. The variation and distribution of such factors among different cell types could be an important basis for the coordinate expression of the TTR and {alpha}1-AT genes in the liver or the discordant transcriptional activation of these genes in a few other cell types. The identification of such cell-enriched factors is a necessary prelude to understanding the basis for cell specificity.

  4. Applying horizontal gene transfer phenomena to enhance non-viral gene therapy

    PubMed Central

    Elmer, Jacob J.; Christensen, Matthew D.; Rege, Kaushal

    2014-01-01

    Horizontal gene transfer (HGT) is widespread amongst prokaryotes, but eukaryotes tend to be far less promiscuous with their genetic information. However, several examples of HGT from pathogens into eukaryotic cells have been discovered and mimicked to improve non-viral gene delivery techniques. For example, several viral proteins and DNA sequences have been used to significantly increase cytoplasmic and nuclear gene delivery. Plant genetic engineering is routinely performed with the pathogenic bacterium Agrobacterium tumefaciens and similar pathogens (e.g. Bartonella henselae) may also be able to transform human cells. Intracellular parasites like Trypanosoma cruzi may also provide new insights into overcoming cellular barriers to gene delivery. Finally, intercellular nucleic acid transfer between host cells will also be briefly discussed. This article will review the unique characteristics of several different viruses and microbes and discuss how their traits have been successfully applied to improve non-viral gene delivery techniques. Consequently, pathogenic traits that originally caused diseases may eventually be used to treat many genetic diseases. PMID:23994344

  5. Comparison between Agrobacterium-mediated and direct gene transfer using the gene gun.

    PubMed

    Gao, Caixia; Nielsen, Klaus K

    2013-01-01

    Agrobacterium-mediated transformation and direct gene transfer using the gene gun (microparticle -bombardment) are the two most widely used methods for plant genetic modification. The Agrobacterium method has been successfully practiced in dicots for many years, but only recently have efficient protocols been developed for grasses. Microparticle bombardment has evolved as a method delivering exogenous nucleic acids into plant genome and is a commonly employed technique in plant science. Here these two systems are compared for transformation efficiency, transgene integration, and transgene expression when used to transform tall fescue (Festuca arundinacea Schreb.). The tall fescue transformation protocols lead to the production of large numbers of fertile, independent transgenic lines. PMID:23104329

  6. Antisense SNF1-related (SnRK1) protein kinase gene represses transient activity of an alpha-amylase (alpha-Amy2) gene promoter in cultured wheat embryos.

    PubMed

    Laurie, Sophie; McKibbin, Rowan S; Halford, Nigel G

    2003-02-01

    A DNA fragment corresponding to part of an SNF1 (sucrose non-fermenting-1)-related protein kinase (SnRK1) transcript was amplified by a polymerase chain reaction (PCR) from a wheat (Triticum aestivum) endosperm cDNA library. It was used to construct a chimaeric gene, pUasSnRKN, comprising a ubiquitin promoter, the SnRK1 PCR product in the antisense orientation and the nopaline synthase (Nos) gene terminator. This construct was used in transient gene expression experiments in cultured wheat embryos together with a series of reporter gene constructs. These included the wheat alpha amylase gene alpha-Amy2 promoter with UidA (Gus) coding region (palpha2GT), rice actin promoter with Gus (pActIDGus), ubiquitin promoter with Gus (pAHC25) and actin promoter with green fluorescent protein (GFP) gene (pAct1Is-GFP1). All of the reporter genes were found to be active when bombarded into scutellae isolated from immature grains at 25 d post-anthesis and incubated on MS medium for 24 h prior to bombardment. However, co-bombardment of palpha2GT with equimolar amounts of pUasSnRKN resulted in no detectable Gus activity, indicating that the antisense SnRK1 construct repressed the alpha-Amy2 promoter. Co-bombardment with pUasSnRKN had no effect on the activity of the other promoters used in the study. A triple bombardment with palpha2GT, pAct1Is-GFP-1 and pUasSnRKN resulted in clear green fluorescence, indicating that the bombarded cells were still viable, but no Gus activity. RT-PCR analysis showed clearly that the antisense SnRK1 gene was expressing. Northern and RT-PCR analyses confirmed that SnRK1 and both alpha-amylase genes, alpha-Amy1 and alpha-Amy2, are expressed in cultured wheat embryos harvested from grain 25 d post-anthesis. Expression of alpha-Amy1 and alpha-Amy2 was up-regulated by sugar starvation. PMID:12554717

  7. PGC-1alpha induces dynamic protein interactions on the ERRalpha gene multi-hormone response element nucleosome in kidney cells.

    PubMed

    Wang, Liangli; Li, Yin; Hu, Peng; Teng, Christina T

    2008-12-15

    ERR (oestrogen-related receptor)-alpha modulates the oestrogen signalling pathway and regulates genes participating in the physiological energy balance programme. Oestrogen and PGC-1alpha (peroxisome proliferator-activated receptor-gamma coactivator-1alpha), the master regulator of the energy homoeostasis programme, both regulate the expression of ERRalpha through the MHRE (multi-hormone response element) of the ERRalpha gene. Although the molecular mechanism of oestrogen action on ERRalpha regulation is well characterized, the mechanism of PGC-1alpha induction is unclear. In this study, we examine chromatin structural changes and protein interactions at the MHRE nucleosome in response to PGC-1alpha expression in HK2 human kidney cells. We mapped the nucleosome positions of the ERRalpha gene promoter and examined the changes of histone acetylation in response to PGC-1alpha expression. The interactions of DNA-binding proteins, ERRalpha and ERRgamma, co-activators {CBP [CREB (cAMP-response-element-binding protein)-binding protein], p300, PCAF (p300/CBP-associated factor)}, co-repressor [RIP140 (receptor-interacting protein of 140 kDa)] and RNA polymerase II at the MHRE nucleosome region were investigated over time before and after PGC-1alpha expression in the HK2 cells. We found a dynamic cyclic interaction of these proteins shortly after PGC-1alpha expression and a slower cycling interaction, with fewer proteins involved, 20 h later. By using the siRNA (small interfering RNA) knockdown approach, we discovered that ERRgamma was involved in the initial phase, but not in the later phase, of PGC-1alpha-induced ERRalpha expression. PMID:18673300

  8. Functional cloning of an endo-alpha-1,5-L-arabinanase gene from a metagenomic library

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel endo-alpha-L-arabinanase gene (arn2) was isolated, and expressed in E. coli in active form. The recombinant enzyme (ARN2) had optimum activity at pH 6.0 and 45o-50oC with stability between pH 5.0-8.0 and at temperatures up to 45oC . The recombinant ARN2 catalyzed internal cleavage of alpha-...

  9. Multiple Inter-Kingdom Horizontal Gene Transfers in the Evolution of the Phosphoenolpyruvate Carboxylase Gene Family

    PubMed Central

    Wang, Wen; Su, Bing

    2012-01-01

    Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC) while the other as bacteria-type pepcase (BTPC) because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT) from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought. PMID:23251445

  10. Multiple inter-kingdom horizontal gene transfers in the evolution of the phosphoenolpyruvate carboxylase gene family.

    PubMed

    Peng, Yingmei; Cai, Jing; Wang, Wen; Su, Bing

    2012-01-01

    Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC) while the other as bacteria-type pepcase (BTPC) because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT) from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought. PMID:23251445

  11. Improved gene transfer with histidine-functionalized mesoporous silica nanoparticles.

    PubMed

    Brevet, David; Hocine, Ouahiba; Delalande, Anthony; Raehm, Laurence; Charnay, Clarence; Midoux, Patrick; Durand, Jean-Olivier; Pichon, Chantal

    2014-08-25

    Mesoporous silica nanoparticles (MSN) were functionalized with aminopropyltriethoxysilane (MSN-NH2) then L-histidine (MSN-His) for pDNA delivery in cells and in vivo. The complexation of pDNA with MSN-NH2 and MSN-His was first studied with gel shift assay. pDNA complexed with MSN-His was better protected from DNase degradation than with MSN-NH2. An improvement of the transfection efficiency in cells was observed with MSN-His/pDNA compared to MSN-NH2/pDNA, which could be explained by a better internalization of MSN-His. The improvement of the transfection efficiency with MSN-His was also observed for gene transfer in Achilles tendons in vivo. PMID:24853464

  12. Statistical Mechanics of Horizontal Gene Transfer in Evolutionary Ecology

    NASA Astrophysics Data System (ADS)

    Chia, Nicholas; Goldenfeld, Nigel

    2011-04-01

    The biological world, especially its majority microbial component, is strongly interacting and may be dominated by collective effects. In this review, we provide a brief introduction for statistical physicists of the way in which living cells communicate genetically through transferred genes, as well as the ways in which they can reorganize their genomes in response to environmental pressure. We discuss how genome evolution can be thought of as related to the physical phenomenon of annealing, and describe the sense in which genomes can be said to exhibit an analogue of information entropy. As a direct application of these ideas, we analyze the variation with ocean depth of transposons in marine microbial genomes, predicting trends that are consistent with recent observations using metagenomic surveys.

  13. Alpha-fetoprotein-targeted reporter gene expression imaging in hepatocellular carcinoma

    PubMed Central

    Kim, Kwang Il; Chung, Hye Kyung; Park, Ju Hui; Lee, Yong Jin; Kang, Joo Hyun

    2016-01-01

    Hepatocellular carcinoma (HCC) is one of the most common cancers in Eastern Asia, and its incidence is increasing globally. Numerous experimental models have been developed to better our understanding of the pathogenic mechanism of HCC and to evaluate novel therapeutic approaches. Molecular imaging is a convenient and up-to-date biomedical tool that enables the visualization, characterization and quantification of biologic processes in a living subject. Molecular imaging based on reporter gene expression, in particular, can elucidate tumor-specific events or processes by acquiring images of a reporter gene’s expression driven by tumor-specific enhancers/promoters. In this review, we discuss the advantages and disadvantages of various experimental HCC mouse models and we present in vivo images of tumor-specific reporter gene expression driven by an alpha-fetoprotein (AFP) enhancer/promoter system in a mouse model of HCC. The current mouse models of HCC development are established by xenograft, carcinogen induction and genetic engineering, representing the spectrum of tumor-inducing factors and tumor locations. The imaging analysis approach of reporter genes driven by AFP enhancer/promoter is presented for these different HCC mouse models. Such molecular imaging can provide longitudinal information about carcinogenesis and tumor progression. We expect that clinical application of AFP-targeted reporter gene expression imaging systems will be useful for the detection of AFP-expressing HCC tumors and screening of increased/decreased AFP levels due to disease or drug treatment. PMID:27468205

  14. Cloning the mouse homologue of the human lysosomal acid {alpha}-glucosidase gene

    SciTech Connect

    Ding, J.H.; Yang, B.Z.; Liu, H.M.

    1994-09-01

    Pompe disease (GSD II) is an autosomal recessive disorder caused by a deficiency of lysosomal acid {alpha}-glucosidase (GAA). In an attempt to create a mouse model for Pompe disease, we isolated and characterized the gene encoding the mouse homologue of the human GAA. Twenty clones that extend from exon 2 to the poly(A) tail were isolated from a mouse liver cDNA library, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-2 were cloned by RACE from mouse liver RNA. The full-length liver GAA cDNA contains 3365 nucleotides with a coding region of 2859 nucleotides and a 394 base pair 3{prime}-nontranslated region. The deduced amino acid sequence of the mouse GAA shows 84% identity to the human GAA. Southern blot analysis demonstrated that the mouse GAA was encoded by a single copy gene. Then six bacteriophages containing DNA from the GAA gene were isolated by screening 10{sup 6} phage plaques of a mouse 129 genomic library using a mouse GAA cDNA as a probe. From one of these bacteriophages, an 11-kilobase EcoRI fragment containing exons 3 to 15 was subcloned and sequenced. Work is in progress using this genomic clone to disrupt the GAA gene in murine embryonic stem cells in order to create GSD II mice.

  15. The retinoid-related orphan receptor alpha (RORA) gene and fear-related psychopathology

    PubMed Central

    Miller, Mark W.; Wolf, Erika J.; Logue, Mark W.; Baldwin, Clinton T.

    2013-01-01

    Background This study followed on findings from a recent genome-wide association study of PTSD that implicated the retinoid-related orphan receptor alpha (RORA) gene (Logue et al, 2012) by examining its relationship to broader array of disorders. Methods Using data from the same cohort (N = 540), we analyzed patterns of association between 606 single nucleotide polymorphisms (SNPs) spanning the RORA gene and comorbidity factors termed fear, distress (i.e., internalizing factors) and externalizing. Results Results showed that rs17303244 was associated with the fear component of internalizing (i.e., defined by symptoms of panic, agoraphobia, specific phobia, and obsessive-compulsive disorder) at a level of significance that withstood correction for gene-wide multiple testing. Limitations The primary limitations were the modest size of the cohort and the absence of a replication sample. Conclusions Results add to a growing literature implicating the RORA gene in a wide range of neuropsychiatric disorders and offer new insight into possible molecular mechanisms of the effects of traumatic stress on the brain and the role of genetic factors in those processes. PMID:24007783

  16. Detecting rare gene transfer events in bacterial populations.

    PubMed

    Nielsen, Kaare M; Bøhn, Thomas; Townsend, Jeffrey P

    2014-01-01

    Horizontal gene transfer (HGT) enables bacteria to access, share, and recombine genetic variation, resulting in genetic diversity that cannot be obtained through mutational processes alone. In most cases, the observation of evolutionary successful HGT events relies on the outcome of initially rare events that lead to novel functions in the new host, and that exhibit a positive effect on host fitness. Conversely, the large majority of HGT events occurring in bacterial populations will go undetected due to lack of replication success of transformants. Moreover, other HGT events that would be highly beneficial to new hosts can fail to ensue due to lack of physical proximity to the donor organism, lack of a suitable gene transfer mechanism, genetic compatibility, and stochasticity in tempo-spatial occurrence. Experimental attempts to detect HGT events in bacterial populations have typically focused on the transformed cells or their immediate offspring. However, rare HGT events occurring in large and structured populations are unlikely to reach relative population sizes that will allow their immediate identification; the exception being the unusually strong positive selection conferred by antibiotics. Most HGT events are not expected to alter the likelihood of host survival to such an extreme extent, and will confer only minor changes in host fitness. Due to the large population sizes of bacteria and the time scales involved, the process and outcome of HGT are often not amenable to experimental investigation. Population genetic modeling of the growth dynamics of bacteria with differing HGT rates and resulting fitness changes is therefore necessary to guide sampling design and predict realistic time frames for detection of HGT, as it occurs in laboratory or natural settings. Here we review the key population genetic parameters, consider their complexity and highlight knowledge gaps for further research. PMID:24432015

  17. Detecting rare gene transfer events in bacterial populations

    PubMed Central

    Nielsen, Kaare M.; Bøhn, Thomas; Townsend, Jeffrey P.

    2014-01-01

    Horizontal gene transfer (HGT) enables bacteria to access, share, and recombine genetic variation, resulting in genetic diversity that cannot be obtained through mutational processes alone. In most cases, the observation of evolutionary successful HGT events relies on the outcome of initially rare events that lead to novel functions in the new host, and that exhibit a positive effect on host fitness. Conversely, the large majority of HGT events occurring in bacterial populations will go undetected due to lack of replication success of transformants. Moreover, other HGT events that would be highly beneficial to new hosts can fail to ensue due to lack of physical proximity to the donor organism, lack of a suitable gene transfer mechanism, genetic compatibility, and stochasticity in tempo-spatial occurrence. Experimental attempts to detect HGT events in bacterial populations have typically focused on the transformed cells or their immediate offspring. However, rare HGT events occurring in large and structured populations are unlikely to reach relative population sizes that will allow their immediate identification; the exception being the unusually strong positive selection conferred by antibiotics. Most HGT events are not expected to alter the likelihood of host survival to such an extreme extent, and will confer only minor changes in host fitness. Due to the large population sizes of bacteria and the time scales involved, the process and outcome of HGT are often not amenable to experimental investigation. Population genetic modeling of the growth dynamics of bacteria with differing HGT rates and resulting fitness changes is therefore necessary to guide sampling design and predict realistic time frames for detection of HGT, as it occurs in laboratory or natural settings. Here we review the key population genetic parameters, consider their complexity and highlight knowledge gaps for further research. PMID:24432015

  18. Widespread impact of horizontal gene transfer on plant colonization of land

    PubMed Central

    Yue, Jipei; Hu, Xiangyang; Sun, Hang; Yang, Yongping; Huang, Jinling

    2012-01-01

    In complex multicellular eukaryotes such as animals and plants, horizontal gene transfer is commonly considered rare with very limited evolutionary significance. Here we show that horizontal gene transfer is a dynamic process occurring frequently in the early evolution of land plants. Our genome analyses of the moss Physcomitrella patens identified 57 families of nuclear genes that were acquired from prokaryotes, fungi or viruses. Many of these gene families were transferred to the ancestors of green or land plants. Available experimental evidence shows that these anciently acquired genes are involved in some essential or plant-specific activities such as xylem formation, plant defence, nitrogen recycling as well as the biosynthesis of starch, polyamines, hormones and glutathione. These findings suggest that horizontal gene transfer had a critical role in the transition of plants from aquatic to terrestrial environments. On the basis of these findings, we propose a model of horizontal gene transfer mechanism in nonvascular and seedless vascular plants. PMID:23093189

  19. Determination of the kappa anti-alpha(1,3) dextran immune response difference by A gene(s) in the VKappa-locus of mice

    PubMed Central

    1979-01-01

    Mice lacking the V(alpha(1,3) (h gamma1)-gene do not produce a gamma1 anti-alpha(1,3) dextran response. However, on hyperimmunization some strains mount a kappa-anti-alpha(1,3) dextran response, whereas other remain nonresponder. Responsiveness in dominant. The kappa-anti- alpha(1,3) response difference is linked to the Ly-3 locus on chromosone 6 and is likely the result of a structural Vkappa-gene(s). In conjunction with previous work, three Vkappa-allogroups can now be distinguished. At present, this is the only example of an immune responsiveness difference associated with the Vkappa-locus. PMID:109565

  20. Multiple losses and transfers to the nucleus of two mitochondrial succinate dehydrogenase genes during angiosperm evolution.

    PubMed Central

    Adams, K L; Rosenblueth, M; Qiu, Y L; Palmer, J D

    2001-01-01

    Unlike in animals, the functional transfer of mitochondrial genes to the nucleus is an ongoing process in plants. All but one of the previously reported transfers in angiosperms involve ribosomal protein genes. Here we report frequent transfer of two respiratory genes, sdh3 and sdh4 (encoding subunits 3 and 4 of succinate dehydrogenase), and we also show that these genes are present and expressed in the mitochondria of diverse angiosperms. Southern hybridization surveys reveal that sdh3 and sdh4 have been lost from the mitochondrion about 40 and 19 times, respectively, among the 280 angiosperm genera examined. Transferred, functional copies of sdh3 and sdh4 were characterized from the nucleus in four and three angiosperm families, respectively. The mitochondrial targeting presequences of two sdh3 genes are derived from preexisting genes for anciently transferred mitochondrial proteins. On the basis of the unique presequences of the nuclear genes and the recent mitochondrial gene losses, we infer that each of the seven nuclear sdh3 and sdh4 genes was derived from a separate transfer to the nucleus. These results strengthen the hypothesis that angiosperms are experiencing a recent evolutionary surge of mitochondrial gene transfer to the nucleus and reveal that this surge includes certain respiratory genes in addition to ribosomal protein genes. PMID:11454775

  1. Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases: An NHLBI Resource for the Gene Therapy Community

    PubMed Central

    Skarlatos, Sonia I.

    2012-01-01

    Abstract The goals of the National Heart, Lung, and Blood Institute (NHLBI) Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases are to conduct gene transfer studies in monkeys to evaluate safety and efficiency; and to provide NHLBI-supported investigators with expertise, resources, and services to actively pursue gene transfer approaches in monkeys in their research programs. NHLBI-supported projects span investigators throughout the United States and have addressed novel approaches to gene delivery; “proof-of-principle”; assessed whether findings in small-animal models could be demonstrated in a primate species; or were conducted to enable new grant or IND submissions. The Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases successfully aids the gene therapy community in addressing regulatory barriers, and serves as an effective vehicle for advancing the field. PMID:22974119

  2. Polymorphism and structure of the gene coding for the alpha 1 subunit of the Artemia franciscana Na/K-ATPase.

    PubMed Central

    García-Sáez, A; Perona, R; Sastre, L

    1997-01-01

    Genomic clones coding for one of the two identified Artemia franciscana Na/K-ATPase alpha subunits, the alpha 1 subunit, have been isolated. Several overlapping clones were obtained, although their restriction maps showed a large heterogeneity. Sequencing of their exons showed that they differ in up to 3.46% of their nucleotides in translated regions and 8.18% in untranslated regions. Southern blot analysis of DNA purified from different lots of A. franciscana cysts and from isolated individuals suggests that the variation is due to the existence of multiple Na/K-ATPase alpha 1 subunit alleles in A. franciscana. The Na/K-ATPase alpha 1 subunit gene is divided into 15 exons. Ten of the 14 introns are located in identical positions in this gene as in the human Na/K-ATPase alpha 3 subunit gene. Analysis of the 5' flanking region of the gene has allowed identification of the transcription-initiation sites. The adjacent upstream region has been shown to have functional promoter activity in cultured mammalian cells, suggesting the evolutionary conservation of some of the promoter regulatory sequences. PMID:9020888

  3. [Expression of the human interferon alpha F gene in the obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida].

    PubMed

    Chistoserdov, A Iu; Eremashvili, M R; Mashko, S V; Lapidus, A L; Skvortsova, M A

    1987-08-01

    The expression of human leucocyte interferon alpha F gene in plasmid pLM-IFN alpha F-273 is controlled by a hybrid tac (trp-lac) promoter. A structural gene for interferon alpha F is a component of the hybrid operon lacZ'-IFN alpha F-TcR, that contains an E. coli trp-operon intercystronic region. Plasmid pLM IFN alpha F-273--directed interferon synthesis allows to obtain about 10(7) IU/l. This plasmid was cloned in broad-host-range vector plasmid pAYC31. The hybrid bi-repliconed plasmid containing interferon gene as well as its single-repliconed deletion derivatives obtained by the in vivo recombination, were introduced into obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida PpG6. Methylotrophic strain and Pseudomonas were able to transcribe the interferon gene from E. coli tac promoter, the yield of interferon being 2-4-fold higher as compared with the one in the initial host. PMID:3119998

  4. The tryptophanase gene cluster of Haemophilus influenzae type b: evidence for horizontal gene transfer.

    PubMed

    Martin, K; Morlin, G; Smith, A; Nordyke, A; Eisenstark, A; Golomb, M

    1998-01-01

    Among strains of Haemophilus influenzae, the ability to catabolize tryptophan (as detected by indole production) varies and is correlated with pathogenicity. Tryptophan catabolism is widespread (70 to 75%) among harmless respiratory isolates but is nearly universal (94 to 100%) among strains causing serious disease, including meningitis. As a first step in investigating the relationship between tryptophan catabolism and virulence, we have identified genes in pathogenic H. influenzae which are homologous to the tryptophanase (tna) operon of Escherichia coli. The tna genes are located on a 3.1-kb fragment between nlpD and mutS in the H. influenzae type b (Eagan) genome, are flanked by 43-bp direct repeats of an uptake signal sequence downstream from nlpD, and appear to have been inserted as a mobile unit within this sequence. The organization of this insertion is reminiscent of pathogenicity islands. The tna cluster is found at the same map location in all indole-positive strains of H. influenzae surveyed and is absent from reference type d and e genomes. In contrast to H. influenzae, most other Haemophilus species lack tna genes. Phylogenetic comparisons suggest that the tna cluster was acquired by intergeneric lateral transfer, either by H. influenzae or a recent ancestor, and that E. coli may have acquired its tnaA gene from a related source. Genomes of virulent H. influenzae resemble those of pathogenic enterics in having an island of laterally transferred DNA next to mutS. PMID:9422600

  5. Gene-transfer study approval awaits more data

    SciTech Connect

    Marwick, C.

    1988-11-18

    Approval of the gene-transfer study in cancer patients has been delayed. The proposal was recommended for approval by a National Institutes of Health (NIH) advisory committee, but has been put on hold by James B. Wyngaarden, MD, NIH director, pending submission in writing of further information. Some of this information, now forthcoming, had been withheld because data on preliminary studies had been submitted to peer-reviewed journals. The study involves placing the gene for neomycin-resistance to tumor-infiltrating lymphocytes as a marker. When these cells are injected into the patient, the presence of the marker should enable their fate to be studied over a prolonged period and an improved antitumor regimen could result. The use of tumor-infiltrating lymphocytes as immunotherapy has been studied for two years at the NIH's National Cancer Institute. The patients' tumors are removed and the tumor-infiltrating lymphocytes are cultivated to obtain several billion cells. These cells are then injected back into the patient. Early clinical experience has shown a substantial decease in tumor size in some patients, but not in all, an no one knows why.

  6. Adenoviral-mediated gene transfer to rabbit synovium in vivo.

    PubMed Central

    Roessler, B J; Allen, E D; Wilson, J M; Hartman, J W; Davidson, B L

    1993-01-01

    Currently, treatment for rheumatoid arthritis and other inflammatory arthropathies is often ineffective in ameliorating the progression of the disease, particularly the invasive destruction of cartilage and bone by rheumatoid synovium. Multiple aspects of this inflammatory process are mediated by the synovial lining cells (synoviocytes). Genetic modification of these cells in vivo represents a potential method for the treatment of these conditions. In this report, we describe a novel technique for the genetic transduction of synovial lining cells in vivo using recombinant adenoviral vectors and intraarticular injection techniques. Purified high titer suspensions of a recombinant adenoviral vector containing the gene for Escherichia coli beta-galactosidase (AdCMVlacZ) were directly injected into the hind knees of New Zealand white rabbits. Synovial tissues were then examined for transgenic lacZ expression using a combination of in situ staining for beta-galactosidase activity, immunohistochemical staining, and transmission electron microscopy. High efficiency gene transfer and lacZ expression was observed in both type A and type B synoviocytes throughout the articular and periarticular synovium of the rabbit knee, with continued expression of transgenic lacZ detected for > or = 8 wk after infection. Images PMID:8349791

  7. Passive Immunization against HIV/AIDS by Antibody Gene Transfer

    PubMed Central

    Yang, Lili; Wang, Pin

    2014-01-01

    Despite tremendous efforts over the course of many years, the quest for an effective HIV vaccine by the classical method of active immunization remains largely elusive. However, two recent studies in mice and macaques have now demonstrated a new strategy designated as Vectored ImmunoProphylaxis (VIP), which involves passive immunization by viral vector-mediated delivery of genes encoding broadly neutralizing antibodies (bnAbs) for in vivo expression. Robust protection against virus infection was observed in preclinical settings when animals were given VIP to express monoclonal neutralizing antibodies. This unorthodox approach raises new promise for combating the ongoing global HIV pandemic. In this article, we survey the status of antibody gene transfer, review the revolutionary progress on isolation of extremely bnAbs, detail VIP experiments against HIV and its related virus conduced in humanized mice and macaque monkeys, and discuss the pros and cons of VIP and its opportunities and challenges towards clinical applications to control HIV/AIDS endemics. PMID:24473340

  8. Passive immunization against HIV/AIDS by antibody gene transfer.

    PubMed

    Yang, Lili; Wang, Pin

    2014-02-01

    Despite tremendous efforts over the course of many years, the quest for an effective HIV vaccine by the classical method of active immunization remains largely elusive. However, two recent studies in mice and macaques have now demonstrated a new strategy designated as Vectored ImmunoProphylaxis (VIP), which involves passive immunization by viral vector-mediated delivery of genes encoding broadly neutralizing antibodies (bnAbs) for in vivo expression. Robust protection against virus infection was observed in preclinical settings when animals were given VIP to express monoclonal neutralizing antibodies. This unorthodox approach raises new promise for combating the ongoing global HIV pandemic. In this article, we survey the status of antibody gene transfer, review the revolutionary progress on isolation of extremely bnAbs, detail VIP experiments against HIV and its related virus conduced in humanized mice and macaque monkeys, and discuss the pros and cons of VIP and its opportunities and challenges towards clinical applications to control HIV/AIDS endemics. PMID:24473340

  9. Horizontal transference of S-layer genes within Thermus thermophilus.

    PubMed Central

    Fernández-Herrero, L A; Olabarría, G; Castón, J R; Lasa, I; Berenguer, J

    1995-01-01

    The S-layers of Thermus thermophilus HB27 and T. thermophilus HB8 are composed of protein units of 95 kDa (P95) and 100 kDa (P100), respectively. We have selected S-layer deletion mutants from both strains by complete replacement of the slpA gene. Mutants of the two strains showed similar defects in growth and morphology and overproduced an external cell envelope inside of which cells remained after division. However, the nature of this external layer is strain specific, being easily stained and regular in the HB8 delta slpA derivative and amorphous and poorly stained in the HB27 delta slpA strain. The addition of chromosomic DNA from T. thermophilus HB8 to growing cultures of T. thermophilus HB27 delta slpA led to the selection of a new strain, HB27C8, which expressed a functional S-layer composed of the P100 protein. Conversely, the addition of chromosomic DNA from T. thermophilus HB27 to growing cultures of T. thermophilus HB8 delta slpA allowed the isolation of strain HB8C27, which expressed a functional S-layer composed of the P95 protein. The driving force which selected the transference of the S-layer genes in these experiments was the difference in growth rates, one of the main factors leading to selection in natural environments. PMID:7559330

  10. Cloning of a novel human nonmuscle alpha-actinin gene and characterization of alpha-actinin expression in nemaline rod myopathy (NRM)

    SciTech Connect

    Beggs, A.H.; Knoll, J.H.M.; Kunkel, L.M.

    1994-09-01

    The {alpha}-actinins are a group of actin-binding and crosslinking proteins that are evolutionarily related to the spectrins and dystrophin. Muscle isoforms of {alpha}-actinin are localized at the Z-lines where they anchor actin filaments in a constitutive manner. In contrast, nonmuscle (cytoskeletal) isoforms are found in all tissues at adherens type junctions where they may play a role in organizing the actin cytoskeleton. In addition to the previously identified nonmuscle/smooth muscle ACTN1 gene and the skeletal/cardiac isoforms, ACTN2 and ACTN3, we have identified a fourth gene, ACTN4, which maps to chromosome 19q13.1 and is expressed at similar levels in all tissues tested. Sequence analysis reveals that all four human genes are collinear with about 77% amino acid identity overall. The ACTN4 gene is alternatively spliced at the first EF-hand to generate two alternative isoforms, both of which are predicted to be calcium insensitive. By indirect immunofluorescence, ACTN1 and ACTN4 are both expressed in association with the actin cytoskeleton of cultured fibroblasts. Additionally, both are found in vascular smooth muscle, however, ACTN4, but not ACTN1, was also found in some capillaries in skeletal muscle. ACTN2 was found at Z-lines in all muscle fiber types while ACTN3 expression is limited to Z-lines in a subset of fast (Type II) fibers. Interestingly, both ACTN2 and ACTN3 isoforms were identified in nemaline rods from a patient with NRM, suggesting that the genetic mutation responsible for this disorder may not be in an {alpha}-actinin gene.

  11. Genome-scale phylogenetic analysis finds extensive gene transfer among fungi

    PubMed Central

    Szöllősi, Gergely J.; Davín, Adrián Arellano; Tannier, Eric; Daubin, Vincent; Boussau, Bastien

    2015-01-01

    Although the role of lateral gene transfer is well recognized in the evolution of bacteria, it is generally assumed that it has had less influence among eukaryotes. To explore this hypothesis, we compare the dynamics of genome evolution in two groups of organisms: cyanobacteria and fungi. Ancestral genomes are inferred in both clades using two types of methods: first, Count, a gene tree unaware method that models gene duplications, gains and losses to explain the observed numbers of genes present in a genome; second, ALE, a more recent gene tree-aware method that reconciles gene trees with a species tree using a model of gene duplication, loss and transfer. We compare their merits and their ability to quantify the role of transfers, and assess the impact of taxonomic sampling on their inferences. We present what we believe is compelling evidence that gene transfer plays a significant role in the evolution of fungi. PMID:26323765

  12. Frequent, independent transfers of a catabolic gene from bacteria to contrasted filamentous eukaryotes

    PubMed Central

    Bruto, Maxime; Prigent-Combaret, Claire; Luis, Patricia; Moënne-Loccoz, Yvan; Muller, Daniel

    2014-01-01

    Even genetically distant prokaryotes can exchange genes between them, and these horizontal gene transfer events play a central role in adaptation and evolution. While this was long thought to be restricted to prokaryotes, certain eukaryotes have acquired genes of bacterial origin. However, gene acquisitions in eukaryotes are thought to be much less important in magnitude than in prokaryotes. Here, we describe the complex evolutionary history of a bacterial catabolic gene that has been transferred repeatedly from different bacterial phyla to stramenopiles and fungi. Indeed, phylogenomic analysis pointed to multiple acquisitions of the gene in these filamentous eukaryotes—as many as 15 different events for 65 microeukaryotes. Furthermore, once transferred, this gene acquired introns and was found expressed in mRNA databases for most recipients. Our results show that effective inter-domain transfers and subsequent adaptation of a prokaryotic gene in eukaryotic cells can happen at an unprecedented magnitude. PMID:24990676

  13. Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen

    PubMed Central

    Dhillon, Braham; Feau, Nicolas; Aerts, Andrea L.; Beauseigle, Stéphanie; Bernier, Louis; Copeland, Alex; Foster, Adam; Gill, Navdeep; Henrissat, Bernard; Herath, Padmini; LaButti, Kurt M.; Levasseur, Anthony; Lindquist, Erika A.; Majoor, Eline; Ohm, Robin A.; Pangilinan, Jasmyn L.; Pribowo, Amadeus; Saddler, John N.; Sakalidis, Monique L.; de Vries, Ronald P.; Grigoriev, Igor V.; Goodwin, Stephen B.; Tanguay, Philippe; Hamelin, Richard C.

    2015-01-01

    Some of the most damaging tree pathogens can attack woody stems, causing lesions (cankers) that may be lethal. To identify the genomic determinants of wood colonization leading to canker formation, we sequenced the genomes of the poplar canker pathogen, Mycosphaerella populorum, and the closely related poplar leaf pathogen, M. populicola. A secondary metabolite cluster unique to M. populorum is fully activated following induction by poplar wood and leaves. In addition, genes encoding hemicellulose-degrading enzymes, peptidases, and metabolite transporters were more abundant and were up-regulated in M. populorum growing on poplar wood-chip medium compared with M. populicola. The secondary gene cluster and several of the carbohydrate degradation genes have the signature of horizontal transfer from ascomycete fungi associated with wood decay and from prokaryotes. Acquisition and maintenance of the gene battery necessary for growth in woody tissues and gene dosage resulting in gene expression reconfiguration appear to be responsible for the adaptation of M. populorum to infect, colonize, and cause mortality on poplar woody stems. PMID:25733908

  14. Towards liver-directed gene therapy: retrovirus-mediated gene transfer into human hepatocytes.

    PubMed

    Grossman, M; Raper, S E; Wilson, J M

    1991-11-01

    Liver-directed gene therapy is being considered in the treatment of inherited metabolic diseases. One approach we are considering is the transplantation of autologous hepatocytes that have been genetically modified with recombinant retroviruses ex vivo. We describe, in this report, techniques for isolating human hepatocytes and efficiently transducing recombinant genes into primary cultures. Hepatocytes were isolated from tissue of four different donors, plated in primary culture, and exposed to recombinant retroviruses expressing either the LacZ reporter gene or the cDNA for rabbit LDL receptor. The efficiency of gene transfer under optimal conditions, as determined by Southern blot analysis, varied from a maximum of one proviral copy per cell to a minimum of 0.1 proviral copy per cell. Cytochemical assays were used to detect expression of the recombinant derived proteins, E. coli beta-galactosidase and rabbit LDL receptor. Hepatocytes transduced with the LDL receptor gene expressed levels of receptor protein that exceeded the normal endogenous levels. The ability to isolate and genetically modify human hepatocytes, as described in this report, is an important step towards the development of liver-directed gene therapies in humans. PMID:1767337

  15. Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen.

    PubMed

    Dhillon, Braham; Feau, Nicolas; Aerts, Andrea L; Beauseigle, Stéphanie; Bernier, Louis; Copeland, Alex; Foster, Adam; Gill, Navdeep; Henrissat, Bernard; Herath, Padmini; LaButti, Kurt M; Levasseur, Anthony; Lindquist, Erika A; Majoor, Eline; Ohm, Robin A; Pangilinan, Jasmyn L; Pribowo, Amadeus; Saddler, John N; Sakalidis, Monique L; de Vries, Ronald P; Grigoriev, Igor V; Goodwin, Stephen B; Tanguay, Philippe; Hamelin, Richard C

    2015-03-17

    Some of the most damaging tree pathogens can attack woody stems, causing lesions (cankers) that may be lethal. To identify the genomic determinants of wood colonization leading to canker formation, we sequenced the genomes of the poplar canker pathogen, Mycosphaerella populorum, and the closely related poplar leaf pathogen, M. populicola. A secondary metabolite cluster unique to M. populorum is fully activated following induction by poplar wood and leaves. In addition, genes encoding hemicellulose-degrading enzymes, peptidases, and metabolite transporters were more abundant and were up-regulated in M. populorum growing on poplar wood-chip medium compared with M. populicola. The secondary gene cluster and several of the carbohydrate degradation genes have the signature of horizontal transfer from ascomycete fungi associated with wood decay and from prokaryotes. Acquisition and maintenance of the gene battery necessary for growth in woody tissues and gene dosage resulting in gene expression reconfiguration appear to be responsible for the adaptation of M. populorum to infect, colonize, and cause mortality on poplar woody stems. PMID:25733908

  16. The Fusarium graminearum Genome Reveals More Secondary Metabolite Gene Clusters and Hints of Horizontal Gene Transfer

    PubMed Central

    Wong, Philip; Münsterkötter, Martin; Mewes, Hans-Werner; Schmeitzl, Clemens; Varga, Elisabeth; Berthiller, Franz; Adam, Gerhard; Güldener, Ulrich

    2014-01-01

    Fungal secondary metabolite biosynthesis genes are of major interest due to the pharmacological properties of their products (like mycotoxins and antibiotics). The genome of the plant pathogenic fungus Fusarium graminearum codes for a large number of candidate enzymes involved in secondary metabolite biosynthesis. However, the chemical nature of most enzymatic products of proteins encoded by putative secondary metabolism biosynthetic genes is largely unknown. Based on our analysis we present 67 gene clusters with significant enrichment of predicted secondary metabolism related enzymatic functions. 20 gene clusters with unknown metabolites exhibit strong gene expression correlation in planta and presumably play a role in virulence. Furthermore, the identification of conserved and over-represented putative transcription factor binding sites serves as additional evidence for cluster co-regulation. Orthologous cluster search provided insight into the evolution of secondary metabolism clusters. Some clusters are characteristic for the Fusarium phylum while others show evidence of horizontal gene transfer as orthologs can be found in representatives of the Botrytis or Cochliobolus lineage. The presented candidate clusters provide valuable targets for experimental examination. PMID:25333987

  17. Improved efficiency of the walnut somatic embryo gene transfer system.

    PubMed

    McGranahan, G H; Leslie, C A; Uratsu, S L; Dandekar, A M

    1990-01-01

    AnAgrobacterium-mediated gene transfer system which relies on repetitive embryogenesis to regenerate transgenic walnut plants has been made more efficient by using a more virulent strain ofAgrobacterium and vectors containing genes for both kanamycin resistance and beta-glucuronidase (GUS) activity to facilitate early screening and selection. Two plasmids (pCGN7001 and pCGN7314) introduced individually into the disarmedAgrobacterium host strain EHA101 were used as inoculum. Embryos maintained on medium containing 100 mg/l kanamycin after co-cultivation produced more transformed secondary embryos than embryos maintained on kanamycin-free medium. Of the 186 GUS-positive secondary embryo lines identified, 70% were regenerated from 3 out of 16 primary embryos inoculated with EHA101/pCGN7314 and grown on kanamycin- containing medium, 28% from 4 out of 17 primary embryos inoculated with EHA101/ pCGN7001 and grown on kanamycin medium, and 2% from one out of 13 primary embryos inoculated with EHA101/pCGN7001 but not exposed to kanamycin. Because kanamycin inhibits but does not completely block new embryo formation in controls, identification of transformants formerly required repetitive selection on kanamycin for several months. Introduction of the GUS marker gene allowed positive identification of transformant secondary embryos as early as 5-6 weeks after inoculation. DNA analysis of a representative subset of lines (n=13) derived from secondary embryos confirmed transformation and provided evidence for multiple insertion events in single inoculated primary embryos. PMID:24226275

  18. Cloning of genes encoding alpha-L-arabinofuranosidase and beta-xylosidase from Trichoderma reesei by expression in Saccharomyces cerevisiae.

    PubMed Central

    Margolles-Clark, E; Tenkanen, M; Nakari-Setälä, T; Penttilä, M

    1996-01-01

    A cDNA expression library of Trichoderma reesei RutC-30 was constructed in the yeast Saccharomyces cerevisiae. Two genes, abf1 and bxl1, were isolated by screening the yeast library for extracellular alpha-L-arabinofuranosidase activity with the substrate p-nitrophenyl-alpha-L-arabinofuranoside. The genes abf1 and bxl1 encode 500 and 758 amino acids, respectively, including the signal sequences. The deduced amino acid sequence of ABFI displays high-level similarity to the alpha-L-arabinofuranosidase B of Aspergillus niger, and the two can form a new family of glycosyl hydrolases. The deduced amino acid sequence of BXLI shows similarities to the beta-glucosidases grouped in family 3. The yeast-produced enzymes were tested for enzymatic activities against different substrates. ABFI released L-arabinose from p-nitrophenyl-alpha-L-arabinofuranoside and arabinoxylans and showed some beta-xylosidase activity toward p-nitrophenyl-beta-D-xylopyranoside. BXLI did not release L-arabinose from arabinoxylan. It showed alpha-L-arabinofuranosidase, alpha-L-arabinopyranosidase, and beta-xylosidase activities against p-nitrophenyl-alpha-L-arabinofuranosidase, p-nitrophenyl-alpha-L-arabinopyranoside, and p-nitrophenyl-beta-D- xylopyranoside, respectively, with the last activity being the highest. It was also able to hydrolyze xylobiose and slowly release xylose from polymeric xylan. ABFI and BXLI correspond to a previously purified alpha-L-arabinofuranosidase and a beta-xylosidase from T. reesei, respectively, as confirmed by partial amino acid sequencing of the Trichoderma-produced enzymes. Both enzymes produced in yeasts displayed hydrolytic properties similar to those of the corresponding enzymes purified from T. reesei. PMID:8837440

  19. PPAR{alpha} does not suppress muscle-associated gene expression in brown adipocytes but does influence expression of factors that fingerprint the brown adipocyte

    SciTech Connect

    Walden, Tomas B.; Petrovic, Natasa; Nedergaard, Jan

    2010-06-25

    Brown adipocytes and myocytes develop from a common adipomyocyte precursor. PPAR{alpha} is a nuclear receptor important for lipid and glucose metabolism. It has been suggested that in brown adipose tissue, PPAR{alpha} represses the expression of muscle-associated genes, in this way potentially acting to determine cell fate in brown adipocytes. To further understand the possible role of PPAR{alpha} in these processes, we measured expression of muscle-associated genes in brown adipose tissue and brown adipocytes from PPAR{alpha}-ablated mice, including structural genes (Mylpf, Tpm2, Myl3 and MyHC), regulatory genes (myogenin, Myf5 and MyoD) and a myomir (miR-206). However, in our hands, the expression of these genes was not influenced by the presence or absence of PPAR{alpha}, nor by the PPAR{alpha} activator Wy-14,643. Similarly, the expression of genes common for mature brown adipocyte and myocytes (Tbx15, Meox2) were not affected. However, the brown adipocyte-specific regulatory genes Zic1, Lhx8 and Prdm16 were affected by PPAR{alpha}. Thus, it would not seem that PPAR{alpha} represses muscle-associated genes, but PPAR{alpha} may still play a role in the regulation of the bifurcation of the adipomyocyte precursor into a brown adipocyte or myocyte phenotype.

  20. Risk of open angle glaucoma due to tumor necrosis factor alpha gene polymorphisms

    PubMed Central

    Hamid, Mona Abdel; Moemen, Leqaa; Labib, Hany; Helmy, Hazem; Elsergany, Tarek

    2016-01-01

    Introduction Axonal degeneration and retinal ganglion cell apoptosis in glaucoma is associated with tumor necrosis factor alpha (TNF-α), which is an important pro-inflammatory cytokine. The aim of this study was to determine the association between the risk of open angle glaucoma (OAG) in the Egyptian population and tumor necrosis factor alpha (TNF-α) gene polymorphisms. Methods Sixty OAG patients and 26 healthy unrelated controls were used to analyze TNF-α polymorphism G-308A using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Results the GG genotype was found at a higher frequency in the controls than in the patients, and the AA and GA genotypes were associated strongly with OAG. Conclusion In this study, we found that the TNF-α polymorphism G-308A was associated significantly with OAG in the Egyptian population. However, there is a need for population-based studies with large numbers of subjects. Also, long-term follow up is required to verify the association between TNF-α polymorphism G-308A and glaucoma susceptibility. PMID:27054008

  1. Transcriptional promoter of the human alpha 1(V) collagen gene (COL5A1).

    PubMed Central

    Lee, S; Greenspan, D S

    1995-01-01

    We have characterized the 5' region of the human alpha 1(V) collagen gene (COL5A1). The transcriptional promoter is shown to have a number of features characteristic of the promoters of 'housekeeping' and growth-control-related genes. It lacks obvious TATA and CAAT boxes, has multiple transcription start sites, has a high GC content, lies within a well-defined CpG island and has a number of consensus sites for the potential binding of transcription factor Sp1. This type of promoter structure, while unusual for a collagen gene, is consistent with the broad distribution of expression of COL5A1 and is reminiscent of the promoter structures of the genes encoding type VI collagen, which has a similarly broad distribution of expression. Stepwise deletion of COL5A1 5' sequences, placed upstream of a heterologous reporter gene, yielded a gradual decrease in promoter activity, indicating that the COL5A1 promoter is composed of an array of cis-acting elements. A minimal promoter region contained within the 212 bp immediately upstream of the major transcription start site contained no consensus sequences for the binding of known transcription factors, but gel mobility shift assays showed this region to bind nuclear factors, including Sp1, at a number of sites. The major transcription start site is flanked by an upstream 34-bp oligopurine/oligopyrimidine stretch, or 'GAGA' box, and a downstream 56-bp GAGA box which contains a 10-bp mirror repeat and is sensitive to cleavage with S1 nuclease. Images Figure 1 Figure 3 Figure 4 Figure 6 PMID:7646438

  2. Liver-Specific Alpha 2 Interferon Gene Expression Results in Protection from Induced Hepatitis

    PubMed Central

    Aurisicchio, Luigi; Delmastro, Paola; Salucci, Valentina; Paz, Odalys Gonzalez; Rovere, Patrizia; Ciliberto, Gennaro; La Monica, Nicola; Palombo, Fabio

    2000-01-01

    The current therapy for hepatitis B and C is based on systemic administration of recombinant human alpha interferon (r-hIFN-α). However, systemic delivery of r-hIFN-α is associated with severe side effects, but more importantly, it is effective in only a small percentage of patients. In an effort to maximize IFN-α antiviral efficacy, we have explored the therapeutic potential of murine IFN-α2 (mIFNα2) selectively expressed in the liver. To this end, we have developed a helper-dependent adenovirus vector (HD) containing the mIFN-α2 gene under the control of the liver-specific transthyretin promoter (HD-IFN). Comparison with a first-generation adenovirus carrying the same mIFN-α2 expression cassette indicates that at certain HD-IFN doses, induction of antiviral genes can be achieved in the absence of detectable circulating mIFN-α2. Challenge of injected mice with mouse hepatitis virus type 3 showed that HD-IFN provides high liver protection. Moreover, liver protection was also observed in acute nonviral liver inflammation hepatitis induced by concanavalin A at 1 month postinfection. These results hold promise for the development of a gene therapy treatment for chronic viral hepatitis based on liver-restricted expression of IFN-α2. PMID:10775620

  3. Genetic polymorphism of estrogen receptor alpha gene in Egyptian women with type II diabetes mellitus

    PubMed Central

    Motawi, Tarek M.K.; El-Rehany, Mahmoud A.; Rizk, Sherine M.; Ramzy, Maggie M.; el-Roby, Doaa M.

    2015-01-01

    Estrogen might play an important role in type 2 diabetes mellitus pathogenesis. A number of polymorphisms have been reported in the estrogen receptor alpha gene including the XbaI and PvuII restriction enzyme polymorphisms. The aim of this study was to determine if ESRα gene polymorphisms are associated with type 2 diabetes mellitus and correlated with lipid profile. Ninety diabetic Egyptian patients were compared with forty healthy controls. ESRα genotyping of PvuII and XbaI was performed using restriction fragment length polymorphism analysis. Our study showed that there is more significant difference in the frequency of C and G polymorphic allele between patients and control groups in PvuII and XbaI respectively. Also carriers of minor C and G alleles of PvuII and XbaI gene polymorphisms were associated with increased fasting blood glucose and disturbance in lipid profile as there is an increase in total cholesterol, triglycerides and Low density lipoprotein. So findings of present study suggest the possibility that PvuII and XbaI polymorphisms in ERα are related to T2DM and with increased serum lipids among Egyptian population. PMID:26401488

  4. Estrogen receptor alpha gene amplification in breast cancer: 25 years of debate

    PubMed Central

    Holst, Frederik

    2016-01-01

    Twenty-five years ago, Nembrot and colleagues reported amplification of the estrogen receptor alpha gene (ESR1) in breast cancer, initiating a broad and still ongoing scientific debate on the prevalence and clinical significance of this genetic aberration, which affects one of the most important genes in breast cancer. Since then, a multitude of studies on this topic has been published, covering a wide range of divergent results and arguments. The reported prevalence of this alteration in breast cancer ranges from 0% to 75%, suggesting that ESR1 copy number analysis is hampered by technical and interpreter issues. To date, two major issues related to ESR1 amplification remain to be conclusively addressed: (1) The extent to which abundant amounts of messenger RNA can mimic amplification in standard fluorescence in situ hybridization assays in the analysis of strongly expressed genes like ESR1, and (2) the clinical relevance of ESR1 amplification: Such relevance is strongly disputed, with data showing predictive value for response as well as for resistance of the cancer to anti-estrogen therapies, or for subsequent development of cancers in the case of precursor lesions that display amplification of ESR1. This review provides a comprehensive summary of the various views on ESR1 amplification, and highlights explanations for the contradictions and conflicting data that could inform future ESR1 research. PMID:27081639

  5. Association of transforming growth-factor alpha gene polymorphisms with nonsyndromic cleft palate only (CPO)

    SciTech Connect

    Shiang, R. ); Lidral, A.C.; Ardinger, H.H.; Murray, J.C.; Romitti, P.A.; Munger, R.G.; Buetow, K.H.

    1993-10-01

    Genetic analysis and tissue-specific expression studies support a role for transforming growth-factor alpha (TGFA) in craniofacial development. Previous studies have confirmed an association of alleles for TGFA with nonsyndromic cleft lip with or without cleft palate (CL/P) in humans. The authors carried out a retrospective association study to determine whether specific allelic variants of the TGFA gene are also associated with cleft palate only (CPO). The PCR products from 12 overlapping sets of primers to the TGFA cDNA were examined by using single-strand conformational polymorphism analysis. Four DNA polymorphic sites for TGFA were identified in the 3[prime] untranslated region of the TGFA gene. These variants, as well as previously identified RFLPs for TGFA, were characterized in case and control populations for CPO by using X[sup 2] analysis. A significant association between alleles of TGFA and CPO was identified which further supports a role for this gene as one of the genetic determinants of craniofacial development. Sequence analysis of the variants disclosed a cluster of three variable sites within 30 bp of each other in the 3[prime] untranslated region previously associated with an antisense transcript. These studies extend the role for TGFA in craniofacial morphogenesis and support an interrelated mechanism underlying nonsyndromic forms of CL/P. 46 refs., 3 figs., 3 tabs.

  6. Expression and regulation of the macrophage inflammatory protein-1 alpha gene by nicotine in rat alveolar macrophages.

    PubMed

    Chong, Inn-Wen; Lin, Shiu-Ru; Hwang, Jhi-Jhu; Huang, Ming-Shyan; Wang, Tung-Heng; Hung, Jen-Yu; Paulauskis, Joseph D

    2002-01-01

    Cigarette smoking causes inflammation mainly confined to the airway and lung. Nicotine is one of the primary constituents in cigarette smoke. Alveolar macrophages apparently play a pivotal role in mediating pulmonary inflammation via the production of chemokines. Macrophage inflammatory protein-1 alpha (MIP-1 alpha), a member of CC chemokines, has been shown to contribute to monocyte/macrophage and neutrophil chemotaxis and activation. Our previous work demonstrated that MIP-1 alpha mRNA expression in macrophages is induced by a variety of stimuli. In the present study, we further investigate whether nicotine can regulate the gene expression of MIP-1 alpha in macrophages and determine the mechanism leading to increased expression. A rat alveolar macrophage (RAM) cell line, NR8383, was treated with nicotine at a dose of 3.1, 31, 310 microM, or 3.1 mM. Northern blot analysis showed that the induction of MIP-1 alpha mRNA expression was dose-dependent. To define the time course of the inflammatory response, RAM cells were exposed to 31 microM nicotine, MIP-1 alpha mRNA was induced as early as 1 h after treatment, was maximally expressed at 4 and 6 hours, and reduced by 8 hours. Western blot analysis demonstrated a single band with an estimated molecular weight of 10 kD for MIP-1 alpha which was induced after nicotine treatment, suggesting that expression of MIP-1 alpha mRNA could reflect in protein synthesis. In addition. the increase in MIP-1 alpha mRNA expression induced by nicotine was attenuated by co-treatment with the antioxidant N-acetylcysteine (NAC), at doses of 10 and 20 mM, suggesting that the induction of MIP-1 alpha mRNA is mediated via the generation of reactive oxygen species (ROS). To further investigate transcriptional regulation of the MIP-1 alpha gene expression, RAM cells were exposed to nicotine. MIP-1 alpha mRNA levels were significantly increased in nuclear RNA preparations, indicating that transcriptional activation is involved in increased

  7. A single gene directs synthesis of a precursor protein with beta- and alpha-amylase activities in Bacillus polymyxa.

    PubMed Central

    Uozumi, N; Sakurai, K; Sasaki, T; Takekawa, S; Yamagata, H; Tsukagoshi, N; Udaka, S

    1989-01-01

    The Bacillus polymyxa amylase gene comprises 3,588 nucleotides. The mature amylase comprises 1,161 amino acids with a molecular weight of 127,314. The gene appeared to be divided into two portions by the direct-repeat sequence located at almost the middle of the gene. The 5' region upstream of the direct-repeat sequence was shown to be responsible for the synthesis of beta-amylase. The 3' region downstream of the direct-repeat sequence contained four sequences homologous with those in other alpha-amylases, such as Taka-amylase A. The 48-kilodalton (kDa) amylase isolated from B. polymyxa was proven to have alpha-amylase activity. The amino acid sequences of the peptides generated from the 48-kDa amylase showed complete agreement with the predicted amino acid sequence of the C-terminal portion. The B. polymyxa amylase gene was therefore concluded to contain in-phase beta- and alpha-amylase-coding sequences in the 5' and 3' regions, respectively. A precursor protein, a 130-kDa amylase, directed by a plasmid, pYN520, carrying the entire amylase gene, had both beta- and alpha-amylase activities. This represents the first report of a single protein precursor in procaryotes that gives rise to two enzymes. Images PMID:2464578

  8. Regulatory elements in the first intron contribute to transcriptional control of the human. cap alpha. 1(I) collagen gene

    SciTech Connect

    Bornstein, P.; McKay, J.; Morishima, J.K.; Devarayalu, S.; Gelinas, R.E.

    1987-12-01

    Several lines of evidence have suggested that the regulation of type I collagen gene transcription is complex and that important regulatory elements reside 5' to, and within, the first intron of the ..cap alpha..1(I) gene. The authors therefore sequenced a 2.3-kilobase HindIII fragment that encompasses 804 base pairs of 5' flanking sequence, the first exon, and most of the first intron of the ..cap alpha..1(I) human collagen gene. A 274-base-pair intronic sequence, flanked by Ava I sites (A274), contained a sequence identical to a high-affinity decanucleotide binding site for transcription factor Sp1 and a viral core enhancer sequence. DNase I protection experiments indicated zones of protection that corresponded to these motifs. When A274 was cloned 5' to the chloramphenicol acetyltransferase (CAT) gene, driven by an ..cap alpha..1(I) collagen promoter sequence, and expression was assessed by transfection, significant orientation-specific inhibition of CAT activity was observed. This effect was most apparent in chicken tendon fibroblasts, which modulate their level of collagen synthesis in culture. They propose that normal regulation of ..cap alpha..1(I) collagen gene transcription results from an interplay of positive and negative elements present in the promoter region and within the first intron.

  9. Regulation of pT alpha gene expression by a dosage of E2A, HEB, and SCL.

    PubMed

    Tremblay, Mathieu; Herblot, Sabine; Lecuyer, Eric; Hoang, Trang

    2003-04-11

    The expression of the pT alpha gene is required for effective selection, proliferation, and survival of beta T-cell receptor (beta TCR)-expressing immature thymocytes. Here, we have identified two phylogenetically conserved E-boxes within the pT alpha enhancer sequence that are required for optimal enhancer activity and for its stage-specific activity in immature T cells. We have shown that the transcription factors E2A and HEB associate with high affinity to these E-boxes. Moreover, we have identified pT alpha as a direct target of E2A-HEB heterodimers in immature thymocytes because they specifically occupy the enhancer in vivo. In these cells, pT alpha mRNA levels are determined by the presence of one or two functional E2A or HEB alleles. Furthermore, E2A/HEB transcriptional activity is repressed by heterodimerization with SCL, a transcription factor that is turned off in differentiating thymocytes exactly at a stage when pT alpha is up-regulated. Taken together, our observations suggest that the dosage of E2A, HEB, and SCL determines pT alpha gene expression in immature T cells. PMID:12566462

  10. Multimodality Imaging of Gene Transfer with a Receptor-Based Reporter Gene

    PubMed Central

    Chen, Ron; Parry, Jesse J.; Akers, Walter J.; Berezin, Mikhail Y.; El Naqa, Issam M.; Achilefu, Samuel; Edwards, W. Barry; Rogers, Buck E.

    2010-01-01

    Gene therapy trials have traditionally used tumor and tissue biopsies for assessing the efficacy of gene transfer. Non-invasive imaging techniques offer a distinct advantage over tissue biopsies in that the magnitude and duration of gene transfer can be monitored repeatedly. Human somatostatin receptor subtype 2 (SSTR2) has been used for the nuclear imaging of gene transfer. To extend this concept, we have developed a somatostatin receptor–enhanced green fluorescent protein fusion construct (SSTR2-EGFP) for nuclear and fluorescent multimodality imaging. Methods An adenovirus containing SSTR2-EGFP (AdSSTR2-EGFP) was constructed and evaluated in vitro and in vivo. SCC-9 human squamous cell carcinoma cells were infected with AdEGFP, AdSSTR2, or AdSSTR2-EGFP for in vitro evaluation by saturation binding, internalization, and fluorescence spectroscopy assays. In vivo biodistribution and nano-SPECT imaging studies were conducted with mice bearing SCC-9 tumor xenografts directly injected with AdSSTR2-EGFP or AdSSTR2 to determine the tumor localization of 111In-diethylenetriaminepentaacetic acid (DTPA)-Tyr3-octreotate. Fluorescence imaging was conducted in vivo with mice receiving intratumoral injections of AdSSTR2, AdSSTR2-EGFP, or AdEGFP as well as ex vivo with tissues extracted from mice. Results The similarity between AdSSTR2-EGFP and wild-type AdSSTR2 was demonstrated in vitro by the saturation binding and internalization assays, and the fluorescence emission spectra of cells infected with AdSSTR2-EGFP was almost identical to the spectra of cells infected with wild-type AdEGFP. Biodistribution studies demonstrated that the tumor uptake of 111In-DTPA-Tyr3-octreotate was not significantly different (P > 0.05) when tumors (n = 5) were injected with AdSSTR2 or AdSSTR2-EGFP but was significantly greater than the uptake in control tumors. Fluorescence was observed in tumors injected with AdSSTR2-EGFP and AdEGFP in vivo and ex vivo but not in tumors injected with AdSSTR2

  11. Sanfilippo syndrome type B: cDNA and gene encoding human {alpha}-N-acetylglucosaminidase

    SciTech Connect

    Zhao, H.G.; Lopez, R.; Rennecker, J.

    1994-09-01

    Deficiency of the lysosomal enzyme {alpha}-N-acetlyglucosaminidase underlies the type B Sanfilippo syndrome (MPS III B), a mucopolysaccharide storage disease with profound neurologic deterioration. We are acquiring tools to study the molecular basis of the disorder. The enzyme was purified from bovine testis; after ConA-, DEAE- and phenyl-Sepharose chromatography, it was subjected to SDS-PAGE without preheating. Of two bands of activity detected on the gel, 170 kDa and 87 kDa, the larger one, which coincided with a well-defined Coomassie blue band, was selected for sequence analysis. Degenerate 17-base oligonucleotides, corresponding to the ends of an internal 23 amino acid sequence, were used for RT-PCR of RNA from human fibroblasts. A 41-mer was synthesized from the sequence of the RT-PCR product and used to screen a human testis cDNA library. A number of cDNA inserts were isolated, all lacking the 5{prime} end and none longer than 1.7 kb. An additional 300 bp segment has been obtained by RACE. The cDNA sequence accounts for 9 of 11 peptides, allowing for species difference. Northern analysis of fibroblast RNA with a 1.5 kb cDNA probe showed the presence of a 3 kb mRNA; marked deficiency of this mRNA in two MPS III B fibroblast lines confirmed the authenticity of the cloned cDNA. While no homologous amino acid sequence has been found in a search of GenBank, the nucleotide sequence (interrupted by 4 introns) is present in a flanking region upstream of an unrelated gene on chromosome 17q11-21 (human 17{beta}-hydroxysteroid dehydrogenase). This must therefore be the chromosomal locus of the {alpha}-N-acetylglucosaminidase gene and of MPS III B.

  12. Persistent Gene Expression in Mouse Nasal Epithelia following Feline Immunodeficiency Virus-Based Vector Gene Transfer

    PubMed Central

    Sinn, Patrick L.; Burnight, Erin R.; Hickey, Melissa A.; Blissard, Gary W.; McCray, Paul B.

    2005-01-01

    Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 106 transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 107 to 109 TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (∼109 TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for ∼1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity. PMID:16188984

  13. Tumour necrosis factor-alpha up-regulates decay-accelerating factor gene expression in human intestinal epithelial cells.

    PubMed Central

    Andoh, A; Fujiyama, Y; Sumiyoshi, K; Sakumoto, H; Okabe, H; Bamba, T

    1997-01-01

    The increased expression of decay-accelerating factor (DAF) has been detected in intestinal epithelial cells at the inflamed mucosa. In this study, we examined the effects of tumour necrosis factor (TNF)-alpha on DAF expression in three intestinal epithelial cell lines. DAF mRNA expression was evaluated by Northern blot analysis, and DAF protein expression was analysed by biotin labelling and immunoprecipitation. TNF-alpha induced a marked increase in DAF mRNA and protein expression in HT-29, T84 and Caco-2 cells. In HT-29 cells, the effects of TNF-a on DAF mRNA accumulation were observed in a dose-dependent manner; DAF mRNA accumulation reached a maximum at 3-6 hr, and then gradually decreased. These effects of TNF-alpha required de novo protein synthesis. Messenger RNA stability studies suggested that TNF-alpha partially regulated DAF gene expression by a posttranscriptional mechanism. Moreover, the combination of TNF-alpha and interleukin (IL)-4 induced an additive increase in DAF mRNA accumulation in HT-29 and T84 cells. In human intestinal epithelial cells, TNF-alpha acts as a potent inducer of DAF mRNA expression, indicating an important role for TNF-alpha in the regulation of DAF expression at the inflamed mucosa. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 Figure 8 PMID:9155641

  14. Horizontal gene transfer of a Chlamydial tRNA-guanine transglycosylase gene to eukaryotic microbes.

    PubMed

    Manna, Sam; Harman, Ashley

    2016-01-01

    tRNA-guanine transglycosylases are found in all domains of life and mediate the base exchange of guanine with queuine in the anticodon loop of tRNAs. They can also regulate virulence in bacteria such as Shigella flexneri, which has prompted the development of drugs that inhibit the function of these enzymes. Here we report a group of tRNA-guanine transglycosylases in eukaryotic microbes (algae and protozoa) which are more similar to their bacterial counterparts than previously characterized eukaryotic tRNA-guanine transglycosylases. We provide evidence demonstrating that the genes encoding these enzymes were acquired by these eukaryotic lineages via horizontal gene transfer from the Chlamydiae group of bacteria. Given that the S. flexneri tRNA-guanine transglycosylase can be targeted by drugs, we propose that the bacterial-like tRNA-guanine transglycosylases could potentially be targeted in a similar fashion in pathogenic amoebae that possess these enzymes such as Acanthamoeba castellanii. This work also presents ancient prokaryote-to-eukaryote horizontal gene transfer events as an untapped resource of potential drug target identification in pathogenic eukaryotes. PMID:26435002

  15. Structural organization of a 17 KB segment of the alpha 2 collagen gene: evaluation by R loop mapping.

    PubMed Central

    Schafer, M P; Boyd, C D; Tolstoshev, P; Crystal, R G

    1980-01-01

    A recombinant phage, SpC3, containing a 17 kb genomic DNA insert representing approximately 60% of the 3' portion of the sheep collagen alpha 2 gene, was evaluated by electron microscopic R loop analysis. A minimum of 17 intervening sequences (introns) and 18 alpha 2 coding sequences (exons) were mapped. With the exception of the 850 base pair exon located at the extreme 3' end of the insert, all exons contained 250 base pairs or less. The total length of all the exons in SpC3 was 3,014 base pairs. The length distribution of the 17 introns ranged from 300 to 1600 base pairs; together, all of the introns comprised 14,070 base pairs of SpC3 DNA. Thus, the DNA region required for coding the interspersed 3 kb of alpha 2 collagen genetic information was 5.6 fold longer than the corresponding alpha 2 mRNA coding sequences. Images PMID:7433091

  16. cDNA cloning and disruption of the major vault protein alpha gene (mvpA) in Dictyostelium discoideum.

    PubMed

    Vasu, S K; Kedersha, N L; Rome, L H

    1993-07-25

    Vaults are large cytoplasmic ribonucleoprotein particles found in nearly all eukaryotic cells. Dictyostelium vaults contain two major proteins, MVP alpha (94.2 kDa) and MVP beta (approximately 92 kDa). Using an anti-rat vault antibody, we screened a Dictyostelium cDNA expression library and isolated a 2.8-kilobase pair clone that contained a single full-length reading frame. The identity of the clone was established by the presence of a predicted 20-amino acid sequence identical to that found in a peptide sequenced from purified MVP alpha. We have disrupted the single copy gene using homologous recombination and have demonstrated a loss of MVP alpha. Although the cells still produce MVP beta, they do not contain characteristic vault particles, suggesting that MVP alpha is required for normal vault structure. These cells should be a valuable tool for elucidating the function of vaults. PMID:8340365

  17. Transduction-like gene transfer in the methanogen Methanococcus voltae

    NASA Technical Reports Server (NTRS)

    Bertani, G.

    1999-01-01

    Strain PS of Methanococcus voltae (a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (voltae transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the M. voltae cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 x 10(-5) (BES resistance) to a maximum of 10(-3) (histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the AluI restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of M. voltae.

  18. Transduction-Like Gene Transfer in the Methanogen Methanococcus voltae

    PubMed Central

    Bertani, Giuseppe

    1999-01-01

    Strain PS of Methanococcus voltae (a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (voltae transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the M. voltae cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 × 10−5 (BES resistance) to a maximum of 10−3 (histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the AluI restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of M. voltae. PMID:10321998

  19. Calfection: a novel gene transfer method for suspension cells.

    PubMed

    Lindell, Jeanette; Girard, Philippe; Müller, Natalie; Jordan, Martin; Wurm, Florian

    2004-01-20

    We have developed a novel method called Calfection for gene delivery to and protein expression from suspension-cultivated mammalian cells. Plasmid DNA was simply diluted into a calcium chloride solution and then added to the cell culture for transfection. We evaluated and optimized this approach using suspension-adapted HEK293 cells grown in 12-well plates that were shaken on an orbital shaker. Highest expression levels were obtained when cells were transfected at a density of 5x10(5) cells/ml in the presence of 9 mM calcium and 5 microg/ml of plasmid DNA while maintaining a culture pH of 7.6 at the time of transfection. Suspension-adapted BHK 21 and CHO DG 44 cells could also be transfected using this method. Calfection differs from the widely known calcium phosphate coprecipitation technique. The physico-chemical composition of the DNA interacting complexes is not yet known. The transfection cocktail, DNA in a calcium chloride solution, remained highly efficient during long-term storage at temperatures ranging from room temperature to -80 degrees C. In contrast, calcium phosphate-DNA cocktails are only efficient for gene transfer when prepared fresh. Furthermore, passing the calcium-plasmid DNA mixture through a 0.2-microm filter did not compromise protein expression, whereas calcium phosphate-DNA coprecipitates were retained by the filter. High protein expression levels, a limited number of manipulations and the possibility to filter the cocktail make the Calfection approach suitable for both large-scale transfection in bioreactors and for high-throughput transfection experiments in microtiter plates. PMID:14746910

  20. Microbial Evolution Is in the Cards: Horizontal Gene Transfer in the Classroom

    ERIC Educational Resources Information Center

    Kagle, Jeanne; Hay, Anthony G.

    2007-01-01

    Horizontal gene transfer, the exchange of genetic material between bacteria, is a potentially important factor in the degradation of synthetic compounds introduced to the environment and in the acquisition of other characteristics including antibiotic resistance. This game-based activity illustrates the role of horizontal gene transfer in the…

  1. Sp1 trans-activates the murine H(+)-K(+)-ATPase alpha(2)-subunit gene.

    PubMed

    Yu, Zhiyuan; Li, Mei; Zhang, Dongyu; Xu, William; Kone, Bruce C

    2009-07-01

    The H(+)-K(+)-ATPase alpha(2) (HKalpha2) gene of the renal collecting duct and distal colon plays a central role in potassium and acid-base homeostasis, yet its transcriptional control remains poorly characterized. We previously demonstrated that the proximal 177 bp of its 5'-flanking region confers basal transcriptional activity in murine inner medullary collecting duct (mIMCD3) cells and that NF-kappaB and CREB-1 bind this region to alter transcription. In the present study, we sought to determine whether the -144/-135 Sp element influences basal HKalpha2 gene transcription in these cells. Electrophoretic mobility shift and supershift assays using probes for -154/-127 revealed Sp1-containing DNA-protein complexes in nuclear extracts of mIMCD3 cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that Sp1, but not Sp3, binds to this promoter region of the HKalpha2 gene in mIMCD3 cells in vivo. HKalpha2 minimal promoter-luciferase constructs with point mutations in the -144/-135 Sp element exhibited much lower activity than the wild-type promoter in transient transfection assays. Overexpression of Sp1, but not Sp3, trans-activated an HKalpha2 proximal promoter-luciferase construct in mIMCD3 cells as well as in SL2 insect cells, which lack Sp factors. Conversely, small interfering RNA knockdown of Sp1 inhibited endogenous HKalpha2 mRNA expression, and binding of Sp1 to chromatin associated with the proximal HKalpha2 promoter without altering the binding or regulatory influence of NF-kappaB p65 or CREB-1 on the proximal HKalpha2 promoter. We conclude that Sp1 plays an important and positive role in controlling basal HKalpha2 gene expression in mIMCD3 cells in vivo and in vitro. PMID:19420113

  2. Opposing action of estrogen receptors alpha and beta on cyclin D1 gene expression.

    PubMed

    Liu, Meng-Min; Albanese, Chris; Anderson, Carol M; Hilty, Kristin; Webb, Paul; Uht, Rosalie M; Price, Richard H; Pestell, Richard G; Kushner, Peter J

    2002-07-01

    Induction of cyclin D1 gene transcription by estrogen receptor alpha (ERalpha) plays an important role in estrogen-mediated proliferation. There is no classical estrogen response element in the cyclin D1 promoter, and induction by ERalpha has been mapped to an alternative response element, a cyclic AMP-response element at -57, with possible participation of an activating protein-1 site at -954. The action of ERbeta at the cyclin D1 promoter is unknown, although evidence suggests that ERbeta may inhibit the proliferative action of ERalpha. We examined the response of cyclin D1 promoter constructs by luciferase assay and the response of the endogenous protein by Western blot in HeLa cells transiently expressing ERalpha, ERalphaK206A (a derivative that is superactive at alternative response elements), or ERbeta. In each case, ER activation at the cyclin D1 promoter is mediated by both the cyclic AMP-response element and the activating protein-1 site, which play partly redundant roles. The activation by ERbeta occurs only with antiestrogens. Estrogens, which activate cyclin D1 gene expression with ERalpha, inhibit expression with ERbeta. Strikingly, the presence of ERbeta completely inhibits cyclin D1 gene activation by estrogen and ERalpha or even by estrogen and the superactive ERalphaK206A. The observation of the opposing action and dominance of ERbeta over ERalpha in activation of cyclin D1 gene expression has implications for the postulated role of ERbeta as a modulator of the proliferative effects of estrogen. PMID:11986316

  3. Targeting Mycobacterium tuberculosis Tumor Necrosis Factor Alpha-Downregulating Genes for the Development of Antituberculous Vaccines

    PubMed Central

    Olsen, Aaron; Chen, Yong; Ji, Qingzhou; Zhu, Guofeng; De Silva, Aruna Dharshan; Vilchèze, Catherine; Weisbrod, Torin; Li, Weimin; Xu, Jiayong; Larsen, Michelle; Zhang, Jinghang; Porcelli, Steven A.; Jacobs, William R.

    2016-01-01

    ABSTRACT Tumor necrosis factor alpha (TNF) plays a critical role in the control of Mycobacterium tuberculosis, in part by augmenting T cell responses through promoting macrophage phagolysosomal fusion (thereby optimizing CD4+ T cell immunity by enhancing antigen presentation) and apoptosis (a process that can lead to cross-priming of CD8+ T cells). M. tuberculosis can evade antituberculosis (anti-TB) immunity by inhibiting host cell TNF production via expression of specific mycobacterial components. We hypothesized that M. tuberculosis mutants with an increased capacity to induce host cell TNF production (TNF-enhancing mutants) and thus with enhanced immunogenicity can be useful for vaccine development. To identify mycobacterial genes that regulate host cell TNF production, we used a TNF reporter macrophage clone to screen an H37Rv M. tuberculosis cosmid library constructed in M. smegmatis. The screen has identified a set of TNF-downregulating mycobacterial genes that, when deleted in H37Rv, generate TNF-enhancing mutants. Analysis of mutants disrupted for a subset of TNF-downregulating genes, annotated to code for triacylglycerol synthases and fatty acyl-coenzyme A (acyl-CoA) synthetase, enzymes that concern lipid biosynthesis and metabolism, has revealed that these strains can promote macrophage phagolysosomal fusion and apoptosis better than wild-type (WT) bacilli. Immunization of mice with the TNF-enhancing M. tuberculosis mutants elicits CD4+ and CD8+ T cell responses that are superior to those engendered by WT H37Rv. The results suggest that TNF-upregulating M. tuberculosis genes can be targeted to enhance the immunogenicity of mycobacterial strains that can serve as the substrates for the development of novel anti-TB vaccines. PMID:27247233

  4. Cloning of the LamA3 gene encoding the alpha 3 chain of the adhesive ligand epiligrin. Expression in wound repair.

    PubMed

    Ryan, M C; Tizard, R; VanDevanter, D R; Carter, W G

    1994-09-01

    We have isolated cDNA clones encoding the entire 170-kDa chain of epiligrin (alpha 3Ep) and a genomic clone encoding the alpha 3Ep gene (LamA3). Analysis of multiple cDNA clones revealed two distinct transcripts (alpha 3EpA and alpha 3EpB). Sequencing of the alpha 3EpA transcript indicated sequence and structural homology to laminin alpha 1 and alpha 2 chains that extend from domain IIIa through the carboxyl-terminal G domain. The alpha 3EpB transcript encodes a larger amino-terminal domain and contains additional epidermal growth factor repeats and sequences corresponding to domain IV of alpha 1 laminin. Fluorescence in situ hybridization indicated that the LamA3 gene is located on chromosome 18q11.2, a locus distinct from the LamA1 gene (18p11.3). The G domain of the epiligrin alpha 3 chain contains five subdomains that are individually related to the G subdomains reported for Drosophila and vertebrate laminin alpha chains. Sequence divergence within the G domain of alpha 3 epiligrin suggests that it is functionally distinct from laminin, consistent with our previous report showing that epiligrin interacts with different integrin adhesion receptors. Analysis of RNA from human foreskin keratinocytes (HFKs) identified multiple epiligrin transcripts that were down-regulated by viral transformation and differentiation. In contrast, epiligrin expression was up-regulated in wound sites of human skin. PMID:8077230

  5. The spectrum of low molecular weight alpha-amylase/protease inhibitor genes expressed in the US bread wheat Butte 86

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complement of genes encoding alpha-amylase/protease inhibitors expressed in Triticum aestivum cv. Butte 86 was characterized by transcript and proteomic analysis. Coding sequences for 18 distinct proteins were identified among a collection of expressed sequence tags (ESTs) from Butte 86 developi...

  6. The significance and effect of tandem repeats within the Mycobacterium tuberculosis leuA gene on alpha-isopropylmalate synthase.

    PubMed

    Chanchaem, Wimon; Palittapongarnpim, Prasit

    2008-09-01

    The 57-bp tandem repeats located in the Mycobacterium tuberculosis leuA gene code for the alpha-isopropylmalate synthase (alpha-IPMS). It is unique to this pathogen. It was previously demonstrated that the leuA-coding sequence Rv3710, containing the tandem repeats, can be translated to an active alpha-IPMS. The objective of the present study was to investigate the significance and effect of the two 57-bp tandem repeats upon gene expression and the general properties of alpha-IPMS. The putative M. tuberculosis H37Rv leuA gene with and without the tandem repeats was cloned by PCR and expressed in an Escherichia coli host. The enzyme product was studied for general properties, comparing that from a native leuA gene containing two repeats and that from the 57-bp tandem repeats deletion mutant. Upon deletion of the two 57-bp tandem repeats, the expression level of leuA from M. tuberculosis H37Rv was comparable with that of the native form. The general properties of the two types of enzymes were similar. They were both functional with the same range of optimal temperature and optimal pH for activity and with similar enzyme stability. Deletion of the repeats had no detectable effect on leuA expression level or the general properties of the enzyme product. PMID:18647358

  7. Horizontal gene transfer in the acquisition of novel traits by metazoans

    PubMed Central

    Boto, Luis

    2014-01-01

    Horizontal gene transfer is accepted as an important evolutionary force modulating the evolution of prokaryote genomes. However, it is thought that horizontal gene transfer plays only a minor role in metazoan evolution. In this paper, I critically review the rising evidence on horizontally transferred genes and on the acquisition of novel traits in metazoans. In particular, I discuss suspected examples in sponges, cnidarians, rotifers, nematodes, molluscs and arthropods which suggest that horizontal gene transfer in metazoans is not simply a curiosity. In addition, I stress the scarcity of studies in vertebrates and other animal groups and the importance of forthcoming studies to understand the importance and extent of horizontal gene transfer in animals. PMID:24403327

  8. Gene transfer in the evolution of parasite nucleotide biosynthesis.

    PubMed

    Striepen, Boris; Pruijssers, Andrea J P; Huang, Jinling; Li, Catherine; Gubbels, Marc-Jan; Umejiego, Nwakaso N; Hedstrom, Lizbeth; Kissinger, Jessica C

    2004-03-01

    Nucleotide metabolic pathways provide numerous successful targets for antiparasitic chemotherapy, but the human pathogen Cryptosporidium parvum thus far has proved extraordinarily refractory to classical treatments. Given the importance of this protist as an opportunistic pathogen afflicting immunosuppressed individuals, effective treatments are urgently needed. The genome sequence of C. parvum is approaching completion, and we have used this resource to critically assess nucleotide biosynthesis as a target in C. parvum. Genomic analysis indicates that this parasite is entirely dependent on salvage from the host for its purines and pyrimidines. Metabolic pathway reconstruction and experimental validation in the laboratory further suggest that the loss of pyrimidine de novo synthesis is compensated for by possession of three salvage enzymes. Two of these, uridine kinase-uracil phosphoribosyltransferase and thymidine kinase, are unique to C. parvum within the phylum Apicomplexa. Phylogenetic analysis suggests horizontal gene transfer of thymidine kinase from a proteobacterium. We further show that the purine metabolism in C. parvum follows a highly streamlined pathway. Salvage of adenosine provides C. parvum's sole source of purines. This renders the parasite susceptible to inhibition of inosine monophosphate dehydrogenase, the rate-limiting enzyme in the multistep conversion of AMP to GMP. The inosine 5' monophosphate dehydrogenase inhibitors ribavirin and mycophenolic acid, which are already in clinical use, show pronounced anticryptosporidial activity. Taken together, these data help to explain why widely used drugs fail in the treatment of cryptosporidiosis and suggest more promising targets. PMID:14973196

  9. Neprilysin gene transfer: A promising therapeutic approach for Alzheimer's disease.

    PubMed

    Li, Yuanli; Wang, Junqing; Zhang, Shenghao; Liu, Zhaohui

    2015-09-01

    Alzheimer's disease (AD) is characterized by widespread neurodegeneration throughout the association cortex and limbic system, deposition of amyloid-β (Aβ) in the neuropil and around blood vessels, and formation of neurofibrillary tangles. Aβ accumulation is considered the major pathological change in AD progression. In recent years, several therapeutic strategies for treating AD have focused on reducing the Aβ burden in the brain. Among these approaches, the expression of Aβ-degrading enzymes in the brain has been effective but, so far, impractical for treating patients. Neprilysin (NEP), the most prominent of the Aβ-degrading enzymes in vivo, has been successfully delivered intracranially by viral vectors and is a promising therapeutic approach for reducing Aβ accumulation and treating AD. However, some challenges are associated with the use of viral and nonviral vectors, including secondary toxicity, activation of the immune response, and low efficiency. Therefore, safe and efficient NEP delivery systems that could avoid the viral problems with minor injury and high transfection efficiency are required to deliver AD medical applications. This Mini-Review summarizes NEP gene transfer technologies that use viral and nonviral vectors and discusses the rationale and benefits of these delivery systems for AD treatment trials, providing a reference for basic and clinical studies on AD. PMID:26096375

  10. Gene transfer in the evolution of parasite nucleotide biosynthesis

    PubMed Central

    Striepen, Boris; Pruijssers, Andrea J. P.; Huang, Jinling; Li, Catherine; Gubbels, Marc-Jan; Umejiego, Nwakaso N.; Hedstrom, Lizbeth; Kissinger, Jessica C.

    2004-01-01

    Nucleotide metabolic pathways provide numerous successful targets for antiparasitic chemotherapy, but the human pathogen Cryptosporidium parvum thus far has proved extraordinarily refractory to classical treatments. Given the importance of this protist as an opportunistic pathogen afflicting immunosuppressed individuals, effective treatments are urgently needed. The genome sequence of C. parvum is approaching completion, and we have used this resource to critically assess nucleotide biosynthesis as a target in C. parvum. Genomic analysis indicates that this parasite is entirely dependent on salvage from the host for its purines and pyrimidines. Metabolic pathway reconstruction and experimental validation in the laboratory further suggest that the loss of pyrimidine de novo synthesis is compensated for by possession of three salvage enzymes. Two of these, uridine kinase-uracil phosphoribosyltransferase and thymidine kinase, are unique to C. parvum within the phylum Apicomplexa. Phylogenetic analysis suggests horizontal gene transfer of thymidine kinase from a proteobacterium. We further show that the purine metabolism in C. parvum follows a highly streamlined pathway. Salvage of adenosine provides C. parvum's sole source of purines. This renders the parasite susceptible to inhibition of inosine monophosphate dehydrogenase, the rate-limiting enzyme in the multistep conversion of AMP to GMP. The inosine 5′ monophosphate dehydrogenase inhibitors ribavirin and mycophenolic acid, which are already in clinical use, show pronounced anticryptosporidial activity. Taken together, these data help to explain why widely used drugs fail in the treatment of cryptosporidiosis and suggest more promising targets. PMID:14973196

  11. The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1 alpha: molecular cloning, characterization and expression.

    PubMed

    Axelos, M; Bardet, C; Liboz, T; Le Van Thai, A; Curie, C; Lescure, B

    1989-10-01

    The gene family encoding the Arabidopsis thaliana translation elongation factor (EF-1 alpha) was analysed. This family contains four genes (A1-A4) organized in a similar manner in different varieties of Arabidopsis. Based upon both their physical separation and a comparison of their sequences, it is suggested that the A4 gene and the A1, A2, and A3 genes constitute two distinct subfamilies within the genome. By introducing chimaeric gene constructs into Arabidopsis cells, we showed that the A1 gene promoter mediates a transient expression about twofold higher than that obtained using the CaMV 35 S promoter. This expression depends on a 348 bp DNA fragment extending from -982 to -634 bp upstream of the initiation codon. This element contains a characteristic telomeric sequence (AACCCTAA) which is also found in the promoters of the A2 and A4 genes as well as in the promoters of the Drosophila EF-1 alpha F1 gene and of several highly expressed plant genes. PMID:2615757

  12. Cloning and Characterization of an alpha-amylase Gene from the Hyperthermophilic Archaeon Thermococcus Thioreducens

    NASA Technical Reports Server (NTRS)

    Bernhardsdotter, Eva C. M. J.; Pusey, Mark L.; Ng, Joseph D.; Garriott, Owen K.

    2004-01-01

    The gene encoding an extracellular alpha-amylase, TTA, from the hyperthermophilic archaeon Thermococcus thioreducens was cloned and expressed in Escherichia coli. Primary structural analysis revealed high similarity with other a-amylases from the Thermococcus and Pyrococcus genera, as well as the four highly conserved regions typical for a-amylases. The 1374 bp gene encodes a protein of 457 amino acids, of which 435 constitute the mature protein preceded by a 22 amino acid signal peptide. The molecular weight of the purified recombinant enzyme was estimated to be 43 kDa by denaturing gel electrophoresis. Maximal enzymatic activity of recombinant TTA was observed at 90 C and pH 5.5 in the absence of exogenous Ca(2+), and the enzyme was considerably stable even after incubation at 90 C for 2 hours. The thermostability at 90 and 102 C was enhanced in the presence of 5 mM Ca(2+). The extraordinarily high specific activity (about 7.4 x 10(exp 3) U/mg protein at 90 C, pH 5.5 with soluble starch as substrate) together with its low pH optimum makes this enzyme an interesting candidate for starch processing applications.

  13. Ancient allelism at the cytosolic chaperonin-alpha-encoding gene of the zebrafish.

    PubMed Central

    Takami, K; Figueroa, F; Mayer, W E; Klein, J

    2000-01-01

    The T-complex protein 1, TCP1, gene codes for the CCT-alpha subunit of the group II chaperonins. The gene was first described in the house mouse, in which it is closely linked to the T locus at a distance of approximately 11 cM from the Mhc. In the zebrafish, Danio rerio, in which the T homolog is linked to the class I Mhc loci, the TCP1 locus segregates independently of both the T and the Mhc loci. Despite its conservation between species, the zebrafish TCP1 locus is highly polymorphic. In a sample of 15 individuals and the screening of a cDNA library, 12 different alleles were found, and some of the allelic pairs were found to differ by up to nine nucleotides in a 275-bp-long stretch of sequence. The substitutions occur in both translated and untranslated regions, but in the former they occur predominantly at synonymous codon sites. Phylogenetically, the alleles fall into two groups distinguished also by the presence or absence of a 10-bp insertion/deletion in the 3' untranslated region. The two groups may have diverged as long as 3.5 mya, and the polymorphic differences may have accumulated by genetic drift in geographically isolated populations. PMID:10628990

  14. Three missense variants of metabolic syndrome-related genes are associated with alpha-1 antitrypsin levels

    PubMed Central

    Setoh, Kazuya; Terao, Chikashi; Muro, Shigeo; Kawaguchi, Takahisa; Tabara, Yasuharu; Takahashi, Meiko; Nakayama, Takeo; Kosugi, Shinji; Sekine, Akihiro; Yamada, Ryo; Mishima, Michiaki; Matsuda, Fumihiko

    2015-01-01

    Alpha-1 antitrypsin (AAT) encoded by SERPINA1 is an acute-phase inflammation marker, and AAT deficiency (AATD) is known as one of the common genetic disorders in European populations. However, no genetic determinants to AAT levels apart from the SERPINA gene clusters have been identified to date. Here we perform a genome-wide association study of serum AAT levels followed by a two-staged replication study recruiting a total of 9,359 Japanese community-dwelling population. Three missense variants of metabolic syndrome-related genes, namely, rs671 in ALDH2, rs1169288 in HNF1A and rs1260326 in GCKR, significantly associate with AAT levels (P≤1.5 × 10−12). Previous reports have shown the functional relevance of ALDH2 and HNF1A to AAT. We observe a significant interaction of rs671 and alcohol consumption on AAT levels. We confirm the association between AAT and rs2896268 in SERPINA1, which is independent of known causative variants of AATD. These findings would support various AAT functions including metabolic processes. PMID:26174136

  15. Cell type-specific transcriptional regulation of the gene encoding importin-{alpha}1

    SciTech Connect

    Kamikawa, Yasunao; Yasuhara, Noriko; Yoneda, Yoshihiro; Department of Biochemistry, Graduate School of Medicine, Osaka University; JST, CREST, Graduate School of Frontier Biosciences, Osaka University, Yamada-oka, Suita, Osaka 565-0871

    2011-08-15

    Importin-{alpha}1 belongs to a receptor family that recognizes classical nuclear localization signals. Encoded by Kpna2, this receptor subtype is highly expressed in mouse embryonic stem (ES) cells. In this study, we identified a critical promoter region in Kpna2 and showed that the expression of this gene is differentially regulated in ES cells and NIH3T3 cells. Conserved CCAAT boxes are required for Kpna2 promoter activity in both ES and NIH3T3 cells. Interestingly, deletion of the region from nucleotide position - 251 to - 179 bp resulted in a drastic reduction in Kpna2 transcriptional activity only in ES cells. This region contains Krueppel-like factor (Klf) binding sequences and is responsible for transactivation of the gene by Klf2 and Klf4. Accordingly, endogenous Kpna2 mRNA levels decreased in response to depletion of Klf2 and Klf4 in ES cells. Our results suggest that Klf2 and Klf4 function redundantly to drive high level of Kpna2 expression in ES cells. -- Research Highlights: {yields} We showed the cell type-specific transcriptional regulation of Kpna2 encoding importin-al. {yields} NF-Y binds the CCAAT boxes to activate Kpna2 transcription in NIH3T3 cells. {yields} Klf2 and Klf4 redundantly activate the expression of Kpna2 in ES cells.

  16. Estrogen Receptor Alpha (ESR1) Gene Polymorphisms in Pre-eclamptic Saudi Patients

    PubMed Central

    El-Beshbishy, Hesham A.; Tawfeek, Manal A.; Al-Azhary, Nevin M.; Mariah, Reham A.; Habib, Fawzia A.; Aljayar, Lamya; Alahmadi, Abrar F.

    2015-01-01

    Objectives: Pre-eclampsia causes maternal mortality worldwide. Estrogen receptor alpha (ESR1) gene polymorphisms were responsible for cardiovascular diseases. This case control study was conducted to investigate whether 2 polymorphic genes of ESR1 are associated with pre-eclampsia among Saudi women in Madina city, Saudi Arabia. Methods: Blood samples from 97 pre-eclamptic and 94 healthy pregnant women were analyzed using restriction fragment length polymorphism-polymerase chain reaction method. All the subjects were recruited randomly from outpatient clinics of Madina Maternity Children Hospital (MMCH), Madina, Saudi Arabia, between Dec. 2012 and Jan. 2014. Results: There was no association between pre-eclampsia and PvuII and XbaI ESR1 gene polymorphisms individually. TT/AA and TT/AG genotype combination existed significantly in pre-eclamptic patients compared to control. The frequency of PvuII and XbaI combined TT/AA genotypes between pre-eclamptic women was 36.1% vs 9.6%, however, frequency of PvuII and XbaI combined TT/AG genotypes between pre-eclamptic women was 3.1% vs 17%, compared to control. The homozygous T-A haplotype carriers showed high pre-eclampsia risk, independent of pregnancy, BMI and smoking status (adjusted odds ratio (OR): 3.26, 95% confidence interval (CI):1.71-9.21). The heterozygous T-A haplotype carriers did not differ from that of non-carriers (adjusted OR: 1.12, 95% CI: 0.47-2.75). No association was observed between pre-eclampsia and T-G, C-G and C-A haplotype of PvuII and XbaIESR1 gene polymorphisms. Conclusions: T-A haplotype of homozygous associated with pre eclampsia not heterozygous carriers of ESR 1 PvuII and XbaI gene polymorphisms elicited high risk of pre-eclampsia. GG genotype of XbaI polymorphism decreased pre-eclampsia risk. Further studies using larger sample size are recommended to investigate the ESR 1 gene polymorphisms associated with pre-eclampsia. PMID:26430422

  17. Identification of the class I genes of the mouse major histocompatibility complex by DNA-mediated gene transfer.

    PubMed

    Goodenow, R S; McMillan, M; Nicolson, M; Sher, B T; Eakle, K; Davidson, N; Hood, L

    1982-11-18

    DNA-mediated gene transfer was used to identify cloned class I genes from the major histocompatibility complex of the BALB/c mouse. Three genes encoding the transplantation antigens H-2 Kd, Dd and Ld were identified as well as genes encoding the Qa-2,3 and two TL differentiation antigens. As many as 10 putative novel class I genes were detected by the association of their gene products with beta 2-microglobulin. Alloantiserum prepared to one of the novel antigens was used to demonstrate the expression of the previously undetected antigen on spleen cells of various inbred, congeneic, and recombinant congeneic strains of mice. PMID:6815535

  18. Cultured Ito cells of rat liver express the alpha 2-macroglobulin gene.

    PubMed

    Andus, T; Ramadori, G; Heinrich, P C; Knittel, T; Meyer zum Büschenfelde, K H

    1987-11-01

    Ito cells were isolated from rat liver and kept in culture for up to 13 days. The capability of the Ito cells to synthesize alpha 2-macroglobulin was analyzed at different times after isolation and by pulse-chase experiments. Newly synthesized alpha 2-macroglobulin was determined by immunoprecipitation followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. alpha 2-Macroglobulin synthesis was hardly detectable in Ito cells and their media 3 days after plating. However, 5-11 days after the isolation of the cells, increasing amounts of alpha 2-macroglobulin were synthesized. The results of pulse-chase experiments performed on day 7 showed that radioactively labeled alpha 2-macroglobulin decreased in the intracellular compartment and increased in the culture medium. alpha 2-Macroglobulin was identified by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions. Furthermore, when unlabeled alpha 2-macroglobulin was added during the immunoprecipitation, a competition was observed. Incubation of pancreatic elastase with culture medium of rat Ito cells or rat hepatocytes led to the same cleavage products as found with alpha 2-macroglobulin. alpha 2-Macroglobulin-specific mRNA could be demonstrated by Northern blot analysis of total RNA extracted from rat Ito cells. Under the conditions where alpha 2-macroglobulin was synthesized in Ito cells, no synthesis of alpha 1-macroglobulin, alpha 1-inhibitor 3, alpha 1-proteinase inhibitor, alpha 1-acid glycoprotein, alpha 1-acute-phase globulin (T-kininogen) and albumin could be demonstrated. It is concluded that alpha 2-macroglobulin is a true secretory protein of rat Ito cells in culture. This could be of importance for collagen metabolism in liver diseases. PMID:2444437

  19. Hepatocyte nuclear factor-1alpha is required for expression but dispensable for histone acetylation of the lactase-phlorizin hydrolase gene in vivo.

    PubMed

    Bosse, Tjalling; van Wering, Herbert M; Gielen, Marieke; Dowling, Lauren N; Fialkovich, John J; Piaseckyj, Christina M; Gonzalez, Frank J; Akiyama, Taro E; Montgomery, Robert K; Grand, Richard J; Krasinski, Stephen D

    2006-05-01

    Hepatocyte nuclear factor-1alpha (HNF-1alpha) is a modified homeodomain-containing transcription factor that has been implicated in the regulation of intestinal genes. To define the importance and underlying mechanism of HNF-1alpha for the regulation of intestinal gene expression in vivo, we analyzed the expression of the intestinal differentiation markers and putative HNF-1alpha targets lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) in hnf1alpha null mice. We found that in adult jejunum, LPH mRNA in hnf1alpha(-/-) mice was reduced 95% compared with wild-type controls (P < 0.01, n = 4), whereas SI mRNA was virtually identical to that in wild-type mice. Furthermore, SI mRNA abundance was unchanged in the absence of HNF-1alpha along the length of the adult mouse small intestine as well as in newborn jejunum. We found that HNF-1alpha occupies the promoters of both the LPH and SI genes in vivo. However, in contrast to liver and pancreas, where HNF-1alpha regulates target genes by recruitment of histone acetyl transferase activity to the promoter, the histone acetylation state of the LPH and SI promoters was not affected by the presence or absence of HNF-1alpha. Finally, we showed that a subset of hypothesized intestinal target genes is regulated by HNF-1alpha in vivo and that this regulation occurs in a defined tissue-specific and developmental context. These data indicate that HNF-1alpha is an activator of a subset of intestinal genes and induces these genes through an alternative mechanism in which it is dispensable for chromatin remodeling. PMID:16223943

  20. Cytotoxicity associated with artemis overexpression after lentiviral vector-mediated gene transfer.

    PubMed

    Multhaup, Megan; Karlen, Andrea D; Swanson, Debra L; Wilber, Andrew; Somia, Nikunj V; Cowan, Morton J; McIvor, R Scott

    2010-07-01

    Artemis is a hairpin-opening endonuclease involved in nonhomologous end-joining and V(D)J recombination. Deficiency of Artemis results in radiation-sensitive severe combined immunodeficiency (SCID) characterized by complete absence of T and B cells due to an arrest at the receptor recombination stage. We have generated several lentiviral vectors for transduction of the Artemis sequence, intending to complement the deficient phenotype. We found that transduction by a lentiviral vector in which Artemis is regulated by a strong EF-1alpha promoter resulted in a dose-dependent loss of cell viability due to perturbed cell cycle distribution, increased DNA damage, and increased apoptotic cell frequency. This toxic response was not observed in cultures exposed to identical amounts of control vector. Loss of cell viability was also observed in cells transfected with an Artemis expression construct, indicating that toxicity is independent of lentiviral transduction. Reduced toxicity was observed when cells were transduced with a moderate-strength phosphoglycerate kinase promoter to regulate Artemis expression. These results present a novel challenge in the establishment of conditions that support Artemis expression at levels that are nontoxic yet sufficient to correct the T(-)B(-) phenotype, crucial for preclinical studies and clinical application of Artemis gene transfer in the treatment of human SCID-A. PMID:20163250

  1. The human intestinal fatty acid binding protein (hFABP2) gene is regulated by HNF-4{alpha}

    SciTech Connect

    Klapper, Maja . E-mail: klapper@molnut.uni-kiel.de; Boehme, Mike; Nitz, Inke; Doering, Frank

    2007-04-27

    The cytosolic human intestinal fatty acid binding protein (hFABP2) is proposed to be involved in intestinal absorption of long-chain fatty acids. The aim of this study was to investigate the regulation of hFABP2 by the endodermal hepatocyte nuclear factor 4{alpha} (HNF-4{alpha}), involved in regulation of genes of fatty acid metabolism and differentiation. Electromobility shift assays demonstrated that HNF-4{alpha} binds at position -324 to -336 within the hFABP2 promoter. Mutation of this HNF-4 binding site abolished the luciferase reporter activity of hFABP2 in postconfluent Caco-2 cells. In HeLa cells, this mutation reduced the activation of the hFABP2 promoter by HNF-4{alpha} by about 50%. Thus, binding element at position -336/-324 essentially determines the transcriptional activity of promoter and may be important in control of hFABP2 expression by dietary lipids and differentiation. Studying genotype interactions of hFABP2 and HNF-4{alpha}, that are both candidate genes for diabetes type 2, may be a powerful approach.

  2. Association between estrogen receptor alpha (ESR1) gene polymorphisms and severe preeclampsia.

    PubMed

    Molvarec, Attila; Vér, Agota; Fekete, Andrea; Rosta, Klára; Derzbach, László; Derzsy, Zoltán; Karádi, István; Rigó, János

    2007-03-01

    Associations have been reported between estrogen receptor alpha (ESR1) gene polymorphisms and various pathological conditions, including cardiovascular diseases. Our aim was to investigate whether two polymorphisms of the ESR1 gene (ESR1 c.454 -397T>C: PvuII restriction site and c.454 -351A>G: XbaI restriction site) are associated with preeclampsia. In a case-control study, we analyzed blood samples from 119 severely preeclamptic patients and 103 normotensive, healthy pregnant women using the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. All of the women were Caucasian. There was no association between severe preeclampsia and the PvuII and XbaI ESR1 gene polymorphisms separately. However, with the simultaneous carriage of both polymorphisms, the TT/AA genotype combination was significantly more frequent in severely preeclamptic patients than in healthy control subjects (24.4% vs. 9.7%, p=0.003), whereas the TT/AG combination was significantly less frequent in the severely preeclamptic group than in the control group (5.0% vs. 18.4%, p=0.002). According to the haplotype estimation, the homozygous T-A haplotype carriers had an increased risk of severe preeclampsia independent of maternal age, prepregnancy BMI, primiparity and smoking status (adjusted odds ratio [OR]: 4.36, 95% confidence interval [CI]: 1.65-11.53). The GG genotype of the XbaI polymorphism was associated with a lower risk of fetal growth restriction in patients with severe preeclampsia (OR: 0.23, 95% CI: 0.07-0.73). In conclusion, the homozygous T-A haplotype carriers of ESR1 PvuII and XbaI polymorphisms showed an increased risk of severe preeclampsia. In addition, the GG genotype of the XbaI polymorphism decreased the risk of fetal growth restriction in severely preeclamptic patients. PMID:17510501

  3. Induction of liver alpha-1 acid glycoprotein gene expression involves both positive and negative transcription factors.

    PubMed Central

    Lee, Y M; Tsai, W H; Lai, M Y; Chen, D S; Lee, S C

    1993-01-01

    Expression of the alpha-1 acid glycoprotein (AGP) gene is liver specific and acute phase responsive. Within the 180-bp region of the AGP promoter, at least five cis elements have been found to interact with trans-acting factors. Four of these elements (A, C, D, and E) interacted with AGP/EBP, a liver-enriched transcription factor, as shown by footprinting analysis and by an anti-AGP/EBP antibody-induced supershift in a gel retardation assay. Modification of these sites by site-directed mutagenesis coupled with transfection analysis indicated that AGP/EBP binding to all of these sites resulted in positive regulation of the promoter. Dose-response data suggest that AGP/EBP binding to these sites results in the cooperative activation of the promoter. In contrast, functional assays showed that element B is a negative regulatory element; this element is recognized by heat-stable DNA-binding factors which are found in many cells and tissues. The regulation of these binding proteins was studied in rat liver treated with lipopolysaccharide (LPS), which induced an acute-phase reaction. We found that LPS treatment resulted in a two- to threefold increase in AGP/EBP activity and a severalfold decrease in the activity of factors that bind to element B in the liver. These results indicate that expression of the AGP gene can be regulated by both positive and negative factors and that the modulation of these factors can account for the LPS induction of the AGP gene. Images PMID:8417341

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

    PubMed

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

    1983-01-01

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

  5. Non-Viral Gene Transfer as a Tool for Studying Transcription Regulation of Xenobiotic Metabolizing Enzymes

    PubMed Central

    Bonamassa, Barbara; Liu, Dexi

    2010-01-01

    Numerous xenobiotic metabolizing enzymes are regulated by nuclear receptors at transcriptional level. The challenge we currently face is to understand how a given nuclear receptor interacts with its xenobiotics, migrates into nucleus, binds to the xenobiotic response element of a target gene, and regulates transcription. Toward this end, new methods have been developed to introduce the nuclear receptor gene into appropriate cells and study its activity in activating reporter gene expression under the control of a promoter containing xenobiotic response elements. The goal of this review is to critically examine the gene transfer methods currently available. We concentrate on the gene transfer mechanism, advantages and limitations of each method when employed for nuclear receptor-mediated gene regulation studies. It is our hope that the information provided highlights the importance of gene transfer in studying the mechanisms by which our body eliminates the potentially harmful substances and maintains the homeostasis. PMID:20713102

  6. Tumor necrosis factor-alpha gene and protein expression in adult feline myocardium after endotoxin administration.

    PubMed Central

    Kapadia, S; Lee, J; Torre-Amione, G; Birdsall, H H; Ma, T S; Mann, D L

    1995-01-01

    TNF alpha mRNA and protein biosynthesis were examined in the adult feline heart after stimulation with endotoxin. When freshly isolated hearts were stimulated with endotoxin in vitro, de novo TNF alpha mRNA expression occurred within 30 min, and TNF alpha protein production was detected within 60-75 min; however, TNF alpha mRNA and protein production were not detected in diluent-treated hearts. Immunohistochemical studies localized TNF alpha to endothelial cells, smooth muscle cells, and cardiac myocytes in the endotoxin-treated hearts, whereas TNF alpha immunostaining was absent in the diluent-treated hearts. To determine whether the cardiac myocyte was a source for TNF alpha production, two studies were performed. First, in situ hybridization studies, using highly specific biotinylated probes, demonstrated TNF alpha mRNA in cardiac myocytes from endotoxin-stimulated hearts; in contrast, TNF alpha mRNA was not expressed in myocytes from diluent-treated hearts. Second, TNF alpha protein production was observed when cultured cardiac myocytes were stimulated with endotoxin, whereas TNF alpha protein production was not detected in the diluent-treated cells. The functional significance of the intramyocardial production of TNF alpha was determined by examining cell motion in isolated cardiac myocytes treated with superfusates from endotoxin- and diluent-stimulated hearts. These studies showed that cell motion was depressed in myocytes treated with superfusates from the endotoxin-treated hearts, but was normal with the superfusates from the diluent-treated hearts; moreover, the negative inotropic effects of the superfusates from the endotoxin-treated hearts could be abrogated completely by pretreatment with an anti-TNF alpha antibody. Finally, endotoxin stimulation was also shown to result in the intramyocardial production of TNF alpha mRNA and protein in vivo. Thus, this study shows for the first time that the adult mammalian myocardium synthesizes biologically active

  7. Human and rat mast cell high-affinity immunoglobulin E receptors: Characterization of putative. alpha. -chain gene products

    SciTech Connect

    Shimizu, Akira; Benfey, P.N.; Leder, P. ); Tepler, I. Brigham and Women's Hospital, Boston, MA ); Berenstein, E.H.; Siraganian, R.P. )

    1988-03-01

    The authors have cloned and determined the entire nucleotide sequence of cDNAs corresponding to the putative {alpha} subunits of the human and rat mast cell high-affinity IgE receptors. Both human and rat cDNAs encode an NH{sub 2}-terminal signal peptide, two immunoglobulin-like extracellular domains (encoded by discrete exons), a hydrophobic transmembrane region, and a positively charged cytoplasmic tail. The human and rat {alpha} subunits share an overall homology with one another and the immunoglobulin gene family, suggesting that they arose from a common ancestral gene and continue to share structural homology with their ligands. In addition, the rat gene is transcribed into at least three distinct forms, each of which yields a somewhat different coding sequence.

  8. Alpha Thalassemia

    MedlinePlus

    ... an apparently normal individual has a child with hemoglobin H disease or alpha thalassemia minor. It can ... gene on one chromosome 25% 25% 25% 25% hemoglobin H disease there is a 25% chance with ...

  9. Apramycin resistance as a selective marker for gene transfer in mycobacteria.

    PubMed Central

    Paget, E; Davies, J

    1996-01-01

    We have explored the potential of using the apramycin resistance gene as a marker in mycobacterial gene transfer studies. Shuttle plasmids available for both electroporation and conjugation studies have been constructed, and we have successfully validated the use of the apramycin resistance gene as a component of cloning vectors for Mycobacterium smegmatis, M. bovis BCG, and M. tuberculosis. PMID:8892841

  10. Migration and horizontal gene transfer divide microbial genomes into multiple niches.

    PubMed

    Niehus, Rene; Mitri, Sara; Fletcher, Alexander G; Foster, Kevin R

    2015-01-01

    Horizontal gene transfer is central to microbial evolution, because it enables genetic regions to spread horizontally through diverse communities. However, how gene transfer exerts such a strong effect is not understood. Here we develop an eco-evolutionary model and show how genetic transfer, even when rare, can transform the evolution and ecology of microbes. We recapitulate existing models, which suggest that asexual reproduction will overpower horizontal transfer and greatly limit its effects. We then show that allowing immigration completely changes these predictions. With migration, the rates and impacts of horizontal transfer are greatly increased, and transfer is most frequent for loci under positive natural selection. Our analysis explains how ecologically important loci can sweep through competing strains and species. In this way, microbial genomes can evolve to become ecologically diverse where different genomic regions encode for partially overlapping, but distinct, ecologies. Under these conditions ecological species do not exist, because genes, not species, inhabit niches. PMID:26592443

  11. Migration and horizontal gene transfer divide microbial genomes into multiple niches

    PubMed Central

    Niehus, Rene; Mitri, Sara; Fletcher, Alexander G.; Foster, Kevin R.

    2015-01-01

    Horizontal gene transfer is central to microbial evolution, because it enables genetic regions to spread horizontally through diverse communities. However, how gene transfer exerts such a strong effect is not understood. Here we develop an eco-evolutionary model and show how genetic transfer, even when rare, can transform the evolution and ecology of microbes. We recapitulate existing models, which suggest that asexual reproduction will overpower horizontal transfer and greatly limit its effects. We then show that allowing immigration completely changes these predictions. With migration, the rates and impacts of horizontal transfer are greatly increased, and transfer is most frequent for loci under positive natural selection. Our analysis explains how ecologically important loci can sweep through competing strains and species. In this way, microbial genomes can evolve to become ecologically diverse where different genomic regions encode for partially overlapping, but distinct, ecologies. Under these conditions ecological species do not exist, because genes, not species, inhabit niches. PMID:26592443

  12. Proteomic profiling of salivary gland after nonviral gene transfer mediated by conventional plasmids and minicircles

    PubMed Central

    Geguchadze, Ramaz; Wang, Zhimin; Zourelias, Lee; Perez-Riveros, Paola; Edwards, Paul C; Machen, Laurie; Passineau, Michael J

    2014-01-01

    In this study, we compared gene transfer efficiency and host response to ultrasound-assisted, nonviral gene transfer with a conventional plasmid and a minicircle vector in the submandibular salivary glands of mice. Initially, we looked at gene transfer efficiency with equimolar amounts of the plasmid and minicircle vectors, corroborating an earlier report showing that minicircle is more efficient in the context of a physical method of gene transfer. We then sought to characterize the physiological response of the salivary gland to exogenous gene transfer using global proteomic profiling. Somewhat surprisingly, we found that sonoporation alone, without a gene transfer vector present, had virtually no effect on the salivary gland proteome. However, when a plasmid vector was used, we observed profound perturbations of the salivary gland proteome that compared in magnitude to that seen in a previous report after high doses of adeno-associated virus. Finally, we found that gene transfer with a minicircle induces only minor proteomic alterations that were similar to sonoporation alone. Using mass spectrometry, we assigned protein IDs to 218 gel spots that differed between plasmid and minicircle. Bioinformatic analysis of these proteins demonstrated convergence on 68 known protein interaction pathways, most notably those associated with innate immunity, cellular stress, and morphogenesis. PMID:25414909

  13. Regulation of glycoprotein D synthesis: does alpha 4, the major regulatory protein of herpes simplex virus 1, regulate late genes both positively and negatively?

    PubMed Central

    Arsenakis, M; Campadelli-Fiume, G; Roizman, B

    1988-01-01

    Earlier studies have described the alpha 4/c113 baby hamster kidney cell line which constitutively expresses the alpha 4 protein, the major regulatory protein of herpes simplex virus 1 (HSV-1). Introduction of the HSV-1 glycoprotein B (gB) gene, regulated as a gamma 1 gene, into these cells yielded a cell line which constitutively expressed both the alpha 4 and gamma 1 gB genes. The expression of the gB gene was dependent on the presence of functional alpha 4 protein. In this article we report that we introduced into the alpha 4/c113 and into the parental BHK cells, the HSV-1 BamHI J fragment, which encodes the domains of four genes, including those of glycoproteins D, G, and I (gD, gG, and gI), and most of the coding sequences of the glycoprotein E (gE) gene. In contrast to the earlier studies, we obtained significant constitutive expression of gD (also a gamma 1 gene) in a cell line (BJ) derived from parental BHK cells, but not in a cell line (alpha 4/BJ) which expresses functional alpha 4 protein. RNA homologous to the gD gene was present in significant amounts in the BJ cell line; smaller amounts of this RNA were detected in the alpha 4/BJ cell line. RNA homologous to gE, presumed to be polyadenylated from signals in the vector sequences, was present in the BJ cells but not in the alpha 4/BJ cells. The expression of the HSV-1 gD and gE genes was readily induced in the alpha 4/BJ cells by superinfection with HSV-2. The BJ cell line was, in contrast, resistant to expression of HSV-1 and HSV-2 genes. The BamHI J DNA fragment copy number was approximately 1 per BJ cell genome equivalent and 30 to 50 per alpha 4/BJ cell genome equivalent. We conclude that (i) the genes specifying gD and gB belong to different viral regulatory gene subsets, (ii) the gD gene is subject to both positive and negative regulation, (iii) both gD and gE mRNAs are subject to translational controls although they may be different, and (iv) the absence of expression of gD in the alpha 4/BJ

  14. Pur-Alpha Induces JCV Gene Expression and Viral Replication by Suppressing SRSF1 in Glial Cells

    PubMed Central

    Sariyer, Ilker Kudret; Sariyer, Rahsan; Otte, Jessica; Gordon, Jennifer

    2016-01-01

    Objective PML is a rare and fatal demyelinating disease of the CNS caused by the human polyomavirus, JC virus (JCV), which occurs in AIDS patients and those on immunosuppressive monoclonal antibody therapies (mAbs). We sought to identify mechanisms that could stimulate reactivation of JCV in a cell culture model system and targeted pathways which could affect early gene transcription and JCV T-antigen production, which are key steps of the viral life cycle for blocking reactivation of JCV. Two important regulatory partners we have previously identified for T-antigen include Pur-alpha and SRSF1 (SF2/ASF). SRSF1, an alternative splicing factor, is a potential regulator of JCV whose overexpression in glial cells strongly suppresses viral gene expression and replication. Pur-alpha has been most extensively characterized as a sequence-specific DNA- and RNA-binding protein which directs both viral gene transcription and mRNA translation, and is a potent inducer of the JCV early promoter through binding to T-antigen. Methods and Results Pur-alpha and SRSF1 both act directly as transcriptional regulators of the JCV promoter and here we have observed that Pur-alpha is capable of ameliorating SRSF1-mediated suppression of JCV gene expression and viral replication. Interestingly, Pur-alpha exerted its effect by suppressing SRSF1 at both the protein and mRNA levels in glial cells suggesting this effect can occur independent of T-antigen. Pur-alpha and SRSF1 were both localized to oligodendrocyte inclusion bodies by immunohistochemistry in brain sections from patients with HIV-1 associated PML. Interestingly, inclusion bodies were typically positive for either Pur-alpha or SRSF1, though some cells appeared to be positive for both proteins. Conclusions Taken together, these results indicate the presence of an antagonistic interaction between these two proteins in regulating of JCV gene expression and viral replication and suggests that they play an important role during viral

  15. T cell receptor alpha-chain gene rearrangements in B-precursor leukemia are in contrast to the findings in T cell acute lymphoblastic leukemia. Comparative study of T cell receptor gene rearrangement in childhood leukemia.

    PubMed Central

    Hara, J; Benedict, S H; Mak, T W; Gelfand, E W

    1987-01-01

    We have analyzed T cell receptor alpha-chain gene configuration using three genomic joining (J) region probes in 64 children with acute lymphoblastic leukemia (ALL). 11 out of 18 T-ALLs were T3 positive; alpha-chain gene rearrangements were demonstrated in only two of 18, indicating that the majority of T-ALLs would have rearrangements involving J alpha segments located upstream of these probes. In contrast, 15 out of 46 B-precursor ALLs showed rearrangements of the alpha-chain gene and J alpha segments located approximately 20-30 kb upstream of the constant region were involved in 13 of these patients. Nine of 15 B-precursor ALLs with rearranged alpha-chain genes had rearrangements of both gamma- and beta-chain genes, whereas the remaining six had no rearrangements of gamma- and beta-chain genes. These findings indicated that alpha-chain gene rearrangement is not specific for T lineage cells and gamma- and/or beta-chain gene rearrangement does not appear essential for alpha-chain gene rearrangement, at least in B-precursor leukemic cells. Images PMID:3500187

  16. Horizontal Gene Transfer of Pectinases from Bacteria Preceded the Diversification of Stick and Leaf Insects

    PubMed Central

    Shelomi, Matan; Danchin, Etienne G. J.; Heckel, David; Wipfler, Benjamin; Bradler, Sven; Zhou, Xin; Pauchet, Yannick

    2016-01-01

    Genes acquired by horizontal transfer are increasingly being found in animal genomes. Understanding their origin and evolution requires knowledge about the phylogenetic relationships from both source and recipient organisms. We used RNASeq data and respective assembled transcript libraries to trace the evolutionary history of polygalacturonase (pectinase) genes in stick insects (Phasmatodea). By mapping the distribution of pectinase genes on a Polyneoptera phylogeny, we identified the transfer of pectinase genes from known phasmatodean gut microbes into the genome of an early euphasmatodean ancestor that took place between 60 and 100 million years ago. This transfer preceded the rapid diversification of the suborder, enabling symbiont-free pectinase production that would increase the insects’ digestive efficiency and reduce dependence on microbes. Bacteria-to-insect gene transfer was thought to be uncommon, however the increasing availability of large-scale genomic data may change this prevailing notion. PMID:27210832

  17. Horizontal Gene Transfer of Pectinases from Bacteria Preceded the Diversification of Stick and Leaf Insects.

    PubMed

    Shelomi, Matan; Danchin, Etienne G J; Heckel, David; Wipfler, Benjamin; Bradler, Sven; Zhou, Xin; Pauchet, Yannick

    2016-01-01

    Genes acquired by horizontal transfer are increasingly being found in animal genomes. Understanding their origin and evolution requires knowledge about the phylogenetic relationships from both source and recipient organisms. We used RNASeq data and respective assembled transcript libraries to trace the evolutionary history of polygalacturonase (pectinase) genes in stick insects (Phasmatodea). By mapping the distribution of pectinase genes on a Polyneoptera phylogeny, we identified the transfer of pectinase genes from known phasmatodean gut microbes into the genome of an early euphasmatodean ancestor that took place between 60 and 100 million years ago. This transfer preceded the rapid diversification of the suborder, enabling symbiont-free pectinase production that would increase the insects' digestive efficiency and reduce dependence on microbes. Bacteria-to-insect gene transfer was thought to be uncommon, however the increasing availability of large-scale genomic data may change this prevailing notion. PMID:27210832

  18. A direct gene transfer strategy via brain internal capsule reverses the biochemical defect in Tay-Sachs disease.

    PubMed

    Martino, S; Marconi, P; Tancini, B; Dolcetta, D; De Angelis, M G Cusella; Montanucci, P; Bregola, G; Sandhoff, K; Bordignon, C; Emiliani, C; Manservigi, R; Orlacchio, A

    2005-08-01

    Therapy for neurodegenerative lysosomal Tay-Sachs (TS) disease requires active hexosaminidase (Hex) A production in the central nervous system and an efficient therapeutic approach that can act faster than human disease progression. We combined the efficacy of a non-replicating Herpes simplex vector encoding for the Hex A alpha-subunit (HSV-T0alphaHex) and the anatomic structure of the brain internal capsule to distribute the missing enzyme optimally. With this gene transfer strategy, for the first time, we re-established the Hex A activity and totally removed the GM2 ganglioside storage in both injected and controlateral hemispheres, in the cerebellum and spinal cord of TS animal model in the span of one month's treatment. In our studies, no adverse effects were observed due to the viral vector, injection site or gene expression and on the basis of these results, we feel confident that the same approach could be applied to similar diseases involving an enzyme defect. PMID:15961412

  19. The functions of a cucumber phospholipase D alpha gene (CsPLDα) in growth and tolerance to hyperosmotic stress.

    PubMed

    Li, Shuzhen; Huang, Meili; Di, Qinghua; Ji, Tuo; Wang, Xiufeng; Wei, Min; Shi, Qinghua; Li, Yan; Gong, Biao; Yang, Fengjuan

    2015-12-01

    Plant phospholipase D (PLD), which can hydrolyze membrane phospholipids to produce phosphatidic acid (PA), a secondary signaling molecule, has been proposed to function in diverse plant stress responses. In this research, a qRT-PCR analysis indicated that the expression of a cucumber phospholipase D alpha gene (CsPLDα) was induced by salt and drought stresses in the roots and leaves. To further study the roles of CsPLDα in regulating plant tolerance to salt, polyethylene glycol (PEG) and abscisic acid (ABA) stresses, transgenic tobacco plants constitutively overexpressing CsPLDα were produced. A qRT-PCR analysis showed that the CsPLDα transcript levels were high in transgenic tobacco lines, whereas no expression was found in wild type (WT) tobacco, indicating that CsPLDα was successfully transferred into the tobacco genome and overexpressed. Under normal conditions for 30 d, seeds of transgenic lines germinated neatly, and the seedlings were robust and bigger than WT plants. When treated with different concentrations of NaCl, PEG and ABA, germination rates and seedling sizes of the transgenic lines were significantly greater than WT. In addition, the germination times for transgenic lines were also remarkably shorter. Further studies indicated that transgenic lines had longer primary roots and more biomass accumulation than WT plants. The water loss in transgenic lines was also much lower than in WT. These findings suggest that the CsPLDα overexpression positively regulates plant tolerance to hyperosmotic stresses, and that CsPLDα is involved in the ABA regulation of stomatal closure and the alleviation of ABA inhibition on seed germination and seedling growth. PMID:26476791

  20. Horizontal gene transfer and the evolution of transcriptionalregulation in Escherichia coli

    SciTech Connect

    Price, Morgan N.; Dehal, Paramvir S.; Arkin, Adam P.

    2007-12-20

    Background: Most bacterial genes were acquired by horizontalgene transfer from other bacteria instead of being inherited bycontinuous vertical descent from an ancient ancestor}. To understand howthe regulation of these {acquired} genes evolved, we examined theevolutionary histories of transcription factors and of regulatoryinteractions from the model bacterium Escherichia coli K12. Results:Although most transcription factors have paralogs, these usually arose byhorizontal gene transfer rather than by duplication within the E. colilineage, as previously believed. In general, most neighbor regulators --regulators that are adjacent to genes that they regulate -- were acquiredby horizontal gene transfer, while most global regulators evolvedvertically within the gamma-Proteobacteria. Neighbor regulators wereoften acquired together with the adjacent operon that they regulate, sothe proximity might be maintained by repeated transfers (like "selfishoperons"). Many of the as-yet-uncharacterized (putative) regulators havealso been acquired together with adjacent genes, so we predict that theseare neighbor regulators as well. When we analyzed the histories ofregulatory interactions, we found that the evolution of regulation byduplication was rare, and surprisingly, many of the regulatoryinteractions that are shared between paralogs result from convergentevolution. Another surprise was that horizontally transferred genes aremore likely than other genes to be regulated by multiple regulators, andmost of this complex regulation probably evolved after the transfer.Conclusions: Our results highlight the rapid evolution of niche-specificgene regulation in bacteria.

  1. Role of horizontal gene transfer in the evolution of photosynthetic eukaryotes and their plastids.

    PubMed

    Keeling, Patrick J

    2009-01-01

    Plastids are the organelles derived from a cyanobacterium through endosymbiosis. Unlike mitochondria, plastids are not found in all eukaryotes, but their evolution has an added layer of complexity since plastids have moved between eukaryotic lineages by secondary and tertiary endosymbiotic events. This complex history, together with the genetic integration between plastids and their host, has led to many opportunities for gene flow between phylogenetically distinct lineages. Some intracellular transfers do not lead to a protein functioning in a new environment, but many others do and the protein makeup of many plastids appears to have been influenced by exogenous sources as well. Here, different evolutionary sources and cellular destinations of gene flow that has affected the plastid lineage are reviewed. Most horizontal gene transfer (HGT) affecting the modern plastid has taken place via the host nucleus, in the form of genes for plastid-targeted proteins. The impact of this varies greatly from lineage to lineage, but in some cases such transfers can be as high as one fifth of analyzed genes. More rarely, genes have also been transferred to the plastid genome itself, and plastid genes have also been transferred to other non-plant, non-algal lineages. Overall, the proteome of many plastids has emerged as a mosaic of proteins from many sources, some from within the same cell (e.g., cytosolic genes or genes left over from the replacement of an earlier plastid), some from the plastid of other algal lineages, and some from completely unrelated sources. PMID:19271204

  2. Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus.

    PubMed

    Monier, Adam; Pagarete, António; de Vargas, Colomban; Allen, Michael J; Read, Betsy; Claverie, Jean-Michel; Ogata, Hiroyuki

    2009-08-01

    Interactions between viruses and phytoplankton, the main primary producers in the oceans, affect global biogeochemical cycles and climate. Recent studies are increasingly revealing possible cases of gene transfers between cyanobacteria and phages, which might have played significant roles in the evolution of cyanobacteria/phage systems. However, little has been documented about the occurrence of horizontal gene transfer in eukaryotic phytoplankton/virus systems. Here we report phylogenetic evidence for the transfer of seven genes involved in the sphingolipid biosynthesis pathway between the cosmopolitan eukaryotic microalga Emiliania huxleyi and its large DNA virus EhV. PCR assays indicate that these genes are prevalent in E. huxleyi and EhV strains isolated from different geographic locations. Patterns of protein and gene sequence conservation support that these genes are functional in both E. huxleyi and EhV. This is the first clear case of horizontal gene transfer of multiple functionally linked enzymes in a eukaryotic phytoplankton-virus system. We examine arguments for the possible direction of the gene transfer. The virus-to-host direction suggests the existence of ancient viruses that controlled the complex metabolic pathway in order to infect primitive eukaryotic cells. In contrast, the host-to-virus direction suggests that the serial acquisition of genes involved in the same metabolic pathway might have been a strategy for the ancestor of EhVs to stay ahead of their closest relatives in the great evolutionary race for survival. PMID:19451591

  3. Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus

    PubMed Central

    Monier, Adam; Pagarete, António; de Vargas, Colomban; Allen, Michael J.; Read, Betsy; Claverie, Jean-Michel; Ogata, Hiroyuki

    2009-01-01

    Interactions between viruses and phytoplankton, the main primary producers in the oceans, affect global biogeochemical cycles and climate. Recent studies are increasingly revealing possible cases of gene transfers between cyanobacteria and phages, which might have played significant roles in the evolution of cyanobacteria/phage systems. However, little has been documented about the occurrence of horizontal gene transfer in eukaryotic phytoplankton/virus systems. Here we report phylogenetic evidence for the transfer of seven genes involved in the sphingolipid biosynthesis pathway between the cosmopolitan eukaryotic microalga Emiliania huxleyi and its large DNA virus EhV. PCR assays indicate that these genes are prevalent in E. huxleyi and EhV strains isolated from different geographic locations. Patterns of protein and gene sequence conservation support that these genes are functional in both E. huxleyi and EhV. This is the first clear case of horizontal gene transfer of multiple functionally linked enzymes in a eukaryotic phytoplankton–virus system. We examine arguments for the possible direction of the gene transfer. The virus-to-host direction suggests the existence of ancient viruses that controlled the complex metabolic pathway in order to infect primitive eukaryotic cells. In contrast, the host-to-virus direction suggests that the serial acquisition of genes involved in the same metabolic pathway might have been a strategy for the ancestor of EhVs to stay ahead of their closest relatives in the great evolutionary race for survival. PMID:19451591

  4. Association of Retinoid X Receptor Alpha Gene Polymorphism with Clinical Course of Chronic Glomerulonephritis.

    PubMed

    Grzegorzewska, Alicja E; Ostromecki, Grzegorz; Zielińska, Paulina; Mostowska, Adrianna; Niemir, Zofia; Polcyn-Adamczak, Magdalena; Pawlik, Magdalena; Sowińska, Anna; Jagodziński, Paweł P

    2015-01-01

    BACKGROUND Vitamin D (VD), VD binding protein, VD receptor (VDR), and retinoids are involved in pathogenesis of chronic glomerulonephritis (ChGN). We aimed to compare distribution of VD pathway gene polymorphisms in ChGN patients showing glomerular filtration rate (GFR) category 1-3, GFR category 5D, and healthy controls in order to elucidate the role of VD-related polymorphisms in the course of ChGN. MATERIAL AND METHODS GFR category 1-3 ChGN patients (n=195), GFR category 5D ChGN patients (n=178), and controls (n=751) underwent testing for polymorphisms of genes encoding VD binding protein (GC, rs2298849, rs7041, rs1155563), VDR (VDR, rs2228570, rs1544410), and retinoid X receptor alpha (RXRA, rs10776909, rs10881578, rs749759). RESULTS Among GFR 1-3 subjects possessing TT genotype of RXRA rs10776909, 75% of patients had nephrotic syndrome, and 37.5% had glomerular hyperfiltration defined as GFR >140 ml/min/1.73 m2, and, consequently, serum creatinine was lower in these patients compared to the remaining subjects (0.67±0.26 vs. 0.94±0.34, P=0.014). In GFR category 5D ChGN patients, frequencies of RXRA rs10776909 allele T (25% vs. 19%) and CT+TT (46% vs. 34%) were higher compared to frequencies of respective variants in controls (Ptrend=0.004, Pgenotype=0.008). CONCLUSIONS RXRA rs10776909 allele T is specifically involved in the pathogenesis of ChGN. This risk allele may be also associated with worse clinical course of ChGN. PMID:26610845

  5. Haplotypes that include the integrin alpha 11 gene are associated with tick burden in cattle

    PubMed Central

    2010-01-01

    Background Infestations on cattle by the ectoparasite Boophilus (Rhipicephalus) microplus (cattle tick) impact negatively on animal production systems. Host resistance to tick infestation has a low to moderate heritability in the range 0.13 - 0.64 in Australia. Previous studies identified a QTL on bovine chromosome 10 (BTA10) linked to tick burden in cattle. Results To confirm these associations, we collected genotypes of 17 SNP from BTA10, including three obtained by sequencing part of the ITGA11 (Integrin alpha 11) gene. Initially, we genotyped 1,055 dairy cattle for the 17 SNP, and then genotyped 557 Brahman and 216 Tropical Composite beef cattle for 11 of the 17 SNP. In total, 7 of the SNP were significantly (P < 0.05) associated with tick burden tested in any of the samples. One SNP, ss161109814, was significantly (P < 0.05) associated with tick burden in both the taurine and the Brahman sample, but the favourable allele was different. Haplotypes for three and for 10 SNP were more significantly (P < 0.001) associated with tick burden than SNP analysed individually. Some of the common haplotypes with the largest sample sizes explained between 1.3% and 1.5% of the residual variance in tick burden. Conclusions These analyses confirm the location of a QTL affecting tick burden on BTA10 and position it close to the ITGA11 gene. The presence of a significant association in such widely divergent animals suggests that further SNP discovery in this region to detect causal mutations would be warranted. PMID:20565915

  6. Fibroblast growth factor 7 inhibits cholesterol 7{alpha}-hydroxylase gene expression in hepatocytes

    SciTech Connect

    Sun, Zhichao; Yu, Xuemei; Wu, Weibin; Jia, Dongwei; Chen, Yinle; Ji, Lingling; Liu, Xijun; Peng, Xiaomin; Li, Yintao; Yang, Lili; Ruan, Yuanyuan; Gu, Jianxin; Ren, Shifang; Zhang, Songwen

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer FGF7 strongly and rapidly down-regulates the expression of CYP7A1 in hepatocytes. Black-Right-Pointing-Pointer FGF7 suppresses the expression of CYP7A1 via FGFR2 and downstream JNK activation. Black-Right-Pointing-Pointer Blocking FGF7 abrogates HSC-induced inhibition of CYP7A1 expression in hepatocytes. -- Abstract: Cholesterol 7{alpha}-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl{sub 4})-induced fibrotic mouse liver, we demonstrated that the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis.

  7. Orofacial clefts, parental cigarette smoking, and transforming growth factor-alpha gene variants

    SciTech Connect

    Shaw, G.M.; Wasserman, C.R.; O`Malley, C.D.

    1996-03-01

    Results of studies determine whether women who smoke during early pregnancy are at increased risk of delivering infants with orofacial clefts have been mixed, and recently a gene-environment interaction between maternal smoking, transforming growth factor-alpha (TGFa), and clefting has been reported. Using a large population-based case-control study, we investigated whether parental periconceptional cigarette smoking was associated with an increased risk for having offspring with orofacial clefts. We also investigated the influence of genetic variation of the TGFa locus on the relation between smoking and clefting. Parental smoking information was obtained from telephone interviews with mothers of 731 (84.7% of eligible) orofacial cleft case infants and with mothers of 734 (78.2%) nonmalformed control infants. DNA was obtained from newborn screening blood spots and genotyped for the allelic variants of TGFa. We found that risks associated with maternal smoking were most elevated for isolated cleft lip with or without cleft palate, (odds ratio 2.1 [95% confidence interval 1.3-3.6]) and for isolated cleft palate (odds ratio 2.2 [1.1-4.5]) when mothers smoked {ge} 20 cigarrettes/d. These risks for white infants ranged from 3-fold to 11-fold across phenotypic groups. Paternal smoking was not associated with clefting among the offspring of nonsmoking mothers, and passive smoke exposures were associated with at most slightly increased risks. This study offers evidence that the risk for orofacial clefting in infants may be influenced by maternal smoke exposures alone as well as in combination (gene-environment interaction) with the presence of the uncommon TGFa allele. 56 refs., 5 tabs.

  8. Horizontally transferred genes in the genome of Pacific white shrimp, Litopenaeus vannamei

    PubMed Central

    2013-01-01

    Background In recent years, as the development of next-generation sequencing technology, a growing number of genes have been reported as being horizontally transferred from prokaryotes to eukaryotes, most of them involving arthropods. As a member of the phylum Arthropoda, the Pacific white shrimp Litopenaeus vannamei has to adapt to the complex water environments with various symbiotic or parasitic microorganisms, which provide a platform for horizontal gene transfer (HGT). Results In this study, we analyzed the genome-wide HGT events in L. vannamei. Through homology search and phylogenetic analysis, followed by experimental PCR confirmation, 14 genes with HGT event were identified: 12 of them were transferred from bacteria and two from fungi. Structure analysis of these genes showed that the introns of the two fungi-originated genes were substituted by shrimp DNA fragment, two genes transferred from bacteria had shrimp specific introns inserted in them. Furthermore, around other three bacteria-originated genes, there were three large DNA segments inserted into the shrimp genome. One segment was a transposon that fully transferred, and the other two segments contained only coding regions of bacteria. Functional prediction of these 14 genes showed that 6 of them might be related to energy metabolism, and 4 others related to defense of the organism. Conclusions HGT events from bacteria or fungi were happened in the genome of L. vannamei, and these horizontally transferred genes can be transcribed in shrimp. This is the first time to report the existence of horizontally transferred genes in shrimp. Importantly, most of these genes are exposed to a negative selection pressure and appeared to be functional. PMID:23914989

  9. Ultrasound -Assisted Gene Transfer to Adipose Tissue-Derived Stem/Progenitor Cells (ASCs)

    NASA Astrophysics Data System (ADS)

    Miyamoto, Yoshitaka; Ueno, Hitomi; Hokari, Rei; Yuan, Wenji; Kuno, Shuichi; Kakimoto, Takashi; Enosawa, Shin; Negishi, Yoichi; Yoshinaka, Kiyoshi; Matsumoto, Yoichiro; Chiba, Toshio; Hayashi, Shuji

    2011-09-01

    In recent years, multilineage adipose tissue-derived stem cells (ASCs) have become increasingly attractive as a promising source for cell transplantation and regenerative medicine. Particular interest has been expressed in the potential to make tissue stem cells, such as ASCs and marrow stromal cells (MSCs), differentiate by gene transfection. Gene transfection using highly efficient viral vectors such as adeno- and sendai viruses have been developed for this purpose. Sonoporation, or ultrasound (US)-assisted gene transfer, is an alternative gene manipulation technique which employs the creation of a jet stream by ultrasonic microbubble cavitation. Sonoporation using non-viral vectors is expected to be a much safer, although less efficient, tool for prospective clinical gene therapy. In this report, we assessed the efficacy of the sonoporation technique for gene transfer to ASCs. We isolated and cultured adipocyets from mouse adipose tissue. ASCs that have the potential to differentiate with transformation into adipocytes or osteoblasts were obtained. Using the US-assisted system, plasmid DNA containing beta-galactosidase (beta-Gal) and green fluorescent protein (GFP) genes were transferred to the ASCs. For this purpose, a Sonopore 4000 (NEPAGENE Co.) and a Sonazoid (Daiichi Sankyo Co.) instrument were used in combination. ASCs were subjected to US (3.1 MHz, 50% duty cycle, burst rate 2.0 Hz, intensity 1.2 W/cm2, exposure time 30 sec). We observed that the gene was more efficiently transferred with increased concentrations of plasmid DNA (5-150 μg/mL). However, further optimization of the US parameters is required, as the gene transfer efficiency was still relatively low. In conclusion, we herein demonstrate that a gene can be transferred to ASCs using our US-assisted system. In regenerative medicine, this system might resolve the current issues surrounding the use of viral vectors for gene transfer.

  10. Clofibrate causes an upregulation of PPAR-{alpha} target genes but does not alter expression of SREBP target genes in liver and adipose tissue of pigs.

    PubMed

    Luci, Sebastian; Giemsa, Beatrice; Kluge, Holger; Eder, Klaus

    2007-07-01

    This study investigated the effect of clofibrate treatment on expression of target genes of peroxisome proliferator-activated receptor (PPAR)-alpha and various genes of the lipid metabolism in liver and adipose tissue of pigs. An experiment with 18 pigs was performed in which pigs were fed either a control diet or the same diet supplemented with 5 g clofibrate/kg for 28 days. Pigs treated with clofibrate had heavier livers, moderately increased mRNA concentrations of various PPAR-alpha target genes in liver and adipose tissue, a higher concentration of 3-hydroxybutyrate, and markedly lower concentrations of triglycerides and cholesterol in plasma and lipoproteins than control pigs (P < 0.05). mRNA concentrations of sterol regulatory element-binding proteins (SREBP)-1 and -2, insulin-induced genes (Insig)-1 and Insig-2, and the SREBP target genes acetyl-CoA carboxylase, 3-methyl-3-hydroxyglutaryl-CoA reductase, and low-density lipoprotein receptor in liver and adipose tissue and mRNA concentrations of apolipoproteins A-I, A-II, and C-III in the liver were not different between both groups of pigs. In conclusion, this study shows that clofibrate treatment activates PPAR-alpha in liver and adipose tissue and has a strong hypotriglyceridemic and hypocholesterolemic effect in pigs. The finding that mRNA concentrations of some proteins responsible for the hypolipidemic action of fibrates in humans were not altered suggests that there were certain differences in the mode of action compared with humans. It is also shown that PPAR-alpha activation by clofibrate does not affect hepatic expression of SREBP target genes involved in synthesis of triglycerides and cholesterol homeostasis in liver and adipose tissue of pigs. PMID:17363680

  11. A 3D Radiative Transfer Simulation of Lyma-alpha Backscatter Intensity Reduced From Voyager’s Ultraviolet Spectrometer

    NASA Astrophysics Data System (ADS)

    Fayock, Brian; Zank, Gary; Heerikhuisen, Jacob

    2014-06-01

    Models of the heliosphere have evolved for the past few decades to fit observations made by a large number of spacecraft. Voyager missions have provided unique in-situ measurements that have proven to be essential for model testing. Lyman-alpha backscatter intensity has been reduced from measurements taken by the ultraviolet spectrometers on board both Voyager spacecraft. We have developed a 3D Monte Carlo radiative transfer code to simulate this backscatter intensity by generating millions of photons from the sun to scatter within a neutral hydrogen distribution resulting from a state-of-the-art 3D MHD-kinetic neutral heliospheric model, both of which have been developed within the Center for Space Physics and Aeronomic Research at the University of Alabama in Huntsville. While many have attempted to simulate the Voyager observations, we are the first to achieve agreement with our results. In this presentation, we will discuss the core mechanisms driving the radiative transfer code, the statistical quantities collected, and the interpretation of the results relative to the spacecraft data.

  12. Chemokine gene expression in the murine renal cell carcinoma, RENCA, following treatment in vivo with interferon-alpha and interleukin-2.

    PubMed Central

    Sonouchi, K.; Hamilton, T. A.; Tannenbaum, C. S.; Tubbs, R. R.; Bukowski, R.; Finke, J. H.

    1994-01-01

    The expression of three chemoattractant cytokine (chemokine) messenger (m)RNAs in the murine renal cell carcinoma (RENCA) from mice treated with a combination of interferon-alpha (IFN-alpha) and interleukin-2 was examined and related to tumor infiltration by inflammatory leukocytes. Using a semi-quantitative reverse transcriptase polymerase chain reaction assay, mRNAs encoding the KC, JE, and IP-10 genes were all elevated in tumor tissue from mice treated systemically with IFN-alpha/interleukin-2 for 4 days. Similarly, the mRNA for tumor necrosis factor-alpha (TNF-alpha) was also increased in tumors from treated as compared to control animals. The same tumors showed a significant increase in Mac-1+ leukocytes, which correlated well with the increase in chemokine and TNF-alpha gene expression. The renal cell carcinoma tumor itself may be responsible for the expression of chemokine genes in the tumor bed following cytokine therapy. Cultures of freshly explanted RENCA cells expressed significant levels of chemokine mRNAs when stimulated in vitro with IFN alpha, IFN gamma, and/or interleukin-2, demonstrating that this tumor cell has potential for expression of these genes in vivo. In contrast, TNF-alpha expression was not detected in cultured tumor cells. Thus TNF-alpha may be expressed by infiltrating monocytes following exposure to recombinant cytokine therapy. Images Figure 1 Figure 2 Figure 4 PMID:8160774

  13. Nanoalumina promotes the horizontal transfer of multiresistance genes mediated by plasmids across genera

    PubMed Central

    Qiu, Zhigang; Yu, Yunmei; Chen, Zhaoli; Jin, Min; Yang, Dong; Zhao, Zuguo; Wang, Jingfeng; Shen, Zhiqiang; Wang, Xinwei; Qian, Di; Huang, Aihua; Zhang, Buchang; Li, Jun-Wen

    2012-01-01

    Antibiotic resistance is a worldwide public health concern. Conjugative transfer between closely related strains or species of bacteria is an important method for the horizontal transfer of multidrug-resistance genes. The extent to which nanomaterials are able to cause an increase in antibiotic resistance by the regulation of the conjugative transfer of antibiotic-resistance genes in bacteria, especially across genera, is still unknown. Here we show that nanomaterials in water can significantly promote the horizontal conjugative transfer of multidrug-resistance genes mediated by the RP4, RK2, and pCF10 plasmids. Nanoalumina can promote the conjugative transfer of the RP4 plasmid from Escherichia coli to Salmonella spp. by up to 200-fold compared with untreated cells. We also explored the mechanisms behind this phenomenon and demonstrate that nanoalumina is able to induce oxidative stress, damage bacterial cell membranes, enhance the expression of mating pair formation genes and DNA transfer and replication genes, and depress the expression of global regulatory genes that regulate the conjugative transfer of RP4. These findings are important in assessing the risk of nanomaterials to the environment, particularly from water and wastewater treatment systems, and in the estimation of the effect of manufacture and use of nanomaterials on the environment. PMID:22411796

  14. Hepatocyte nuclear factor-6 stimulates transcription of the alpha-fetoprotein gene and synergizes with the retinoic-acid-receptor-related orphan receptor alpha-4.

    PubMed Central

    Nacer-Cherif, Habib; Bois-Joyeux, Brigitte; Rousseau, Guy G; Lemaigre, Frédéric P; Danan, Jean-Louis

    2003-01-01

    The rat alpha-fetoprotein ( afp ) gene is controlled by three enhancers whose function depends on their interaction with liver-enriched transcription factors. The afp enhancer III, located at -6 kb, is composed of three regions that act in synergy. Two of these regions, called s1 and s2, contain a putative binding site for hepatocyte nuclear factor-6 (HNF-6). This factor is the prototype of the ONECUT family of cut-homoeodomain proteins and is a known regulator of liver gene expression in adults and during development. We show here that the two splicing isoforms of HNF-6 bind to a site in the s1 region and in the s2 region. The core sequence of the s1 site corresponds to none of the known HNF-6 binding sites. Nevertheless, the binding properties of the s1 site are identical with those of the s2 site and of previously characterized HNF-6 binding sequences. The HNF-6 consensus should therefore be rewritten as DRRTCVATND. Binding of HNF-6 to the s1 and s2 sites requires both the cut and the homoeo domains, is co-operative and induces DNA bending. HNF-6 strongly stimulates the activity of the afp enhancer III in transient transfection experiments. This effect requires the stereo-specific alignment of the two HNF-6 sites. Moreover, HNF-6 stimulates the enhancer in synergy with the retinoic-acid-receptor-related orphan receptor alpha (RORalpha), which binds to a neighbouring site in the s1 region. Thus expression of the afp gene requires functional interactions between HNF-6 molecules and between HNF-6 and RORalpha. PMID:12379144

  15. Novel intronic promoter in the rat ER alpha gene responsible for the transient transcription of a variant receptor.

    PubMed

    Tiffoche, C; Vaillant, C; Schausi, D; Thieulant, M L

    2001-09-01

    To analyze the molecular origin of an ER variant, the truncated ER product-1, transiently expressed at the proestrus in lactotrope cells, we generated a 2.5-kb sequence of a genomic region upstream and downstream the specific sequence truncated ER product-1. Genomic Southern blot analysis showed that truncated ER product-1 is spliced from a noncoding leader exon localized within the intron 4 of the ER alpha gene. Analysis of the promoter sequence revealed the presence of a major transcriptional start site, a canonical TATA box and putative cis regulatory elements for pituitary specific expression as well as an E-responsive element. In transient transfection, the truncated ER product-1 promoter was transcriptionally the most active in the lactotrope cell lines (MMQ). Analysis of truncated ER product-1 functionality showed that: 1) the protein inhibited ER alpha binding to the E-responsive element in electromobility shift assays, 2) inhibited the E2 binding to ER alpha in binding assays, 3) the truncated ER product-1/ER alpha complex antagonized the transcriptional activity elicited by E2, 4) nuclear localization of green fluorescent protein-ER alpha was altered in Chinese hamster ovary cell lines stably expressing truncated ER product-1. Collectively, these data demonstrated that the protein exerts full dominant negative activity against ER alpha. Moreover, truncated ER product-1/ER alpha complex also repressed the activity of all promoters tested to date, suggesting a general inhibitory effect toward transcription. In conclusion, the data suggest that truncated ER product-1 could regulate estrogen signaling via a specific promoter in lactotrope cells. PMID:11517190

  16. Gene transfer into experimental brain tumors mediated by adenovirus, herpes simplex virus, and retrovirus vectors.

    PubMed

    Boviatsis, E J; Chase, M; Wei, M X; Tamiya, T; Hurford, R K; Kowall, N W; Tepper, R I; Breakefield, X O; Chiocca, E A

    1994-02-01

    Three vectors derived from retrovirus, herpes simplex virus type 1 (HSV), and adenovirus were compared in cultured rat 9L gliosarcoma cells for gene transfer efficiency and in a 9L rat brain tumor model for histologic pattern and distribution of foreign gene delivery, as well as for associated tumor necrosis and inflammation. At a multiplicity of infection of 1, in vitro transfer of a foreign gene (lacZ from Escherichia coli) into cells was more efficient with either the replication-defective retrovirus vector or the replication-conditional thymidine kinase (TK)-deficient HSV vector than with the replication-defective adenovirus vector. In vivo, stereotactic injections of each vector into rat brain tumors revealed three main histopathologic findings: (i) retrovirus and HSV vector-mediated gene transfer was relatively selective for cells within the tumor, whereas adenovirus vector-mediated gene transfer occurred into several types of endogenous neural cells, as well as into cells within the tumor; (ii) gene transfer to multiple infiltrating tumor deposits without apparent gene transfer to intervening normal brain tissue occurred uniquely in one animal inoculated with the HSV vector, and (iii) extensive necrosis and selective inflammation in the tumor were evident with the HSV vector, whereas there was minimal evidence of tumor necrosis and inflammation with either the retrovirus or adenovirus vectors. PMID:8186298

  17. Indirect Fitness Benefits Enable the Spread of Host Genes Promoting Costly Transfer of Beneficial Plasmids

    PubMed Central

    Dimitriu, Tatiana; Misevic, Dusan; Lotton, Chantal; Brown, Sam P.; Lindner, Ariel B.; Taddei, François

    2016-01-01

    Bacterial genes that confer crucial phenotypes, such as antibiotic resistance, can spread horizontally by residing on mobile genetic elements (MGEs). Although many mobile genes provide strong benefits to their hosts, the fitness consequences of the process of transfer itself are less clear. In previous studies, transfer has been interpreted as a parasitic trait of the MGEs because of its costs to the host but also as a trait benefiting host populations through the sharing of a common gene pool. Here, we show that costly donation is an altruistic act when it spreads beneficial MGEs favoured when it increases the inclusive fitness of donor ability alleles. We show mathematically that donor ability can be selected when relatedness at the locus modulating transfer is sufficiently high between donor and recipients, ensuring high frequency of transfer between cells sharing donor alleles. We further experimentally demonstrate that either population structure or discrimination in transfer can increase relatedness to a level selecting for chromosomal transfer alleles. Both mechanisms are likely to occur in natural environments. The simple process of strong dilution can create sufficient population structure to select for donor ability. Another mechanism observed in natural isolates, discrimination in transfer, can emerge through coselection of transfer and discrimination alleles. Our work shows that horizontal gene transfer in bacteria can be promoted by bacterial hosts themselves and not only by MGEs. In the longer term, the success of cells bearing beneficial MGEs combined with biased transfer leads to an association between high donor ability, discrimination, and mobile beneficial genes. However, in conditions that do not select for altruism, host bacteria promoting transfer are outcompeted by hosts with lower transfer rate, an aspect that could be relevant in the fight against the spread of antibiotic resistance. PMID:27270455

  18. Biomaterial-Mediated Retroviral Gene Transfer Using Self-Assembled Monolayers

    PubMed Central

    Gersbach, Charles A.; Coyer, Sean R.; Le Doux, Joseph M.; García, Andrés J.

    2007-01-01

    Biomaterial-mediated gene delivery has recently emerged as a promising alternative to conventional gene transfer technologies that focus on direct delivery of viral vectors or DNA-polymer/matrix complexes. However, biomaterial-based strategies have primarily targeted transient gene expression vehicles, including plasmid DNA and adenovirus particles. This study expands on this work by characterizing biomaterial properties conducive to the surface immobilization of retroviral particles and subsequent transduction of mammalian cells at the cell-material interface. Self-assembled monolayers (SAMs) of functionally-terminated alkanethiols on gold were used to establish biomaterial surfaces of defined chemical composition. Gene transfer was observed to be greater than 90% on NH2-terminated surfaces, approximately 50% on COOH-functionalized surfaces, and undetectable on CH3-terminated SAMs, similar to controls of tissue culture-treated polystyrene. Gene delivery via the NH2-SAM was further characterized as a function of coating time, virus concentration, and cell seeding density. Finally, SAM-mediated gene delivery was comparable to fibronectin- and poly-L-lysine-based methods for gene transfer. This work is significant to establishing safe and effective gene therapy strategies, developing efficient methods for gene delivery, and supporting recent progress in the field of biomaterial-mediated gene transfer. PMID:17698189

  19. Secretion, purification, and characterisation of barley alpha-amylase produced by heterologous gene expression in Aspergillus niger.

    PubMed

    Juge, N; Svensson, B; Williamson, G

    1998-04-01

    Efficient production of recombinant barley alpha-amylase has been achieved in Aspergillus niger. The cDNA encoding alpha-amylase isozyme 1 (AMY1) and its signal peptide was placed under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for alpha-amylase activity and low protease activity. The recombinant AMY1 (reAMY1) was purified to homogeneity and found to be identical to native barley AMY1 with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the endogenous plant signal peptide is correctly processed in A. niger. Electrospray ionisation/mass spectrometry gave a molecular mass for the dominant form of 44,960 Da, in accordance with the loss of the LQRS C-terminal residues; glycosylation apparently did not occur. The activities of recombinant and native barley alpha-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol beta-D-maltoheptaoside and amylose (degree of polymerisation = 17). Barley alpha-amylase is the first plant protein efficiently secreted and correctly processed by A. niger using its own signal sequence. PMID:9615479

  20. A single base mutation in an I-A alpha-chain gene alters T-cell recognition.

    PubMed

    Griffith, I J; Choi, E M; Glimcher, L H

    1987-02-01

    The interaction between the clonally selected T-cell antigen receptor, antigen, and Ia molecule is poorly understood at the molecular level. A cell line bearing an altered I-Ak alpha-chain (Ak alpha) molecule has been examined in order to provide more information about the relationship between Ia structure and function. The cell line, 3J9, was derived from the TA3 B-cell hybridoma through a series of negative and positive immunoselection steps. The 3J9 mutant lacked the binding site recognized by the Ak alpha-specific monoclonal antibody 39J and failed to present antigen to two T-cell hybridomas out of a large panel of I-Ak-restricted T-cell hybridomas examined. Sequence analysis of the mutant Ak alpha gene showed a single base transition (G----A) that resulted in a glutamic acid to lysine substitution at amino acid 75 of the alpha 1 domain. This mutation confirms the importance of amino acid 75 in the expression of the Ia.19 epitope, demonstrates the involvement of this region in the presentation of antigen to specific T cells, and provides a further example of the multiple functional domains on the Ia molecule that are involved in antigen presentation. PMID:3493486

  1. A single base mutation in an I-A alpha-chain gene alters T-cell recognition.

    PubMed Central

    Griffith, I J; Choi, E M; Glimcher, L H

    1987-01-01

    The interaction between the clonally selected T-cell antigen receptor, antigen, and Ia molecule is poorly understood at the molecular level. A cell line bearing an altered I-Ak alpha-chain (Ak alpha) molecule has been examined in order to provide more information about the relationship between Ia structure and function. The cell line, 3J9, was derived from the TA3 B-cell hybridoma through a series of negative and positive immunoselection steps. The 3J9 mutant lacked the binding site recognized by the Ak alpha-specific monoclonal antibody 39J and failed to present antigen to two T-cell hybridomas out of a large panel of I-Ak-restricted T-cell hybridomas examined. Sequence analysis of the mutant Ak alpha gene showed a single base transition (G----A) that resulted in a glutamic acid to lysine substitution at amino acid 75 of the alpha 1 domain. This mutation confirms the importance of amino acid 75 in the expression of the Ia.19 epitope, demonstrates the involvement of this region in the presentation of antigen to specific T cells, and provides a further example of the multiple functional domains on the Ia molecule that are involved in antigen presentation. PMID:3493486

  2. Blood pressure in patients with primary aldosteronism is influenced by bradykinin B(2) receptor and alpha-adducin gene polymorphisms.

    PubMed

    Mulatero, Paolo; Williams, Tracy A; Milan, Alberto; Paglieri, Cristina; Rabbia, Franco; Fallo, Francesco; Veglio, Franco

    2002-07-01

    Primary aldosteronism (PA) is the most common cause of endocrine hypertension. PA is most frequently presented as moderate to severe hypertension, but the clinical and biochemical features vary widely. The aim of our study was to identify genetic variants that influence the phenotype of patients with PA. We hypothesized that genetic variants potentially affecting aldosterone production (aldosterone synthase, CYP11B2), renal proximal tubule reabsorption (alpha-adducin), or the mechanisms of counterbalance leading to vasodilatation and sodium excretion (bradykinin B(2)-receptor, B(2)R) could influence the clinical and biochemical characteristics of patients with PA. We studied three polymorphisms of these genes (C-344T of CYP11B2, G460W of alpha-adducin, and C-58T of B(2)R) in 167 primary aldosteronism patients (56 with aldosterone-producing adenoma and 111 with idiopathic hyperaldosteronism). B(2)R and alpha-adducin genotypes were strong independent predictors of both systolic and diastolic blood pressure levels; plasma renin activity and aldosterone also play a marginal role on BP levels. Body mass index, age, sex, and CYP11B2 genotype displayed no significant effect on the clinical parameters of our population. In particular, alpha-adducin and B(2)R polymorphisms accounted for 13.2% and 11.0% of the systolic and diastolic blood pressure variance, respectively. These data suggest that genetic variants of alpha-adducin and the bradykinin B(2)-R influence the blood pressure levels in patients with primary aldosteronism. PMID:12107246

  3. Two bi-allelic single nucleotide polymorphisms within the promoter region of the horse tumour necrosis factor alpha gene.

    PubMed

    Matiasovic, J; Lukeszová, L; Horín, P

    2002-08-01

    Primers based on GenBank sequences within the 5' untranslated region (UTR) of the human and horse tumour necrosis factor alpha (TNF-alpha) genes were designed and used to amplify a 522-bp product. Sequencing of five clones derived from five independent PCRs obtained from three different animals of three different breeds (Old Kladruber, Akhal-Teke and Shetland Pony) revealed a high level of sequence identity to the TNF-alpha promoter regions of other species. The existing GenBank horse sequences were confirmed and extended upstream by 230 nucleotides. Based on the sequence obtained, a new horse-specific forward primer was designed to amplify a 213-bp PCR product, which was screened for polymorphism using single-strand conformation polymorphism (SSCP). Three allelic variants of the horse TNF-alpha gene were identified and sequenced (GenBank accession numbers ADF 349558-60). Two single nucleotide polymorphisms explained the existence of the three SSCP alleles detected: C/T and T/C single base pair substitutions at positions 137 and 147, respectively. Differences in allelic frequencies between Old Kladruber and Akhal-Teke breeds were observed. PMID:12121271

  4. Cloning and expression of canine interferon-alpha genes in Escherichia coli.

    PubMed

    Taira, Osamu; Watanugi, Itsuki; Hagiwara, Yuko; Takahashi, Masaki; Arai, Setsuo; Sato, Hisaaki; Maehara, Nobutoshi

    2005-10-01

    We cloned five new subtypes of cDNA encoding canine interferon-alpha (CaIFN-alpha) from a canine epithelial cell line. CaIFN-alphas were divided into two groups by amino acid sequences and a molecular phylogenic tree. Two subtypes of them were expressed in Escherichia coli, and IFN proteins were purified. Recombinant CaIFN-alphas were highly species-specific and showed antiviral activity against Vesicular stomatitis New Jersey virus and canine adenovirus-1 , but not against canine herpesvirus-1. PMID:16276065

  5. Splicing mutation in the ATR-X gene can lead to a dysmorphic mental retardation phenotype without {alpha}-thalassemia

    SciTech Connect

    Villard, L.; Lossi, A.M.; Fontes, M.

    1996-03-01

    We have previously reported the isolation of a gene from Xq13 that codes for a putative regulator of transcription (XNP) and has now been shown to be the gene involved in the X-linked {alpha}-thalassemia with mental retardation (ATR-X) syndrome. The widespread expression and numerous domains present in the putative protein suggest that this gene could be involved in other phenotypes. The predominant expression of the gene in the developing brain, as well as its association with neuron differentiation, indicates that mutations of this gene might result in a mental retardation (MR) phenotype. In this paper we present a family with a splice junction mutation in XNP that results in the skipping of an exon and in the introduction of a stop codon in the middle of the XNP-coding sequence. Only the abnormal transcript is expressed in two first cousins presenting the classic ATR-X phenotype (with {alpha}-thalassemia and HbH inclusions). In a distant cousin presenting a similar dysmorphic MR phenotype but not having thalassemia, {approximately}30% of the XNP transcripts are normal. These data demonstrate that the mode of action of the XNP gene product on globin expression is distinct from its mode of action in brain development and facial morphogenesis and suggest that other dysmorphic mental retardation phenotypes, such as Juberg-Marsidi or some sporadic cases of Coffin-Lowry, could be due to mutations in XNP. 20 refs., 5 figs., 2 tabs.

  6. Chromosomal localization, genomic structure, and allelic polymorphism of the human CD79a (lg-{alpha}/mb-1) gene

    SciTech Connect

    Hashimoto, S.; Gregersen, P.K.; Chiorazzi, N. |; Mohrenweiser, H.W.

    1994-12-31

    The germline DNA sequence of the human CD79a (Ig-{alpha}/mb-1) gene was determined by polymerase chain reaction sequencing of a cosmid clone derived from an arrayed human chromosome 19 library. The CD79a gene was localized to chromosome 19q13.2; this localization places the gene within the CEA-like gene cluster with the following gene order: -CEA-CGM1-CD79a-RPS11-ATP1A3-BGP-CGM9-. The genomic organization of the human CD79a gene resembles the mouse counterpart with five exons interrupted by four introns. Computer analyses suggest the presence of transcription regulatory elements known to be important in the regulation of mouse CD79a (AP-1, EBF, AP-2, MUF2, and SP-1 sites), as well as elements not found in the mouse gene (an NK-kB binding site and a series of E-box motifs). Similar to the mouse gene, the 5{prime} flanking region of human CD79a lacks a TATA box; however, unlike mouse CD79a, a classical octamer motif could not be identified in the human gene. Finally, a new Rsa I restriction fragment length polymorphism was defined in the non-coding regions of the human gene. 64 refs., 4 figs., 2 tabs.

  7. An ancient horizontal gene transfer between mosquito and the endosymbiotic bacterium Wolbachia pipientis.

    PubMed

    Woolfit, Megan; Iturbe-Ormaetxe, Iñaki; McGraw, Elizabeth A; O'Neill, Scott L

    2009-02-01

    The extent and biological relevance of horizontal gene transfer (HGT) in eukaryotic evolution remain highly controversial. Recent studies have demonstrated frequent and large-scale HGT from endosymbiotic bacteria to their hosts, but the great majority of these transferred genes rapidly become nonfunctional in the recipient genome. Here, we investigate an ancient HGT between a host metazoan and an endosymbiotic bacterium, Wolbachia pipientis. The transferred gene has so far been found only in mosquitoes and Wolbachia. In mosquitoes, it is a member of a gene family encoding candidate receptors required for malaria sporozoite invasion of the mosquito salivary gland. The gene copy in Wolbachia has substantially diverged in sequence from the mosquito homolog, is evolving under purifying selection, and is expressed, suggesting that this gene is also functional in the bacterial genome. Several lines of evidence indicate that the gene may have been transferred from eukaryotic host to bacterial endosymbiont. Regardless of the direction of transfer, however, these results demonstrate that interdomain HGT may give rise to functional, persistent, and possibly evolutionarily significant new genes. PMID:18988686

  8. SAFETY AND EFFICIENCY OF MODULATING PARACELLULAR PERMEABILITY TO ENHANCE AIRWAY EPITHELIAL GENE TRANSFER IN VIVO

    EPA Science Inventory


    ABSTRACT

    We evaluated the safety of agents that enhance gene transfer by modulating paracellular permeability. Lactate dehydrogenase (LDH) and cytokine release were measured in polarized primary human airway epithelial (HAE) cells after luminal application of vehicle, ...

  9. Long-Range Electron Transfer across Cytochrome-Hematite (alpha-Fe2O3) Interfaces.

    SciTech Connect

    Grantham, Ms. Meg; Wigginton, Nicholas S; Rosso, Kevin M; Stack, Andrew G; HochellaJr., Michael F

    2008-10-01

    Electrochemical scanning tunneling microscopy was used to assess the distance dependence of electron transfer facilitated by a bacterial multiheme cytochrome to a single crystal iron oxide surface. We measured tunneling current-distance (I-s) profiles across the nanoscale space between Au STM tips and the basal (001) surface of a hematite (R-Fe2O3) crystal and compared them to the case in which an intervening small tetraheme cytochrome (STC) from Shewanella oneidensis was covalently linked to the end of the Au tip. Tunneling profiles were collected at constant surface potentials in solutions having a range of ionic strengths. For the case without intervening cytochrome, at short tip-sample separation, the distance dependence of the tunneling current shows a quasi-linear behavior, whereas at longer distances, near-exponential decay is observed. The different regions can be understood first in terms of reduction of interfacial water and ion layers in the electrical double layer associated with the hematite surface, followed by electron tunneling through bulk water. The effective tunneling range and the transition between the two conduction mechanisms are substantially increased when STC is present in the tunneling junction, suggesting that cytochrome molecules provide enhanced tunneling pathways and stronger electronic coupling to the hematite surface. On the basis of these results, cytochrome-mediated electron transfer during bacterial metal reduction may be possible at distances farther than originally speculated. In addition, as multiheme cytochromes and other similar molecules gain attention for their promising role in fuel cells and molecular electronics, we demonstrate that the solution conditions and surface properties of the substrate must be carefully considered.

  10. Tissue-specific loss of fucosylated glycolipids in mice with targeted deletion of alpha(1,2)fucosyltransferase genes.

    PubMed Central

    Iwamori, Masao; Domino, Steven E

    2004-01-01

    Glycolipids in epithelial tissues of the gastrointestinal tract act as receptors for enteric bacteria and are implicated in the activation of the intestinal immune system. To clarify the genes involved in the fucosylation of the major glycolipids, substrate glycolipids and fucosylated products were measured in tissues of wild-type and mutant mice lacking alpha(1,2)fucosyltransferase genes FUT1 or FUT2. Quantitative determination was performed by TLC-immunostaining for GA1 (Gg4Cer), FGA1 (fucosyl GA1), GM1 (II3NeuAc-Gg4Cer), FGM1 (fucosyl GM1), and Forssman glycolipids. Both FGM1 and FGA1 completely disappeared from the antrum, cecum, and colon of FUT2-null mice, but not those of FUT1-null and wild-type mice. Precursor glycolipids, GM1 and GA1, accumulated in tissues of FUT2-null mice, indicating that the FUT2-encoded enzyme preferentially participates in the fucosylation of GA1 and GM1 in these tissues. Female reproductive organs were similarly found to utilize FUT2 for the fucosylation of glycolipids FGA1 (uterus and cervix), and FGM1 (ovary), due to their absence in FUT2-null mice. In FUT1-null mice FGA1 was lost from the pancreas, but was present in wild-type and FUT2-null mice, indicating that FUT1 is essential for fucosylation of GA1 in the pancreas. Ulex europaeus agglutinin-I lectin histochemistry for alpha(1,2)fucose residues confirmed the absence of alpha(1,2)fucose residues from the apical surface of pancreatic acinar glands of FUT1-null mice. Ileum, epididymis, and testis retained specific fucosylated glycolipids, irrespective of targeted deletion of either gene, indicating either compensation for or redundancy of the alpha(1,2)fucosyltransferase genes in these tissues. PMID:14967068

  11. Tissue-specific loss of fucosylated glycolipids in mice with targeted deletion of alpha(1,2)fucosyltransferase genes.

    PubMed

    Iwamori, Masao; Domino, Steven E

    2004-05-15

    Glycolipids in epithelial tissues of the gastrointestinal tract act as receptors for enteric bacteria and are implicated in the activation of the intestinal immune system. To clarify the genes involved in the fucosylation of the major glycolipids, substrate glycolipids and fucosylated products were measured in tissues of wild-type and mutant mice lacking alpha(1,2)fucosyltransferase genes FUT1 or FUT2. Quantitative determination was performed by TLC-immunostaining for GA1 (Gg4Cer), FGA1 (fucosyl GA1), GM1 (II3NeuAc-Gg4Cer), FGM1 (fucosyl GM1), and Forssman glycolipids. Both FGM1 and FGA1 completely disappeared from the antrum, cecum, and colon of FUT2-null mice, but not those of FUT1-null and wild-type mice. Precursor glycolipids, GM1 and GA1, accumulated in tissues of FUT2-null mice, indicating that the FUT2-encoded enzyme preferentially participates in the fucosylation of GA1 and GM1 in these tissues. Female reproductive organs were similarly found to utilize FUT2 for the fucosylation of glycolipids FGA1 (uterus and cervix), and FGM1 (ovary), due to their absence in FUT2-null mice. In FUT1-null mice FGA1 was lost from the pancreas, but was present in wild-type and FUT2-null mice, indicating that FUT1 is essential for fucosylation of GA1 in the pancreas. Ulex europaeus agglutinin-I lectin histochemistry for alpha(1,2)fucose residues confirmed the absence of alpha(1,2)fucose residues from the apical surface of pancreatic acinar glands of FUT1-null mice. Ileum, epididymis, and testis retained specific fucosylated glycolipids, irrespective of targeted deletion of either gene, indicating either compensation for or redundancy of the alpha(1,2)fucosyltransferase genes in these tissues. PMID:14967068

  12. An XMRV Derived Retroviral Vector as a Tool for Gene Transfer

    PubMed Central

    2011-01-01

    Background Retroviral vectors are widely used tools for gene delivery and gene therapy. They are useful for gene expression studies and genetic manipulation in vitro and in vivo. Many retroviral vectors are derived from the mouse gammaretrovirus, murine leukemia virus (MLV). These vectors have been widely used in gene therapy clinical trials. XMRV, initially found in prostate cancer tissue, was the first human gammaretrovirus described. Findings We developed a new retroviral vector based on XMRV called pXC. It was developed for gene transfer to human cells and is produced by transient cotransfection of LNCaP cells with pXC and XMRV-packaging plasmids. Conclusions We demonstrated that pXC mediates expression of inserted transgenes in cell lines. This new vector will be a useful tool for gene transfer in human and non-human cell lines, including gene therapy studies. PMID:21651801

  13. Evolutionary Advantage Conferred by an Eukaryote-to-Eukaryote Gene Transfer Event in Wine Yeasts

    PubMed Central

    Marsit, Souhir; Mena, Adriana; Bigey, Frédéric; Sauvage, François-Xavier; Couloux, Arnaud; Guy, Julie; Legras, Jean-Luc; Barrio, Eladio; Dequin, Sylvie; Galeote, Virginie

    2015-01-01

    Although an increasing number of horizontal gene transfers have been reported in eukaryotes, experimental evidence for their adaptive value is lacking. Here, we report the recent transfer of a 158-kb genomic region between Torulaspora microellipsoides and Saccharomyces cerevisiae wine yeasts or closely related strains. This genomic region has undergone several rearrangements in S. cerevisiae strains, including gene loss and gene conversion between two tandemly duplicated FOT genes encoding oligopeptide transporters. We show that FOT genes confer a strong competitive advantage during grape must fermentation by increasing the number and diversity of oligopeptides that yeast can utilize as a source of nitrogen, thereby improving biomass formation, fermentation efficiency, and cell viability. Thus, the acquisition of FOT genes has favored yeast adaptation to the nitrogen-limited wine fermentation environment. This finding indicates that anthropic environments offer substantial ecological opportunity for evolutionary diversification through gene exchange between distant yeast species. PMID:25750179

  14. Effect of alpha-tocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis related genes.

    PubMed

    Jeong, Yeon-Ji; Kim, Mi-Kyeong; Song, Hye-Jin; Kang, Eun-Ju; Ock, Sun-A; Kumar, B Mohana; Balasubramanian, S; Rho, Gyu-Jin

    2009-04-01

    Boar semen is extremely vulnerable to cold shock and sensitive to peroxidative damage due to high content of unsaturated fatty acids in the phospholipids of the plasma membrane and the relatively low antioxidant capacity of seminal plasma. The present study evaluated the influence of alpha-tocopherol supplementation at various concentrations in the boar semen extender during cryopreservation on post-thawed sperm motility characteristics (total sperm motility, MOT; local motility, LCM; curvilinear velocity, VCL; straight linear velocity, VSL; and average path velocity, VAP), sperm qualities (viability, acrosomal integrity and apoptosis), expression of stress protein (HSP70), and the expression of pro-apoptotic (Bax and Bak) and anti-apoptotic (Bcl-2l and Bcl-xl) genes. Semen collected from 10 Duroc boars was cryopreserved in lactose-egg yolk buffer supplemented with various concentrations of alpha-tocopherol (0, 100, 200, 400, 600 and 800 microM) using the straw-freezing procedure and stored at -196 degrees C for a minimum period of one month. In frozen-thawed groups, sperm motility was significantly (P<0.05) lower than that of fresh sperm. In fresh sperm, HSP70 immunoreactivity expression was observed in the equatorial region, but in frozen-thawed groups, expressions were mostly observed in the sperm head. Higher apoptosis rates were observed in 600 and 800 microM alpha-tocopherol supplemented frozen-thawed groups. In alpha-tocopherol supplemented frozen-thawed groups immediately after thawing, the expression was similar to that of fresh group. But after incubation at 37 degrees C for 3h, the expression in 200 and 800 microM alpha-tocopherol supplemented groups was higher than that of others. Expression of pro-apoptotic genes was significantly higher and anti-apoptotic genes was significantly (P<0.01) lower in alpha-tocopherol supplemented frozen-thawed groups compared to fresh sperm group. In conclusion, alpha-tocopherol, supplemented at 200 microM concentration

  15. Identification and characterization of an alternative promoter of the human PGC-1{alpha} gene

    SciTech Connect

    Yoshioka, Toyo; Inagaki, Kenjiro; Noguchi, Tetsuya; Sakai, Mashito; Ogawa, Wataru; Hosooka, Tetsuya; Iguchi, Haruhisa; Watanabe, Eijiro; Matsuki, Yasushi; Hiramatsu, Ryuji; Kasuga, Masato

    2009-04-17

    The transcriptional regulator peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) controls mitochondrial biogenesis and energy homeostasis. Although physical exercise induces PGC-1{alpha} expression in muscle, the underlying mechanism of this effect has remained incompletely understood. We recently identified a novel muscle-enriched isoform of PGC-1{alpha} transcript (designated PGC-1{alpha}-b) that is derived from a previously unidentified first exon. We have now cloned and characterized the human PGC-1{alpha}-b promoter. The muscle-specific transcription factors MyoD and MRF4 transactivated this promoter through interaction with a proximal E-box motif. Furthermore, either forced expression of Ca{sup 2+}- and calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A, or the p38 mitogen-activated protein kinase (p38 MAPK) kinase MKK6 or the intracellular accumulation of cAMP activated the PGC-1{alpha}-b promoter in cultured myoblasts through recruitment of cAMP response element (CRE)-binding protein (CREB) to a putative CRE located downstream of the E-box. Our results thus reveal a potential molecular basis for isoform-specific regulation of PGC-1{alpha} expression in contracting muscle.

  16. In vivo regulation of gene transcription by alpha- and gamma-Tocopherol in murine T lymphocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Of the 8 different analogues (alpha-, beta-, gamma-, delta-tocopherols and tocotrienols) designated as vitamin E, alpha-tocopherol (a-T) has been mostly studied, together with gamma-tocopherol (g-T) which is abundant in the US diet. We compared the effect of dietary supplementation with adequate or ...

  17. CTLA-4 +49 and TNF-alpha-308 gene polymorphisms in celiac patients with exocrine pancreatic insufficiency.

    PubMed

    Licul, Vanja; Cizmarević, Nada Starcević; Ristić, Smiljana; Mikolasević, Ivana; Mijandrusić, Brankica Sincić

    2013-12-01

    Celiac disease (CD) is a life-long gluten sensitive autoimmune disease of the small intestine affecting genetically susceptible individuals. Human leukocyte antigen (HLA) genotype contributes to the genetic risk for CD, but "non-HLA" genes also play a role. Clinical presentation could be classical, but majority of patients present with non-classical, atypical signs and symptoms. Endocrine and/or exocrine pancreatic insufficiency (EXPI) is common in celiac patients. The aim of our study was to assess EXPI among our CD patients by measurement of faecal pancreatic elastase (FE1) and to find potential association of CTLA-4 +49 and TNF-alpha-308 gene polymorphism and EXPI. Eighty three patients entered the study. Tissue transglutaminase antibodies (anti-TTG), faecal elastase-1 (FE1) assays and genotyping for the CTLA-4 +49A/G and TNF-alpha308 were performed. Of 83 patients with CD EXPI had 13 (15.6 %). There was no statistically significant difference in frequency of polymorphisms for both genes (CTL-4 +49 i TNF-alpha-308) in the group with and without EXPI. In conclusion, EXPI is common in symptomatic CD patients, but further genetic studies with larger number of patients are needed. PMID:24611333

  18. Direct gene transfer into human cultured cells facilitated by laser micropuncture of the cell membrane

    SciTech Connect

    Tao, W.; Wilkinson, J.; Stanbridge, E.J.; Berns, M.W.

    1987-06-01

    The selective alteration of the cellular genome by laser microbeam irradiation has been extensively applied in cell biology. We report here the use of the third harmonic (355 nm) of an yttrium-aluminum garnet laser to facilitate the direct transfer of the neo gene into cultured human HT1080-6TG cells. The resultant transformants were selected in media containing an aminoglycoside antibiotic, G418. Integration of the neo gene into individual chromosomes and expression of the gene were demonstrated by Southern blot analyses, microcell-mediated chromosome transfer, and chromosome analyses. The stability of the integrated neo gene in the transformants was shown by a comparative growth assay in selective and nonselective media. Transformation and incorporation of the neo gene into the host genome occurred at a frequency of 8x10-4-3x10-3. This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.

  19. Direct Gene Transfer into Human Cultured Cells Facilitated by Laser Micropuncture of the Cell Membrane

    NASA Astrophysics Data System (ADS)

    Tao, Wen; Wilkinson, Joyce; Stanbridge, Eric J.; Berns, Michael W.

    1987-06-01

    The selective alteration of the cellular genome by laser microbeam irradiation has been extensively applied in cell biology. We report here the use of the third harmonic (355 nm) of an yttrium-aluminum garnet laser to facilitate the direct transfer of the neo gene into cultured human HT1080-6TG cells. The resultant transformants were selected in medium containing an aminoglycoside antibiotic, G418. Integration of the neo gene into individual human chromosomes and expression of the gene were demonstrated by Southern blot analyses, microcell-mediated chromosome transfer, and chromosome analyses. The stability of the integrated neo gene in the transformants was shown by a comparative growth assay in selective and nonselective media. Transformation and incorporation of the neo gene into the host genome occurred at a frequency of 8 × 10-4-3 × 10-3. This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.

  20. Quantitative analysis of recombination between YFP and CFP genes of FRET biosensors introduced by lentiviral or retroviral gene transfer.

    PubMed

    Komatsubara, Akira T; Matsuda, Michiyuki; Aoki, Kazuhiro

    2015-01-01

    Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have been developed to visualize spatio-temporal dynamics of signalling molecules in living cells. Many of them adopt a backbone of intramolecular FRET biosensor with a cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) as donor and acceptor, respectively. However, there remains the difficulty of establishing cells stably expressing FRET biosensors with a YFP and CFP pair by lentiviral or retroviral gene transfer, due to the high incidence of recombination between YFP and CFP genes. To address this, we examined the effects of codon-diversification of YFP on the recombination of FRET biosensors introduced by lentivirus or retrovirus. The YFP gene that was fully codon-optimized to E.coli evaded the recombination in lentiviral or retroviral gene transfer, but the partially codon-diversified YFP did not. Further, the length of spacer between YFP and CFP genes clearly affected recombination efficiency, suggesting that the intramolecular template switching occurred in the reverse-transcription process. The simple mathematical model reproduced the experimental data sufficiently, yielding a recombination rate of 0.002-0.005 per base. Together, these results show that the codon-diversified YFP is a useful tool for expressing FRET biosensors by lentiviral or retroviral gene transfer. PMID:26290434

  1. Location of the alpha-amylase gene in rumen Streptococcus bovis strains distinguished by unstable amylase activity.

    PubMed

    Mareková, M; Jonecová, Z; Kmeĭ, V

    1995-01-01

    Genetic stability of amylase activity after serial subcultivation experiments with amylolytic ruminal Streptococcus bovis strains was investigated. Two strains Amy+ and Amy- were obtained. Loss of amylase activity connected with the loss of plasmid DNA was not found in these strains. The presence of the gene responsible for the amylase activity in the chromosome of these strains was revealed by hybridization of the alpha-amylase gene on pJK108 against chromosomal DNA of S. bovis and Bacillus subtilis after a complete restriction with EcoRI. PMID:8851562

  2. Precise mapping of the brain [alpha][sub 2]-adrenergic receptor gene within chromosome 4p16

    SciTech Connect

    Riess, O.; Siedlaczck, I.; Potisek, S.; Epplen, J.T. ); Thies, U. ); Graham, R.; Theilmann, J.; Hayden, M.R. ); Grimm, T. )

    1994-01-15

    The gene encoding the brain [alpha][sub 2]-adrenergic receptor (ADRA2C) is located on human chromosome 4. It has been circumstantially associated with a number of human disorders, including Parkinson disease, panic disorders, and Huntington disease (HD). Using somatic cell hybrids, the authors localized the gene to chromosome 4p16 distal to P8 (D4S62). To investigate this locus further, they isolated several cosmid clones covering the entire gene. The gene was found to be intronless. Two (GT)[sub n] repeats in close proximity to the ADRAC2 gene were analyzed and used to define its precise location. Linkage disequilibrium studies of one microsatellite in HD families showed strong nonrandom association to the HD mutation, indicating its tight linkage to the HD gene. The investigation of families carrying recombinant chromosomes, pulsed-field analysis, and genomic walking mapped the ADRAC2 gene adjacent to D4S81, 500 kb proximal to the HD gene. The newly defined microsatellites at the ADRAC2 locus, its precise localization within 4p16, and the detailed PCR conditions facilitate the identification of any defect caused by this gene. 22 refs., 4 figs., 2 tabs.

  3. Cytokine-inducible expression in endothelial cells of an I kappa B alpha-like gene is regulated by NF kappa B.

    PubMed Central

    de Martin, R; Vanhove, B; Cheng, Q; Hofer, E; Csizmadia, V; Winkler, H; Bach, F H

    1993-01-01

    The transient expression of many different genes is mediated by the inducible transcription factor p50-p65 NF kappa B, which in turn is regulated by complex formation with its inhibitor I kappa B alpha. We describe here that in porcine aortic endothelial cells, either IL-1 alpha, TNF alpha or LPS upregulates an inhibitor of NF kappa B which we refer to as ECI-6. ECI-6 is by structural and functional criteria an I kappa B alpha protein, the porcine homologue of MAD-3, pp40 and RL/IF-1. We have studied the promoter of the ECI-6/I kappa B alpha gene and provide three lines of evidence that its expression is directly regulated by NF kappa B. First, the 5' regulatory region of ECI-6/I kappa B alpha contains two sites that bind NF kappa B in electrophoretic mobility shift assays. Second, expression following transfection of an ECI-6/I kappa B alpha promoter-luciferase reporter construct is dependent on a co-transfected NF kappa B-p65 subunit. Third, pretreatment of endothelial cells with antioxidants, agents that inhibit activation of NF kappa B, inhibit the expression of ECI-6/I kappa B alpha. We conclude that the regulated expression of ECI-6/I kappa B alpha could represent a novel feedback mechanism by which NF kappa B downregulates its own activity after transient activation of target genes has been achieved. Images PMID:8334993

  4. Development of second- and third-generation bovine immunodeficiency virus-based gene transfer systems.

    PubMed

    Matukonis, Meghan; Li, Mengtao; Molina, Rene P; Paszkiet, Brian; Kaleko, Michael; Luo, Tianci

    2002-07-20

    Lentivirus-based gene transfer systems have demonstrated their utility in mediating gene transfer to dividing and nondividing cells both in vitro and in vivo. An early-generation gene transfer system developed from bovine immunodeficiency virus (BIV) has been described (Berkowitz et al., J. Virol. 2001;75:3371-3382). In this paper, we describe the development of second-generation (three-plasmid) and third-generation (four-plasmid) BIV-based systems. All accessory genes (vif, vpw, vpy, and tmx) and the regulatory gene tat were deleted or largely truncated from the packaging construct. Furthermore, we split the packaging function into two constructs by expressing Rev in a separate plasmid. Together with our minimal BIV transfer vector construct and a vesicular stomatitis virus G glycoprotein-expressing plasmid, the BIV vectors were generated. The vectors produced by the three- and four-plasmid systems had titers greater than 1 x 10(6) transducing units per milliliter and were fully functional as indicated by their ability to efficiently transduce both dividing and nondividing cells. These results suggest that the accessory genes vif, vpw, vpy, and tmx are dispensable for functional BIV vector development. The modifications made to the packaging constructs improve the safety profile of the vector system. Finally, BIV vectors provide an alternative to human immunodeficiency virus-based gene transfer systems. PMID:12162812

  5. Exploration of horizontal gene transfer between transplastomic tobacco and plant-associated bacteria.

    PubMed

    Demanèche, Sandrine; Monier, Jean-Michel; Dugat-Bony, Eric; Simonet, Pascal

    2011-10-01

    The likelihood of gene transfer from transgenic plants to bacteria is dependent on the transgene copy number and on the presence of homologous sequences for recombination. The large number of chloroplast genomes in a plant cell as well as the prokaryotic origin of the transgene may thus significantly increase the likelihood of gene transfer from transplastomic plants to bacteria. In order to assess the probability of such a transfer, bacterial isolates, screened for their ability to colonize decaying tobacco plant tissue and possessing DNA sequence similarity to the chloroplastic genes accD and rbcL flanking the transgene (aadA), were tested for their ability to take up extracellular DNA (broad host-range pBBR1MCS-3-derived plasmid, transplastomic plant DNA and PCR products containing the genes accD-aadA-rbcL) by natural or electrotransformation. The results showed that among the 16 bacterial isolates tested, six were able to accept foreign DNA and acquire the spectinomycin resistance conferred by the aadA gene on plasmid, but none of them managed to integrate transgenic DNA in their chromosome. Our results provide no indication that the theoretical gene transfer-enhancing properties of transplastomic plants cause horizontal gene transfer at rates above those found in other studies with nuclear transgenes. PMID:21564143

  6. Skeletal muscle microcirculatory response to rat alpha-calcitonin gene-related peptide.

    PubMed

    Arden, W A; Fiscus, R R; Beihn, L D; Derbin, M; Oremus, R; Gross, D R

    1994-07-01

    We used in vivo video microscopy to determine the effect of increasing doses of rat alpha-calcitonin gene-related peptide (rCGRP) on rat cremaster muscle arterioles in the presence or absence of the nitric oxide synthase inhibitor N-omega-nitro-L-arginine (L-NNA). Male Sprague-Dawley rats (118-148 g) were anaesthetized with pentobarbital, and neurovascularly intact cremaster muscles were imaged. Changes in the diameter, erythrocyte velocity and volume flow in second-(A2), third-(A3), and fourth-(A4) order arterioles were determined. To produce uniform arteriolar tone, the cremaster preparation was challenged with norepinephrine (NE: 10(-7) M). L-NNA (10(-4) M), which was shown to inhibit acetylcholine-(ACh: 10(-6) M) induced arteriolar dilations, was added to 16 of the preparations. Preparations were then challenged by adding cumulative log concentrations of rCGRP (10(-12)-10-7) M; n = 16) or an equivalent volume of vehicle (n = 19) to the bath. Following rCGRP challenge, arterioles were maximally dilated with 10(-5) M nitroprusside (NP). rCGRP caused significant dose-dependent increases in erythrocyte velocity and volume flow in A2 arterioles, and in diameter, velocity, and volume flow in A3 and A4 arterioles, by 10(-8) M, when compared with vehicle-treated controls. L-NNA had no significant effect on rCGRP-induced responses. These data indicate that rCGRP causes dose-dependent dilation of skeletal muscle resistance arterioles at a concentration similar to that observed in larger vessels. This dilation does not appear to be dependent on the vascular production of nitric oxide from L-arginine. PMID:7526261

  7. Mutations in the PCCA gene encoding the {alpha} subunit of propionyl-CoA carboxylase in patients with propionic acidemia

    SciTech Connect

    Campeau, E.; Leon-Del-Rio, A.; Gravel, R.A.

    1994-09-01

    Propionic acidemia is a rare autosomal recessive disorder characterized by a deficiency of the mitochondrial biotin-dependent enzyme, propionyl-CoA carboxylase (PCC). PCC has the structure {alpha}{sub 4}{beta}{sub 4}, with the {alpha} subunit containing the biotin prosthetic group. This study is concerned with defining the spectrum of mutations occurring in the PCCA gene encoding the {alpha} subunit. Mutations were initially assigned to this gene through complementation experiments done after somatic fusion of patient fibroblasts. The analyses were performed on PCR-amplified reverse transcripts of fibroblast RNA. The mutations were identified by single strand conformational polymorphism analysis and direct sequencing of PCR products. Three candidate disease-causing mutations and one DNA polymorphism were identified in the {alpha} subunit sequence in different patients: (1) a 3 bp deletion {triangle}CTG{sub 2058-2060}, which eliminates Cys687 near the biotin binding site (Lys669); (2) T{sub 611}{r_arrow}A which converts Met204 to Lys in a highly conserved region matching that of an ATP binding site; (3) An {approximately}50 bp deletion near the 3{prime} end of the cDNA which likely corresponds to the loss of an exon due to a splicing defect; and (4) a 3 bp insertion, +CAG{sub 2203}, located downstream of the stop codon, which is likely a DNA polymorphism. In order to determine the effect of the Cys687 deletion on the biotinylation of PCC, we expressed the mutation in a 67 amino acid C-terminal fragment of the PCC {alpha} subunit in E. coli in which biotinylation is directed by the bacterial biotin ligase. While the mutant peptide was expressed at about half-normal levels, the biotinylation of the peptide that was present was reduced to only {approximately}20% normal. We suggest, therefore, that the absence of PCC activity due to {triangle}Cys687 results at least in part from defective biotinylation of an unstable protein.

  8. Chloroplast-like transfer RNA genes expressed in wheat mitochondria.

    PubMed Central

    Joyce, P B; Gray, M W

    1989-01-01

    In the course of a systematic survey of wheat mitochondrial tRNA genes, we have sequenced chloroplast-like serine (trnS-GGA), phenylalanine (trnF-GAA) and cysteine (trnC-GCA) tRNA genes and their flanking regions. These genes are remnants of 'promiscuous' chloroplast DNA that has been incorporated into wheat mtDNA in the course of its evolution. Each gene differs by one or a few nucleotides from the authentic chloroplast homolog previously characterized in wheat or other plants, and each could potentially encode a functional tRNA whose secondary structure shows no deviations from the generalized model. To determine whether these chloroplast-like tRNA genes are actually expressed, wheat mitochondrial tRNAs were resolved by a series of polyacrylamide gel electrophoreses, after being specifically end-labeled in vitro by 3'-CCA addition mediated by wheat tRNA nucleotidyltransferase. Subsequent direct RNA sequence analysis identified prominent tRNA species corresponding to the mitochondrial and not the chloroplast trnS, trnF and trnC genes. This analysis also revealed chloroplast-like elongator methionine, asparagine and tryptophan tRNAs. Our results suggest that at least some chloroplast-like tRNA genes in wheat mtDNA are transcribed, with transcripts undergoing processing, post-transcriptional modification and 3'-CCA addition, to produce mature tRNAs that may participate in mitochondrial protein synthesis. Images PMID:2762145

  9. Single nucleotide polymorphism in the tumor necrosis factor-alpha gene affects inflammatory bowel diseases risk

    PubMed Central

    Ferguson, Lynnette R; Huebner, Claudia; Petermann, Ivonne; Gearry, Richard B; Barclay, Murray L; Demmers, Pieter; McCulloch, Alan; Han, Dug Yeo

    2008-01-01

    AIM: To investigate the role that single nucleotide polymorphisms (SNPs) in the promoter of the tumour necrosis factor-alpha (TNF-α) gene play in the risk of inflammatory bowel diseases (IBDs) in a New Zealand population, in the context of international studies. METHODS: DNA samples from 388 patients with Crohn’s disease (CD), 405 ulcerative colitis (UC), 27 indeterminate colitis (IC) and 201 randomly selected controls, from Canterbury, New Zealand were screened for 3 common polymorphisms in the TNF-α receptor: -238 G→A, -308 G→A and -857C→T, using a TaqmanR assay. A meta-analysis was performed on the data obtained on these polymorphisms combined with that from other published studies. RESULTS: Individuals carrying the -308 G/A allele had a significantly (OR = 1.91, χ2 = 17.36, P < 0.0001) increased risk of pancolitis, and a 1.57-fold increased risk (OR = 1.57, χ2 = 4.34, P = 0.037) of requiring a bowel resection in UC. Carrying the -857 C/T variant decreased the risk of ileocolonic CD (OR = 0.56, χ2 = 4.32, P = 0.037), and the need for a bowel resection (OR = 0.59, χ2 = 4.85, P = 0.028). The risk of UC was reduced in individuals who were smokers at diagnosis, (OR = 0.48, χ2 = 4.86, P = 0.028). CONCLUSION: TNF-α is a key cytokine known to play a role in inflammatory response, and the locus for the gene is found in the IBD3 region on chromosome 6p21, known to be associated with an increased risk for IBD. The -308 G/A SNP in the TNF-α promoter is functional, and may account in part for the increased UC risk associated with the IBD3 genomic region. The -857 C/T SNP may decrease IBD risk in certain groups. Pharmaco- or nutrigenomic approaches may be desirable for individuals with such affected genotypes. PMID:18698679

  10. Characterization and regulation of the NADP-linked 7 alpha-hydroxysteroid dehydrogenase gene from Clostridium sordellii.

    PubMed Central

    Coleman, J P; Hudson, L L; Adams, M J

    1994-01-01

    A bile acid-inducible NADP-linked 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) from Clostridium sordellii ATCC 9714 was purified 310-fold by ion-exchange, gel filtration, and dye-ligand affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed one predominant peptide band (30,000 Da). The N-terminal sequence was determined, and the corresponding oligonucleotides were synthesized and used to screen EcoRI and HindIII genomic digests of C. sordellii. Two separate fragments (4,500 bp, EcoRI; 3,200 bp, HindIII) were subsequently cloned by ligation to pUC19 and transformation into Escherichia coli DH5 alpha-MCR. The EcoRI fragment was shown to contain a truncated 7 alpha-HSDH gene, while the HindIII fragment contained the entire coding region. E. coli clones containing the HindIII insert expressed high levels of an NADP-linked 7 alpha-HSDH. Nucleotide sequence analyses suggest that the 7 alpha-HSDH is encoded by a monocistronic transcriptional unit, with DNA sequence elements resembling rho-independent terminators located in both the upstream and downstream flanking regions. The transcriptional start site was located by primer extension analysis. Northern (RNA) blot analysis indicated that induction is mediated at the transcriptional level in response to the presence of bile acid in the growth medium. In addition, growth-phase-dependent expression is observed in uninduced cultures. Analysis of the predicted protein sequence indicates that the enzyme can be classified in the short-chain dehydrogenase group. Images PMID:8050999

  11. Lentiviral vector-mediated gene transfer and RNA silencing technology in neuronal dysfunctions.

    PubMed

    Dreyer, Jean-Luc

    2011-02-01

    Lentiviral-mediated gene transfer in vivo or in cultured mammalian neurons can be used to address a wide variety of biological questions, to design animals models for specific neurodegenerative pathologies, or to test potential therapeutic approaches in a variety of brain disorders. Lentiviruses can infect non-dividing cells, thereby allowing stable gene transfer in post-mitotic cells such as mature neurons. An important contribution has been the use of inducible vectors: the same animal can thus be used repeatedly in the doxycycline-on or -off state, providing a powerful mean for assessing the function of a gene candidate in a disorder within a specific neuronal circuit. Furthermore, lentivirus vectors provide a unique tool to integrate siRNA expression constructs with the aim to locally knockdown expression of a specific gene, enabling to assess the function of a gene in a very specific neuronal pathway. Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in the brain. Therefore, the use of lentiviruses for stable expression of siRNA in brain is a powerful aid to probe gene functions in vivo and for gene therapy of diseases of the central nervous system. In this chapter I review the applications of lentivirus-mediated gene transfer in the investigation of specific gene candidates involved in major brain disorders and neurodegenerative processes. Major applications have been in polyglutamine disorders, such as synucleinopathies and Parkinson's disease, or in investigating gene function in Huntington's disease, dystonia, or muscular dystrophy. Recently, lentivirus gene transfer has been an invaluable tool for evaluation of gene function in behavioral disorders such as drug addiction and attention-deficit hyperactivity disorder or in learning and cognition. PMID:20862616

  12. Decreased expression of hepatocyte nuclear factor 3 alpha during the acute-phase response influences transthyretin gene transcription.

    PubMed Central

    Qian, X; Samadani, U; Porcella, A; Costa, R H

    1995-01-01

    Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (alpha, beta, and gamma) are known to regulate the transcription of numerous liver-specific genes. The HNF-3 proteins bind to DNA as monomers through a winged-helix motif, which is also utilized by a number of developmental regulators, including the Drosophila homeotic fork head (fkh) protein. We have previously characterized a strong-affinity HNF-3S site in the transthyretin (TTR) promoter region which is essential for expression in human hepatoma (HepG2) cells. In the current study, we identify an activating protein 1 (AP-1) site which partially overlaps the HNF-3S sequence in the TTR promoter. We show that in HepG2 cells the AP-1 sequence confers 12-O-tetradecanoylphorbol-13-acetate inducibility to the TTR promoter and contributes to normal TTR transcriptional activity. We also demonstrate that the HNF-3 proteins and AP-1 bind independently to the TTR AP-1-HNF-3 site, and cotransfection experiments suggest that they do not cooperate to activate an AP-1-HNF-3 reporter construct. In addition, 12-O-tetradecanoylphorbol-13-acetate exposure of HepG2 cells results in a reciprocal decrease in HNF-3 alpha and -3 gamma expression which may facilitate interaction of AP-1 with the TTR AP-1-HNF-3 site. In order to explore the role of HNF-3 in the liver, we have examined expression patterns of TTR and HNF-3 during the acute-phase response and liver regeneration. Partial hepatectomy produced minimal fluctuation in HNF-3 and TTR expression, suggesting that HNF-3 expression is not influenced by proliferative signals induced during liver regeneration. In acute-phase livers, we observed a dramatic reduction in HNF-3 alpha expression which correlates with a decrease in the expression of its target gene, the TTR gene. Furthermore, consistent with previous studies, the acute-phase livers are induced for c-jun but not c-fos expression. We propose that the reduction in TTR gene expression during the acute phase is likely due

  13. Complete structural organization of the human {alpha}1(V) collagen gene (COL5A1): Divergence from the conserved organization of other characterized fibrillar collagen genes

    SciTech Connect

    Takahara, Kazuhiko; Hoffman, G.G.; Greenspan, D.S.

    1995-10-10

    Genes that encode the vertebrate fibrillar collagen types I-III have previously been shown to share a highly conserved intron/exon organization, thought to reflect common ancestry and evolutionary pressures at the protein level. We report here the complete intron/exon organization of COL5A1, the human gene that encodes the {alpha}1 chain of fibrillar collagen type V. The structure of COL5A1 is shown to be considerably diverged from the conserved structure of the genes for fibrillar collagen types I-III. COL5A1 has 66 exons, which is greater than the number of exons found in the genes for collagen types I-III. The increased number of exons is partly due to the increased size of the pro-{alpha}1(V) N-propeptide, relative to the sizes of the N-propeptides of the types I-III procollagen molecules. In addition, however, the increased number of exons is due to differences in the intron/exon organization of the triple-helix coding region of COL5A1 compared to the organization of the triple-helix coding regions of the genes for collagen types I-III. Of particular interest is the increase of 54 bp exons in this region of COL5A1, strongly supporting the proposal that the triple-helix coding regions of fibrillar collagen genes evolved from duplication of a 54 bp primordial genetic element. Moreover, comparison of the structure of COL5A1 to the highly conserved structure of the genes of collagen types I-III provides insights into the probable structure of the ancestral gene that gave rise to what appears to be two classes of vertebrate fibrillar collagen genes. 50 refs., 5 figs.

  14. Gene transfer into hematopoietic progenitor and stem cells: progress and problems.

    PubMed

    Dunbar, C E; Emmons, R V

    1994-11-01

    Gene transfer to hematopoietic cells for the purpose of "gene therapy" is a new and rapidly developing field with clinical trials in progress. A fundamental goal of research in this field is the incorporation of exogenous genes into the chromosomes of the most primitive hematopoietic progenitor cells--stem cells. Recombinantly engineered retroviral vectors are the best characterized and are currently the only vector type in clinical trials directed at the hematopoietic system. High efficiency gene transfer and expression in murine stem cells and their progeny is now routine, but in larger animal models such as dogs or primates and preliminary clinical trials, gene transfer has been less successful. Problems such as retroviral efficiency, gene expression, insertional mutagenesis and helper virus contamination are being addressed. A promising new vector, the adeno-associated virus (AAV), has shown promise and may allow production of high titer, stable, recombinant virions without helper contamination and with potentially better safety parameters. However, the technology for AAV gene transfer is currently underdeveloped, and issues related to the reproducible production of vectors must be addressed. Other non-viral vector systems are being explored, but little data are available on applications to hematopoietic cells. Better preclinical models are needed to study gene targeting and expression in human cells. An overview of recombinant retroviral and adeno-associated viral vector production, preclinical data and preliminary clinical data will be given, and problems needing to be addressed at all stages of development before broad clinical utility can be achieved will be discussed. PMID:7881358

  15. Evolutionary transfer of the chloroplast tufA gene to the nucleus.

    PubMed

    Baldauf, S L; Palmer, J D

    1990-03-15

    Evolutionary gene transfer is a basic corollary of the now widely accepted endosymbiotic theory, which proposes that mitochondria and chloroplasts originated from once free-living eubacteria. The small organellar chromosomes are remnants of larger bacterial genomes, with most endosymbiont genes having been either transferred to the nucleus soon after endosymbiosis or lost entirely, with some being functionally replaced by pre-existing nuclear genes. Several lines of evidence indicate that relocation of some organelle genes could have been more recent. These include the abundance of non-functional organelle sequences of recent origin in nuclear DNA, successful artificial transfer of functional organelle genes to the nucleus, and several examples of recently lost organelle genes, although none of these is known to have been replaced by a nuclear homologue that is clearly of organellar ancestry. We present gene sequence and molecular phylogenetic evidence for the transfer of the chloroplast tufA gene to the nucleus in the green algal ancestor of land plants. PMID:2314461

  16. Mutant HNF-1{alpha} and mutant HNF-1{beta} identified in MODY3 and MODY5 downregulate DPP-IV gene expression in Caco-2 cells

    SciTech Connect

    Gu Ning; Adachi, Tetsuya; Matsunaga, Tetsuro; Takeda, Jun; Tsujimoto, Gozoh; Ishihara, Akihiko; Yasuda, Koichiro; Tsuda, Kinsuke . E-mail: jinkan@tom.life.h.kyoto-u.ac.jp

    2006-08-04

    Dipeptidylpeptidase IV (DPP-IV) is a well-documented drug target for the treatment of type 2 diabetes. Hepatocyte nuclear factors (HNF)-1{alpha} and HNF-1{beta}, known as the causal genes of MODY3 and MODY5, respectively, have been reported to be involved in regulation of DPP-IV gene expression. But, it is not completely clear (i) that they play roles in regulation of DPP-IV gene expression, and (ii) whether DPP-IV gene activity is changed by mutant HNF-1{alpha} and mutant HNF-1{beta} in MODY3 and MODY5. To explore these questions, we investigated transactivation effects of wild HNF-1{alpha} and 13 mutant HNF-1{alpha}, as well as wild HNF-1{beta} and 2 mutant HNF-1{beta}, on DPP-IV promoter luciferase gene in Caco-2 cells by means of a transient experiment. Both wild HNF-1{alpha} and wild HNF-1{beta} significantly transactivated DPP-IV promoter, but mutant HNF-1{alpha} and mutant HNF-1{beta} exhibited low transactivation activity. Moreover, to study whether mutant HNF-1{alpha} and mutant HNF-1{beta} change endogenous DPP-IV enzyme activity, we produced four stable cell lines from Caco-2 cells, in which wild HNF-1{alpha} or wild HNF-1{beta}, or else respective dominant-negative mutant HNF-1{alpha}T539fsdelC or dominant-negative mutant HNF-1{beta}R177X, was stably expressed. We found that DPP-IV gene expression and enzyme activity were significantly increased in wild HNF-1{alpha} cells and wild HNF-1{beta} cells, whereas they decreased in HNF-1{alpha}T539fsdelC cells and HNF-1{beta}R177X cells, compared with DPP-IV gene expression and enzyme activity in Caco-2 cells. These results suggest that both wild HNF-1{alpha} and wild HNF-1{beta} have a stimulatory effect on DPP-IV gene expression, but that mutant HNF-1{alpha} and mutant HNF-1{beta} attenuate the stimulatory effect.

  17. Novel ring A stereoisomers of 2-methyl-1alpha,25-dihydroxyvitamin D(3) and 2-methyl-20-epi-1alpha,25-dihydroxyvitamin D(3): transactivation of target genes and modulation of differentiation in human promyelocytic leukemia (HL-60) cells.

    PubMed

    Nakagawa, K; Kurobe, M; Ozono, K; Konno, K; Fujishima, T; Takayama, H; Okano, T

    2000-03-15

    We evaluated the biological activity of two sets of ring A stereoisomers of 2-methyl-1alpha,25-dihydroxyvitamin D(3) (2-methyl-1alpha,25(OH)(2)D(3)) and 2-methyl-20-epi-1alpha, 25-dihydroxyvitamin D(3) (2-methyl-20-epi-1alpha,25(OH)(2)D(3)) in terms of the following: transactivation of a rat 25-hydroxyvitamin D(3)-24-hydroxylase gene promoter including two vitamin D response elements (VDREs) and a human osteocalcin gene promoter including a VDRE in transfected human osteosarcoma (MG-63) cells; a vitamin D receptor (VDR)-mediated response using a VDR-GAL4 one-hybrid luciferase reporter system and a retinoid X receptor alpha (RXRalpha)-mediated response using an expressed VDR/RXRalpha-GAL4 modified two-hybrid luciferase reporter system in transfected human epitheloid carcinoma, cervix (HeLa) cells; and modulation of cell surface CD11b antigen expression in human leukemia (HL-60) cells. All the diastereomers of both analogues exhibited unique biological activity profiles depending upon the configurations of the C-1 and C-3 hydroxyl groups, the C-2 methyl group in ring A, and the C-20 methyl group in the side chain. Of the eight possible diastereomers of the 2-methyl analogues, 2alpha-methyl-1alpha,25(OH)(2)D(3) was the most potent and exhibited comparable or even greater biological potency than 1alpha,25(OH)(2)D(3). Of the eight possible diastereomers of the 2-methyl-20-epi analogues, 2alpha-methyl-20-epi-1alpha,25(OH)(2)D(3) was the most potent and exhibited 100- to 200-fold higher transcriptional potencies than 1alpha,25(OH)(2)D(3) and exceptionally high cell regulatory activities. 2beta-methyl-20-epi-1alpha,25(OH)(2)D(3) was nearly as potent as its 2-epimer, 2alpha-methyl-20-epi-1alpha,25(OH)(2)D(3), whereas its 20-epimer, 2beta-methyl-1alpha,25(OH)(2)D(3), was almost completely biologically inactive. In these respects, it can be postulated that the double modification of 2-methyl substitution and 20-epimerization to 1alpha,25(OH)(2)D(3) induces remarkable changes

  18. Physical linkage of the human growth hormone gene cluster and the skeletal muscle sodium channel {alpha}-subunit gene (SCN4A) on chromosome 17

    SciTech Connect

    Bennani-Baiti, I.M.; Jones, B.K.; Liebhaber, S.A.; Cooke, N.E.

    1995-10-10

    The human growth hormone (GH) locus, a cluster of five genes, spans 47 kb on chromosome 17q22-q24. The skeletal muscle sodium channel {alpha}-subunit locus (SCN4A), a 32.5-kb gene, has previously been mapped to 17q23.1-q25.3. We demonstrate that both the GH gene cluster and the SCN4A gene colocalize to a single 525-kb yeast artificial chromosome (YAC) containing DNA derived from human chromosome 17. Restriction maps of two cosmids encompassing the 5{prime} terminus of the GH locus and including up to 40 kb of 5{prime}-flanking sequences demonstrate a perfect 20-kb overlap with previously published maps of the SCN4A gene. A 720-bp DNA segment, encompassing sequences 32.3 to 31.6 kb 5{prime} to GH, was sequenced and found to be identical to exon 14 of SCN4A. These data demonstrate that the SCN4A gene and the entire GH gene cluster are contained within 100 kb on chromosome 17 and are separated by only 21.5 kb. Remarkably, this physical linkage between GH and SCN4A also reveals that multiple elements critical to tissue-specific transcriptional activation of the GH gene lie within the SCN4A gene. 48 refs., 5 figs.

  19. Physical mapping of four serpin genes: [alpha][sub 1]-antitrypsin, [alpha][sub 1]-antichymotrypsin, corticosteroid-binding globulin, and protein C inhibitor, within a 280-kb region on chromosome 14q32. 1

    SciTech Connect

    Billingsley, G.D.; Cox, D.W. Univ. of Toronto, Ontario ); Walter, M.A. ); Hammond, G.L. )

    1993-02-01

    Alpha[sub 1]-antitrypsin ([alpha]1AT; protease inhibitor [PI] locus), [alpha][sub 1]-antichymotrypsin ([alpha]1ACT; AACT locus), corticosteroid-binding globulin (CBG; CBG locus), and protein C inhibitor (PCI; PCI locus) are members of the serine protease inhibitor (serpin) superfamily. A noncoding PI-like (PIL) gene has been located 12 kb 3[prime] of the PI gene. The PI, PIL, and AACT loci have been localized to 14q32.1, the CBG locus has been localized to 14q31-14q32.1, and PCI has been mapped to chromosome 14. Genetic linkage analysis suggests tight linkage between PI and AACT. The authors have used pulsed-field gel electrophoresis to generate a physical map linking these five serpin genes. The order of the genetic loci is AACT/PCI-PI-PIL-CBG, with a maximum distance of about 220 kb between the AACT/PCI and PI genes. These genes form a PI cluster at 14q32.1, similar to that of the homologous genes on murine chromosome 12. The close proximity of these genes has implications for disease-association studies. 44 refs., 6 figs., 2 tabs.

  20. Horizontal Gene Transfers from Bacteria to Entamoeba Complex: A Strategy for Dating Events along Species Divergence.

    PubMed

    Romero, Miguel; Cerritos, R; Ximenez, Cecilia

    2016-01-01

    Horizontal gene transfer has proved to be relevant in eukaryotic evolution, as it has been found more often than expected and related to adaptation to certain niches. A relatively large list of laterally transferred genes has been proposed and evaluated for the parasite Entamoeba histolytica. The goals of this work were to elucidate the importance of lateral gene transfer along the evolutionary history of some members of the genus Entamoeba, through identifying donor groups and estimating the divergence time of some of these events. In order to estimate the divergence time of some of the horizontal gene transfer events, the dating of some Entamoeba species was necessary, following an indirect dating strategy based on the fossil record of plausible hosts. The divergence between E. histolytica and E. nuttallii probably occurred 5.93 million years ago (Mya); this lineage diverged from E. dispar 9.97 Mya, while the ancestor of the latter separated from E. invadens 68.18 Mya. We estimated times for 22 transferences; the most recent occurred 31.45 Mya and the oldest 253.59 Mya. Indeed, the acquisition of genes through lateral transfer may have triggered a period of adaptive radiation, thus playing a major role in the evolution of the Entamoeba genus. PMID:27239333

  1. Horizontal Gene Transfers from Bacteria to Entamoeba Complex: A Strategy for Dating Events along Species Divergence

    PubMed Central

    Romero, Miguel; Ximenez, Cecilia

    2016-01-01

    Horizontal gene transfer has proved to be relevant in eukaryotic evolution, as it has been found more often than expected and related to adaptation to certain niches. A relatively large list of laterally transferred genes has been proposed and evaluated for the parasite Entamoeba histolytica. The goals of this work were to elucidate the importance of lateral gene transfer along the evolutionary history of some members of the genus Entamoeba, through identifying donor groups and estimating the divergence time of some of these events. In order to estimate the divergence time of some of the horizontal gene transfer events, the dating of some Entamoeba species was necessary, following an indirect dating strategy based on the fossil record of plausible hosts. The divergence between E. histolytica and E. nuttallii probably occurred 5.93 million years ago (Mya); this lineage diverged from E. dispar 9.97 Mya, while the ancestor of the latter separated from E. invadens 68.18 Mya. We estimated times for 22 transferences; the most recent occurred 31.45 Mya and the oldest 253.59 Mya. Indeed, the acquisition of genes through lateral transfer may have triggered a period of adaptive radiation, thus playing a major role in the evolution of the Entamoeba genus. PMID:27239333

  2. Kinetics of conjugative gene transfer on surfaces in granular porous media

    NASA Astrophysics Data System (ADS)

    Massoudieh, A.; Crain, C.; Lambertini, E.; Nelson, K. E.; Barkouki, T.; L'Amoreaux, P.; Loge, F. J.; Ginn, T. R.

    2010-03-01

    The transfer of genetic material among bacteria in the environment can occur both in the planktonic and attached state. Given the propensity of organisms to exist in sessile microbial communities in oligotrophic subsurface conditions, and that such conditions typify the subsurface, this study focuses on exploratory modeling of horizontal gene transfer among surface-associated Escherichiacoli in the subsurface. The mathematics so far used to describe the kinetics of conjugation in biofilms are developed largely from experimental observations of planktonic gene transfer, and are absent of lags or plasmid stability that appear experimentally. We develop a model and experimental system to quantify bacterial filtration and gene transfer in the attached state, on granular porous media. We include attachment kinetics described in Nelson et al. (2007) using the filtration theory approach of Nelson and Ginn (2001, 2005) with motility of E. coli described according to Biondi et al. (1998).

  3. Kinetics of conjugative gene transfer on surfaces in granular porous media

    NASA Astrophysics Data System (ADS)

    Ginn, T.; Massoudieh, A.; Nelson, K.; Mathew, A.; Lambertini, E.

    2005-12-01

    The transfer of genetic material among bacteria in the environment can occur both in the planktonic and attached state. Given the propensity of organisms to exist in sessile microbial communities in oligotrophic conditions, and that such conditions typify the subsurface, this study focuses on exploratory modeling of horizontal gene transfer among surface-associated E. coli in the subsurface. The mathematics so far used to describe the kinetics of conjugation in biofilms are developed largely from experimental observations of planktonic gene transfer, and are absent of lags or plasmid stability that appear experimentally. We develop a model for bacterial filtration and gene transfer in the attached state, for the early stages of biofilm formation using a recently revised filtration theory approach (Nelson and Ginn, 2005) with motility of E. coli described as a continuous time random walk according to data from microflow chamber experiments (Biondi et al., 2002).

  4. Growth hormone-releasing hormone resistance in pseudohypoparathyroidism type ia: new evidence for imprinting of the Gs alpha gene.

    PubMed

    Mantovani, Giovanna; Maghnie, Mohamad; Weber, Giovanna; De Menis, Ernesto; Brunelli, Valeria; Cappa, Marco; Loli, Paola; Beck-Peccoz, Paolo; Spada, Anna

    2003-09-01

    Heterozygous inactivating mutations in the Gs alpha gene cause Albright's hereditary osteodystrophy. Consistent with the observation that only maternally inherited mutations lead to resistance to hormone action [pseudohypoparathyroidism type Ia (PHP Ia)], recent studies provided evidence for a predominant maternal origin of Gs alpha transcripts in endocrine organs, such as thyroid, gonad, and pituitary. The aim of this study was to investigate the presence of pituitary resistance to hypothalamic hormones acting via Gs alpha-coupled receptors in patients with PHP Ia. Six of nine patients showed an impaired GH responsiveness to GHRH plus arginine, consistent with a complete GH deficiency (GH peak from 2.6-8.6 microg/liter, normal > 16.5), and partial (GH peak 13.9 and 13.6 microg/liter) and normal responses were found in two and one patient, respectively. Accordingly, IGF-I levels were below and in the low-normal range in seven and two patients. All patients had a normal cortisol response to 1 microg ACTH test, suggesting a normal corticotroph function that was confirmed by a normal ACTH and cortisol response to CRH test in three patients. In conclusion, we report that in addition to PTH and TSH resistance, patients with PHP Ia display variable degrees of GHRH resistance, consistent with Gs alpha imprinting in human pituitary. PMID:12970263

  5. An amino acid substitution in the pyruvate dehydrogenase E1{alpha} gene, affecting mitochondrial import of the precursor protein

    SciTech Connect

    Takakubo, F.; Thorburn, D.R.; Dahl, H.H.M.

    1995-10-01

    A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1{alpha} deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1{alpha}immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1{alpha} targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1{alpha} sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease. 58 refs., 5 figs., 1 tab.

  6. Limitations of the murine nose in the development of nonviral airway gene transfer.

    PubMed

    Griesenbach, Uta; Sumner-Jones, Stephanie G; Holder, Emma; Munkonge, Felix M; Wodehouse, Theresa; Smith, Stephen N; Wasowicz, Marguerite Y; Pringle, Ian; Casamayor, Isabel; Chan, Mario; Coles, Rebecca; Cornish, Nikki; Dewar, Ann; Doherty, Ann; Farley, Raymond; Green, Anne-Marie; Jones, Bryony L; Larsen, Mia D B; Lawton, Anna E; Manvell, Michelle; Painter, Hazel; Singh, Charanjit; Somerton, Lucinda; Stevenson, Barbara; Varathalingam, Anusha; Siegel, Craig; Scheule, Ronald K; Cheng, Seng H; Davies, Jane C; Porteous, David J; Gill, Deborah R; Boyd, A Christopher; Hyde, Steve C; Alton, Eric W F W

    2010-07-01

    A clinical program to assess whether lipid GL67A-mediated gene transfer can ameliorate cystic fibrosis (CF) lung disease is currently being undertaken by the UK CF Gene Therapy Consortium. We have evaluated GL67A gene transfer to the murine nasal epithelium of wild-type and CF knockout mice to assess this tissue as a test site for gene transfer agents. The plasmids used were regulated by either (1) the commonly used short-acting cytomegalovirus promoter/enhancer or (2) the ubiquitin C promoter. In a study of approximately 400 mice with CF, vector-specific CF transmembrane conductance regulator (CFTR) mRNA was detected in nasal epithelial cells of 82% of mice treated with a cytomegalovirus-plasmid (pCF1-CFTR), and 62% of mice treated with an ubiquitin C-plasmid. We then assessed whether CFTR gene transfer corrected a panel of CFTR-specific endpoint assays in the murine nose, including ion transport, periciliary liquid height, and ex vivo bacterial adherence. Importantly, even with the comparatively large number of animals assessed, the CFTR function studies were only powered to detect changes of more than 50% toward wild-type values. Within this limitation, no significant correction of the CF phenotype was detected. At the current levels of gene transfer efficiency achievable with nonviral vectors, the murine nose is of limited value as a stepping stone to human trials. PMID:19648474

  7. Complexity of genetic sequences modified by horizontal gene transfer and degraded-DNA uptake

    NASA Astrophysics Data System (ADS)

    Tremberger, George; Dehipawala, S.; Nguyen, A.; Cheung, E.; Sullivan, R.; Holden, T.; Lieberman, D.; Cheung, T.

    2015-09-01

    Horizontal gene transfer has been a major vehicle for efficient transfer of genetic materials among living species and could be one of the sources for noncoding DNA incorporation into a genome. Our previous study of lnc- RNA sequence complexity in terms of fractal dimension and information entropy shows a tight regulation among the studied genes in numerous diseases. The role of sequence complexity in horizontal transferred genes was investigated with Mealybug in symbiotic relation with a 139K genome microbe and Deinococcus radiodurans as examples. The fractal dimension and entropy showed correlation R-sq of 0.82 (N = 6) for the studied Deinococcus radiodurans sequences. For comparison the Deinococcus radiodurans oxidative stress tolerant catalase and superoxide dismutase genes under extracellular dGMP growth condition showed R-sq ~ 0.42 (N = 6); and the studied arsenate reductase horizontal transferred genes for toxicity survival in several microorganisms showed no correlation. Simulation results showed that R-sq < 0.4 would be improbable at less than one percent chance, suggestive of additional selection pressure when compared to the R-sq ~ 0.29 (N = 21) in the studied transferred genes in Mealybug. The mild correlation of R-sq ~ 0.5 for fractal dimension versus transcription level in the studied Deinococcus radiodurans sequences upon extracellular dGMP growth condition would suggest that lower fractal dimension with less electron density fluctuation favors higher transcription level.

  8. Targeted gene transfer into rat facial muscles by nanosecond pulsed laser-induced stress waves.

    PubMed

    Kurita, Akihiro; Matsunobu, Takeshi; Satoh, Yasushi; Ando, Takahiro; Sato, Shunichi; Obara, Minoru; Shiotani, Akihiro

    2011-09-01

    We investigate the feasibility of using nanosecond pulsed laser-induced stress waves (LISWs) for gene transfer into rat facial muscles. LISWs are generated by irradiating a black natural rubber disk placed on the target tissue with nanosecond pulsed laser light from the second harmonics (532 nm) of a Q-switched Nd:YAG laser, which is widely used in head and neck surgery and proven to be safe. After injection of plasmid deoxyribose nucleic acid (DNA) coding for Lac Z into rat facial muscles, pulsed laser is used to irradiate the laser target on the skin surface without incision or exposure of muscles. Lac Z expression is detected by X-gal staining of excised rat facial skin and muscles. Strong Lac Z expression is observed seven days after gene transfer, and sustained for up to 14 days. Gene transfer is achieved in facial muscles several millimeters deep from the surface. Gene expression is localized to the tissue exposed to LISWs. No tissue damage from LISWs is observed. LISW is a promising nonviral target gene transfer method because of its high spatial controllability, easy applicability, and minimal invasiveness. Gene transfer using LISW to produce therapeutic proteins such as growth factors could be used to treat nerve injury and paralysis. PMID:21950944

  9. Gene Transfers Shaped the Evolution of De Novo NAD+ Biosynthesis in Eukaryotes

    PubMed Central

    Ternes, Chad M.; Schönknecht, Gerald

    2014-01-01

    NAD+ is an essential molecule for life, present in each living cell. It can function as an electron carrier or cofactor in redox biochemistry and energetics, and serves as substrate to generate the secondary messenger cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate. Although de novo NAD+ biosynthesis is essential, different metabolic pathways exist in different eukaryotic clades. The kynurenine pathway starting with tryptophan was most likely present in the last common ancestor of all eukaryotes, and is active in fungi and animals. The aspartate pathway, detected in most photosynthetic eukaryotes, was probably acquired from the cyanobacterial endosymbiont that gave rise to chloroplasts. An evolutionary analysis of enzymes catalyzing de novo NAD+ biosynthesis resulted in evolutionary trees incongruent with established organismal phylogeny, indicating numerous gene transfers. Endosymbiotic gene transfers probably introduced the aspartate pathway into eukaryotes and may have distributed it among different photosynthetic clades. In addition, several horizontal gene transfers substituted eukaryotic genes with bacterial orthologs. Although horizontal gene transfer is accepted as a key mechanism in prokaryotic evolution, it is supposed to be rare in eukaryotic evolution. The essential metabolic pathway of de novo NAD+ biosynthesis in eukaryotes was shaped by numerous gene transfers. PMID:25169983

  10. Targeted gene transfer into rat facial muscles by nanosecond pulsed laser-induced stress waves

    NASA Astrophysics Data System (ADS)

    Kurita, Akihiro; Matsunobu, Takeshi; Satoh, Yasushi; Ando, Takahiro; Sato, Shunichi; Obara, Minoru; Shiotani, Akihiro

    2011-09-01

    We investigate the feasibility of using nanosecond pulsed laser-induced stress waves (LISWs) for gene transfer into rat facial muscles. LISWs are generated by irradiating a black natural rubber disk placed on the target tissue with nanosecond pulsed laser light from the second harmonics (532 nm) of a Q-switched Nd:YAG laser, which is widely used in head and neck surgery and proven to be safe. After injection of plasmid deoxyribose nucleic acid (DNA) coding for Lac Z into rat facial muscles, pulsed laser is used to irradiate the laser target on the skin surface without incision or exposure of muscles. Lac Z expression is detected by X-gal staining of excised rat facial skin and muscles. Strong Lac Z expression is observed seven days after gene transfer, and sustained for up to 14 days. Gene transfer is achieved in facial muscles several millimeters deep from the surface. Gene expression is localized to the tissue exposed to LISWs. No tissue damage from LISWs is observed. LISW is a promising nonviral target gene transfer method because of its high spatial controllability, easy applicability, and minimal invasiveness. Gene transfer using LISW to produce therapeutic proteins such as growth factors could be used to treat nerve injury and paralysis.

  11. Molecular cloning of the mouse gene coding for {alpha}{sub 2}-macroglobulin and targeting of the gene in embryonic stem cells

    SciTech Connect

    Umans, L.; Serneels, L.; Hilliker, C.

    1994-08-01

    The authors have cloned the mouse gene coding for {alpha}{sub 2}-macroglobulin in overlapping {lambda} clones and have analyzed its structure. The gene contains 36 exons, coding for the 4.8-kb cDNA that we cloned previously. Including putative control elements in the 5{prime} flanking region, the gene covers about 45 kb. A region of 3.8 kb, stretching from 835 bases upstream of the cDNA start site to exon 4, including all intervening sequences, was sequenced completely. The analysis demonstrated that the putative promoter region of the mouse A2M gene differed considerably from the known promoter sequences of the human A2M gene and of the rat acute-phas A2M gene. Comparison of the exon-intron structure of all known genes of the A2M family confirmed that the rat acute phase A2M gene is more closely related to the human gene than to the mouse A2M gene. To generate mice with the A2M gene inactivated, an insertion type of construct containing 7.5 kb of genomic DNA of the mouse strain 129/J, encompassing exons 16 to 19, was synthesized. A hygromycin marker gene was embedded in intron 17. After electroporation, 198 hygromycin-resistant ES cell lines were isolated and analyzed by Southern blotting. Five ES cell lines were obtained with one allele of the mouse A2M gene targeted by this insertion construct, demonstrating that the position and the characteristics of the vector served the intended goal.

  12. Synthetic gene transfer vectors II: back to the future.

    PubMed

    Behr, Jean-Paul

    2012-07-17

    The discovery of RNA interference has given a new lease on life to both the chemistry of oligonucleotides and chemical approaches for the intracellular delivery of nucleic acids. In particular, delivery of siRNA, whether in vitro for screening and target validation purposes or in humans as a new class of drugs, may revolutionize our approach to therapy. Their impact could equal that of the bioproduction and various uses of monoclonal antibodies today. Unfortunately, global pharmaceutical companies again seem to be waiting to buy the next Genentech or Genzyme of gene silencing rather than investing research and development into this promising area of research. Gene silencing encounters barriers similar to gene addition and hence may benefit from the extra decade of experience brought by gene therapy. "Chemical" transfection of cells in culture has become routine, and this Account discusses some of the reasons this success has not extended to nonviral gene therapy trials, most of which do not progress beyond the phase 2 stage. The author also discusses a (much debated) mechanism of nucleic acid cell entry and subsequent release of the polycationic particles into the cytoplasm. Both topics should be useful to those interested in delivery of siRNA. The move from gene therapy toward siRNA as an oligonucleotide-based therapy strategy provides a much wider range of druggable targets. Even though these molecules are a hundredfold smaller than a gene, they are delivered via similar cellular mechanisms. Their complexes with cationic polymers are less stable than those with a higher number of phosphate groups, which may be compensated by siRNA concatemerization or by chemical conjugation with the cationic carrier. Thus chemistry is again desperately needed. PMID:22311735

  13. Optical fiber-based photomechanical gene transfer system for in vivo application.

    PubMed

    Sato, Shunichi; Ando, Takahiro; Obara, Minoru

    2011-12-01

    We developed an optical-fiber-based photomechanical gene transfer system for endoscopic or catheter-based application. A fiber tip with a laser-absorbing film covered with a transparent plastic disk for plasma confinement was attached to a quartz fiber; the film was irradiated with nanosecond laser pulses transmitted through the fiber to generate photomechanical waves (PMWs). Characteristics of PMWs emitted from the fiber tip were examined to confirm the necessary conditions for gene transfer. We then attempted to transfer reporter genes to the rat skin as a test tissue in vivo with the fiber system, and the results showed significantly high protein levels and spatially selective pinpoint gene expressions in the tissue. PMID:22139237

  14. The agricultural antibiotic carbadox induces phage-mediated gene transfer in Salmonella

    PubMed Central

    Bearson, Bradley L.; Allen, Heather K.; Brunelle, Brian W.; Lee, In Soo; Casjens, Sherwood R.; Stanton, Thaddeus B.

    2013-01-01

    Antibiotics are used for disease therapeutic or preventative effects in humans and animals, as well as for enhanced feed conversion efficiency in livestock. Antibiotics can also cause undesirable effects in microbial populations, including selection for antibiotic resistance, enhanced pathogen invasion, and stimulation of horizontal gene transfer. Carbadox is a veterinary antibiotic used in the US during the starter phase of swine production for improved feed efficiency and control of swine dysentery and bacterial swine enteritis. Carbadox has been shown in vitro to induce phage-encoded Shiga toxin in Shiga toxin-producing Escherichia coli (STEC) and a phage-like element transferring antibiotic resistance genes in Brachyspira hyodysenteriae, but the effect of carbadox on prophages in other bacteria is unknown. This study examined carbadox exposure on prophage induction and genetic transfer in Salmonella enterica serovar Typhimurium, a human foodborne pathogen that frequently colonizes swine without causing disease. S. Typhimurium LT2 exposed to carbadox induced prophage production, resulting in bacterial cell lysis and release of virions that were visible by electron microscopy. Carbadox induction of phage-mediated gene transfer was confirmed by monitoring the transduction of a sodCIII::neo cassette in the Fels-1 prophage from LT2 to a recipient Salmonella strain. Furthermore, carbadox frequently induced generalized transducing phages in multidrug-resistant phage type DT104 and DT120 isolates, resulting in the transfer of chromosomal and plasmid DNA that included antibiotic resistance genes. Our research indicates that exposure of Salmonella to carbadox induces prophages that can transfer virulence and antibiotic resistance genes to susceptible bacterial hosts. Carbadox-induced, phage-mediated gene transfer could serve as a contributing factor in bacterial evolution during animal production, with prophages being a reservoir for bacterial fitness genes in the

  15. Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time.

    PubMed Central

    Zabner, J; Zeiher, B G; Friedman, E; Welsh, M J

    1996-01-01

    The efficiency of adenovirus-mediated gene transfer to airway epithelia will be an important factor in determining whether recombinant adenoviruses can be developed as vectors for transferring cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to patients with cystic fibrosis. Current understanding of the biology of CF lung disease suggests that vectors should express transgene in mature, ciliated airway epithelia. We evaluated the efficiency of adenovirus-mediated gene transfer to primary cultures of normal and CF human airway epithelia. Our studies showed that the airway cells developed from an undifferentiated epithelium with markers characteristic of basal cells and a surface covered by short microvilli 3 days after seeding to a mature epithelium whose apical surface was covered with cilia by 10 to 14 days. The ability of adenovirus vectors to express a reporter gene and to correct defective cyclic AMP-stimulated Cl- transport in CF epithelia was correlated inversely with the state of differentiation. However, the inefficiency of adenovirus-mediated gene transfer could be partially corrected when the contact time between vector and epithelium was prolonged. After prolonged contact, we observed complete correction of the CF Cl- transport defect in differentiated CF airway epithelia in culture and of the Cl- transport defect in the nasal epithelia of mice homozygous for the deltaF508 mutation. The fact that gene transfer to airway epithelia required prolonged incubation with vector contrasts with the rapid infection observed in cell models such as 293 and HeLa cells, which are commonly used to study adenovirus infection. Gene transfer observed after prolonged incubation may result from mechanisms different from those that mediate infection of 293 cells. These observations suggest that interventions that either increase the contact time or alter the epithelium or the vector may be required to facilitate gene transfer to ciliated respiratory epithelia

  16. Identification and characterization of the Trichoderma harzianum gene encoding alpha-1,3-glucanase involved in streptococcal mutan degradation.

    PubMed

    Wiater, Adrian; Janczarek, Monika; Pleszczyńska, Małgorzata; Szczodrak, Janusz

    2011-01-01

    alpha-1,3-Glucanases (mutanases) are currently of great interest due to their potential use in the field of dental care. These enzymes have been reported in several bacteria, yeasts and fungi, but up to now, characterization of this family of proteins has been relatively poor. In this study, we identify and characterize a mutanase gene from Trichoderma harzianum CCM F-340. Sequence analysis, on the nucleotide and amino acid levels reveals that this alpha-1,3-glucanase is highly homologous to alpha-1,3-glucanases from T harzianum isolate CBS 243.71 and T asperellum CECT 20539. T. harzianum CCM F-340 mutanase is a 634-aa residue protein with a calculated molecular mass of 67.65 kDa, composed of two distinct, highly conserved domains (a long N-terminal catalytic domain and a short C-terminal polysaccharide-binding domain) separated by a less conserved Pro-Ser-Thr-rich linker region. The mutanase gene was expressed in an E. coli BL21 (DE3) host, under the transcriptional control of T7 promoter. The purified enzyme migrated as a band of about 68 kDa after SDS-polyacrylamide gel electrophoresis, which coincided with the predicted size based on the amino acid sequence. Our data indicate that this enzyme is highly conserved in Trichoderma and can be produced in active form in such heterologous expression system. PMID:22390063

  17. Familial Albright`s hereditary osteodystrophy with hypoparathyroidism: Normal structural G{sub s}{alpha} gene

    SciTech Connect

    Shapira, H.; Friedman, E.; Farfel, Z.

    1996-04-01

    Albright`s hereditary osteodystrophy (AHO) is a characteristic skeletal phenotype, including short stature, obesity, round face, and brachydactyly. AHO appears in patients with pseudohypoparathyroidism (PHP) who have resistance to PTH and in their eumetabolic family members who have pseudohypoparathyroidism (PPHP). The differential diagnosis of AHO in families without PHP includes brachydactyly E, whose existence as a distinct entity has been questioned. We studied a patient with familial AHO who presented with hypocalcemia. To our surprise, PTH levels were low, and the response to PTH administration was normal. This is the first case of familial AHO with hypoparathyroidism. The proband`s family included 22 affected subjects spanning 3 generations, who had variable degrees of AHO manifestations, with an autosomal dominant inheritance trait. The metacarpophalangeal pattern profile was typical of that of PHP-PPHP. As deficient activity and inactivating mutations of G{sub s}{alpha} were described in PHP as well as in PPHP, we measured the biological activity of G{sub s} in family members, which was normal. To exclude subtle abnormalities in the G{sub s}{alpha} gene, we sequenced the entire coding region of G{alpha} in the propositus, which was normal. We conclude that hypocalcemia should be adequately evaluated even in the presence of familial AHO, and that familial AHO can occur with a normal coding structural Ga gene. Identification of the molecular defect in familial AHO without PHP will shed light on the pathogenesis of AHO in general. 20 refs., 3 figs.

  18. Haploinsufficiency in the PPAR{alpha} and LDL receptor genes leads to gender- and age-specific obesity and hyperinsulinemia

    SciTech Connect

    Sugiyama, Eiko . E-mail: eikoyoko@nagano-kentan.ac.jp; Tanaka, Naoki; Nakajima, Tamie; Kamijo, Yuji; Yokoyama, Shin; Li Yufeng; Gonzalez, Frank J.; Aoyama, Toshifumi

    2006-11-17

    When preparing peroxisome proliferator-activated receptor (PPAR){alpha}:low-density lipoprotein receptor (LDLR) (-/-) double knockout mice, we unexpectedly found a unique gender- and age-specific obesity in the F1 generation, PPAR{alpha} (+/-):LDLR (+/-), even in mice fed standard chow. Body weights of the male heterozygous mice increased up to about 60 g at 75 weeks of age, then decreased by about 30 g at 100 weeks of age. More than 95% of the heterozygous mice between 35- and 75-week-olds were overweight. Of interest, the obese heterozygous mice also exhibited hyperinsulinemia correlating with moderate insulin resistance. Hepatic gene expression of LDLR was lower than expected in the heterozygous mice, particularly at 50 and 75 weeks of age. In contrast, the hepatic expression of PPAR{alpha} was higher than expected in obese heterozygous mice, but decreased in non-obese older heterozygous mice. Modulated expression of these genes may be partially associated with the onset of the hyperinsulinemia.

  19. Mutational diversity and hot spots in the alpha-sarcoglycan gene in autosomal recessive muscular dystrophy (LGMD2D).

    PubMed Central

    Carrié, A; Piccolo, F; Leturcq, F; de Toma, C; Azibi, K; Beldjord, C; Vallat, J M; Merlini, L; Voit, T; Sewry, C; Urtizberea, J A; Romero, N; Tomé, F M; Fardeau, M; Sunada, Y; Campbell, K P; Kaplan, J C; Jeanpierre, M

    1997-01-01

    Sarcoglycanopathies are a genetically heterogeneous group of autosomal recessive muscular dystrophies in which the primary defect may reside in any of the genes coding for the different partners of the sarcolemmal sarcoglycan (SG) complex: the alpha-SG (LGMD2D at 17q21.2), the beta-SG (LGMD2E at 4q12), the gamma-SG (LGMD2C at 13q12), and the delta-SG (LGMD2F at 5q33). We report a series of 20 new unrelated families with 14 different mutations in the alpha-SG gene. Along with the mutations that we previously reported this brings our cohort of patients with alpha-sarcoglycanopathy to a total of 31 unrelated patients, carrying 25 different mutations. The missense mutations reside in the extracellular domain of the protein. Five of 15 missense mutations, carried by unrelated subjects on different haplotype backgrounds and of widespread geographical origins, account for 58% of the mutated chromosomes, with a striking prevalence of the R77C substitution (32%). The severity of the disease varies strikingly and correlates at least in part with the amount of residual protein and the type of mutation. The recurrent R284C substitution is associated with a benign disease course. Images PMID:9192266

  20. Prokaryotic genes in eukaryotic genome sequences: when to infer horizontal gene transfer and when to suspect an actual microbe.

    PubMed

    Artamonova, Irena I; Lappi, Tanya; Zudina, Liudmila; Mushegian, Arcady R

    2015-07-01

    Assessment of phylogenetic positions of predicted gene and protein sequences is a routine step in any genome project, useful for validating the species' taxonomic position and for evaluating hypotheses about genome evolution and function. Several recent eukaryotic genome projects have reported multiple gene sequences that were much more similar to homologues in bacteria than to any eukaryotic sequence. In the spirit of the times, horizontal gene transfer from bacteria to eukaryotes has been invoked in some of these cases. Here, we show, using comparative sequence analysis, that some of those bacteria-like genes indeed appear likely to have been horizontally transferred from bacteria to eukaryotes. In other cases, however, the evidence strongly indicates that the eukaryotic DNA sequenced in the genome project contains a sample of non-integrated DNA from the actual bacteria, possibly providing a window into the host microbiome. Recent literature suggests also that common reagents, kits and laboratory equipment may be systematically contaminated with bacterial DNA, which appears to be sampled by metagenome projects non-specifically. We review several bioinformatic criteria that help to distinguish putative horizontal gene transfers from the admixture of genes from autonomously replicating bacteria in their hosts' genome databases or from the reagent contamination. PMID:25919787

  1. Functional expression of Desulfovibrio vulgaris Hildenborough cytochrome c3 in Desulfovibrio desulfuricans G200 after conjugational gene transfer from Escherichia coli.

    PubMed Central

    Voordouw, G; Pollock, W B; Bruschi, M; Guerlesquin, F; Rapp-Giles, B J; Wall, J D

    1990-01-01

    Plasmid pJRDC800-1, containing the cyc gene encoding cytochrome c3 from Desulfovibrio vulgaris subsp. vulgaris Hildenborough, was transferred by conjugation from Escherichia coli DH5 alpha to Desulfovibrio desulfuricans G200. The G200 strain produced an acidic cytochrome c3 (pI = 5.8), which could be readily separated from the Hildenborough cytochrome c3 (pI = 10.5). The latter was indistinguishable from cytochrome c3 produced by D. vulgaris subsp. vulgaris Hildenborough with respect to a number of chemical and physical criteria. Images PMID:2170341

  2. Two Horizontally Transferred Xenobiotic Resistance Gene Clusters Associated with Detoxification of Benzoxazolinones by Fusarium Species

    PubMed Central

    Glenn, Anthony E.; Davis, C. Britton; Gao, Minglu; Gold, Scott E.; Mitchell, Trevor R.; Proctor, Robert H.; Stewart, Jane E.; Snook, Maurice E.

    2016-01-01

    Microbes encounter a broad spectrum of antimicrobial compounds in their environments and often possess metabolic strategies to detoxify such xenobiotics. We have previously shown that Fusarium verticillioides, a fungal pathogen of maize known for its production of fumonisin mycotoxins, possesses two unlinked loci, FDB1 and FDB2, necessary for detoxification of antimicrobial compounds produced by maize, including the γ-lactam 2-benzoxazolinone (BOA). In support of these earlier studies, microarray analysis of F. verticillioides exposed to BOA identified the induction of multiple genes at FDB1 and FDB2, indicating the loci consist of gene clusters. One of the FDB1 cluster genes encoded a protein having domain homology to the metallo-β-lactamase (MBL) superfamily. Deletion of this gene (MBL1) rendered F. verticillioides incapable of metabolizing BOA and thus unable to grow on BOA-amended media. Deletion of other FDB1 cluster genes, in particular AMD1 and DLH1, did not affect BOA degradation. Phylogenetic analyses and topology testing of the FDB1 and FDB2 cluster genes suggested two horizontal transfer events among fungi, one being transfer of FDB1 from Fusarium to Colletotrichum, and the second being transfer of the FDB2 cluster from Fusarium to Aspergillus. Together, the results suggest that plant-derived xenobiotics have exerted evolutionary pressure on these fungi, leading to horizontal transfer of genes that enhance fitness or