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2016 |
Kowalska, J; Martin, F; Jemielity, J Synthetic Capped mRNAs for Cap-Specific Photo-Cross-Linking Experiments. incollection Rhoads, R (Ed.): Synthetic mRNA: Production, Introduction Into Cells, and Physiological Consequences, 1428 , p. 31-43, Springer Protocols, Humana Press, 2016. Résumé | BibTeX | Étiquettes: 5′, 6-thioguanosine, analogs, Binding, Cap, ERIANI, Histone, mRNA, Photo-cross-linking, stability, Transcription @incollection{, title = {Synthetic Capped mRNAs for Cap-Specific Photo-Cross-Linking Experiments.}, author = { J. Kowalska and F. Martin and J. Jemielity}, editor = { R. Rhoads}, year = {2016}, date = {2016-01-01}, booktitle = {Synthetic mRNA: Production, Introduction Into Cells, and Physiological Consequences}, volume = {1428}, pages = {31-43}, publisher = {Springer Protocols, Humana Press}, series = {Methods in Molecular Biology}, abstract = {he 7-methylguanosine triphosphate cap present at the 5' ends of eukaryotic mRNAs plays numerous roles in mRNA expression and metabolism. The identification and studies on cap-binding partners can be significantly advanced using tailored chemical tools such as synthetic cap analogues or RNAs carrying modified cap structures. Here we provide protocols for the production of mRNAs specifically labeled within the 5' cap with a nucleoside capable of being photo-activated, either 6-thioguanosine or 7-methyl-6-thioguanosine, which can be used in photo-cross-linking experiments to identify or characterize cap-binding biomolecules. We also describe a protocol for the cross-linking experiments with capped RNAs to map histone H4 cap-binding pocket.}, keywords = {5′, 6-thioguanosine, analogs, Binding, Cap, ERIANI, Histone, mRNA, Photo-cross-linking, stability, Transcription}, pubstate = {published}, tppubtype = {incollection} } he 7-methylguanosine triphosphate cap present at the 5' ends of eukaryotic mRNAs plays numerous roles in mRNA expression and metabolism. The identification and studies on cap-binding partners can be significantly advanced using tailored chemical tools such as synthetic cap analogues or RNAs carrying modified cap structures. Here we provide protocols for the production of mRNAs specifically labeled within the 5' cap with a nucleoside capable of being photo-activated, either 6-thioguanosine or 7-methyl-6-thioguanosine, which can be used in photo-cross-linking experiments to identify or characterize cap-binding biomolecules. We also describe a protocol for the cross-linking experiments with capped RNAs to map histone H4 cap-binding pocket. |
2008 |
Deddouche, Safia; Matt, Nicolas; Budd, Aidan; Mueller, Stefanie; Kemp, Cordula; Galiana-Arnoux, Delphine; Dostert, Catherine; Antoniewski, Christophe; Hoffmann, Jules A; Imler, Jean-Luc The DExD/Ħ-box helicase Dicer-2 mediates the induction of antiviral activity in drosophila Article de journal Nature Immunology, 9 (12), p. 1425–1432, 2008, ISSN: 1529-2916. Résumé | Liens | BibTeX | Étiquettes: Amino Acid, Animals, Electrophoresis, Fat Body, Gene Expression Regulation, Genetic, Genetically Modified, hoffmann, Humans, imler, M3i, matt, Phylogeny, Polyacrylamide Gel, Reverse Transcriptase Polymerase Chain Reaction, Ribonuclease III, RNA Helicases, Sequence Homology, Transcription, Virus Diseases @article{deddouche_dexd/h-box_2008, title = {The DExD/Ħ-box helicase Dicer-2 mediates the induction of antiviral activity in drosophila}, author = {Safia Deddouche and Nicolas Matt and Aidan Budd and Stefanie Mueller and Cordula Kemp and Delphine Galiana-Arnoux and Catherine Dostert and Christophe Antoniewski and Jules A Hoffmann and Jean-Luc Imler}, doi = {10.1038/ni.1664}, issn = {1529-2916}, year = {2008}, date = {2008-12-01}, journal = {Nature Immunology}, volume = {9}, number = {12}, pages = {1425--1432}, abstract = {Drosophila, like other invertebrates and plants, relies mainly on RNA interference for its defense against viruses. In flies, viral infection also triggers the expression of many genes. One of the genes induced, Vago, encodes a 18-kilodalton cysteine-rich polypeptide. Here we provide genetic evidence that the Vago gene product controlled viral load in the fat body after infection with drosophila C virus. Induction of Vago was dependent on the helicase Dicer-2. Dicer-2 belongs to the same DExD/H-box helicase family as do the RIG-I-like receptors, which sense viral infection and mediate interferon induction in mammals. We propose that this family represents an evolutionary conserved set of sensors that detect viral nucleic acids and direct antiviral responses.}, keywords = {Amino Acid, Animals, Electrophoresis, Fat Body, Gene Expression Regulation, Genetic, Genetically Modified, hoffmann, Humans, imler, M3i, matt, Phylogeny, Polyacrylamide Gel, Reverse Transcriptase Polymerase Chain Reaction, Ribonuclease III, RNA Helicases, Sequence Homology, Transcription, Virus Diseases}, pubstate = {published}, tppubtype = {article} } Drosophila, like other invertebrates and plants, relies mainly on RNA interference for its defense against viruses. In flies, viral infection also triggers the expression of many genes. One of the genes induced, Vago, encodes a 18-kilodalton cysteine-rich polypeptide. Here we provide genetic evidence that the Vago gene product controlled viral load in the fat body after infection with drosophila C virus. Induction of Vago was dependent on the helicase Dicer-2. Dicer-2 belongs to the same DExD/H-box helicase family as do the RIG-I-like receptors, which sense viral infection and mediate interferon induction in mammals. We propose that this family represents an evolutionary conserved set of sensors that detect viral nucleic acids and direct antiviral responses. |
Roetzer, Andreas; Gregori, Christa; Jennings, Ann Marie; Quintin, Jessica; Ferrandon, Dominique; Butler, Geraldine; Kuchler, Karl; Ammerer, Gustav; Schüller, Christoph Candida glabrata environmental stress response involves Saccharomyces cerevisiae Msn2/4 orthologous transcription factors Article de journal Mol. Microbiol., 69 (3), p. 603–620, 2008, ISSN: 1365-2958. Résumé | Liens | BibTeX | Étiquettes: Animals, Candida glabrata, Candidiasis, DNA-Binding Proteins, ferrandon, Fungal, Fungal Proteins, Gene Expression Profiling, Gene Expression Regulation, Genetic, Humans, M3i, Oligonucleotide Array Sequence Analysis, Osmotic Pressure, Regulon, Saccharomyces cerevisiae Proteins, Transcription, Transcription Factors, Virulence, Yeasts @article{roetzer_candida_2008b, title = {Candida glabrata environmental stress response involves Saccharomyces cerevisiae Msn2/4 orthologous transcription factors}, author = {Andreas Roetzer and Christa Gregori and Ann Marie Jennings and Jessica Quintin and Dominique Ferrandon and Geraldine Butler and Karl Kuchler and Gustav Ammerer and Christoph Schüller}, doi = {10.1111/j.1365-2958.2008.06301.x}, issn = {1365-2958}, year = {2008}, date = {2008-01-01}, journal = {Mol. Microbiol.}, volume = {69}, number = {3}, pages = {603--620}, abstract = {We determined the genome-wide environmental stress response (ESR) expression profile of Candida glabrata, a human pathogen related to Saccharomyces cerevisiae. Despite different habitats, C. glabrata, S. cerevisiae, Schizosaccharomyces pombe and Candida albicans have a qualitatively similar ESR. We investigate the function of the C. glabrata syntenic orthologues to the ESR transcription factor Msn2. The C. glabrata orthologues CgMsn2 and CgMsn4 contain a motif previously referred to as HD1 (homology domain 1) also present in Msn2 orthologues from fungi closely related to S. cerevisiae. We show that regions including this motif confer stress-regulated intracellular localization when expressed in S. cerevisiae. Site-directed mutagenesis confirms that nuclear export of CgMsn2 in C. glabrata requires an intact HD1. Transcript profiles of CgMsn2/4 mutants and CgMsn2 overexpression strains show that they regulate a part of the CgESR. CgMsn2 complements a S. cerevisiae msn2 null mutant and in stressed C. glabrata cells, rapidly translocates from the cytosol to the nucleus. CgMsn2 is required for full resistance against severe osmotic stress and rapid and full induction of trehalose synthesis genes (TPS1, TPS2). Constitutive activation of CgMsn2 is detrimental for C. glabrata. These results establish an Msn2-regulated general stress response in C. glabrata.}, keywords = {Animals, Candida glabrata, Candidiasis, DNA-Binding Proteins, ferrandon, Fungal, Fungal Proteins, Gene Expression Profiling, Gene Expression Regulation, Genetic, Humans, M3i, Oligonucleotide Array Sequence Analysis, Osmotic Pressure, Regulon, Saccharomyces cerevisiae Proteins, Transcription, Transcription Factors, Virulence, Yeasts}, pubstate = {published}, tppubtype = {article} } We determined the genome-wide environmental stress response (ESR) expression profile of Candida glabrata, a human pathogen related to Saccharomyces cerevisiae. Despite different habitats, C. glabrata, S. cerevisiae, Schizosaccharomyces pombe and Candida albicans have a qualitatively similar ESR. We investigate the function of the C. glabrata syntenic orthologues to the ESR transcription factor Msn2. The C. glabrata orthologues CgMsn2 and CgMsn4 contain a motif previously referred to as HD1 (homology domain 1) also present in Msn2 orthologues from fungi closely related to S. cerevisiae. We show that regions including this motif confer stress-regulated intracellular localization when expressed in S. cerevisiae. Site-directed mutagenesis confirms that nuclear export of CgMsn2 in C. glabrata requires an intact HD1. Transcript profiles of CgMsn2/4 mutants and CgMsn2 overexpression strains show that they regulate a part of the CgESR. CgMsn2 complements a S. cerevisiae msn2 null mutant and in stressed C. glabrata cells, rapidly translocates from the cytosol to the nucleus. CgMsn2 is required for full resistance against severe osmotic stress and rapid and full induction of trehalose synthesis genes (TPS1, TPS2). Constitutive activation of CgMsn2 is detrimental for C. glabrata. These results establish an Msn2-regulated general stress response in C. glabrata. |
2002 |
Imler, Jean-Luc; Hoffmann, Jules A Toll receptors in Drosophila: a family of molecules regulating development and immunity Article de journal Current Topics in Microbiology and Immunology, 270 , p. 63–79, 2002, ISSN: 0070-217X. Résumé | BibTeX | Étiquettes: Animals, Cell Surface, Genetic, Gram-Negative Bacteria, hoffmann, imler, M3i, Receptors, Signal Transduction, Toll-Like Receptors, Transcription @article{imler_toll_2002, title = {Toll receptors in Drosophila: a family of molecules regulating development and immunity}, author = {Jean-Luc Imler and Jules A Hoffmann}, issn = {0070-217X}, year = {2002}, date = {2002-01-01}, journal = {Current Topics in Microbiology and Immunology}, volume = {270}, pages = {63--79}, abstract = {In recent years, Toll-like receptors (TLRs) have emerged as key receptors which detect microbes and initiate an inflammatory response. The Toll receptor was originally identified and characterized 14 years ago for its role in the embryonic development of the fruit-fly Drosophila melanogaster. Subsequently, it was also shown to be an essential component of the signaling pathway mediating the anti-fungal host defense in this model organism. New factors involved in the activation of the Toll receptor or in intracytoplasmic signaling during the immune response in Drosophila have recently been identified. The existence of significant functional differences between mammalian TLRs and Drosophila Toll receptors is also becoming apparent.}, keywords = {Animals, Cell Surface, Genetic, Gram-Negative Bacteria, hoffmann, imler, M3i, Receptors, Signal Transduction, Toll-Like Receptors, Transcription}, pubstate = {published}, tppubtype = {article} } In recent years, Toll-like receptors (TLRs) have emerged as key receptors which detect microbes and initiate an inflammatory response. The Toll receptor was originally identified and characterized 14 years ago for its role in the embryonic development of the fruit-fly Drosophila melanogaster. Subsequently, it was also shown to be an essential component of the signaling pathway mediating the anti-fungal host defense in this model organism. New factors involved in the activation of the Toll receptor or in intracytoplasmic signaling during the immune response in Drosophila have recently been identified. The existence of significant functional differences between mammalian TLRs and Drosophila Toll receptors is also becoming apparent. |
2001 |
Levashina, Elena A; Moita, L F; Blandin, Stéphanie A; Vriend, G; Lagueux, Marie; Kafatos, F C Conserved role of a complement-like protein in phagocytosis revealed by dsRNA knockout in cultured cells of the mosquito, Anopheles gambiae Article de journal Cell, 104 (5), p. 709–718, 2001, ISSN: 0092-8674. Résumé | BibTeX | Étiquettes: alpha-Macroglobulins, Animals, Anopheles, blandin, Cells, Cloning, Complement C3, Cultured, DNA Fragmentation, Double-Stranded, Female, Genetic, Gram-Negative Bacteria, Hemocytes, Insect Proteins, M3i, Molecular, Nucleic Acid Denaturation, Phagocytosis, Protein Structure, RNA, Tertiary, Transcription @article{levashina_conserved_2001, title = {Conserved role of a complement-like protein in phagocytosis revealed by dsRNA knockout in cultured cells of the mosquito, Anopheles gambiae}, author = {Elena A Levashina and L F Moita and Stéphanie A Blandin and G Vriend and Marie Lagueux and F C Kafatos}, issn = {0092-8674}, year = {2001}, date = {2001-01-01}, journal = {Cell}, volume = {104}, number = {5}, pages = {709--718}, abstract = {We characterize a novel hemocyte-specific acute phase glycoprotein from the malaria vector, Anopheles gambiae. It shows substantial structural and functional similarities, including the highly conserved thioester motif, to both a central component of mammalian complement system, factor C3, and to a pan-protease inhibitor, alpha2-macroglobulin. Most importantly, this protein serves as a complement-like opsonin and promotes phagocytosis of some Gram-negative bacteria in a mosquito hemocyte-like cell line. Chemical inactivation by methylamine and depletion by double-stranded RNA knockout demonstrate that this function is dependent on the internal thioester bond. This evidence of a complement-like function in a protostome animal adds substantially to the accumulating evidence of a common ancestry of immune defenses in insects and vertebrates.}, keywords = {alpha-Macroglobulins, Animals, Anopheles, blandin, Cells, Cloning, Complement C3, Cultured, DNA Fragmentation, Double-Stranded, Female, Genetic, Gram-Negative Bacteria, Hemocytes, Insect Proteins, M3i, Molecular, Nucleic Acid Denaturation, Phagocytosis, Protein Structure, RNA, Tertiary, Transcription}, pubstate = {published}, tppubtype = {article} } We characterize a novel hemocyte-specific acute phase glycoprotein from the malaria vector, Anopheles gambiae. It shows substantial structural and functional similarities, including the highly conserved thioester motif, to both a central component of mammalian complement system, factor C3, and to a pan-protease inhibitor, alpha2-macroglobulin. Most importantly, this protein serves as a complement-like opsonin and promotes phagocytosis of some Gram-negative bacteria in a mosquito hemocyte-like cell line. Chemical inactivation by methylamine and depletion by double-stranded RNA knockout demonstrate that this function is dependent on the internal thioester bond. This evidence of a complement-like function in a protostome animal adds substantially to the accumulating evidence of a common ancestry of immune defenses in insects and vertebrates. |
2000 |
Lagueux, Marie; Perrodou, E; Levashina, Elena A; Capovilla, Maria; Hoffmann, Jules A Constitutive expression of a complement-like protein in toll and JAK gain-of-function mutants of Drosophila Article de journal Proc. Natl. Acad. Sci. U.S.A., 97 (21), p. 11427–11432, 2000, ISSN: 0027-8424. Résumé | Liens | BibTeX | Étiquettes: alpha-Macroglobulins, Amino Acid, Animals, Cell Surface, Complement C3, Esters, Genetic, hoffmann, Insect Proteins, Janus Kinases, M3i, Membrane Glycoproteins, Mutation, Protein-Tyrosine Kinases, Proteins, Receptors, Sequence Homology, Sulfhydryl Compounds, Toll-Like Receptors, Transcription, Transcription Factors @article{lagueux_constitutive_2000, title = {Constitutive expression of a complement-like protein in toll and JAK gain-of-function mutants of Drosophila}, author = {Marie Lagueux and E Perrodou and Elena A Levashina and Maria Capovilla and Jules A Hoffmann}, doi = {10.1073/pnas.97.21.11427}, issn = {0027-8424}, year = {2000}, date = {2000-10-01}, journal = {Proc. Natl. Acad. Sci. U.S.A.}, volume = {97}, number = {21}, pages = {11427--11432}, abstract = {We show that Drosophila expresses four genes encoding proteins with significant similarities with the thiolester-containing proteins of the complement C3/alpha(2)-macroglobulin superfamily. The genes are transcribed at a low level during all stages of development, and their expression is markedly up-regulated after an immune challenge. For one of these genes, which is predominantly expressed in the larval fat body, we observe a constitutive expression in gain-of-function mutants of the Janus kinase (JAK) hop and a reduced inducibility in loss-of-function hop mutants. We also observe a constitutive expression in gain-of-function Toll mutants. We discuss the possible roles of these novel complement-like proteins in the Drosophila host defense.}, keywords = {alpha-Macroglobulins, Amino Acid, Animals, Cell Surface, Complement C3, Esters, Genetic, hoffmann, Insect Proteins, Janus Kinases, M3i, Membrane Glycoproteins, Mutation, Protein-Tyrosine Kinases, Proteins, Receptors, Sequence Homology, Sulfhydryl Compounds, Toll-Like Receptors, Transcription, Transcription Factors}, pubstate = {published}, tppubtype = {article} } We show that Drosophila expresses four genes encoding proteins with significant similarities with the thiolester-containing proteins of the complement C3/alpha(2)-macroglobulin superfamily. The genes are transcribed at a low level during all stages of development, and their expression is markedly up-regulated after an immune challenge. For one of these genes, which is predominantly expressed in the larval fat body, we observe a constitutive expression in gain-of-function mutants of the Janus kinase (JAK) hop and a reduced inducibility in loss-of-function hop mutants. We also observe a constitutive expression in gain-of-function Toll mutants. We discuss the possible roles of these novel complement-like proteins in the Drosophila host defense. |
1999 |
Lowenberger, C A; Kamal, S; Chiles, J; Paskewitz, S; Bulet, Philippe; Hoffmann, Jules A; Christensen, B M Mosquito-Plasmodium interactions in response to immune activation of the vector Article de journal Exp. Parasitol., 91 (1), p. 59–69, 1999, ISSN: 0014-4894. Résumé | Liens | BibTeX | Étiquettes: Aedes, Animals, Anopheles, Culicidae, Defensins, Digestive System, Escherichia coli, Female, Genetic, Hemolymph, hoffmann, Insect Vectors, M3i, messenger, Micrococcus luteus, Plasmodium, Plasmodium berghei, Plasmodium gallinaceum, Proteins, Reverse Transcriptase Polymerase Chain Reaction, RNA, Transcription @article{lowenberger_mosquito-plasmodium_1999, title = {Mosquito-Plasmodium interactions in response to immune activation of the vector}, author = {C A Lowenberger and S Kamal and J Chiles and S Paskewitz and Philippe Bulet and Jules A Hoffmann and B M Christensen}, doi = {10.1006/expr.1999.4350}, issn = {0014-4894}, year = {1999}, date = {1999-01-01}, journal = {Exp. Parasitol.}, volume = {91}, number = {1}, pages = {59--69}, abstract = {During the development of Plasmodium sp. within the mosquito midgut, the parasite undergoes a series of developmental changes. The elongated ookinete migrates through the layers of the midgut where it forms the oocyst under the basal lamina. We demonstrate here that if Aedes aegypti or Anopheles gambiae, normally susceptible to Plasmodium gallinaceum and P. berghei, respectively, are immune activated by the injection of bacteria into the hemocoel, and subsequently are fed on an infectious bloodmeal, there is a significant reduction in the prevalence and mean intensity of infection of oocysts on the midgut. Only those mosquitoes immune activated prior to, or immediately after, parasite ingestion exhibit this reduction in parasite development. Mosquitoes immune activated 2-5 days after bloodfeeding show no differences in parasite burdens compared with naive controls. Northern analyses reveal that transcriptional activity for mosquito defensins is not detected in the whole bodies of Ae. aegypti from 4 h to 10 days after ingesting P. gallinaceum, suggesting that parasite ingestion, passage from the food bolus through the midgut, oocyst formation, and subsequent release of sporozoites into the hemolymph do not induce the production of defensin. However, reverse transcriptase-PCR of RNA isolated solely from the midguts of Ae. aegypti indicates that transcription of mosquito defensins occurs in the midguts of naive mosquitoes and those ingesting an infectious or noninfectious bloodmeal. Bacteria-challenged Ae. aegypti showed high levels of mature defensin in the hemolymph that correlate with a lower prevalence and mean intensity of infection with oocysts. Because few oocysts were found on the midgut of immune-activated mosquitoes, the data suggest that some factor, induced by bacterial challenge, kills the parasite at a preoocyst stage.}, keywords = {Aedes, Animals, Anopheles, Culicidae, Defensins, Digestive System, Escherichia coli, Female, Genetic, Hemolymph, hoffmann, Insect Vectors, M3i, messenger, Micrococcus luteus, Plasmodium, Plasmodium berghei, Plasmodium gallinaceum, Proteins, Reverse Transcriptase Polymerase Chain Reaction, RNA, Transcription}, pubstate = {published}, tppubtype = {article} } During the development of Plasmodium sp. within the mosquito midgut, the parasite undergoes a series of developmental changes. The elongated ookinete migrates through the layers of the midgut where it forms the oocyst under the basal lamina. We demonstrate here that if Aedes aegypti or Anopheles gambiae, normally susceptible to Plasmodium gallinaceum and P. berghei, respectively, are immune activated by the injection of bacteria into the hemocoel, and subsequently are fed on an infectious bloodmeal, there is a significant reduction in the prevalence and mean intensity of infection of oocysts on the midgut. Only those mosquitoes immune activated prior to, or immediately after, parasite ingestion exhibit this reduction in parasite development. Mosquitoes immune activated 2-5 days after bloodfeeding show no differences in parasite burdens compared with naive controls. Northern analyses reveal that transcriptional activity for mosquito defensins is not detected in the whole bodies of Ae. aegypti from 4 h to 10 days after ingesting P. gallinaceum, suggesting that parasite ingestion, passage from the food bolus through the midgut, oocyst formation, and subsequent release of sporozoites into the hemolymph do not induce the production of defensin. However, reverse transcriptase-PCR of RNA isolated solely from the midguts of Ae. aegypti indicates that transcription of mosquito defensins occurs in the midguts of naive mosquitoes and those ingesting an infectious or noninfectious bloodmeal. Bacteria-challenged Ae. aegypti showed high levels of mature defensin in the hemolymph that correlate with a lower prevalence and mean intensity of infection with oocysts. Because few oocysts were found on the midgut of immune-activated mosquitoes, the data suggest that some factor, induced by bacterial challenge, kills the parasite at a preoocyst stage. |
1998 |
Levashina, Elena A; Ohresser, S; Lemaitre, Bruno; Imler, Jean-Luc Two distinct pathways can control expression of the gene encoding the Drosophila antimicrobial peptide metchnikowin Article de journal Journal of Molecular Biology, 278 (3), p. 515–527, 1998, ISSN: 0022-2836. Résumé | Liens | BibTeX | Étiquettes: Animals, Anti-Infective Agents, Antimicrobial Cationic Peptides, Base Sequence, Cloning, Gene Expression Regulation, Genes, Genetic, Genetically Modified, Glycopeptides, imler, Insect, Insect Proteins, Larva, M3i, Molecular, Mutation, Peptides, Promoter Regions, Recombinant Fusion Proteins, Reporter, Restriction Mapping, Transcription @article{levashina_two_1998, title = {Two distinct pathways can control expression of the gene encoding the Drosophila antimicrobial peptide metchnikowin}, author = {Elena A Levashina and S Ohresser and Bruno Lemaitre and Jean-Luc Imler}, doi = {10.1006/jmbi.1998.1705}, issn = {0022-2836}, year = {1998}, date = {1998-01-01}, journal = {Journal of Molecular Biology}, volume = {278}, number = {3}, pages = {515--527}, abstract = {Metchnikowin is a recently discovered proline-rich peptide from Drosophila with antibacterial and antifungal properties. Like most other antimicrobial peptides from insects, its expression is immune-inducible. Here we present evidence that induction of metchnikowin gene expression can be mediated either by the TOLL pathway or by the imd gene product. We show that the gene remains inducible in Toll-deficient mutants, in which the antifungal response is blocked, as well as in imd mutants, which fail to mount an antibacterial response. However, in Toll-deficient;imd double mutants, metchnikowin gene expression can no longer be detected after immune challenge. Our results suggest that expression of this peptide with dual activity can be triggered by signals generated by either bacterial or fungal infection. Cloning of the metchnikowin gene revealed the presence in the 5' flanking region of several putative cis-regulatory motifs characterized in the promoters of insect immune genes: namely, Rel sites, GATA motifs, interferon consensus response elements and NF-IL6 response elements. Establishment of transgenic fly lines in which the GFP reporter gene was placed under the control of 1.5 kb of metchnikowin gene upstream sequences indicates that this fragment is able to confer full immune inducibility and tissue specificity of expression on the transgene.}, keywords = {Animals, Anti-Infective Agents, Antimicrobial Cationic Peptides, Base Sequence, Cloning, Gene Expression Regulation, Genes, Genetic, Genetically Modified, Glycopeptides, imler, Insect, Insect Proteins, Larva, M3i, Molecular, Mutation, Peptides, Promoter Regions, Recombinant Fusion Proteins, Reporter, Restriction Mapping, Transcription}, pubstate = {published}, tppubtype = {article} } Metchnikowin is a recently discovered proline-rich peptide from Drosophila with antibacterial and antifungal properties. Like most other antimicrobial peptides from insects, its expression is immune-inducible. Here we present evidence that induction of metchnikowin gene expression can be mediated either by the TOLL pathway or by the imd gene product. We show that the gene remains inducible in Toll-deficient mutants, in which the antifungal response is blocked, as well as in imd mutants, which fail to mount an antibacterial response. However, in Toll-deficient;imd double mutants, metchnikowin gene expression can no longer be detected after immune challenge. Our results suggest that expression of this peptide with dual activity can be triggered by signals generated by either bacterial or fungal infection. Cloning of the metchnikowin gene revealed the presence in the 5' flanking region of several putative cis-regulatory motifs characterized in the promoters of insect immune genes: namely, Rel sites, GATA motifs, interferon consensus response elements and NF-IL6 response elements. Establishment of transgenic fly lines in which the GFP reporter gene was placed under the control of 1.5 kb of metchnikowin gene upstream sequences indicates that this fragment is able to confer full immune inducibility and tissue specificity of expression on the transgene. |
1996 |
Lowenberger, C A; Ferdig, M T; Bulet, Philippe; Khalili, S; Hoffmann, Jules A; Christensen, B M Aedes aegypti: induced antibacterial proteins reduce the establishment and development of Brugia malayi Article de journal Exp. Parasitol., 83 (2), p. 191–201, 1996, ISSN: 0014-4894. Résumé | Liens | BibTeX | Étiquettes: Aedes, Analysis of Variance, Animals, Anti-Bacterial Agents, Base Sequence, Blood Proteins, Blotting, Brugia malayi, Culicidae, Defensins, DNA, Escherichia coli, Fat Body, Genetic, Gerbillinae, hoffmann, M3i, Micrococcus luteus, Microfilaria, Northern, RNA, Transcription @article{lowenberger_aedes_1996, title = {Aedes aegypti: induced antibacterial proteins reduce the establishment and development of Brugia malayi}, author = {C A Lowenberger and M T Ferdig and Philippe Bulet and S Khalili and Jules A Hoffmann and B M Christensen}, doi = {10.1006/expr.1996.0066}, issn = {0014-4894}, year = {1996}, date = {1996-07-01}, journal = {Exp. Parasitol.}, volume = {83}, number = {2}, pages = {191--201}, abstract = {The effect of host immune activation on the development of Brugia malayi in one susceptible and four refractory strains of Aedes aegypti and in Armigeres subalbatus was assessed. A. aegypti that were immune activated by the injection of saline or bacteria 24 hr before feeding on a B. malayi-infected gerbil had significantly reduced prevalences and mean intensities of infection from those of naive controls when exposed to bloodmeals with low (105 mf/20 microliters) and medium (160 mf/20 microliters) microfilaremias. At a higher microfilaremia (237 mf/20 microliters) there were no significant differences in mean intensities, suggesting that the number of parasites ingested may affect the host's ability to mount an effective defense response. Because the major immune proteins in A. aegypti are defensins, we did Northern analyses of fat body RNA 8 hr after immune activation or bloodfeeding. All mosquitoes demonstrated rapid transcriptional activity for defensins following immune activation by intrathoracic inoculation with either saline or bacteria. However, no strain of A. aegypti, susceptible or refractory to B. malayi, nor Ar. subalbatus produced defensin transcripts after bloodfeeding on an uninfected or a B. malayi-infected gerbil. These data suggest that inducible immune proteins of mosquitoes can reduce the prevalence and mean intensity of infections with ingested parasites, but these proteins are not expressed routinely after parasite ingestion and midgut penetration and probably do not contribute to existing refractory mechanisms. Immune proteins such as defensins, however, represent potential candidates to genetically engineer mosquitoes for resistance to filarial worms.}, keywords = {Aedes, Analysis of Variance, Animals, Anti-Bacterial Agents, Base Sequence, Blood Proteins, Blotting, Brugia malayi, Culicidae, Defensins, DNA, Escherichia coli, Fat Body, Genetic, Gerbillinae, hoffmann, M3i, Micrococcus luteus, Microfilaria, Northern, RNA, Transcription}, pubstate = {published}, tppubtype = {article} } The effect of host immune activation on the development of Brugia malayi in one susceptible and four refractory strains of Aedes aegypti and in Armigeres subalbatus was assessed. A. aegypti that were immune activated by the injection of saline or bacteria 24 hr before feeding on a B. malayi-infected gerbil had significantly reduced prevalences and mean intensities of infection from those of naive controls when exposed to bloodmeals with low (105 mf/20 microliters) and medium (160 mf/20 microliters) microfilaremias. At a higher microfilaremia (237 mf/20 microliters) there were no significant differences in mean intensities, suggesting that the number of parasites ingested may affect the host's ability to mount an effective defense response. Because the major immune proteins in A. aegypti are defensins, we did Northern analyses of fat body RNA 8 hr after immune activation or bloodfeeding. All mosquitoes demonstrated rapid transcriptional activity for defensins following immune activation by intrathoracic inoculation with either saline or bacteria. However, no strain of A. aegypti, susceptible or refractory to B. malayi, nor Ar. subalbatus produced defensin transcripts after bloodfeeding on an uninfected or a B. malayi-infected gerbil. These data suggest that inducible immune proteins of mosquitoes can reduce the prevalence and mean intensity of infections with ingested parasites, but these proteins are not expressed routinely after parasite ingestion and midgut penetration and probably do not contribute to existing refractory mechanisms. Immune proteins such as defensins, however, represent potential candidates to genetically engineer mosquitoes for resistance to filarial worms. |
Gross, I; Georgel, Philippe; Kappler, Christine; Reichhart, Jean-Marc; Hoffmann, Jules A Drosophila immunity: a comparative analysis of the Rel proteins dorsal and Dif in the induction of the genes encoding diptericin and cecropin Article de journal Nucleic Acids Res., 24 (7), p. 1238–1245, 1996, ISSN: 0305-1048. Résumé | BibTeX | Étiquettes: Animals, Antimicrobial Cationic Peptides, Base Sequence, DNA Primers, DNA-Binding Proteins, Gene Expression Regulation, Genetic, hoffmann, Insect Hormones, Insect Proteins, M3i, NF-kappa B, Nuclear Proteins, Peptides, Phosphoproteins, reichhart, Transcription, Transcription Factors, Transcriptional Activation @article{gross_drosophila_1996, title = {Drosophila immunity: a comparative analysis of the Rel proteins dorsal and Dif in the induction of the genes encoding diptericin and cecropin}, author = {I Gross and Philippe Georgel and Christine Kappler and Jean-Marc Reichhart and Jules A Hoffmann}, issn = {0305-1048}, year = {1996}, date = {1996-04-01}, journal = {Nucleic Acids Res.}, volume = {24}, number = {7}, pages = {1238--1245}, abstract = {In Drosophila, bacterial challenge induces the rapid transcription of several genes encoding potent antibacterial peptides. The upstream sequences of the diptericin and cecropin Al genes, which have been investigated in detail, contain two, respectively one sequence element homologous to the binding site of the mammalian nuclear factor kappaB. These elements have been shown to be mandatory for immune-induced transcription of both genes. Functional studies have shown that these kappaB-related elements can be the target for the Drosophila Rel proteins dorsal and Dif. Here we present a comparative analysis of the transactivating capacities of these proteins on reporter genes fused to either the diptericin or the cecropin kappaB-related motifs. We conclude from our results: (i) the kappaB motifs of the diptericin and cecropin genes are not functionally equivalent; (ii) the dorsal and Dif proteins have distinct DNA-binding characteristics; (iii) dorsal and Dif can heterodimerize in vitro; (iv) mutants containing no copies of dorsal and a single copy of Dif retain their full capacity to express the diptericin and cecropin genes in response to challenge.}, keywords = {Animals, Antimicrobial Cationic Peptides, Base Sequence, DNA Primers, DNA-Binding Proteins, Gene Expression Regulation, Genetic, hoffmann, Insect Hormones, Insect Proteins, M3i, NF-kappa B, Nuclear Proteins, Peptides, Phosphoproteins, reichhart, Transcription, Transcription Factors, Transcriptional Activation}, pubstate = {published}, tppubtype = {article} } In Drosophila, bacterial challenge induces the rapid transcription of several genes encoding potent antibacterial peptides. The upstream sequences of the diptericin and cecropin Al genes, which have been investigated in detail, contain two, respectively one sequence element homologous to the binding site of the mammalian nuclear factor kappaB. These elements have been shown to be mandatory for immune-induced transcription of both genes. Functional studies have shown that these kappaB-related elements can be the target for the Drosophila Rel proteins dorsal and Dif. Here we present a comparative analysis of the transactivating capacities of these proteins on reporter genes fused to either the diptericin or the cecropin kappaB-related motifs. We conclude from our results: (i) the kappaB motifs of the diptericin and cecropin genes are not functionally equivalent; (ii) the dorsal and Dif proteins have distinct DNA-binding characteristics; (iii) dorsal and Dif can heterodimerize in vitro; (iv) mutants containing no copies of dorsal and a single copy of Dif retain their full capacity to express the diptericin and cecropin genes in response to challenge. |
1995 |
Levashina, Elena A; Ohresser, S; Bulet, Philippe; Reichhart, Jean-Marc; Hetru, Charles; Hoffmann, Jules A Metchnikowin, a novel immune-inducible proline-rich peptide from Drosophila with antibacterial and antifungal properties Article de journal Eur. J. Biochem., 233 (2), p. 694–700, 1995, ISSN: 0014-2956. Résumé | BibTeX | Étiquettes: Animals, Anti-Bacterial Agents, Antifungal Agents, Antimicrobial Cationic Peptides, bacteria, Base Sequence, Cells, Chromosome Mapping, Cloning, Cultured, Genetic, hoffmann, M3i, Molecular, Peptides, Proline, reichhart, Transcription @article{levashina_metchnikowin_1995, title = {Metchnikowin, a novel immune-inducible proline-rich peptide from Drosophila with antibacterial and antifungal properties}, author = {Elena A Levashina and S Ohresser and Philippe Bulet and Jean-Marc Reichhart and Charles Hetru and Jules A Hoffmann}, issn = {0014-2956}, year = {1995}, date = {1995-10-01}, journal = {Eur. J. Biochem.}, volume = {233}, number = {2}, pages = {694--700}, abstract = {One of the characteristics of the host defense of higher insects is the rapid and transient synthesis of a variety of potent antimicrobial peptides. To date, several distinct inducible antimicrobial peptides or peptide families have been totally or partially characterized. We present here the isolation and characterization of a novel 26-residue proline-rich immune-inducible peptide from Drosophila, which exhibits both antibacterial (Gram-positive) and antifungal activities. Peptide sequencing and cDNA cloning indicate the presense of two isoforms in our Drosophila Oregon strain, which differ by one residue (His compared to Arg) as a consequence of a single nucleotide change. The gene, which maps in position 52A1-2 on the right arm of the second chromosome, is expressed in the fat body after immune challenge. The novel peptide, which we propose to name metchnikowin, is a member of a family of proline-rich peptides, and we discuss the possible evolutionary relationships within this family.}, keywords = {Animals, Anti-Bacterial Agents, Antifungal Agents, Antimicrobial Cationic Peptides, bacteria, Base Sequence, Cells, Chromosome Mapping, Cloning, Cultured, Genetic, hoffmann, M3i, Molecular, Peptides, Proline, reichhart, Transcription}, pubstate = {published}, tppubtype = {article} } One of the characteristics of the host defense of higher insects is the rapid and transient synthesis of a variety of potent antimicrobial peptides. To date, several distinct inducible antimicrobial peptides or peptide families have been totally or partially characterized. We present here the isolation and characterization of a novel 26-residue proline-rich immune-inducible peptide from Drosophila, which exhibits both antibacterial (Gram-positive) and antifungal activities. Peptide sequencing and cDNA cloning indicate the presense of two isoforms in our Drosophila Oregon strain, which differ by one residue (His compared to Arg) as a consequence of a single nucleotide change. The gene, which maps in position 52A1-2 on the right arm of the second chromosome, is expressed in the fat body after immune challenge. The novel peptide, which we propose to name metchnikowin, is a member of a family of proline-rich peptides, and we discuss the possible evolutionary relationships within this family. |
Heyman, T; Agoutin, B; Friant, S; Wilhelm, F X; Wilhelm, M L Plus-strand DNA synthesis of the yeast retrotransposon Ty1 is initiated at two sites, PPT1 next to the 3' LTR and PPT2 within the pol gene. PPT1 is sufficient for Ty1 transposition Article de journal J Mol Biol, 253 (2), p. 291-303, 1995, (0022-2836 Journal Article). Résumé | BibTeX | Étiquettes: *DNA, *Genes, *Repetitive, *Retroelements, Acid, Base, C/analysis, cerevisiae/genetics/*virology, Chain, Cloning, Data, DNA, Fungal, Fungal/biosynthesis, Genes, Genetic, Genome, Gov't, Mapping, Molecular, Non-U.S., Nucleic, pol, Poly, Polymerase, Primers, Reaction, Replication, Restriction, Saccharomyces, Sequence, Sequences, Support, Transcription, Viral, Viral/*biosynthesis @article{, title = {Plus-strand DNA synthesis of the yeast retrotransposon Ty1 is initiated at two sites, PPT1 next to the 3' LTR and PPT2 within the pol gene. PPT1 is sufficient for Ty1 transposition}, author = { T. Heyman and B. Agoutin and S. Friant and F. X. Wilhelm and M. L. Wilhelm}, year = {1995}, date = {1995-01-01}, journal = {J Mol Biol}, volume = {253}, number = {2}, pages = {291-303}, abstract = {Long terminal repeat elements and retroviruses require primers for initiation of minus and plus-strand DNA synthesis by reverse transcriptase. Here we demonstrate genetically that plus-strand DNA synthesis of the yeast Ty1 element is initiated at two sites located at the 5' boundary of the 3' long terminal repeat (PPT1) and near the middle of the pol gene in the integrase coding sequence (PPT2). A consequence of the presence of two PPTs is that Ty1 plus-strand DNA exists as segments at some time during replication. Three fragments have been identified: the plus-strand strong-stop DNA initiated at PPT1, a downstream fragment initiated at PPT2 and an upstream fragment spanning the 5'-terminal part of Ty1 and a portion of the TyB gene. Characterization of the 3' ends of the plus-strand DNA fragments reveals (1) that the upstream fragment is elongated beyond PPT2 creating a plus-strand overlap and (2) that the majority of plus-strand strong-stop DNA fragments bear a copy of the minus-strand primer binding site in agreement with the accepted model of retroviral genomic RNA reverse transcription. The two polypurine tracts, PPT1 and PPT2, have an identical sequence GGGTGGTA. Mutations replacing purines by pyrimidines in this sequence significantly diminish or abolish initiation of plus-strand synthesis. Ty1 elements bearing a mutated PPT2 sequence are not defective for transposition whereas mutations in PPT1 abolish transposition.}, note = {0022-2836 Journal Article}, keywords = {*DNA, *Genes, *Repetitive, *Retroelements, Acid, Base, C/analysis, cerevisiae/genetics/*virology, Chain, Cloning, Data, DNA, Fungal, Fungal/biosynthesis, Genes, Genetic, Genome, Gov't, Mapping, Molecular, Non-U.S., Nucleic, pol, Poly, Polymerase, Primers, Reaction, Replication, Restriction, Saccharomyces, Sequence, Sequences, Support, Transcription, Viral, Viral/*biosynthesis}, pubstate = {published}, tppubtype = {article} } Long terminal repeat elements and retroviruses require primers for initiation of minus and plus-strand DNA synthesis by reverse transcriptase. Here we demonstrate genetically that plus-strand DNA synthesis of the yeast Ty1 element is initiated at two sites located at the 5' boundary of the 3' long terminal repeat (PPT1) and near the middle of the pol gene in the integrase coding sequence (PPT2). A consequence of the presence of two PPTs is that Ty1 plus-strand DNA exists as segments at some time during replication. Three fragments have been identified: the plus-strand strong-stop DNA initiated at PPT1, a downstream fragment initiated at PPT2 and an upstream fragment spanning the 5'-terminal part of Ty1 and a portion of the TyB gene. Characterization of the 3' ends of the plus-strand DNA fragments reveals (1) that the upstream fragment is elongated beyond PPT2 creating a plus-strand overlap and (2) that the majority of plus-strand strong-stop DNA fragments bear a copy of the minus-strand primer binding site in agreement with the accepted model of retroviral genomic RNA reverse transcription. The two polypurine tracts, PPT1 and PPT2, have an identical sequence GGGTGGTA. Mutations replacing purines by pyrimidines in this sequence significantly diminish or abolish initiation of plus-strand synthesis. Ty1 elements bearing a mutated PPT2 sequence are not defective for transposition whereas mutations in PPT1 abolish transposition. |
Lemaitre, Bruno; Meister, Marie; Govind, S; Georgel, Philippe; Steward, R; Reichhart, Jean-Marc; Hoffmann, Jules A Functional analysis and regulation of nuclear import of dorsal during the immune response in Drosophila Article de journal EMBO J., 14 (3), p. 536–545, 1995, ISSN: 0261-4189. Résumé | BibTeX | Étiquettes: Animals, Anti-Bacterial Agents, Anti-Infective Agents, Antimicrobial Cationic Peptides, Biological Transport, Cell Nucleus, Cell Surface, DNA-Binding Proteins, Fat Body, Gene Expression Regulation, Genetic, hoffmann, Immunity, Immunohistochemistry, Insect Hormones, Insect Proteins, M3i, Melanins, Membrane Glycoproteins, Mutation, Neoplasms, Nuclear Proteins, Phosphoproteins, Receptors, reichhart, Signal Transduction, Toll-Like Receptors, Transcription, Transcription Factors @article{lemaitre_functional_1995, title = {Functional analysis and regulation of nuclear import of dorsal during the immune response in Drosophila}, author = {Bruno Lemaitre and Marie Meister and S Govind and Philippe Georgel and R Steward and Jean-Marc Reichhart and Jules A Hoffmann}, issn = {0261-4189}, year = {1995}, date = {1995-01-01}, journal = {EMBO J.}, volume = {14}, number = {3}, pages = {536--545}, abstract = {In addition to its function in embryonic development, the NF-kappa B/rel-related gene dorsal (dl) of Drosophila is expressed in larval and adult fat body where its RNA expression is enhanced upon injury. Injury also leads to a rapid nuclear translocation of dl from the cytoplasm in fat body cells. Here we present data which strongly suggest that the nuclear localization of dl during the immune response is controlled by the Toll signaling pathway, comprising gene products that participate in the intracellular part of the embryonic dorsoventral pathway. We also report that in mutants such as Toll or cactus, which exhibit melanotic tumor phenotypes, dl is constitutively nuclear. Together, these results point to a potential link between the Toll signaling pathway and melanotic tumor induction. Although dl has been shown previously to bind to kappa B-related motifs within the promoter of the antibacterial peptide coding gene diptericin, we find that injury-induced expression of diptericin can occur in the absence of dl. Furthermore, the melanotic tumor phenotype of Toll and cactus is not dl dependent. These data underline the complexity of the Drosophila immune response. Finally, we observed that like other rel proteins, dl can control the level of its own transcription.}, keywords = {Animals, Anti-Bacterial Agents, Anti-Infective Agents, Antimicrobial Cationic Peptides, Biological Transport, Cell Nucleus, Cell Surface, DNA-Binding Proteins, Fat Body, Gene Expression Regulation, Genetic, hoffmann, Immunity, Immunohistochemistry, Insect Hormones, Insect Proteins, M3i, Melanins, Membrane Glycoproteins, Mutation, Neoplasms, Nuclear Proteins, Phosphoproteins, Receptors, reichhart, Signal Transduction, Toll-Like Receptors, Transcription, Transcription Factors}, pubstate = {published}, tppubtype = {article} } In addition to its function in embryonic development, the NF-kappa B/rel-related gene dorsal (dl) of Drosophila is expressed in larval and adult fat body where its RNA expression is enhanced upon injury. Injury also leads to a rapid nuclear translocation of dl from the cytoplasm in fat body cells. Here we present data which strongly suggest that the nuclear localization of dl during the immune response is controlled by the Toll signaling pathway, comprising gene products that participate in the intracellular part of the embryonic dorsoventral pathway. We also report that in mutants such as Toll or cactus, which exhibit melanotic tumor phenotypes, dl is constitutively nuclear. Together, these results point to a potential link between the Toll signaling pathway and melanotic tumor induction. Although dl has been shown previously to bind to kappa B-related motifs within the promoter of the antibacterial peptide coding gene diptericin, we find that injury-induced expression of diptericin can occur in the absence of dl. Furthermore, the melanotic tumor phenotype of Toll and cactus is not dl dependent. These data underline the complexity of the Drosophila immune response. Finally, we observed that like other rel proteins, dl can control the level of its own transcription. |
1994 |
Dimarcq, Jean-Luc; Hoffmann, Danièle; Meister, Marie; Bulet, Philippe; Lanot, R; Reichhart, Jean-Marc; Hoffmann, Jules A Characterization and transcriptional profiles of a Drosophila gene encoding an insect defensin. A study in insect immunity Article de journal Eur. J. Biochem., 221 (1), p. 201–209, 1994, ISSN: 0014-2956. Résumé | BibTeX | Étiquettes: Animals, Base Sequence, Blood Proteins, Chromosome Mapping, Cloning, Complementary, Defensins, DNA, Gene Expression, Genetic, Gram-Positive Bacteria, hoffmann, Larva, M3i, Molecular, Molecular Structure, Nucleic Acid, Protein Precursors, Regulatory Sequences, reichhart, Transcription @article{dimarcq_characterization_1994, title = {Characterization and transcriptional profiles of a Drosophila gene encoding an insect defensin. A study in insect immunity}, author = {Jean-Luc Dimarcq and Danièle Hoffmann and Marie Meister and Philippe Bulet and R Lanot and Jean-Marc Reichhart and Jules A Hoffmann}, issn = {0014-2956}, year = {1994}, date = {1994-04-01}, journal = {Eur. J. Biochem.}, volume = {221}, number = {1}, pages = {201--209}, abstract = {Insect defensins are a family of 4-kDa, cationic, inducible antibacterial peptides which bear six cysteine residues engaged in three intramolecular disulfide bridges. They owe their name to certain sequence similarities with defensins from mammalian neutrophiles and macrophages. We report the characterization of a novel defensin isoform from Drosophila and the cloning of the gene encoding a preprodefensin. The gene, which is intronless and present in a single copy/haploid genome, maps at position 46CD on the right arm of the second chromosome. The analysis of the upstream region of the gene reveals the presence of multiple putative cis-regulatory sequences similar to mammalian regulatory motifs of acute-phase-response genes. Transcriptional profiles indicate that the Drosophila defensin gene is induced by bacterial challenge with acute-phase kinetics. It is also expressed in the absence of immune challenge during metamorphosis. These and other data on the Drosophila defensin gene lead us to suggest that insect and mammalian defensins have evolved independently.}, keywords = {Animals, Base Sequence, Blood Proteins, Chromosome Mapping, Cloning, Complementary, Defensins, DNA, Gene Expression, Genetic, Gram-Positive Bacteria, hoffmann, Larva, M3i, Molecular, Molecular Structure, Nucleic Acid, Protein Precursors, Regulatory Sequences, reichhart, Transcription}, pubstate = {published}, tppubtype = {article} } Insect defensins are a family of 4-kDa, cationic, inducible antibacterial peptides which bear six cysteine residues engaged in three intramolecular disulfide bridges. They owe their name to certain sequence similarities with defensins from mammalian neutrophiles and macrophages. We report the characterization of a novel defensin isoform from Drosophila and the cloning of the gene encoding a preprodefensin. The gene, which is intronless and present in a single copy/haploid genome, maps at position 46CD on the right arm of the second chromosome. The analysis of the upstream region of the gene reveals the presence of multiple putative cis-regulatory sequences similar to mammalian regulatory motifs of acute-phase-response genes. Transcriptional profiles indicate that the Drosophila defensin gene is induced by bacterial challenge with acute-phase kinetics. It is also expressed in the absence of immune challenge during metamorphosis. These and other data on the Drosophila defensin gene lead us to suggest that insect and mammalian defensins have evolved independently. |
Wilhelm, M; Wilhelm, F X; Keith, G; Agoutin, B; Heyman, T Yeast Ty1 retrotransposon: the minus-strand primer binding site and a cis-acting domain of the Ty1 RNA are both important for packaging of primer tRNA inside virus-like particles Article de journal Nucleic Acids Res, 22 (22), p. 4560-5, 1994, (0305-1048 Journal Article). Résumé | BibTeX | Étiquettes: Acid, Amino, Base, Binding, cerevisiae/*genetics, Cloning, Data, Fungal/*genetics, Genetic, Gov't, Met/*genetics, Molecular, Mutation/physiology, Non-U.S., Retroelements/*genetics/physiology, Retroviridae/genetics, RNA, RNA/*genetics, Saccharomyces, Sequence, Sites, Support, Transcription, Transfer @article{, title = {Yeast Ty1 retrotransposon: the minus-strand primer binding site and a cis-acting domain of the Ty1 RNA are both important for packaging of primer tRNA inside virus-like particles}, author = { M. Wilhelm and F. X. Wilhelm and G. Keith and B. Agoutin and T. Heyman}, year = {1994}, date = {1994-01-01}, journal = {Nucleic Acids Res}, volume = {22}, number = {22}, pages = {4560-5}, abstract = {Reverse transcription of the yeast retrotransposon Ty1 is primed by the cytoplasmic initiator methionine tRNA (tRNA(iMet)). The primer tRNA(iMet) is packaged inside virus-like particles (VLPs) and binds to a 10 nucleotides minus-strand primer binding site, the (-)PBS, complementary to its 3' acceptor stem. We have found that three short sequences of the Ty1 RNA (box 1, box 2.1 and box 2.2) located 3' to the (-)PBS are complementary to other regions of the primer tRNA(iMet) (T psi C and DHU stems and loops). Reconstitution of reverse transcription in vitro with T7 transcribed Ty1 RNA species and tRNA(iMet) purified from yeast cells shows that the boxes do not affect the efficiency of reverse transcription. Thus the role of the boxes on packaging of the primer tRNA(iMet) into the VLPs was investigated by analysing the level of tRNA(iMet) packaged into mutant VLPs. Specific nucleotide changes in the (-)PBS or in the boxes that do not change the protein coding sequence but disrupt the complementarity with the primer tRNA(iMet) within the VLPs. We propose that base pairing between the primer tRNA(iMet) and the Ty1 RNA is of major importance for tRNA(iMet) packaging into the VLPs. Moreover the intactness of the boxes is essential for retrotransposition as shown by the transposition defect of a Ty1 element harboring an intact (-)PBS and mutated boxes.}, note = {0305-1048 Journal Article}, keywords = {Acid, Amino, Base, Binding, cerevisiae/*genetics, Cloning, Data, Fungal/*genetics, Genetic, Gov't, Met/*genetics, Molecular, Mutation/physiology, Non-U.S., Retroelements/*genetics/physiology, Retroviridae/genetics, RNA, RNA/*genetics, Saccharomyces, Sequence, Sites, Support, Transcription, Transfer}, pubstate = {published}, tppubtype = {article} } Reverse transcription of the yeast retrotransposon Ty1 is primed by the cytoplasmic initiator methionine tRNA (tRNA(iMet)). The primer tRNA(iMet) is packaged inside virus-like particles (VLPs) and binds to a 10 nucleotides minus-strand primer binding site, the (-)PBS, complementary to its 3' acceptor stem. We have found that three short sequences of the Ty1 RNA (box 1, box 2.1 and box 2.2) located 3' to the (-)PBS are complementary to other regions of the primer tRNA(iMet) (T psi C and DHU stems and loops). Reconstitution of reverse transcription in vitro with T7 transcribed Ty1 RNA species and tRNA(iMet) purified from yeast cells shows that the boxes do not affect the efficiency of reverse transcription. Thus the role of the boxes on packaging of the primer tRNA(iMet) into the VLPs was investigated by analysing the level of tRNA(iMet) packaged into mutant VLPs. Specific nucleotide changes in the (-)PBS or in the boxes that do not change the protein coding sequence but disrupt the complementarity with the primer tRNA(iMet) within the VLPs. We propose that base pairing between the primer tRNA(iMet) and the Ty1 RNA is of major importance for tRNA(iMet) packaging into the VLPs. Moreover the intactness of the boxes is essential for retrotransposition as shown by the transposition defect of a Ty1 element harboring an intact (-)PBS and mutated boxes. |
1993 |
Pfeiffer, P; Jung, J L; Heitzler, J; Keith, G Unusual structure of the double-stranded RNA associated with the '447' cytoplasmic male sterility in Vicia faba Article de journal J Gen Virol, 74 (Pt 6), p. 1167-73, 1993, (0022-1317 Journal Article). Résumé | BibTeX | Étiquettes: *Plants, &, Base, Bodies, Cytoplasm, Data, Extrachromosomal, Fabaceae/*genetics, Genetic, Inclusion, Infertility/genetics, Inheritance/*genetics, Medicinal, Molecular, Pollen/genetics, purification, Replicase/metabolism, RNA, RNA/*genetics/isolation, Sequence, Transcription, Viral @article{, title = {Unusual structure of the double-stranded RNA associated with the '447' cytoplasmic male sterility in Vicia faba}, author = { P. Pfeiffer and J. L. Jung and J. Heitzler and G. Keith}, year = {1993}, date = {1993-01-01}, journal = {J Gen Virol}, volume = {74}, number = {Pt 6}, pages = {1167-73}, abstract = {The 16.7 kbp dsRNA specific to the '447' cytoplasmic male sterility (CMS) line of Vicia faba was labelled in vitro with [alpha-32P]ATP and poly(A) polymerase, and by T4 RNA ligase-mediated addition of [32P]pCp. Analysis of the reaction products under denaturing conditions revealed in both cases extensive labelling of a 4.5 kb ssRNA, already detected in previous experiments in which the RNA-dependent RNA polymerase associated with the dsRNA was allowed to pursue RNA synthesis on preinitiated complexes. Mobility shift analysis of total pCp-labelled dsRNA revealed not two but three different 3' termini. The most prominent sequencing pattern corresponded to the 4.5 kb ssRNA, indicating that this RNA species has a preferentially accessible, free 3' OH extremity. Northern blot analysis of the denatured dsRNA confirmed that the 4.5 kb ssRNA is a subgenomic mRNA and detected its counterpart of about 12 kb. Nearly all 16.7 kbp dsRNA molecules featured an interrupted positive-sense strand, indicating a marked prevalence of transcription over replication complexes. This unusual strategy of transcription by a strand displacement mechanism, following initiation at an internal discontinuity, is compared with that of other dsRNA viruses or defective viruses, and is discussed in relation to the expression of the CMS trait.}, note = {0022-1317 Journal Article}, keywords = {*Plants, &, Base, Bodies, Cytoplasm, Data, Extrachromosomal, Fabaceae/*genetics, Genetic, Inclusion, Infertility/genetics, Inheritance/*genetics, Medicinal, Molecular, Pollen/genetics, purification, Replicase/metabolism, RNA, RNA/*genetics/isolation, Sequence, Transcription, Viral}, pubstate = {published}, tppubtype = {article} } The 16.7 kbp dsRNA specific to the '447' cytoplasmic male sterility (CMS) line of Vicia faba was labelled in vitro with [alpha-32P]ATP and poly(A) polymerase, and by T4 RNA ligase-mediated addition of [32P]pCp. Analysis of the reaction products under denaturing conditions revealed in both cases extensive labelling of a 4.5 kb ssRNA, already detected in previous experiments in which the RNA-dependent RNA polymerase associated with the dsRNA was allowed to pursue RNA synthesis on preinitiated complexes. Mobility shift analysis of total pCp-labelled dsRNA revealed not two but three different 3' termini. The most prominent sequencing pattern corresponded to the 4.5 kb ssRNA, indicating that this RNA species has a preferentially accessible, free 3' OH extremity. Northern blot analysis of the denatured dsRNA confirmed that the 4.5 kb ssRNA is a subgenomic mRNA and detected its counterpart of about 12 kb. Nearly all 16.7 kbp dsRNA molecules featured an interrupted positive-sense strand, indicating a marked prevalence of transcription over replication complexes. This unusual strategy of transcription by a strand displacement mechanism, following initiation at an internal discontinuity, is compared with that of other dsRNA viruses or defective viruses, and is discussed in relation to the expression of the CMS trait. |
1992 |
Lavignon, M; Tounekti, N; Rayner, B; Imbach, J L; Keith, G; Paoletti, J; Malvy, C Inhibition of murine leukemia viruses by nuclease-resistant alpha-oligonucleotides Article de journal Antisense Res Dev, 2 (4), p. 315-24, 1992, (1050-5261 Journal Article). Résumé | BibTeX | Étiquettes: 3T3, Animals, Antisense/metabolism/*pharmacology, Base, Binding, Cells, Culture, Data, effects, effects/genetics/physiology, Friend, Genetic/drug, Gov't, leukemia, Media, Messenger/chemistry/genetics/metabolism, Mice, Molecular, Moloney, murine, Non-U.S., oligonucleotides, RNA, Sequence, Sites, Support, Transcription, Translation, Viral/chemistry/genetics/metabolism, virus/*drug @article{, title = {Inhibition of murine leukemia viruses by nuclease-resistant alpha-oligonucleotides}, author = { M. Lavignon and N. Tounekti and B. Rayner and J. L. Imbach and G. Keith and J. Paoletti and C. Malvy}, year = {1992}, date = {1992-01-01}, journal = {Antisense Res Dev}, volume = {2}, number = {4}, pages = {315-24}, abstract = {We studied the antiviral activity of nuclease-resistant alpha-anomeric oligonucleotides. An alpha-oligonucleotide (20-mer) targeted to the primer binding site (PBS) of murine retroviruses inhibited viral spreading. The inhibition only occurred when the cells had been electropermeabilized in the presence of the oligonucleotide. The PBS sequence is involved in reverse transcription and in translation. The data suggest that the oligonucleotide could perturb reverse transcription activity. Thus, either the oligonucleotide induced a decrease in initiation or it inhibited the extension of the minus or plus strands DNA during reverse transcription. These results show that reverse transcription may be an interesting target for antisense oligonucleotides.}, note = {1050-5261 Journal Article}, keywords = {3T3, Animals, Antisense/metabolism/*pharmacology, Base, Binding, Cells, Culture, Data, effects, effects/genetics/physiology, Friend, Genetic/drug, Gov't, leukemia, Media, Messenger/chemistry/genetics/metabolism, Mice, Molecular, Moloney, murine, Non-U.S., oligonucleotides, RNA, Sequence, Sites, Support, Transcription, Translation, Viral/chemistry/genetics/metabolism, virus/*drug}, pubstate = {published}, tppubtype = {article} } We studied the antiviral activity of nuclease-resistant alpha-anomeric oligonucleotides. An alpha-oligonucleotide (20-mer) targeted to the primer binding site (PBS) of murine retroviruses inhibited viral spreading. The inhibition only occurred when the cells had been electropermeabilized in the presence of the oligonucleotide. The PBS sequence is involved in reverse transcription and in translation. The data suggest that the oligonucleotide could perturb reverse transcription activity. Thus, either the oligonucleotide induced a decrease in initiation or it inhibited the extension of the minus or plus strands DNA during reverse transcription. These results show that reverse transcription may be an interesting target for antisense oligonucleotides. |
1990 |
Hoffmann, Jules A; Hoffmann, Danièle The inducible antibacterial peptides of dipteran insects Article de journal Res. Immunol., 141 (9), p. 910–918, 1990, ISSN: 0923-2494. BibTeX | Étiquettes: Animals, Antimicrobial Cationic Peptides, Defensins, Diptera, Gene Expression Regulation, Genetic, hoffmann, Insect Hormones, Insect Proteins, M3i, Nucleic Acid, Proteins, Sequence Homology, Transcription @article{hoffmann_inducible_1990, title = {The inducible antibacterial peptides of dipteran insects}, author = {Jules A Hoffmann and Danièle Hoffmann}, issn = {0923-2494}, year = {1990}, date = {1990-12-01}, journal = {Res. Immunol.}, volume = {141}, number = {9}, pages = {910--918}, keywords = {Animals, Antimicrobial Cationic Peptides, Defensins, Diptera, Gene Expression Regulation, Genetic, hoffmann, Insect Hormones, Insect Proteins, M3i, Nucleic Acid, Proteins, Sequence Homology, Transcription}, pubstate = {published}, tppubtype = {article} } |
1989 |
Reichhart, Jean-Marc; Essrich, M; Dimarcq, Jean-Luc; Hoffmann, Danièle; Hoffmann, Jules A; Lagueux, Marie Insect immunity. Isolation of cDNA clones corresponding to diptericin, an inducible antibacterial peptide from Phormia terranovae (Diptera). Transcriptional profiles during immunization Article de journal Eur. J. Biochem., 182 (2), p. 423–427, 1989, ISSN: 0014-2956. Résumé | BibTeX | Étiquettes: Animals, Anti-Bacterial Agents, Bacterial Proteins, Base Sequence, Blotting, Diptera, DNA, Endoribonucleases, Enterobacter, Enterobacteriaceae, Gene Expression Regulation, Genes, Genetic, hoffmann, Insect Hormones, Insect Proteins, M3i, messenger, MHC Class II, Northern, reichhart, Ribonuclease H, RNA, Transcription @article{reichhart_insect_1989, title = {Insect immunity. Isolation of cDNA clones corresponding to diptericin, an inducible antibacterial peptide from Phormia terranovae (Diptera). Transcriptional profiles during immunization}, author = {Jean-Marc Reichhart and M Essrich and Jean-Luc Dimarcq and Danièle Hoffmann and Jules A Hoffmann and Marie Lagueux}, issn = {0014-2956}, year = {1989}, date = {1989-01-01}, journal = {Eur. J. Biochem.}, volume = {182}, number = {2}, pages = {423--427}, abstract = {We have previously isolated and characterized a family of novel 8-kDa cationic antibacterial peptides synthesized by larvae of Phormia terranovae (Diptera) in response to various injuries. These molecules have been named diptericins. The peptide sequence of diptericin A was used to prepare oligonucleotides for screening cDNA libraries and we report in the present paper the isolation of several cDNA clones encoding diptericin. The analysis of the nucleotide sequences indicates that diptericin is synthesized as a prepeptide which is matured in two steps: (a) cleavage of a signal peptide and (b) amidation of the C-terminal residue. Interestingly, the 3' untranslated region of the mRNA contains a consensus sequence TTATTTAT which is also observed in the mRNA of another insect antibacterial peptide (attacin-related sarcotoxin IIA) and in mRNAs encoding proteins related to the inflammatory response in mammals. Our data illustrate that diptericins form a polymorphic family of immune peptides. The transcription of the diptericin genes is rapidly induced in the fat body after inoculation of bacteria, as evidenced by the transcriptional profile.}, keywords = {Animals, Anti-Bacterial Agents, Bacterial Proteins, Base Sequence, Blotting, Diptera, DNA, Endoribonucleases, Enterobacter, Enterobacteriaceae, Gene Expression Regulation, Genes, Genetic, hoffmann, Insect Hormones, Insect Proteins, M3i, messenger, MHC Class II, Northern, reichhart, Ribonuclease H, RNA, Transcription}, pubstate = {published}, tppubtype = {article} } We have previously isolated and characterized a family of novel 8-kDa cationic antibacterial peptides synthesized by larvae of Phormia terranovae (Diptera) in response to various injuries. These molecules have been named diptericins. The peptide sequence of diptericin A was used to prepare oligonucleotides for screening cDNA libraries and we report in the present paper the isolation of several cDNA clones encoding diptericin. The analysis of the nucleotide sequences indicates that diptericin is synthesized as a prepeptide which is matured in two steps: (a) cleavage of a signal peptide and (b) amidation of the C-terminal residue. Interestingly, the 3' untranslated region of the mRNA contains a consensus sequence TTATTTAT which is also observed in the mRNA of another insect antibacterial peptide (attacin-related sarcotoxin IIA) and in mRNAs encoding proteins related to the inflammatory response in mammals. Our data illustrate that diptericins form a polymorphic family of immune peptides. The transcription of the diptericin genes is rapidly induced in the fat body after inoculation of bacteria, as evidenced by the transcriptional profile. |