Publications
2013 |
Fukuyama, Hidehiro; Verdier, Yann; Guan, Yongsheng; Makino-Okamura, Chieko; Shilova, Victoria; Liu, Xi; Maksoud, Elie; Matsubayashi, Jun; Haddad, Iman; Spirohn, Kerstin; Ono, Kenichiro; Hetru, Charles; Rossier, Jean; Ideker, Trey; Boutros, Michael; Vinh, Joëlle; Hoffmann, Jules A Landscape of protein-protein interactions in Drosophila immune deficiency signaling during bacterial challenge Article de journal Proc. Natl. Acad. Sci. U.S.A., 110 (26), p. 10717–10722, 2013, ISSN: 1091-6490. Résumé | Liens | BibTeX | Étiquettes: Amino Acid, Animals, Chromatin Assembly and Disassembly, Escherichia coli, functional proteomics, Genes, Genetically Modified, Histone Acetyltransferases, hoffmann, Host-Pathogen Interactions, Humans, IMD interactome, Insect, M3i, Models, Molecular, Protein Interaction Maps, Sequence Homology, Signal Transduction, small ubiquitin-like modifier @article{fukuyama_landscape_2013, title = {Landscape of protein-protein interactions in Drosophila immune deficiency signaling during bacterial challenge}, author = {Hidehiro Fukuyama and Yann Verdier and Yongsheng Guan and Chieko Makino-Okamura and Victoria Shilova and Xi Liu and Elie Maksoud and Jun Matsubayashi and Iman Haddad and Kerstin Spirohn and Kenichiro Ono and Charles Hetru and Jean Rossier and Trey Ideker and Michael Boutros and Joëlle Vinh and Jules A Hoffmann}, doi = {10.1073/pnas.1304380110}, issn = {1091-6490}, year = {2013}, date = {2013-06-01}, journal = {Proc. Natl. Acad. Sci. U.S.A.}, volume = {110}, number = {26}, pages = {10717--10722}, abstract = {The Drosophila defense against pathogens largely relies on the activation of two signaling pathways: immune deficiency (IMD) and Toll. The IMD pathway is triggered mainly by Gram-negative bacteria, whereas the Toll pathway responds predominantly to Gram-positive bacteria and fungi. The activation of these pathways leads to the rapid induction of numerous NF-κB-induced immune response genes, including antimicrobial peptide genes. The IMD pathway shows significant similarities with the TNF receptor pathway. Recent evidence indicates that the IMD pathway is also activated in response to various noninfectious stimuli (i.e., inflammatory-like reactions). To gain a better understanding of the molecular machinery underlying the pleiotropic functions of this pathway, we first performed a comprehensive proteomics analysis to identify the proteins interacting with the 11 canonical members of the pathway initially identified by genetic studies. We identified 369 interacting proteins (corresponding to 291 genes) in heat-killed Escherichia coli-stimulated Drosophila S2 cells, 92% of which have human orthologs. A comparative analysis of gene ontology from fly or human gene annotation databases points to four significant common categories: (i) the NuA4, nucleosome acetyltransferase of H4, histone acetyltransferase complex, (ii) the switching defective/sucrose nonfermenting-type chromatin remodeling complex, (iii) transcription coactivator activity, and (iv) translation factor activity. Here we demonstrate that sumoylation of the IκB kinase homolog immune response-deficient 5 plays an important role in the induction of antimicrobial peptide genes through a highly conserved sumoylation consensus site during bacterial challenge. Taken together, the proteomics data presented here provide a unique avenue for a comparative functional analysis of proteins involved in innate immune reactions in flies and mammals.}, keywords = {Amino Acid, Animals, Chromatin Assembly and Disassembly, Escherichia coli, functional proteomics, Genes, Genetically Modified, Histone Acetyltransferases, hoffmann, Host-Pathogen Interactions, Humans, IMD interactome, Insect, M3i, Models, Molecular, Protein Interaction Maps, Sequence Homology, Signal Transduction, small ubiquitin-like modifier}, pubstate = {published}, tppubtype = {article} } The Drosophila defense against pathogens largely relies on the activation of two signaling pathways: immune deficiency (IMD) and Toll. The IMD pathway is triggered mainly by Gram-negative bacteria, whereas the Toll pathway responds predominantly to Gram-positive bacteria and fungi. The activation of these pathways leads to the rapid induction of numerous NF-κB-induced immune response genes, including antimicrobial peptide genes. The IMD pathway shows significant similarities with the TNF receptor pathway. Recent evidence indicates that the IMD pathway is also activated in response to various noninfectious stimuli (i.e., inflammatory-like reactions). To gain a better understanding of the molecular machinery underlying the pleiotropic functions of this pathway, we first performed a comprehensive proteomics analysis to identify the proteins interacting with the 11 canonical members of the pathway initially identified by genetic studies. We identified 369 interacting proteins (corresponding to 291 genes) in heat-killed Escherichia coli-stimulated Drosophila S2 cells, 92% of which have human orthologs. A comparative analysis of gene ontology from fly or human gene annotation databases points to four significant common categories: (i) the NuA4, nucleosome acetyltransferase of H4, histone acetyltransferase complex, (ii) the switching defective/sucrose nonfermenting-type chromatin remodeling complex, (iii) transcription coactivator activity, and (iv) translation factor activity. Here we demonstrate that sumoylation of the IκB kinase homolog immune response-deficient 5 plays an important role in the induction of antimicrobial peptide genes through a highly conserved sumoylation consensus site during bacterial challenge. Taken together, the proteomics data presented here provide a unique avenue for a comparative functional analysis of proteins involved in innate immune reactions in flies and mammals. |
Baron, Olga Lucia; van West, Pieter; Industri, Benoit; Ponchet, Michel; Dubreuil, Géraldine; Gourbal, Benjamin; Reichhart, Jean-Marc; Coustau, Christine Parental transfer of the antimicrobial protein LBP/BPI protects Biomphalaria glabrata eggs against oomycete infections Article de journal PLoS Pathog., 9 (12), p. e1003792, 2013, ISSN: 1553-7374. Résumé | Liens | BibTeX | Étiquettes: Acute-Phase Proteins, Animals, Antimicrobial Cationic Peptides, Biomphalaria, Blood Proteins, Carrier Proteins, Cell Membrane, Cell Membrane Permeability, Cloning, Escherichia coli, Female, Immunity, infection, M3i, Maternally-Acquired, Membrane Glycoproteins, Microbial Sensitivity Tests, Molecular, Oomycetes, Recombinant Proteins, reichhart, Zygote @article{baron_parental_2013, title = {Parental transfer of the antimicrobial protein LBP/BPI protects Biomphalaria glabrata eggs against oomycete infections}, author = {Olga Lucia Baron and Pieter van West and Benoit Industri and Michel Ponchet and Géraldine Dubreuil and Benjamin Gourbal and Jean-Marc Reichhart and Christine Coustau}, doi = {10.1371/journal.ppat.1003792}, issn = {1553-7374}, year = {2013}, date = {2013-01-01}, journal = {PLoS Pathog.}, volume = {9}, number = {12}, pages = {e1003792}, abstract = {Vertebrate females transfer antibodies via the placenta, colostrum and milk or via the egg yolk to protect their immunologically immature offspring against pathogens. This evolutionarily important transfer of immunity is poorly documented in invertebrates and basic questions remain regarding the nature and extent of parental protection of offspring. In this study, we show that a lipopolysaccharide binding protein/bactericidal permeability increasing protein family member from the invertebrate Biomphalaria glabrata (BgLBP/BPI1) is massively loaded into the eggs of this freshwater snail. Native and recombinant proteins displayed conserved LPS-binding, antibacterial and membrane permeabilizing activities. A broad screening of various pathogens revealed a previously unknown biocidal activity of the protein against pathogenic water molds (oomycetes), which is conserved in human BPI. RNAi-dependent silencing of LBP/BPI in the parent snails resulted in a significant reduction of reproductive success and extensive death of eggs through oomycete infections. This work provides the first functional evidence that a LBP/BPI is involved in the parental immune protection of invertebrate offspring and reveals a novel and conserved biocidal activity for LBP/BPI family members.}, keywords = {Acute-Phase Proteins, Animals, Antimicrobial Cationic Peptides, Biomphalaria, Blood Proteins, Carrier Proteins, Cell Membrane, Cell Membrane Permeability, Cloning, Escherichia coli, Female, Immunity, infection, M3i, Maternally-Acquired, Membrane Glycoproteins, Microbial Sensitivity Tests, Molecular, Oomycetes, Recombinant Proteins, reichhart, Zygote}, pubstate = {published}, tppubtype = {article} } Vertebrate females transfer antibodies via the placenta, colostrum and milk or via the egg yolk to protect their immunologically immature offspring against pathogens. This evolutionarily important transfer of immunity is poorly documented in invertebrates and basic questions remain regarding the nature and extent of parental protection of offspring. In this study, we show that a lipopolysaccharide binding protein/bactericidal permeability increasing protein family member from the invertebrate Biomphalaria glabrata (BgLBP/BPI1) is massively loaded into the eggs of this freshwater snail. Native and recombinant proteins displayed conserved LPS-binding, antibacterial and membrane permeabilizing activities. A broad screening of various pathogens revealed a previously unknown biocidal activity of the protein against pathogenic water molds (oomycetes), which is conserved in human BPI. RNAi-dependent silencing of LBP/BPI in the parent snails resulted in a significant reduction of reproductive success and extensive death of eggs through oomycete infections. This work provides the first functional evidence that a LBP/BPI is involved in the parental immune protection of invertebrate offspring and reveals a novel and conserved biocidal activity for LBP/BPI family members. |
2011 |
Aoun, Richard Bou; Hetru, Charles; Troxler, Laurent; Doucet, Daniel; Ferrandon, Dominique; Matt, Nicolas Analysis of thioester-containing proteins during the innate immune response of Drosophila melanogaster Article de journal J Innate Immun, 3 (1), p. 52–64, 2011, ISSN: 1662-8128. Résumé | Liens | BibTeX | Étiquettes: Animals, bioinformatic, DNA, Evolution, ferrandon, Gene Expression Regulation, Hemocytes, Immunity, In Situ Hybridization, Innate, M3i, matt, Molecular, Mutation, Phylogeny, Sequence Analysis @article{bou_aoun_analysis_2011, title = {Analysis of thioester-containing proteins during the innate immune response of Drosophila melanogaster}, author = {Richard Bou Aoun and Charles Hetru and Laurent Troxler and Daniel Doucet and Dominique Ferrandon and Nicolas Matt}, doi = {10.1159/000321554}, issn = {1662-8128}, year = {2011}, date = {2011-01-01}, journal = {J Innate Immun}, volume = {3}, number = {1}, pages = {52--64}, abstract = {Thioester-containing proteins (TEPs) are conserved proteins among insects that are thought to be involved in innate immunity. In Drosophila, the Tep family is composed of 6 genes named Tep1-Tep6. In this study, we investigated the phylogeny, expression pattern and roles of these genes in the host defense of Drosophila. Protostomian Tep genes are clustered in 3 distinct branches, 1 of which is specific to mosquitoes. Most D. melanogaster Tep genes are expressed in hemocytes, can be induced in the fat body, and are expressed in specific regions of the hypodermis. This expression pattern is consistent with a role in innate immunity. However, we find that TEP1, TEP2, and TEP4 are not strictly required in the body cavity to fight several bacterial and fungal infections. One possibility is that Drosophila TEPs act redundantly or that their absence can be compensated by other components of the immune response. TEPs may thus provide a subtle selective advantage during evolution. Alternatively, they may be required in host defense against specific as yet unidentified natural pathogens of Drosophila.}, keywords = {Animals, bioinformatic, DNA, Evolution, ferrandon, Gene Expression Regulation, Hemocytes, Immunity, In Situ Hybridization, Innate, M3i, matt, Molecular, Mutation, Phylogeny, Sequence Analysis}, pubstate = {published}, tppubtype = {article} } Thioester-containing proteins (TEPs) are conserved proteins among insects that are thought to be involved in innate immunity. In Drosophila, the Tep family is composed of 6 genes named Tep1-Tep6. In this study, we investigated the phylogeny, expression pattern and roles of these genes in the host defense of Drosophila. Protostomian Tep genes are clustered in 3 distinct branches, 1 of which is specific to mosquitoes. Most D. melanogaster Tep genes are expressed in hemocytes, can be induced in the fat body, and are expressed in specific regions of the hypodermis. This expression pattern is consistent with a role in innate immunity. However, we find that TEP1, TEP2, and TEP4 are not strictly required in the body cavity to fight several bacterial and fungal infections. One possibility is that Drosophila TEPs act redundantly or that their absence can be compensated by other components of the immune response. TEPs may thus provide a subtle selective advantage during evolution. Alternatively, they may be required in host defense against specific as yet unidentified natural pathogens of Drosophila. |
2009 |
Kemp, Cordula; Imler, Jean-Luc Antiviral immunity in drosophila Article de journal Current Opinion in Immunology, 21 (1), p. 3–9, 2009, ISSN: 1879-0372. Résumé | Liens | BibTeX | Étiquettes: Animals, Argonaute Proteins, Caspases, DEAD-box RNA Helicases, Evolution, Gene Expression Regulation, Host-Pathogen Interactions, imler, M3i, Membrane Proteins, Molecular, Nuclear Proteins, Ribonuclease III, RNA, RNA Helicases, RNA Interference, RNA Virus Infections, RNA Viruses, RNA-Induced Silencing Complex, Viral, Virulence @article{kemp_antiviral_2009, title = {Antiviral immunity in drosophila}, author = {Cordula Kemp and Jean-Luc Imler}, doi = {10.1016/j.coi.2009.01.007}, issn = {1879-0372}, year = {2009}, date = {2009-02-01}, journal = {Current Opinion in Immunology}, volume = {21}, number = {1}, pages = {3--9}, abstract = {Genetic analysis of the drosophila antiviral response indicates that RNA interference plays a major role. This contrasts with the situation in mammals, where interferon-induced responses mediate innate antiviral host-defense. An inducible response also contributes to antiviral immunity in drosophila, and similarities in the sensing and signaling of viral infection are becoming apparent between drosophila and mammals. In particular, DExD/H box helicases appear to play a crucial role in the cytosolic detection of viral RNAs in flies and mammals.}, keywords = {Animals, Argonaute Proteins, Caspases, DEAD-box RNA Helicases, Evolution, Gene Expression Regulation, Host-Pathogen Interactions, imler, M3i, Membrane Proteins, Molecular, Nuclear Proteins, Ribonuclease III, RNA, RNA Helicases, RNA Interference, RNA Virus Infections, RNA Viruses, RNA-Induced Silencing Complex, Viral, Virulence}, pubstate = {published}, tppubtype = {article} } Genetic analysis of the drosophila antiviral response indicates that RNA interference plays a major role. This contrasts with the situation in mammals, where interferon-induced responses mediate innate antiviral host-defense. An inducible response also contributes to antiviral immunity in drosophila, and similarities in the sensing and signaling of viral infection are becoming apparent between drosophila and mammals. In particular, DExD/H box helicases appear to play a crucial role in the cytosolic detection of viral RNAs in flies and mammals. |
Garrett, Matthew; Fullaondo, Ane; Troxler, Laurent; Micklem, Gos; Gubb, David Identification and analysis of serpin-family genes by homology and synteny across the 12 sequenced Drosophilid genomes Article de journal BMC Genomics, 10 , p. 489, 2009, ISSN: 1471-2164. Résumé | Liens | BibTeX | Étiquettes: Animals, bioinformatic, Comparative Genomic Hybridization, Conserved Sequence, DNA, Drosophilidae, Evolution, Genome, Insect, Molecular, Multigene Family, Sequence Alignment, Sequence Analysis, Serpins, Synteny @article{garrett_identification_2009, title = {Identification and analysis of serpin-family genes by homology and synteny across the 12 sequenced Drosophilid genomes}, author = {Matthew Garrett and Ane Fullaondo and Laurent Troxler and Gos Micklem and David Gubb}, doi = {10.1186/1471-2164-10-489}, issn = {1471-2164}, year = {2009}, date = {2009-01-01}, journal = {BMC Genomics}, volume = {10}, pages = {489}, abstract = {BACKGROUND: The Drosophila melanogaster genome contains 29 serpin genes, 12 as single transcripts and 17 within 6 gene clusters. Many of these serpins have a conserved "hinge" motif characteristic of active proteinase inhibitors. However, a substantial proportion (42%) lacks this motif and represents non-inhibitory serpin-fold proteins of unknown function. Currently, it is not known whether orthologous, inhibitory serpin genes retain the same target proteinase specificity within the Drosophilid lineage, nor whether they give rise to non-inhibitory serpin-fold proteins or other, more diverged, proteins. RESULTS: We collated 188 orthologues to the D. melanogaster serpins from the other 11 Drosophilid genomes and used synteny to find further family members, raising the total to 226, or 71% of the number of orthologues expected assuming complete conservation across all 12 Drosophilid species. In general the sequence constraints on the serpin-fold itself are loose. The critical Reactive Centre Loop (RCL) sequence, including the target proteinase cleavage site, is strongly conserved in inhibitory serpins, although there are 3 exceptional sets of orthologues in which the evolutionary constraints are looser. Conversely, the RCL of non-inhibitory serpin orthologues is less conserved, with 3 exceptions that presumably bind to conserved partner molecules. We derive a consensus hinge motif, for Drosophilid inhibitory serpins, which differs somewhat from that of the vertebrate consensus. Three gene clusters appear to have originated in the melanogaster subgroup, Spn28D, Spn77B and Spn88E, each containing one inhibitory serpin orthologue that is present in all Drosophilids. In addition, the Spn100A transcript appears to represent a novel serpin-derived fold. CONCLUSION: In general, inhibitory serpins rarely change their range of proteinase targets, except by a duplication/divergence mechanism. Non-inhibitory serpins appear to derive from inhibitory serpins, but not the reverse. The conservation of different family members varied widely across the 12 sequenced Drosophilid genomes. An approach considering synteny as well as homology was important to find the largest set of orthologues.}, keywords = {Animals, bioinformatic, Comparative Genomic Hybridization, Conserved Sequence, DNA, Drosophilidae, Evolution, Genome, Insect, Molecular, Multigene Family, Sequence Alignment, Sequence Analysis, Serpins, Synteny}, pubstate = {published}, tppubtype = {article} } BACKGROUND: The Drosophila melanogaster genome contains 29 serpin genes, 12 as single transcripts and 17 within 6 gene clusters. Many of these serpins have a conserved "hinge" motif characteristic of active proteinase inhibitors. However, a substantial proportion (42%) lacks this motif and represents non-inhibitory serpin-fold proteins of unknown function. Currently, it is not known whether orthologous, inhibitory serpin genes retain the same target proteinase specificity within the Drosophilid lineage, nor whether they give rise to non-inhibitory serpin-fold proteins or other, more diverged, proteins. RESULTS: We collated 188 orthologues to the D. melanogaster serpins from the other 11 Drosophilid genomes and used synteny to find further family members, raising the total to 226, or 71% of the number of orthologues expected assuming complete conservation across all 12 Drosophilid species. In general the sequence constraints on the serpin-fold itself are loose. The critical Reactive Centre Loop (RCL) sequence, including the target proteinase cleavage site, is strongly conserved in inhibitory serpins, although there are 3 exceptional sets of orthologues in which the evolutionary constraints are looser. Conversely, the RCL of non-inhibitory serpin orthologues is less conserved, with 3 exceptions that presumably bind to conserved partner molecules. We derive a consensus hinge motif, for Drosophilid inhibitory serpins, which differs somewhat from that of the vertebrate consensus. Three gene clusters appear to have originated in the melanogaster subgroup, Spn28D, Spn77B and Spn88E, each containing one inhibitory serpin orthologue that is present in all Drosophilids. In addition, the Spn100A transcript appears to represent a novel serpin-derived fold. CONCLUSION: In general, inhibitory serpins rarely change their range of proteinase targets, except by a duplication/divergence mechanism. Non-inhibitory serpins appear to derive from inhibitory serpins, but not the reverse. The conservation of different family members varied widely across the 12 sequenced Drosophilid genomes. An approach considering synteny as well as homology was important to find the largest set of orthologues. |
2008 |
Geary, C; Baudrey, S; Jaeger, L Comprehensive features of natural and in vitro selected GNRA tetraloop-binding receptors Article de journal Nucleic Acids Res, 36 (4), p. 1138-52, 2008, (1362-4962 (Electronic) Journal Article Research Support, N.I.H., Extramural Research Support, U.S. Gov't, Non-P.H.S.). Résumé | BibTeX | Étiquettes: Acid, Adenine/chemistry, Analysis, Base, Conformation, Data, dimerization, directed, Evolution, KROL, Models, Molecular, Nucleic, RNA, RNA/*chemistry/classification, Sequence, Thermodynamics @article{, title = {Comprehensive features of natural and in vitro selected GNRA tetraloop-binding receptors}, author = { C. Geary and S. Baudrey and L. Jaeger}, year = {2008}, date = {2008-01-01}, journal = {Nucleic Acids Res}, volume = {36}, number = {4}, pages = {1138-52}, abstract = {Specific recognitions of GNRA tetraloops by small helical receptors are among the most widespread long-range packing interactions in large ribozymes. However, in contrast to GYRA and GAAA tetraloops, very few GNRA/receptor interactions have yet been identified to involve GGAA tetraloops in nature. A novel in vitro selection scheme based on a rigid self-assembling tectoRNA scaffold designed for isolation of intermolecular interactions with A-minor motifs has yielded new GGAA tetraloop-binding receptors with affinity in the nanomolar range. One of the selected receptors is a novel 12 nt RNA motif, (CCUGUG. AUCUGG), that recognizes GGAA tetraloop hairpin with a remarkable specificity and affinity. Its physical and chemical characteristics are comparable to those of the well-studied '11nt' GAAA tetraloop receptor motif. A second less specific motif (CCCAGCCC. GAUAGGG) binds GGRA tetraloops and appears to be related to group IC3 tetraloop receptors. Mutational, thermodynamic and comparative structural analysis suggests that natural and in vitro selected GNRA receptors can essentially be grouped in two major classes of GNRA binders. New insights about the evolution, recognition and structural modularity of GNRA and A-minor RNA-RNA interactions are proposed.}, note = {1362-4962 (Electronic) Journal Article Research Support, N.I.H., Extramural Research Support, U.S. Gov't, Non-P.H.S.}, keywords = {Acid, Adenine/chemistry, Analysis, Base, Conformation, Data, dimerization, directed, Evolution, KROL, Models, Molecular, Nucleic, RNA, RNA/*chemistry/classification, Sequence, Thermodynamics}, pubstate = {published}, tppubtype = {article} } Specific recognitions of GNRA tetraloops by small helical receptors are among the most widespread long-range packing interactions in large ribozymes. However, in contrast to GYRA and GAAA tetraloops, very few GNRA/receptor interactions have yet been identified to involve GGAA tetraloops in nature. A novel in vitro selection scheme based on a rigid self-assembling tectoRNA scaffold designed for isolation of intermolecular interactions with A-minor motifs has yielded new GGAA tetraloop-binding receptors with affinity in the nanomolar range. One of the selected receptors is a novel 12 nt RNA motif, (CCUGUG. AUCUGG), that recognizes GGAA tetraloop hairpin with a remarkable specificity and affinity. Its physical and chemical characteristics are comparable to those of the well-studied '11nt' GAAA tetraloop receptor motif. A second less specific motif (CCCAGCCC. GAUAGGG) binds GGRA tetraloops and appears to be related to group IC3 tetraloop receptors. Mutational, thermodynamic and comparative structural analysis suggests that natural and in vitro selected GNRA receptors can essentially be grouped in two major classes of GNRA binders. New insights about the evolution, recognition and structural modularity of GNRA and A-minor RNA-RNA interactions are proposed. |
2007 |
Mandin, P; Repoila, F; Vergassola, M; Geissmann, T; Cossart, P Identification of new noncoding RNAs in Listeria monocytogenes and prediction of mRNA targets Article de journal Nucleic Acids Res, 35 (3), p. 962-74, 2007, (1362-4962 (Electronic) Journal Article Research Support, Non-U.S. Gov't). Résumé | BibTeX | Étiquettes: 5', Assay, Bacterial, Base, Biology, Computational, Data, DNA, Electrophoretic, Flanking, Genes, Genomics, Intergenic/chemistry, Listeria, Messenger/chemistry/*metabolism, Mobility, Molecular, monocytogenes/*genetics/metabolism, Region, RNA, ROMBY, Sequence, Shift, Untranslated/analysis/*genetics/metabolism @article{, title = {Identification of new noncoding RNAs in Listeria monocytogenes and prediction of mRNA targets}, author = { P. Mandin and F. Repoila and M. Vergassola and T. Geissmann and P. Cossart}, year = {2007}, date = {2007-01-01}, journal = {Nucleic Acids Res}, volume = {35}, number = {3}, pages = {962-74}, abstract = {To identify noncoding RNAs (ncRNAs) in the pathogenic bacterium Listeria monocytogenes, we analyzed the intergenic regions (IGRs) of strain EGD-e by in silico-based approaches. Among the twelve ncRNAs found, nine are novel and specific to the Listeria genus, and two of these ncRNAs are expressed in a growth-dependent manner. Three of the ncRNAs are transcribed in opposite direction to overlapping open reading frames (ORFs), suggesting that they act as antisense on the corresponding mRNAs. The other ncRNA genes appear as single transcription units. One of them displays five repeats of 29 nucleotides. Five of these new ncRNAs are absent from the non-pathogenic species L. innocua, raising the possibility that they might be involved in virulence. To predict mRNA targets of the ncRNAs, we developed a computational method based on thermodynamic pairing energies and known ncRNA-mRNA hybrids. Three ncRNAs, including one of the putative antisense ncRNAs, were predicted to have more than one mRNA targets. Several of them were shown to bind efficiently to the ncRNAs suggesting that our in silico approach could be used as a general tool to search for mRNA targets of ncRNAs.}, note = {1362-4962 (Electronic) Journal Article Research Support, Non-U.S. Gov't}, keywords = {5', Assay, Bacterial, Base, Biology, Computational, Data, DNA, Electrophoretic, Flanking, Genes, Genomics, Intergenic/chemistry, Listeria, Messenger/chemistry/*metabolism, Mobility, Molecular, monocytogenes/*genetics/metabolism, Region, RNA, ROMBY, Sequence, Shift, Untranslated/analysis/*genetics/metabolism}, pubstate = {published}, tppubtype = {article} } To identify noncoding RNAs (ncRNAs) in the pathogenic bacterium Listeria monocytogenes, we analyzed the intergenic regions (IGRs) of strain EGD-e by in silico-based approaches. Among the twelve ncRNAs found, nine are novel and specific to the Listeria genus, and two of these ncRNAs are expressed in a growth-dependent manner. Three of the ncRNAs are transcribed in opposite direction to overlapping open reading frames (ORFs), suggesting that they act as antisense on the corresponding mRNAs. The other ncRNA genes appear as single transcription units. One of them displays five repeats of 29 nucleotides. Five of these new ncRNAs are absent from the non-pathogenic species L. innocua, raising the possibility that they might be involved in virulence. To predict mRNA targets of the ncRNAs, we developed a computational method based on thermodynamic pairing energies and known ncRNA-mRNA hybrids. Three ncRNAs, including one of the putative antisense ncRNAs, were predicted to have more than one mRNA targets. Several of them were shown to bind efficiently to the ncRNAs suggesting that our in silico approach could be used as a general tool to search for mRNA targets of ncRNAs. |
Croker, Ben; Crozat, Karine; Berger, Michael; Xia, Yu; Sovath, Sosathya; Schaffer, Lana; Eleftherianos, Ioannis; Imler, Jean-Luc; Beutler, Bruce ATP-sensitive potassium channels mediate survival during infection in mammals and insects Article de journal Nature Genetics, 39 (12), p. 1453–1460, 2007, ISSN: 1546-1718. Résumé | Liens | BibTeX | Étiquettes: Animals, ATP-Binding Cassette Transporters, Cloning, Coronary Vessels, Crosses, Ethylnitrosourea, Genetic, Homozygote, imler, infection, Inwardly Rectifying, KATP Channels, Lipopolysaccharides, M3i, Mice, Molecular, Mutagenesis, Potassium Channels, Sulfonylurea Receptors @article{croker_atp-sensitive_2007, title = {ATP-sensitive potassium channels mediate survival during infection in mammals and insects}, author = {Ben Croker and Karine Crozat and Michael Berger and Yu Xia and Sosathya Sovath and Lana Schaffer and Ioannis Eleftherianos and Jean-Luc Imler and Bruce Beutler}, doi = {10.1038/ng.2007.25}, issn = {1546-1718}, year = {2007}, date = {2007-01-01}, journal = {Nature Genetics}, volume = {39}, number = {12}, pages = {1453--1460}, abstract = {Specific homeostatic mechanisms confer stability in innate immune responses, preventing injury or death from infection. Here we identify, from a screen of N-ethyl-N-nitrosourea-mutagenized mice, a mutation causing both profound susceptibility to infection by mouse cytomegalovirus and approximately 20,000-fold sensitization to lipopolysaccharide (LPS), poly(I.C) and immunostimulatory (CpG) DNA. The LPS hypersensitivity phenotype is not suppressed by mutations in Myd88, Trif, Tnf, Tnfrsf1a, Ifnb, Ifng or Stat1, genes contributing to LPS responses, and results from an abnormality extrinsic to hematopoietic cells. The phenotype is due to a null allele of Kcnj8, encoding Kir6.1, a protein that combines with SUR2 to form an ATP-sensitive potassium channel (K(ATP)) expressed in coronary artery smooth muscle and endothelial cells. In Drosophila melanogaster, suppression of dSUR by RNA interference similarly causes hypersensitivity to infection by flock house virus. Thus, K(ATP) evolved to serve a homeostatic function during infection, and in mammals it prevents coronary artery vasoconstriction induced by cytokines dependent on TLR and/or MDA5 immunoreceptors.}, keywords = {Animals, ATP-Binding Cassette Transporters, Cloning, Coronary Vessels, Crosses, Ethylnitrosourea, Genetic, Homozygote, imler, infection, Inwardly Rectifying, KATP Channels, Lipopolysaccharides, M3i, Mice, Molecular, Mutagenesis, Potassium Channels, Sulfonylurea Receptors}, pubstate = {published}, tppubtype = {article} } Specific homeostatic mechanisms confer stability in innate immune responses, preventing injury or death from infection. Here we identify, from a screen of N-ethyl-N-nitrosourea-mutagenized mice, a mutation causing both profound susceptibility to infection by mouse cytomegalovirus and approximately 20,000-fold sensitization to lipopolysaccharide (LPS), poly(I.C) and immunostimulatory (CpG) DNA. The LPS hypersensitivity phenotype is not suppressed by mutations in Myd88, Trif, Tnf, Tnfrsf1a, Ifnb, Ifng or Stat1, genes contributing to LPS responses, and results from an abnormality extrinsic to hematopoietic cells. The phenotype is due to a null allele of Kcnj8, encoding Kir6.1, a protein that combines with SUR2 to form an ATP-sensitive potassium channel (K(ATP)) expressed in coronary artery smooth muscle and endothelial cells. In Drosophila melanogaster, suppression of dSUR by RNA interference similarly causes hypersensitivity to infection by flock house virus. Thus, K(ATP) evolved to serve a homeostatic function during infection, and in mammals it prevents coronary artery vasoconstriction induced by cytokines dependent on TLR and/or MDA5 immunoreceptors. |
2004 |
Burnouf, D Y; Olieric, V; Wagner, J; Fujii, S; Reinbolt, J; Fuchs, R P; Dumas, P Structural and biochemical analysis of sliding clamp/ligand interactions suggest a competition between replicative and translesion DNA polymerases Article de journal J Mol Biol, 335 (5), p. 1187-97, 2004, (0022-2836 Journal Article). Résumé | BibTeX | Étiquettes: *Binding, Antigen/metabolism, Bacterial/genetics, beta/*chemistry/genetics/*metabolism, Binding, Cell, coli/*enzymology, Competitive, Crystallization, DNA, DUMAS, Escherichia, Fragments/*metabolism, I/metabolism, III/metabolism, Kinetics, ligands, Models, Molecular, Nuclear, Peptide, Polymerase, Proliferating, Protein, Proteins/chemistry/metabolism, Recombinant, Replication/*genetics, Subunits @article{, title = {Structural and biochemical analysis of sliding clamp/ligand interactions suggest a competition between replicative and translesion DNA polymerases}, author = { D. Y. Burnouf and V. Olieric and J. Wagner and S. Fujii and J. Reinbolt and R. P. Fuchs and P. Dumas}, year = {2004}, date = {2004-01-01}, journal = {J Mol Biol}, volume = {335}, number = {5}, pages = {1187-97}, abstract = {Most DNA polymerases interact with their cognate processive replication factor through a small peptide, this interaction being absolutely required for their function in vivo. We have solved the crystal structure of a complex between the beta sliding clamp of Escherichia coli and the 16 residue C-terminal peptide of Pol IV (P16). The seven C-terminal residues bind to a pocket located at the surface of one beta monomer. This region was previously identified as the binding site of another beta clamp binding protein, the delta subunit of the gamma complex. We show that peptide P16 competitively prevents beta-clamp-mediated stimulation of both Pol IV and alpha subunit DNA polymerase activities, suggesting that the site of interaction of the alpha subunit with beta is identical with, or overlaps that of Pol IV. This common binding site for delta, Pol IV and alpha subunit is shown to be formed by residues that are highly conserved among many bacterial beta homologs, thus defining an evolutionarily conserved hydrophobic crevice for sliding clamp ligands and a new target for antibiotic drug design.}, note = {0022-2836 Journal Article}, keywords = {*Binding, Antigen/metabolism, Bacterial/genetics, beta/*chemistry/genetics/*metabolism, Binding, Cell, coli/*enzymology, Competitive, Crystallization, DNA, DUMAS, Escherichia, Fragments/*metabolism, I/metabolism, III/metabolism, Kinetics, ligands, Models, Molecular, Nuclear, Peptide, Polymerase, Proliferating, Protein, Proteins/chemistry/metabolism, Recombinant, Replication/*genetics, Subunits}, pubstate = {published}, tppubtype = {article} } Most DNA polymerases interact with their cognate processive replication factor through a small peptide, this interaction being absolutely required for their function in vivo. We have solved the crystal structure of a complex between the beta sliding clamp of Escherichia coli and the 16 residue C-terminal peptide of Pol IV (P16). The seven C-terminal residues bind to a pocket located at the surface of one beta monomer. This region was previously identified as the binding site of another beta clamp binding protein, the delta subunit of the gamma complex. We show that peptide P16 competitively prevents beta-clamp-mediated stimulation of both Pol IV and alpha subunit DNA polymerase activities, suggesting that the site of interaction of the alpha subunit with beta is identical with, or overlaps that of Pol IV. This common binding site for delta, Pol IV and alpha subunit is shown to be formed by residues that are highly conserved among many bacterial beta homologs, thus defining an evolutionarily conserved hydrophobic crevice for sliding clamp ligands and a new target for antibiotic drug design. |
Delarue, M; Dumas, P On the use of low-frequency normal modes to enforce collective movements in refining macromolecular structural models Article de journal Proc Natl Acad Sci U S A, 101 (18), p. 6957-62, 2004, (0027-8424 Journal Article). Résumé | BibTeX | Étiquettes: *Models, Carrier, coli, Computer, Diffraction, DUMAS, Escherichia, Gov't, Molecular, Non-U.S., Proteins/*chemistry, Proteins/chemistry, Simulation, Support, X-Ray @article{, title = {On the use of low-frequency normal modes to enforce collective movements in refining macromolecular structural models}, author = { M. Delarue and P. Dumas}, year = {2004}, date = {2004-01-01}, journal = {Proc Natl Acad Sci U S A}, volume = {101}, number = {18}, pages = {6957-62}, abstract = {As more and more structures of macromolecular complexes get solved in different conditions, it has become apparent that flexibility is an inherent part of their biological function. Normal mode analysis using simplified models of proteins such as the elastic network model has proved very effective in showing that many of the structural transitions derived from a survey of the Protein Data Bank can be explained by just a few of the lowest-frequency normal modes. In this work, normal modes are used to carry out medium- or low-resolution structural refinement, enforcing collective and large-amplitude movements that are beyond the reach of existing methods. Refinement is carried out in reciprocal space with respect to the normal mode amplitudes, by using standard conjugate-gradient minimization. Several tests on synthetic diffraction data whose mode concentration follows the one of real movements observed in the Protein Data Bank have shown that the radius of convergence is larger than the one of rigid-body refinement. Tests with experimental diffraction data for the same protein in different environments also led to refined structural models showing drastic reduction of the rms deviation with the target model. Because the structural transition is described by very few parameters, over-fitting of real experimental data is easily detected by using a cross-validation test. The method has also been applied to the refinement of atomic models into molecular envelopes and could readily be used to fit large macromolecular complex rearrangements into cryo-electron microscopy-reconstructed images as well as small-angle x-ray scattering-derived envelopes.}, note = {0027-8424 Journal Article}, keywords = {*Models, Carrier, coli, Computer, Diffraction, DUMAS, Escherichia, Gov't, Molecular, Non-U.S., Proteins/*chemistry, Proteins/chemistry, Simulation, Support, X-Ray}, pubstate = {published}, tppubtype = {article} } As more and more structures of macromolecular complexes get solved in different conditions, it has become apparent that flexibility is an inherent part of their biological function. Normal mode analysis using simplified models of proteins such as the elastic network model has proved very effective in showing that many of the structural transitions derived from a survey of the Protein Data Bank can be explained by just a few of the lowest-frequency normal modes. In this work, normal modes are used to carry out medium- or low-resolution structural refinement, enforcing collective and large-amplitude movements that are beyond the reach of existing methods. Refinement is carried out in reciprocal space with respect to the normal mode amplitudes, by using standard conjugate-gradient minimization. Several tests on synthetic diffraction data whose mode concentration follows the one of real movements observed in the Protein Data Bank have shown that the radius of convergence is larger than the one of rigid-body refinement. Tests with experimental diffraction data for the same protein in different environments also led to refined structural models showing drastic reduction of the rms deviation with the target model. Because the structural transition is described by very few parameters, over-fitting of real experimental data is easily detected by using a cross-validation test. The method has also been applied to the refinement of atomic models into molecular envelopes and could readily be used to fit large macromolecular complex rearrangements into cryo-electron microscopy-reconstructed images as well as small-angle x-ray scattering-derived envelopes. |
Martineau, Y; Bec, Le C; Monbrun, L; Allo, V; Chiu, I M; Danos, O; Moine, H; Prats, H; Prats, A C Internal ribosome entry site structural motifs conserved among mammalian fibroblast growth factor 1 alternatively spliced mRNAs Article de journal Mol Cell Biol, 24 (17), p. 7622-35, 2004, (0270-7306 Journal Article). Résumé | BibTeX | Étiquettes: (Genetics), *5', *Alternative, *Nucleic, *Promoter, 1/*genetics, Acid, Alignment, Animals, Base, Cell, Conformation, Data, EHRESMANN, Factor, Fibroblast, Gene, Genes, Genetic, Gov't, Growth, Human, Line, Messenger/chemistry/*genetics/metabolism, Mice, Molecular, Muscle, Mutagenesis, Non-U.S., Regions, Ribosomes/*metabolism, RNA, Sequence, Site-Directed, Skeletal/cytology/physiology, Splicing, Structural/genetics, Support, Techniques, Transfer, Untranslated, Vectors @article{, title = {Internal ribosome entry site structural motifs conserved among mammalian fibroblast growth factor 1 alternatively spliced mRNAs}, author = { Y. Martineau and C. Le Bec and L. Monbrun and V. Allo and I. M. Chiu and O. Danos and H. Moine and H. Prats and A. C. Prats}, year = {2004}, date = {2004-01-01}, journal = {Mol Cell Biol}, volume = {24}, number = {17}, pages = {7622-35}, abstract = {Fibroblast growth factor 1 (FGF-1) is a powerful angiogenic factor whose gene structure contains four promoters, giving rise to a process of alternative splicing resulting in four mRNAs with alternative 5' untranslated regions (5' UTRs). Here we have identified, by using double luciferase bicistronic vectors, the presence of internal ribosome entry sites (IRESs) in the human FGF-1 5' UTRs, particularly in leaders A and C, with distinct activities in mammalian cells. DNA electrotransfer in mouse muscle revealed that the IRES present in the FGF-1 leader A has a high activity in vivo. We have developed a new regulatable TET OFF bicistronic system, which allowed us to rule out the possibility of any cryptic promoter in the FGF-1 leaders. FGF-1 IRESs A and C, which were mapped in fragments of 118 and 103 nucleotides, respectively, are flexible in regard to the position of the initiation codon, making them interesting from a biotechnological point of view. Furthermore, we show that FGF-1 IRESs A of murine and human origins show similar IRES activity profiles. Enzymatic and chemical probing of the FGF-1 IRES A RNA revealed a structural domain conserved among mammals at both the nucleotide sequence and RNA structure levels. The functional role of this structural motif has been demonstrated by point mutagenesis, including compensatory mutations. These data favor an important role of IRESs in the control of FGF-1 expression and provide a new IRES structural motif that could help IRES prediction in 5' UTR databases.}, note = {0270-7306 Journal Article}, keywords = {(Genetics), *5', *Alternative, *Nucleic, *Promoter, 1/*genetics, Acid, Alignment, Animals, Base, Cell, Conformation, Data, EHRESMANN, Factor, Fibroblast, Gene, Genes, Genetic, Gov't, Growth, Human, Line, Messenger/chemistry/*genetics/metabolism, Mice, Molecular, Muscle, Mutagenesis, Non-U.S., Regions, Ribosomes/*metabolism, RNA, Sequence, Site-Directed, Skeletal/cytology/physiology, Splicing, Structural/genetics, Support, Techniques, Transfer, Untranslated, Vectors}, pubstate = {published}, tppubtype = {article} } Fibroblast growth factor 1 (FGF-1) is a powerful angiogenic factor whose gene structure contains four promoters, giving rise to a process of alternative splicing resulting in four mRNAs with alternative 5' untranslated regions (5' UTRs). Here we have identified, by using double luciferase bicistronic vectors, the presence of internal ribosome entry sites (IRESs) in the human FGF-1 5' UTRs, particularly in leaders A and C, with distinct activities in mammalian cells. DNA electrotransfer in mouse muscle revealed that the IRES present in the FGF-1 leader A has a high activity in vivo. We have developed a new regulatable TET OFF bicistronic system, which allowed us to rule out the possibility of any cryptic promoter in the FGF-1 leaders. FGF-1 IRESs A and C, which were mapped in fragments of 118 and 103 nucleotides, respectively, are flexible in regard to the position of the initiation codon, making them interesting from a biotechnological point of view. Furthermore, we show that FGF-1 IRESs A of murine and human origins show similar IRES activity profiles. Enzymatic and chemical probing of the FGF-1 IRES A RNA revealed a structural domain conserved among mammals at both the nucleotide sequence and RNA structure levels. The functional role of this structural motif has been demonstrated by point mutagenesis, including compensatory mutations. These data favor an important role of IRESs in the control of FGF-1 expression and provide a new IRES structural motif that could help IRES prediction in 5' UTR databases. |
Blandin, Stephanie A; Shiao, Shin-Hong; Moita, Luis F; Janse, Chris J; Waters, Andrew P; Kafatos, Fotis C; Levashina, Elena A Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae Article de journal Cell, 116 (5), p. 661–670, 2004, ISSN: 0092-8674. Résumé | BibTeX | Étiquettes: Animals, Anopheles, blandin, Female, Genetic, Humans, Insect Proteins, Insect Vectors, M3i, Malaria, Models, Molecular, Plasmodium berghei, Polymorphism, Protein Structure, RNA, Sequence Alignment, Tertiary @article{blandin_complement-like_2004, title = {Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae}, author = {Stephanie A Blandin and Shin-Hong Shiao and Luis F Moita and Chris J Janse and Andrew P Waters and Fotis C Kafatos and Elena A Levashina}, issn = {0092-8674}, year = {2004}, date = {2004-01-01}, journal = {Cell}, volume = {116}, number = {5}, pages = {661--670}, abstract = {Anopheles mosquitoes are major vectors of human malaria in Africa. Large variation exists in the ability of mosquitoes to serve as vectors and to transmit malaria parasites, but the molecular mechanisms that determine vectorial capacity remain poorly understood. We report that the hemocyte-specific complement-like protein TEP1 from the mosquito Anopheles gambiae binds to and mediates killing of midgut stages of the rodent malaria parasite Plasmodium berghei. The dsRNA knockdown of TEP1 in adults completely abolishes melanotic refractoriness in a genetically selected refractory strain. Moreover, in susceptible mosquitoes this knockdown increases the number of developing parasites. Our results suggest that the TEP1-dependent parasite killing is followed by a TEP1-independent clearance of dead parasites by lysis and/or melanization. Further elucidation of the molecular mechanisms of TEP1-mediated parasite killing will be of great importance for our understanding of the principles of vectorial capacity in insects.}, keywords = {Animals, Anopheles, blandin, Female, Genetic, Humans, Insect Proteins, Insect Vectors, M3i, Malaria, Models, Molecular, Plasmodium berghei, Polymorphism, Protein Structure, RNA, Sequence Alignment, Tertiary}, pubstate = {published}, tppubtype = {article} } Anopheles mosquitoes are major vectors of human malaria in Africa. Large variation exists in the ability of mosquitoes to serve as vectors and to transmit malaria parasites, but the molecular mechanisms that determine vectorial capacity remain poorly understood. We report that the hemocyte-specific complement-like protein TEP1 from the mosquito Anopheles gambiae binds to and mediates killing of midgut stages of the rodent malaria parasite Plasmodium berghei. The dsRNA knockdown of TEP1 in adults completely abolishes melanotic refractoriness in a genetically selected refractory strain. Moreover, in susceptible mosquitoes this knockdown increases the number of developing parasites. Our results suggest that the TEP1-dependent parasite killing is followed by a TEP1-independent clearance of dead parasites by lysis and/or melanization. Further elucidation of the molecular mechanisms of TEP1-mediated parasite killing will be of great importance for our understanding of the principles of vectorial capacity in insects. |
2003 |
Luna, C; Hoa, N T; Zhang, J; Kanzok, S M; Brown, S E; Imler, Jean-Luc; Knudson, D L; Zheng, L Characterization of three Toll-like genes from mosquito Aedes aegypti Article de journal Insect Molecular Biology, 12 (1), p. 67–74, 2003, ISSN: 0962-1075. Résumé | BibTeX | Étiquettes: Aedes, Animals, Base Sequence, Cell Surface, Chimera, Cloning, Developmental, Female, Gene Expression Regulation, Genetic, imler, Insect Proteins, M3i, Male, messenger, Models, Molecular, Mutagenesis, Promoter Regions, Receptors, Reverse Transcriptase Polymerase Chain Reaction, RNA, Sequence Alignment, Signal Transduction, Site-Directed, Transfection @article{luna_characterization_2003, title = {Characterization of three Toll-like genes from mosquito Aedes aegypti}, author = {C Luna and N T Hoa and J Zhang and S M Kanzok and S E Brown and Jean-Luc Imler and D L Knudson and L Zheng}, issn = {0962-1075}, year = {2003}, date = {2003-02-01}, journal = {Insect Molecular Biology}, volume = {12}, number = {1}, pages = {67--74}, abstract = {Three Toll-related genes (AeToll1A, AeToll1B and AeToll5) were cloned and characterized from the yellow fever vector mosquito, Aedes aegypti. All three genes exhibited high levels of amino acid sequence similarity with Drosophila melanogaster (Dm)Toll1 and DmTehao (Toll5). AeToll1A and AeToll1B are 1124 and 1076 amino acid residues long, respectively. Both contain a carboxyl extension downstream of the Toll/interleukin-1 receptor (TIR) domain. AeToll5 is 1007 residues long and, like DmTehao, lacks the carboxyl terminal extension. Expression of these three genes was examined throughout development and after immune challenge. Both AeToll1A and AeToll5, like their Drosophila counterparts, activate transcription of drosomycin promoter in both Aedes and Drosophila cell lines. Deletion of the carboxyl extension of AeToll1A did not result in a further elevated level of the antifungal response. The intracellular signalling process appears to be species specific based on two observations. (1) DmToll is completely inactive in an Aedes cell line, suggesting a higher specificity requirement for DmToll in the intracellular signalling process. (2) Only one of three amino acid residues essential for DmToll function is required for AeToll1A function.}, keywords = {Aedes, Animals, Base Sequence, Cell Surface, Chimera, Cloning, Developmental, Female, Gene Expression Regulation, Genetic, imler, Insect Proteins, M3i, Male, messenger, Models, Molecular, Mutagenesis, Promoter Regions, Receptors, Reverse Transcriptase Polymerase Chain Reaction, RNA, Sequence Alignment, Signal Transduction, Site-Directed, Transfection}, pubstate = {published}, tppubtype = {article} } Three Toll-related genes (AeToll1A, AeToll1B and AeToll5) were cloned and characterized from the yellow fever vector mosquito, Aedes aegypti. All three genes exhibited high levels of amino acid sequence similarity with Drosophila melanogaster (Dm)Toll1 and DmTehao (Toll5). AeToll1A and AeToll1B are 1124 and 1076 amino acid residues long, respectively. Both contain a carboxyl extension downstream of the Toll/interleukin-1 receptor (TIR) domain. AeToll5 is 1007 residues long and, like DmTehao, lacks the carboxyl terminal extension. Expression of these three genes was examined throughout development and after immune challenge. Both AeToll1A and AeToll5, like their Drosophila counterparts, activate transcription of drosomycin promoter in both Aedes and Drosophila cell lines. Deletion of the carboxyl extension of AeToll1A did not result in a further elevated level of the antifungal response. The intracellular signalling process appears to be species specific based on two observations. (1) DmToll is completely inactive in an Aedes cell line, suggesting a higher specificity requirement for DmToll in the intracellular signalling process. (2) Only one of three amino acid residues essential for DmToll function is required for AeToll1A function. |
Salah, Ben R; Zouari, N; Reinbolt, J; Mejdoub, H Purification of turkey pancreatic phospholipase A2 Article de journal Biosci Biotechnol Biochem, 67 (10), p. 2139-44, 2003, (0916-8451 Journal Article). Résumé | BibTeX | Étiquettes: *Turkeys, &, A/*isolation, Acid, acids, Amino, Ammonium, and, Animals, Bile, Calcium, Chromatography, Concentration, Data, Hydrogen-Ion, Molecular, Pancreas/*enzymology, Phospholipases, purification, Salts, Sequence, Sulfate, Temperature, Weight @article{, title = {Purification of turkey pancreatic phospholipase A2}, author = { R. Ben Salah and N. Zouari and J. Reinbolt and H. Mejdoub}, year = {2003}, date = {2003-01-01}, journal = {Biosci Biotechnol Biochem}, volume = {67}, number = {10}, pages = {2139-44}, abstract = {Turkey pancreatic phospholipase (TPP) has been purified from delipidated pancreases. The purification included ammonium sulfate fractionation, acidic (pH 5) treatment, followed by sequencial column chromatographies on DEAE-cellulose, Sephadex G-75, and reverse phase high pressure liquid chromatography. The purified enzyme was found to be a monomeric protein with molecular mass of 14 kDa. The optimal activity was measured at pH 8 and 37 degrees C using egg yolk emulsion as substrate. Our results show that the enzyme (TPP) was not stable for 1 h at 60 degrees C, and that bile salt and Ca2+ were required for the expression of the purified enzyme. The sequence of the N-terminal amino acids of the purified enzyme shows a very close similarity between TPP and all other known pancreatic phospholipases.}, note = {0916-8451 Journal Article}, keywords = {*Turkeys, &, A/*isolation, Acid, acids, Amino, Ammonium, and, Animals, Bile, Calcium, Chromatography, Concentration, Data, Hydrogen-Ion, Molecular, Pancreas/*enzymology, Phospholipases, purification, Salts, Sequence, Sulfate, Temperature, Weight}, pubstate = {published}, tppubtype = {article} } Turkey pancreatic phospholipase (TPP) has been purified from delipidated pancreases. The purification included ammonium sulfate fractionation, acidic (pH 5) treatment, followed by sequencial column chromatographies on DEAE-cellulose, Sephadex G-75, and reverse phase high pressure liquid chromatography. The purified enzyme was found to be a monomeric protein with molecular mass of 14 kDa. The optimal activity was measured at pH 8 and 37 degrees C using egg yolk emulsion as substrate. Our results show that the enzyme (TPP) was not stable for 1 h at 60 degrees C, and that bile salt and Ca2+ were required for the expression of the purified enzyme. The sequence of the N-terminal amino acids of the purified enzyme shows a very close similarity between TPP and all other known pancreatic phospholipases. |
Bonnal, S; Schaeffer, C; Creancier, L; Clamens, S; Moine, H; Prats, A C; Vagner, S A single internal ribosome entry site containing a G quartet RNA structure drives fibroblast growth factor 2 gene expression at four alternative translation initiation codons Article de journal J Biol Chem, 278 (41), p. 39330-6, 2003, (0021-9258 Journal Article). Résumé | BibTeX | Étiquettes: 2/*genetics, Acid, Alternative, Base, Cell, Chain, Codon, Complementary/genetics, Conformation, Data, Deletion, DNA, Expression, Factor, Fibroblast, Gene, Gov't, Growth, Human, initiation, Initiator/genetics, Line, Messenger/*chemistry/*genetics, Molecular, Non-U.S., Nucleic, Peptide, Ribosomes/*metabolism, RNA, Sequence, Splicing, Support, Transfection @article{, title = {A single internal ribosome entry site containing a G quartet RNA structure drives fibroblast growth factor 2 gene expression at four alternative translation initiation codons}, author = { S. Bonnal and C. Schaeffer and L. Creancier and S. Clamens and H. Moine and A. C. Prats and S. Vagner}, year = {2003}, date = {2003-01-01}, journal = {J Biol Chem}, volume = {278}, number = {41}, pages = {39330-6}, abstract = {The 484-nucleotide (nt) alternatively translated region (ATR) of the human fibroblast growth factor 2 (FGF-2) mRNA contains four CUG and one AUG translation initiation codons. Although the 5'-end proximal CUG codon is initiated by a cap-dependent translation process, the other four initiation codons are initiated by a mechanism of internal entry of ribosomes. We undertook here a detailed analysis of the cis-acting elements defining the FGF-2 internal ribosome entry site (IRES). A thorough deletion analysis study within the 5'-ATR led us to define a 176-nt region as being necessary and sufficient for IRES function at four codons present in a downstream 308-nt RNA segment. Unexpectedly, a single IRES module is therefore responsible for translation initiation at four distantly localized codons. The determination of the FGF-2 5'-ATR RNA secondary structure by enzymatic and chemical probing experiments showed that the FGF-2 IRES contained two stem-loop regions and a G quartet motif that constitute novel structural determinants of IRES function.}, note = {0021-9258 Journal Article}, keywords = {2/*genetics, Acid, Alternative, Base, Cell, Chain, Codon, Complementary/genetics, Conformation, Data, Deletion, DNA, Expression, Factor, Fibroblast, Gene, Gov't, Growth, Human, initiation, Initiator/genetics, Line, Messenger/*chemistry/*genetics, Molecular, Non-U.S., Nucleic, Peptide, Ribosomes/*metabolism, RNA, Sequence, Splicing, Support, Transfection}, pubstate = {published}, tppubtype = {article} } The 484-nucleotide (nt) alternatively translated region (ATR) of the human fibroblast growth factor 2 (FGF-2) mRNA contains four CUG and one AUG translation initiation codons. Although the 5'-end proximal CUG codon is initiated by a cap-dependent translation process, the other four initiation codons are initiated by a mechanism of internal entry of ribosomes. We undertook here a detailed analysis of the cis-acting elements defining the FGF-2 internal ribosome entry site (IRES). A thorough deletion analysis study within the 5'-ATR led us to define a 176-nt region as being necessary and sufficient for IRES function at four codons present in a downstream 308-nt RNA segment. Unexpectedly, a single IRES module is therefore responsible for translation initiation at four distantly localized codons. The determination of the FGF-2 5'-ATR RNA secondary structure by enzymatic and chemical probing experiments showed that the FGF-2 IRES contained two stem-loop regions and a G quartet motif that constitute novel structural determinants of IRES function. |
Heyman, T; Wilhelm, M; Wilhelm, F X The central PPT of the yeast retrotransposon Ty1 is not essential for transposition Article de journal J Mol Biol, 331 (2), p. 315-20, 2003, (0022-2836 Journal Article). Résumé | BibTeX | Étiquettes: Base, cerevisiae/genetics, Data, DNA/*biosynthesis, Genetic, Gov't, Models, Molecular, Mutation, Non-U.S., Purines/*chemistry, Retroelements/*genetics, Saccharomyces, Sequence, Support @article{, title = {The central PPT of the yeast retrotransposon Ty1 is not essential for transposition}, author = { T. Heyman and M. Wilhelm and F. X. Wilhelm}, year = {2003}, date = {2003-01-01}, journal = {J Mol Biol}, volume = {331}, number = {2}, pages = {315-20}, abstract = {The yeast retrotransposon Ty1 has structural and functional similarities to retroviruses. We report here that, as in retroviruses, the plus-strand DNA of Ty1 is synthesized as two segments. A central DNA flap is formed during reverse transcription consecutive to elongation (with strand displacement) of the upstream segment beyond the central polypurine tract (cPPT) until the replication machinery is stopped at the central termination sequence. Comparison of wild-type and cPPT-mutant Ty1 elements shows that the mutant element lacking the central DNA flap is only twofold defective in transposition.}, note = {0022-2836 Journal Article}, keywords = {Base, cerevisiae/genetics, Data, DNA/*biosynthesis, Genetic, Gov't, Models, Molecular, Mutation, Non-U.S., Purines/*chemistry, Retroelements/*genetics, Saccharomyces, Sequence, Support}, pubstate = {published}, tppubtype = {article} } The yeast retrotransposon Ty1 has structural and functional similarities to retroviruses. We report here that, as in retroviruses, the plus-strand DNA of Ty1 is synthesized as two segments. A central DNA flap is formed during reverse transcription consecutive to elongation (with strand displacement) of the upstream segment beyond the central polypurine tract (cPPT) until the replication machinery is stopped at the central termination sequence. Comparison of wild-type and cPPT-mutant Ty1 elements shows that the mutant element lacking the central DNA flap is only twofold defective in transposition. |
Wilhelm, F X; Wilhelm, M; Gabriel, A Extension and cleavage of the polypurine tract plus-strand primer by Ty1 reverse transcriptase Article de journal J Biol Chem, 278 (48), p. 47678-84, 2003, (0021-9258 Journal Article). Résumé | BibTeX | Étiquettes: Base, Calf, Data, DNA, DNA/chemistry, Genetic, Gov't, H, Messenger/metabolism, Models, Molecular, Non-U.S., P.H.S., Polymerase/*chemistry, Primers, Proteins/chemistry, Purines/*chemistry, Recombinant, Replication, Retroelements/*genetics, Ribonuclease, RNA, RNA-Directed, RNA/chemistry, Sequence, Support, Templates, Thymus/chemistry, U.S., Viral @article{, title = {Extension and cleavage of the polypurine tract plus-strand primer by Ty1 reverse transcriptase}, author = { F. X. Wilhelm and M. Wilhelm and A. Gabriel}, year = {2003}, date = {2003-01-01}, journal = {J Biol Chem}, volume = {278}, number = {48}, pages = {47678-84}, abstract = {Using hybrid RNA/DNA substrates containing the polypurine tract (PPT) plus-strand primer, we have examined the interaction between the Ty1 reverse transcriptase (RT) and the plus-strand initiation complex. We show here that, although the PPT sequence is relatively resistant to RNase H cleavage, it can be cleaved internally by the polymerase-independent RNase H activity of Ty1 RT. Alternatively, this PPT can be used to initiate plus-strand DNA synthesis. We demonstrate that cleavage at the PPT/DNA junction occurs only after at least 9 nucleotides are extended. Cleavage leaves a nick between the RNA primer and the nascent plus-strand DNA. We show that Ty1 RT has a strand displacement activity beyond a gap but that the PPT is not efficiently re-utilized in vitro for another round of DNA synthesis after a first plus-strand DNA has been synthesized and cleaved at the PPT/U3 junction.}, note = {0021-9258 Journal Article}, keywords = {Base, Calf, Data, DNA, DNA/chemistry, Genetic, Gov't, H, Messenger/metabolism, Models, Molecular, Non-U.S., P.H.S., Polymerase/*chemistry, Primers, Proteins/chemistry, Purines/*chemistry, Recombinant, Replication, Retroelements/*genetics, Ribonuclease, RNA, RNA-Directed, RNA/chemistry, Sequence, Support, Templates, Thymus/chemistry, U.S., Viral}, pubstate = {published}, tppubtype = {article} } Using hybrid RNA/DNA substrates containing the polypurine tract (PPT) plus-strand primer, we have examined the interaction between the Ty1 reverse transcriptase (RT) and the plus-strand initiation complex. We show here that, although the PPT sequence is relatively resistant to RNase H cleavage, it can be cleaved internally by the polymerase-independent RNase H activity of Ty1 RT. Alternatively, this PPT can be used to initiate plus-strand DNA synthesis. We demonstrate that cleavage at the PPT/DNA junction occurs only after at least 9 nucleotides are extended. Cleavage leaves a nick between the RNA primer and the nascent plus-strand DNA. We show that Ty1 RT has a strand displacement activity beyond a gap but that the PPT is not efficiently re-utilized in vitro for another round of DNA synthesis after a first plus-strand DNA has been synthesized and cleaved at the PPT/U3 junction. |
2002 |
Perederina, A; Nevskaya, N; Nikonov, O; Nikulin, A; Dumas, P; Yao, M; Tanaka, I; Garber, M; Gongadze, G; Nikonov, S Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex Article de journal RNA, 8 (12), p. 1548-57, 2002, (1355-8382 Journal Article). Résumé | BibTeX | Étiquettes: 5S/*chemistry/*metabolism, Acid, Amino, Bacterial, Base, Binding, Bonding, coli/genetics, Conformation, Data, Escherichia, Fragments/chemistry/metabolism, Gov't, Hydrogen, Models, Molecular, Non-U.S., Nucleic, Peptide, Protein, Proteins/*chemistry/*metabolism, Proteins/chemistry/metabolism, Ribosomal, RNA, Sequence, Sites, Support @article{, title = {Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex}, author = { A. Perederina and N. Nevskaya and O. Nikonov and A. Nikulin and P. Dumas and M. Yao and I. Tanaka and M. Garber and G. Gongadze and S. Nikonov}, year = {2002}, date = {2002-01-01}, journal = {RNA}, volume = {8}, number = {12}, pages = {1548-57}, abstract = {The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit.}, note = {1355-8382 Journal Article}, keywords = {5S/*chemistry/*metabolism, Acid, Amino, Bacterial, Base, Binding, Bonding, coli/genetics, Conformation, Data, Escherichia, Fragments/chemistry/metabolism, Gov't, Hydrogen, Models, Molecular, Non-U.S., Nucleic, Peptide, Protein, Proteins/*chemistry/*metabolism, Proteins/chemistry/metabolism, Ribosomal, RNA, Sequence, Sites, Support}, pubstate = {published}, tppubtype = {article} } The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit. |
Wilhelm, M; Fishman, J A; Pontikis, R; Aubertin, A M; Wilhelm, F X Susceptibility of recombinant porcine endogenous retrovirus reverse transcriptase to nucleoside and non-nucleoside inhibitors Article de journal Cell Mol Life Sci, 59 (12), p. 2184-90, 2002, (1420-682x Journal Article). Résumé | BibTeX | Étiquettes: Acid, Amino, Animals, Calf, Chloride/metabolism, Chlorides/metabolism, Cloning, Compounds/metabolism, Data, DNA, DNA-Directed, endogenous, Gov't, H, Human, Inhibitors/*pharmacology, Magnesium, Manganese, Molecular, Non-U.S., Nucleosides/chemistry/*metabolism, P.H.S., Polymerase/chemistry/genetics/*metabolism, Polymerase/metabolism, Proteins/metabolism, Recombinant, Retroviruses/*enzymology, Reverse, Ribonuclease, RNA-Directed, Sequence, Sodium, structure, Support, Swine, Thymus/metabolism, Transcriptase, U.S. @article{, title = {Susceptibility of recombinant porcine endogenous retrovirus reverse transcriptase to nucleoside and non-nucleoside inhibitors}, author = { M. Wilhelm and J. A. Fishman and R. Pontikis and A. M. Aubertin and F. X. Wilhelm}, year = {2002}, date = {2002-01-01}, journal = {Cell Mol Life Sci}, volume = {59}, number = {12}, pages = {2184-90}, abstract = {Transplantation of organs, tissues or cells from pigs to humans could be a potential solution to the shortage of human organs for transplantation. Porcine endogenous retroviruses (PERVs) remain a major safety concern for porcine xenotransplantation. Thus, finding drugs that could be used as virological prophylaxis (or therapy) against PERV replication would be desirable. One of the most effective ways to block retroviral multiplication is to inhibit the enzyme reverse transcriptase (RT) which catalyzes the reverse transcription of viral RNA to proviral double-stranded DNA. We report here the cloning and expression of PERV RT and its susceptibility to several inhibitors. Our data demonstrate PERV susceptibility in vitro to the triphosphorylated nucleoside analog of zidovudine (AZT) and to ddGTP and to a lesser extent to ddTTP but almost no susceptibility to the non-nucleoside RT inhibitors tested.}, note = {1420-682x Journal Article}, keywords = {Acid, Amino, Animals, Calf, Chloride/metabolism, Chlorides/metabolism, Cloning, Compounds/metabolism, Data, DNA, DNA-Directed, endogenous, Gov't, H, Human, Inhibitors/*pharmacology, Magnesium, Manganese, Molecular, Non-U.S., Nucleosides/chemistry/*metabolism, P.H.S., Polymerase/chemistry/genetics/*metabolism, Polymerase/metabolism, Proteins/metabolism, Recombinant, Retroviruses/*enzymology, Reverse, Ribonuclease, RNA-Directed, Sequence, Sodium, structure, Support, Swine, Thymus/metabolism, Transcriptase, U.S.}, pubstate = {published}, tppubtype = {article} } Transplantation of organs, tissues or cells from pigs to humans could be a potential solution to the shortage of human organs for transplantation. Porcine endogenous retroviruses (PERVs) remain a major safety concern for porcine xenotransplantation. Thus, finding drugs that could be used as virological prophylaxis (or therapy) against PERV replication would be desirable. One of the most effective ways to block retroviral multiplication is to inhibit the enzyme reverse transcriptase (RT) which catalyzes the reverse transcription of viral RNA to proviral double-stranded DNA. We report here the cloning and expression of PERV RT and its susceptibility to several inhibitors. Our data demonstrate PERV susceptibility in vitro to the triphosphorylated nucleoside analog of zidovudine (AZT) and to ddGTP and to a lesser extent to ddTTP but almost no susceptibility to the non-nucleoside RT inhibitors tested. |
2001 |
Carnicelli, D; Brigotti, M; Rizzi, S; Keith, G; Montanaro, L; Sperti, S Nucleotides U28-A42 and A37 in unmodified yeast tRNA(Trp) as negative identity elements for bovine tryptophanyl-tRNA synthetase Article de journal FEBS Lett, 492 (3), p. 238-41, 2001, (0014-5793 Journal Article). Résumé | BibTeX | Étiquettes: Acid, Adenine/chemistry, Animals, Base, Cattle, cerevisiae/genetics, Conformation, Data, Fungal/genetics/metabolism, Gov't, Kinetics, Ligase/*metabolism, Molecular, Non-U.S., Nucleic, RNA, Saccharomyces, Sequence, Species, Specificity, Substrate, Support, Transfer, Trp/chemistry/*metabolism, Tryptophan-tRNA, Uridine/chemistry @article{, title = {Nucleotides U28-A42 and A37 in unmodified yeast tRNA(Trp) as negative identity elements for bovine tryptophanyl-tRNA synthetase}, author = { D. Carnicelli and M. Brigotti and S. Rizzi and G. Keith and L. Montanaro and S. Sperti}, year = {2001}, date = {2001-01-01}, journal = {FEBS Lett}, volume = {492}, number = {3}, pages = {238-41}, abstract = {Wild-type bovine and yeast tRNA(Trp) are efficiently aminoacylated by tryptophanyl-tRNA synthetase both from beef and from yeast. Upon loss of modified bases in the synthetic transcripts, mammalian tRNA(Trp) retains the double recognition by the two synthetases, while yeast tRNA(Trp) loses its substrate properties for the bovine enzyme and is recognised only by the cognate synthetase. By testing chimeric bovine-yeast transcripts with tryptophanyl-tRNA synthetase purified from beef pancreas, the nucleotides responsible for the loss of charging of the synthetic yeast transcript have been localised in the anticodon arm. A complete loss of charging akin to that observed with the yeast transcript requires substitution in the bovine backbone of G37 in the anticodon loop with yeast A37 and of C28-G42 in the anticodon stem with yeast U28-A42. Since A37 does not prevent aminoacylation of the wild-type yeast tRNA(Trp) by the beef enzyme, a negative combination apparently emerges in the synthetic transcript after unmasking of U28 by loss of pseudourydilation.}, note = {0014-5793 Journal Article}, keywords = {Acid, Adenine/chemistry, Animals, Base, Cattle, cerevisiae/genetics, Conformation, Data, Fungal/genetics/metabolism, Gov't, Kinetics, Ligase/*metabolism, Molecular, Non-U.S., Nucleic, RNA, Saccharomyces, Sequence, Species, Specificity, Substrate, Support, Transfer, Trp/chemistry/*metabolism, Tryptophan-tRNA, Uridine/chemistry}, pubstate = {published}, tppubtype = {article} } Wild-type bovine and yeast tRNA(Trp) are efficiently aminoacylated by tryptophanyl-tRNA synthetase both from beef and from yeast. Upon loss of modified bases in the synthetic transcripts, mammalian tRNA(Trp) retains the double recognition by the two synthetases, while yeast tRNA(Trp) loses its substrate properties for the bovine enzyme and is recognised only by the cognate synthetase. By testing chimeric bovine-yeast transcripts with tryptophanyl-tRNA synthetase purified from beef pancreas, the nucleotides responsible for the loss of charging of the synthetic yeast transcript have been localised in the anticodon arm. A complete loss of charging akin to that observed with the yeast transcript requires substitution in the bovine backbone of G37 in the anticodon loop with yeast A37 and of C28-G42 in the anticodon stem with yeast U28-A42. Since A37 does not prevent aminoacylation of the wild-type yeast tRNA(Trp) by the beef enzyme, a negative combination apparently emerges in the synthetic transcript after unmasking of U28 by loss of pseudourydilation. |
Wilhelm, M; Uzun, O; Mules, E H; Gabriel, A; Wilhelm, F X Polypurine tract formation by Ty1 RNase H Article de journal J Biol Chem, 276 (50), p. 47695-701, 2001, (0021-9258 Journal Article). Résumé | BibTeX | Étiquettes: *Purines, *Retroelements, Base, Binding, Calf, Data, DNA, DNA/metabolism, Factors, Gov't, H, Hydrolysis, Molecular, Mutation, Non-U.S., P.H.S., Polymerase/*chemistry/*metabolism, Primers/pharmacology, Protein, Proteins/metabolism, Recombinant, Ribonuclease, RNA-Directed, RNA/metabolism, Sequence, Sites, Support, Thymus/*chemistry/*genetics/metabolism, time, U.S. @article{, title = {Polypurine tract formation by Ty1 RNase H}, author = { M. Wilhelm and O. Uzun and E. H. Mules and A. Gabriel and F. X. Wilhelm}, year = {2001}, date = {2001-01-01}, journal = {J Biol Chem}, volume = {276}, number = {50}, pages = {47695-701}, abstract = {To better understand the mechanism by which Ty1 RNase H creates the polypurine tract (PPT) primer, we have demonstrated the polymerase-dependent hydrolytic activity of Ty1 reverse transcriptase (RT) during minus-strand synthesis. Using RNase H and polymerase mutants of the recombinant Ty1 RT protein, we show that the two domains of Ty1 RT can act independently of one another. Our results indicate that RNA/DNA substrates containing a short RNA PPT, which serve as primers for plus-strand DNA synthesis, are relatively resistant to RNase H cleavage. RNA substrates with a correct 5' end but with 3' end extending beyond the plus-strand initiation site were cleaved specifically to generate the correct 3' end of the PPT. Using long RNA/DNA duplexes containing the PPT, we show that Ty1 RT is able to make specific internal cleavages that could generate the plus-strand primer with correct 5' and 3' ends. Long RNA/DNA duplexes with mutations in the PPT or in a U-rich region upstream of the PPT, which abolish plus-strand initiation in vivo, were not cleaved specifically at the 5' end of the PPT. Our work demonstrates that the in vitro enzyme can recapitulate key processes that control proper replication in vivo.}, note = {0021-9258 Journal Article}, keywords = {*Purines, *Retroelements, Base, Binding, Calf, Data, DNA, DNA/metabolism, Factors, Gov't, H, Hydrolysis, Molecular, Mutation, Non-U.S., P.H.S., Polymerase/*chemistry/*metabolism, Primers/pharmacology, Protein, Proteins/metabolism, Recombinant, Ribonuclease, RNA-Directed, RNA/metabolism, Sequence, Sites, Support, Thymus/*chemistry/*genetics/metabolism, time, U.S.}, pubstate = {published}, tppubtype = {article} } To better understand the mechanism by which Ty1 RNase H creates the polypurine tract (PPT) primer, we have demonstrated the polymerase-dependent hydrolytic activity of Ty1 reverse transcriptase (RT) during minus-strand synthesis. Using RNase H and polymerase mutants of the recombinant Ty1 RT protein, we show that the two domains of Ty1 RT can act independently of one another. Our results indicate that RNA/DNA substrates containing a short RNA PPT, which serve as primers for plus-strand DNA synthesis, are relatively resistant to RNase H cleavage. RNA substrates with a correct 5' end but with 3' end extending beyond the plus-strand initiation site were cleaved specifically to generate the correct 3' end of the PPT. Using long RNA/DNA duplexes containing the PPT, we show that Ty1 RT is able to make specific internal cleavages that could generate the plus-strand primer with correct 5' and 3' ends. Long RNA/DNA duplexes with mutations in the PPT or in a U-rich region upstream of the PPT, which abolish plus-strand initiation in vivo, were not cleaved specifically at the 5' end of the PPT. Our work demonstrates that the in vitro enzyme can recapitulate key processes that control proper replication in vivo. |
Wilhelm, M; Wilhelm, F X Reverse transcription of retroviruses and LTR retrotransposons Article de journal Cell Mol Life Sci, 58 (9), p. 1246-62, 2001, (1420-682x Journal Article Review Review, Academic). Résumé | BibTeX | Étiquettes: *Retroelements, *Terminal, Acid, Alignment, Amino, Animals, Base, Conformation, Data, DNA, Homology, Human, Molecular, Nucleic, Polymerase/*chemistry/*metabolism, Repeat, Retroviridae/*enzymology/*genetics, RNA-Directed, Sequence, Sequences @article{, title = {Reverse transcription of retroviruses and LTR retrotransposons}, author = { M. Wilhelm and F. X. Wilhelm}, year = {2001}, date = {2001-01-01}, journal = {Cell Mol Life Sci}, volume = {58}, number = {9}, pages = {1246-62}, abstract = {Retroelements are mobile genetic entities that replicate via reverse transcription of a template RNA. A key component to the life cycle of these elements is the enzyme reverse transcriptase (RT), which copies the single-stranded genomic RNA of the element into a linear double-stranded DNA that is ultimately integrated into the host genome by the element-encoded integrase. RT is a multifunctionnal enzyme which possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity that specifically degrades the RNA strand of RNA-DNA duplexes. At some stages of the replication a strand-displacement activity of RT is also necessary. All activities are essential for the conversion of single-stranded genomic RNA into the double-stranded preintegrative DNA. This review focuses on the role of RT in the different steps of the replication process of retroelements. The features of retrotransposon replication which differ from the retroviral ones will be emphasized. In a second part of the review, the biochemical and enzymatic properties of two newly characterized retrotransposon RTs will be described. The role of the integrase domain in reverse transcriptase activity of some retroviral and retrotransposon RTs will be discussed.}, note = {1420-682x Journal Article Review Review, Academic}, keywords = {*Retroelements, *Terminal, Acid, Alignment, Amino, Animals, Base, Conformation, Data, DNA, Homology, Human, Molecular, Nucleic, Polymerase/*chemistry/*metabolism, Repeat, Retroviridae/*enzymology/*genetics, RNA-Directed, Sequence, Sequences}, pubstate = {published}, tppubtype = {article} } Retroelements are mobile genetic entities that replicate via reverse transcription of a template RNA. A key component to the life cycle of these elements is the enzyme reverse transcriptase (RT), which copies the single-stranded genomic RNA of the element into a linear double-stranded DNA that is ultimately integrated into the host genome by the element-encoded integrase. RT is a multifunctionnal enzyme which possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity that specifically degrades the RNA strand of RNA-DNA duplexes. At some stages of the replication a strand-displacement activity of RT is also necessary. All activities are essential for the conversion of single-stranded genomic RNA into the double-stranded preintegrative DNA. This review focuses on the role of RT in the different steps of the replication process of retroelements. The features of retrotransposon replication which differ from the retroviral ones will be emphasized. In a second part of the review, the biochemical and enzymatic properties of two newly characterized retrotransposon RTs will be described. The role of the integrase domain in reverse transcriptase activity of some retroviral and retrotransposon RTs will be discussed. |
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 |
Wilhelm, M; Boutabout, M; Wilhelm, F X Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities Article de journal Biochem J, 348 (Pt 2), p. 337-42, 2000, (0264-6021 Journal Article). Résumé | BibTeX | Étiquettes: &, Acid, affinity, Alignment, Amino, Calf, cerevisiae/*enzymology/*genetics, Chromatography, Cloning, Codon, coli, Comparative, Data, DNA, DNA/metabolism, Escherichia, Frames, Fusion, Genetic, Gov't, H, Heteroduplexes/metabolism, HIV-1, Homology, Integrases/chemistry/metabolism, Kinetics, Molecular, Non-U.S., Nucleic, Open, Polymerase/chemistry/isolation, Proteins/chemistry/isolation, purification/*metabolism, purification/metabolism, Reading, Recombinant, Retroelements/*genetics, Reverse, Ribonuclease, RNA-Directed, RNA/metabolism, Saccharomyces, Sequence, Study, Support, Templates, Terminator, Thymus/isolation, Transcriptase/chemistry @article{, title = {Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities}, author = { M. Wilhelm and M. Boutabout and F. X. Wilhelm}, year = {2000}, date = {2000-01-01}, journal = {Biochem J}, volume = {348}, number = {Pt 2}, pages = {337-42}, abstract = {Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl(2), NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.}, note = {0264-6021 Journal Article}, keywords = {&, Acid, affinity, Alignment, Amino, Calf, cerevisiae/*enzymology/*genetics, Chromatography, Cloning, Codon, coli, Comparative, Data, DNA, DNA/metabolism, Escherichia, Frames, Fusion, Genetic, Gov't, H, Heteroduplexes/metabolism, HIV-1, Homology, Integrases/chemistry/metabolism, Kinetics, Molecular, Non-U.S., Nucleic, Open, Polymerase/chemistry/isolation, Proteins/chemistry/isolation, purification/*metabolism, purification/metabolism, Reading, Recombinant, Retroelements/*genetics, Reverse, Ribonuclease, RNA-Directed, RNA/metabolism, Saccharomyces, Sequence, Study, Support, Templates, Terminator, Thymus/isolation, Transcriptase/chemistry}, pubstate = {published}, tppubtype = {article} } Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl(2), NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase. |
1999 |
Lowenberger, C A; Smartt, C T; Bulet, Philippe; Ferdig, M T; Severson, D W; Hoffmann, Jules A; Christensen, B M Insect immunity: molecular cloning, expression, and characterization of cDNAs and genomic DNA encoding three isoforms of insect defensin in Aedes aegypti Article de journal Insect Mol. Biol., 8 (1), p. 107–118, 1999, ISSN: 0962-1075. Résumé | BibTeX | Étiquettes: Aedes, Amino Acid, Animals, Base Sequence, Blotting, Chromosome Mapping, Cloning, Complementary, Defensins, DNA, Gene Expression, Hemolymph, hoffmann, M3i, Molecular, Northern, Protein Isoforms, Proteins, Sequence Homology @article{lowenberger_insect_1999, title = {Insect immunity: molecular cloning, expression, and characterization of cDNAs and genomic DNA encoding three isoforms of insect defensin in Aedes aegypti}, author = {C A Lowenberger and C T Smartt and Philippe Bulet and M T Ferdig and D W Severson and Jules A Hoffmann and B M Christensen}, issn = {0962-1075}, year = {1999}, date = {1999-02-01}, journal = {Insect Mol. Biol.}, volume = {8}, number = {1}, pages = {107--118}, abstract = {Aedes aegypti were immune activated by injection with bacteria, and the expression of insect defensins was measured over time. Northern analyses indicated that defensin transcriptional activity continued for at least 21 days after bacterial injection, and up to 10 days after saline inoculation. Mature defensin levels in the haemolymph reached approximately 45 microM at 24 h post inoculation. cDNAs encoding the preprodefensins of three previously described mature Ae. aegypti defensins were amplified by PCR, cloned and sequenced. Genomic clones were amplified using primers designed against the cDNA sequence. Sequence comparison indicates that there is significant inter- and intra-isoform variability in the signal peptide and prodefensin sequences of defensin genes. Preprodefensin sequences of isoforms A and B are very similar, consisting of a signal peptide region of twenty amino acids, a prodefensin region of thirty-eight amino acids and a forty amino acid mature peptide domain. The sequence encoding isoform C is significantly different, comprising a signal peptide region of twenty-three amino acids, a prodefensin region of thirty-six amino acids, and the mature protein domain of forty amino acids. Analysis of the genomic clones of each isoform revealed one intron spatially conserved in the prodefensin region of all sequences. The intron in isoforms A and B is 64 nt long, and except for a 4 nt substitution in one clone, these intron sequences are identical. The intron in isoform C is 76 nt long and does not share significant identity with the intron sequences of isoforms A or B. The defensin gene mapped to chromosome 3, between two known loci, blt and LF168.}, keywords = {Aedes, Amino Acid, Animals, Base Sequence, Blotting, Chromosome Mapping, Cloning, Complementary, Defensins, DNA, Gene Expression, Hemolymph, hoffmann, M3i, Molecular, Northern, Protein Isoforms, Proteins, Sequence Homology}, pubstate = {published}, tppubtype = {article} } Aedes aegypti were immune activated by injection with bacteria, and the expression of insect defensins was measured over time. Northern analyses indicated that defensin transcriptional activity continued for at least 21 days after bacterial injection, and up to 10 days after saline inoculation. Mature defensin levels in the haemolymph reached approximately 45 microM at 24 h post inoculation. cDNAs encoding the preprodefensins of three previously described mature Ae. aegypti defensins were amplified by PCR, cloned and sequenced. Genomic clones were amplified using primers designed against the cDNA sequence. Sequence comparison indicates that there is significant inter- and intra-isoform variability in the signal peptide and prodefensin sequences of defensin genes. Preprodefensin sequences of isoforms A and B are very similar, consisting of a signal peptide region of twenty amino acids, a prodefensin region of thirty-eight amino acids and a forty amino acid mature peptide domain. The sequence encoding isoform C is significantly different, comprising a signal peptide region of twenty-three amino acids, a prodefensin region of thirty-six amino acids, and the mature protein domain of forty amino acids. Analysis of the genomic clones of each isoform revealed one intron spatially conserved in the prodefensin region of all sequences. The intron in isoforms A and B is 64 nt long, and except for a 4 nt substitution in one clone, these intron sequences are identical. The intron in isoform C is 76 nt long and does not share significant identity with the intron sequences of isoforms A or B. The defensin gene mapped to chromosome 3, between two known loci, blt and LF168. |
Auxilien, S; Keith, G; Grice, Le S F; Darlix, J L Role of post-transcriptional modifications of primer tRNALys,3 in the fidelity and efficacy of plus strand DNA transfer during HIV-1 reverse transcription Article de journal J Biol Chem, 274 (7), p. 4412-20, 1999, (0021-9258 Journal Article). Résumé | BibTeX | Étiquettes: *RNA, *Transcription, Acid, Base, Calf, Conformation, Data, DNA, Genetic, Gov't, H, HIV-1, HIV-1/*physiology, Lys/*metabolism, Molecular, Non-U.S., Nucleic, post-transcriptional, Processing, Reverse, Ribonuclease, RNA, Sequence, Support, Templates, Thymus/metabolism, Transcriptase/metabolism, Transfer, Viral/*metabolism, Viral/metabolism @article{, title = {Role of post-transcriptional modifications of primer tRNALys,3 in the fidelity and efficacy of plus strand DNA transfer during HIV-1 reverse transcription}, author = { S. Auxilien and G. Keith and S. F. Le Grice and J. L. Darlix}, year = {1999}, date = {1999-01-01}, journal = {J Biol Chem}, volume = {274}, number = {7}, pages = {4412-20}, abstract = {During HIV reverse transcription, (+) strand DNA synthesis is primed by an RNase H-resistant sequence, the polypurine tract, and continues as far as a 18-nt double-stranded RNA region corresponding to the 3' end of tRNALys,3 hybridized to the viral primer binding site (PBS). Before (+) strand DNA transfer, reverse transcriptase (RT) needs to unwind the double-stranded tRNA-PBS RNA in order to reverse-transcribe the 3' end of primer tRNALys,3. Since the detailed mechanism of (+) strand DNA transfer remains incompletely understood, we developed an in vitro system to closely examine this mechanism, composed of HIV 5' RNA, natural modified tRNALys,3, synthetic unmodified tRNALys,3 or oligonucleotides (RNA or DNA) complementary to the PBS, as well as the viral proteins RT and nucleocapsid protein (NCp7). Prior to (+) strand DNA transfer, RT stalls at the double-stranded tRNA-PBS RNA complex and is able to reverse-transcribe modified nucleosides of natural tRNALys,3. Modified nucleoside m1A-58 of natural tRNALys,3 is only partially effective as a stop signal, as RT can transcribe as far as the hyper-modified adenosine (ms2t6A-37) in the anticodon loop. m1A-58 is almost always transcribed into A, whereas other modified nucleosides are transcribed correctly, except for m7G-46, which is sometimes transcribed into T. In contrast, synthetic tRNALys,3, an RNA PBS primer, and a DNA PBS primer are completely reverse-transcribed. In the presence of an acceptor template, (+) strand DNA transfer is efficient only with templates containing natural tRNALys,3 or the RNA PBS primer. Sequence analysis of transfer products revealed frequent errors at the transfer site with synthetic tRNALys,3, not observed with natural tRNALys,3. Thus, modified nucleoside m1A-58, present in all retroviral tRNA primers, appears to be important for both efficacy and fidelity of (+) strand DNA transfer. We show that other factors such as the nature of the (-) PBS of the acceptor template and the RNase H activity of RT also influence the efficacy of (+) strand DNA transfer.}, note = {0021-9258 Journal Article}, keywords = {*RNA, *Transcription, Acid, Base, Calf, Conformation, Data, DNA, Genetic, Gov't, H, HIV-1, HIV-1/*physiology, Lys/*metabolism, Molecular, Non-U.S., Nucleic, post-transcriptional, Processing, Reverse, Ribonuclease, RNA, Sequence, Support, Templates, Thymus/metabolism, Transcriptase/metabolism, Transfer, Viral/*metabolism, Viral/metabolism}, pubstate = {published}, tppubtype = {article} } During HIV reverse transcription, (+) strand DNA synthesis is primed by an RNase H-resistant sequence, the polypurine tract, and continues as far as a 18-nt double-stranded RNA region corresponding to the 3' end of tRNALys,3 hybridized to the viral primer binding site (PBS). Before (+) strand DNA transfer, reverse transcriptase (RT) needs to unwind the double-stranded tRNA-PBS RNA in order to reverse-transcribe the 3' end of primer tRNALys,3. Since the detailed mechanism of (+) strand DNA transfer remains incompletely understood, we developed an in vitro system to closely examine this mechanism, composed of HIV 5' RNA, natural modified tRNALys,3, synthetic unmodified tRNALys,3 or oligonucleotides (RNA or DNA) complementary to the PBS, as well as the viral proteins RT and nucleocapsid protein (NCp7). Prior to (+) strand DNA transfer, RT stalls at the double-stranded tRNA-PBS RNA complex and is able to reverse-transcribe modified nucleosides of natural tRNALys,3. Modified nucleoside m1A-58 of natural tRNALys,3 is only partially effective as a stop signal, as RT can transcribe as far as the hyper-modified adenosine (ms2t6A-37) in the anticodon loop. m1A-58 is almost always transcribed into A, whereas other modified nucleosides are transcribed correctly, except for m7G-46, which is sometimes transcribed into T. In contrast, synthetic tRNALys,3, an RNA PBS primer, and a DNA PBS primer are completely reverse-transcribed. In the presence of an acceptor template, (+) strand DNA transfer is efficient only with templates containing natural tRNALys,3 or the RNA PBS primer. Sequence analysis of transfer products revealed frequent errors at the transfer site with synthetic tRNALys,3, not observed with natural tRNALys,3. Thus, modified nucleoside m1A-58, present in all retroviral tRNA primers, appears to be important for both efficacy and fidelity of (+) strand DNA transfer. We show that other factors such as the nature of the (-) PBS of the acceptor template and the RNase H activity of RT also influence the efficacy of (+) strand DNA transfer. |
Perreau, V M; Keith, G; Holmes, W M; Przykorska, A; Santos, M A; Tuite, M F The Candida albicans CUG-decoding ser-tRNA has an atypical anticodon stem-loop structure Article de journal J Mol Biol, 293 (5), p. 1039-53, 1999, (0022-2836 Journal Article). Résumé | BibTeX | Étiquettes: *Nucleic, Acid, albicans/*genetics, Anticodon/*chemistry/*genetics/metabolism, Base, Candida, cerevisiae/genetics, Code/genetics, Conformation, Evolution, Fungal/chemistry/genetics/metabolism, Genetic, Gov't, Imidazoles/metabolism, Lead/metabolism, Methylation, Methyltransferases/metabolism, Molecular, Mutation/genetics, Non-P.H.S., Non-U.S., Nucleosides/genetics/metabolism, P.H.S., Ribonucleases/metabolism, RNA, Saccharomyces, Sequence, Ser/*chemistry/*genetics/metabolism, Solutions, Support, Transfer, tRNA, U.S. @article{, title = {The Candida albicans CUG-decoding ser-tRNA has an atypical anticodon stem-loop structure}, author = { V. M. Perreau and G. Keith and W. M. Holmes and A. Przykorska and M. A. Santos and M. F. Tuite}, year = {1999}, date = {1999-01-01}, journal = {J Mol Biol}, volume = {293}, number = {5}, pages = {1039-53}, abstract = {In many Candida species, the leucine CUG codon is decoded by a tRNA with two unusual properties: it is a ser-tRNA and, uniquely, has guanosine at position 33 (G33). Using a combination of enzymatic (V1 RNase, RnI nuclease) and chemical (Pb(2+), imidazole) probing of the native Candida albicans ser-tRNACAG, we demonstrate that the overall tertiary structure of this tRNA resembles that of a ser-tRNA rather than a leu-tRNA, except within the anticodon arm where there is considerable disruption of the anticodon stem. Using non-modified in vitro transcripts of the C. albicans ser-tRNACAG carrying G, C, U or A at position 33, we demonstrate that it is specifically a G residue at this position that induces the atypical anticodon stem structure. Further quantitative evidence for an unusual structure in the anticodon arm of the G33-tRNA is provided by the observed change in kinetics of methylation of the G at position 37, by purified Escherichia coli m(1)G37 methyltransferase. We conclude that the anticodon arm distortion, induced by a guanosine base at position 33 in the anticodon loop of this novel tRNA, results in reduced decoding ability which has facilitated the evolution of this tRNA without extinction of the species encoding it.}, note = {0022-2836 Journal Article}, keywords = {*Nucleic, Acid, albicans/*genetics, Anticodon/*chemistry/*genetics/metabolism, Base, Candida, cerevisiae/genetics, Code/genetics, Conformation, Evolution, Fungal/chemistry/genetics/metabolism, Genetic, Gov't, Imidazoles/metabolism, Lead/metabolism, Methylation, Methyltransferases/metabolism, Molecular, Mutation/genetics, Non-P.H.S., Non-U.S., Nucleosides/genetics/metabolism, P.H.S., Ribonucleases/metabolism, RNA, Saccharomyces, Sequence, Ser/*chemistry/*genetics/metabolism, Solutions, Support, Transfer, tRNA, U.S.}, pubstate = {published}, tppubtype = {article} } In many Candida species, the leucine CUG codon is decoded by a tRNA with two unusual properties: it is a ser-tRNA and, uniquely, has guanosine at position 33 (G33). Using a combination of enzymatic (V1 RNase, RnI nuclease) and chemical (Pb(2+), imidazole) probing of the native Candida albicans ser-tRNACAG, we demonstrate that the overall tertiary structure of this tRNA resembles that of a ser-tRNA rather than a leu-tRNA, except within the anticodon arm where there is considerable disruption of the anticodon stem. Using non-modified in vitro transcripts of the C. albicans ser-tRNACAG carrying G, C, U or A at position 33, we demonstrate that it is specifically a G residue at this position that induces the atypical anticodon stem structure. Further quantitative evidence for an unusual structure in the anticodon arm of the G33-tRNA is provided by the observed change in kinetics of methylation of the G at position 37, by purified Escherichia coli m(1)G37 methyltransferase. We conclude that the anticodon arm distortion, induced by a guanosine base at position 33 in the anticodon loop of this novel tRNA, results in reduced decoding ability which has facilitated the evolution of this tRNA without extinction of the species encoding it. |
1998 |
Uttenweiler-Joseph, S; Moniatte, M; Lagueux, Marie; Dorsselaer, Van A; Hoffmann, Jules A; Bulet, Philippe Differential display of peptides induced during the immune response of Drosophila: a matrix-assisted laser desorption ionization time-of-flight mass spectrometry study Article de journal Proc. Natl. Acad. Sci. U.S.A., 95 (19), p. 11342–11347, 1998, ISSN: 0027-8424. Résumé | BibTeX | Étiquettes: Animals, bacteria, Chromatography, Cloning, Hemolymph, High Pressure Liquid, hoffmann, Immunity, Insect Proteins, M3i, Mass, Matrix-Assisted Laser Desorption-Ionization, messenger, Molecular, Peptides, Protein Precursors, RNA, Sequence Analysis, Spectrometry, Time Factors @article{uttenweiler-joseph_differential_1998, title = {Differential display of peptides induced during the immune response of Drosophila: a matrix-assisted laser desorption ionization time-of-flight mass spectrometry study}, author = {S Uttenweiler-Joseph and M Moniatte and Marie Lagueux and Van A Dorsselaer and Jules A Hoffmann and Philippe Bulet}, issn = {0027-8424}, year = {1998}, date = {1998-09-01}, journal = {Proc. Natl. Acad. Sci. U.S.A.}, volume = {95}, number = {19}, pages = {11342--11347}, abstract = {We have developed an approach based on a differential mass spectrometric analysis to detect molecules induced during the immune response of Drosophila, regardless of their biological activities. For this, we have applied directly matrix-assisted laser desorption/ionization MS to hemolymph samples from individual flies before and after an immune challenge. This method provided precise information on the molecular masses of immune-induced molecules and allowed the detection, in the molecular range of 1.5-11 kDa, of 24 Drosophila immune-induced molecules (DIMs). These molecules are all peptides, and four correspond to already characterized antimicrobial peptides. We have further analyzed the induction of the various peptides by immune challenge in wild-type flies and in mutants with a compromised antimicrobial response. We also describe a methodology combining matrix-assisted laser desorption ionization time-of-flight MS, HPLC, and Edman degradation, which yielded the peptide sequence of three of the DIMs. Finally, molecular cloning and Northern blot analyses revealed that one of the DIMs is produced as a prepropeptide and is inducible on a bacterial challenge.}, keywords = {Animals, bacteria, Chromatography, Cloning, Hemolymph, High Pressure Liquid, hoffmann, Immunity, Insect Proteins, M3i, Mass, Matrix-Assisted Laser Desorption-Ionization, messenger, Molecular, Peptides, Protein Precursors, RNA, Sequence Analysis, Spectrometry, Time Factors}, pubstate = {published}, tppubtype = {article} } We have developed an approach based on a differential mass spectrometric analysis to detect molecules induced during the immune response of Drosophila, regardless of their biological activities. For this, we have applied directly matrix-assisted laser desorption/ionization MS to hemolymph samples from individual flies before and after an immune challenge. This method provided precise information on the molecular masses of immune-induced molecules and allowed the detection, in the molecular range of 1.5-11 kDa, of 24 Drosophila immune-induced molecules (DIMs). These molecules are all peptides, and four correspond to already characterized antimicrobial peptides. We have further analyzed the induction of the various peptides by immune challenge in wild-type flies and in mutants with a compromised antimicrobial response. We also describe a methodology combining matrix-assisted laser desorption ionization time-of-flight MS, HPLC, and Edman degradation, which yielded the peptide sequence of three of the DIMs. Finally, molecular cloning and Northern blot analyses revealed that one of the DIMs is produced as a prepropeptide and is inducible on a bacterial challenge. |
Bergdoll, M; Eltis, L D; Cameron, A D; Dumas, P; Bolin, J T All in the family: structural and evolutionary relationships among three modular proteins with diverse functions and variable assembly Article de journal Protein Sci, 7 (8), p. 1661-70, 1998, (0961-8368 Journal Article). Résumé | BibTeX | Étiquettes: *Acetyltransferases, *Evolution, Acid, Amino, Bacterial, Burkholderia/*chemistry, Crystallography, Data, Genetic, Gov't, Homology, Human, Lactoylglutathione, Lyase/*chemistry, Models, Molecular, Non-U.S., Oxygenases/chemistry, P.H.S., Phylogeny, Protein, Proteins/*chemistry, Secondary, Sequence, structure, Support, U.S., X-Ray @article{, title = {All in the family: structural and evolutionary relationships among three modular proteins with diverse functions and variable assembly}, author = { M. Bergdoll and L. D. Eltis and A. D. Cameron and P. Dumas and J. T. Bolin}, year = {1998}, date = {1998-01-01}, journal = {Protein Sci}, volume = {7}, number = {8}, pages = {1661-70}, abstract = {The crystal structures of three proteins of diverse function and low sequence similarity were analyzed to evaluate structural and evolutionary relationships. The proteins include a bacterial bleomycin resistance protein, a bacterial extradiol dioxygenase, and human glyoxalase I. Structural comparisons, as well as phylogenetic analyses, strongly indicate that the modern family of proteins represented by these structures arose through a rich evolutionary history that includes multiple gene duplication and fusion events. These events appear to be historically shared in some cases, but parallel and historically independent in others. A significant early event is proposed to be the establishment of metal-binding in an oligomeric ancestor prior to the first gene fusion. Variations in the spatial arrangements of homologous modules are observed that are consistent with the structural principles of three-dimensional domain swapping, but in the unusual context of the formation of larger monomers from smaller dimers or tetramers. The comparisons support a general mechanism for metalloprotein evolution that exploits the symmetry of a homooligomeric protein to originate a metal binding site and relies upon the relaxation of symmetry, as enabled by gene duplication, to establish and refine specific functions.}, note = {0961-8368 Journal Article}, keywords = {*Acetyltransferases, *Evolution, Acid, Amino, Bacterial, Burkholderia/*chemistry, Crystallography, Data, Genetic, Gov't, Homology, Human, Lactoylglutathione, Lyase/*chemistry, Models, Molecular, Non-U.S., Oxygenases/chemistry, P.H.S., Phylogeny, Protein, Proteins/*chemistry, Secondary, Sequence, structure, Support, U.S., X-Ray}, pubstate = {published}, tppubtype = {article} } The crystal structures of three proteins of diverse function and low sequence similarity were analyzed to evaluate structural and evolutionary relationships. The proteins include a bacterial bleomycin resistance protein, a bacterial extradiol dioxygenase, and human glyoxalase I. Structural comparisons, as well as phylogenetic analyses, strongly indicate that the modern family of proteins represented by these structures arose through a rich evolutionary history that includes multiple gene duplication and fusion events. These events appear to be historically shared in some cases, but parallel and historically independent in others. A significant early event is proposed to be the establishment of metal-binding in an oligomeric ancestor prior to the first gene fusion. Variations in the spatial arrangements of homologous modules are observed that are consistent with the structural principles of three-dimensional domain swapping, but in the unusual context of the formation of larger monomers from smaller dimers or tetramers. The comparisons support a general mechanism for metalloprotein evolution that exploits the symmetry of a homooligomeric protein to originate a metal binding site and relies upon the relaxation of symmetry, as enabled by gene duplication, to establish and refine specific functions. |
Brigotti, M; Keith, G; Pallanca, A; Carnicelli, D; Alvergna, P; Dirheimer, G; Montanaro, L; Sperti, S Identification of the tRNAs which up-regulate agrostin, barley RIP and PAP-S, three ribosome-inactivating proteins of plant origin Article de journal FEBS Lett, 431 (2), p. 259-62, 1998, (0014-5793 Journal Article). Résumé | BibTeX | Étiquettes: &, Acid, Adenosine, Base, Conformation, Data, effects/*metabolism, Gov't, Hordeum/metabolism, Hydrolases/*metabolism, Molecular, N-Glycosyl, Non-U.S., Nucleic, Plant, Plant/chemistry/isolation, Proteins/drug, purification/*metabolism, RNA, Sequence, Support, Transfer/chemistry/isolation, Triphosphate/pharmacology, Up-Regulation @article{, title = {Identification of the tRNAs which up-regulate agrostin, barley RIP and PAP-S, three ribosome-inactivating proteins of plant origin}, author = { M. Brigotti and G. Keith and A. Pallanca and D. Carnicelli and P. Alvergna and G. Dirheimer and L. Montanaro and S. Sperti}, year = {1998}, date = {1998-01-01}, journal = {FEBS Lett}, volume = {431}, number = {2}, pages = {259-62}, abstract = {Ribosome-inactivating proteins (RIP) are RNA-N-glycosidases widely diffused in plants which depurinate ribosomal RNA at a specific universally conserved position, A4324 in rat ribosomes. A small group of RIPs (cofactor-dependent RIPs) require ATP and tRNA to reach maximal activity on isolated ribosomes. The tRNA which stimulates gelonin was identified as tRNA(Trp). The present paper reports the identification of three other tRNAs which stimulate agrostin (tRNA(Ala)), barley RIP (tRNA(Ala), tRNA(Val)) and PAP-S (tRNA(Gly)), while for tritin-S no particular stimulating tRNA emerged. The sequences of tRNA(Val) and tRNA(Gly) correspond to the already known ones (rabbit and man, respectively). The tRNA(Ala) (anticodon IGC) identifies a new isoacceptor. Only the stimulating activity of the tRNA(Ala) for agrostin approaches the specificity previously observed for the couple gelonin-tRNA(Trp).}, note = {0014-5793 Journal Article}, keywords = {&, Acid, Adenosine, Base, Conformation, Data, effects/*metabolism, Gov't, Hordeum/metabolism, Hydrolases/*metabolism, Molecular, N-Glycosyl, Non-U.S., Nucleic, Plant, Plant/chemistry/isolation, Proteins/drug, purification/*metabolism, RNA, Sequence, Support, Transfer/chemistry/isolation, Triphosphate/pharmacology, Up-Regulation}, pubstate = {published}, tppubtype = {article} } Ribosome-inactivating proteins (RIP) are RNA-N-glycosidases widely diffused in plants which depurinate ribosomal RNA at a specific universally conserved position, A4324 in rat ribosomes. A small group of RIPs (cofactor-dependent RIPs) require ATP and tRNA to reach maximal activity on isolated ribosomes. The tRNA which stimulates gelonin was identified as tRNA(Trp). The present paper reports the identification of three other tRNAs which stimulate agrostin (tRNA(Ala)), barley RIP (tRNA(Ala), tRNA(Val)) and PAP-S (tRNA(Gly)), while for tritin-S no particular stimulating tRNA emerged. The sequences of tRNA(Val) and tRNA(Gly) correspond to the already known ones (rabbit and man, respectively). The tRNA(Ala) (anticodon IGC) identifies a new isoacceptor. Only the stimulating activity of the tRNA(Ala) for agrostin approaches the specificity previously observed for the couple gelonin-tRNA(Trp). |
Friant, S; Heyman, T; Bystrom, A S; Wilhelm, M; Wilhelm, F X Interactions between Ty1 retrotransposon RNA and the T and D regions of the tRNA(iMet) primer are required for initiation of reverse transcription in vivo Article de journal Mol Cell Biol, 18 (2), p. 799-806, 1998, (0270-7306 Journal Article). Résumé | BibTeX | Étiquettes: *Retroelements, *Transcription, Acid, Base, Binding, cerevisiae, Conformation, Data, DNA, Fungal/*metabolism, Fungal/biosynthesis, Genetic, Gov't, Met/*metabolism, Molecular, Mutagenesis, Non-U.S., Nucleic, Primers, Replication, RNA, Saccharomyces, Sequence, Sites, Support, Transfer @article{, title = {Interactions between Ty1 retrotransposon RNA and the T and D regions of the tRNA(iMet) primer are required for initiation of reverse transcription in vivo}, author = { S. Friant and T. Heyman and A. S. Bystrom and M. Wilhelm and F. X. Wilhelm}, year = {1998}, date = {1998-01-01}, journal = {Mol Cell Biol}, volume = {18}, number = {2}, pages = {799-806}, abstract = {Reverse transcription of the Saccharomyces cerevisiae Ty1 retrotransposon is primed by tRNA(iMet) base paired to the primer binding site (PBS) near the 5' end of Ty1 genomic RNA. The 10-nucleotide PBS is complementary to the last 10 nucleotides of the acceptor stem of tRNA(iMet). A structural probing study of the interactions between the Ty1 RNA template and the tRNA(iMet) primer showed that besides interactions between the PBS and the 3' end of tRNA(iMet), three short regions of Ty1 RNA, named boxes 0, 1, and 2.1, interact with the T and D stems and loops of tRNA(iMet). To determine if these sequences are important for the reverse transcription pathway of the Ty1 retrotransposon, mutant Ty1 elements and tRNA(iMet) were tested for the ability to support transposition. We show that the Ty1 boxes and the complementary sequences in the T and D stems and loops of tRNA(iMet) contain bases that are critical for Ty1 retrotransposition. Disruption of 1 or 2 bp between tRNA(iMet) and box 0, 1, or 2.1 dramatically decreases the level of transposition. Compensatory mutations which restore base pairing between the primer and the template restore transposition. Analysis of the reverse transcription intermediates generated inside Ty1 virus-like particles indicates that initiation of minus-strand strong-stop DNA synthesis is affected by mutations disrupting complementarity between Ty1 RNA and primer tRNA(iMet).}, note = {0270-7306 Journal Article}, keywords = {*Retroelements, *Transcription, Acid, Base, Binding, cerevisiae, Conformation, Data, DNA, Fungal/*metabolism, Fungal/biosynthesis, Genetic, Gov't, Met/*metabolism, Molecular, Mutagenesis, Non-U.S., Nucleic, Primers, Replication, RNA, Saccharomyces, Sequence, Sites, Support, Transfer}, pubstate = {published}, tppubtype = {article} } Reverse transcription of the Saccharomyces cerevisiae Ty1 retrotransposon is primed by tRNA(iMet) base paired to the primer binding site (PBS) near the 5' end of Ty1 genomic RNA. The 10-nucleotide PBS is complementary to the last 10 nucleotides of the acceptor stem of tRNA(iMet). A structural probing study of the interactions between the Ty1 RNA template and the tRNA(iMet) primer showed that besides interactions between the PBS and the 3' end of tRNA(iMet), three short regions of Ty1 RNA, named boxes 0, 1, and 2.1, interact with the T and D stems and loops of tRNA(iMet). To determine if these sequences are important for the reverse transcription pathway of the Ty1 retrotransposon, mutant Ty1 elements and tRNA(iMet) were tested for the ability to support transposition. We show that the Ty1 boxes and the complementary sequences in the T and D stems and loops of tRNA(iMet) contain bases that are critical for Ty1 retrotransposition. Disruption of 1 or 2 bp between tRNA(iMet) and box 0, 1, or 2.1 dramatically decreases the level of transposition. Compensatory mutations which restore base pairing between the primer and the template restore transposition. Analysis of the reverse transcription intermediates generated inside Ty1 virus-like particles indicates that initiation of minus-strand strong-stop DNA synthesis is affected by mutations disrupting complementarity between Ty1 RNA and primer tRNA(iMet). |
Motorin, Y; Keith, G; Simon, C; Foiret, D; Simos, G; Hurt, E; Grosjean, H The yeast tRNA:pseudouridine synthase Pus1p displays a multisite substrate specificity Article de journal RNA, 4 (7), p. 856-69, 1998, (1355-8382 Journal Article). Résumé | BibTeX | Étiquettes: *RNA, cerevisiae, Cloning, Fractions/metabolism, Fungal, Fungal/metabolism, Gov't, Hydro-Lyases/biosynthesis/genetics/*metabolism, Molecular, Mutation, Non-U.S., Plant/metabolism, post-transcriptional, Precursors/*metabolism, Processing, Proteins/biosynthesis, Proteins/biosynthesis/genetics/metabolism, Pseudouridine/*biosynthesis, Recombinant, RNA, Saccharomyces, Specificity, Subcellular, Substrate, Support, Transfer/*metabolism @article{, title = {The yeast tRNA:pseudouridine synthase Pus1p displays a multisite substrate specificity}, author = { Y. Motorin and G. Keith and C. Simon and D. Foiret and G. Simos and E. Hurt and H. Grosjean}, year = {1998}, date = {1998-01-01}, journal = {RNA}, volume = {4}, number = {7}, pages = {856-69}, abstract = {We have previously shown that the yeast gene PUS1 codes for a tRNA:pseudouridine synthase and that recombinant Pus1p catalyzes, in an intron-dependent way, the formation of psi34 and psi36 in the anticodon loop of the yeast minor tRNA(Ile) in vitro (Simos G et al., 1996, EMBO J 15:2270-2284). Using a set of T7 transcripts of different tRNA genes, we now demonstrate that yeast pseudouridine synthase 1 catalyzes in vitro pseudouridine formation at positions 27 and/or 28 in several yeast cytoplasmic tRNAs and at position 35 in the intron-containing tRNA(Tyr) (anticodon GUA). Thus, Pus1p not only displays a broad specificity toward the RNA substrates, but is also capable of catalyzing the pseudouridine (psi) formation at distinct noncontiguous sites within the same tRNA molecule. The cell-free extract prepared from the yeast strain bearing disrupted gene PUS1 is unable to catalyze the formation of psi27, psi28, psi34, and psi36 in vitro, however, psi35 formation in the intron-containing tRNA(Tyr)(GUA) remains unaffected. Thus, in yeast, only one gene product accounts for tRNA pseudouridylation at positions 27, 28, 34, and 36, whereas for position 35 in tRNA(Tyr), another site-specific tRNA:pseudouridine synthase with overlapping specificity exists. Mapping of pseudouridine residues present in various tRNAs extracted from the PUS1-disrupted strain confirms the in vitro data obtained with the recombinant Pus1p. In addition, they suggest that Pus1p is implicated in modification at positions U26, U65, and U67 in vivo.}, note = {1355-8382 Journal Article}, keywords = {*RNA, cerevisiae, Cloning, Fractions/metabolism, Fungal, Fungal/metabolism, Gov't, Hydro-Lyases/biosynthesis/genetics/*metabolism, Molecular, Mutation, Non-U.S., Plant/metabolism, post-transcriptional, Precursors/*metabolism, Processing, Proteins/biosynthesis, Proteins/biosynthesis/genetics/metabolism, Pseudouridine/*biosynthesis, Recombinant, RNA, Saccharomyces, Specificity, Subcellular, Substrate, Support, Transfer/*metabolism}, pubstate = {published}, tppubtype = {article} } We have previously shown that the yeast gene PUS1 codes for a tRNA:pseudouridine synthase and that recombinant Pus1p catalyzes, in an intron-dependent way, the formation of psi34 and psi36 in the anticodon loop of the yeast minor tRNA(Ile) in vitro (Simos G et al., 1996, EMBO J 15:2270-2284). Using a set of T7 transcripts of different tRNA genes, we now demonstrate that yeast pseudouridine synthase 1 catalyzes in vitro pseudouridine formation at positions 27 and/or 28 in several yeast cytoplasmic tRNAs and at position 35 in the intron-containing tRNA(Tyr) (anticodon GUA). Thus, Pus1p not only displays a broad specificity toward the RNA substrates, but is also capable of catalyzing the pseudouridine (psi) formation at distinct noncontiguous sites within the same tRNA molecule. The cell-free extract prepared from the yeast strain bearing disrupted gene PUS1 is unable to catalyze the formation of psi27, psi28, psi34, and psi36 in vitro, however, psi35 formation in the intron-containing tRNA(Tyr)(GUA) remains unaffected. Thus, in yeast, only one gene product accounts for tRNA pseudouridylation at positions 27, 28, 34, and 36, whereas for position 35 in tRNA(Tyr), another site-specific tRNA:pseudouridine synthase with overlapping specificity exists. Mapping of pseudouridine residues present in various tRNAs extracted from the PUS1-disrupted strain confirms the in vitro data obtained with the recombinant Pus1p. In addition, they suggest that Pus1p is implicated in modification at positions U26, U65, and U67 in vivo. |
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. |
1997 |
Bergdoll, M; Remy, M H; Cagnon, C; Masson, J M; Dumas, P Proline-dependent oligomerization with arm exchange Article de journal Structure, 5 (3), p. 391-401, 1997, (0969-2126 Journal Article). Résumé | BibTeX | Étiquettes: *Acetyltransferases, *Dimerization, *Protein, Acid, Alignment, Amino, Aminotransferases/chemistry, Animals, Aspartate, ATPase/chemistry, Bacterial, Binding, Cattle, Chickens, Comparative, Conformation, Data, Folding, Heart/enzymology, Human, mitochondria, Models, Molecular, Mutagenesis, Na(+)-K(+)-Exchanging, Pancreatic/chemistry, Plant, Proline/*physiology, Protein, Proteins/chemistry, Pyrophosphatases/chemistry, Ribonuclease, Sequence, Site-Directed, Structural, Study, Viral, Viruses/chemistry @article{, title = {Proline-dependent oligomerization with arm exchange}, author = { M. Bergdoll and M. H. Remy and C. Cagnon and J. M. Masson and P. Dumas}, year = {1997}, date = {1997-01-01}, journal = {Structure}, volume = {5}, number = {3}, pages = {391-401}, abstract = {BACKGROUND: Oligomerization is often necessary for protein activity or regulation and its efficiency is fundamental for the cell. The quaternary structure of a large number of oligomers consists of protomers tightly anchored to each other by exchanged arms or swapped domains. However, nothing is known about how the arms can be kept in a favourable conformation before such an oligomerization. RESULTS: Upon examination of such quaternary structures, we observe an extremely frequent occurrence of proline residues at the point where the arm leaves the protomer. Sequence alignment and site-directed mutagenesis confirm the importance of these prolines. The conservation of these residues at the hinge regions can be explained by the constraints that they impose on polypeptide conformation and dynamics: by rigidifying the mainchain, prolines favour extended conformations of arms thus favouring oligomerization, and may prevent interaction of the arms with the core of the protomer. CONCLUSIONS: Hinge prolines can be considered as 'quaternary structure helpers'. The presence of a proline should be considered when searching for a determinant of oligomerization with arm exchange and could be used to engineer synthetic oligomers or to displace a monomers to oligomers equilibrium by mutation of this proline residue.}, note = {0969-2126 Journal Article}, keywords = {*Acetyltransferases, *Dimerization, *Protein, Acid, Alignment, Amino, Aminotransferases/chemistry, Animals, Aspartate, ATPase/chemistry, Bacterial, Binding, Cattle, Chickens, Comparative, Conformation, Data, Folding, Heart/enzymology, Human, mitochondria, Models, Molecular, Mutagenesis, Na(+)-K(+)-Exchanging, Pancreatic/chemistry, Plant, Proline/*physiology, Protein, Proteins/chemistry, Pyrophosphatases/chemistry, Ribonuclease, Sequence, Site-Directed, Structural, Study, Viral, Viruses/chemistry}, pubstate = {published}, tppubtype = {article} } BACKGROUND: Oligomerization is often necessary for protein activity or regulation and its efficiency is fundamental for the cell. The quaternary structure of a large number of oligomers consists of protomers tightly anchored to each other by exchanged arms or swapped domains. However, nothing is known about how the arms can be kept in a favourable conformation before such an oligomerization. RESULTS: Upon examination of such quaternary structures, we observe an extremely frequent occurrence of proline residues at the point where the arm leaves the protomer. Sequence alignment and site-directed mutagenesis confirm the importance of these prolines. The conservation of these residues at the hinge regions can be explained by the constraints that they impose on polypeptide conformation and dynamics: by rigidifying the mainchain, prolines favour extended conformations of arms thus favouring oligomerization, and may prevent interaction of the arms with the core of the protomer. CONCLUSIONS: Hinge prolines can be considered as 'quaternary structure helpers'. The presence of a proline should be considered when searching for a determinant of oligomerization with arm exchange and could be used to engineer synthetic oligomers or to displace a monomers to oligomers equilibrium by mutation of this proline residue. |
Friant, S; Heyman, T; Poch, O; Wilhelm, M; Wilhelm, F X Sequence comparison of the Ty1 and Ty2 elements of the yeast genome supports the structural model of the tRNAiMet-Ty1 RNA reverse transcription initiation complex Article de journal Yeast, 13 (7), p. 639-45, 1997, (0749-503x Journal Article). Résumé | BibTeX | Étiquettes: *Sequence, Acid, Alignment, Amino, Analysis, Base, Data, DNA, Elements/*genetics, Fungal/genetics, Gov't, Met/*chemistry/*genetics, Molecular, Non-U.S., RNA, Sequence, structure, Support, Transfer, Transposable, Yeasts/*genetics @article{, title = {Sequence comparison of the Ty1 and Ty2 elements of the yeast genome supports the structural model of the tRNAiMet-Ty1 RNA reverse transcription initiation complex}, author = { S. Friant and T. Heyman and O. Poch and M. Wilhelm and F. X. Wilhelm}, year = {1997}, date = {1997-01-01}, journal = {Yeast}, volume = {13}, number = {7}, pages = {639-45}, abstract = {In the reverse transcription initiation complex of the yeast Ty1 retrotransposon, interaction between the template RNA and primer tRNAiMet is not limited to base pairing of the primer binding site (PBS) with ten nucleotides at the 3' end of tRNAiMet, but three regions named boxes O, 1 and 2.1 interact with the T and D stems and loops of tRNAiMet. Sequence comparison of 33 Ty1 elements and 13 closely related Ty2 elements found in the yeast genome shows that the nucleotide sequence of all elements is highly conserved in the region spanning the PBS and the three boxes. Since the domain of the template RNA encodes a portion of protein TyA, we have calculated its amino acid profile and its nucleotide profile to evaluate the role played by nucleotide sequence conservation in the selection for TyA function and in the maintenance of base pairing interactions for the priming function of Ty1 RNA. Our results show that the nucleotide sequence conservation of Ty1 RNA is constrained not only by selection for Ty1 function but also by maintenance of a given nucleotide sequence able to base pair with the tRNAiMet in the primer-template initiation complex.}, note = {0749-503x Journal Article}, keywords = {*Sequence, Acid, Alignment, Amino, Analysis, Base, Data, DNA, Elements/*genetics, Fungal/genetics, Gov't, Met/*chemistry/*genetics, Molecular, Non-U.S., RNA, Sequence, structure, Support, Transfer, Transposable, Yeasts/*genetics}, pubstate = {published}, tppubtype = {article} } In the reverse transcription initiation complex of the yeast Ty1 retrotransposon, interaction between the template RNA and primer tRNAiMet is not limited to base pairing of the primer binding site (PBS) with ten nucleotides at the 3' end of tRNAiMet, but three regions named boxes O, 1 and 2.1 interact with the T and D stems and loops of tRNAiMet. Sequence comparison of 33 Ty1 elements and 13 closely related Ty2 elements found in the yeast genome shows that the nucleotide sequence of all elements is highly conserved in the region spanning the PBS and the three boxes. Since the domain of the template RNA encodes a portion of protein TyA, we have calculated its amino acid profile and its nucleotide profile to evaluate the role played by nucleotide sequence conservation in the selection for TyA function and in the maintenance of base pairing interactions for the priming function of Ty1 RNA. Our results show that the nucleotide sequence conservation of Ty1 RNA is constrained not only by selection for Ty1 function but also by maintenance of a given nucleotide sequence able to base pair with the tRNAiMet in the primer-template initiation complex. |
1996 |
Richman, A M; Bulet, Philippe; Hetru, Charles; Barillas-Mury, Carolina; Hoffmann, Jules A; Kafalos, Fotis C Inducible immune factors of the vector mosquito Anopheles gambiae: biochemical purification of a defensin antibacterial peptide and molecular cloning of preprodefensin cDNA Article de journal Insect Mol. Biol., 5 (3), p. 203–210, 1996, ISSN: 0962-1075. Résumé | BibTeX | Étiquettes: Amino Acid, Animals, Anopheles, Base Sequence, Blood Bactericidal Activity, Blood Proteins, Cloning, Complementary, Defensins, DNA, Escherichia coli, Female, Gene Expression, Genes, hoffmann, Insect, Insect Vectors, Larva, M3i, Micrococcus luteus, Molecular, Sequence Homology @article{richman_inducible_1996, title = {Inducible immune factors of the vector mosquito Anopheles gambiae: biochemical purification of a defensin antibacterial peptide and molecular cloning of preprodefensin cDNA}, author = {A M Richman and Philippe Bulet and Charles Hetru and Carolina Barillas-Mury and Jules A Hoffmann and Fotis C Kafalos}, issn = {0962-1075}, year = {1996}, date = {1996-08-01}, journal = {Insect Mol. Biol.}, volume = {5}, number = {3}, pages = {203--210}, abstract = {Larvae of the mosquito vector of human malaria, Anopheles gambiae, were inoculated with bacteria and extracts were biochemically fractionated by reverse-phase HPLC. Multiple induced polypeptides and antibacterial activities were observed following bacterial infection, including a member of the insect defensin family of antibacterial proteins. A cDNA encoding An. gambiae preprodefensin was isolated using PCR primers based on phylogenetically conserved sequences. The mature peptide is highly conserved, but the signal and propeptide segments are not, relative to corresponding defensin sequences of other insects. Defensin expression is induced in response to bacterial infection, in both adult and larval stages. In contrast, pupae express defensin mRNA constitutively. Defensin expression may prove a valuable molecular marker to monitor the An. gambiae host response to infection by parasitic protozoa of medical importance.}, keywords = {Amino Acid, Animals, Anopheles, Base Sequence, Blood Bactericidal Activity, Blood Proteins, Cloning, Complementary, Defensins, DNA, Escherichia coli, Female, Gene Expression, Genes, hoffmann, Insect, Insect Vectors, Larva, M3i, Micrococcus luteus, Molecular, Sequence Homology}, pubstate = {published}, tppubtype = {article} } Larvae of the mosquito vector of human malaria, Anopheles gambiae, were inoculated with bacteria and extracts were biochemically fractionated by reverse-phase HPLC. Multiple induced polypeptides and antibacterial activities were observed following bacterial infection, including a member of the insect defensin family of antibacterial proteins. A cDNA encoding An. gambiae preprodefensin was isolated using PCR primers based on phylogenetically conserved sequences. The mature peptide is highly conserved, but the signal and propeptide segments are not, relative to corresponding defensin sequences of other insects. Defensin expression is induced in response to bacterial infection, in both adult and larval stages. In contrast, pupae express defensin mRNA constitutively. Defensin expression may prove a valuable molecular marker to monitor the An. gambiae host response to infection by parasitic protozoa of medical importance. |
Friant, S; Heyman, T; Wilhelm, F X; Wilhelm, M Role of RNA primers in initiation of minus-strand and plus-strand DNA synthesis of the yeast retrotransposon Ty1 Article de journal Biochimie, 78 (7), p. 674-80, 1996, (0300-9084 Journal Article). Résumé | BibTeX | Étiquettes: *DNA, Acid, Bacterial/*metabolism, Complementary/metabolism, Conformation, Data, DNA, Elements, Gov't, Molecular, Mutagenesis, Non-U.S., Nucleic, Replication, RNA, RNA/*metabolism, Sequence, Support, Transposable @article{, title = {Role of RNA primers in initiation of minus-strand and plus-strand DNA synthesis of the yeast retrotransposon Ty1}, author = { S. Friant and T. Heyman and F. X. Wilhelm and M. Wilhelm}, year = {1996}, date = {1996-01-01}, journal = {Biochimie}, volume = {78}, number = {7}, pages = {674-80}, abstract = {The Ty1 retrotransposon of the yeast Saccharomyces cerevisiae is a long terminal repeat mobile genetic element that transposes through an RNA intermediate. Initiation of minus-strand and plus-strand DNA synthesis are two critical steps during reverse transcription of the retrotransposon genome. Initiation of minus-strand DNA synthesis of the Ty1 element is primed by the cytoplasmic initiator methionine tRNA base paired to the primer binding site near the 5' end of the genomic RNA. A structural probing study of the primer tRNA-Ty1 RNA binary complex reveals that besides interactions between the primer binding site and the last 10 nucleotides at the 3' end of the primer tRNA, three short regions of Ty1 RNA named box 0, box 1 and box 2.1 interact with the T and D stems and loops of the primer tRNA. Some in vivo results underline the functional importance of the nucleotide sequence of the boxes and suggest that extended interactions between genomic Ty1 RNA and the primer tRNA play a role in the reverse transcription pathway. Plus-strand DNA synthesis is initiated from an RNase H resistant oligoribonucleotide spanning a purine-rich sequence, the polypurine tract (PPT). Two sites of initiation located at the 5' boundary of the 3' long terminal repeat (PPT1) and near the middle of the TyB (pol) gene in the integrase coding sequence (PPT2) have been identified in the genome of Ty1. The two PPTs have an identical sequence, TGGGTGGTA. Mutations replacing purines by pyrimidines in this sequence significantly diminish or abolish initiation of plus-strand DNA synthesis. Ty1 elements bearing a mutated PPT2 sequence are not defective for transposition whereas mutations in PPT1 abolish transposition.}, note = {0300-9084 Journal Article}, keywords = {*DNA, Acid, Bacterial/*metabolism, Complementary/metabolism, Conformation, Data, DNA, Elements, Gov't, Molecular, Mutagenesis, Non-U.S., Nucleic, Replication, RNA, RNA/*metabolism, Sequence, Support, Transposable}, pubstate = {published}, tppubtype = {article} } The Ty1 retrotransposon of the yeast Saccharomyces cerevisiae is a long terminal repeat mobile genetic element that transposes through an RNA intermediate. Initiation of minus-strand and plus-strand DNA synthesis are two critical steps during reverse transcription of the retrotransposon genome. Initiation of minus-strand DNA synthesis of the Ty1 element is primed by the cytoplasmic initiator methionine tRNA base paired to the primer binding site near the 5' end of the genomic RNA. A structural probing study of the primer tRNA-Ty1 RNA binary complex reveals that besides interactions between the primer binding site and the last 10 nucleotides at the 3' end of the primer tRNA, three short regions of Ty1 RNA named box 0, box 1 and box 2.1 interact with the T and D stems and loops of the primer tRNA. Some in vivo results underline the functional importance of the nucleotide sequence of the boxes and suggest that extended interactions between genomic Ty1 RNA and the primer tRNA play a role in the reverse transcription pathway. Plus-strand DNA synthesis is initiated from an RNase H resistant oligoribonucleotide spanning a purine-rich sequence, the polypurine tract (PPT). Two sites of initiation located at the 5' boundary of the 3' long terminal repeat (PPT1) and near the middle of the TyB (pol) gene in the integrase coding sequence (PPT2) have been identified in the genome of Ty1. The two PPTs have an identical sequence, TGGGTGGTA. Mutations replacing purines by pyrimidines in this sequence significantly diminish or abolish initiation of plus-strand DNA synthesis. Ty1 elements bearing a mutated PPT2 sequence are not defective for transposition whereas mutations in PPT1 abolish transposition. |
Friant, S; Heyman, T; Wilhelm, M L; Wilhelm, F X Extended interactions between the primer tRNAi(Met) and genomic RNA of the yeast Ty1 retrotransposon Article de journal Nucleic Acids Res, 24 (3), p. 441-9, 1996, (0305-1048 Journal Article). Résumé | BibTeX | Étiquettes: Acid, Base, cerevisiae, Conformation, Data, Gov't, Met/genetics/*metabolism, Molecular, Mutation, Non-U.S., Nucleic, Retroelements/*genetics, RNA, RNA/genetics/*metabolism, Saccharomyces, Sequence, structure, Support, Transfer @article{, title = {Extended interactions between the primer tRNAi(Met) and genomic RNA of the yeast Ty1 retrotransposon}, author = { S. Friant and T. Heyman and M. L. Wilhelm and F. X. Wilhelm}, year = {1996}, date = {1996-01-01}, journal = {Nucleic Acids Res}, volume = {24}, number = {3}, pages = {441-9}, abstract = {Reverse transcription of the yeast Ty1 retrotransposon is primed by tRNAi(Met) base paired to the primer binding site near the 5'-end of Ty1 genomic RNA. To understand the molecular basis of the tRNAi(Met)-Ty1 RNA interaction the secondary structure of the binary complex was analysed. Enzymatic probes were used to test the conformation of tRNAi(Met) and of Ty1 RNA in the free form and in the complex. A secondary structure model of the tRNAi(Met) Ty1 RNA complex consistent with the probing data was constructed with the help of a computer program. The model shows that besides interactions between the primer binding site and the last 10 nt at the 3'-end of tRNAi(Met), three short regions of Ty1 RNA named boxes 0, 1 and 2.1 interact with the T and D stems and loops of tRNAiMet. Mutations were made in the boxes or in the complementary sequences of tRNAi(Met) to study the contribution of these sequences to formation of the complex. We find that interaction with at least one of the two boxes 0 or 1 is absolutely required for efficient annealing of the two RNAs. Sequence comparison showing that the primary sequence of the boxes is strictly conserved in Ty1 and Ty2 elements and previously published in vivo results underline the functional importance of the primary sequence of the boxes and suggest that extended interactions between genomic Ty1 RNA and the primary tRNAi(Met) play a role in the reverse transcription pathway.}, note = {0305-1048 Journal Article}, keywords = {Acid, Base, cerevisiae, Conformation, Data, Gov't, Met/genetics/*metabolism, Molecular, Mutation, Non-U.S., Nucleic, Retroelements/*genetics, RNA, RNA/genetics/*metabolism, Saccharomyces, Sequence, structure, Support, Transfer}, pubstate = {published}, tppubtype = {article} } Reverse transcription of the yeast Ty1 retrotransposon is primed by tRNAi(Met) base paired to the primer binding site near the 5'-end of Ty1 genomic RNA. To understand the molecular basis of the tRNAi(Met)-Ty1 RNA interaction the secondary structure of the binary complex was analysed. Enzymatic probes were used to test the conformation of tRNAi(Met) and of Ty1 RNA in the free form and in the complex. A secondary structure model of the tRNAi(Met) Ty1 RNA complex consistent with the probing data was constructed with the help of a computer program. The model shows that besides interactions between the primer binding site and the last 10 nt at the 3'-end of tRNAi(Met), three short regions of Ty1 RNA named boxes 0, 1 and 2.1 interact with the T and D stems and loops of tRNAiMet. Mutations were made in the boxes or in the complementary sequences of tRNAi(Met) to study the contribution of these sequences to formation of the complex. We find that interaction with at least one of the two boxes 0 or 1 is absolutely required for efficient annealing of the two RNAs. Sequence comparison showing that the primary sequence of the boxes is strictly conserved in Ty1 and Ty2 elements and previously published in vivo results underline the functional importance of the primary sequence of the boxes and suggest that extended interactions between genomic Ty1 RNA and the primary tRNAi(Met) play a role in the reverse transcription pathway. |
de Barros, Pais J P; Keith, G; Adlouni, El C; Glasser, A L; Mack, G; Dirheimer, G; Desgres, J 2'-O-methyl-5-formylcytidine (f5Cm), a new modified nucleotide at the 'wobble' of two cytoplasmic tRNAs Leu (NAA) from bovine liver Article de journal Nucleic Acids Res, 24 (8), p. 1489-96, 1996, (0305-1048 Journal Article). Résumé | BibTeX | Étiquettes: &, Acid, Acyl/*chemistry/isolation, Amino, Animals, Base, Borohydrides/chemistry, Cattle, Cells, Conformation, Cytidine/*analogs, Cytoplasm, Data, derivatives/chemistry/isolation, Fragmentography, Gov't, Hela, Human, Liver/*chemistry, Mass, Molecular, Non-U.S., Nucleic, purification, RNA, Sequence, structure, Support, Transfer @article{, title = {2'-O-methyl-5-formylcytidine (f5Cm), a new modified nucleotide at the 'wobble' of two cytoplasmic tRNAs Leu (NAA) from bovine liver}, author = { J. P. Pais de Barros and G. Keith and C. El Adlouni and A. L. Glasser and G. Mack and G. Dirheimer and J. Desgres}, year = {1996}, date = {1996-01-01}, journal = {Nucleic Acids Res}, volume = {24}, number = {8}, pages = {1489-96}, abstract = {The nucleotide analysis of a cytoplasmic tRNA(Leu) isolated from bovine liver revealed the presence of an unknown modified nucleotide N. The corresponding N nucleoside was isolated by different enzymatic and chromatographic protocols from a partially purified preparation of this tRNA(Leu). Its chemical characterization was determined from its chromatographic properties, UV-absorption spectroscopy and mass spectrometric measurements, as well as from those of the borohydride reduced N nucleoside and its etheno-trimethylsilyl derivative. The structure of N was established as 2'-O-methyl-5-formylcytidine (f5CM), and its reduced derivative as 2'-O-methyl-5-hydroxy-methylcytidine (om5Cm). By sequencing the bovine liver tRNA(Leu), the structure of the anticodon was determined as f5CmAA. In addition, the nucleotide sequence showed two primary structures differing only by the nucleotide 47c which is either uridine or adenosine. The two slightly differing bovine liver tRNAs-Leu(f5CmAA) are the only tRNAs so far sequenced which contain f5Cm. The role of such a modified cytidine at the first position of the anticodon is discussed in terms of decoding properties for the UUG and UUA leucine codons. Recently, precise evidence was obtained for the presence of f5Cm at the same position in tRNAs(Leu)(NAA) isolated from rabbit and lamb liver. Therefore, the 2'-O-methyl-5-formyl modification of cytidine at position 34 could be a general feature of cytoplasmic tRNAs(Leu)(NAA) in mammals.}, note = {0305-1048 Journal Article}, keywords = {&, Acid, Acyl/*chemistry/isolation, Amino, Animals, Base, Borohydrides/chemistry, Cattle, Cells, Conformation, Cytidine/*analogs, Cytoplasm, Data, derivatives/chemistry/isolation, Fragmentography, Gov't, Hela, Human, Liver/*chemistry, Mass, Molecular, Non-U.S., Nucleic, purification, RNA, Sequence, structure, Support, Transfer}, pubstate = {published}, tppubtype = {article} } The nucleotide analysis of a cytoplasmic tRNA(Leu) isolated from bovine liver revealed the presence of an unknown modified nucleotide N. The corresponding N nucleoside was isolated by different enzymatic and chromatographic protocols from a partially purified preparation of this tRNA(Leu). Its chemical characterization was determined from its chromatographic properties, UV-absorption spectroscopy and mass spectrometric measurements, as well as from those of the borohydride reduced N nucleoside and its etheno-trimethylsilyl derivative. The structure of N was established as 2'-O-methyl-5-formylcytidine (f5CM), and its reduced derivative as 2'-O-methyl-5-hydroxy-methylcytidine (om5Cm). By sequencing the bovine liver tRNA(Leu), the structure of the anticodon was determined as f5CmAA. In addition, the nucleotide sequence showed two primary structures differing only by the nucleotide 47c which is either uridine or adenosine. The two slightly differing bovine liver tRNAs-Leu(f5CmAA) are the only tRNAs so far sequenced which contain f5Cm. The role of such a modified cytidine at the first position of the anticodon is discussed in terms of decoding properties for the UUG and UUA leucine codons. Recently, precise evidence was obtained for the presence of f5Cm at the same position in tRNAs(Leu)(NAA) isolated from rabbit and lamb liver. Therefore, the 2'-O-methyl-5-formyl modification of cytidine at position 34 could be a general feature of cytoplasmic tRNAs(Leu)(NAA) in mammals. |
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. |
Cornet, B; Bonmatin, J M; Hetru, Charles; Hoffmann, Jules A; Ptak, M; Vovelle, F Refined three-dimensional solution structure of insect defensin A Article de journal Structure, 3 (5), p. 435–448, 1995, ISSN: 0969-2126. Résumé | BibTeX | Étiquettes: Amino Acid, Animals, Bacteriolysis, Chemistry, Defensins, Diptera, Gram-Positive Bacteria, hoffmann, Hydrogen Bonding, Insect Hormones, M3i, Magnetic Resonance Spectroscopy, Models, Molecular, Physical, Physicochemical Phenomena, Protein Conformation, Recombinant Proteins, Sequence Homology, Solutions, Structure-Activity Relationship @article{cornet_refined_1995, title = {Refined three-dimensional solution structure of insect defensin A}, author = {B Cornet and J M Bonmatin and Charles Hetru and Jules A Hoffmann and M Ptak and F Vovelle}, issn = {0969-2126}, year = {1995}, date = {1995-05-01}, journal = {Structure}, volume = {3}, number = {5}, pages = {435--448}, abstract = {BACKGROUND: Insect defensin A is a basic 4 kDa protein secreted by Phormia terranovae larvae in response to bacterial challenges or injuries. Previous biological tests suggest that the bacterial cytoplasmic membrane is the target of defensin A. The structural study of this protein is the first step towards establishing a structure-activity relationship and forms the basis for understanding its antibiotic activity at the molecular level. RESULTS: We describe a refined model of the three-dimensional structure of defensin A derived from an extensive analysis of 786 inter-proton nuclear Overhauser effects. The backbone fold involves an N-terminal loop and an alpha-helical fragment followed by an antiparallel beta-structure. The helix and the beta-structure are connected by two of the three disulphide bridges present in defensin A, forming a so-called 'cysteine-stabilized alpha beta' (CS alpha beta) motif. The N-terminal loop, which is locally well defined, can occupy different positions with respect to the other moieties of the molecule. CONCLUSIONS: The CS alpha beta motif, which forms the core of the defensin A structure, appears to be a common organization for several families of small proteins with toxic properties. The distribution of amino acid side chains in the protein structure creates several hydrophobic or hydrophilic patches. This leads us to propose that the initial step in the action of positively charged defensin A molecules with cytoplasmic membranes may involve interactions with acidic phospholipids.}, keywords = {Amino Acid, Animals, Bacteriolysis, Chemistry, Defensins, Diptera, Gram-Positive Bacteria, hoffmann, Hydrogen Bonding, Insect Hormones, M3i, Magnetic Resonance Spectroscopy, Models, Molecular, Physical, Physicochemical Phenomena, Protein Conformation, Recombinant Proteins, Sequence Homology, Solutions, Structure-Activity Relationship}, pubstate = {published}, tppubtype = {article} } BACKGROUND: Insect defensin A is a basic 4 kDa protein secreted by Phormia terranovae larvae in response to bacterial challenges or injuries. Previous biological tests suggest that the bacterial cytoplasmic membrane is the target of defensin A. The structural study of this protein is the first step towards establishing a structure-activity relationship and forms the basis for understanding its antibiotic activity at the molecular level. RESULTS: We describe a refined model of the three-dimensional structure of defensin A derived from an extensive analysis of 786 inter-proton nuclear Overhauser effects. The backbone fold involves an N-terminal loop and an alpha-helical fragment followed by an antiparallel beta-structure. The helix and the beta-structure are connected by two of the three disulphide bridges present in defensin A, forming a so-called 'cysteine-stabilized alpha beta' (CS alpha beta) motif. The N-terminal loop, which is locally well defined, can occupy different positions with respect to the other moieties of the molecule. CONCLUSIONS: The CS alpha beta motif, which forms the core of the defensin A structure, appears to be a common organization for several families of small proteins with toxic properties. The distribution of amino acid side chains in the protein structure creates several hydrophobic or hydrophilic patches. This leads us to propose that the initial step in the action of positively charged defensin A molecules with cytoplasmic membranes may involve interactions with acidic phospholipids. |
Gabryszuk, J; Keith, G; Monko, M; Kuligowska, E; Dirheimer, G; Szarkowski, J W; Przykorska, A Structural specificity of nuclease from wheat chloroplasts stroma Article de journal Nucleic Acids Symp Ser, (33), p. 115-9, 1995, (0261-3166 Journal Article). Résumé | BibTeX | Étiquettes: &, Acid, Asp/chemistry/genetics/metabolism, Base, Binding, Chloroplasts/*enzymology, Conformation, Data, Endonucleases/isolation, Fungal/chemistry/genetics/metabolism, Gov't, Molecular, Non-U.S., Nucleic, Phe/chemistry/genetics/metabolism, purification/*metabolism, RNA, RNA/chemistry/metabolism, Sequence, Sites, Specificity, Substrate, Support, Transfer, Triticum/*enzymology @article{, title = {Structural specificity of nuclease from wheat chloroplasts stroma}, author = { J. Gabryszuk and G. Keith and M. Monko and E. Kuligowska and G. Dirheimer and J. W. Szarkowski and A. Przykorska}, year = {1995}, date = {1995-01-01}, journal = {Nucleic Acids Symp Ser}, number = {33}, pages = {115-9}, abstract = {A single-strand-specific nuclease from wheat chloroplasts (ChS nuclease) was tested as a tool for RNA secondary and tertiary structure investigations, using yeast tRNA(Phe) and yeast tRNA(Asp) as models. In tRNA(Phe) the nuclease introduced main primary cleavages at positions U33, A35 and A36 in the anticodon-loop and G18 and G19 in the D-loop. In tRNA(Asp) the main primary cleavages occurred at positions U33, G34 and U35 in the anticodon-loop and the lower one at position C20:1 in the D-loop. No primary cleavages were observed within the double-stranded stems. Because ChS nuclease has (i) a low molecular weight, (ii) a wide pH range of action (5.0 to 7.5) (iii) no divalent cation requirement in the reaction mixture and (iv) can be obtained as a pure protein in rather large quantities it appeared to be a very good tool for secondary and tertiary structural studies of RNAs.}, note = {0261-3166 Journal Article}, keywords = {&, Acid, Asp/chemistry/genetics/metabolism, Base, Binding, Chloroplasts/*enzymology, Conformation, Data, Endonucleases/isolation, Fungal/chemistry/genetics/metabolism, Gov't, Molecular, Non-U.S., Nucleic, Phe/chemistry/genetics/metabolism, purification/*metabolism, RNA, RNA/chemistry/metabolism, Sequence, Sites, Specificity, Substrate, Support, Transfer, Triticum/*enzymology}, pubstate = {published}, tppubtype = {article} } A single-strand-specific nuclease from wheat chloroplasts (ChS nuclease) was tested as a tool for RNA secondary and tertiary structure investigations, using yeast tRNA(Phe) and yeast tRNA(Asp) as models. In tRNA(Phe) the nuclease introduced main primary cleavages at positions U33, A35 and A36 in the anticodon-loop and G18 and G19 in the D-loop. In tRNA(Asp) the main primary cleavages occurred at positions U33, G34 and U35 in the anticodon-loop and the lower one at position C20:1 in the D-loop. No primary cleavages were observed within the double-stranded stems. Because ChS nuclease has (i) a low molecular weight, (ii) a wide pH range of action (5.0 to 7.5) (iii) no divalent cation requirement in the reaction mixture and (iv) can be obtained as a pure protein in rather large quantities it appeared to be a very good tool for secondary and tertiary structural studies of RNAs. |
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. |
Keith, G; Dirheimer, G Postlabeling: a sensitive method for studying DNA adducts and their role in carcinogenesis Article de journal Curr Opin Biotechnol, 6 (1), p. 3-11, 1995, (0958-1669 Journal Article Review Review, Academic). Résumé | BibTeX | Étiquettes: *Cell, *Genome, Adducts/*analysis, and, Animals, Base, Cell, Conditions/genetics/pathology, Data, Dilution, Division, DNA, Genetic, Gov't, Human, Models, Molecular, Mutagenesis, Neoplasms/*genetics/pathology, Neoplastic, Non-U.S., Phosphorus, Precancerous, Radioisotope, Radioisotopes, Sensitivity, Sequence, Specificity, Support, Technique, Transformation, Xenobiotics @article{, title = {Postlabeling: a sensitive method for studying DNA adducts and their role in carcinogenesis}, author = { G. Keith and G. Dirheimer}, year = {1995}, date = {1995-01-01}, journal = {Curr Opin Biotechnol}, volume = {6}, number = {1}, pages = {3-11}, abstract = {The covalent binding of xenobiotics to DNA is an important trigger of the multistage process that leads to carcinogenesis. 32P-postlabeling represents a highly sensitive method for biomonitoring exposure to genotoxic agents and for cancer risk assessment; it is capable of detecting less than one DNA adduct per human genome. Recent improvements to the technique have shown that the resistance of adducted DNA to enzyme digestion may lead to an overestimation of the number of different adducts present in a sample.}, note = {0958-1669 Journal Article Review Review, Academic}, keywords = {*Cell, *Genome, Adducts/*analysis, and, Animals, Base, Cell, Conditions/genetics/pathology, Data, Dilution, Division, DNA, Genetic, Gov't, Human, Models, Molecular, Mutagenesis, Neoplasms/*genetics/pathology, Neoplastic, Non-U.S., Phosphorus, Precancerous, Radioisotope, Radioisotopes, Sensitivity, Sequence, Specificity, Support, Technique, Transformation, Xenobiotics}, pubstate = {published}, tppubtype = {article} } The covalent binding of xenobiotics to DNA is an important trigger of the multistage process that leads to carcinogenesis. 32P-postlabeling represents a highly sensitive method for biomonitoring exposure to genotoxic agents and for cancer risk assessment; it is capable of detecting less than one DNA adduct per human genome. Recent improvements to the technique have shown that the resistance of adducted DNA to enzyme digestion may lead to an overestimation of the number of different adducts present in a sample. |
O'Connor, M; Brunelli, C A; Firpo, M A; Gregory, S T; Lieberman, K R; Lodmell, J S; Moine, H; Ryk, Van D I; Dahlberg, A E Genetic probes of ribosomal RNA function Article de journal Biochem Cell Biol, 73 (11-12), p. 859-68, 1995, (0829-8211 Journal Article Review Review, Tutorial). Résumé | BibTeX | Étiquettes: 16S/genetics, Acid, Base, Conformation, Data, Gov't, Messenger/genetics, Molecular, Mutation, Non-U.S., Nucleic, P.H.S., Probes, Ribosomal, Ribosomal/*genetics, RNA, Sequence, Support, Transfer/genetics, U.S. @article{, title = {Genetic probes of ribosomal RNA function}, author = { M. O'Connor and C. A. Brunelli and M. A. Firpo and S. T. Gregory and K. R. Lieberman and J. S. Lodmell and H. Moine and D. I. Van Ryk and A. E. Dahlberg}, year = {1995}, date = {1995-01-01}, journal = {Biochem Cell Biol}, volume = {73}, number = {11-12}, pages = {859-68}, abstract = {We have used a genetic approach to uncover the functional roles of rRNA in protein synthesis. Mutations were constructed in a cloned rrn operon by site-directed mutagenesis or isolated by genetic selections following random mutagenesis. We have identified mutations that affect each step in the process of translation. The data are consistent with the results of biochemical and phylogenetic analyses but, in addition, have provided novel information on regions of rRNA not previously investigated.}, note = {0829-8211 Journal Article Review Review, Tutorial}, keywords = {16S/genetics, Acid, Base, Conformation, Data, Gov't, Messenger/genetics, Molecular, Mutation, Non-U.S., Nucleic, P.H.S., Probes, Ribosomal, Ribosomal/*genetics, RNA, Sequence, Support, Transfer/genetics, U.S.}, pubstate = {published}, tppubtype = {article} } We have used a genetic approach to uncover the functional roles of rRNA in protein synthesis. Mutations were constructed in a cloned rrn operon by site-directed mutagenesis or isolated by genetic selections following random mutagenesis. We have identified mutations that affect each step in the process of translation. The data are consistent with the results of biochemical and phylogenetic analyses but, in addition, have provided novel information on regions of rRNA not previously investigated. |
1994 |
Fehlbaum, P; Bulet, Philippe; Michaut, L; Lagueux, Marie; Broekaert, W F; Hetru, Charles; Hoffmann, Jules A Insect immunity. Septic injury of Drosophila induces the synthesis of a potent antifungal peptide with sequence homology to plant antifungal peptides Article de journal J. Biol. Chem., 269 (52), p. 33159–33163, 1994, ISSN: 0021-9258. Résumé | BibTeX | Étiquettes: Amino Acid, Animals, Antifungal Agents, Base Sequence, Cloning, Complementary, DNA, hoffmann, Insect Proteins, M3i, Male, messenger, Microbial Sensitivity Tests, Molecular, Peptide Biosynthesis, Peptides, Plants, Protein Biosynthesis, Protein Precursors, Proteins, RNA, Sequence Homology @article{fehlbaum_insect_1994, title = {Insect immunity. Septic injury of Drosophila induces the synthesis of a potent antifungal peptide with sequence homology to plant antifungal peptides}, author = {P Fehlbaum and Philippe Bulet and L Michaut and Marie Lagueux and W F Broekaert and Charles Hetru and Jules A Hoffmann}, issn = {0021-9258}, year = {1994}, date = {1994-12-01}, journal = {J. Biol. Chem.}, volume = {269}, number = {52}, pages = {33159--33163}, abstract = {In response to a septic injury (pricking with a bacteria-soaked needle) larvae and adults of Drosophila produce considerable amounts of a 44-residue peptide containing 8 cysteines engaged in intramolecular disulfide bridges. The peptide is synthesized in the fat body, a functional homologue of the mammalian liver, and secreted into the blood of the insect. It exhibits potent antifungal activity but is inactive against bacteria. This novel inducible peptide, which we propose to name drosomycin, shows a significant homology with a family of 5-kDa cysteine-rich plant antifungal peptides recently isolated from seeds of Brassicaceae. This finding underlines that plants and insects can rely on similar molecules in their innate host defense.}, keywords = {Amino Acid, Animals, Antifungal Agents, Base Sequence, Cloning, Complementary, DNA, hoffmann, Insect Proteins, M3i, Male, messenger, Microbial Sensitivity Tests, Molecular, Peptide Biosynthesis, Peptides, Plants, Protein Biosynthesis, Protein Precursors, Proteins, RNA, Sequence Homology}, pubstate = {published}, tppubtype = {article} } In response to a septic injury (pricking with a bacteria-soaked needle) larvae and adults of Drosophila produce considerable amounts of a 44-residue peptide containing 8 cysteines engaged in intramolecular disulfide bridges. The peptide is synthesized in the fat body, a functional homologue of the mammalian liver, and secreted into the blood of the insect. It exhibits potent antifungal activity but is inactive against bacteria. This novel inducible peptide, which we propose to name drosomycin, shows a significant homology with a family of 5-kDa cysteine-rich plant antifungal peptides recently isolated from seeds of Brassicaceae. This finding underlines that plants and insects can rely on similar molecules in their innate host defense. |
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. |
Dumas, P; Bergdoll, M; Cagnon, C; Masson, J M Crystal structure and site-directed mutagenesis of a bleomycin resistance protein and their significance for drug sequestering Article de journal EMBO J, 13 (11), p. 2483-92, 1994, (0261-4189 Journal Article). Résumé | BibTeX | Étiquettes: *Acetyltransferases, &, Acid, Amino, Bacterial, Bacterial/*genetics, Base, Binding, Bleomycin/*metabolism/pharmacology, Conformation, Crystallization, Crystallography, Data, Drug, Fusion, Genes, Gov't, Microbial/genetics, Models, Molecular, Mutagenesis, Non-U.S., Protein, Proteins/*chemistry/genetics/isolation, Proteins/isolation, purification, purification/metabolism, Recombinant, Relationship, Resistance, Secondary, Sequence, Site-Directed, Sites, Structural, structure, Structure-Activity, Support, X-Ray @article{, title = {Crystal structure and site-directed mutagenesis of a bleomycin resistance protein and their significance for drug sequestering}, author = { P. Dumas and M. Bergdoll and C. Cagnon and J. M. Masson}, year = {1994}, date = {1994-01-01}, journal = {EMBO J}, volume = {13}, number = {11}, pages = {2483-92}, abstract = {The antibiotic bleomycin, a strong DNA cutting agent, is naturally produced by actinomycetes which have developed a resistance mechanism against such a lethal compound. The crystal structure, at 2.3 A resolution, of a bleomycin resistance protein of 14 kDa reveals a structure in two halves with the same alpha/beta fold despite no sequence similarity. The crystal packing shows compact dimers with a hydrophobic interface and involved in mutual chain exchange. Two independent solution studies (analytical centrifugation and light scattering) showed that this dimeric form is not a packing artefact but is indeed the functional one. Furthermore, light scattering also showed that one dimer binds two antibiotic molecules as expected. A crevice located at the dimer interface, as well as the results of a site-directed mutagenesis study, led to a model wherein two bleomycin molecules are completely sequestered by one dimer. This provides a novel insight into antibiotic resistance due to drug sequestering, and probably also into drug transport and excretion.}, note = {0261-4189 Journal Article}, keywords = {*Acetyltransferases, &, Acid, Amino, Bacterial, Bacterial/*genetics, Base, Binding, Bleomycin/*metabolism/pharmacology, Conformation, Crystallization, Crystallography, Data, Drug, Fusion, Genes, Gov't, Microbial/genetics, Models, Molecular, Mutagenesis, Non-U.S., Protein, Proteins/*chemistry/genetics/isolation, Proteins/isolation, purification, purification/metabolism, Recombinant, Relationship, Resistance, Secondary, Sequence, Site-Directed, Sites, Structural, structure, Structure-Activity, Support, X-Ray}, pubstate = {published}, tppubtype = {article} } The antibiotic bleomycin, a strong DNA cutting agent, is naturally produced by actinomycetes which have developed a resistance mechanism against such a lethal compound. The crystal structure, at 2.3 A resolution, of a bleomycin resistance protein of 14 kDa reveals a structure in two halves with the same alpha/beta fold despite no sequence similarity. The crystal packing shows compact dimers with a hydrophobic interface and involved in mutual chain exchange. Two independent solution studies (analytical centrifugation and light scattering) showed that this dimeric form is not a packing artefact but is indeed the functional one. Furthermore, light scattering also showed that one dimer binds two antibiotic molecules as expected. A crevice located at the dimer interface, as well as the results of a site-directed mutagenesis study, led to a model wherein two bleomycin molecules are completely sequestered by one dimer. This provides a novel insight into antibiotic resistance due to drug sequestering, and probably also into drug transport and excretion. |
Heyman, T; Agoutin, B; Fix, C; Dirheimer, G; Keith, G Yeast serine isoacceptor tRNAs: variations of their content as a function of growth conditions and primary structure of the minor tRNA(Ser)GCU Article de journal FEBS Lett, 347 (2-3), p. 143-6, 1994, (0014-5793 Journal Article). Résumé | BibTeX | Étiquettes: &, Acid, Anticodon, Base, cerevisiae/*genetics/*growth, Conformation, Culture, Data, development, Fungal/*chemistry, Galactose, Hybridization, Media, Molecular, Nucleic, Probes, RNA, Saccharomyces, Sequence, Ser/analysis/*chemistry, Transfer, Transfer/*chemistry @article{, title = {Yeast serine isoacceptor tRNAs: variations of their content as a function of growth conditions and primary structure of the minor tRNA(Ser)GCU}, author = { T. Heyman and B. Agoutin and C. Fix and G. Dirheimer and G. Keith}, year = {1994}, date = {1994-01-01}, journal = {FEBS Lett}, volume = {347}, number = {2-3}, pages = {143-6}, abstract = {The primary structure of Saccharomyces cerevisiae tRNA(Ser)GCU is presented (EMBL database accession No. X74268 S. cerevisiae tRNA-Ser). In addition, quantitation of the relative amounts of serine isoaccepting tRNAs in yeast grown on different media showed that the minor tRNA(Ser)GCU decreased while the major tRNA(Ser)AGA increased as the growth rate and the cellular protein content increased. The minor species, tRNA(Ser)CGA and tRNA(Ser)UGA, were not separated by our gel system, however, taken together they appeared to vary in the same way as tRNA(Ser)GCU. These data suggest a growth rate dependence of yeast tRNAs similar to that previously described for E. coli tRNAs.}, note = {0014-5793 Journal Article}, keywords = {&, Acid, Anticodon, Base, cerevisiae/*genetics/*growth, Conformation, Culture, Data, development, Fungal/*chemistry, Galactose, Hybridization, Media, Molecular, Nucleic, Probes, RNA, Saccharomyces, Sequence, Ser/analysis/*chemistry, Transfer, Transfer/*chemistry}, pubstate = {published}, tppubtype = {article} } The primary structure of Saccharomyces cerevisiae tRNA(Ser)GCU is presented (EMBL database accession No. X74268 S. cerevisiae tRNA-Ser). In addition, quantitation of the relative amounts of serine isoaccepting tRNAs in yeast grown on different media showed that the minor tRNA(Ser)GCU decreased while the major tRNA(Ser)AGA increased as the growth rate and the cellular protein content increased. The minor species, tRNA(Ser)CGA and tRNA(Ser)UGA, were not separated by our gel system, however, taken together they appeared to vary in the same way as tRNA(Ser)GCU. These data suggest a growth rate dependence of yeast tRNAs similar to that previously described for E. coli tRNAs. |
Moine, H; Dahlberg, A E Mutations in helix 34 of Escherichia coli 16 S ribosomal RNA have multiple effects on ribosome function and synthesis Article de journal J Mol Biol, 243 (3), p. 402-12, 1994, (0022-2836 Journal Article). Résumé | BibTeX | Étiquettes: *Mutation, *Nucleic, *Translation, &, 16S/*chemistry/genetics, Acid, Base, beta-Galactosidase/genetics, Codon, coli/*genetics/growth, Conformation, Data, development, Escherichia, Genetic, Gov't, Molecular, Non-U.S., P.H.S., Ribosomal, Ribosomes/*metabolism, RNA, Sequence, Support, Terminator, U.S. @article{, title = {Mutations in helix 34 of Escherichia coli 16 S ribosomal RNA have multiple effects on ribosome function and synthesis}, author = { H. Moine and A. E. Dahlberg}, year = {1994}, date = {1994-01-01}, journal = {J Mol Biol}, volume = {243}, number = {3}, pages = {402-12}, abstract = {Helix 34 of E. coli 16 S rRNA (1046 to 1067 and 1189 to 1211) has been proposed to participate directly in the termination of translation at UGA stop codons. We have constructed mutations in this helix in plasmid-encoded rDNA to explore the specific functional roles of the sequence UCAUCA (1199 to 1204) and a secondary structure also involving positions 1054 and 1057-1058. The rRNA mutations were analyzed for their effects on in vivo translational accuracy (stop codon readthrough and frameshifting) as well as growth rate, ribosome synthesis and incorporation into polysomes. Mutations at positions 1054, 1057, 1058, 1199 and 1200 had significant effects on translational accuracy, causing non-specific readthrough of all three stop codons as well as enhanced +1 and -1 frameshifting. Mutations at 1202 and 1203, however, had no effect. The incorporation of deleterious mutant subunits into 70 S ribosomes and polysomes was severely reduced and was associated with a slower growth rate and increased synthesis of host-encoded ribosomes. These data support the proposal that helix 34 is an essential component of the decoding center of the 30 S ribosomal subunit and is not restricted in function to UGA-codon specific termination.}, note = {0022-2836 Journal Article}, keywords = {*Mutation, *Nucleic, *Translation, &, 16S/*chemistry/genetics, Acid, Base, beta-Galactosidase/genetics, Codon, coli/*genetics/growth, Conformation, Data, development, Escherichia, Genetic, Gov't, Molecular, Non-U.S., P.H.S., Ribosomal, Ribosomes/*metabolism, RNA, Sequence, Support, Terminator, U.S.}, pubstate = {published}, tppubtype = {article} } Helix 34 of E. coli 16 S rRNA (1046 to 1067 and 1189 to 1211) has been proposed to participate directly in the termination of translation at UGA stop codons. We have constructed mutations in this helix in plasmid-encoded rDNA to explore the specific functional roles of the sequence UCAUCA (1199 to 1204) and a secondary structure also involving positions 1054 and 1057-1058. The rRNA mutations were analyzed for their effects on in vivo translational accuracy (stop codon readthrough and frameshifting) as well as growth rate, ribosome synthesis and incorporation into polysomes. Mutations at positions 1054, 1057, 1058, 1199 and 1200 had significant effects on translational accuracy, causing non-specific readthrough of all three stop codons as well as enhanced +1 and -1 frameshifting. Mutations at 1202 and 1203, however, had no effect. The incorporation of deleterious mutant subunits into 70 S ribosomes and polysomes was severely reduced and was associated with a slower growth rate and increased synthesis of host-encoded ribosomes. These data support the proposal that helix 34 is an essential component of the decoding center of the 30 S ribosomal subunit and is not restricted in function to UGA-codon specific termination. |