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Wilma Ziebuhr - One of the best experts on this subject based on the ideXlab platform.
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the bacterial Insertion Sequence element is256 occurs preferentially in nosocomial staphylococcus epidermidis isolates association with biofilm formation and resistance to aminoglycosides
Infection and Immunity, 2004Co-Authors: Svetlana Kozitskaya, Katja Dietrich, Reinhard Marre, Kurt G Naber, Wilma ZiebuhrAbstract:Staphylococcus epidermidis is a normal constituent of the healthy human microflora, but it is also the most common cause of nosocomial infections associated with the use of indwelling medical devices. Isolates from device-associated infections are known for their pronounced phenotypic and genetic variability, and in this study we searched for factors that might contribute to this flexibility. We show that mutator phenotypes, which exhibit elevated spontaneous mutation rates, are rare among both pathogenic and commensal S. epidermidis strains. However, the study revealed that, in contrast to those of commensal strains, the genomes of clinical S. epidermidis strains carry multiple copies of the Insertion Sequence IS256, while other typical staphylococcal Insertion Sequences, such as IS257 and IS1272, are distributed equally among saprophytic and clinical isolates. Moreover, detection of IS256 was found to be associated with biofilm formation and the presence of the icaADBC operon as well as with gentamicin and oxacillin resistance in the clinical strains. The data suggest that IS256 is a characteristic element in the genome of multiresistant nosocomial S. epidermidis isolates that might be involved in the flexibility and adaptation of the genome in clinical isolates.
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a novel mechanism of phase variation of virulence in staphylococcus epidermidis evidence for control of the polysaccharide intercellular adhesin synthesis by alternating Insertion and excision of the Insertion Sequence element is256
Molecular Microbiology, 1999Co-Authors: Wilma Ziebuhr, Vanessa Krimmer, Shwan Rachid, Isabel Lossner, Friedrich Gotz, Jorg HackerAbstract:Biofilm formation of Staphylococcus epidermidis on smooth polymer surfaces has been shown to be mediated by the ica operon. Upon activation of this operon, a polysaccharide intercellular adhesin (PIA) is synthesized that supports bacterial cell-to-cell contacts and triggers the production of thick, multilayered biofilms. Thus, the ica gene cluster represents a genetic determinant that significantly contributes to the virulence of specific Staphylococcus epidermidis strains. PIA synthesis has been reported recently to undergo a phase variation process. In this study, biofilm-forming Staphylococcus epidermidis strains and their PIA-negative phase variants were analysed genetically to investigate the molecular mechanisms of phase variation. We have characterized biofilm-negative variants by Southern hybridization with ica-specific probes, polymerase chain reaction and nucleotide sequencing. The data obtained in these analyses suggested that in ≈30% of the variants the missing biofilm formation was due to the inactivation of either the icaA or the icaC gene by the Insertion of the Insertion Sequence element IS256. Furthermore, it was shown that the transposition of IS256 into the ica operon is a reversible process. After repeated passages of the PIA-negative Insertional mutants, the biofilm-forming phenotype could be restored. Nucleotide Sequence analyses of the revertants confirmed the complete excision of IS256, including the initially duplicated 8 bp target sites. These results elucidate, for the first time, a molecular mechanism mediating phase variation in staphylcocci, and they demonstrate that a naturally occurring Insertion Sequence element is actively involved in the modulation of expression of a Staphylococcus virulence factor.
Paul R Copeland - One of the best experts on this subject based on the ideXlab platform.
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processive incorporation of multiple selenocysteine residues is driven by a novel feature of the selenocysteine Insertion Sequence
Journal of Biological Chemistry, 2018Co-Authors: Sumangala P Shetty, Ryan J Sturts, Michael B Vetick, Paul R CopelandAbstract:: RNA stem loop structures have been frequently shown to regulate essential cellular processes. The selenocysteine Insertion Sequence (SECIS) element, found in the 3' UTRs of all selenoprotein mRNAs, is an example of such a structure, as it is required for the incorporation of the 21st amino acid, selenocysteine (Sec). Selenoprotein synthesis poses a mechanistic challenge because Sec is incorporated during translation in response to a stop codon (UGA). Although it is known that a SECIS-binding protein (SBP2) is required for Sec Insertion, the mechanism of action remains elusive. Additional complexity is present in the synthesis of selenoprotein P (SELENOP), which is the only selenoprotein that contains multiple UGA codons and possesses two SECIS elements in its 3' UTR. Thus, full-length SELENOP synthesis requires processive Sec incorporation. Using zebrafish Selenop, in vitro translation assays, and 75Se labeling in HEK293 cells, we found here that processive Sec incorporation is an intrinsic property of the SECIS elements. Specifically, we identified critical features of SECIS elements that are required for processive Sec incorporation. A screen of the human SECIS elements revealed that most of these elements support processive Sec incorporation in vitro; however, we also found that the processivity of Sec incorporation into Selenop in cells is tightly regulated. We propose a model for processive Sec incorporation that involves differential recruitment of SECIS-binding proteins.
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selenocysteine Insertion Sequence secis binding protein 2 alters conformational dynamics of residues involved in trna accommodation in 80 s ribosomes
Journal of Biological Chemistry, 2012Co-Authors: Kelvin Caban, Paul R CopelandAbstract:Abstract Sec-tRNASec is site-specifically delivered at defined UGA codons in selenoprotein mRNAs. This recoding event is specified by the selenocysteine Insertion Sequence (SECIS) element and requires the selenocysteine (Sec)-specific elongation factor, eEFSec, and the SECIS binding protein, SBP2. Sec-tRNASec is delivered to the ribosome by eEFSec-GTP, but this ternary complex is not sufficient for Sec incorporation, indicating that its access to the ribosomal A-site is regulated. SBP2 stably associates with ribosomes, and mutagenic analysis indicates that this interaction is essential for Sec incorporation. However, the ribosomal function of SBP2 has not been elucidated. To shed light on the functional relevance of the SBP2-ribosome interaction, we screened the functional centers of the 28 S rRNA in translationally competent 80 S ribosomes using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). We demonstrate that SBP2 specifically alters the reactivity of specific residues in Helix 89 (H89) and expansion segment 31 (ES31). These results are indicative of a conformational change in response to SBP2 binding. Based on the known functions of H89 during translation, we propose that SBP2 allows Sec incorporation by either promoting Sec-tRNASec accommodation into the peptidyltransferase center and/or by stimulating the ribosome-dependent GTPase activity of eEFSec.
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a novel protein domain induces high affinity selenocysteine Insertion Sequence binding and elongation factor recruitment
Journal of Biological Chemistry, 2008Co-Authors: Jesse Donovan, Kelvin Caban, Ruchira Ranaweera, Jonathan N Gonzalezflores, Paul R CopelandAbstract:Abstract Selenocysteine (Sec) is incorporated at UGA codons in mRNAs possessing a Sec Insertion Sequence (SECIS) element in their 3′-untranslated region. At least three additional factors are necessary for Sec incorporation: SECIS-binding protein 2 (SBP2), Sec-tRNASec, and a Sec-specific translation elongation factor (eEFSec). The C-terminal half of SBP2 is sufficient to promote Sec incorporation in vitro, which is carried out by the concerted action of a novel Sec incorporation domain and an L7Ae RNA-binding domain. Using alanine scanning mutagenesis, we show that two distinct regions of the Sec incorporation domain are required for Sec incorporation. Physical separation of the Sec incorporation and RNA-binding domains revealed that they are able to function in trans and established a novel role of the Sec incorporation domain in promoting SECIS and eEFSec binding to the SBP2 RNA-binding domain. We propose a model in which SECIS binding induces a conformational change in SBP2 that recruits eEFSec, which in concert with the Sec incorporation domain gains access to the ribosomal A site.
Vadim N. Gladyshev - One of the best experts on this subject based on the ideXlab platform.
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a 4 selenocysteine 2 selenocysteine Insertion Sequence secis element methionine sulfoxide reductase from metridium senile reveals a non catalytic function of selenocysteines
Journal of Biological Chemistry, 2011Co-Authors: Alexey V Lobanov, Stefano M Marino, Alaattin Kaya, Javier Seravalli, Dolph L. Hatfield, Vadim N. GladyshevAbstract:Abstract Selenocysteine (Sec) residues occur in thiol oxidoreductase families, and functionally characterized selenoenzymes typically have a single Sec residue used directly for redox catalysis. However, how new Sec residues evolve and whether non-catalytic Sec residues exist in proteins is not known. Here, we computationally identified several genes with multiple Sec Insertion Sequence (SECIS) elements, one of which was a methionine-R-sulfoxide reductase (MsrB) homolog from Metridium senile that has four in-frame UGA codons and two nearly identical SECIS elements. One of the UGA codons corresponded to the conserved catalytic Sec or Cys in MsrBs, whereas the three other UGA codons evolved recently and had no homologs with Sec or Cys in these positions. Metabolic 75Se labeling showed that all four in-frame UGA codons supported Sec Insertion and that both SECIS elements were functional and collaborated in Sec Insertion at each UGA codon. Interestingly, recombinant M. senile MsrB bound iron, and further analyses suggested the possibility of binding an iron-sulfur cluster by the protein. These data show that Sec residues may appear transiently in genes containing SECIS elements and be adapted for non-catalytic functions.
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a highly efficient form of the selenocysteine Insertion Sequence element in protozoan parasites and its use in mammalian cells
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Sergey V Novoselov, Dolph L. Hatfield, Alexey V Lobanov, Marina V Kasaikina, Vadim N. GladyshevAbstract:Selenoproteins are an elite group of proteins containing a rare amino acid, selenocysteine (Sec), encoded by the codon, UGA. In eukaryotes, incorporation of Sec requires a Sec Insertion Sequence (SECIS) element, a stem–loop structure located in the 3′-untranslated regions of selenoprotein mRNAs. Here we report identification of a noncanonical form of SECIS element in Toxoplasma gondii and Neospora canine, single-celled apicomplexan parasites of humans and domestic animals. This SECIS has a GGGA Sequence in the SBP2-binding site in place of AUGA previously considered invariant. Using a combination of computational and molecular techniques, we show that Toxoplasma and Neospora possess both canonical and noncanonical SECIS elements. The GGGA-type SECIS element supported Sec Insertion in mammalian HEK 293 and NIH 3T3 cells and did so more efficiently than the natural mammalian SECIS elements tested. In addition, mammalian type I and type II SECIS elements mutated into the GGGA forms were functional but manifested decreased Sec Insertion efficiency. We carried out computational searches for both AUGA and GGGA forms of SECIS elements in Toxoplasma and detected five selenoprotein genes, including one coding for a previously undescribed selenoprotein, designated SelQ, and two containing the GGGA form of the SECIS element. In contrast, the GGGA-type SECIS elements were not detected in mammals and nematodes. As a practical outcome of the study, we developed pSelExpress1, a vector for convenient expression of selenoproteins in mammalian cells. It contains an SBP2 gene and the most efficient tested SECIS element: an AUGA mutant of the GGGA-type Toxoplasma SelT structure.
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an algorithm for identification of bacterial selenocysteine Insertion Sequence elements and selenoprotein genes
Bioinformatics, 2005Co-Authors: Yan Zhang, Vadim N. GladyshevAbstract:Motivation: Incorporation of selenocysteine (Sec) into proteins in response to UGA codons requires a cis-acting RNA structure, Sec Insertion Sequence (SECIS) element. Whereas SECIS elements in Escherichia coli are well characterized, a bacterial SECIS consensus structure is lacking. Results: We developed a bacterial SECIS consensus model, the key feature of which is a conserved guanosine in a small apical loop of the properly positioned structure. This consensus was used to build a computational tool, bSECISearch, for detection of bacterial SECIS elements and selenoprotein genes in Sequence databases. The program identified 96.5% of known selenoprotein genes in completely Sequenced bacterial genomes and predicted several new selenoprotein genes. Further analysis revealed that the size of bacterial selenoproteomes varied from 1 to 11 selenoproteins. Formate dehydrogenase was present in most selenoproteomes, often as the only selenoprotein family, whereas the occurrence of other selenoproteins was limited. The availability of the bacterial SECIS consensus and the tool for identification of these structures should help in correct annotation of selenoprotein genes and characterization of bacterial selenoproteomes. Availability: The web server interface is freely accessible to users at http://genomics.unl.edu/bSECISearch/ Contact: vgladyshev1@unl.edu Supplementary information:http://genomics.unl.edu/bSECISearch/supplement.html (includes detailed Methods and Figures S1--S3).
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Mammalian selenoprotein in which selenocysteine (Sec) incorporation is supported by a new form of Sec Insertion Sequence element.
2002Co-Authors: Konstantin V Korotkov, Sergey V Novoselov, Dolph L. Hatfield, Vadim N. GladyshevAbstract:Selenocysteine (Sec), the 21st amino acid in protein, is encoded by UGA. The Sec Insertion Sequence (SECIS) element, which is the stem-loop structure present in 3' untranslated regions (UTRs) of eukaryotic selenoprotein-encoding genes, is essential for recognition of UGA as a codon for Sec rather than as a stop signal. We now report the identification of a new eukaryotic selenoprotein, designated selenoprotein M (SelM). The 3-kb human SelM-encoding gene has five exons and is located on chromosome 22 but has not been correctly identified by either Celera or the public Human Genome Project. We characterized human and mouse SelM cDNA Sequences and expressed the selenoprotein in various mammalian cell lines. The 3" UTR of the human, mouse, and rat SelM-encoding genes lacks a canonical SECIS element. Instead, Sec is incorporated in response to a conserved mRNA structure, in which cytidines are present in place of the adenosines previously considered invariant. Substitution of adenosines for cytidines did not alter Sec incorporation; however, other mutant structures did not support selenoprotein synthesis, demonstrating that this new form of SECIS element is functional. SelM is expressed in a variety of tissues, with increased levels in the brain. It is localized to the perinuclear structures, and its N-terminal signal peptide is necessary for protein translocation.
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new mammalian selenocysteine containing proteins identified with an algorithm that searches for selenocysteine Insertion Sequence elements
Journal of Biological Chemistry, 1999Co-Authors: Gregory V Kryukov, Valentin M Kryukov, Vadim N. GladyshevAbstract:Next Section Abstract Mammalian selenium-containing proteins identified thus far contain selenium in the form of a selenocysteine residue encoded by UGA. These proteins lack common amino acid Sequence motifs, but 3′-untranslated regions of selenoprotein genes contain a common stem-loop structure, selenocysteine Insertion Sequence (SECIS) element, that is necessary for decoding UGA as selenocysteine rather than a stop signal. We describe here a computer program, SECISearch, that identifies mammalian selenoprotein genes by recognizing SECIS elements on the basis of their primary and secondary structures and free energy requirements. When SECISearch was applied to search human dbEST, two new mammalian selenoproteins, designated SelT and SelR, were identified. We determined their cDNA Sequences and expressed them in a monkey cell line as fusion proteins with a green fluorescent protein. Incorporation of selenium into new proteins was confirmed by metabolic labeling with 75Se, and expression of SelT was additionally documented in immunoblot assays. SelT and SelR did not have homology to previously characterized proteins, but their putative homologs were detected in various organisms. SelR homologs were present in every organism characterized by complete genome sequencing. The data suggest applicability of SECISearch for identification of new selenoprotein genes in nucleotide data bases.
Donna M Driscoll - One of the best experts on this subject based on the ideXlab platform.
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altered rna binding activity underlies abnormal thyroid hormone metabolism linked to a mutation in selenocysteine Insertion Sequence binding protein 2
Journal of Biological Chemistry, 2007Co-Authors: Jodi L Bubenik, Donna M DriscollAbstract:The expression of selenoproteins requires the translational recoding of the UGA stop codon to selenocysteine. In eukaryotes, this requires an RNA stem loop structure in the 3'-untranslated region, termed a selenocysteine Insertion Sequence (SECIS), and SECIS-binding protein 2 (SBP2). This study implicates SBP2 in dictating the hierarchy of selenoprotein expression, because it is the first to show that SBP2 distinguishes between SECIS elements in vitro. Using RNA electrophoretic mobility shift assays, we demonstrate that a naturally occurring mutation in SBP2, which correlates with abnormal thyroid hormone function in humans, lies within a novel, bipartite RNA-binding domain. This mutation alters the RNA binding affinity of SBP2 such that it no longer stably interacts with a subset of SECIS elements. Assays performed under competitive conditions to mimic intracellular conditions suggest that the differential affinity of SBP2 for various SECIS elements will determine the expression pattern of the selenoproteome. We hypothesize that the selective loss of a subset of selenoproteins, including some involved in thyroid hormone homeostasis, is responsible for the abnormal thyroid hormone metabolism previously observed in the affected individuals.
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an rna binding protein recognizes a mammalian selenocysteine Insertion Sequence element required for cotranslational incorporation of selenocysteine
Molecular and Cellular Biology, 1997Co-Authors: Andrea Lesoon, Anuradha Mehta, Rajnish Singh, Guy M Chisolm, Donna M DriscollAbstract:In mammalian selenoprotein mRNAs, the recognition of UGA as selenocysteine requires selenocysteine Insertion Sequence (SECIS) elements that are contained in a stable stem-loop structure in the 3' untranslated region (UTR). In this study, we investigated the SECIS elements and cellular proteins required for selenocysteine Insertion in rat phospholipid hydroperoxide glutathione peroxidase (PhGPx). We developed a translational readthrough assay for selenoprotein biosynthesis by using the gene for luciferase as a reporter. Insertion of a UGA or UAA codon into the coding region of luciferase abolished luciferase activity. However, activity was restored to the UGA mutant, but not to the UAA mutant, upon Insertion of the PhGPx 3' UTR. The 3' UTR of rat glutathione peroxidase (GPx) also allowed translational readthrough, whereas the PhGPx and GPx antisense 3' UTRs did not. Deletion of two conserved SECIS elements in the PhGPx 3' UTR (AUGA in the 5' stem or AAAAC in the terminal loop) abolished readthrough activity. UV cross-linking studies identified a 120-kDa protein in rat testis that binds specifically to the sense strands of the PhGPx and GPx 3' UTRs. Direct cross-linking and competition experiments with deletion mutant RNAs demonstrated that binding of the 120-kDa protein requires the AUGA SECIS element but not AAAAC. Point mutations in the AUGA motif that abolished protein binding also prevented readthrough of the UGA codon. Our results suggest that the 120-kDa protein is a significant component of the mechanism of selenocysteine incorporation in mammalian cells.
Jeremy W. Dale - One of the best experts on this subject based on the ideXlab platform.
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Insertion Sequence typing of Mycobacterium tuberculosis: characterization of a widespread subtype with a single copy of IS6110
Tubercle and Lung Disease, 1994Co-Authors: N.g. Fomukong, Thean-hock Tang, S. Al-maamary, W.a. Ibrahim, S. Ramayah, M. Yates, Zainul F. Zainuddin, Jeremy W. DaleAbstract:DNA fingerprinting with the Insertion Sequence IS6110 (also known as IS986) has become established as a major tool for investigating the spread of tuberculosis. Most strains of Mycobacterium tuberculosis have multiple copies of IS6110, but a small minority carry a single copy only. We have examined selected strains from Malaysia, Tanzania and Oman, in comparison with M. bovis isolates and BCG strains carrying one or two copies of IS6110. The Insertion Sequence appears to be present in the same position in all these strains, which suggests that in these organisms the element is defective in transposition and that the loss of transposability may have occurred at an early stage in the evolution of the M. tuberculosis complex.
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characterization of a mycobacterium tuberculosis Insertion Sequence belonging to the is3 family
Molecular Microbiology, 1990Co-Authors: R A Mcadam, P W M Hermans, D Van Soolingen, Z F Zainuddin, D Catty, J D A Van Embden, Jeremy W. DaleAbstract:: A repetitive element (IS986), previously isolated from Mycobacterium tuberculosis and shown to detect multiple restriction fragment-length polymorphisms (RFLPs), has been Sequenced. It consists of a potential Insertion Sequence of 1358bp, with 30-bp inverted repeat ends. IS986 has four potentially significant open reading frames (ORFs): ORFa1, ORFa2 and ORFb on one strand and ORFc on the complementary strand. The Sequences of the potential translated products identify IS986 as a member of the IS3 family, with an apparent frameshift between ORFa1 and ORFa2. IS986 has potential as a highly specific probe for detection and typing of M. tuberculosis, as well as for transposon mutagenesis of mycobacteria. The Sequence of IS986 is virtually identical to that of another recently described element, IS6110 (Thierry et al., 1990).