Artykuły w czasopismach na temat „SEC insertion sequence”
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Copeland, Paul R., Vincent A. Stepanik i Donna M. Driscoll. "Insight into Mammalian Selenocysteine Insertion: Domain Structure and Ribosome Binding Properties of Sec Insertion Sequence Binding Protein 2". Molecular and Cellular Biology 21, nr 5 (1.03.2001): 1491–98. http://dx.doi.org/10.1128/mcb.21.5.1491-1498.2001.
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ABSTRACT The cotranslational incorporation of the unusual amino acid selenocysteine (Sec) into both prokaryotic and eukaryotic proteins requires the recoding of a UGA stop codon as one specific for Sec. The recognition of UGA as Sec in mammalian selenoproteins requires a Sec insertion sequence (SECIS) element in the 3′ untranslated region as well as the SECIS binding protein SBP2. Here we report a detailed analysis of SBP2 structure and function using truncation and site-directed mutagenesis. We have localized the RNA binding domain to a conserved region shared with several ribosomal proteins and eukaryotic translation termination release factor 1. We also identified a separate and novel functional domain N-terminal to the RNA binding domain which was required for Sec insertion but not for SECIS binding. Conversely, we showed that the RNA binding domain was necessary but not sufficient for Sec insertion and that the conserved glycine residue within this domain was required for SECIS binding. Using glycerol gradient sedimentation, we found that SBP2 was stably associated with the ribosomal fraction of cell lysates and that this interaction was not dependent on its SECIS binding activity. This interaction also occurred with purified components in vitro, and we present data which suggest that the SBP2-ribosome interaction occurs via 28S rRNA. SBP2 may, therefore, have a distinct function in selecting the ribosomes to be used for Sec insertion.
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Peiter, Nils, i Michael Rother. "In vivo probing of SECIS-dependent selenocysteine translation in Archaea". Life Science Alliance 6, nr 1 (31.10.2022): e202201676. http://dx.doi.org/10.26508/lsa.202201676.
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Cotranslational insertion of selenocysteine (Sec) proceeds by recoding UGA to a sense codon. This recoding is governed by the Sec insertion sequence (SECIS) element, an RNA structure on the mRNA, but size, location, structure determinants, and mechanism differ for Bacteria, Eukarya, and Archaea. For Archaea, the structure–function relation of the SECIS is poorly understood, as only rather laborious experimental approaches are established. Furthermore, these methods do not allow for quantitative probing of Sec insertion. In order to overcome these limitations, we engineered bacterial β-lactamase into an archaeal selenoprotein, thereby establishing a reporter system, which correlates enzyme activity to Sec insertion. Using this system, in vivo Sec insertion depending on the availability of selenium and the presence of a SECIS element was assessed inMethanococcus maripaludis. Furthermore, a minimal SECIS element required for Sec insertion inM. maripaludiswas defined and a conserved structural motif shown to be essential for function. Besides developing a convenient tool for selenium research, converting a bacterial enzyme into an archaeal selenoprotein provides proof of concept that novel selenoproteins can be engineered in Archaea.
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Labunskyy, Vyacheslav M., Dolph L. Hatfield i Vadim N. Gladyshev. "Selenoproteins: Molecular Pathways and Physiological Roles". Physiological Reviews 94, nr 3 (lipiec 2014): 739–77. http://dx.doi.org/10.1152/physrev.00039.2013.
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Selenium is an essential micronutrient with important functions in human health and relevance to several pathophysiological conditions. The biological effects of selenium are largely mediated by selenium-containing proteins (selenoproteins) that are present in all three domains of life. Although selenoproteins represent diverse molecular pathways and biological functions, all these proteins contain at least one selenocysteine (Sec), a selenium-containing amino acid, and most serve oxidoreductase functions. Sec is cotranslationally inserted into nascent polypeptide chains in response to the UGA codon, whose normal function is to terminate translation. To decode UGA as Sec, organisms evolved the Sec insertion machinery that allows incorporation of this amino acid at specific UGA codons in a process requiring a cis-acting Sec insertion sequence (SECIS) element. Although the basic mechanisms of Sec synthesis and insertion into proteins in both prokaryotes and eukaryotes have been studied in great detail, the identity and functions of many selenoproteins remain largely unknown. In the last decade, there has been significant progress in characterizing selenoproteins and selenoproteomes and understanding their physiological functions. We discuss current knowledge about how these unique proteins perform their functions at the molecular level and highlight new insights into the roles that selenoproteins play in human health.
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Korotkov, Konstantin V., Sergey V. Novoselov, Dolph L. Hatfield i Vadim N. Gladyshev. "Mammalian Selenoprotein in Which Selenocysteine (Sec) Incorporation Is Supported by a New Form of Sec Insertion Sequence Element". Molecular and Cellular Biology 22, nr 5 (1.03.2002): 1402–11. http://dx.doi.org/10.1128/mcb.22.5.1402-1411.2002.
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ABSTRACT 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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Shetty, Sumangala P., Nora T. Kiledjian i Paul R. Copeland. "The selenoprotein P 3’ untranslated region is an RNA binding protein platform that fine tunes selenocysteine incorporation". PLOS ONE 17, nr 7 (29.07.2022): e0271453. http://dx.doi.org/10.1371/journal.pone.0271453.
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Selenoproteins contain the 21st amino acid, selenocysteine (Sec), which is incorporated at select UGA codons when a specialized hairpin sequence, the Sec insertion sequence (SECIS) element, is present in the 3’ UTR. Aside from the SECIS, selenoprotein mRNA 3’ UTRs are not conserved between different selenoproteins within a species. In contrast, the 3’-UTR of a given selenoprotein is often conserved across species, which supports the hypothesis that cis-acting elements in the 3’-UTR other than the SECIS exert post-transcriptional control on selenoprotein expression. In order to determine the function of one such SECIS context, we chose to focus on the plasma selenoprotein, SELENOP, which is required to maintain selenium homeostasis as a selenium transport protein that contains 10 Sec residues. It is unique in that its mRNA contains two SECIS elements in the context of a highly conserved 843-nucleotide 3’ UTR. Here we have used RNA affinity chromatography and identified PTBP1 as the major RNA binding protein that specifically interacts with the sequence between the two SECIS elements. We then used CRISPR/Cas9 genome editing to delete two regions surrounding the first SECIS element. We found that these sequences are involved in regulating SELENOP mRNA and protein levels, which are inversely altered as a function of selenium concentrations.
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Mukai, Takahito. "Bioinformatic Prediction of an tRNASec Gene Nested inside an Elongation Factor SelB Gene in Alphaproteobacteria". International Journal of Molecular Sciences 22, nr 9 (27.04.2021): 4605. http://dx.doi.org/10.3390/ijms22094605.
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In bacteria, selenocysteine (Sec) is incorporated into proteins via the recoding of a particular codon, the UGA stop codon in most cases. Sec-tRNASec is delivered to the ribosome by the Sec-dedicated elongation factor SelB that also recognizes a Sec-insertion sequence element following the codon on the mRNA. Since the excess of SelB may lead to sequestration of Sec-tRNASec under selenium deficiency or oxidative stress, the expression levels of SelB and tRNASec should be regulated. In this bioinformatic study, I analyzed the Rhizobiales SelB species because they were annotated to have a non-canonical C-terminal extension. I found that the open reading frame (ORF) of diverse Alphaproteobacteria selB genes includes an entire tRNASec sequence (selC) and overlaps with the start codon of the downstream ORF. A remnant tRNASec sequence was found in the Sinorhizobium melilotiselB genes whose products have a shorter C-terminal extension. Similar overlapping traits were found in Gammaproteobacteria and Nitrospirae. I hypothesized that once the tRNASec moiety is folded and processed, the expression of the full-length SelB may be repressed. This is the first report on a nested tRNA gene inside a protein ORF in bacteria.
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Hilal, Tarek, Benjamin Y. Killam, Milica Grozdanović, Malgorzata Dobosz-Bartoszek, Justus Loerke, Jörg Bürger, Thorsten Mielke, Paul R. Copeland, Miljan Simonović i Christian M. T. Spahn. "Structure of the mammalian ribosome as it decodes the selenocysteine UGA codon". Science 376, nr 6599 (17.06.2022): 1338–43. http://dx.doi.org/10.1126/science.abg3875.
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The elongation of eukaryotic selenoproteins relies on a poorly understood process of interpreting in-frame UGA stop codons as selenocysteine (Sec). We used cryo-electron microscopy to visualize Sec UGA recoding in mammals. A complex between the noncoding Sec-insertion sequence (SECIS), SECIS-binding protein 2 (SBP2), and 40 S ribosomal subunit enables Sec-specific elongation factor eEFSec to deliver Sec. eEFSec and SBP2 do not interact directly but rather deploy their carboxyl-terminal domains to engage with the opposite ends of the SECIS. By using its Lys-rich and carboxyl-terminal segments, the ribosomal protein eS31 simultaneously interacts with Sec-specific transfer RNA (tRNA Sec ) and SBP2, which further stabilizes the assembly. eEFSec is indiscriminate toward l -serine and facilitates its misincorporation at Sec UGA codons. Our results support a fundamentally distinct mechanism of Sec UGA recoding in eukaryotes from that in bacteria.
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Francetic, Olivera, Nienke Buddelmeijer, Shawn Lewenza, Carol A. Kumamoto i Anthony P. Pugsley. "Signal Recognition Particle-Dependent Inner Membrane Targeting of the PulG Pseudopilin Component of a Type II Secretion System". Journal of Bacteriology 189, nr 5 (8.12.2006): 1783–93. http://dx.doi.org/10.1128/jb.01230-06.
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ABSTRACT The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The β-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.
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Ito, Koreaki, Naomi Shimokawa-Chiba i Shinobu Chiba. "Sec translocon has an insertase-like function in addition to polypeptide conduction through the channel". F1000Research 8 (20.12.2019): 2126. http://dx.doi.org/10.12688/f1000research.21065.1.
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The Sec translocon provides a polypeptide-conducting channel, which is insulated from the hydrophobic lipidic environment of the membrane, for translocation of hydrophilic passenger polypeptides. Its lateral gate allows a downstream hydrophobic segment (stop-transfer sequence) to exit the channel laterally for integration into the lipid phase. We note that this channel model only partly accounts for the translocon function. The other essential role of translocon is to facilitate de novo insertion of the N-terminal topogenic segment of a substrate polypeptide into the membrane. Recent structural studies suggest that de novo insertion does not use the polypeptide-conducting channel; instead, it takes place directly at the lateral gate, which is prone to opening. We propose that the de novo insertion process, in concept, is similar to that of insertases (such as YidC in bacteria and EMC3 in eukaryotes), in which an intramembrane surface of the machinery provides the halfway point of insertion.
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FUJIWARA, Noriko, Tsuneko FUJII, Junichi FUJII i Naoyuki TANIGUCHI. "Functional expression of rat thioredoxin reductase: selenocysteine insertion sequence element is essential for the active enzyme". Biochemical Journal 340, nr 2 (25.05.1999): 439–44. http://dx.doi.org/10.1042/bj3400439.
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Mammalian thioredoxin reductase (TR) is a flavoprotein catalysing reduction of oxidized thioredoxin in an NADPH-dependent manner, and contains a selenocysteine (Sec) residue near the C-terminus. We observed that TR activity was decreased in A549 cells by the lowering of the fetal bovine serum content in the culture medium and was recovered by the addition of selenium. To study the role of Sec in TR activity, we have isolated a full-length clone of the rat TR cDNA (3.3 kb) and have expressed it in COS-1 cells in a transient-expression system. TR activities in COS-1 cells expressing rat TR were increased in accordance with supplemented sodium selenite concentrations, whereas levels of TR protein, examined by Western blotting, were not affected by sodium selenite concentrations. We introduced various deletions into the 3ʹ-untranslated region of the TR cDNA to localize and examine the role of a Sec insertion-sequence (SECIS) element in the functional expression of TR. TR activities were observed only in COS-1 cells transfected with the TR cDNAs containing the putative SECIS element located between 1856 and 1915 bp in the correct orientation. We also carried out radiolabelling of proteins by incubation of the cDNA-transfected cells with sodium [75Se]selenite. 75Se was incorporated into the expressed TR protein of the cells transfected with the SECIS element-containing cDNAs, but not into those without the SECIS element or with an inverted SECIS element. These data clearly showed a requirement of selenium for the formation of functional TR protein.
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Cubas-Gaona, Liliana L., Patricia de Francisco, Ana Martín-González i Juan Carlos Gutiérrez. "Tetrahymena Glutathione Peroxidase Family: A Comparative Analysis of These Antioxidant Enzymes and Differential Gene Expression to Metals and Oxidizing Agents". Microorganisms 8, nr 7 (5.07.2020): 1008. http://dx.doi.org/10.3390/microorganisms8071008.
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In the present work, an extensive analysis of the putative glutathione peroxidases (GPx) of the eukaryotic microorganism model Tetrahymena thermophila is carried out. A comparative analysis with GPx present in other Tetrahymena species and other very taxonomically diverse ciliates is also performed. A majority of ciliate GPx have replaced the selenocysteine (Sec) by Cys in its catalytic center, so they can be considered as phospholipid hydroperoxide glutathione peroxidases (PHGPx). Selenocysteine insertion sequence (SECIS) elements have been detected in several ciliate GPx that do not incorporate Sec in their amino acid sequences, and conversely, in other ciliate GPx with Sec, no SECIS elements are detected. These anomalies are analyzed and discussed. From the phylogenetic analysis using the ciliate GPx amino acid sequences, the existence of extensive intra- and interspecific gene duplications that produced multiple GPx isoforms in each species is inferred. The ancestral character of the selenoproteins is also corroborated. The analysis by qRT-PCR of six selected T. thermophila GPx genes has shown a quantitative differential expression between them, depending on the stressor (oxidizing agents, apoptotic inducer or metals) and the time of exposure.
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Caban, Kelvin, Scott A. Kinzy i Paul R. Copeland. "The L7Ae RNA Binding Motif Is a Multifunctional Domain Required for the Ribosome-Dependent Sec Incorporation Activity of Sec Insertion Sequence Binding Protein 2". Molecular and Cellular Biology 27, nr 18 (16.07.2007): 6350–60. http://dx.doi.org/10.1128/mcb.00632-07.
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ABSTRACT The decoding of specific UGA codons as selenocysteine is specified by the Sec insertion sequence (SECIS) element. Additionally, Sec-tRNA[Ser]Sec and the dedicated Sec-specific elongation factor eEFSec are required but not sufficient for nonsense suppression. SECIS binding protein 2 (SBP2) is also essential for Sec incorporation, but its precise role is unknown. In addition to binding the SECIS element, SBP2 binds stably and quantitatively to ribosomes. To determine the function of the SBP2-ribosome interaction, conserved amino acids throughout the SBP2 L7Ae RNA binding motif were mutated to alanine in clusters of five. Mutant proteins were analyzed for ribosome binding, SECIS element binding, and Sec incorporation activity, allowing us to identify two distinct but interdependent sites within the L7Ae motif: (i) a core L7Ae motif required for SECIS binding and ribosome binding and (ii) an auxiliary motif involved in physical and functional interactions with the ribosome. Structural modeling of SBP2 based on the 15.5-kDa protein-U4 snRNA complex strongly supports a two-site model for L7Ae domain function within SBP2. These results provide evidence that the SBP2-ribosome interaction is essential for Sec incorporation.
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Rapoport, Tom A., Kent E. S. Matlack, Kathrin Plath, Benjamin Misselwitz i Oliver Staeck. "Posttranslational Protein Translocation Across the Membrane of the Endoplasmic Reticulum". Biological Chemistry 380, nr 10 (12.10.1999): 1143–50. http://dx.doi.org/10.1515/bc.1999.145.
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AbstractPosttranslational protein translocation across the membrane of the endoplasmic reticulum is mediated by the Sec complex. This complex includes a transmembrane channel formed by multiple copies of the Sec61 protein. Translocation of a polypeptide begins when the signal sequence binds at a specific site within the channel. Binding results in the insertion of the substrate into the channel, possibly as a loop with a small segment exposed to the lumen. While bound, the signal sequence is in contact with both protein components of the channel and the lipid of the membrane. Subsequent movement of the polypeptide through the channel occurs when BiP molecules interact transiently with a luminal domain of the Sec complex, hydrolyze ATP, and bind to the substrate. Bound BiP promotes translocation by preventing the substrate from diffusing backwards through the channel, and thus acts as a molecular ratchet.
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Novoselov, Sergey V., Deame Hua, Alexey V. Lobanov i Vadim N. Gladyshev. "Identification and characterization of Fep15, a new selenocysteine-containing member of the Sep15 protein family". Biochemical Journal 394, nr 3 (24.02.2006): 575–79. http://dx.doi.org/10.1042/bj20051569.
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Sec (selenocysteine) is a rare amino acid in proteins. It is co-translationally inserted into proteins at UGA codons with the help of SECIS (Sec insertion sequence) elements. A full set of selenoproteins within a genome, known as the selenoproteome, is highly variable in different organisms. However, most of the known eukaryotic selenoproteins are represented in the mammalian selenoproteome. In addition, many of these selenoproteins have cysteine orthologues. Here, we describe a new selenoprotein, designated Fep15, which is distantly related to members of the 15 kDa selenoprotein (Sep15) family. Fep15 is absent in mammals, can be detected only in fish and is present in these organisms only in the selenoprotein form. In contrast with other members of the Sep15 family, which contain a putative active site composed of Sec and cysteine, Fep15 has only Sec. When transiently expressed in mammalian cells, Fep15 incorporated Sec in an SECIS- and SBP2 (SECIS-binding protein 2)-dependent manner and was targeted to the endoplasmic reticulum by its N-terminal signal peptide. Phylogenetic analyses of Sep15 family members suggest that Fep15 evolved by gene duplication.
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Vindry, Caroline, Olivia Guillin, Philippe E. Mangeot, Théophile Ohlmann i Laurent Chavatte. "A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA[Ser]Sec Gene". Cells 8, nr 6 (11.06.2019): 574. http://dx.doi.org/10.3390/cells8060574.
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The translation of selenoprotein mRNAs involves a non-canonical ribosomal event in which an in-frame UGA is recoded as a selenocysteine (Sec) codon instead of being read as a stop codon. The recoding machinery is centered around two dedicated RNA components: The selenocysteine insertion sequence (SECIS) located in the 3′ UTR of the mRNA and the selenocysteine-tRNA (Sec-tRNA[Ser]Sec). This translational UGA-selenocysteine recoding event by the ribosome is a limiting stage of selenoprotein expression. Its efficiency is controlled by the SECIS, the Sec-tRNA[Ser]Sec and their interacting protein partners. In the present work, we used a recently developed CRISPR strategy based on murine leukemia virus-like particles (VLPs) loaded with Cas9-sgRNA ribonucleoproteins to inactivate the Sec-tRNA[Ser]Sec gene in human cell lines. We showed that these CRISPR-Cas9-VLPs were able to induce efficient genome-editing in Hek293, HepG2, HaCaT, HAP1, HeLa, and LNCaP cell lines and this caused a robust reduction of selenoprotein expression. The alteration of selenoprotein expression was the direct consequence of lower levels of Sec-tRNA[Ser]Sec and thus a decrease in translational recoding efficiency of the ribosome. This novel strategy opens many possibilities to study the impact of selenoprotein deficiency in hard-to-transfect cells, since these CRISPR-Cas9-VLPs have a wide tropism.
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Kiledjian, Nora T., Rushvi Shah, Michael B. Vetick i Paul R. Copeland. "The expression of essential selenoproteins during development requires SECIS-binding protein 2–like". Life Science Alliance 5, nr 5 (24.02.2022): e202101291. http://dx.doi.org/10.26508/lsa.202101291.
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The dietary requirement for selenium is based on its incorporation into selenoproteins, which contain the amino acid selenocysteine (Sec). The Sec insertion sequence (SECIS) is an RNA structure found in the 3′ UTR of all selenoprotein mRNAs, and it is required to convert in-frame UGA codons from termination to Sec-incorporating codons. SECIS-binding protein 2 (Sbp2) is required for Sec incorporation, but its paralogue, SECIS-binding protein 2–like (Secisbp2l), while conserved, has no known function. Here we determined the relative roles of Sbp2 and Secisbp2l by introducing CRISPR mutations in both genes in zebrafish. By monitoring selenoprotein synthesis with 75Se labeling during embryogenesis, we found that sbp2−/− embryos still make a select subset of selenoproteins but secisbp2l−/− embryos retain the full complement. Abrogation of both genes completely prevents selenoprotein synthesis and juveniles die at 14 days post fertilization. Embryos lacking Sbp2 are sensitive to oxidative stress and express the stress marker Vtg1. We propose a model where Secisbp2l is required to promote essential selenoprotein synthesis when Sbp2 activity is compromised.
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Mita, Yuichiro, Risa Uchida, Sayuri Yasuhara, Kohei Kishi, Takayuki Hoshi, Yoshitaka Matsuo, Tadashi Yokooji i in. "Identification of a novel endogenous long non-coding RNA that inhibits selenoprotein P translation". Nucleic Acids Research 49, nr 12 (18.06.2021): 6893–907. http://dx.doi.org/10.1093/nar/gkab498.
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Abstract Selenoprotein P (SELENOP) is a major plasma selenoprotein that contains 10 Sec residues, which is encoded by the UGA stop codon. The mRNA for SELENOP has the unique property of containing two Sec insertion sequence (SECIS) elements, which is located in the 3′ untranslated region (3′UTR). Here, we coincidentally identified a novel gene, CCDC152, by sequence analysis. This gene was located in the antisense region of the SELENOP gene, including the 3′UTR region in the genome. We demonstrated that this novel gene functioned as a long non-coding RNA (lncRNA) that decreased SELENOP protein levels via translational rather than transcriptional, regulation. We found that the CCDC152 RNA interacted specifically and directly with the SELENOP mRNA and inhibited its binding to the SECIS-binding protein 2, resulting in the decrease of ribosome binding. We termed this novel gene product lncRNA inhibitor of SELENOP translation (L-IST). Finally, we found that epigallocatechin gallate upregulated L-IST in vitro and in vivo, to suppress SELENOP protein levels. Here, we provide a new regulatory mechanism of SELENOP translation by an endogenous long antisense ncRNA.
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Papp, Laura V., Jun Lu, Frank Striebel, Derek Kennedy, Arne Holmgren i Kum Kum Khanna. "The Redox State of SECIS Binding Protein 2 Controls Its Localization and Selenocysteine Incorporation Function". Molecular and Cellular Biology 26, nr 13 (1.07.2006): 4895–910. http://dx.doi.org/10.1128/mcb.02284-05.
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ABSTRACT Selenoproteins are central controllers of cellular redox homeostasis. Incorporation of selenocysteine (Sec) into selenoproteins employs a unique mechanism to decode the UGA stop codon. The process requires the Sec insertion sequence (SECIS) element, tRNASec, and protein factors including the SECIS binding protein 2 (SBP2). Here, we report the characterization of motifs within SBP2 that regulate its subcellular localization and function. We show that SBP2 shuttles between the nucleus and the cytoplasm via intrinsic, functional nuclear localization signal and nuclear export signal motifs and that its nuclear export is dependent on the CRM1 pathway. Oxidative stress induces nuclear accumulation of SBP2 via oxidation of cysteine residues within a redox-sensitive cysteine-rich domain. These modifications are efficiently reversed in vitro by human thioredoxin and glutaredoxin, suggesting that these antioxidant systems might regulate redox status of SBP2 in vivo. Depletion of SBP2 in cell lines using small interfering RNA results in a decrease in Sec incorporation, providing direct evidence for its requirement for selenoprotein synthesis. Furthermore, Sec incorporation is reduced substantially after treatment of cells with agents that cause oxidative stress, suggesting that nuclear sequestration of SBP2 under such conditions may represent a mechanism to regulate the expression of selenoproteins.
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Bohleber, Simon, Noelia Fradejas-Villar, Wenchao Zhao, Uschi Reuter i Ulrich Schweizer. "High-Resolution Ribosome Profiling Reveals Gene-Specific Details of UGA Re-Coding in Selenoprotein Biosynthesis". Biomolecules 12, nr 10 (17.10.2022): 1504. http://dx.doi.org/10.3390/biom12101504.
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Co-translational incorporation of selenocysteine (Sec) into selenoproteins occurs at UGA codons in a process in which translational elongation competes with translational termination. Selenocysteine insertion sequence-binding protein 2 (SECISBP2) greatly enhances Sec incorporation into selenoproteins by interacting with the mRNA, ribosome, and elongation factor Sec (EFSEC). Ribosomal profiling allows to study the process of UGA re-coding in the physiological context of the cell and at the same time for all individual selenoproteins expressed in that cell. Using HAP1 cells expressing a mutant SECISBP2, we show here that high-resolution ribosomal profiling can be used to assess read-through efficiency at the UGA in all selenoproteins, including those with Sec close to the C-terminus. Analysis of ribosomes with UGA either at the A-site or the P-site revealed, in a transcript-specific manner, that SECISBP2 helps to recruit tRNASec and stabilize the mRNA. We propose to assess the effect of any perturbation of UGA read-through by determining the proportion of ribosomes carrying UGA in the P-site, pUGA. An additional, new observation is frameshifting that occurred 3′ of the UGA/Sec codon in SELENOF and SELENOW in SECISBP2-mutant HAP1 cells, a finding corroborated by reanalysis of neuron-specific Secisbp2R543Q-mutant brains.
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ZHANG, YING, YUAN HE, LI HE, HONGYING ZONG i GUOBIN CAI. "Expression and characterization of a phospholipid hydroperoxide glutathione peroxidase gene inSchistosoma japonicum". Parasitology 142, nr 13 (18.08.2015): 1595–604. http://dx.doi.org/10.1017/s0031182015001055.
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SUMMARYPhospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) is a major antioxidant enzyme, which plays unique roles in the protection of cells against oxidative stress by catalysing reduction of lipid hydroperoxides. We isolated and characterized a full-length cDNA sequence encodingGPxgene from a blood fluke,Schistosoma japonicum(designatedSjGPx), which contained an in-frame TGA codon for selenocysteine (Sec) and a concurrent Sec insertion sequence in its 3′-untranslated region. Protein encoded bySjGPxdemonstrated a primary structure characteristic to the PHGPx family, including preservation of catalytic domains and absence of the subunit interaction domains. Semi-quantitative reverse transcription PCR and Western blotting showed that the SjGPx was mainly expressed in the female adults and eggs. RNA interference approach was employed to investigate the effects of knockdown ofSjGPx. SjGPx expression level was significantly reduced on the 5th day post-RNAi. Significantly reduction in GPx enzyme activities, as well as obvious changes in morphology of intrauterine eggs followed the reduction inSjGPxtranscript level. We observed a 63·04% reduction in GPx activity and the eggs severely deformed. Our results revealed that SjGPx protein might be involved in the provision of enzyme activity during egg production.
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Kalmokoff, M. L., S. K. Banerjee, T. Cyr, M. A. Hefford i T. Gleeson. "Identification of a New Plasmid-Encodedsec-Dependent Bacteriocin Produced by Listeria innocua 743". Applied and Environmental Microbiology 67, nr 9 (1.09.2001): 4041–47. http://dx.doi.org/10.1128/aem.67.9.4041-4047.2001.
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ABSTRACT Listeria innocua 743 produces an inhibitory activity demonstrating broad-spectrum inhibition of Listeria monocytogenes isolates. Gel-electrophoretic analysis of culture supernatants indicated that two inhibitors with different molecular weights were produced by this strain. Insertion of Tn917into a 2.9 Kb plasmid (pHC743) generated mutants with either an impaired ability or a loss in ability to produce one of the inhibitors. Sequence analysis of the transposon insertion regions revealed the presence of two continuous open reading frames, the first encoding a new pediocin-like bacteriocin (lisA) and the second encoding a protein homologous with genes involved in immunity toward other bacteriocins (lisB). Translation of the bacteriocin gene (lisA) initiates from a noncanonical start codon and encodes a 71-amino-acid prebacteriocin which lacked the double glycine leader peptidase processing site common in other type II bacteriocins. Alignment of the sequence with the processed N termini of related bacteriocins suggests that the mature bacteriocin consists of 43 amino acids, with a predicted molecular mass of 4,484 Da. Mutants containing insertions into lisA were sensitive to the inhibitor, indicating that lisAB forms a single operon and that lisB represents the immunity protein. Cloning of an amplicon containing the lisAB operon intoEscherichia coli resulted in expression and export of the bacteriocin. This finding confirms that the phenotype is dependent on the structural and immunity gene only and that export of this bacteriocin is sec dependent. This is the first confirmation of bacteriocin production in a Listeriaspp., and it is of interest that this bacteriocin is closely related to the pediocin family of bacteriocins produced by lactic acid bacteria.
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Fitzgerald, J. Ross, Steven R. Monday, Timothy J. Foster, Gregory A. Bohach, Patrick J. Hartigan, William J. Meaney i Cyril J. Smyth. "Characterization of a Putative Pathogenicity Island from Bovine Staphylococcus aureus Encoding Multiple Superantigens". Journal of Bacteriology 183, nr 1 (1.01.2001): 63–70. http://dx.doi.org/10.1128/jb.183.1.63-70.2001.
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ABSTRACT Previous studies have demonstrated that a proportion ofStaphylococcus aureus isolates from bovine mastitis coproduce toxic shock syndrome toxin (TSST) and staphylococcal enterotoxin C (SEC). In this study, molecular genetic analysis of one such strain, RF122, revealed the presence of a 15,891-bp putative pathogenicity island (SaPIbov) encoding the genes for TSST (tst), the SEC bovine variant (sec-bovine), and a gene (sel) which encodes an enterotoxin-like protein. The island contains 21 open reading frames specifying hypothetical proteins longer than 60 amino acids including an integrase-like gene. The element is bordered by 74-bp direct repeats at the left and right junctions, and the integration site lies adjacent to the 3′ end of the GMP synthase gene (gmps) in the S. aureuschromosome. SaPIbov contains a central region of sequence identity with the previously characterized tst pathogenicity island SaPI1 (J. A. Lindsay et al., Mol. Microbiol. 29:527–543, 1998). A closely related strain, RF120, of the same multilocus enzyme electrophoretic type, random amplified polymorphic DNA type, and ribotype, does not contain the island, implying that the element is mobile and that a recent insertion/deletion event has taken place. TSST and TSST/SEC-deficient mutants of S. aureus strain RF122 were constructed by allele replacement. In vitro bovine Vβ-specific lymphocyte expansion analysis by culture supernatants of wild-type strains and of tst and sec-bovine allele replacement mutants revealed that TSST stimulates BTB13-specific T cells whereas SEC-bovine stimulates BTB93-specific T cells. This suggests that the presence of SaPIbov may contribute to modulation of the bovine immune response.
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Sanders, Jo P., Serge Van der Geyten, Ellen Kaptein, Veerle M. Darras, Eduard R. Kühn, Jack L. Leonard i Theo J. Visser. "Characterization of a Propylthiouracil-Insensitive Type I Iodothyronine Deiodinase*". Endocrinology 138, nr 12 (1.12.1997): 5153–60. http://dx.doi.org/10.1210/endo.138.12.5581.
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Abstract Mammalian type I iodothyronine deiodinase (D1) activates and inactivates thyroid hormone by outer ring deiodination (ORD) and inner ring deiodination (IRD), respectively, and is potently inhibited by propylthiouracil (PTU). Here we describe the cloning and characterization of a complementary DNA encoding a PTU-insensitive D1 from teleost fish (Oreochromis niloticus, tilapia). This complementary DNA codes for a protein of 248 amino acids, including a putative selenocysteine (Sec) residue, encoded by a TGA triplet, at position 126. The 3′ untranslated region contains two putative Sec insertion sequence (SECIS) elements. Recombinant enzyme expressed in COS-1 cells catalyzes both ORD of T4 and rT3 and IRD of T3 and T3 sulfate with the same substrate specificity as native tilapia D1 (tD1), i.e. rT3 ≫ T4 > T3 sulfate > T3. Native and recombinant tD1 show equally low sensitivities to inhibition by PTU, iodoacetate, and gold thioglucose compared with the potent inhibitions observed with mammalian D1s. Because the residue 2 positions downstream from Sec is Pro in tD1 and in all (PTU-insensitive) type II and type III iodothyronine deiodinases but Ser in all PTU-sensitive D1s, we prepared the Pro128Ser mutant of tD1. The mutant enzyme showed strongly decreased ORD and somewhat increased IRD activity, but was still insensitive to PTU. These results provide new information about the structure-activity relationship of D1 concerning two characteristic properties, i.e. catalysis of both ORD and IRD, and inhibition by PTU.
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Carle, Patricia, Colette Saillard, Nathalie Carr�re, S�bastien Carr�re, Sybille Duret, Sandrine Eveillard, Patrice Gaurivaud i in. "Partial Chromosome Sequence of Spiroplasma citri Reveals Extensive Viral Invasion and Important Gene Decay". Applied and Environmental Microbiology 76, nr 11 (2.04.2010): 3420–26. http://dx.doi.org/10.1128/aem.02954-09.
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ABSTRACT The assembly of 20,000 sequencing reads obtained from shotgun and chromosome-specific libraries of the Spiroplasma citri genome yielded 77 chromosomal contigs totaling 1,674 kbp (92%) of the 1,820-kbp chromosome. The largest chromosomal contigs were positioned on the physical and genetic maps constructed from pulsed-field gel electrophoresis and Southern blot hybridizations. Thirty-eight contigs were annotated, resulting in 1,908 predicted coding sequences (CDS) representing an overall coding density of only 74%. Cellular processes, cell metabolism, and structural-element CDS account for 29% of the coding capacity, CDS of external origin such as viruses and mobile elements account for 24% of the coding capacity, and CDS of unknown function account for 47% of the coding capacity. Among these, 21% of the CDS group into 63 paralog families. The organization of these paralogs into conserved blocks suggests that they represent potential mobile units. Phage-related sequences were particularly abundant and include plectrovirus SpV1 and SVGII3 and lambda-like SpV2 sequences. Sixty-nine copies of transposases belonging to four insertion sequence (IS) families (IS30, IS481, IS3, and ISNCY) were detected. Similarity analyses showed that 21% of chromosomal CDS were truncated compared to their bacterial orthologs. Transmembrane domains, including signal peptides, were predicted for 599 CDS, of which 58 were putative lipoproteins. S. citri has a Sec-dependent protein export pathway. Eighty-four CDS were assigned to transport, such as phosphoenolpyruvate phosphotransferase systems (PTS), the ATP binding cassette (ABC), and other transporters. Besides glycolytic and ATP synthesis pathways, it is noteworthy that S. citri possesses a nearly complete pathway for the biosynthesis of a terpenoid.
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KIM, S. H., G. B. CAI, Y. A. BAE, E. G. LEE, Y. S. LEE i Y. KONG. "Two novel phospholipid hydroperoxide glutathione peroxidase genes of Paragonimus westermani induced by oxidative stress". Parasitology 136, nr 5 (5.03.2009): 553–65. http://dx.doi.org/10.1017/s0031182009005654.
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SUMMARYPhospholipid hydroperoxide glutathione peroxidase (PHGPx; GPx4) plays unique roles in the protection of cells against oxidative stress by catalysing reduction of lipid hydroperoxides. We characterized 2 novel GPx genes from a lung fluke, Paragonimus westermani (designated PwGPx1 and PwGPx2). These single copy genes spanned 6559 and 12 371 bp, respectively, and contained each of 5 intervening introns. The PwGPx2 harboured a codon for Sec and a Sec insertion sequence motif. Proteins encoded by the Paragonimus genes demonstrated a primary structure characteristic to the PHGPx family, including preservation of catalytic and glutathione-binding domains and absence of the subunit interaction domain. Expression of PwGPx1 increased gradually as the parasite matured, whereas that of PwGPx2 was temporally regulated. PwGPx2 was expressed at the basal level from the metacercariae to the 3-week-old juveniles; however, the expression was significantly induced in the 7-week-old immature worms and reached a plateau in the 12-week-old adults and eggs. PwGPx1 and PwGPx2 were largely localized in vitellocytes within vitelline glands and eggs. Oxidative stress-inducible paraquat, juglone and H2O2 substantially augmented the PwGPx1 and PwGPx2 expressions in viable worms by 1·5- to 11-fold. Our results strongly suggested that PwGPxs may actively participate in detoxification of oxidative hazards in P. westermani.
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de Jesus, Lucia A., Peter R. Hoffmann, Tanya Michaud, Erin P. Forry, Andrea Small-Howard, Robert J. Stillwell, Nadya Morozova, John W. Harney i Marla J. Berry. "Nuclear Assembly of UGA Decoding Complexes on Selenoprotein mRNAs: a Mechanism for Eluding Nonsense-Mediated Decay?" Molecular and Cellular Biology 26, nr 5 (1.03.2006): 1795–805. http://dx.doi.org/10.1128/mcb.26.5.1795-1805.2006.
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ABSTRACT Recoding of UGA from a stop codon to selenocysteine poses a dilemma for the protein translation machinery. In eukaryotes, two factors that are crucial to this recoding process are the mRNA binding protein of the Sec insertion sequence, SBP2, and the specialized elongation factor, EFsec. We sought to determine the subcellular localization of these selenoprotein synthesis factors in mammalian cells and thus gain insight into how selenoprotein mRNAs might circumvent nonsense-mediated decay. Intriguingly, both EFsec and SBP2 localization differed depending on the cell line but significant colocalization of the two proteins was observed in cells where SBP2 levels were detectable. We identify functional nuclear localization and export signals in both proteins, demonstrate that SBP2 undergoes nucleocytoplasmic shuttling, and provide evidence that SBP2 levels and localization may influence EFsec localization. Our results suggest a mechanism for the nuclear assembly of the selenocysteine incorporation machinery that could allow selenoprotein mRNAs to circumvent nonsense-mediated decay, thus providing new insights into the mechanism of selenoprotein translation.
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Schachtsiek, Martina, Walter P. Hammes i Christian Hertel. "Characterization of Lactobacillus coryniformis DSM 20001T Surface Protein Cpf Mediating Coaggregation with and Aggregation among Pathogens". Applied and Environmental Microbiology 70, nr 12 (grudzień 2004): 7078–85. http://dx.doi.org/10.1128/aem.70.12.7078-7085.2004.
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ABSTRACT Phenotypic characterization of aggregation phenotypes of Lactobacillus coryniformis revealed that strain DSM 20001T coaggregated with Escherichia coli K88, Campylobacter coli, and Campylobacter jejuni but not with other human pathogens. In addition, cells of these pathogens aggregated in the presence of the spent culture supernatant (SCS) of strain DSM 20001T. Cells of E. coli K88 remained viable in the coaggregates and aggregates for up to 24 h. Both coaggregation and aggregation (co/aggregation) occurred at pH 3.5 to 7.5 and was sensitive to heat (85°C for 15 min) and proteinase K. The co/aggregation-promoting factor (Cpf) was purified, and the gene was identified by PCR with degenerate primers derived from internal amino acid sequences. The cpf gene encoded a 19.9-kDa preprotein with a sec-dependent leader and an isoelectric point of 4.4. The amino acid sequence had no significant similarity to proteins with known functions. Northern analysis revealed not only major transcription from the promoter of cpf but also major transcription from the promoter of the preceding insertion element, ISLco1 belonging to the IS3 family. Recombinant Cpf produced in E. coli mediated aggregation of pathogens comparable to the aggregation obtained with purified Cpf or SCS of strain DSM 20001T. Cpf could be removed from cells of strain DSM 20001T by treatment with 5 M LiCl and could be subsequently reattached to the cell surface by using SCS or recombinant Cpf, which resulted in restoration of the co/aggregation property. These results together with those of the amino acid sequence analysis suggest that Cpf is a novel surface protein of L. coryniformis that mediates co/aggregation of some pathogens.
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Handy, Diane E., Yufeng Zhang i Joseph Loscalzo. "Homocysteine Down-regulates Cellular Glutathione Peroxidase (GPx1) by Decreasing Translation". Journal of Biological Chemistry 280, nr 16 (25.02.2005): 15518–25. http://dx.doi.org/10.1074/jbc.m501452200.
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Hyperhomocysteinemia contributes to vascular dysfunction and an increase in the risk of cardiovascular disease. An elevated level of homocysteinein vivoand in cell culture systems results in a decrease in the activity of cellular glutathione peroxidase (GPx1), an intracellular antioxidant enzyme that reduces hydrogen peroxide and lipid peroxides. In this study, we show that homocysteine interferes with GPx1 protein expression without affecting transcript levels. Expression of the selenocysteine (SEC)-containing GPx1 protein requires special translational cofactors to “read-through” a UGA-stop codon that specifies SEC incorporation at the active site of the enzyme. These factors include a selenocysteine incorporation sequence (SECIS) in the 3′-untranslated region of the GPx1 mRNA and cofactors involved in the biosynthesis and translational insertion of SEC. To monitor SEC incorporation, we used a reporter gene system that has a UGA codon within the protein-coding region of the luciferase mRNA. Addition of either the GPx1 or GPx3 SECIS element in the 3′-untranslated region of the luciferase gene stimulated read-through by 6–11-fold in selenium-replete cells; absence of selenium prevented translation. To alter cellular homocysteine production, we used methionine in the presence of aminopterin, a folate antagonist, co-administered with hypoxanthine and thymidine (HAT/Met). This treatment increased homocysteine levels in the media by 30% (p< 0.01) and decreased GPx1 enzyme activity by 45% (p= 0.0028). HAT/Met treatment decreased selenium-mediated read-through significantly (p< 0.001) in luciferase constructs containing the GPx1 or GPx3 SECIS element; most importantly, the suppression of selenium-dependent read-through was similar whether an SV40 promoter or the GPx1 promoter was used to drive transcription of the SECIS-containing constructs. Furthermore, HAT/Met had no effect on steady-state GPx1 mRNA levels but decreased GPx1 protein levels, suggesting that this effect is not transcriptionally mediated. These data support the conclusion that homocysteine decreases GPx1 activity by altering the translational mechanism essential for the synthesis of this selenocysteine-containing protein.
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Bing, Xiao-Li, Dian-Shu Zhao, Jing-Tao Sun, Kai-Jun Zhang i Xiao-Yue Hong. "Genomic Analysis of Wolbachia from Laodelphax striatellus (Delphacidae, Hemiptera) Reveals Insights into Its “Jekyll and Hyde” Mode of Infection Pattern". Genome Biology and Evolution 12, nr 2 (20.01.2020): 3818–31. http://dx.doi.org/10.1093/gbe/evaa006.
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Abstract Wolbachia is a widely distributed intracellular bacterial endosymbiont among invertebrates. The wStriCN, the Wolbachia strain that naturally infects an agricultural pest Laodelphax striatellus, has a “Jekyll and Hyde” mode of infection pattern with positive and negative effects: It not only kills many offspring by inducing cytoplasmic incompatibility (CI) but also significantly increases host fecundity. In this study, we assembled the draft genome of wStriCN and compared it with other Wolbachia genomes to look for clues to its Jekyll and Hyde characteristics. The assembled wStriCN draft genome is 1.79 Mb in size, which is the largest Wolbachia genome in supergroup B. Phylogenomic analysis showed that wStriCN is closest to Wolbachia from Asian citrus psyllid Diaphorina citri. These strains formed a monophylogentic clade within supergroup B. Compared with other Wolbachia genomes, wStriCN contains the most diverse insertion sequence families, the largest amount of prophage sequences, and the most ankyrin domain protein coding genes. The wStriCN genome encodes components of multiple secretion systems, including Types I, II, IV, VI, Sec, and Tac. We detected three pairs of homologs for CI factors CifA and CifB. These proteins harbor the catalytic domains responsible for CI phenotypes but are phylogenetically and structurally distinct from all known Cif proteins. The genome retains pathways for synthesizing biotin and riboflavin, which may explain the beneficial roles of wStriCN in its host planthoppers, which feed on nutrient-poor plant sap. Altogether, the genomic sequencing of wStriCN provides insight into understanding the phylogeny and biology of Wolbachia.
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Squires, Jeffrey E., Ilko Stoytchev, Erin P. Forry i Marla J. Berry. "SBP2 Binding Affinity Is a Major Determinant in Differential Selenoprotein mRNA Translation and Sensitivity to Nonsense-Mediated Decay". Molecular and Cellular Biology 27, nr 22 (10.09.2007): 7848–55. http://dx.doi.org/10.1128/mcb.00793-07.
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ABSTRACT Selenoprotein mRNAs are potential targets for degradation via nonsense-mediated decay due to the presence of in-frame UGA codons that can be decoded as either selenocysteine or termination codons. When UGA decoding is inefficient, as occurs when selenium is limiting, termination occurs at these positions. Based on the predicted exon-intron structure, 14 of the 25 human selenoprotein mRNAs are predicted to be sensitive to nonsense-mediated decay. Among these, sensitivity varies widely, resulting in a hierarchy of preservation or degradation of selenoprotein mRNAs and, thus, of selenoprotein synthesis. Potential factors in dictating the hierarchy of selenoprotein synthesis are the Sec insertion sequence RNA-binding proteins, SBP2 and nucleolin. To investigate the mechanistic basis for this hierarchy and the role of these two proteins, we carried out knockdowns of SBP2 expression and assessed the effects on selenoprotein mRNA levels. We also investigated in vivo binding of selenoprotein mRNAs by SBP2 and nucleolin via immunoprecipitation of the proteins and quantitation of bound mRNAs. We report that SBP2 exhibits strong preferential binding to some selenoprotein mRNAs over others, whereas nucleolin exhibits minimal differences in binding. Thus, SBP2 is a major determinant in dictating the hierarchy of selenoprotein synthesis via differential selenoprotein mRNA translation and sensitivity to nonsense-mediated decay.
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Rose, R. Wesley, i Mechthild Pohlschröder. "In Vivo Analysis of an Essential Archaeal Signal Recognition Particle in Its Native Host". Journal of Bacteriology 184, nr 12 (15.06.2002): 3260–67. http://dx.doi.org/10.1128/jb.184.12.3260-3267.2002.
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ABSTRACT The evolutionarily conserved signal recognition particle (SRP) plays an integral role in Sec-mediated cotranslational protein translocation and membrane protein insertion, as it has been shown to target nascent secretory and membrane proteins to the bacterial and eukaryotic translocation pores. However, little is known about its function in archaea, since characterization of the SRP in this domain of life has thus far been limited to in vitro reconstitution studies of heterologously expressed archaeal SRP components identified by sequence comparisons. In the present study, the genes encoding the SRP54, SRP19, and 7S RNA homologs (hv54h, hv19h, and hv7Sh, respectively) of the genetically and biochemically tractable archaeon Haloferax volcanii were cloned, providing the tools to analyze the SRP in its native host. As part of this analysis, an hv54h knockout strain was created. In vivo characterization of this strain revealed that the archaeal SRP is required for viability, suggesting that cotranslational protein translocation is an essential process in archaea. Furthermore, a method for the purification of this SRP employing nickel chromatography was developed in H. volcanii, allowing the successful copurification of (i) Hv7Sh with a histidine-tagged Hv54h, as well as (ii) Hv54h and Hv7Sh with a histidine-tagged Hv19h. These results provide the first in vivo evidence that these components interact in archaea. Such copurification studies will provide insight into the significance of the similarities and differences of the protein-targeting systems of the three domains of life, thereby increasing knowledge about the recognition of translocated proteins in general.
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Huang, Ying, Xiaodong Liu, Zheng Cui, Daniel Wiegmann, Giuliana Niro, Christian Ducho, Yuan Song, Zhaoyong Yang i Steven G. Van Lanen. "Pyridoxal-5′-phosphate as an oxygenase cofactor: Discovery of a carboxamide-forming, α-amino acid monooxygenase-decarboxylase". Proceedings of the National Academy of Sciences 115, nr 5 (17.01.2018): 974–79. http://dx.doi.org/10.1073/pnas.1718667115.
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Capuramycins are antimycobacterial antibiotics that consist of a modified nucleoside named uridine-5′-carboxamide (CarU). Previous biochemical studies have revealed that CarU is derived from UMP, which is first converted to uridine-5′-aldehyde in a reaction catalyzed by the dioxygenase CapA and subsequently to 5′-C-glycyluridine (GlyU), an unusual β–hydroxy-α-amino acid, in a reaction catalyzed by the pyridoxal-5′-phosphate (PLP)-dependent transaldolase CapH. The remaining steps that are necessary to furnish CarU include decarboxylation, O atom insertion, and oxidation. We demonstrate that Cap15, which has sequence similarity to proteins annotated as bacterial, PLP-dependent l-seryl-tRNA(Sec) selenium transferases, is the sole catalyst responsible for complete conversion of GlyU to CarU. Using a complementary panel of in vitro assays, Cap15 is shown to be dependent upon substrates O2 and (5′S,6′R)-GlyU, the latter of which was unexpected given that (5′S,6′S)-GlyU is the isomeric product of the transaldolase CapH. The two products of Cap15 are identified as the carboxamide-containing CarU and CO2. While known enzymes that catalyze this type of chemistry, namely α-amino acid 2-monooxygenase, utilize flavin adenine dinucleotide as the redox cofactor, Cap15 remarkably requires only PLP. Furthermore, Cap15 does not produce hydrogen peroxide and is shown to directly incorporate a single O atom from O2 into the product CarU and thus is an authentic PLP-dependent monooxygenase. In addition to these unusual discoveries, Cap15 activity is revealed to be dependent upon the inclusion of phosphate. The biochemical characteristics along with initiatory mechanistic studies of Cap15 are reported, which has allowed us to assign Cap15 as a PLP-dependent (5′S,6′R)-GlyU:O2 monooxygenase-decarboxylase.
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Hoffman-Liebermann, B., D. Liebermann, L. H. Kedes i S. N. Cohen. "TU elements: a heterogeneous family of modularly structured eucaryotic transposons". Molecular and Cellular Biology 5, nr 5 (maj 1985): 991–1001. http://dx.doi.org/10.1128/mcb.5.5.991-1001.1985.
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We describe here a family of foldback transposons found in the genome of the higher eucaryote, the sea urchin Strongylocentrotus purpuratus. Two major classes of TU elements have been identified by analysis of genomic DNA and TU element clones. One class consists of largely similar elements with long terminal inverted repeats (IVRs) containing outer and inner domains and sharing a common middle segment that can undergo deletions. Some of these elements contain insertions. The second class is highly heterogeneous, with many different middle segments nonhomologous to those of the first-class and variable-sized inverted repeats that contain only an outer domain. The middle and insertion segments of both classes carry sequences that also are found unassociated from the inverted repeats at many other genomic locations. We conclude that the TU elements are modular structures composed of inverted repeats plus other sequence domains that are themselves members of different families of dispersed repetitive sequences. Such modular elements may have a role in the dispersion and rearrangement of genomic DNA segments.
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Hoffman-Liebermann, B., D. Liebermann, L. H. Kedes i S. N. Cohen. "TU elements: a heterogeneous family of modularly structured eucaryotic transposons." Molecular and Cellular Biology 5, nr 5 (maj 1985): 991–1001. http://dx.doi.org/10.1128/mcb.5.5.991.
Pełny tekst źródłaStreszczenie:
We describe here a family of foldback transposons found in the genome of the higher eucaryote, the sea urchin Strongylocentrotus purpuratus. Two major classes of TU elements have been identified by analysis of genomic DNA and TU element clones. One class consists of largely similar elements with long terminal inverted repeats (IVRs) containing outer and inner domains and sharing a common middle segment that can undergo deletions. Some of these elements contain insertions. The second class is highly heterogeneous, with many different middle segments nonhomologous to those of the first-class and variable-sized inverted repeats that contain only an outer domain. The middle and insertion segments of both classes carry sequences that also are found unassociated from the inverted repeats at many other genomic locations. We conclude that the TU elements are modular structures composed of inverted repeats plus other sequence domains that are themselves members of different families of dispersed repetitive sequences. Such modular elements may have a role in the dispersion and rearrangement of genomic DNA segments.
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Kamae, Tsuyoshi, Kazunobu Kiyomizu, Tsuyoshi Nakazawa, Seiji Tadokoro, Shigenori Honda, Hirokazu Kashiwagi, Yuzuru Kanakura i Yoshiaki Tomiyama. "Bleeding Tendency and Impaired Platelet Function In a Patient Carrying a Heterozygous Mutation In Thromboxane A2 Receptor". Blood 116, nr 21 (19.11.2010): 2524. http://dx.doi.org/10.1182/blood.v116.21.2524.2524.
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Abstract Abstract 2524 Characterization of inherited platelet function disorders has revealed critical molecules and receptors for hemostasis and thrombosis. Thromboxane A2 (TXA2) is one of the major platelet agonists, and thromboxane A2 receptors (TP) are seven transmembrane small G protein coupled receptor. There are two isoforms, α and β, which differ only their C-terminus. TPα is mainly expressed in platelet. In general, platelet receptor abnormalities such as Glanzmann thrombasthenia and P2Y12 deficiency are inherited in an autosomal recessive manner. In other words, a subject carrying a heterozygous mutation does not show any platelet functional abnormality. In this study, we examined a patient with bleeding tendency and platelet dysfunction, and demonstrated that the patient is heterozygous for a novel mutation in TP, G insertion at nt.167-8. The proband (OSP-2) was a 7-year-old Japanese girl, and had repeated nasal bleeding and mild purpura from 3 years old. The nasal bleeding was sometimes severe and continued for 1 hour to be stopped. She was born from non-consanguineous parents. She was referred to Osaka University Hospital because of suspected platelet dysfunction. There were no abnormalities in blood cell counts and coagulation. Bleeding time was slightly prolonged (7.5 minutes) and ADP-induced platelet aggregation was impaired. U46619 (2.5 mM)-induced platelet aggregation was remarkably impaired in the proband. The impaired platelet aggregation was still observed even at high concentration of U46619 (10 mM). Essentially the same platelet functional abnormality was observed in her father. Informed consent was obtained from the patient and her family members. We firstly confirmed that there is no abnormality in the expression of platelet glycoproteins such as αIIbβ3, GPIb-IX, GPVI and GPIa. Nucleotide sequence analysis of TP cDNA obtained from the proband revealed a novel mutation, G insertion between nt.167 and nt.168 leading to a frame shift. No other mutation was detected in the coding region of TP. Sequencing of genomic DNA showed that the mutation was heterozygous in her father as well as the proband. Thus, the abnormal allele was inherited from her father. The wild-type TP expression construct or the mutant construct in pcDNA3.1 vector indicated that the expression of the mutant TP was markedly impaired. Consistent with molecular genetic analysis, immnoblotting with polyclonal antibody against cytoplasmic domain demonstrated that the amount of TP in platelets from the proband and her father were approximately half of controls. P-selectin expression as well as PAC-1 binding on platelets obtained from the proband was markedly impaired when stimulated with U46619 but not with 0.5 U/ml thrombin or 20 μM ADP. Essentially the same defects were obtained in platelets from her father. To evaluate more precisely the dynamic changes in the αIIbβ3 activation, we performed initial velocity analysis for PAC-1 binding. In brief, FITC-PAC-1 was added to the activated platelets at the indicated time points after stimulation and incubated for only 30 s. Interestingly, we detected PAC-1 binding about 36∼42% of control on platelets from the proband and her father at 5 sec after stimulation with 5μM U46619. However, at 1 min and 5 min PAC1 binding velocity rapidly decreased, suggesting that the sustained αIIbβ3 activation rather than initiation of αIIbβ3 activation was impaired. Consistent with these data, Rap1 activation at 10 sec, 1 min and 5 min after stimulation of U46610 were 50%, 17%, and 11% of control, respectively. We have shown that the P2Y12 plays a critical role in the sustained αIIbβ3 activation (T Kamae, et al. J Thromb Haemost 2006; 4:1379–87) From these data, we assume that the impaired αIIbβ3 activation may be due to a reduction in ADP release from the proband. Indeed, small amount of exogenous ADP (0.5μM) significantly improved the impaired αIIbβ3 activation induced by U46619. Our data demonstrate that the impaired platelet function to U46619 observed in the patient with heterozygous mutation in TP is, at least in part, due to a decrease in granular secretion, especially ADP. Disclosures: No relevant conflicts of interest to declare.
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Premadasa, Lakmini, Gabrielle Dailey, Jan A. Ruzicka i Ethan Will Taylor. "Selenium-Dependent Read Through of the Conserved 3’-Terminal UGA Stop Codon of HIV-1 nef". American Journal of Biopharmacy and Pharmaceutical Sciences 1 (1.11.2021): 1. http://dx.doi.org/10.25259/ajbps_6_2021.
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Objectives: The HIV-1 nef gene terminates in a 3’-UGA stop codon, which is highly conserved in the main group of HIV-1 subtypes, along with a downstream potential coding region that could extend the nef protein by 33 amino acids, if readthrough of the stop codon occurs. It has been proposed that antisense tethering interactions (ATIs) between a viral mRNA and a host selenoprotein mRNA are a potential viral strategy for the capture of a host selenocysteine insertion sequence (SECIS) element. This mRNA hijacking mechanism could enable the expression of virally encoded selenoprotein modules, through translation of in-frame UGA stop codons as selenocysteine (Sec). Here, our aim was to assess whether readthrough of the 3’-terminal UGA codon of nef occurs during translation of HIV-1 nef expression constructs in transfected cells, and whether selenium-based mechanisms might be involved. Material and Methods: To assess UGA codon readthrough, we used fluorescence microscopy image analysis and flow cytometry of HEK 293 cells transfected with full length HIV-1 nef gene expression constructs including the 3’-UGA stop codon and a predicted thioredoxin reductase 1 (TXNRD1) antisense region spanning the UGA codon, engineered with a downstream in-frame green fluorescent protein (GFP) reporter gene. These were designed so that GFP can only be expressed by translational recoding of the UGA codon, that is, if the UGA codon is translated as an amino acid or bypassed by ribosomal hopping. To assess readthrough efficiency, appropriate mutant control constructs were used for 100% and 0% readthrough. We used anti-TXNRD1 siRNA to assess the possible role of the proposed antisense interaction in this event, by knockdown of TXNRD1 mRNA levels. Results: UGA stop codon readthrough efficiency for the wild-type nef construct was estimated by flow cytometry to be about 19% (P < 0.0001). siRNA knockdown of TXNRD1 mRNA resulted in a 67% decrease in GFP expression in this system relative to control cells (P < 0.0001), presumably due to reduced availability of the components involved in selenocysteine incorporation for the stop codon readthrough (i.e. the TXNRD1 SECIS element). Addition of 20 nM sodium selenite to the media enhanced stop codon readthrough in the pNefATI1 plasmid construct by >100% (P < 0.0001), that is, more than doubled the amount of readthrough product, supporting the hypothesis that selenium is involved in the UGA readthrough mechanism. Conclusion: Our results show that readthrough of the 3’-terminal UGA codon of nef occurs during translation of HIV-1 nef expression constructs in transfected cells, that this is dependent on selenium concentration, and the presence of TXNRD1 mRNA, supporting the proposed antisense tethering interaction.
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Lange, Sascha, W. Trent Franks, Nandhakishore Rajagopalan, Kristina Döring, Michel A. Geiger, Arne Linden, Barth-Jan van Rossum, Günter Kramer, Bernd Bukau i Hartmut Oschkinat. "Structural analysis of a signal peptide inside the ribosome tunnel by DNP MAS NMR". Science Advances 2, nr 8 (sierpień 2016): e1600379. http://dx.doi.org/10.1126/sciadv.1600379.
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Proteins are synthesized in cells by ribosomes and, in parallel, prepared for folding or targeting. While ribosomal protein synthesis is progressing, the nascent chain exposes amino-terminal signal sequences or transmembrane domains that mediate interactions with specific interaction partners, such as the signal recognition particle (SRP), the SecA–adenosine triphosphatase, or the trigger factor. These binding events can set the course for folding in the cytoplasm and translocation across or insertion into membranes. A distinction of the respective pathways depends largely on the hydrophobicity of the recognition sequence. Hydrophobic transmembrane domains stabilize SRP binding, whereas less hydrophobic signal sequences, typical for periplasmic and outer membrane proteins, stimulate SecA binding and disfavor SRP interactions. In this context, the formation of helical structures of signal peptides within the ribosome was considered to be an important factor. We applied dynamic nuclear polarization magic-angle spinning nuclear magnetic resonance to investigate the conformational states of the disulfide oxidoreductase A (DsbA) signal peptide stalled within the exit tunnel of the ribosome. Our results suggest that the nascent chain comprising the DsbA signal sequence adopts an extended structure in the ribosome with only minor populations of helical structure.
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Cohen, J. B., B. Hoffman-Liebermann i L. Kedes. "Structure and unusual characteristics of a new family of transposable elements in the sea urchin Strongylocentrotus purpuratus". Molecular and Cellular Biology 5, nr 10 (październik 1985): 2804–13. http://dx.doi.org/10.1128/mcb.5.10.2804-2813.1985.
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The transposable element family TU of the sea urchin Strongylocentrotus purpuratus, a higher eucaryote, has recently been described (D. Liebermann, B. Hoffman-Liebermann, J. Weinthal, G. Childs, R. Maxson, A. Mauron, S.N. Cohen, and L. Kedes, Nature [London] 306:342-347, 1983). A member of this family, TU4, has an insertion, called ISTU4, of non-TU DNA. ISTU4 is a member of a family of repetitive sequences, which are present in some 1,000 copies per haploid S. purpuratus genome (B. Hoffman-Liebermann, D. Liebermann, L.H. Kedes, and S.N. Cohen, Mol. Cell. Biol. 5:991-1001, 1985). We analyzed this insertion to determine whether it is itself a transposable element. The nucleotide sequence of ISTU4 was determined and showed an unusual structure. There are four, approximately 150 nucleotides long, imperfect direct repeats followed by a single truncated version of these repeats. This region is bounded at either side by approximately 100-nucleotide-long sequences that are not related to each other or to the repeats. Nucleotide sequences at the boundaries of ISTU4-homologous and flanking regions in five genomic clones show that ISTU4 represents a family of sequences with discrete ends, which we call Tsp elements. We showed that the genomic locus that carries a Tsp element in one individual was empty in other individuals and conclude that Tsp elements are a new and different type of transposable element. Tsp elements lack two features common to most other transposable elements: Tsp integration does not result in the duplication of host DNA, and there are no inverted repeats at their termini, although short inverted repeats are present at a distance from the termini.
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Cohen, J. B., B. Hoffman-Liebermann i L. Kedes. "Structure and unusual characteristics of a new family of transposable elements in the sea urchin Strongylocentrotus purpuratus." Molecular and Cellular Biology 5, nr 10 (październik 1985): 2804–13. http://dx.doi.org/10.1128/mcb.5.10.2804.
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The transposable element family TU of the sea urchin Strongylocentrotus purpuratus, a higher eucaryote, has recently been described (D. Liebermann, B. Hoffman-Liebermann, J. Weinthal, G. Childs, R. Maxson, A. Mauron, S.N. Cohen, and L. Kedes, Nature [London] 306:342-347, 1983). A member of this family, TU4, has an insertion, called ISTU4, of non-TU DNA. ISTU4 is a member of a family of repetitive sequences, which are present in some 1,000 copies per haploid S. purpuratus genome (B. Hoffman-Liebermann, D. Liebermann, L.H. Kedes, and S.N. Cohen, Mol. Cell. Biol. 5:991-1001, 1985). We analyzed this insertion to determine whether it is itself a transposable element. The nucleotide sequence of ISTU4 was determined and showed an unusual structure. There are four, approximately 150 nucleotides long, imperfect direct repeats followed by a single truncated version of these repeats. This region is bounded at either side by approximately 100-nucleotide-long sequences that are not related to each other or to the repeats. Nucleotide sequences at the boundaries of ISTU4-homologous and flanking regions in five genomic clones show that ISTU4 represents a family of sequences with discrete ends, which we call Tsp elements. We showed that the genomic locus that carries a Tsp element in one individual was empty in other individuals and conclude that Tsp elements are a new and different type of transposable element. Tsp elements lack two features common to most other transposable elements: Tsp integration does not result in the duplication of host DNA, and there are no inverted repeats at their termini, although short inverted repeats are present at a distance from the termini.
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Elbehery, Ali H. A., Ramy K. Aziz i Rania Siam. "Insertion sequences enrichment in extreme Red sea brine pool vent". Extremophiles 21, nr 2 (3.12.2016): 271–82. http://dx.doi.org/10.1007/s00792-016-0900-4.
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Schulze, E., S. Nagel, K. Gavenis i U. Grossbach. "Structurally divergent histone H1 variants in chromosomes containing highly condensed interphase chromatin." Journal of Cell Biology 127, nr 6 (15.12.1994): 1789–98. http://dx.doi.org/10.1083/jcb.127.6.1789.
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Condensed and late-replicating interphase chromatin in the Dipertan insect Chironomus contains a divergent type of histone H1 with an inserted KAP-KAP repeat that is conserved in single H1 variants of Caenorhabditis elegans and Volvox carteri. H1 peptides comprising the insertion interact specifically with DNA. The Chironomid Glyptotendipes exhibits a corresponding correlation between the presence of condensed chromosome sections and the appearance of a divergent H1 subtype. The centromere regions and other sections of Glyptotendipes barbipes chromosomes are inaccessible to immunodecoration by anti-H2B and anti-H1 antibodies one of which is known to recognize nine different epitopes in all domains of the H1 molecule. Microelectrophoresis of the histones from manually isolated unfixed centromeres revealed the presence of H1 and core histones. H1 genes of G. barpipes were sequenced and found to belong to two groups. H1 II and H1 III are rather similar but differ remarkably from H1 I. About 30% of the deduced amino acid residues were found to be unique to H1 I. Most conspicuous is the insertion, SPAKSPGR, in H1 I that is lacking in H1 II and H1 III and at its position gives rise to the sequence repeat SPAKSPAKSPGR. The homologous H1 I gene in Glyptotendipes salinus encodes the very similar repeat TPAKSPAKSPGR. Both sequences are structurally related to the KAPKAP repeat in H1 I-1 specific for condensed chromosome sites in Chironomus and to the SPKKSPKK repeat in sea urchin sperm H1, lie at almost the same distance from the central globular domain, and could interact with linker DNA in packaging condensed chromatin.
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Galbraith, James D., Alastair J. Ludington, Kate L. Sanders, Alexander Suh i David L. Adelson. "Horizontal transfer and subsequent explosive expansion of a DNA transposon in sea kraits ( Laticauda )". Biology Letters 17, nr 9 (wrzesień 2021): 20210342. http://dx.doi.org/10.1098/rsbl.2021.0342.
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Transposable elements (TEs) are self-replicating genetic sequences and are often described as important ‘drivers of evolution’. This driving force is because TEs promote genomic novelty by enabling rearrangement, and through exaptation as coding and regulatory elements. However, most TE insertions potentially lead to neutral or harmful outcomes, therefore host genomes have evolved machinery to suppress TE expansion. Through horizontal transposon transfer (HTT) TEs can colonize new genomes, and since new hosts may not be able to regulate subsequent replication, these TEs may proliferate rapidly. Here, we describe HTT of the Harbinger-Snek DNA transposon into sea kraits ( Laticauda ), and its subsequent explosive expansion within Laticauda genomes. This HTT occurred following the divergence of Laticauda from terrestrial Australian elapids approximately 15–25 Mya. This has resulted in numerous insertions into introns and regulatory regions, with some insertions into exons which appear to have altered UTRs or added sequence to coding exons. Harbinger-Snek has rapidly expanded to make up 8–12% of Laticauda spp. genomes; this is the fastest known expansion of TEs in amniotes following HTT. Genomic changes caused by this rapid expansion may have contributed to adaptation to the amphibious-marine habitat.
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Collinson, I. "The structure of the bacterial protein translocation complex SecYEG". Biochemical Society Transactions 33, nr 6 (26.10.2005): 1225–30. http://dx.doi.org/10.1042/bst0331225.
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Proteins destined for secretion, membrane insertion or organellar import contain signal sequences that direct them to the membrane. Once there, transport machines receive and translocate them appropriately across or into the membrane. The related SecY and Sec61 protein translocation complexes are ubiquitous components of machines that are essential for protein transport. They co-operate with various partners such that the substrate polypeptide is pulled or pushed through the membrane by post- or co-translational mechanisms. In bacteria and archaea, the SecY complex (SecYEG/SecYEβ) is a heterotrimer, which associates with ribosomes so that the polypeptide is threaded through the channel during its synthesis. Bacteria possess an additional pathway, whereby the newly synthesized substrate protein is maintained in an unfolded conformation and is engaged by the ATPase SecA and delivered to the translocon. Recent medium- (cryo-electron microscopy) and high-resolution (X-ray) structures of the Sec complex have dramatically increased our understanding about how proteins pass through membranes, but have posed a number of new questions. The Sec complex is active as an oligomer, but the structure indicates that the protein-conducting channel is formed by a monomer of SecYEG. Structures of the membrane-bound dimer of Escherichia coli SecYEG and the detergent-solubilized monomer of Methanococcus jannaschii SecYEβ will be described and discussed in the context of the mechanism that underlies protein secretion and membrane insertion.
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Johansen, Steinar Daae, Sylvia I. Chi, Arseny Dubin i Tor Erik Jørgensen. "The Mitochondrial Genome of the Sea Anemone Stichodactyla haddoni Reveals Catalytic Introns, Insertion-Like Element, and Unexpected Phylogeny". Life 11, nr 5 (28.04.2021): 402. http://dx.doi.org/10.3390/life11050402.
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A hallmark of sea anemone mitochondrial genomes (mitogenomes) is the presence of complex catalytic group I introns. Here, we report the complete mitogenome and corresponding transcriptome of the carpet sea anemone Stichodactyla haddoni (family Stichodactylidae). The mitogenome is vertebrate-like in size, organization, and gene content. Two mitochondrial genes encoding NADH dehydrogenase subunit 5 (ND5) and cytochrome c oxidase subunit I (COI) are interrupted with complex group I introns, and one of the introns (ND5-717) harbors two conventional mitochondrial genes (ND1 and ND3) within its sequence. All the mitochondrial genes, including the group I introns, are expressed at the RNA level. Nonconventional and optional mitochondrial genes are present in the mitogenome of S. haddoni. One of these gene codes for a COI-884 intron homing endonuclease and is organized in-frame with the upstream COI exon. The insertion-like orfA is expressed as RNA and translocated in the mitogenome as compared with other sea anemones. Phylogenetic analyses based on complete nucleotide and derived protein sequences indicate that S. haddoni is embedded within the family Actiniidae, a finding that challenges current taxonomy.
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Katokhin, A. V., i Yu M. Kornyychuk. "First data of ITS1-genotyping of the Black Sea trematodes Cainocreadium and Helicometra (Trematoda: Opecoelidae)". Marine Biological Journal 3, nr 4 (28.12.2018): 36–42. http://dx.doi.org/10.21072/mbj.2018.03.4.04.
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Data of genetic analysis of the Black Sea trematodes of Cainocreadium genus and Helicometra fasciata were obtained for the first time. The nucleotide sequences of ITS1 rRNA gene cluster of Cainocreadium flesi from Platichthys flesus (GenBank entries MG980645, MG980646) and Cainocreadium sp. from Gaidropsarus mediterraneus (MG980643, MG980644, MK248037, MK248038) off Crimean Black Sea coast were found to be identical. Nevertheless, they have not been synonymized because of morphological differences known between these hostal morphs. Sequences of Cainocreadium from the Black Sea fish turned out to differ, by 4 positions, from similar sequences of a closely related Mediterranean congener, C. labracis (cercaria). Five insertions-deletions and 38 nucleotide sequences distinguish the ITS1 sequences of the Black Sea trematodes, C. flesi and Cainocreadium sp., from ITS1 sequences of another Mediterranean congener, C. dentecis. The ITS1 sequences of the Black Sea and Mediterranean Helicometra fasciata samples also differ: 5 nucleotide changes and 11 insertions-deletions were identified. Supplementary data associated with this article can be found in online version at https://doi.org/10.21072/mbj.2018.03.4.04.
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Yager, Lawrence N., John F. Kaumeyer, Insong Lee i Eric S. Weinberg. "Insertion of an intermediate repetitive sequence into a sea urchin histone-gene spacer". Journal of Molecular Evolution 24, nr 4 (kwiecień 1987): 346–56. http://dx.doi.org/10.1007/bf02134133.
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Jeong, Min-A., Ye-Jin Jeong i Kwang-Il Kim. "Complete Genome Sequences and Pathogenicity Analysis of Two Red Sea Bream Iridoviruses Isolated from Cultured Fish in Korea". Fishes 6, nr 4 (15.12.2021): 82. http://dx.doi.org/10.3390/fishes6040082.
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In Korea, red sea bream iridovirus (RSIV), especially subtype II, has been the main causative agent of red sea bream iridoviral disease since the 1990s. Herein, we report two Korean RSIV isolates with different subtypes based on the major capsid protein and adenosine triphosphatase genes: 17SbTy (RSIV mixed subtype I/II) from Japanese seabass (Lateolabrax japonicus) and 17RbGs (RSIV subtype II) from rock bream (Oplegnathus fasciatus). The complete genome sequences of 17SbTy and 17RbGs were 112,360 and 112,235 bp long, respectively (115 and 114 open reading frames [ORFs], respectively). Based on nucleotide sequence homology with sequences of representative RSIVs, 69 of 115 ORFs of 17SbTy were most closely related to subtype II (98.48–100% identity), and 46 were closely related to subtype I (98.77–100% identity). In comparison with RSIVs, 17SbTy and 17RbGs carried two insertion/deletion mutations (ORFs 014R and 102R on the basis of 17SbTy) in regions encoding functional proteins (a DNA-binding protein and a myristoylated membrane protein). Notably, survival rates differed significantly between 17SbTy-infected and 17RbGs-infected rock breams, indicating that the genomic characteristics and/or adaptations to their respective original hosts might influence pathogenicity. Thus, this study provides complete genome sequences and insights into the pathogenicity of two newly identified RSIV isolates classified as a mixed subtype I/II and subtype II.
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Matsumoto, Gen, Takayuki Homma, Hiroyuki Mori i Koreaki Ito. "A Mutation in secY That Causes Enhanced SecA Insertion and Impaired Late Functions in Protein Translocation". Journal of Bacteriology 182, nr 12 (15.06.2000): 3377–82. http://dx.doi.org/10.1128/jb.182.12.3377-3382.2000.
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ABSTRACT A cold-sensitive secY mutant (secY125) with an amino acid substitution in the first periplasmic domain causes in vivo retardation of protein export. Inverted membrane vesicles prepared from this mutant were as active as the wild-type membrane vesicles in translocation of a minute amount of radioactive preprotein. The mutant membrane also allowed enhanced insertion of SecA, and this SecA insertion was dependent on the SecD and SecF functions. These and other observations suggested that the early events in translocation, such as SecA-dependent insertion of the signal sequence region, is actually enhanced by the SecY125 alteration. In contrast, since the mutant membrane vesicles had decreased capacity to translocate chemical quantity of pro-OmpA and since they were readily inactivated by pretreatment of the vesicles under the conditions in which a pro-OmpA translocation intermediate once accumulated, the late translocation functions appear to be impaired. We conclude that this periplasmicsecY mutation causes unbalanced early and late functions in translocation, compromising the translocase's ability to catalyze multiple rounds of reactions.
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Terwilliger, David P., Katherine M. Buckley, Dhruti Mehta, Priya G. Moorjani i L. Courtney Smith. "Unexpected diversity displayed in cDNAs expressed by the immune cells of the purple sea urchin, Strongylocentrotus purpuratus". Physiological Genomics 26, nr 2 (lipiec 2006): 134–44. http://dx.doi.org/10.1152/physiolgenomics.00011.2006.
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We recently identified a unique family of transcripts, the 185/333 family, that comprise ∼60% of the mRNAs induced by coelomocytes from the purple sea urchin in response to immunological challenge from lipopolysaccharide. An analysis of 81 full-length cDNAs revealed 67 unique nucleotide sequences encoding 64 different proteins. Diversity of the transcripts was based on 25 sequence blocks, or “elements,” which resulted in 22 different element patterns based on their presence or absence. Furthermore, there was a high level of nucleotide variation within elements, including single nucleotide polymorphisms and insertions/deletions, both of which resulted in amino acid sequence variability. The deduced 185/333 proteins contained an NH2-terminal leader, a glycine-rich region with an RGD motif, a histidine-rich region, and a COOH-terminal region. Two 185/333 genes, identified in the partially assembled Strongylocentrotus purpuratus genome, have two exons. The first encoded the leader, and the second encoded the remainder of the predicted protein. Estimates from quantitative PCR indicated that there were ∼100 alleles in the diploid genome. These results suggested that the purple sea urchin may have mechanisms for generating high levels of diversity in response to immunological challenge that have not been considered previously.
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Sabath, DE, EA Spangler, EM Rubin i G. Stamatoyannopoulos. "Analysis of the human zeta-globin gene promoter in transgenic mice". Blood 82, nr 9 (1.11.1993): 2899–905. http://dx.doi.org/10.1182/blood.v82.9.2899.2899.
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Abstract zeta-Globin is the embryonic form of the alpha chain of hemoglobin. Transgenic mice generated with zeta-globin constructs containing the zeta-globin gene, 557 bp of 5′ flanking sequence, and 2-kb of 3′ flanking sequence linked to the beta-globin locus control region hypersensitive site 2 (HS2) expressed human zeta-globin only in embryonic yolk sac erythroid tissue, and not in definitive erythroid tissue in the fetal liver or in adult peripheral blood. To determine what sequences in the 5′ flanking region of the zeta-globin gene might be important for developmental specificity, a series of 5′ deletion constructs of the zeta-globin gene were made and used to generate transgenic mice. The 5′ ends of these constructs were located 417, 207, and 128 bp 5′ to the zeta-globin transcriptional start site, and HS2 was included to increase the level of erythroid-specific expression. In all lines of mice tested, human zeta-globin was expressed only in embryonic tissue, and not in fetal livers or in adult peripheral blood. Expression was independent of copy number and appeared to be dependent on the site of transgene insertion. These data suggest that the proximal 128 bp of the zeta-globin promoter is sufficient to properly regulate zeta-globin expression during development.